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Pathology: Introduction & Neoplasia

Table of Contents
Interface of Pathology and Clinical Medicine .............................................................................................................1
Cellular Pathology I .....................................................................................................................................................2
Cellular Pathology II ....................................................................................................................................................4
Inflammation I – Acute Inflammation ........................................................................................................................6
Inflammation II – Chronic Inflammation ....................................................................................................................9
Biology of Human Neoplasia: Introduction and Overview ...................................................................................... 12
Pathology of Human Neoplasia (The Practical Issues) ............................................................................................ 15
Molecular genetics of cancer .................................................................................................................................. 18

Interface of Pathology and Clinical Medicine

Pathology: pathos = suffering; logos = the study of

Identical clinical presentations can be caused by dramatically different pathologies; different pathologies will
require different treatments.

Famous people with pathology include Hubert Humphrey, Sergi Grinchov, the anonymous roofer, Elvis, and JFK.

Osler is responsible for 95% of all medical aphorisms.

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Cellular Pathology I

Four pillars of pathology:


 Etiology: what initiates a process?
 Pathogenesis: what is its mechanism?
 Morphology: how is it recognized?
 Functional consequences: how does it produce disease?

Disturbance of homeostasis:
 Stress or increased demand can be met by adaptation; injurious stimuli may lead to cell injury / death.
 Failure to adapt can lead to injury/death as well: e.g. adaptations to long-standing hypertension can
predispose to sudden MI.

 Hypoxia: reduction or absence of a normal oxygen supply to an organ


(may result from ischemia = ischemic hypoxia)
 Ischemia: reduction / absence of blood supply to an organ or tissue
 Infarction: death of portion of tissue as result of ischemia (infarction = process, infarct = result)
 White infarct: organs where there’s one blood supply (liver, kidneys, spleen) – wedge shaped infart
downstream of blockage
 Red infarct: main blood supply cut off, reperfused by secondary blood supply (e.g. lung)

Mechanisms of cell injury (inter-dependent & synergistic):


 Decrease in ATP
o Example toxin: cyanide
 Increased (or dec.) intracellular Ca+2
o Increase because Ca/Mg pumps shut down (↓ATP)
o Leads to overactive enzymes (phospholipase, endonuclease, ATPase, protease) – damage
membrane, DNA, etc.
o Example toxin: glutamate excitotoxicity in neurons
 Reactive oxygen species – unpaired e- in outer orbit; leads to oxidative damage
o Superoxide, H2O2, OH- or reactive nitrogen species too
o Endogenous sources (metabolism, enzymes, ox-phos, inflammatory cells)
o Exogenous sources (O2 toxicity, chemicals, radiation, reperfusion injury)
o Usually in balance, but oxidative stress may occur if endogenous anti-oxidants overwhelmed
 Aging, diabetes, alzheimer’s, smoking, cancer, atherosclerosis, etc.
o Example toxin: acetaminophen in liver
 Membrane damage  if irreversible damange, considered the “point of no return”
o Example toxin: complement from immune system

Reversible injury: shut down ox-phos, ↓ATP. Morphological features:


 Swelling of organelles – ER, mito – and membrane blebbing (Na/K pumps shut down). Organelle
changes a.k.a. hydropic/vacuolar degeneration
 Clumping of chromatin (↑anaerobic glycolisis (lactic acid),↓pH, chromatin begins to clump)
 Lipid deposition (↓ protein synthesis; lipids can’t be attached to proteins & build up in cell). A.k.a. fatty
change (steatosis), seen in liver & myocardium
Irreversible when membrane damage starts to occur; time to “point of no return” depends on tissue.

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Reperfusion injury: previously ischemic area reperfused; inflammatory cells all enter at once; big influx of ROS
and calcium (pumps damaged) – may cause irreversible changes in cells.

Necrosis: morphological changes in nucleus & cytoplasm occurring after cell death in a living tissue. (two key
points: cell now dead but host was alive when it happened). Features:
 Eosinophilia (loss of RNA/ribosomes; proteins denatured). Looks more pink.
 Nuclear features: pyknosis (dark, shrunken), karyorrhexis (broken down), karyolysis (totally dissolved)
 Interstitial features: inflammation (need to be alive for this to happen

Subtypes of necrosis:
 Coagulative: after infarction (ischemic cell death) in solid tissues except brain. Most common.
o Tissue architecture looks same, “tombstones” of hyper-eosinophilic cells (more pink)
o Usually resolves as scar after neutrophils, macrophages scavange
 Liquefactive: after infarction in brain.
o Tissue architecture lost, complete hydrolysis / digestion of dead cells
o Resolves by cyst/cavity formation
o Abscess: Liquefactive necrosis as result of localized bacterial infections (fungal, parasitic)
 Accumulation of neutrophils within abscess cavity (making hydrolytic enzymes)
o Pus: dead neutrophils / cell debris
 Requires surgical drainage
 Caseous: “cheese-like”; after TB/fungal infections in immunocompetent individual
 Granuloma: Necrotic center surrounded by rim of inflammatory cells
 Fat necrosis: post-release of pancreatic lipase
o Membrane lipids broken down to FFAs, add calcium = saponification (calcium/fat deposits)

Amount of tissue damaged permanently can depend on quickness of reperfusion (e.g. post-MI or stroke).
Functional consequences can vary for same etiology, pathology, etc.

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Cellular Pathology II

Hyperplasia: increase in the number of cells in tissue/organ. May or may not include hypertrophy.
 Physiologic: e.g. compensatory hyperplasia (e.g. liver), lactating breast.
 Pathologic
o endometrial hyperplasia (pituitary-gonadal axis abnormalities, menorrhagia = heavy bleeding),
o benign prostatic hyperplasia (includes secondary hypertrophy of bladder muscle)

Hypertrophy: increase in individual cell mass, leading to increase in organ mass. Reversible, response to stimulus
 Physiologic: muscle hypertrophy after working out
 Pathologic: hypertrophic myocardium (↑cytoplasm, ↑nucleus size = “boxcar nucleus”). Could be from
chronic hypertension, aortic valve disease, or some other chronic hemodynamic overload.
 Etiology:
o hormone-induced (uterus & breast in pregnancy),
o increased workload (pumping iron or pathologic / cardiac muscle)
o genetic causes (myostatin mutation)

Atrophy: cellular shrinkage due to loss of substance.


 Denervation (e.g. poliovirus infecting neurons innervating skeletal mm)
 Disuse (e.g. hand with a cast on)
 Hormonal: menopause (↓estrogen, endometrium from proliferative to cystic atrophy, can lead to
irritation & atrophic vaginitis)
 Senile atrophy (brain decreases in size with age)
 Nutritional atrophy
o Marasmus: protein-calorie malnutrition, swollen stomach b/c of lowered oncotic pressure
o Cachexia: severe muscle wasting (AIDS, cancer, other chronic inflammation)
Cellular atrophy may include progressive cell loss so tissue / organ can shrink as well (gross scale atrophy too)

Metaplasia: reversible replacement of one differentiated cell type by another differentiated cell type.
Adaptive substitution (new cells can better withstand environment)
 Smoking-associated squamous metaplasia – better able to withstand tobacco insult
o Reserve cell metaplasia (change in reserve cell population, which are reprogrammed over time
to develop into squamous cells rather than columnar epithelium)
o May undergo neoplastic progression (normal  metaplasia  dysplasia  cancer) especially if
insult continues.
o Example: Barrett esophagus (squamous  columnar to withstand acid at gastroesophageal
junction).

Dysplasia: epithelium starts to exhibit abnormal changes; pre-cancerous but mutations starting to occur

Intracellular accumulations: cells can accumulate exogenous or endogenous substances


 Anthracosis: universal finding in people who have lived in city. Black streaks = macrophages that
phagocytosed carbon. No clinical importance
 Lipofuscin: golden brown “wear-and-tear” pigment; tombstone of lipid peroxidation
 Fatty change: absolute increase in intracellular lipids. Potentially reversible. Most common in liver
o Causes: Morbid obesity (diffuse), alcohol abuse (may have central lobule sparing)

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o Does have clinical implications – can be irreversible if hepatocytes die  fibrosis  cirrhosis
Apoptosis: programmed cell death.
 Physiologic: embryogenesis, hormone-dependent (menstruation), mature tissue homeostasis
 Pathologic:
o Response to DNA damage from radiation, free radicals, etc. (via p53)
o Viral infections (viral hepatitis)
o Cytotoxic T-cell mediated injury (transplant rejection or autoimmune conditions)
 Mediators (KNOW THIS)
o Caspases: cysteine proteases that play essential role in execution phase of apoptosis. Require
activation from inactive form via activation cascade
o Bcl-2: anti-apoptotic protein (but bcl-2 family contains both pro- and anti-apoptotic proteins)
o p53: stops cell division in response to DNA damage to facilitate recovery; if recovery fails 
apoptosis.
Morphology of apoptosis:
Characteristic Apoptosis Necrosis
 Specific cells affected Stimulus Usually physiologic Pathologic
(necrosis = sheet of cells)
Involvement Single cells Groups of cells
 Organized process;
Chromatin Uniformly dense masses No pattern
systematic breakdown of
DNA fragmentation Inter-nucleosomal Random
DNA (necrosis = smear)
Cell morphology Apoptotic bodies Swelling, degen.
Inflammation Absent Present

Inhibit apoptosis = facilitate tumorigenesis


 HPV: carcinogen (squamous cell carcinoma of cervix) – HPV abrogates function of p53, p21
 Follicular lymphoma – constitutive activation of Bcl-2

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Inflammation I – Acute Inflammation

Inflammation: a complex response of vascularized tissues to various stimuli, leading to the accumulation of
fluids and leukocytes in the extravascular tissues.

Triggers include trauma, ischemia, neoplasm, infection, foreign matter, immune rxns, etc.

Edema: excess of fluid in interstitial spaces or serous cavities (e.g. pleuroa, pericardium, peritoneum)
 Transudate: edema with low protein content due to ↑ hydrostatic pressure
 Exudate: edema with high protein content, often containing blood cells, due to ↑hydrostatic pressure
and ↑ vascular permeability
o Serous: exudate with few inflammatory cells (pale yellow)
o Serosanginous: exudate with erythrocytes (red tinged)
o Fibrinous: contains large amounts of fibrin (after coagulation of clotting factors)
o Purulent: high inflammatory cell content (often with bacterial infections)
o Supperative: purulent exudate with significant pus (liquefactive necrosis)

What is inflammation trying to do? Deliver effector cells and molecules, provide physical barrier via
microvascular coagulation to prevent spread, promote repair of offending tissue.

Acute inflammation: early & immediate response (minutes to days).


 Characteristics:
o Edema (exudate of fluids & plasma proteins)
o Emigration of leukocytes (esp. neutrophils)
 Triple response of Lewis (histidine mediated):
1. Transient vasoconstriction
2. Wheal (fluid leakage)
3. Flare (vasodilation)
 Steps:
1. Changes in vascular caliber and flow
 Continuity equation: velocity = flow rate / cross sec. area (wider = slower velocity)
 Poiseuille’s law: flow rate increases with r4 (wider = more flow through)
 Bernoulli’s principle: velocity & pressure related inversely
 In acute inflammation: transient vasoconstriction of arterioles followed by vasodilation
of arterioles & capillary beds
 Blood flow increases (Poiseuille’s): heat & redness
 Blood velocity decreases (continuity): blood stasis
 Hydrostatic pressure increases (Bernouilli): extravasation & exudate
2. Increased vascular permeability (leakage)
 Plasma proteins lost so intravascular oncotic pressure drops (higher now in interstitum).
Flow of water out (oncotic & hydrostatic pressure too) when normal balance disrupted
 Results in tumor = swelling (edema)
 Mechanisms
 Contraction of endothelial cells in venules (most common: separation of
junctions; chemically mediated by histidine, reversible & short-lived – 15-30m)
 Reorganization of cytoskeleton in endothelial cells, transcytosis, direct
endothelial injury can play a role too

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3. Leukocytes extravasate & phagocytose
 Want to kill microbes, ingest offending agents, degrade necrotic tissue
 Lots of blood cells extravasate, not just leukocytes (RBC, platelets, etc)
 Glucocorticoids help reduce inflammation by decreasing extravasation

Role of leukocytes (extravasation & phagocytosis)


Granulocytes are key in acute inflammation
 Neutrophils especially (eosinophils for allergies / parasitic infections, basophils & mast cells release
histadine for allergic hypersensitivity, monocytes in chronic inflammation)
 Time course:
1. Neutrophils (6-24h)
2. Monocytes / macrophages (24-48 hrs)
3. Lymphocytes (end, except viral infections where they can be first)
Sequence:
1. Extravasation
a. Margination: larger cells pushed to edge of vessel (RBC in central column) – pushed out even
farther during inflammation
b. Rolling: tumbling & transient halting.
i. Selectins: bone-marrow-derived & endothelial cell expression only
1. Slow down leukocyte under flow & signalling properties (rolling)
2. Ca dependent, carbohydrate binding proteins
3. Example: l-selectin on leukocyte, binds to GlyCam1 addressin (lymph node HEV)
ii. Addressins: expressed on endothelial cells on different sites, bind to homing receptors
on lymphocytes
c. Adhesion: firm attachement to endothelial surfaces. Mediated by complementary molecules.
i. Integrins: α/ß subunit heterodimers from IgG family with lots of types.
1. Classical examples are VLA4 (leukocyte) – VCAM1 (endothelial cell) and LFA1
(leukocyte) – ICAM1 (endothelial cell).
2. LFA1-ICAM1 binding is Mg dependent (↑affinity) and Ca dependent (↑avidity)
d. Diapedesis: passage across endothelium through intercellular junctions
i. Happens in venules (no smooth mm in wall)
ii. Endothelium to basement membrane, where they secrete collagenase to break down
e. Chemotaxis: direction to site of injury under influence of chemotactic agents; move using
pseudopod
i. Exogenous: bacterial products
ii. Endogenous: complement (c5a), lipoxygenase pathway products, chemokines (specific
for cell types, lots known)
2. Phagocytosis
a. What to eat?
i. Opsonic phagocytosis: target covered by something (Fc receptors for Ab Fc segment,
complement receptors for C3b)
ii. Non-opsonic phagocytosis: pathogen associated molecular patterns (PAMPs) (Mannose,
formyl-peptide, toll-like receptors for LPS, etc)
b. Target identified, internalized via Ca-dependent process, phagosome fuses with lysosomes &
secretory vessels to destroy & excrete waste.
3. Microbial killing
a. Neutrophils make microbicidal free radicals
i. NADPH oxidase reduces O2 to superoxide anion

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ii. Superoxide converted hydrogen peroxide by spontaneous dismutation
iii. H2O2 is killing molecule (and other ROS/RNS)
b. Microbial response: catalase degrades H2O2 to H2O and O2
i. Pts with Chronic Granulomatous Disease lack NADPH oxidase system genes, susceptible
to infections by catalase positive microganisms

Chemical mediators of inflammation: vasoactive amines (histamine, serotonin), plasma proteases


(complement, kinins, clotting), arachadonic acid metabolites (prostaglandins, leukotrienes), cytokines – most
involved in vascular permeability regulation too (no surprise)

Complement puts holes in the target, kinin is involved in vasodilation and smooth mm relaxation, clotting
involves fibrin depositing, cyclooxygenase is involved in prostaglandin formation (COX), cytokines & others get
in play too.

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Inflammation II – Chronic Inflammation

Outcomes of acute inflammation:


1. Complete resolution
2. Healing by connective tissue replacment (granulation tissue / organization; fibrosis)
3. Abcess formation
4. Chronic inflammation

Granulation tissue / organization


 After big tissue damage / fibrin exudation
 Buds of endothelial cells  grow and canalize / anastamose
 Macrophages migrate in
 Myofibroblasts & fibroblasts appear, proliferate, form collagen fibers
o Fibrosis: excessive deposition of collagen fibers
 Appearance: packed with cells, juicy, capillaries, collagen with fibroblasts, macrophages removing stuff
 Organization: granulation tissue replacing damaged tissue
o Inflammation
o Wound healing
o Infarct
o Thrombus

Fibroblasts: cytokine-mediated; produce collagen and ECM proteins


Keloid: excessive formation of collagenous tissue resulting in raised area of scar tissue (broad bands of
collagen replacing normal dermal structures
Cirrhosis in liver is result of inflammation  fibrous tissue (collagen) process

Example of organization: pleura following pneumonia, fibrin exudate, forms pleural adhesion

Abcess: focal, localized collection of pus in a newly formed cavity


 Pus in other cavities has different names (empyema in lungs, pyosalpinx in fallopian tubes, etc.)
 Pus = purulent exudate with neutrophils, necrotic cells, edema fluid
 Typically caused by Staphylococci (pyogenic bacteria)
 Appearance:
o Central: necrotic white cells & tissue cells
o Around that: preserved neutrophils
o Outer region: vascular dilation and fibroblastic proliferation (“pyogenic membrane”)
 Can become walled off by connective tissue (body can’t access)
 Can empty through fistula: pathologic channel connecting abcess to internal cavity / body surface

Phlegmon = cellulitis = opposite of an abcess (acute, overwhelming infection spreading along skin – S.
aureus & group A streptococci)

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Chronic Inflammation:
a prolonged process where acute inflammation and destruction
proceed at the same time as healing / immune response (balance)

Causes: anything that causes acute inflammation (if it persists), infections, autoimmunity (most common in US),
alloimmunity (transplants), foreign materials (insoluble, inanimate)

Clinical classification:
 Primary: de novo cause (no clinically evident acute inflammation)
 Secondary to acute inflammation
Histological classification:
 Macrophagic (diffuse or granulomatous), e.g. TB
 Lymphocytic (diffuse or focal / follicle formation), e.g. autoimmunity
 Supperative (lots of neutrophils, abcess formation) e.g. osteomyelitis

Macrophages: most important cell in chronic inflammation


 From bone marrow precursor
 Circulate in blood as monocytes (half life of 1 day)
 Migrate into tissue, transform into macrophages (half life of several months)
 Roles of macrophages / monocytes: phagocytosis, induce immune reactions via antigen presentation,
release signalling molecules
 Activation: macrophages with increased inflammatory capacities; main function is phagocytosis but also
release lots more substances (NO, ROS, proteases, cytokines, enzymes, grotwh factors, complement…)\
 Can also cause significant tissue damage (hallmark of chronic inflammation)

Macrophagic infiltration:
1. Granulomatous: macrophages arranged into compact masses (follicles); epitheloid appearance like a
fence or barracade.
a. Granuloma = focal area of granulomatous inflammation.
i. Small cluster of epitheloid cells surrounded by lymphocytes
ii. Caseation in middle, then epitheloid layer & macrophagic giant cells; ring of
lymphocytes then fibrous tissue walling off on outside.
iii. E.g. tuberculosis
b. Epitheloid cells: pale, pink, granular cytoplasm & indistinct cell boundaries; hypodense
elongated nucleous
c. Giant cells: fusion of 6-8 macrophages (epitheloid); can contain 20+ small nuclei
i. Langhans giant cell (peripheral/horse-shoe nuclei arrangement): chronic immune
granulomata like TB or sarcoidosis
ii. Foreign body giant cell (scattered nuclei throughout cytoplasm) – e.g. asbestosis
d. Foreign body granuloma: particulate mater in middle (too large for phagocytosis by one Mφ)
e. Immune granuloma: inducing cell-mediated immmunity, Mφ present to T-cells, T-cells produce
cytokines to transform Mφ to epitheloid & giant cells
i. E.g. TB: granuloma (“tubercle”) caused by M. tuberculosis (acid-fast), usually caseating
f. GRANULOMA ≠ GRANULATION TISSUE

2. Non-granulomatous: diffuse spread of macrophages

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Lymphocytic infiltration: hallmark of autoimmune diseases
 Collection of lymphocytes in an organ that doesn’t usually have them
 Diffuse or focal lymphocytic infiltrations
o Focal infiltrations: “ectopic follicles” – look just like lymph node follicles but elsewhere in body
 B-cells in center, T-cells in cortex
 Hashimoto’s thyroiditis: autoimmune reaction against thyroid (focal)
 MS: collection of lymphocytes like follicle in brain

Systemic effects of inflammation


 Fever
o Improve efficiency of leukocyte killing, impair replication of microorganisms
o Coordinated by hypothalamus
 Leukocytosis
o WBC >11,000/uL blood
o Accelerated release of cells from bone marrow (immature neutrophils = bands; “left shift”)
 Bacterial infection: neutrophilia
 Viral infection: lymphocytosis
 Parasitic infection: eosinophilia
 Leukopenia
o WBC < 4,000 / uL
o Typhoid fever, other infections, or when pts overwhelmed (disseminated TB, cancer, HIV)

INFLAMMATION SUMMARY
ACUTE CHRONIC
DURATION Short (days) Long (months-years)
ONSET Acute Insidious
INFLAMMATORY CELLS Neutrophils, macrophages Macrophages, Lymphocytes, Fibroblasts
VASCULAR CHANGES Vasodilation, leakage Angiogenesis (granulation tissue)
EDEMA Yes Usually no
CARDINAL CLINICAL SIGNS Yes Usually no
TISSUE NECROSIS No Yes (ongoing)
FIBROSIS No Yes (ongoing)
SYSTEMIC EFFECTS High fever Low-grade fever, weight loss, anemia
BLOOD CHANGES Neutrophilia, lymphocytosis Variable. Polyclonal
hypergammaglobulinemia

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Biology of Human Neoplasia: Introduction and Overview

Neoplasia: clonal proliferation of cells with somatic genetic alterations and aberrant regulation of growth
 Benign: don’t threaten life of neoplasm
 Malignant (cancer): ability to invade into normal tissues and metastasize into distant tissues

Neoplasms generally form masses (tumors) but some (e.g. pre-invasive or in situ neoplasms) don’t form visible
masses.

Key genetic defects in cancer cells:


 activate genes that stimulate cell replication (e.g. growth factor receptor kinases)
 inactivate genes that suppress cell replication

Many cancers do have increased growth (↑mitotic figures & growth fraction = proportion of cycling cells).
Others replicate at normal rate & suppress apoptosis (p53, bcl-2, BAX). So if tx only focuses on proliferating cells,
may miss these that are suppressing apoptosis.

Some of these pathways do both (regulate replication & apoptosis) – so these oncogenes can be very important;
blocking their functions can even lead to regression of cancer via ↑apoptosis (“oncogene addiction”)

Invasion & Metastasis


Invasion:
1. Penetrate basement membrane, degrade ECM, migrate in stroma
a. Cancer cells have active role (matrix metalloproteinases & other proteolytic enzymes)
b. Invasion leads to tissue remodeling: stromal reaction (akin to chronic inflammation)
c. Reactive stroma key to diagnosing invasion (is this tissue somewhere where it shouldn’t be?)
d. Invasive cancer actively migrates (reprogramming of integrin gene expression  changes in cell-
substrate adhesions & cytoskeletal dynamics)
e. Microenvironmental cues (oxygen tension, pH) may guide cancer cells to specific stromal structures
2. Adapt to foreign environment
a. Loss of cell-surface receptors (e.g. ↓e-cadherin, ↑other cadherins)
b. Both begin to be able to bind to ECM & lose requirement to be bound to each other
c. Called “epithelial-mesenchymal transition” (EMT) although not as complete as in embryology
(invasive cells still look like epithelial cells of origin)

Metastasis: spread of cancer to distant sites of body (not surgically treatable)


1. Vascular dissemination of malignant cells
a. Spread via lymphatics or blood vessels
b. Pre-requisites: invasion into vascular space & ability to survive in circulation
i. Most non-hematopoietic cells / non-metastatic cancer cells can’t survive shear stress in
circulation. But small % of cancer cells have “stem-like” properties & can survive in circulation.
ii. Most non-malignant cells undergo apoptosis when not attached to solid matrix (“anoikis”). But
resistance to apoptosis already present in cancer cells.
c. Adhere to endothelium & extravasate through vascular walls (processes not well understood
d. Usually inefficient (e.g. peritoneal-venous shunts in cancer pts don’t lead to widespread metastasis)
2. Growth of tumor in secondary site

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a. Determined in part by routes of vascular & lymphatic drainage
(GI to mesenteric LN / liver, others to regional lymph nodes & lungs)
b. Not entirely dictated by drainage (breast, prostate, lung  bone; breast/lung  CNS)
3. Paget (1889) – “dependence of seed on the soil” (cancer cell on organ)
a. current research: chemokines from cancers & chemokine receptors in receptor organ tissue

Cancer progression (classical paradigm): confined neoplasms  invasive  metastatic.


 Classical: metastasis happens late
 But there is big variability among neoplasms. Probably more likely that certain neoplasms are
programmed to be aggressive or benign from the start depending on mutations. Even small tumors in
some cancers can invade / metastasize early.
 One explanation of why screening hasn’t resulted in huge reduction in cancer mortality

Ability to modify the host environment


 Tumor not just cancer cells: stroma, inflammatory cells, blood vessels
 Angiogenesis: many cancers produce angiogenic substances (e.g. vascular endothelial growth factor,
VEGF; fibroblastic growth factor, FGF); some also produce anti-angiogenic factors.
o Anti-VEGF Ab (bevacizumab) – Tx for some types of cancers
o Low vascularity (hypoxic environment) helps some cancers (e.g. pancreatic) grow. Also makes
resistant to chemotherapeutic drugs because they don’t reach hypoxic areas
 Suppression of immune surveillance: from “self” but altered enough to produce immune response
o Cancer cells evade by secreting things (proteins to inhibit immune cells, cytokines /
prostaglandins to suppress immune response, decoy antigens) or express dummy receptors
 Systemic effects of human cancers: May also secrete humoral factors that affect host physiology
o Ectopic hormones (ACTH, parathyroid-related proteins)
o Cachexins (e.g. TGF) – most not identified yet

Extended doubling potential of tumor cells


 Normal cells: replicate, then eventually reach senescence (telomere shortening). Telomere important in
chromosomal integrity
 Cancer cells: immortality. Increased expression of telomerase to extend the telomeres.
o Kicks in late so cancer cells still have reduced telomere length.
o Could maybe detect cancer via telomerase expression or inhibit telomerase for therapy.

Genomic instability
 Many somatic genetic mutations  Multiple phenotypic alterations
 Most cancer cells aneuploid (abnormal # & structure of chromosomes)
 Continuous rearrangement as cancer cells divide
o Shortening of telomeres – “anaphase bridging” where ends stick together in anaphase
o Inadequate mitotic spindle checkpoint (imperfect alignment & segregation)
o Problems maintaining structure: from defective DNA repair mechanisms (p53, BRCA1&2)
 Leads to non-homologous recombination of broken chromosomes & translocatiosn
o Defective mismatch repair: ↑mutations at sequence level
 Increased microsatellite instability (MSI)
Genomic instability important in carcinogenesis (need many mutations to make cancer) & development of
resistance to chemotherapy.

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Morphology: abnormal, with hyperchromatism (increased chromosomal material) & abnormal, irregular
shape. Structurally abnormal mitosis.

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Pathology of Human Neoplasia (The Practical Issues)

Classification & nomenclature


 Prefixes = tissue of origin
Benign tumors
 -oma (chondroma = cartilage, adenoma = glandular epithelial)
 Hepatoma, melanoma, astrocytoma are exceptions (malignant)
Malignant tumors
 -carcinoma = epithelial origin
 -sarcoma = mesenchymal (stromal) origin

Ways to characterize:
1. Patterns of differentiation (Epithelial, Mesenchymal, Hematopoetic, Melanocytic, Glial)
2. Sub-types: e.g. for epithelial neoplasm: squamous, glandular (adeno), basal/basaloid, transitional
(urothelial), undifferentiated. Each pattern of differentiation has its own sub-types
3. Morphology: papillary, cystic, polypoid, mucinous, etc.
4. Benign (have very minimal risk of progressing to malignancy) and malignant tumors

Borderline or low malignant potential tumors: don’t fall into these categories well
 E.g. carcinoid tumor – neuroendocrine differentiation; respiratory / digestive systems; big range of
malignancy.
Multiple patterns of differentiation:
 epithelial + mesenchymal = fibroadenoma (benign) or carcinosarcoma (malignant).
 Tetroma: more than one germ cell layer from pleuripotential cells

Pattern of differentiation Benign Malignant


Epithelial
Glandular/ ductal epithelium Adenoma Adenocarcinoma
Squamous epithelium Squamous papilloma Squamous cell carcinoma or epidermoid carcinoma
Liver Hepatic adenoma Hepatoma (a.k.a., hepatocellular carcinoma)
Mesenchymal
Smooth muscle Leiomyoma Leiomyosarcoma
Adipocytes Lipoma Liposarcoma
Cartilage Chondroma Chondrosarcoma
Bone Osteoma Osteosarcoma
Endothelial hemangioma Hemangiosarcoma
Melanocytic Melanocytic nevus Melanoma
Glial Astrocytoma, ependymoma, oligodendroglioma
Hematopoetic Leukemia, lymphoma

Morphological characteristics of neoplasms:


 Solid tissues form tumors (except in situ neoplasia, which is more spread out)
 See below for benign vs. malignant cells

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Characteristics of benign cells Characteristics of malignant cells
Relatively low nuclear: cytoplasmic ratio Increased nuclear size (high N:C ratio)
Round nucleus, even distribution of chromatin, small or Irregular nuclear shape, irregular distribution of
inconspicuous nucleoli chromatin, prominent nucleoli
Maintenance of cellular polarity and differentiation Loss of cellular polarity and variable loss of
differentiation
Mitoses are uncommon, are located in usual location Mitoses are common, located above basal cell layer,
(e.g., basal layer), and have typical appearance and have atypical appearance.

These are descriptive characteristics, not rules (consideration of all features in context is important)
Cytopathology: characterizing malignancy, etc. based on these features (e.g. Pap smear for aspirated cervical tissue)

Clinical considerations for diagnosis of malignancy (based on clinical experience)


1. Site: smooth mm. tumor in uterus with certain # mitosis = leiomyoma, in colon = leiomyosarcoma
2. Gender: teratoma in ovary with benign appearance = benign course; in testis of adult male = high metastasis potential
3. Age: Benign-appearing teratoma in testis of child = benign course, benign appearance in adult male = malignant

Pre-invasive neoplasia (defies traditional definitions)


 Tubular adenoma – precursor to colorectal cancer, low potential to invade (if excised, good prognosis)
 Carcinoma in situ = “severe dysplasia of squamous mucosa” (e.g. cervix) – high % develops to invasive

Grading & staging neoplasia


 Grade: degree to which cells have malignant features.
o Low grade = close to normal, high grade = large, irregular nuclei & atypical mitosis
o Poorly differentiated / well differentiated (how well does it resemble normal tissue)? Well
differentiated = low grade, poorly differentiated = high grade
o Standardized criteria (differentiation + nuclear features; nuclear dominate). Has variable
predictive validity depending on type of cancer
 Stage: extent of spread of cancer. Better clinical predictor.
o American Joint Committee on Cancer: TNM staging (Tumor, Lymph Nodes, Metastasis)
o Combine information to make TNM grouping (T1, N2, M0 for example) – cut-offs depend on the
type of cancer
o Grouped staging: 0, I-IV (0 = in situ, no invasion; IV = metastatic) – for surgery, etc.

Ancillary techniques
Immunohistochemistry
 No single marker but some are useful (e.g. p63 for normal basal cell layer in prostate; if missing =
cancerous; AMACR overexpressed in most prostate cancers)
 Mostly not helpful for benign vs malignant but can be used to phenotype tumor (e.g. heomatopoetic
neoplasms – use on suspended cells post-flow-cytometry.

Detection of Cancer via Molecular Markers


 Ideal situation: detect & monitor cancer via small markers with minimal invasiveness
 Current: secreted proteins (tumor-specific antigens, e.g. prostate specific antigen/PSA)

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o PSA: mortality has declined post-PSA introduction
o Associated with over-Dx and over-Tx of disease
o Some mixed studies on benefits in terms of mortality
 Research: better early detection, monitoring disease (mass spec, DNA from cancer cells).
 Still need tissue diagnosis before Tx currently (limits of sensitivity & specificity)

Predictive markers / molecular classification


 Want markers to predict response to therapy or subclassify tumors
 E.g.: Estrogen receptor (ER) in breast cancer: predicts good response to anti-estrogen therapy & better
prognosis.
 Subclassify according to molecular features: e.g. measure multiple genes in parallel (microarray) to form
prognostic indices & determine need for chemotherapy.

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Molecular genetics of cancer
Theory of genetic basis: need social controls on cells; have a high mutational load of a complex organism.
Average human gene mutated 1010 times in lifetime (almost all somatic)
We handle our mutational load well:
 Protect the germline cells (separation from somatic cells early in embryological development)
 Innate resistance to tumorigenesis (single mutation inadequate)

Neoplasia requires accumulation of somatic mutations: a microevolutionary process


 5-7 rate-limiting events needed (but how?)

 Clonal selection theory: tumorigenesis occurs as serial expansion of successive clones of cells,
punctuated by acquisition of certain mutations which give a cell and progeny a selective growth
advantage over neighboring cells
o Why are so many mutations needed? Downregulation mechanisms protect cells (need multiple
mutations to inactivate downregulatory syndromes & accumulate small effects to cause
selective advantage)
o Clonal changes (present in all cells of a neoplasm) indicate important events
o Genetic / epigenetic heterogeneity arises (even though genetic instability not universal in
neoplasms) – genetic instability just accelerates
o Clone is population that derives from single cell; offspring (subclones) compete to see who can
dominate neoplasm (with selection). Otherwise you’d just end up with heterogenous group &
benign neoplasm. Have to select each time one by one or else tumor mass would be huge
o Subsets with worse prognosis = those with more mutations
o Neoplasms arise from chance events so genetic profile varies from pt to pt (individualize
therapy)
 Pediatric tumors may be exception (arise in window of opportunity & don’t resemble
adults: maybe need fewer mutations & not as many steps)

 Mitogenesis is as important as mutagenesis in tumorigenesis


o Not just environmental exposure to mutagens, but also inflammation & regeneratory processes
 Changes in production of stem cells & ability to differentiate are key for neoplasia (commonly mutated)

Frequently mutated genes:


1. Dominant oncogenes: function activated by mutations
o Think about the signal transduction system from outside to nucleus
o Growth factors
o Growth factor receptors (e.g. EGFR)
o Signal transducers (e.g. RAS, ABL-BCR)
o Nuclear oncoproteins
o Agonists of apoptosis (BCL-2)
o Antagonists of tumor-suppressors (e.g. antagonists of p53)
2. Tumor-suppressor genes: function inactivated by mutations (selective advantage)
o Cell-cell, cell-ECM, differentiation-inducing interactions (e.g. E-cadherin)
o Cytoskeletal
o Regulators of signal transduction
o Cell-division cycle regulation (e.g. p53)
o Apoptosis (ultimate negative regulation: p53, BAX)

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o Chromatin structure
3. DNA-maintenance genes: genes inactivated by mutations, often before tumorigenesis (need second hit
for selective advantage)
o DNA repair genes (e.g. xeroderma pigmentosum genes)
o Chromosome stability genes (BRCA2, etc.)
4. Passenger mutations: have no special meaning in the neoplasm

Types of mutations:
 Amplification: overproduction of proteins
 Rearrangement / translocation: fusion of two genes from different proteins or oncogene placed behind
strong promoter
 Small mutation: e.g. point mutation, can activate or inactivate gene
 Large deletion: often a second hit – cause inactivation of suppressor gene, or loss of heterozygosity
(LOH), exposing first hit’s mutation
 Viral insertion: can allow viral oncogenes to continue to be expressed
 Telomere shortening: can cause genetic instability, deletions, translocations. Re-activation in
malignancy helps prevent extreme of this process (cell death) in malignant tumor cells

Inherited syndromes: can be due to germline mutations in:


1. Dominant oncogenes (examples are rare: embryonic lethal?)
2. Tumor suppressor genes (more common). Two-hit model: maybe the first hit is inherited, increasing
rate of neoplasm’s occurrence. E.g. familial adenomatous polyposis (FAP)
a. For instance: recessive gene but get LOH with second hit
b. Other examples: hereditary retinoblastoma (RB1), familial breast/ovarian cancer (BRCA 1), Li-
Fraumeni syndrome (p53 – lots of cancers possible)
3. DNA maintenance genes
a. True examples of higher mutation rates (“genomic instability”)
b. Hard to find “genetic instability” in lab, but some conditions do have true chromosomal
instability
c. Examples:
i. Xeroderma pigmentosa (inadequate repair of UV-induced DNA damage if have 2 mutant
copies of the gene)
ii. Hereditary nonpolyposis colorectal carcinoma (can be heterozygous) – inherited cause
of cancer susceptibility
iii. Ataxia telangiectasia (2 mutant copies)
iv. Fanconi anemia & familial breast/ovarian/pancreatic cancer (BRCA2). Two mutant
copies = highest risk, Fanconi anemia. One mutant copy still increases risk (get LOH in
neoplasm)
4. Susceptibility genes: may be very common & are being studied currently

Rational therapy:
Old model: screen all kinds of toxins for ability to kill cancer cells in culture
New model: look for specific biochemical properties

New ideas: augment deficient function(p53 – hard); replace function (hard without gene therapy working);
inactivate a function (Gleevec – very successful); take advantage of neoplastic defect; re-express genes;
augment immune responses

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Acquired drug resistance via mutations: from mutations in drug-binding pocket; mutations causing
compensatory increase in activity, or mutations eliminating cell’s toxic response

Carcinogens
 Dietary / environmental:
o can use Ames assay (expose to potential mutagen; count colonies on plate that have mutated,
subtract background rate), others (cheaper in bacteria, really expensive in animal models).
o End up only screening things that are pretty certain to be carcinogenic
o Some are suspected carcinogens
o Example: Aflatoxin causes p53 mutations in hepatocellular carcinomas in Africa & China
 Infectious causes:
o Indirect mechanism: mitogenesis & inflammation (e.g. HBV & hepatocellular carcinoma, H.
pylori & gastric cancer)
o Direct mechanism: viral proteins that inactivate tumor-suppressor genes (e.g. HPV & cervical
cancer)

Non-mutated genes can also play a role (may be over- or under-expressed in neoplasms & provide good
background for neoplastic development

What is a neoplasm
“A clone of cells distinguished from other tissues by autonomous growth and somatic mutations”
 Mutations in growth-controlling genes
 Supporting, reactive tissues accompany tumor growth
 Grow in conditions that would otherwise be limiting

Caveats:
 All neoplasms have been found to have somatic mutations
 Inciting stimulus usually not shown for neoplasms
 Neoplasms often do control their own proliferation, but control is altered & cell # increases (evidence:
most neoplasms are benign)
 Other masses & proliferations
o Keloids, developmental abnormalities, granulation tissue, synovitis, etc.
o As long as it’s not clonal, it’s not a neoplasm
 Neoplasms are not always masses (e.g. leukemias, etc.)
 Neoplasms are not just a proliferative abnormality (this would just be hyperplasia) but rather a large
increase in stem cell # (clonal)
 Mutations in growth controlling genes can be inherited rather than acquired (insufficient to cause
neoplasms on their own). ADDITIONAL SOMATIC MUTATIONS ALWAYS REQUIRED.

FAP VS HNPCC (Familial Adenomatous Polyposis vs. hereditary nonpolyposis colorectal cancer)
 FAP: first change occurs quickly (lots of early adenomas) but it takes around 20 years to accumulate more hits
 HNPCC: first change occurs slowly, but fast progression afterwards (2 years) – harder to treat

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Pathophysiology: Neoplasia
Table of Contents
Pathophysiology of Cancer: Basic Principles ........................................................................................................................... 1
Cancer screening and prevention ........................................................................................................................................... 6
Breast Cancer Symposium ...................................................................................................................................................... 8
Radiobiology as applied to the clinic .................................................................................................................................... 11

Pathophysiology of Cancer: Basic Principles

Cancer: 2nd leading cause of mortality in US (23% all deaths). More cancer survivors now than ever.
Women: lung, breast, colon, rectum = 50% cancers; Men: lung, prostate, colorectal

Metastasis: set of host-tumor interactions involved. A failure in any step will halt metastasis.
(proliferation, angiogenesis of primary tumor  detatchment, invasion of lymphatics or blood  embolism &
circulation  transport & survival  arrest in organs  adherence & extravasation  survival in new tissue,
proliferation, angiogenesis)

Natural history of cancer


1. Formation of the primary tumor
a. Cross-talk between
cancer cells & host Cell type Promotion of metastasis Inhibition of metastasis
stromal cells (up or Tumor Activation of growth factor pathways Antigenicity
down-regulated Angiogenic factors Angiogenesis inhibitors
Motility/invasiveness Cohesion (E-cadherin)
gene expression in
Aggregation/deformability Tissue inhibitors of proteolysis
both)
Host Paracrine/endocrine growth factors Tissue barriers
b. Nutrients supplied
Neovascularization Endothelial cells/blood turbulence
by simple diffusion Platelets Tissue inhibitors of proteolysis
(avascular tumor) Immune cells Immune cells
c. Balanced rate of Antiproliferative factors
tumor cell Inhibitors of angiogenesis
proliferation &
death (until “switch” to pro-angiogenic phenotype) – see table
2. Progressive growth & angiogenesis
a. Tumor cells can either directly secrete angiogeneic substances or release / activate them from ECM
i. Can also recruit lymphocytes, macrophages (release angiogenic substances too)
ii. Leads to activation of endothelial cells & neovascularization
b. Process of angiogenesis
i. Capillary basement membrane degraded (vascular deformity in existing vessel)
ii. Endothelial cells migrate out (pro-angiogenic stimulus)
iii. Proliferation @ leading edge of endothelial cell column
iv. Reorganization and canalization of endothelial cell tube
v. Anastamosis to establish blood flow
c. Proangiogenic substances include fibroblast growth factors, epidermal growth factors, vascular
endothelial growth factor (VEGF)
d. Antiangiogenic factors include the statins (angiostatin, endostatin, etc).
e. Tumor angiogenesis is different from physiological angiogenesis
i. Aberrant vasculature & blood flow formed
ii. Altered endothelial cell-pericite interactions
iii. Increased permeability
3. Invasion
a. Tumor cells & invading mononuclear cells from host produce degradative enzymes which facilitate
invasion of stroma
b. Host response: lay down fibrous ECM (desmoplasmic response)
c. Tumor cell downregulates adhesion molecules (e.g. E-cadherin) which normally need to be stimulated
to promote cell survival
d. Invasion: thin-walled vessels (lymphatics, capillaries, venules) – easily penetrated
4. Embolization & transport
a. Single cells or aggregates
b. Blood stream is hostile environment (shear forces, host hematopoetic defenses, etc.)
i. Most die & those that survive rarely produce metastases
ii. Aggregates more likely to survive & become trapped in microvasculature of distant organs
(“safety in numbers”)
c. Not always a simple picture (e.g. malignant ascites from ovarian cancer treated with peritoneavenous
shunts, dumping tons of cancer cells into jugular vein, but no increase in lung metastases).
5. Arrest, adhesion, extravasation
a. Adhere to capillary endothelial cells or subendothelial basement membrane if exposed – primarily a
mechanical process
b. Extravasate & invade stroma
c. Metastasis often but not always explained by drainage patterns to areas of microvasculature (via blood
or lymph drainage)
i. Colon cancer  liver metastases (portal circ)
ii. Breast cancer  lung metastases (systemic circ)
6. Subsequent growth
a. Inefficient & dependent on suitability of “soil” organ (not all observed metastasis sites are compatible
with simple drainage hypothesis)
i. E.g. melanoma to brain, liver, bowel; prostate carcinoma to bone, testicular carcinoma to liver
b. Factors at play:
i. “Seed” = tumor cell: growth factor expression, specific chemokine receptors, cell adhesion
molecules
ii. “Soil” = target organ: chemokine milieu (match of chemokine receptors), etc.
1. Example: breast cancer cells express CXCR4, CCR7 receptors; their normal target organs
express the conjugate chemokine ligands
iii. Certain tumor cell subsets may be genetically predisposed to metastatic phenotype
1. Inactivation of metastasis suppressor genes may be key (genes which prevent
metastasis without impacting growth of the primary tumor)
7. Progressive growth
a. Cancer cells need to grow further to actually establish a metastasis:
i. Establish a microenvironment
ii. Proliferate
iii. Begin angiogenesis (tpo grow beyond 1-2 mm in diameter)
iv. Evade host immune system

Dormancy: can have a relapse decades after primary treatment (e.g. breast cancer, melanoma) – not well understood
 Possible mechanisms: persistent pre-angiogenic micrometastases (dividing & apoptosing @ same rate) and then
undergo angiogenic shift; or maybe persistence of solitary tumor cells in secondary organs

How does cancer make people sick?


1. local effects (compression of vital structures, replacement of tissues)
a. Headaches, seizures, change in personality: brain involvement?
b. Hematuria: urinary tract?
c. Productive cough – postobstructive pneumonia?
d. Bone pain: bone metastases?
2. systemic effects (humoral factors)
3. metastasis

Oncologic emergencies:

1. Spinal cord compression


a. 10-40% pts presenting with acute SCC have undiagnosed cancer
b. Most important: status @ presentation (80% ambulatory pts will retain ability to walk vs 25% non-
ambulatory)
c. Motor function & sphincter control are two big issues
d. Pathophysiology: compression of anterior spinal cord / nerve roots from vertebral body collapse = most
common. Can also have invasion of paraspinous tumor through vertebral foramen
e. Thoracic = 70%; lumbrosacral (20%); cervical (10%)
f. Most from prostate, breast, lung cancer.
g. Back pain is initial symptom in 95% of pts. RESPECT ANY COMPLAINT OF BACK PAIN IN KNOWN
CANCER PTS. THINK SCC!
h. MRI with gadolinium contrast is imaging modality of choice. Plain films can be abnormal if SCC present,
but do not exclude SCC if normal
i. Treatment: steroids, local radiation; anterior decompression & rod placement if unstable

2. Superior Vena Cava Syndrome


a. 80% pts with SVC syndrome have underlying cancer (most frequently small cell lung cancer).
b. Pathophysiology: obstruction of blood flow in SVC from thrombosis or external compression. Both can
occur simultaneously.
c. Symptoms: dyspnea, facial puffiness, fullness in head / dusky complexion, cough
d. Findings: engorgement of neck veins, development of collaterals over chest wall, facial edema (50%),
plethora / peripheral cyanosis (25%)
e. Evaluate with chest radiograph and/or CT to look for chest mass. If chemosensitive; give chemotherapy;
if not, give radiotherapy. If thrombosis, give blood thinners
f. Only use emergency radiotherapy empirically if symptoms are rapidly progressive and there’s no time
for a tissue diagnosis.

3. Leukostasis
a. True oncologic emergency (requires expert management by oncologist with significant experience
b. Most common in AML (acute myelogenous leukemia) or accelerated / blast phases of chronic myelocytic
leukemia (CML)
c. Pathophysiology: plugging of capillaries with immature leukocytes (organ dysfunction results)
i. Not just simple obstruction: white cell thrombi also compete for oxygen, causing further hypoxia
ii. Endothelial injury  invasion of surrounding tissue  pulmonary edema, hypoemia in lung /
risk for hemorrhage in brain
d. Symptoms: dyspnea, tachypnea, cough, chest pain, progressive hypoxemia, fever, headache, dizziness,
visual change, tinnitus, ataxia, lethargy, stupor, somnolence, seizure, coma. Think: microvasculature
(lungs, brain, retina)
e. Findings: tachypnea, bilateral crackles, papilledema, retinal vein distension
f. Treatment: hydration, urine alkalinization & allopurinal (prevent tumor lysis syndrome & uric acid
buildup), chemotherapy. May need leukapheresis to reduce WBC count quickly

4. Hypercalcemia of malignancy
a. Most common life-threatening metabolic disorder in cancer patients
b. Most common causes of hypercalcemia: hyperparathyroidism (45%), malignancy (45%)
i. Multiple myeloma and breast cancer are big two cancers
c. Pathophysiology: direct involvement of cancer metastatic to bone, or humoral secretion at distant cyte.
d. Humoral mediators:
i. 1,25-dihydroxyvitamin D in hypercalcemia associated with melanoma, multiple myeloma,
Hodgkins / Non-Hodgkin’s lymphoma
ii. PTHrP (parathyroid hormone-related peptide) associated with squamous cell carcinoma of
lung, small cell / anaplastic lung carcinoma, melanoma, prostate cancer, breast cancer, renal
carcinoma
e. Immobilization can also play a role in some pts with advanced cancer (bone loss)
f. Symptoms: fatigue, weakness, confusion, lethargy, constipation, nausea, vomiting, polyurea.
g. Normal Ca = 8.4-10.5
h. Therapy: IV fluids, diuretics (lasix), bisphosphonates to inhibit Ca relase from bone (interfere with
osteoclasts). Steroids used too – but need to treat the underlying tumor.
Paraneoplastic syndromes: systemic effects of cancer that are not related to direct invasion or compression from tumor
or to metastatic spread (endocrine, neurologic, hematologic derangements).

Endocrine paraneoplastic syndromes:


 Ectopic ACTH syndrome (most common)
o Results in Cushing’s-type manifestation (weight gain in trunk/face, “buffalo hump”, weight loss
elsewhere, hypertension, hyperpigmentation, hypokalemia, metabolic alkalosis, excess sweating,
hirstutism, etc) with a pulmonary mass usually
o ½ due to small cell lung cancer, remainder pheochromocytoma, thymoma, medullary thyroid cacner,
carcinoid tumors

Neurologic paraneoplastic syndromes:


 Cerebellar and neuropsychiatric are most common; subacute but progressive
 Eaton Lambert myasthenic syndrome: rare manifestation of small cell lung cancer (<1% pts)
o Proximal mm weakness (pelvic girdle, thighs)
o Muscle strength improves with repeated activity; response to edrophonium chloride poor (opposite of
real myasthenia gravis)

Hematologic manifestations of cancer: erythrocytosis, anemia, granulocytosis, granulocytopenia, thrombocytopenia,


eosinophilia, basophilia, thrombocytopenia, thrombo-phlebitis, coagulopathies

Trousseau’s syndrome: thrombophlebitis (venous blood clots) with cancer – Trousseau diagnosed himself.
 Risk is highest for migratory thrombophlebitis in pancreatic cancer but can also be present in other
adenocarcinomas (breast, prostate, ovarian cancers)
 Manage with anticoagulation & tx of underlying malignancy

Gastronintestinal manifestations of cancer: anorexia, cachexia, protein-losing enteropathy


 Significant weight loss associated with shorter survival
 Can result from neurologic dysfunction, GI dysfunction, alterations in taste, depression

Cancer pain
 Grossly undertreated: 90% can be well controlled
 Most due to direct tumor infiltration
 Three types of pain
o Somatic pain: dull, aching, well-localized. E.g. metastatic bone pain
o Visceral pain: deep, squeezing, pressure-like, poorly localized. Infiltration / compression of viscera. Can
be associated with nausea/vomiting if acute (e.g. pancreatic cancer)
o Neuropathic pain: direct injury to PNS/CNS from direct tumor infiltration or compression, or therapy-
related injury.
 Assessment: believe the patient (esp. in cancer); do Hx & physical exam, use pain assessment tools as fifth vital
sign, individualize approach, educate patient
 Treatment: assess & treat underlying cause (including palliative treatment of tumors), match medications to
type of pain, titrate medication to patient response, give on schedule rather than prn basis, use oral meds if
possible, anticipate side effects and treat, be aware of tolerance
o Tolerance: ability to take large dose of drug without ill effect (reduction of desired effects from
continued use)
o Dependence: physical, biological need for a drug to prevent withdrawal syndrome
o Addiction: psychological craving for drug; rare result in appropriate cancer pain management
 Performance status: global assessment of ability to conduct activities of daily living (not QOL).
o Major prognostic factor for pts with cancer, predictor of toxicity of treatment, indicator of comorbid
disease and other host factors.
o Two major scales: Karnofsky performance status (0-100), Eastern Cooperative Oncology Group (0-4)
Cancer screening and prevention
Fundamental assumptions:
1. Disease can be identified in pre-clinical phase
2. Treatment is more effective at earlier rather than usual time of Dx
3. In absence of intervention, all cases proceed to clinical disease
and eventual mortality
4. Disease follows a natural history course
a. DPCP = detectable pre-clinical period (1st point detectable
by screening to first onset of symptoms)

Screening: classification of healthy (asymptomatic) individuals who


probably have a disease from those who probably do not. (vs.
diagnostic test: already have signs & symptoms; surveillance: follow-
up in those already diagnosed)
 Sensitivity: correctly determine patients with the disease;
specificity: correctly identify pts without the disease
 Type I error: false positive; Type II error: false negative
 Validity (accuracy): identification of correct results,
Reliability (precision): reproducibility of results
 Positive predictive value: likelihood that an individual has a
disease if test comes back positive. Depends on prevalence
and specificity

Study types & designs from cancer screening:


 Randomized clinical trial is gold standard (esp. double-blind
& matched experimental / control groups) but expensive &
difficult
 Cohort study: good for more common conditions; groups
unified by a characteristic & entered into study before
appearance of disease in question; control from general
population
 Case-control: compare pts with disease to group of healthy patients; less expensive & difficult (but not as good)
 Cross-sectional study: observing a subset of population at a specific time

More frequent screening is not always better (more false positives = more $$, more unnecessary procedures, etc.)
 Should be determined from rate of disease progression and sensitivity of test
 Also need to determine when to stop screening – balance benefits of screening vs. decreasing life expectancy
from other causes in the aging population
 Screening may not be good in cancers where there’s a good prognosis for disease, in people with a limited life
expectancy, or if you can’t detect disease during a preclinical phase (or short pre-clinical phase)

Identifying high-risk populations (fam Hx, BRCA mutations, exposure to carcinogen like smoking or afalotoxin) causes a
higher “prevalence” in your screened population and therefore increases positive predictive value.

Biases in cancer screening trials


 Volunteer bias: people who volunteer can be different from general pop (those who choose screening = better
health behaviors: screening looks beneficial, or poorer risk profiles so more worried: screening looks worse)
 Lead time bias:
o Lead time: period between early detection of disease from screening program & normal detection time
o Screening catches disease earlier, so survival appears longer even if it’s not: need to correct for lead
time using natural history information & compare age-specific mortality rates instead of 5yr survivals
 Length bias:
o DPCP = detectable pre-clinical phase
o Screening can catch more cases with longer DPCP which can be less aggressive disease
o Better prognosis, so screening appears too beneficial
o Affects case-fatality rate (CFR)
 Over-diagnosis bias:
o Diagnose cases with screening that would normally be undetected in normal lifetime (would not
progress / regress over time)
b. Can cause extra psychological stress, unneeded interventions / treatments

Where should we focus our resources?


 Non-aggressive cancers
 More prevalent cancers
 Effective current treatments

When should we screen? Need to consider the properties of the test & the population effectiveness
 Burden (mortality/morbidity)
 Good detectable clinical phase, early detection beneficial & effective
 Screening test is safe & effective
 Prevalent in population
 Feasible & acceptable to patient and physician
 Want a high precision, high PPV, high accuracy (specificity is especially important because most cancers are rare)

Example: PSA & prostate cancer. Multiple studies:


 no difference in mortality in US study but maybe the PSA cut-off was too high, PSA screening was common in
control group, improvements in Tx masked difference, longer-follow-up needed?
 European study showed some benefit: maybe better control because PSA screening isn’t as common?
Breast Cancer Symposium
Breast cancer: 15% of cancer-related deaths; most common Dx & second most common cause of cancer death in women
1:100 Male:Female; 1 in 50 women by age 50 (1:8 lifetime)

Risk factors
 Reproductive: prolonged estrogen exposure
o early menarche, late menopause, nulliparity / late first pregnancy, lactation (?)
 Environmental: radiation=yes (not pesticides or electromagnetic fields
 Lifestyle: Diet, alcohol, physical activity, tobacco use (big cancer risk)
 Endogenous hormones: high hormone levels, post menopausal obesity (metabolic syndrome), increased bone
density (may be marker for high E instead of risk factor)
 Exogenous hormones: hormone replacement therapy=yes (estrogen replacement therapy = ?, oral
contraceptives = no)

Pathology: atypical ductal or lobular hyperplasia; lobular carcinoma in situ are risk factors

Inheritance: family history important; major inherited susceptibility (DNA repair defects are key)
 About 75% breast cancer has no family influence
 15-20% “clustered” (no clear inheritance); 5-10% directly inheritable
 BRCA1 (20-40%), BRCA2 (10-30%) are two big players in heritability (% of heritability attributable)
 Undiscovered genes (30-70%) are still very important

BRCA MUTATIONS
 BRCA-1
Increased likelihood of having BRCA mutation:
o 50-80% lifetime risk of BC; often early age at onset; o Multiple cases of early onset BC in family
40-60% chance of having second primary BC o Ovarian cancer with FHx of breast/ovarian cancer
o Ovarian cancer 15-45% risk o Breast & ovarian cancer in same woman
o Possible increase in risk of other cancers o Bilateral breast cancer
 BRCA-2 o Ashkenazi Jewish heritage
o 50-85% lifetime risk of BC; 6% male breast cancer o Male breast cancer
o Ovarian cancer 10-20% risk
o Increased risk prostate, laryngeal, pancreatic cancers
 Management: test other relatives; increase surveillance & institute lifestyle changes, chemoprevention
(tamoxifen), possibly prophylaxic surgery if lots of risk
 Risk assessment:
o Gail model (age, repro history, benign breast disease history, FHx in first degree). Doesn’t incorporate
age @ Dx, other cancers, other relatives
o Claus tables: only considers FHx of breast cancer

Prevention:
 Lifestyle changes (exercise, alcohol, tobacco) Signs & Symptoms of Breast Cancer
 Chemoprevention (tamoxifen, raloxifene)  Breast lump or thickening
 Skin or nipple changes
Screening:  Nipple discharge
 Breast self exam (beginning in 20s; some negative RCT)  Regional adenopathy
 Clinical breast exam (annual)  Abnormal mammogram
 Mammography: (good evidence for 50-69, uncertain in 40-
49yo, no data 70+)
o Looking for calcification
 New approaches: ductoscopy (endoscope in duct); ductal lavage (inject fluid, aspirate to get some cells)
Diagnostic tests: bilateral mammography, ultrasound/MRI, biopsy
Pathophysiology & Treatment
 Ductal epithelial cells are site of origin for 95% BC
 Classification:
o Invasive vs non-invasive
o Ductal vs lobular (“origin”)
o Histiologic grade
o Special stains (ER = estrogen receptor, PR = progesterone receptor, HER-2 = human epidermal growth
factor receptor 2 = more aggressive cancer)
 Staging: establish prognosis & guide therapy Staging Breast Cancer
o Use: hx, physical exam; mammorgraphy, CBC/chemistry,
 I: T < 2 cm, N0
X-rays/scans if symptoms, pathological exam on axillary
 II: T > 2 cm – 5 cm or N1
nodes after surgery, analysis of tumor for ER/PR/HER-2
 III: locally advanced breast cancer
 Prognostic factors: predict natural history for individual dz.
 IV: metastases
Nodal status, tumor size, steroid receptors, grade/subtype,
proliferation, age.
 Predictive factors: predict how well tumor will respond to specific therapies. Steroid receptors, HER-2 presence
 Tumor subtypes: different natural histories, different types of responses to therapy
o Best is “luminal A”
o Worst is “basal” & “HER-2+”
 Good example of personalized medicine: “Oncotype dx” – figure out what therapy in combination will work best
for patient’s tumor algorithmically & then apply.
 Surgery & radiation with adjuvant systemic chemo is generally how treatment works.

Surgery
 Halsted pioneered the radical mastectomy
 Less surgery causes less lymphedema, less mobility problems, etc.
 RCTs: mastectomy vs. lumpectomy & radiation showed no survival difference.
 Breast conserving therapy (BCT) is now preferred unless contraindicated (multifocal, poor cosmetic outcome,
patient preference, previous radiation, etc.)

Systemic treatment
 Chemotherapy:
o Alkylators (cyclophosphamide, etc.)
o Antimetabolites (MTX, 5-FU, etc)
o Topoisomerase inhibitors (doxorubicin)
o Antimitotics too
 Hormonal: endocrine therapy
o Ovarian ablation (surgery/radiation or LHRH agonists)
o SERMs like tamoxifen antagonize ER gene products in breast tissue
o Aromatase inhibitors (if post-menopausal)

o Progestins, estrogens, androgens are bad


o Note: pre-menopausal women have ovarian estrogen as primary source, so ovarian ablation is good
therapy. Post-menopausal women have androgens as primary source of e, so aromatase inhibitors are
indicated.
 Growth-factor-receptor targeted (trastuzumab & lapatinib)
o trastuzumab
 For HER-2 positive tumors (15-20%)
 Blocks the mutated, constitutive activation of epidermal growth factor receptor on tumor cells
 Cytotoxic & inhibitory (host immune response, promotes other chemotherapies, etc.)
o lapatinib
 small molecule inhibitor; prevents phosphorylation of HER-2 and EGFR receptors & blocks
signaling in that way
 Anti-angiogenesis (bevacizumab)
o Targets VEGF (vascular endothelial growth factor)

Treating metastatic breast cancer (Stage IV)


 Want to relieve / prevent symptoms from tumor (usually can’t cure)
 Chronic therapy – balance side effects of disease & therapy
 Common sites: lung, liver, bone, soft tissue

Treating locally advanced breast cancer (Stage III)


 Note: swelling of nipple (lymph blocked), peau d’orange (like skin of orange)
 Want to control local disease & eradicate micrometasteses
 Surgery, radiation, chemotherapy – want to be aggressive (eg neoadjuvant therapy - shrink & then surgery)

Treating early breast cancer (Stage I & II)


 Eradicate micrometastatic disease
 Give drugs around time of surgery
 Need effective drugs & high risk population
 Intent: cure (tolerate toxicity because you can beat it)

ER (-) pts don’t’ usually benefit from endocrine therapy


Post-menopausal patients with ER (+) usually don’t give chemo (less aggressive dz; may not respond as well)
Adjuvant chemotherapy: important to keep to dose schedule; short duration (months)
Adjuvant endocrine therapy: need steroid receptor (+) patients; long duration of therapy (years)

Toxicities of adjuvant therapy:


 Acute: nausea, vomiting, hair loss, bone marrow suppression, weight gain, mucositis, fatigue
 Chronic: ovarian failure, late end organ damage, second malignancy, cognitive dysfunction (“chemo brain”)
Radiobiology as applied to the clinic

Basic physics:
 X-ray is a stream of photos with energy inversely proportional to wave length. Ionizing if it can knock an orbital
election out from an atom that it encounters (electron cloud is big, so most likely to hit an electron)
 Electron flies out and deposits energy distant to site of ejection
o Energy reaches a maximum at some distance from site of ejection (distance to maximum depends on
energy)
o this is “sparing” of high dose to tissue right under surface (so for instance skin not always harmed
because it’s closer than the maximum distance

 Indirect action on DNA:


o Tissues are mostly water, so electron hits usually hits water and generates a hydroxyl radical
o Hydroxyl radical leads to base damage, breakage of phosphodiester backbone
 Direct action on DNA also possible: hits DNA, protein, etc. directly (less frequent)

DNA strand breaks / chromosomal aberrations (what happens after DNA gets damaged)
 Single strand break = easily repaired (use complementary strand)
 Double strand break = irreparable
o Need two breaks close to each other in time and space
o Chromosomal aberrations: sticky ends at each broken part
 Common formations: dicentrics, rings (sticky ends stick together – lethal)
 May fail to rejoin: deletion(lethal)
 Rate of aberrations (and resultant cell death) increases with
amount of radiation

Cell survival curves:


 Low doses: less radiation; unlikely to get DSB (two breaks near each
other in time and space). Lethal events caused by single hit (varies
with Dose=D)
 High doses: more likely to get DSB; two events will interact (varies
with Dose2 = D2)
 Curve can therefore be defined by “linear-quadratic formula”.

Four “R’s” of Radiobiology


Most clinical radiation is delivered in a fractionated scheme where total dose is delivered in many small doses instead of
several large doses: why?
Early experiment: try to sterilize a French goat. Big doses hurt the scrotum; smaller doses achieve same sterilization but
without all the agony.

1. Sublethal damage Repair:


a. Increase in cell survival if you split a dose into two fractions with a time interval (the normal cells can
repair themselves)
b. Tumor cells repair less well, with less fidelity (part of why they become tumors in the first place) but still
repair themselves somewhat.
2. Reassortment
a. Cells redistribute into radiosensitive phases of the cell cycle after DNA-damaging events (e.g. G2/M
checkpoint – highly radiosensitive & also arrest checkpoint)
b. If you hit cells with more radiation after they redistribute, they’ll mostly be at the G2/M checkpoint and
will be more vulnerable (killing increases).
3. Repopulation
a. Cells can divide after doses of radiation, increasing
their apparent survival (tumor & normal)
b. See graph on right: first three R’s
i. Survival increases if you deliver the first
dose after a little break (repair)
ii. Then survival decreases if you wait until
reassortment happens
iii. “Survival” increases if you wait until later
(repopulated)
4. Reoxygenation
a. Cells that are hypoxic at time of radiation become
oxygenated afterwards
b. Oxygen is required to make DNA damage permanent
c. Some solid tumors have hypoxic/anoxic areas in the center (farther away from vasculature; tumor cells
are using increased amounts of oxygen)
d. As you hit the cells with radiation, the oxygenated outer cells die & tumor shrinks, allowing oxygenation
of formerly hypoxic cells (ready to be killed with next round)

So in summary: Fractionated irradiation spares normal tissue by allowing repair of sublethal damage and repopulation
of cells. Fractionation increases tumor damage by allowing reassortment of cells into more radiosensitive cell cycle
phases and permits reoxygenation to occur in order to make DNA damage permanent.

Why is exposure to radiation different in tumors vs. normal tissue?


 Early-responding tissue: tumor or normal tissue that when irradiated shows reactions early during the course of
treatment (e.g. skin, mucosa)
 Late-responding tissue: normal tissue where proliferative
rate is low (peripheral nerves, spinal cord)

For early-responding tissue, there’s a beginning of repopulation early


on in treatment. If your time of treatment is lengthy, you would
theoretically have to increase the dose to get the same effect. For
late-responding tissues, that point is much later.

So longer treatment time spares some of the early-responding


tissue (skin, tumor) but has no significant difference in late-
responding tissue (brain spinal cord).

You might think that you’d want to get the treatment done ASAP then
(bigger dose per day to decrease # fractions) to avoid sparing the early
responding tissue (including the tumor).

At higher doses per day, though, effect on late-responding tissues is


proportionally greater, since it has a more curved dose-response curve
(hurts therapeutic index).

If you multifractionate your regimen, you can reduce this problem (top
graph shows that your killing of early-responding tissue is slightly less, but
bottom graph shows that your killing of late-responding tissue is much
less) – improving your therapeutic ratio.

A few new radiation treatment designs:


 Hyperfractionation: use twice the number of fractions & same
amount of time).
o Dose per fraction decreased; total dose increased to give same overall tumor killing.
o Decreases side effects in late-responding normal tissue (not to increase tumor killing significantly)
 Accelerated treatment: get the treatment time done ASAP (avoid repopulation in tumor)
o Increased killing of early-responding tissue but also more side effects in early-responding normal tissue
o Study: use accelerated in pts with high doubling times for tumors: showed much better amount of local
control for pts with fast-dividing tumors
 Trade-off: decreased late side effects (hyperfractionation) vs. increased tumor control with increased acute side
effects (accelerated)
Pharmacology : Introduction & Neoplasia

Table of Contents:
Overview of pharmacology ........................................................................................................................................2
Drugs and enzymes.....................................................................................................................................................3
Receptors: Targets for Drug Action ............................................................................................................................4
Drug Metabolism ........................................................................................................................................................6
Principles of drug development .................................................................................................................................8
Complementary & alternative medicines ............................................................................................................... 10
Molecular Imaging ................................................................................................................................................... 11
Pharmacokinetics .................................................................................................................................................... 12
Autonomic Pharmacology I - Parasympathetic ....................................................................................................... 16
Autonomic Pharmacology II - Sympathetic ............................................................................................................. 19
An Overview of Cancer Chemotherapy ................................................................................................................... 23
Principles of antibody therapy for cancer ............................................................................................................... 26
Mechanisms and uses of antimetabolite drugs, signal transduction inhibitors, and anti-angiogenesis drugs in
antineoplastic therapy............................................................................................................................................. 28
Cancer Chemoprevention........................................................................................................................................ 32
Antineoplastic alkylating agents and platinum compounds ................................................................................... 34
DNA Topoisomerase-targeted drugs and mitotic spindle poisons .......................................................................... 39

1
Overview of pharmacology

Egyptians had all kinds of prescriptions: active constituents, carriers, formulations, deliveries.
Greeks & Romans used drugs like juniper oil (diuretic & abortifacient). Homer described opiates, Socrates took
hemlock (glycine receptor agonist), Hippocraties didn’t like drugs, and Pedanius discordies (physician for Nero)
wrote about 900+ drugs. Claudius Galenius made one of the best discoveries: don’t use urine & feces as drugs,
but also advocated polypharmacy (which didn’t work, so people stopped studying drugs)

16th-18th c.: Ascorbate (vit. C) for scurvy , Quinine for malaria, Digitalis for dropsy (=CHF edema) from foxglove.

Modern approaches:
1. Natural products – through 1960, usually studying extracts’ effects on animals or disease models, and
then purifying compounds to study (in vivo / cellular, etc.). Analogs then synthesized & optimized
2. Drug target screens with synthetic compounds – “magic bullets” (Ehrlich’s approach, 1900-present).
Screen compounds against organisms / receptors / enzymes & use “hits” as basis for more specific /
potent analogs. E.g. salvarsan for syphilis. Precursor of combinatorial chemistry
3. Rational drug design (1970-present). Examine molecular target (protein target, ligand, substrate) &
design high affinity molecules (e.g. ACE inhibitors, protease inhibitors). Manipulate synthetically as
needed.
4. Biotechnology (1980-present). Genetically engineer proteins (e.g. recombinant insulin), monoclonal Ab
(e.g. rituximab), maybe gene therapy in the future.

Challenges:
 Few well-validated human gene targets identified
 Need “blockbusters” for big pharma to invest
 Need to expand “chemical space” (more new classes of drugs)
 Need rational approaches to ADME (absorption, distribution, metabolism, excretion)

2
Drugs and enzymes

Constant Percent Effect


 Many systems: function on a % effect basis (that is, constant % of cellular units affected per unit time
regardless of # of units in the system.
 E.g. same % of enzyme inhibited if you keep inhibitor concentration the same, same % of max velocity
obtained if you keep substrate the same, same % of cancer cells killed if you keep chemo or radiation
the same.
 If X>>A (drug >> target, etc.), then it doesn’t matter how many targets there are – the same % will be
affected (“field effect”). Absolute numbers will differ, however

Graphing:
 You can plot [AX] vs [X] (e.g. velocity vs.
substrate, response vs. drug, binding vs.
receptor) and get a saturated curve
(rectangular hyperbola). If you use
log([X]), you get a sigmoid curve (semilog
plot).
 The point where you’re at half max (inflection in semi-log plot) goes by different names: Km for enzyme,
Ki for inhibitor, ED50 for drug, Kd for receptor binding. But it’s all the same – a measure of how the
thing you’re analyzing works & at what concentration it’s effective.
 If you vary the enzyme or target, when you’ve got a high amount of substrate, you see zero order
kinetics (e.g. the amount doesn’t matter because you’re usually saturated).
 In a rectangular hyperbola example, you’re usually in zero order situations at the point of saturation –
you’re processing a constant amount of something per unit time.
 When you’re in the linear early part of the curve, you’re in first order kinetics: processing a constant
percentage per unit time.

Inhibitors:
 Competitive inhibitors change Km, not Vmax (changing how well it binds, but can be overwhelmed by
more drug). Efficacy is the same (maximum effect)
 Noncompetitive inhibitors change Vmax, not Km (same binding, just taking some enzyme out of
picture). Efficacy is lowered.
 So look at the graph: is Vmax changing? Then it’s noncompetitive. Is Km changing? Competitive.
 Potency means you can get the same effect if you just use more of a dose (effect per dose)

Scatchard plot has drug bound/ drug free on Y axis, drug bound on X-axis.
 The x-intercept is Bmax, the highest amount of bound drug possible.
 The slope is -1/Kd
 Scatchard plot and Eadie-hofstee plots are reciprocals of the same graph.

Therapeutic index: LD50/ED50. You want a big therapeutic index

Kinetics: first order rate means that a constant % is removed per unit time. 𝐷𝑡 = 𝐷0 𝑒 −𝑘𝑡
Example: ethanol. One of very few substances that gets into zero order metabolism (constant amount) because
you ingest in grams scale.

3
Receptors: Targets for Drug Action

Receptor attributes: discrimination (selective subtypes), sensitivity Random interesting fact:


(respond to range of concentrations; range varies based on receptor),  KD is an intrinsic / fundamental
amplification (big downstream effects). relationship (KI too)
 ED50 is an experimentally
Have different targets: membrane receptors, nuclear receptors observed relationship (IC50 too)
Different signals: hormones, neurotransmitters, drugs, toxins

Model for receptor action:


R + L ↔ RL ↔ RL   E (receptor binds ligand, undergoes conformational change, downstream effect results)

Occupancy theory: biological effect (E) proportional to the concentration of the receptor-ligand complex ([RL])

Bioassay and ligand binding

Effect of a drug/hormone/ligand is based on:


1. Ligand concentration around receptor
2. Receptor concentration
3. Affinity between ligand & receptor
4. Nature of post-binding cellular response

Bioassay: quantitative analysis of Agonist action


 Vary total ligand concentration (Lt) and measure 𝜸 = occupancy
biological effect (E) KD = equilibrium dissociation constant
 Assume: occupancy theory holds, only one class of Emax = maximum biological effect
receptor, each receptor is independent Rt= total receptor concentration
𝑹𝑳 𝑬 𝑳
 𝜸= 𝑹𝒕
=𝑬 =𝑲
max 𝑫+ 𝑳

 Plot results as log dose response curve (LDR)


At 1/10th KD, you get 10% of the max response
o X-axis: log ([ligand])
At 10 times KD, you get 90% of the max response
o Y-axis: effect
o Estimate KD from ED50: SMALLER KD = MORE POTENT DRUG
o Can use to compare drugs (multiple curves) or analyze
antagonists vs agonists

 Agonist vs. Antagonist vs. Partial agonist


o This only applies to competitive inhibitors.
(noncompetitive inhibitors would lower the maximum
effect – lower maximum on y-axis)
o Competitive ligands compete for binding at the same
receptor
o Biological effect depends on
 Concentration of each ligand
 Their respective binding constants

4
Apply to receptor with no ligand Apply to receptor along with ligand
Agonist Full effect (stimulation) No effect
Antagonist No effect Full effect (inhibition)
Partial agonist Partial effect (some stimulation) Partial effect (some inhibition)

(Note that to detect an antagonist, you need to start


the system with some activity. Otherwise it might just
be inert)

Bioassay for antagonist action


 Add ligand at constant concentration and then
vary inhibitor concentration [I]
 Plot as a log dose inhibition curve (like LDR
curve but with inhibitor concentration on X-
axis on a log scale). IC50 is approximation for KI
 Can use to compare various inhibitors as well
 SMALLER KI = MORE POTENT INHIBITOR

Ligand Binding
 Use labeled (radiolabeled) ligand
 Vary ligand concentration [Lt]
 Measure receptor-bound ligand concentration
([RL]) and free ligand concentration ([L])
 Plot as either rectangular hyperbola or
scatchard plot (RL/L vs RL on linear scale)
where slope = -1/KD, intercept = Bmax (Rt)
o Steeper line = higher affinity

 Can also use the same method to see


binding of inhibitor, but it won’t actually
tell you what it’s doing: need to do a
bioassay.
o This method has no info about
biological effect – could be
agonist, antagonist, or partial
agonist.

Receptors: can belong to different families; ligand binding & effector domains usually linked. Subtypes exist for
most ligands (hormones, neurotransmitters, drugs). Subtypes can have different downstream effects & patterns
of expression (in different tissues or at different times in development.

5
Drug Metabolism
Drug metabolism: generally producing more polar / water soluble conjugates (better excretion). Can sometimes
make more active/toxic compound
Liver is key for majority of drug metabolism.
Important for:
 Toxicity: sometimes you can generate toxic/teratogenic metabolites (e.g. thalidomide)
 Activity: metabolites can be active (e.g. terfenadine)
 Drug interactions (inhibition of metabolism): another drug can increase to toxic levels (e.g.
ketoconazole and terfenadine)
 Drug interactions (induction of metabolism): another drug can decrease to sub-therapeutic levels (e.g.
rifampin and oral contraceptives)

Almost no oral drugs are absorbed in stomach. Most absorbed in small intestine like food & have to pass
through portal circulation to the liver (makes sense – want to detoxify them first.)
First-pass metabolism: metabolism that drug goes through in that first pass through the liver (before reaching
systemic circulation / “central compartment”)
 “high extraction”: drugs that are taken up & heavily metabolized by hepatocytes during first pass; their
hepatic clearance is dependent on liver blood flow
 “low extraction”: negligible first-pass effect
 How to circumvent:
o Change the route of delivery (IV, sublingual, transdermal, rectal – distal colon to IVC)
o Change the rate of metabolism (co-administer inhibitor of metabolism)

Hepatic drug metabolism


 Phase I: oxidation, reduction, hydrolysis. Cytochrome P450 enzymes or mixed-function oxidases.
o CP450s: oxidation rxns, distinct classes, metabolize different groups of drugs
 Similar to e- transport chain
 Vast majority of drugs metabolized by CYP3A4 (and CYP2D6)
 Example: thalidomide (metabolized to teratogenic metabolite by phase I rxn)
 Phase II: glucuronidation (glucuronyl transferases), acetylation (N-acetyltransferases), methylation
(methionine transferases), sulfation (sulfotransferases)
o E.g. sulfanilamide
 Both phases’ enzymes present in microsomal fraction of liver homogenate (located in SER, membrane-
associated)

Non-microsomal & extra-hepatic metabolism


 Cytosolic / mitochondrial fractions: alcohol/aldehyde dehydrogenase, monoamine oxidase, proteases
 Intestinal CYP450s: intestinal enzymes (including 3A4) may contribute to apparently poor oral
bioavailability or high first pass metabolism (up to 50%).
o Good for hepatotoxic toxins (teleologically)
 Also glucuronyl transferases (e.g. kidney) but liver does most work
 N-acetyltransferase in muscle – may play a role for “slow acetylators”

CYP450s & drugs


Drugs can be: substrates, inhibitors, & inducers of CYP450s (one drug can actually do all 3)
 P450 inhibitor: any drug that inhibits the metabolism or biotransformation of another drug by enzymes
in the cytochrome P450 family (competitive & reversible) – usually substrates too

6
o Can inhibit one or several classes of P450s
o Major offenders: cimetidine (anti-ulcer), macrolide antibiotics (erythromycin), antifungal azoles
(ketoconazole)
o Example: terfenadine levels increase if administered with ketoconazole; original form of drug
causes arrhythmias (before metabolized)
 P450 Inducer: Any drug that causes increased production of enzymes responsible for the metabolism or
biotransformation of another drug (drug acts as promoter, increases transcription)
o Increased mRNA levels (drug binds to enhancer elements upstream from enzyme coding region)
o Major offenders: phenobarbitol (anticonvulsant); rifampin (antibiotic) – e.g. with contraceptives

Effects of aging, disease, genetics

Neonates: low levels of functional glucuronosyl transferases (until ~1 mo) – can be particularly susceptible to
toxicity from toxins & drugs that are inactivated / cleared through glucuronidation

Elderly: can have reduced liver blood flow / reduced Phase I capacity (underlying disease, aging)
 Cirrhosis can affect drug clearance via 2 mechanisms
o Liver fibrosis (reduced blood flow through portal circulation: less 1st pass effect, higher systemic
concentrations of parent drugs
o Decrease in functional hepatocytes = less phase I capacity (mostly late in liver dz)
 Phase I reactions: impaired in acute & chronic liver disease;
 Phase II (conjugations) usually only in end-stage liver disease

Pharmacogenetics: polymorphisms in drug metabolism (altered amounts of enzymes or mutations in enzymes),


mostly affecting promoter region (altered amount more common)
 Classic example: Isoniazid (some are rapid metabolizers, some are slow metabolizers).

7
Principles of drug development
Five major players: pharma, regulatory agencies (FDA), consumers, academia, legislature)

History of drug development: chance observation  trial and error  targeted screening (Ehrlich’s “magic
bullet”, pasteur’s “anti-bodies” & “lock & key” model)

Discovery vs Development
 Discovery: identify the “lock”, develop a pattern of chemical “keys”, rapid-throughput screening, in vitro
or in vivo model systems – or can exploit a chance observation (still possible)
 Development: getting the drug discovered, through the system & to the patient.
o 15-20% of overall health care expenditures, but changes from year to year (can regulate – and
there’s a trade-off with savings from reduced hospital days / morbidity / mortality)
o $420B in 2005; increasing market for generics (2/3 Rx in 2009) and worldwide
 1:30,000 chemicals  licensed drug
 1:10 drugs in clinical testing  licensed drug
 1:5 licensed drugs  covers R&D expenditure
o Costs ~ $1B and patent life is 8-10 years so need $50-100M/year
o So pharma focuses on blockbusters (high use & profitability – prevalent chronic conditions)

Phases of drugs
 Pre-clinical drug development
o Efficacy, mechanism of action, toxicology
o Pharmacokinetics (ADME) – Absorption, Distribution, Metabolism, Excretion
o Pharmaceutics (formulation development)
 Phase I: short-term safety & tolerability; pharmacokinetics
o 10s of healthy volunteers; days to weeks
 Phase II: medium-term safety and tolerability; initial evidence of beneficial activity
o 100s of patients; weeks to months
o “proof of concept”
 Phase III: long-term safety and tolerability; clinical efficacy
o 1000s of patients; years
o “proof of effectiveness” – convince FDA that drug’s ready for market
o Very expensive phase – imaging, testing monitoring
 Phase IV: post-marketing surveillance; develop new indications
o study special pt populations, “real-world” effectiveness
o 1000s of patients; often retrospective

DEFINITIONS  KNOW THIS STUFF

Drug: any chemical administered with therapeutic intent.


 Different from foods, health foods (legistlative artifact), GRAS substances

Orphan drugs: intended for conditions affecting <200,000 people (get a little break on some regulations)

IND: Investigational New Drugs: application required for investigational new drugs or approved drugs if:
 Change in drug label (package insert: the only info anywhere that’s FDA approved)
 Significant advertising changes

8
 New route of administration, formulation, dose, pt. population with increase in risk
 IRB asks for it

New Drug Application: data submitted to support marketing approval of investigational new drug
 Reviewed by advisory committee who make recommendation (approve / disapprove)
 FDA not obliged to follow advisory committee recommendations

9
Complementary & alternative medicines
CAMs are important. 11.2% of all out-of-pocket spending in US, 38% of pts use CAMs

Extracts are combinations, supplements, not isolated


Drugs are isolated, pure chemical entities
Herbals are a subset of CAMs (also chiropractics, meditation, acupuncture)

Echinacea is the most common herbal product (cold remedy)


No evidence that any of these are safe or effective.

Healing is belief based and science based. Whereas scientific medicine is evidence based & changing,
traditional medicine is belief-based, anecdotal, static, and authoritarian. Science = good, other stuff = bad.

THIS IS WHAT YOU NEED TO MEMORIZE


ALL MEDICINE (TRADITIONAL, SCIENTIFIC, HOLISTIC) NEEDS TO FOLLOW THESE 3 PRINCIPLES:

1. Product must be standardized & rigorously regulated


2. Product must be proven to be effective for something that is of value to the patient
3. Product must be proven to be acceptably safe

1994: Dietary supplement health and education act (DSHEA) effectively neutered FDA (health food lobby won
out; FDA has very little control)

CAM development circumvents the 8-10 year, $1B drug development process: but at the cost of safety/toxicity
studies, effectiveness proven, standardization of the product

Example: PC-SPES (for prostate cancer – even in NEJM) but turned out to have synthetic drugs (hoax)

Herbal medicines generally not standardized (so you can’t study them well or refute claims). Many are
adulterated or inconsistent with their labeling.
 Ethically need to be able to know what you’re giving patients.
 Scientifically need to be able to replicate a study.
NEEDS TO BE EFFECTIVE & ACCEPTABLY SAFE

Only one herbal medicine has been approved (veregen for topical tx of genital & perianal warts in
immunosuppressed patients) – and even that doesn’t have great results

Several others (witch hazel = cuts & scrapes, senna & psyillum = laxatives) have been approved for modest value
in some illnesses.

CAMS can cause drug interactions.


E.g. St. John’s Wort increases metabolism of cyclosporin (an immunosuppressant) by ↑P450 3A4, which caused
transplant patients to lose their new hearts (immune response & rejection)

10
Molecular Imaging
Molecular imaging: the remote sensing of cellular processes at the molecular level in vivo
 (people or lab animals)
 Allows early detection of changes in tissue; manage changes in real time for patient (personalized
medicine), facilitates drug development (where is drug going?)

Modalities:
 Keys: spatial resolution of techniques, sensitivity (what quantities can be measured – e.g. optical =
picomolar, MRI = micromolar), specificity of probe (just hit target, not normal tissue)
 Can be combined with anatomic techniques like CT
 Four modalities: Optical, Nuclear (radiopharmaceutical), Magnetic Resonance (MR), or Ultrasound

Nuclear imaging:
 PET (Positron emission tomography) – currently the most clinically translatable modality
o More expensive; requires cyclotron, quantitative, 4mm resolution
o Physiologic tracers (15O, 13N, 11C, 18F, 124I)
o Combine with CT: PET-CT to see anatomy
o Receptor/enzyme/transporter mapping; assess metabolism; calculate drug receptor occupancy;
monitor biomarkers for therapy & patient selection for treatments
 SPECT (Single photo emission computed tomography)
o Less expensive ($500k), qualitative, 1.5cm resolution
o Uses chelation chemistry
o Example: image for prostate-specific membrane antigen (PSMA) for prostate cancer (if present
outside of prostate, could be recurrance of cancer

Tracer principle: use concentrations of a probe that are so low that they won’t alter the physiology of the
system under study (no pharmacological effect). Makes use in humans more easily approved. Especially good
for radiopharmaceuticals.

Receptor pre-blockade: saturate the target sites with a normal nonlabeled ligand, then apply your probe /
imaging agent. If there’s any signal detected, that shows non-specific / background signal (should only be
binding to your site of interest). Shows specificity. Could also use knockout animals

Direct imaging: probe binds directly to target (e.g. PSMA)


Indirect imaging: for instance, a reporter gene / reporter probe system (e.g. introduce a certain kinase gene
that will phosphorylate your probe & trap it in the cell, and put it behind a promoter that also controls
your gene of interest. When your gene of interest is turned on, the kinase will also be made, and your
probe will be trapped in those cells to be imaged)

Signal amplification: enzymatic reporters can be useful because they amplify the signal (your probe, for
instance) – keep working inside the cell (especially good for less sensitive modalities like MR)

Example: C. novyi (anaerobic, goes to anoxic center of tumor). How to image bacteria as they home in? image
with 125I (FIAU) – the toxin produced by a bacterial kinase . Turned out to be toxic (5 deaths) so not a Tx now.

Can also image breast cancer using PET (18F-fluorothymididine = FLT-PET) to detect early cancers.

11
Pharmacokinetics
Pharmacodynamics: what the drug does to the body (deciding on the target) – effect vs. concentration
Pharmacokinetics: what the body does to the drug (hitting the target) – concentration vs. time

What we’re really interested in: PK/PD interrelationship (effect vs. time)

Movement across membranes depends on various factors (size, dynamic polarity, water:octanol distribution,
protein binding, pKa, membrane transporters). Needs to be uncharged to get across (low dynamic polar
surface area < 140 angstoms, lower for brain/blood barrier)

Absorption determinants:
 Passive movement: middle ground for hydrophobicity is best to move between compartments
 Protein binding: generally only unbound drug can move across membranes
 Efficiency issue: high binding is not failure (if approved, it has effect)
 Albumin & α-acid glycoprotein are major proteins
 Varies with time (↑ α-a-g with inflammation, ↓ albumin with cirrhosis & nephrotic syndrome) so can
change concentrations of highly bound drugs
 Affinity can also change (uremia in renal failure decreases binding)
 Displacement theoretically possible but no known drug-drug examples

Henderson-Hasselbach Equation
 pH = pKa + log(A-/HA); pH = pKa when HA = A-
 Acid uncharged when pH < pKa (HA+)
 Base uncharged when pH > pKa (HA)
 Disease conditions (alkalosis or acidosis) can change how drug moves across membrane
o Gastric fluid 1.5-7, urine 4.5-7.5, blood etc. 7.4. Some drugs can be trapped depending on pH
o pH can change with other drugs (e.g. gastric fluid pH & omeprazole)

Transporters: active movement


 Can block transporters to keep drug in or to keep from being pumped out, but usually work to pump out
range of drugs
 E.g. organic ion (pumps from cell into lumen); P-glycoprotein (pumps from capillary into cell)

Basic definitions

AUC (mg/mL * hours): drug exposure per time

F: Bioavailability (unitless): fraction reaching systemic circulation. Limited by failure to enter solution, short
exposure to absorptive surface, failure to pass across membrane, pre-systemic metabolism so less than 1 in
almost all circumstances.
𝑨𝑼𝑪
 Bioavailability= 𝑨𝑼𝑪𝑬𝑽 (where EV = after extravascular dose, IV = after intravascular dose)
𝑰𝑽
 EV peaks later, around longer than IV
 Low bioavailability affects ability to dose po (may need frequent doses or IV) & can be a feasibility issue
 F<1.0 because drugs fail to enter solution, have short exposure to absorptive surface, limited passage
across membranes, and/or or pre-systemic metabolism
 Adjust for F when logically required

12
S: Salt (unitless): some drugs are composed of other stuff by weight too
𝒂𝒎𝒐𝒖𝒏𝒕 𝒂𝒄𝒕𝒊𝒗𝒆 𝒅𝒓𝒖𝒈 𝒊𝒏 𝒂𝒅𝒎𝒊𝒏𝒊𝒔𝒕𝒆𝒓𝒆𝒅 𝒇𝒐𝒓𝒎
 𝑺 = 𝒂𝒎𝒐𝒖𝒏𝒕 𝒐𝒇 𝒕𝒐𝒕𝒂𝒍 𝒅𝒓𝒖𝒈 𝒔𝒂𝒍𝒕 𝒊𝒏 𝒂𝒅𝒎𝒊𝒏𝒊𝒔𝒕𝒆𝒓𝒆𝒅 𝒇𝒐𝒓𝒎
 Analogous to F
 E.g. aminophylline is 80% theophyilline by weight

Vd: Volume of distribution (L): Volume into which dose appears to be uniformly distributed
𝑫𝒐𝒔𝒆
 𝑽
= 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏
𝒅
 Affected by drug movement across membrane (less movement = higher conc in central compartment)
 Use population Vd (adjust for weight – given in L/kg) as first estimate. Can be “lean weight” if not fat
soluble
 Generally a theoretical concept: measuring only total, not free drug; only sampling central compartment

ke: Elimination rate constant (1/time): fraction of drug eliminated in a unit of time
 Not the elimination rate
 First order (not saturated) – constant value (constant fraction eliminated); goes down as saturation
occurs (zero order – constant amount eliminated)
 𝑪𝒕 = 𝑪𝟎 𝒆−𝒌𝒕 for elimination
 Plot ln(Concentration) vs. Time and your slope is –k
 Can backwards-extrapolate to get C0, your initial concentration
 𝒌 = 𝑪𝒍/𝑽𝒅

t1/2 : half life (time): how long it takes concentration to drop by half.
 Only works for first order.
 When Ct/C0 = ½ , equation above simplifies
𝟎.𝟕
o 𝒕 =𝒌
𝟏/𝟐
 Another way to look at it
𝟏
o 𝒆−𝒌𝒕 = 𝒏
𝟐
o n = number of half lives elapsed post dose, or number of half lives in dosing interval to find
trough.
 Or:
𝟏 𝒏
o 𝑪𝒕 = 𝑪𝟎
𝟐

Cl: Clearance (L/hr)


 Relates concentration to rate of elimination (First order, saturable process).
 𝑬𝒍𝒊𝒎𝒊𝒏𝒂𝒕𝒊𝒐𝒏 𝒓𝒂𝒕𝒆 = 𝑪𝒍 ∙ 𝑪
 Varies with drug and patient
 Cl = Q * E (depends on flow and extraction ratio across organ)
o E.g. hepatic clearance is a function of hepatic blood flow and extraction ratio, which in turn
depends on metabolizing enzyme function
o Renal clearance varies with protein binding (filtration + secretion – absorption)
 Total clearance = renal + hepatic + other
 “Apparent clearance” = Clearance / bioavailability
𝑪𝒍 𝑫𝒐𝒔𝒆
o 𝑭𝒕𝒐𝒕𝒂𝒍
= 𝑨𝑼𝑪
𝒃𝒍𝒐𝒐𝒅

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Rac: Rate of accumulation
𝐶𝑚𝑎𝑥 ,𝑠𝑠 1 1
 𝑅𝑎𝑐 = 𝐶 = 1−𝑒 −𝑘𝜏 = 1
𝑚𝑎𝑥 ,1𝑠𝑡 𝑑𝑜𝑠𝑒 1− 𝑛
2
 If you’re dosing a drug with a long half life and dosing, say, every 1/5 half life, you can get a big variation
over time. If you’re dosing ever 4-5 half-lives, you don’t get much buildup

Loading dose: achieve a desired drug concentration.


 Assumes instantaneous, uniform distribution throughout body with no account for time
 𝑫𝒐𝒔𝒆 = ∆𝑪 ∙ 𝑽𝒅
 Adjust for F and S as needed

Calculating an individual’s Vd and t1/2


 Measure drug concentrations at two timepoints during decay
 Plot ln(concentration) vs time
 Find slope (-k) and calculate using k = 0.7 / t1/2
 Find intercept = C0 and then calculate Vd (Vd = dose * C0, corrected for F)

Continuous infusion: elimination rate matches infusion rate exactly (quasi-steady-state)


𝑫𝒐𝒔𝒆
 = 𝑪𝒍 ∙ 𝑪𝒔𝒔
𝝉
 Adjust for F as needed
 Note that things that change clearance (other drugs, etc) will affect this concentration at SS

If you give a loading dose & follow up with continuous infusion, the curves sum to be almost square (infusion
accumulates as loading dose decays.

Intermittent & continuous dosing


 Average dose is the same by either method (elimination rate is slow at first for intermittent dosing but
then evens out)
 Fixed daily dose, different interval: peak/trough changes, concentration at steady state doesn’t
 Same dose, different interval: same peak/trough, different Css

Basic half-life equations:


1
o Rise: 𝐶𝑡 = 𝐶𝑠𝑠 1 − 𝑒 −𝑘𝑡 = 𝐶𝑠𝑠 (1 − 2𝑛 )
1
o Fall: 𝐶𝑡 = 𝐶0 𝑒 −𝑘𝑡 = 𝐶0 (2𝑛 )
Time to 95% (up or down)= 4 to 5 half-lives

Concentration-constrained dosing: how to calculate dose & interval for peak & trough
 Shortcut: if you want Cmax / Cmin to be 2, max interval is t(1/2). If you want 4, 2t(1/2) is your interval
 Interval (Rate = ratio) – Cmax/Cmin = 2n
𝐶 𝑚𝑎𝑥 ,𝑠𝑠
ln
𝐶 𝑚𝑖𝑛 ,𝑠𝑠
o 𝜏𝑚𝑎𝑥 = where 𝑘 = 0.7/𝑡1
𝑘 2
 Dose=Difference
o Single loading dose (get up to the max SS)
o 𝐷 = 𝐶𝑚𝑎𝑥 ,𝑠𝑠 − 𝐶𝑝𝑟𝑒𝑠𝑒𝑛𝑡 ∗ 𝑉𝑑
o Correct for F, S when needed
 Intermittent maintenance dose (given every 𝜏𝑚𝑎𝑥 )

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o 𝐷 = 𝐶𝑚𝑎𝑥 ,𝑠𝑠 − 𝐶𝑝𝑟𝑒𝑠𝑒𝑛𝑡 ∗ 𝑉𝑑
o Correct for F, S when needed

A few other topics:


Distribution: at first, both drug moving out of central compartment (distribution) and being eliminated, so rate
of drop in concentration is higher.
 Implications
o Delayed effect if peripheral site of action
o Big distribution phase increases risk for central toxicity (if narrow therapeutic index)
o Need to monitor post-distribution levels (otherwise artificially overestimate initial
concentration)
Short infusions are more common than IV bolus infusions: distribution can apply though

Ka (absorption rate constant) – if a lower Ka, then slower abosorption. Peak is lower, trough is higher, and AUC
is the same. Peak is also later.
 Slower absorption can be good – keeps a narrow window in your peaks and troughs (e.g. extravascular
dosing)

Things to adjust away from population values if needed


 Loading dose: altered Vd (linearly proportional)
 Maintenance intermittent dosing (if t1/2 changes, not linearly proportional)
 Maintenance infusion dosing rate: altered Cl (linearly proportional)
 Time to steady state: linearly proportional to t1/2

Renal clearance: can adjust for creatinine clearance if drug is cleared by the kidney (age, weight, gender taken
into account). If creatinine clearance is lower, you can drop your infusion to get to the same steady state
concentration (but it will take longer! T1/2 is larger)

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Autonomic Pharmacology I - Parasympathetic

Basics:
 Nervous system: afferent (sensory information), integration in ganglia in CNS structure, efferent
(somatic motor = voluntary skeletal mm, autonomic – involuntary smooth mm), mostly antagonistic
 Parasympathetc = homeostasis, sympathetic = fight or flight

Parasympathetic Nervous System


 Anatomy: originates from midbrain, medulla, sacral spinal cord, long presynaptics, ganglia near targets
so stimulation gives localized effects
 Ganglionic receptors = nicotinic AcH (gated ion channel)
 Target receptors = muscarinic AcH (GCPR)

PNS Actions:
 Homeostasis, opposes sympathetic activity generally
 Constricts smooth muscle, increases glandular secretions usually
 Classical actions: pupil constriction, bronchial constriction, decreased heart rate, relax sphincters &
contract GI segmental/longitudinal mm, contract bladder (relax sphincter)

Cholinergic synapse:
 AcH synthesized by choline acetyltransferase
 Ca+ dependent release (+ B-bungarotoxin, black widow venom, - botulism toxin by cleaving SNARE)
 Muscarinic AcH receptor (+pilocarpine, - atropine)
o 7-membrane-spanning GPCR
o M1-M5 subtypes with selective tissue expression, specific functions but overlap
o M2 is cardiac-selective; subtyping hasn’t really been exploited clinically so far
 Acetylcholinesterase breaks it down (- neostigmine, soman)
o This is the turnoff (compare to sympathetic)
o Neuronal AchE and also serum/liver (butrylcholinesterase) so you can’t just inject Ach into gut
 Choline uptake

MUSCARINIC AGONISTS

Generally: choline esters & other cholinomimetics

pilocarpine Mechanism of Action: muscarinic AcH receptor agonist


Effects: generalized muscarinic (incl. ocular-pupillary constriction, fall in intraocular pressure after
sudden rise, miosis = constriction of pupil lasts several hours)
Indications: glaucoma (esp. open angle)
Administration: aqueous opthalmic solution (prevent cardiac effects)

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metoclopramide Mechanism of Action: Muscarinic AcH receptor agonist
Effects: increases gastric emptying; anti-emetic (dopamine receptor agonist), also enhances cholinergic
effects
Indications: First-line gastroparesis & anti-emetic agent
Administration: oral
Other: a.k.a. "Reglan"

MUSCARINIC ANTAGONISTS

atropine Mechanism of Action: competitive antagonist of muscarinic Ach receptors


Effects: dry mouth, constipation, urinary retention, dry skin, flush, bronchodilation, mydriasis (=pupil
dilation), delirium. Mad as a hatter, red as a beet, dry as a bone
Indications: Stop asystole (code blue), diarrhea, antidote to AchE toxins, pupil dilation
Administration: po, opthalmic, or by injection

scopolamine Mechanism of Action: muscarinic Ach receptor antagonist


Indications: motion sickness
Administration: oral or transdermal

ipratropium Mechanism of Action: muscarinic Ach receptor antagonist


bromide Effects: bronchodilation
Indications: asthma, COPD
Administration: metered dose inhaler
Other: a.k.a. atrovent

ACETYLCHOLINESTERASE INHIBITORS
 May be therapeutics (physostigmine for glaucoma), antidotes to poisons (pyridostigmine), or toxins
themselves (sarin) depending on their affinity for AchE, route of administration, dose, etc.
 Many insecticides, nerve gases, etc. fall in this category
 AchE is a serine protease that cleaves Ach to acetate & choline in synaptic cleft
 Found in high molecular weight aggregates (stable = low turnover = really bad if you mess it up)

neostigmine Mechanism of Action: reversible covalent inhibitor of AchE


Indications: can be used for prophylactic protection to AchE inhibitor nerve gases (fills up site, then
wears off unlike sarin, etc.)

sarin Mechanism of Action: "irreversible" covalent inhibitor of AchE


Effects: signs of AchE poisoning: bronchial spasm, salivation, lacrimation, defecation, urination,
bradycardia, hypotension, muscle weakness, death in minutes to hours
Administration: nerve gas / chemical warfare agent
Other: can be reversed with pralidoxime rescue if "aging" doesn't occur first (irreversible complex
formed with AchE over time, preventing palidoxime's nucleophilic attack). Treatment also includes large
quantities of atropine

17
pralidoxime Mechanism of Action: nucleophilic attack on AchE-sarin covalent complex
Effects: Reversal of sarin AchE poisoning
Indications: sarin AchE poisoning
Other: used along with atropine for sarin & other similar AchE poisons

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Autonomic Pharmacology II - Sympathetic
Anatomy:
 From thoracolumbar spinal cord; ganglia are paraverterbral & longer post-synaptic fibers
 Ganglionic receptors: nicotinic cholinergic
 Target receptors: adrenergic
 Primary postganglionic neurotransmitter: norepinephrine
 Adrenal medulla is like a big postganglionic neuron, releases epinephrine
 Stimulation results in a widespread reaction

Physiology:
 “Fight or flight”response
 Smooth mm: relax or constrict
 Classical actions: dilate bronchi, ↑glucose from liver, ↑rate & stroke volume from heart (↑ cardiac
output), ↑blood flow to skeletal mm, ↓motility & ↑ sphincter tone in gut
 Activation via enervation of target and adrenaline (=epinephrine) in blood circulation from adrenals

Adrenergic synapses
 NE synthesized in neuron (tyrDOPA dopamineNE). Epinephrine synthesized in adrenal gland
 Tyrosine hydroxylase is the rate-limiting step of synthesis & activated by sympathetic stim.
o α-methyltyrosine blocks tyrosine hydroxylase, used in management of pheochromocytoma
(usually benign tumor of adrenal that fires out too much catecholamine)
 Release of NE is Ca-dependent
o Some agents can act as false neurotransmitters, replacing NE in secreted vesicle & ↓NE effect
o Tyramine, amphetamine, ephedrine: sympathomimetic (enhance NE secretion)
 Adrenergic receptor (many agonists & antagonists) on post-synaptic side
o GPCR
 NE transporter brings NE back into pre-synaptic neuron (blocked by tricyclic antidepressants)
o Turnoff mechanism is re-uptake (not breakdown like parasympathetic)
 Monoamine oxidase breaks down NE & deaminated products extruded (target of antidepressants)

Adrenal glands have PNMT which converts NE (noradrenaline) to ephinephrine (adrenaline), major adrenal
medulla hormone. NE and ephinephrine have different pharmacological effects

Subtypes of the adrenergic receptors:


 α1 (with 3 subtypes α1A, α1B, α1C): mixed effects
 α2 (with 3 subtypes α2A, α2B, α2C): ↓adenylate cyclase, ↓Ca channels, ↑K channels
 β (with 3 subtypes β1, β2, β3): ↑ adenylate cyclase
 Dopamine receptors (D1-5) in vascular bed (D1) and CNS (all 5) can also bind catecholamine derivatives;
dopamine can bind α-receptor

α-receptor pharmacology

Non-selective α-receptor agonists: epinephrine, NE, phenylephrine, dopamine (see end of section)
Non-selective α-receptor antagonists:

19
phenoxybenzamine Mechanism of Action: nonselective antagonist of α adrenergic receptors
Effects: Reduces NE effect via inhibition
Indications: pheochromocytoma: adrenal tumor producing large amounts of
catecholamines (tumor usually benign; reverse effects)

α1-receptor agonists:
phenylephrine Mechanism of Action: selective agonist of the α1 adrenergic receptor
Effects: induces vasoconstriction
Indications: hypotension, nasal congestion
Administration: po or nasal spray as decongestant

α1-receptor antagonists:
prazosin Mechanism of Action: selective antagonist of the α1 adrenergic receptor
Effects: blocks smooth muscle constriction (relaxes ureters); vasodilation (can help with
hypertension)
Indications: enhances urine flow in benign prostatic hyperplasia, especially with hypertension

α2-receptor agonists:
clonidine Mechanism of Action: selective agonist of the α2 adrenergic receptor.
Effects: decreases NE release pre-synaptically (α2 is on pre-synaptic side, for feedback inhibition).
Indications: hypertension (reduces blood pressure), used for withdrawal in substance abuse
Administration: orally (both indications) or patch (substance abuse)

α2-receptor antagonists: yohimbine but questionable clinical use

β -receptor pharmacology
Non-specific β receptor agonists:
isoproterenol Mechanism of Action: nonselective agonist of the ß adrenergic receptors
Effects: stimulates cardiac output, dilates bronchial smooth muscle, dilates vascular smooth
muscle
Indications: bradychardia or heart block; rarely used to treat asthma

Non-specific β receptor antagonists:


propranolol Mechanism of Action: nonselective antagonist of ß adrenergic receptors (general ß blocker)
Effects: decreased cardiac output, bronchial smooth mm constriction
Indications: used to treat hypertension, angina, anxiety, vasovagal syncope (when heart rate
rises, blood vessels dilate too much, decreased brain perfusion, patient faints), arrythmias

β1 receptor agonists:

20
dobutamine Mechanism of Action: selective agonist of ß1 adrenergic receptor
Effects: increases cardiac rate & force of contraction (increased cardiac output); dilates
coronary arteries to reduce afterload
Indications: cardiomyopathy with CHF, especially in anginal states
Administration: IV
Other: doesn't work indefinitely

β1 receptor antagonists:
metoprolol Mechanism of Action: selective antagonist of ß1 adrenergic receptor (beta-blocker)
Effects: reduces cardiac output, reducing demand on heart
Indications: hypertension & angina
Other: also may promote better filling of coronary arteries by increasing length of diastolic phase

β2 receptor agonists:
albuterol Mechanism of Action: selective agonist of ß2 adrenergic receptor
Effects: dilates bronchial smooth muscle & uterine smooth muscle
Indications: asthma; stopping premature labor
Other: very few cardiac side effects because of ß2 selectivity. Also promotes glycogenolysis in liver
(be careful not to precipitate diabetic state)

β2 receptor antagonists: not useful clinically


Some interest in β3 receptor agonists to reduce adipose tissue in adiposity

Epi, NE, and dopamine


epinephrine Mechanism of Action: predominantly an agonist of ß adrenergic receptors (over α)
Effects: increases cardiac output & systolic arterial blood pressure; large metabolic effect
(increases O2 comsumption & blood glucose)
Indications: stopping anaphylactic response, used in code blue settings

norepinephrine Mechanism of Action: Primarily an agonist of α adrenergic receptors


Effects: Increases systolic and diastolic blood pressure; less effect on cardiac output &
metabolism
Indications: Hypotensive shock (largely replaced by phenylephrine now)

dopamine Mechanism of Action: Acts on D1 receptor in vasculature; also α and ß in high doses
Effects: Pressor (increases blood pressure), used in cardiac situations.
Indications: Low dose (renal dose) can be used to impact D1 receptor only if patient has low renal
perfusion; higher doses impact D, α, and ß receptors and is first pressor in most instances of
shock (low BP)
Misc: Adenosine receptor pharmacology: treating asthma, for example, by antagonizing adenosine at airways;
Nitric oxide signaling is also autonomic.

21
SUMMARY TABLE (autonomic I and II)

22
An Overview of Cancer Chemotherapy

 Current treatment strategies: prevent, cure, or palliate


 Cancer can cause morbidity & mortality by disrupting physiology locally (invade/disrupt vital organ function)
or systemically (metastatic spread) – so treatment can be directed locally or systemically as well
 Important rise in # of cancer survivors in US – have to re-calibrate their health risks (Tx after-effects, etc.)

Local treatments
 Surgery (solid organ malignancy; need systemic disease, need staging before surgery to determine extent)
 Radiation therapy (localized cancers that can’t be removed surgery; also as adjuvant with surgery &
palliation of metastases that can cause morbidity)
 Chemotherapy (sometimes given regionally – e.g. hepatic artery for colonic cancer liver metasteses)
 Other (immunotherapies, etc. – often experimental)
Cancers vary in responsiveness to
Systemic treatments cytotoxic chemotherapy
 Hormone / Growth Factor (remove / antagonize trophic
hormones, e.g. estrogen for breast cancer, androgens for  Highly responsive (Hodgkin’s, Wilm’s
prostate cancer) tumor)
o Tamoxifen (anti-estrogen) – adjuvant after resection of  Responsive (acute leukemia in
breast cancer or systemic for metastatic children, retinoblastoma, testicular
 Chemotherapy: for systemic cancers, for cure (leukemia, testis, cancer)
lymphoma) or pallation (solid organ cancers)  Moderately responsive (acute
o Can be used as adjuvant to improve local treatment leukemia in adults, multiple myeloma,
potential breast cancer, prostate, ovarian)
 Other (immunotherapy, gene therapy, etc.)  Partially responsive (glioblastoma,
colorectal, pancreatic islet cell
Treatments can target: carcinoma, bladder)
 Minimally responsive: (liver,
 Targets unique to cancer cells (mutant gene products,
pancreatic cancer; melanoma)
oncogenic virus products)
 Qualitative/quantitative differences in cancer cells (cell cycle
effectors, macromolecules, biosynthetic enzymes, signal transduction pathway components) that are also
present in normal cells
o Therapeutic index (LD50/ED50)usually low for anti-cancer drugs (bad) because of this

Antineoplastic drugs: cytotoxic mechanisms of action


(based on the idea that cancer cells divide more rapidly; not actually that
simple)
 Damage cancer cell DNA (alkylating agents)
 Limit supply of precursors for DNA synthesis (antimetabolites)
 Interfere with DNA replication (topoisomerase poisons)
 Interfere with mitotic segregation
Cancer cells in an exponential growth phase are more sensitive to cytotoxic
agents than those in plateau phases

Log-linear kill kinetic behavior of antineoplastic drugs:

23
 Fractional cell kill: a specific dose of chemotherapeutic drug kills a specific fraction of tumor cells regardless
of tumor cell population (see logarithmic decline with therapy)

Norton-Simon hypothesis
 Based on observation that improvements in response rates from
chemotherapy doesn’t lead to improvements in survival
 General idea: a more effective treatment will kill lots of cells, get
down to the exponential growth part of the curve; cancer cells
replicate quickly and “catch up” to a similar less-effective
treatment. Mortality occurs way out in plateau phase.

Activation of apoptosis by antineoplastic drugs


 Accumulation of neoplastic cells = ↑division, ↓death, or both
 Cancer cells can escape apoptosis by activating senescence or autophagy
 ↓p53 or ↑Bcl-2 are common mutations to avoid
 Antineoplastic agents can stabilize p53 or use other strategies to induce apoptosis
o Many classes create DNA strand breaks to increase p53 activity & apoptosis
 New pathways for targeting: caspase cascade, other cell-death signal transduction pathways

Antineoplastic drug resistance


 Cancers arise clonally but have heterogeneity in later populations (tumor cell heterogeneity)
 Subclones can be resistant to drugs
o ABC transporter family: pump out lots of kinds of drugs in ATP-dependent manner. Blocking is
difficult therapeutic target (also pump drugs out in kidney, etc.  increased toxicity)

Goldie-Coldman hypothesis
 Luria and Delbruck: in bacteria, rate of genetic variants appearing related to rate of cell division &
probability of variant arising with each division (genetic instability)
 Goldie / Coldman: considered in cancer cells & anti-neoplastic drof genetic instability in population
o Although rate of appearance is independent of total number, absolute number of subclones is
greater for a greater size of cancer cell population (need to detect early!)
 Experiment (L-D Fluctuation Analysis): culture cells and then:
o Plate concentration of mixture of cultured cells on a bunch of plates: about the same amount of
resistance occurs in each plate (Poisson distribution = random)
o Pick out individual cells, grow up clonal population, and plate same concentration. Non-Poisson
distribution in amount of resistance on each plate (mutation can occur early or late in clonal
growth process)

24
So: cancer might respond well but eventually these subclones can emerge and treatment fails.
Rationale for combination chemotherapy, combined modality treatments, debulking surgery: rapidly reduce
the total # cancer cells and absolute # drug-resistant subclones

Why are some cancers readily curable & others poorly responsive?
1. Cancer cell kinetic properties. Curable cancers may have more rapid growth (antineoplastic drugs kill
them better).
2. Cancer cell biochemical properties. Noncurable cancers can detoxify, extrude, or otherwise escape
antineoplastic drugs.
3. Cancer cell phenotypic properties. Related to cells of origin of cancer (not well defined) – e.g. testis
cancer more responsive than prostate.

Cancer “stem cells”


 Normal renewing cell populations have rare stem cells around (differentiate to “transient amplifying”
then fully differentiated, non-proliferative cells)
 Cancer may be similar
 Theory (controversial):
 Tx that kill differentiated cancer cells show treatment response but not improved survival or cure
 Tx that kill stem cells might prolong surviva but not show initial response

Developing new drugs:


 Historically: Phase I (tolerance, dose-limiting toxicities, maximally tolerated dose = MTD), Phase II
(efficacy: complete & partial responses), Phase III (comparative efficacy & toxicity vs current regimens),
Phase IV (integrating into standard treatment after FDA marketing). 10 years, $1 Billion.
 Now: more common Phase I/II combinatorial trials after molecular biomarker use. Cheaper & quicker
 Big bottleneck: not discovery but rather clinical trials / approval

25
Principles of antibody therapy for cancer

Antibody clearance:
 Abs are too big to go through glomeruli (except in renal disease)
 Fab fragments are small and are therefore cleared within hours
 Chimeric Ab have mouse variabble region, human constant region
 Humanized Ab have partially human variable regions too

Polyclonal antibodies (therapeutic uses)


 Anti-venins (against toxic proteins from snakes, spiders)
 Drug overdose (e.g. digoxin OD)
 Immune modulation (e.g. antithymocyte globulin)
 Treat infection (pre-antibiotics)
 Replacement IgG for X-linked agammaglobulinemia (XLA) – administer human IgG every 3-4 weeks (t1/2
= ¾ weeks)

Lymphoma:
 Ron Levy:
o B-cells display “idiotype” (specific B-cell receptor expressed on surface)
o Use the clonal nature of B-cell lymphomas (same idiotype) as target
o Follicular lymphoma: slow growing, could wait for treatment, expected to live several years, IgG
on cell membrane
o Made IgG for each pt’s lymphoma in lab and treated them (1st patient was big success, mixed
results afterwards)
o Serum sickness: immune reaction against foreign antigens
o One problem: Human Anti-Mouse Antibody (HAMA) – retreatment difficult (already have
HAMA)
o Recurrence: anti-idiotype ab no longer bound to tumor cells (somatic hypermutation in B-cells
& in this lymphoma = development of new lymphocytes)
o Precise but time consuming & recurrence possible
 CD20 & Rituximab
o CD20 only expressed on B cells (not plasma cells, not precursor stem cells)
 Lets you regenerate your B cells after treatment
o When anti-CD20 Ab bind, no internalization of complex & Cd20 not downregulated

Rituximab
 Toxicity: More adverse events in first infusion (killing most B-cells at first) – opposite of other Ab
therapies, where serum sickness sets in more with more immune reaction to foreign Abs. Due to large
number of dead B-cells.
 THINGS TO KNOW:
o HAMA don’t develop because rituximab is killing B cells, which would generate the human anti-
mouse antibodies
o Serum IgG levels don’t fall because plasma cells make most of the IgG in serum. No CD20
means they’re not killed
o Why do B cells recover? Hematopoetic stem cells don’t have CD20 so aren’t killed.
 Efficacy of rituximab depends on CD20 expression; often used with other therapies
 Mechanisms of Action:

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o Directly induces apoptosis (lymphocytes’ own self-destruct mechanism triggered by CD20
binding)
o Antibody dependent cell-mediated cytotoxicity (ADCC) – killed by NK cells & others
o Complement fixed & cells lysed.
 Reistance
o CD20 not downregulated & escape mutations are rare
o Major mechanisms:
1. Failure to generate ADCC (genetic variant in Fc binding receptor)
2. Variations in apoptotic pathways (probably most common)
o Overall: Ab still binds to tumor, just doesn’t kill it
 Treatment considerations:
o Everyone eventually relapses
o Maintenance therapy?
1. inhibits ability to generate new IgG
2. ↑ susceptibility to certain infections
o Retreatment generally works if pts have not received rituximab recently (but not if on
maintence)
Nomenclature
Antibody conjugates Ri-tu-xi-mab; 1-2-3-4
 Radioimmunoconjugates 1. Anything
o Murine Ab; target CD20 and conjugated to 2. tu(m) = tumor is target
radioactive isotopes 3. type of antibody
o Crossfire increases action (overcomes resistance) a. o = mouse
because neighboring cells are killed too by b. xi = chimeric
radiation (those which are apoptosis-resistant) c. zu = humanized
4. mab = monoclonal antibody
 Immunotoxins work by a similar idea
 If tumor is radiosensitive, conjugate radioactivity; if not,
conjugate a toxin (but toxin may be antigenic, preventing
Half-lives
retreatment
 Fab = hours
Consequences of Ab therapy  Mouse = days
 Targets  Chimera = days to weeks
o Looking for muntant proteins, only in cancer, etc.  Human = months
– but now maybe some tissues (e.g. b-cells) are
disposable? Thyroid / prostate?

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Mechanisms and uses of antimetabolite drugs, signal transduction inhibitors, and anti-
angiogenesis drugs in antineoplastic therapy

General notes:
 Antimetabolites: In the end, these pretty much all work by leading to DNA double-strand breaks
(↓nucleotide pool or ↓DNA polymerase speed; causing DNApol to get stuck & cause DBS without
repair)
o All pretty much have bone marrow toxicity
 In traditional treatment, all roads lead to DNA (now more of a targeted approach

Antimetabolites:
 Mimic structure of normal metabolic species; inhibit enzymes in both normal and tumor cells
 Administered as prodrugs that require metabolic activation
 Inhibit deoxynucleotide and DNA synthesis; kill cells in S phase

 Targeting tumor cells: take advantage of different transport or enzymatic activation of prodrug, or of
cells that are progressing through cell cycle
 Nonneoplastic cells most affected: rapid cell division
o Hair follicle, bone marrow, intestinal epithelium cells
o Therapy induced leukemia is major complication

 Limitations: drug delivery (central hypoxic zone of solid tumors), not all tumor cells are cycling, fixed
percentage killed with each treatment, need active immune system, drug resistance common

Folate antagonists:
 Inhibit dihydrofolate reductase (DHFR) which reduces folic acid to dihydrofolate (DHF) and
tetrahydrofolate (THF)
 THF is required to carry methyl & methylene (1-C) groups for thymidine and purine biosynthesis

methotrexate Mechanism of Action: Folic antagonist (antimetabolite). Inhibits dihydrofolate reductase (DHFR).
Effects: Inhibits DHFR which is involved in synthesis of THF from folic acid. THF is the methyl/methelyne
carrier for purine and thymidine synthesis.
Indications: wide variety of cancer breast cancer, colorectal cancer, lymphoma
Administration: often paired with leucovorin shortly after MTX given ("leucovorin rescue") - replentishes
folate stores
Toxicity: mucositis, kidney damage, hepatotoxicity
Resistance: Reduced uptake; reduction in enzymes that add polyglutamate; DHFR gene amplification
Other: Actively transported into cells. Requires activation by addition of several glutamates (traps in cell;
enhances inhibition). Aka MTX

Deoxynucleotide synthesis antagonists

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hydroxyurea Mechanism of Action: Inhibits ribonucleotide reductase. Antimetabolite antineoplastic agent.
Effects: Ribonucleotide reductase reduces NDPs to dNDPs for DNA synthesis
Indications: Leukemias; head and neck cancers
Toxicity: Standard (bone marrow, etc.)
Resistance: Overexpression of reductase

Note: Nucleoside analogs (5-FU, 6-percaptopurine, 6-thioguanine, etc.) are able to be transported into cell via
nucleoside transporter, and are then p-lated and trapped in cell. They must fit into a kinase in the cell, however
(resistance mechanism)

Pyrimidine biosynthesis antagonists

5-fluorouracil Mechanism of Action: Covalently modifies thymidylate synthase, the enzyme which converts dUMP to
TMP. Triphosphate form can also be incorporated into DNA and cause strand breaks
Effects: Antimetabolite antineoplastic agent.
Indications: Colorectal and breast cancer
Administration: IV and oral. Often co-administered with leucovorin. TS uses folate as a cofactor (also when
5-FU binding), so adding a folate analog like leucovorin pushese the inhibitory equilibrium through
(LeChatlier's). Also often co-administered with 5-ethynyluracil, which inhibits dihydropyrimidine
dehydrogenase in intestine.
Toxicity: Bone marrow suppression
Resistance: Decreased activity of activating enzyme. Intestinal enzyme dihydropyrimidine dehydrogenase
can inactivate by converting it to dihydroform and preventing absorption.
Other: Transported in via nucleoside transporter, then P-lated and trapped in cell (needs to fit in kinase)

Purine biosynthesis antagonists

6-mercaptopurine, Mechanism of Action: Competitive inhibitor of several enzymes in purine synthesis pathways (looks
6-thioguanine, like guanine); also gets incorporated into DNA
azathioprine Effects: Purine biosynthesis antagonist; antimetabolite antineoplastic agent.
Indications: leukemias
Administration: oral
Toxicity: Bone marrow suppression
Resistance: inactivated by xanthine oxidase (XO). Decrease in HGPRTase activity is common resistance
mechanism.
Other: Must undergo activation to form mononucleotide (add sugar) via HGPRTase. Also inactivation,
elimination in urine pathways competing.

Inhibitors of DNA polymerase

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gemcitabine (2', 2' difluorodeoxycitidine) Mechanism of Action: Inhibits DNA polymerase by blocking DNA strand
elongation (substrate but can't elongage afterwards)
cytosine arabinoside (cytarabine, Ara-C.) Effects: Antimetabolite antineoplastic agent
Indications: pancreatic cancer (gemcitabine), chronic lymphocytic leukemia
fludarabine (arabinosyl-2-fluoroadenine) (CLL – fludarabine), acute myelogenous leukemia (AML – cytosine
arabinoside)
5-azacytidine (5-aza-C) Toxicity: myelosuppression
Resistance: decreased activity of activating enzymes; decreased nucleoside
2-chlorodeoxyadenosine (cladribine, 2-CdA) transport across cell membrane
Other: Must be activated by deoxycytidylate kinase and nucleoside
diphosphate kinase

New strategies: signal transduction inhibitors

Receptor & non-receptor protein kinases

Resistance to all of these is via amplifaction of oncogenic protein kinase gene, resistance mutations in kinase
catalytic domain. Second-generation protein kinase inhibitors have bene developed (active against mutant PKs)

imatinib Mechanism of Action: inhibits tyrosine kinases (BCR-ABL, c-KIT, PGDF receptor kinase)
Effects: Tyrosine kinase inhibitor; antineoplastic agent. BCR-ABL is a hyperactive fusion kinase implicated in
CML (philadelphia chromosome). PGDF RK = platelet-derived growth factor receptor kinase
Indications:CML, gastrointestinal stromal cancer.
Resistance: amplifaction of oncogenic protein kinase gene, resistance mutations in kinase catalytic domain.
Second-generation protein kinase inhibitors have bene developed (active against mutant PKs)
Other: aka Gleevec. BCR-ABL + cells are resistant to apoptosis, proliferate more, and have altered adhesion
properties.

trastuzumab Mechanism of Action: monoclonal antibody that binds to extracellular region of HER2, a
transmembrane receptor tyrosine kinase from epidermal growth factor receptor family
Indications: Antineoplastic agent. Breast cancer (25% invasive primary breast cancers have HER2
overexpression)
Resistance: amplifaction of oncogenic protein kinase gene, resistance mutations in kinase catalytic
domain. Second-generation protein kinase inhibitors have bene developed (active against mutant
PKs)Resistance:
Other: a.k.a. Herceptin

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cetuximab Mechanism of Action: monoclonal antibody against epidermal growth factor receptor (EGFR).
Indications: Antineoplastic agent. Epithelial tumors (colorectal cancer, head and neck tumors)
Resistance: amplifaction of oncogenic protein kinase gene, resistance mutations in kinase catalytic
domain. Second-generation protein kinase inhibitors have bene developed (active against mutant PKs)

gefitinib Mechanism of Action: Tyrosine kinase inhibitor (inhibits epithelial growth factor receptor kinase)
Indications: Non-small-cell lung cancer (NSCLC)
Resistance:amplifaction of oncogenic protein kinase gene, resistance mutations in kinase catalytic domain.
Second-generation protein kinase inhibitors have bene developed (active against mutant PKs)
Other: Higher response if EGFR mutated or overexpressed.

Anti-angiogenesis drugs (investigational)

Basic idea: formation of new blood vessels essential for tumor progression.
 Protease inhibitors block ECM breakdown
 Inhibitors of endothelial cell proliferation (small molecule receptor protein kinase inhibitors like
Gleevec & endogenous peptides)

Proteosome inhibition

Basic idea:
 NF-kappa-B controls expression of stress response genes & others that promote cell survival
 I-kappa-B inhibits NF-kappa-B, degraded in proteosome after ubiquination to activate NF-kappa-B
 Less proteosome activity = more NF-kappa-B inhibition

Velcade Mechanism of Action: Proteosome inhibitor. Antineoplastic agent


Effects: Inhibits the chymotrypsin-like active site of proteosome.
Indications: myeloma
Other: Boronic acid dipeptide; mimics tetrahedral peptidase reaction site.

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Cancer Chemoprevention

As many as 80% cancers in men, 77% in women can be prevented – idea is to detect early for public health
intervention before clinical appearance. For instance, Japanese migrants to US assume US-type cancer risk
profile, and their sons even more so (even without genetic mixing).

Long latency for many cancers helps in potential for chemoprevention.

Tobacco and diet are the big risks (30%, 35% cancer deaths attributable respectively)

Can be synergistic interactions between risk factors (e.g. alcohol & tobacco use in squamous carcinoma of
esophagus – 150x higher risk if heavy use of both)

Principles for control of human cancer:


1. Prevention (requires knowledge of carcinogens). Diet, exercise, tobacco, alcohol, etc.
2. Protection: inhibit or attenuate carcinogen effect. Doesn’t require knowledge of carcinogens
3. Treatment of premalignancy (screening).

Cancer chemoprevention: the use of natural or synthetic agents that retard, block, or reverse carcinogenesis
before invasive malignancy develops. (chemotherapy is for those who already have cancer)

To determine preventive measure, need to consider cancer risk, treatment risk, and treatment benefit: low risk
requires low intervention like lifestyle modification, high risk can require high-level intervention like surgery.
Medium-high risk might require chemoprevention.

Breast cancer and Selective Estrogen Receptor Modifiers (SERMs)

Estrogen has good and bad effects (improves cognition, lowers cholesterol, prevents bone loss… but also
increases breast / endometrial cancer & thromboembolism risk).

Tamoxifen and raloxifene are two examples of SERMS – can act like estrogen in some tissues and antagonize
estrogen in others

Several big randomized trials (MORE, STAR, etc.):


1. Tamoxifen vs. placebo (↓ER+ breast cancer but ↑ endometrial cancer, blood clots, stroke)
2. Raloxifene vs placebo(↓ER+ breast cancer but ↑ endometrial cancer, no change in blood clots, stroke)
3. Tamoxifen vs raloxifene – similar but raloxifene fewer endometrial cancers, blood clots

5-α Reductase Inhibitors and Prostate Cancer

Idea: reduce androgen activity. There are a bunch of inihibitors of all steps of androgen synthesis.
In some prostate cancers, oncogene translocated behind androgen-based promoter.

Finasteride and duasteride approved for male pattern baldness, BPH (PC chemoprevention)
 Two big trials (PCPT & REDUCE): big prevalence of prostate cancer already; couldn’t use PSA because
PSA production related to androgens so had to use biopsy
 Outcomes: both saw reduction in prostate cancer, but one showed increase in high grade cancer.

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 Random area biopsy – maybe shrinking prostate with inhibitors increased chance that high grade cancer
would be found?

Aspirin and Coxibs for Colorectal Cancer Prevention

Basic idea: arachidonic acid, inflammation might be part of adenoma  cancer recurrence in colorectal cancer.
Wanted to use COX-2 inihbitors like celecoxib (Celebrex) and rofexocib (Vioxx) to selectively inhibit COX-2
(NSAIDs, originally for arthritis). COX-2 makes prostaglandins around epithelial cells of GI tract.

Multiple randomized trials


 Aspirin vs. placebo: decrease in colorectal cancer but increased risk of serious GI bleeding
 APC: celecoxib vs placebo. Reduction in polyp recurrence but increase in cardiovascular events 2.6x
 Approve: rofecoxib vs placebo (similar reduction in polyps & increase in CV events 1.8x)

More stuff:
Cruciferous vegetables might help cancer chemoprevention: sulforaphane

Problems that you might run into in chemoprevention


 Toxicity in a large population
 Side effects could be significant & hurt adherence
 No easily identifiable end-point to study (like cholesterol, for instance)
 Need big population and long durations
 Poor definition of the best dose (no dose-response curve easily made)

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Antineoplastic alkylating agents and platinum compounds

Alkylating agents: basic principles

Akylating agents react with certain nucleophilic areas in DNA (e.g. N7 of guanine) with several consequences:
 Mispairing of modified base
 Crosslinking of DNA bases on same strand (intrastrand) or opposite strands (interstrand)
 Crosslinking DNA to proteins, RNA, other macromolecules
 DNA strand scission (weaken sugar-phosphate backbone; endonucleases attack during repair attempt)

Monofunctional alkylating agents have one arm to react with DNA


Bifunctional agents have two and therefore are the only ones that can crosslink. CROSSLINKING IS KEY.
If bifunctional agents react with water, they can behave like monofunctional agents. Bifunctional agents are
more selective for replicative-based effects because they can work via crosslinking mechanisms

Types of alkylating agents


1. Nitrogen mustards: mechlorethamine is basic nitrogen mustard; very active & used up quickly.
mechlorethamine Mechanism of Action: Bifunctional alkylating agent; antineoplastic agent.
Effects: Forms interstrand or intrastrand DNA cross-links. Can also cross-link DNA to other
macromolecules, cause DNA strand scission, or mispairing of modified base.
Indications: Hodgkin's disease, mycosis fungoides (cutaneous lymphoma)
Administration: part of MOPP regimen. IV, very short half-life
Toxicity: nausea, vomiting, phlebitis, bone marrow suppression
Other: nitrogen mustard. Very reactive (reacts with everything even in blood, so have to give IV)

2. Substituted nitrogen mustards (melphalan & chlorambucil) which are less reactive & can be given po.
melphalan, Mechanism of Action: Bifunctional alkylating agent; antineoplastic agent.
chlorambucil Effects: Forms interstrand or intrastrand DNA cross-links. Can also cross-link DNA to other macromolecules,
cause DNA strand scission, or mispairing of modified base
Indications: multiple myeloma (mephlan), chronic lymphocytic leukemia (CLL), indolent lymphomas,
Waldenstrom's macroglobulinemia (chlorambucil)
Administration: can give PO
Toxicity: bone marrow suppression, amenorrhea, sterility
Other: substituted nitrogen mustard; less reactive than mechlorethamine

3. Nitrogen mustards that require metabolic activation (cyclophosphamide & ifosfamide).


Cyclophosphamide is most commonly used.

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cyclophosphamide, Mechanism of Action: Bifunctional alkylating agent
ifosfamide Effects: Must first undergo metabolic activation (P450). Forms interstrand or intrastrand DNA cross-
links. Can also cross-link DNA to other macromolecules, cause DNA strand scission, or mispairing of
modified base.
Indications: Cyclophosphamide: Many human cancers (non-Hodgkin's lymphoma, breast cancer).
Can also be used with bone marrow or peripheral hematopoietic stem cell transplantation therapy
(high-dose). Used to treat auto-immune diseases as well. Ifosfamide: sarcomas & many other cancers
Administration: Administering with 2-mecaptoethane sulfonate and vigorous hydration can help
prevent cystitis
Toxicity: bone marrow suppression, alopecia, gonadal toxicity, and hemorrhagic cystitis (diffuse
inflammation of the bladder leading to dysuria, hematuria, and hemorrhage) resulting from excretion
of a reactive metabolite (acrolein). Ifosfamide has more hemorrhagic cystitis, less bone marrow
suppression than cyclophosphamide
Other: Most commonly used alkylating agent. Stem cells have aldehyde dehydrogenase, which
protects from the active form of cyclophosphamide (which means they won't get killed)

Platinum compounds: basic principles


 Early experiments: cis platinum compounds had effect on E. coli, while trans didn’t (from electrodes)
 DNA is target (interstrand & intrastrand crosslinking of DNA, or crosslinking to other macromolecules).
 Difference is in speed of aquation & elimination. Slower aquation (carboplatin, oxaliplatin) means less
kidney damage than cisplatin.

cisplatin Mechanism of Action: DNA cross-linking agent (antineoplastic platinum compound)


Effects: Intra- and inter-strand crosslinking of DNA; crosslinking of DNA to other molecules.
Indications: Wide variety of cancers, esp. epithelial (carcinomas of lung, bladder, stomach, ovary) & testis
cancer.
Toxicity: nephrotoxicity, ototoxicity, peripheral neuropathy
Resistance: chemical detoxification, DNA repair
Other: more side effects (shorter half-life) than carboplatin, oxaliplatin (which have bone marrow toxicity as dose
limiting side effect rather than nephrotoxicity)

carboplatin, Mechanism of Action: DNA cross-linking agent (antineoplastic platinum compound)


oxaliplatin Effects: Intra- and inter-strand crosslinking of DNA; crosslinking of DNA to other molecules.
Indications: Wide variety of cancers, esp. epithelial (carcinomas of lung, bladder, stomach, ovary) & testis
cancer.
Toxicity: bone marrow suppression
Resistance: chemical detoxification, DNA repair
Other: shorter half life than cisplatin (less nephrotoxicity)

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Mechanisms of resistance to antineoplastic drugs generally fall under two categories:
1. Chemical detoxification. E.g. glutathione, glutathione-S-transferases, etc. Resistance usually comes
from this pathway (think phase II enzymes, etc. – could be upregulated, for instance)
2. DNA repair. Less common – excision repair, mismatch repair, etc.

Side effects related to antineoplastic drug treatment


Think: these are very near to their maximum tolerance, so lots of side effects will happen - low therapeutic index
 Nausea and vomiting
o Profound before; less now
o Complex neurological interactions: chemo triggers pretty much all of these
 Chemoreceptor trigger zone (metabolic disorders, drugs like chemo)
 Peripheral receptors (vagal/splanchnic nerves) – e.g. in gut (damage from chemo)
 Vestibular center (motion sickness)
 Cerebral cortex (anticipatory emesis from chemotherapy)
o All converge to vomiting center in brainstem
o What matters: which drug & how much
 Cisplatin is notorious (as is cyclophosphamide in high doses) for causing nausea
 Less for less dose
o Brainstem receptors use dopamine pathways, so now
selective 5-HT3 receptor agonists work well for
cisplatin nausea and vomiting
 Alopecia
o Usually a few days into 1st or 2nd cycle (1-2 weeks after
chemo)
o Hair will grow back but big psychological effect
o Hair follicle: anagen (growth), categen (involution),
telogen (resting)
 Hair damaged during anagen (growth phase)
 Falls out in order of most growing (most in
anagen) scalp>eyebrows, eyelashes, face, axilla,
body, etc.

 Bone marrow toxicity (granulocytopenia, anemia,


thrombocytopenia, etc.)
o Usually dose limiting toxicities
o Fall & recover in predictable ways
o Chemotherapy-induced neutropenia
 Like clockwork (around 10th day) – PNMs
affected 10-14 days because of speed of
production (stem cells resistant to
cyclophosphamide because of aldehyde
dehydrogenase expression so they don’t die)
 Dose-response doesn’t change timing, just the
depth of the nadir
 Hematopoetic cytokines (e.g. G-CSF) can be
used to shorten the length of the nadir (same

36
depth, just less broad)
o Transfusion of RBC or platelets can sometimes be used

 Gonadal dysfunction
o Lots of ongoing mitosis/meiosis & proliferation in this area
o Testes > ovarian follicles for dysfunction (ongoing spermatogenesis vs arrested ova)
o Important factors:
 Age/gender (pubertal gonads resistant; women < men for dysfunction)
 Chemo agent & dose (alkylating agents & platinum compounds especially bad
 Make sure to bank sperm / store eggs if possible!
 Some regimens have huge differences in recovery rates

 Secondary cancers (leukemia, etc.)


o Really worrisome – genome damage could lead to malignant transformations
o Limited mostly to alkylating agents: monofunctional agents are carcinogens (modify the base &
cause damage but not necessarily lethal)
 If cell doesn’t die (e.g. if only one arm able to fire for bifunctional agent) normal tissues
can get mutations
o Very hard cancers to treat; up to 10% in Hodgkin’s pts with radiation+alkylators can get AML

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Additional drugs (maybe know?) Under “other” (alkylating agents?)

Nitrosureas
 Carmustine: lipophilic; treat brain tumors (drug-implanted wafer: glioblastoma multiforme)
o (toxicities: bone marrow suppression, nausea & vomiting)
 Streptozotocin: antibiotic that is retained in beta-islet cells of pancreas; treats islet cells tumors
o Nephrotoxicity, hepatotoxicity, diabetes

Aziridines
 Thiotepa: breast cancer; instill in bladder for superficial bladder cancer
o Usual toxicities
 Mitomycin C: antibiotic – limited use in recta / pancreatic cancer; superficial bladder cancer as instillate
o Usual toxicities
o Rare but significant: interstitial pneumonitis, nephrotoxicity, hepatic veno-occlusive disease,
hemolytic-uremic syndrome

Alkane sulfonates
 Busulfan: high dose chemo for bone marrow or peripheral hematopoetic stem cell transplants
o Usual toxicities + pulmonary fibrosis, hepatic veno-occlusive disease, skin pigment changes

Methylating agents
 Procarbazine: Hodgkin’s disease. Normal toxicities + peripheral neuropathy, sterility, secondary
leukemia
 Dacarbazine: Hodgkin’s disease. Normal side effects + “flu-like” syndrome, Budd-Chiari syndrome,
photosensitivity

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DNA Topoisomerase-targeted drugs and mitotic spindle poisons

Many of the other drugs (alkylating agents, etc.) were rationally designed; these are screened natural products

Topoisomerase-targeted drugs

All topoisomerases: reversible nucleophilic substitution; active site tyrosine as nucleophile


 Preserves energy of initial phosphodiester bond; prevents loss of end of DNA
 All form covalent enzyme-DNA intermediates (keep ends of DNA together to minimize recombination /
DSB; reversible – after unwinding, DNA can re-attack the tyrosine-DNA linkage & rejoin
 Help relieve supercoiling / superhelical strain from normal DNA activities (transcription, replication,
segregation, chromosone condensation, etc.)

Type I topoisomerase: monomeric, single tyrosine, Type II topoisomerase: dimeric, two tyrosines, cleave both
cleave one strand of DNA, no ATP requirement strands of DNA, ATP / Mg required
 DNA can freely rotate around remaining  Enzyme holds in place – prevents DSB
strand’s axis  Forms gate – for duplexed DNA to pass through
 Removes one supercoil per cleavage  Removes two supercoils per cleavage

Cell killing mechanism of topo poisons:


 Stabilize covalent complex  DNA replication fork arrest & breakage (DSB)
 (also RNA transcription arrested)
 Topo I: G2 cell cycle arrest
 Induce apoptosis through these mechanisms

Type I topoisomerase-targeting drugs


 Stabilize covalent DNA-enzyme complex by:
o DNA intercalation (“direct block”): wedge in between 5’ leaving group and 3’ phosphate to
sterically block the reversal of the covalent enzyme-DNA binding. E.g. camptothecins
camptothecin Mechanism of Action: Topoisomerase I-targeted drug (antineoplastic agent)
Effects: Intercalates into & stabilizes topo-I / DNA covalent complex (sterically blocks DNA-DNA nucleophilic
topotecan attack & reversal of enzyme-DNA complex formation).
Indications: Solid tumors (first line for colorectal cancer in US/europe), also ovarian & other adult
irinotecan maligancies
(CPT-11) Toxicity: myelosuppression, nausea, hair loss, fatigue. Irinotecan: liver toxicity in patients with
gluconconjugation deficiencies. Early and late onset diarrhea.
Resistance: MDR drug efflux pumps (ABC-type)
Other: lactone ring is not stable at neutral or basic pH (converts to inactive form). Irinotecan: Administered
as a prodrug; more bioavailable but gives diarrhea / liver toxicities from cleaved off prodrug part. Must be
activated by carboxyesterase

o DNA bending (“indirect block”): bend DNA in a way that puts active site in conformation where
reversal of attack isn’t favorable. E.g. minor groove binding agents (investigational).
 Curved structure and positive charges let them fit in to minor groove well

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 Increase amount of covalent complex by DNA bending; S-phase specific
 Can increase/decrease effects of other topo I poisons

o Actinomycin D
 Induces DNA bending / structural pertubations
 Precise genomic target not known (maybe RNApol instead of topo I)
actinomycin D Mechanism of Action: "hybrid" minor groove binding antineoplastic agent
Effects: Precise target not known. Probably induces DNA bending / structural pertubations (may target
RNA polymerase instead of topoisomerase I)
Indications: Childhood malignancies (Wilm's tumor, Ewing's sarcoma, embryonal rhabdosarcoma)
Toxicity: Usual (myelosupression, hair loss, oral / GI ulceration)
Resistance: MDR drug efflux pumps (ABC-type)

Type II topoisomerase-targeting drugs


 Probably a similar stabilization of covalent complex (not well understood)

Epipodophylotoxins (non-intercalative)
etoposide Mechanism of Action: Topoisomerase II-targeted antineoplastic agent.
teniposide Effects: Nonintercalative; increases covalent DNA-enzyme complex by unknown structural mechanism
Indications: Many types of cancers
Toxicity: Usual (myelosuppression, mucositis, nausea, anaphylaxis)
Resistance: MDR drug efflux pumps (ABC-type)

Anthracyclines (intercalative)
doxorubicin Mechanism of Action: Topoisomerase-II-targeted antineoplastic agent.
daunorubicin Effects: Intercalates into & stabilizes DNA-topo II covalent complex by direct or indirect interaction
Indications: solid tumors (doxorubicin); ALL, AML (acute leukemias) (daunorubicin)
Toxicity: Dose-limiting acute & chronic cardiotoxicity. Liver toxicity (where metabolism occurs -
hydrophobic, so bile excretion).
Resistance: MDR drug efflux pumps (ABC-type). Cardiotoxicity from quinone groups (generates hydroxyl
radicals)

Mitotic spindle poisons

Spindle basics:
 Made of microtubules (α & β subunits; dynamic structure ; bind GTP & hydrolize to GDP)
 Assemble: heterodimers  protofilaments  microtubules
 Important in mitosis (don’t get good DNA segregation without it)
 Lots of tubulin in neurons for axonal transport  neurologic toxicity of tubulin-binding agents

Basic idea: spindle poisons slow microtubule polymerization dynamics.


 Freezing/slowing down the dynamic equilibrium interferes with spindle action during mitosis

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Vinca alkaloids
 Bind to β-tubulin (as monomer?), which disturbs polymerization & disrupts protofilament structure
vincristine Mechanism of Action: Mitotic spindle poison (antineoplastic agent). Vinca alkaloid
vinblastine Effects: binds to beta-tubulin (distorts protofilament structure / polymerization, slows dynamics, affecting
vinorelbine ability to undergo mitosis)
Indications: ALL, lymphoma, hodgkin's, childhood malignancies (vincristine), germ cell tumors, Hodgkin's
disease (vinblastine), lung, breast cancer (vinorelbine)
Toxicity: liver toxicity, myelosuppression. Vincristine: neurotoxicity (limits dosage)
Resistance:MDR drug efflux pumps (ABC-type)

Taxanes
 Bind to β-tubulin, stabilizing lateral tubulin contacts (freezing filaments in place)
paclitaxel Mechanism of Action: Mitotic spindle poison. Taxane.
docetaxel Effects:Binds to beta-tubulin, maybe stabilizing lateral contacts & freezing protofilament in place. Slows down
microtubule dynamics, hurting ability to undergo mitosis.
Indications: ovarian, breast cancers (paclitaxel), metastatic breast cancer (docetaxel)
Toxicity: dose-limiting myelosuppression. Peripheral neuropathy (paclitaxel more effects than docetaxel)
Resistance: MDR drug efflux pumps (ABC-type)

Mechanisms of drug resistance

1. Multidrug resistance: ATP-dependent transporter proteins (ABC family = ATP-binding cassette).


a. Cellular drug efflux
b. Intracellular redistribution of drug away from target (?)

Drug needs to accumulate in cell before it has an effect.


Also important in antibiotic therapy (bacteria have these too)
These pumps are very non-specific

2. Atypical drug resistance


o Mutation of drug binding site (e.g. HIV drugs)
o Downregulation of gene expression of protein target (e.g. topo I/II)
o Ubquitination & proteolysis of target protein (topo I/II)
o Decreased enzyme activity for activation of prodrug (e.g. carboxylesterase & irinotecan)

3. Detoxification by metabolism (e.g. phase I, phase II enzymes) – overexpress or increase activity

41
Pathology: ID & Micro
Pathology of Bacterial Infections ............................................................................................................................................ 2
Streptococci (I & II) ................................................................................................................................................................. 5
Enterococci.............................................................................................................................................................................. 9
Listeria & Other Gram-Positive Rods .................................................................................................................................... 10
Introduction to Gram-negative Bacilli................................................................................................................................... 14
Fastidious Gram Negative Rods ............................................................................................................................................ 17
Non-fermentative Gram Negative Bacteria .......................................................................................................................... 20
Neisseria Species ................................................................................................................................................................... 23
Anaerobic Gram Positive Bacteria ........................................................................................................................................ 25
Emerging and Re-emerging Bacterial Zoonoses ................................................................................................................... 28
Pathology of Mycobacteria Infection.................................................................................................................................... 31
Vector-Borne Zoonoses ........................................................................................................................................................ 34

1
Pathology of Bacterial Infections
Final outcome determined by host & bacterial pathogen: can direct Tx towards both
Examples: think about what bacteria has to overcome to get in!
 Respiratory entry: eyes (blinking, tears, lysozyme, secreted IgA, lactoferrin sequesters iron); nasopharynx
(microflora, secretions); lungs (resident macrophages)
 GI tract entry: mouth (sloughing cells, flow of saliva, lysozyme, sIgA, microflora, lactoferrin); stomach (low pH,
proteolytic enzymes), small intestine (fast flow, mucous, sloughing cells); colon (slow flow, sloughing cells,
mucus, lots of resident microflora)

Pathologic & histopathologic correlates of bacterial infection


Acute inflammation:
 PMNs (purulent): marginate & extravasate, eventually followed by mononuclear cells (48h: Mφ & plasma cells)
 Can have necrosis (suppuration)

Chronic inflammation:
 Key: Lymphocytes & Macrophages (= histiocytes)
1. Granulomas: aggregates of lymphocytes & histiocytes; often with fibroblasts & giant cells
2. Granulomatas: poorly aggregated infiltrates of lymphocytes / histiocytes
3. Chronic nonspecific inflammation: mostly infiltrates of lymphocytes, fewer histiocytes

Major types of histopathologic inflammatory responses to bacterial pathogen infections

1. Exudative +/- necrosis


o Usually localized
o Vascular permeability ↑; recruitment of PMN & other WBC ↑
o Non-necrotizing: examples
 S. pyogenes
 Pharyngitis (“strep throat”): pus on tonsils; swollen (edema) – lots of PMNs
 S. pneumonia
 Pneumonia: lobar, generally resolves well (not necrosis, just fill up alveoli with exudate
& PMNs. Turn firm because filled up = consolidation)
 Meningitis: meninges / septae too wide (inflammatory cells); inflammation can cause
bystander damage (neurological sequelae). Gross pathology: meninges cloudy (filled
with leukocytes)
o Necrotizing
 Exudative with necrosis = suppuration
 Host damage: can be from bacterial virulence factors
 P. aeruginosa
o Pneumonia: consolidation with infiltrate; makes lots of toxins (cell damage &
death); lots of hemorrhage and necrosis
 Clostridium perfringens
 Myonecrosis (gas gangrene): big gas bubbles produced; inflammation & necrosis

2. Chronic inflammation +/- granulomas


o Granulomas & granulomatous inflammation
 activated epitheliod Mφ, lymphocytes, etc.
 Response to bacteria that withstand PMN phagocytosis
o Granulomas:
 M. tuberculosis: granulomas (nodular) with giant cells; caseous necrosis in center
 Little red bacilli with acid fast stain within or outside cells
2
o Granulomatous inflammation (more diffuse; same cells but less organized)
 M. leprae: foamy Mφ; more diffuse organization; acid-fast rods inside cells
o Interstitial Inflammation
 Nonspecific morphology (chronic, nonspecific inflammation)
 Virus, Mycoplasma, Rickettsia, spirochete infections (if in infectious clinical setting)
 Mycoplasma pneumonia: Interstitial pneumonitis
 Thickening of alveolar spaces, lots of cells & fluid in interstitium (hurts O2 exchange)

o When do granulomas form and when does general chronic inflammation happen?
 Cytokines IL-1B, IFN-γ, CXCL, CCL  granuloma forms
 Different cytokines IL-4, IL-10  chronic inflammation
 Each type of cytokine suppresses the other response

3. Cytopathic
o Most typical of viral infections
o Can be seen with intracellular bacterial infections
 Chlaymydia trachomatis – U/G infections
 Cervix red, swollen; purulent mucoid exudates
 Chlamydia grows within vacuoles intracellularly
4. Cytoproliferative inflammation
o Bartonella spp. (henslae) – angioproliferative responses (cause blood vessels to overgrow)
 Esp. in HIV patients, causes dermis to be replaced by vascular structures
 From cats (also causes cat scratch fever)
5. “Null reaction”
o Absence of inflammatory, necrotizing, or cytopathic responses
o Rare in bacterial infections
o Can occur with neutropenia, immune compromise (HIV, cancer chemo, genetic defects) or by rapid,
unrestricted bacterial growth
o Vibrio vulinficus with neutropenia: tons of Gram (-) bacteria but just a few inflammatory cells
o Bacillus anthracis (anthrax): skin, meninges, inhalational. Bacteria grows faster than immune response
is mounted (also suppress immune response)

Extracellular vs Intracellular bacteria: differences in response


Extracellular:
 Acute inflammation (PMNs) +/- necrosis (depends on virulence factors)
1. Acute inflammation with edema
2. Necrosis  pus formation (suppuration) or abscess formation
 Eventually gives way to mixed acute & chronic inflammation
 Examples: S. pneumonia, S. aureus, P. aeruginosa, most other bacteria

Intracellular: both facultative & obligate


 Chronic nonspecific or granulomatous inflammation
1. Granulomas (e.g. M. tuberculosis)
2. Chronic nonspecific or lymphohistiocitic inflammation (e.g. mycoplasma, rickettsiae, chlamydiae,
spirochetes)
 Initially chronic inflammation +/- acute inflammation (chronic or mixed)
 Can cause necrosis
1. Caseous (e.g. granulomas)
2. Microabscesses (granulomatous inflammation)
3. Host-cell-specific necrosis or apoptosis
 Examples: M. tuberculosis, R. rickettsii (Rocky Mountain Spotted Fever), C. trachomatis (U/G infections)

3
Effects of alterations in host defense, inflammation, immunity

1. Physiologic defects
 Cystic fibrosis (no appropriate mucous production = ↑ lung infiltrates)
 Achlorhydria (can’t generate acid in stomach = ↑ lower GI infections)
2. Host can’t make inflammatory cells: no inflammatory infiltrates (congenital neutropenia, etc.)
3. Host inflammatory cells can’t accumulate: no inflammatory infiltrates
 Leukocyte adhesion molecule deficiency (don’t see WBC at site of infection)
4. Host immune suppressed (HIV, Rxs): modified inflammatory response
 Chronic granulomatous disease (↓ superoxide radicals in phagocytes: see bacteria ingested but not
destroyed)
 Complement deficiency, asplenia = ↑ susceptibility to encapsulated bacteria (need C’ to opsonize)
 Hypogammaglobulinemia = ↓ opsonization
 HIV, cancer therapy, corticosteroid, immune suppressive therapy = ↓ T-cell responses
 Interferon-γ receptor deficiencies = recurrent Mycobacteria and Salmonella infections

Hosts that lack inflammatory response = not well protected against effects of infection

Laboratory tests to identify bacterial infections

I. During active infection: Finding Organism


1. Nonspecific morphological PMNs in gastric mucosa H. pylori
identification (Gram stains, tissue Abscesses with “sulfur granules” Actinomyces spp
sections) Caseating granulomas M. tuberculosis
o Initial inflammatory response Lymphocytic vasculitis R. rickettsii
usually stereotypical and (Rocky Mountain Spotted Fever)
nonspecific
o Can use inflammation type, special stains, anatomic location, other clues
 H. pylori: seagull-shaped; on surface of mucosal epithelium; can use silver stain. PMNs in pits of
gastric epithelium
 Actinomycosis: huge GPR collecting in abscesses with fibrous appearance; sulfur granules
 M. tuberculosis: acid-fast stain, or can use fluorochrome
 R. rickettsii: petichae, lymphocytic vasculitis (lots of lymphocytes around / in vessels & walls; R.
rickettsii in endothelial cells
2. Culture
3. Direct detection in clinical samples (fluorescent Ab, in situ hybridization, PCR)

II. Examining host immune response


 Serological (Ab detection): antibody present, seroconversion – looking at humoral immunity
o Ab titer increase: usually want to look @ onset of illness & follow titer level. Compare acute phase (low
titer) to chronic phase (higher titer) to confirm Dx.
o Fluorescence tests for Abs too (indirect)
o Agglutination (mix serum with antigen & look for agglutination)
o Western blots (HIV, Lyme disease)
 Cellular immunity tests (e.g. PPD)
 Lymphocyte proliferation test
 IFNγ production test

4
Streptococci (I & II)
General Features of Streptococci:
Structure Streptococci: Major Pathogens
 Gram (+) cocci (GPC) in very nice Str. pyogenes Group A Most frequent cause of bacterial
chains and pairs (division in 1 plane). infection globally
Remain attached via thin cell wall Str. agalactiae Group B Peri-natal & opportunistic
bridges Str. pneumoniae Primary cause of pneumonia &
 Non-motile, non-spore bearing meningitis globally
 Gram (+) cell wall +/- capsule (major Viridans group Relative wimps
pathogens do have capsule)
Physiology Organism Normal flora in…
 Fastidious: enriched media, narrow pH/temp ranges Viridans #1 aerobic colonizer of upper
 Aerobic & facultative anaerobes resp tract (#2 aerobic on skin)
 Fermentative metabolism (glucose  lactic acid, not Group A Small #s of individuals
Krebs cycle) Group B Normal below the belt
 Catalase NEGATIVE Str. pneumoniae Depends on country & season
o No cytochrome oxidative system (Gram
negatives, S. aureus, etc. do have)
Classification
 Hemolytic reaction
Hemolysis Examples Blood agar
Beta (produce H2O2, lyse RBC) Groups A & B (also C,D,F,G) Clear
Alpha (degrade Hb to met-Hb) Viridans group Green
Str. pneumoniae
Non-hemolytic (“gamma”) Red
 Immunologic
o Grouping: based on C-polysaccharide antigen on cell wall
o Typing (for Group A): based on M-protein
 Genetic: 45+ strains known

Str. pyogenes
Group A strep virulence factors: Cytolysis and spreading factors

Cell-associated:
 Hyaluronic acid capsule (unusual – most just complex
polysaccharide) – INHIBITS PHAGOCYTOSIS
 M-protein (typing)
1. Inhibits C’ fixation
2. Major adherence factor: binds with LTA so cells can adhere
to pharynx, skin & not be washed away
 Protein F: binds to fibronectin, adherence factor
 Lipoteichoic acid (LTA): binds with M-protein; adherence factor
 Cell-bound peptidase: inhibits C’
 Peptidoglycan layer
 Has fimbrae (M-protein + LTA) to help adhere

Extracellular:
 Hemolysins O (oxygen labile) and S (oxygen stable)
5
o Large zone of beta-hemolysis; most strains have both but if they’ve only got one, it’s O
o O: very antigenic (make Ab)
 Endonucleases: digest nucleic acids, nuclei of leukocytes.
o Fewer WBC around than Staph infections, etc. because of endonucleases
 Streptokinase: liquefies material around cells; dissolves clots
 Pyrogenic exotoxins (A, B, C)
 Toxic shock toxin (like S. aureus toxin)
 Many others (so organism can diffuse throughout body)

Clinical features: Local manifestations: edema, heat, erythema, pain, spreads with extreme rapidity, few PMN-rich
abscesses, systemic manifestations too

Toxigenic strains: toxic shock, scarlet fever

Disease presentations:
 Pharyngitis (also other URI: otitis, sinusitis). Pus; grayish-white discharge; edematous tonsils. Complications:
clotting / obstruction / jugular vv infections. Common in children, closed populations (college, military). 3-5
days: transmissibility decreases
 Skin / soft tissue: erythema, edema, pain
o Impetigo: frequently staph/strep mix; painful, swelling, little clear fluid-filled vesicles if strep only.
Different M-protein types than pharyngitis types
o Celulitis: superficial skin infection. rapid spread, advances up lympathics (reddish streaks). Huge
vesicles with clear fluid later
o Necrotizing fasciitis / myositis (killing skin / muscle cells). Abx don’t reverse damage – need surgical
intervention. Path: dead mm cells, no PMNs
 Puerperal (post-delivery) sepsis – more historically no.
 Scarlet fever
o Str. pyogenes strain producing pyrogenic toxin (plasmid-mediated). Usually pharyngitis-associated
o Scarlatiniform sandpaper rash (upper chest to trunk, extremities); strawberry tongue
o Desquamation follows
 Toxic shock syndrome: strep TSS parallels S. aureus’
 Post-streptococcal diseases (immunologic cross-reactions between strep antigens & heart or kidney)
o Rheumatic fever (9-10d post-infection)
 Any M-protein type; reinfection has same latency
 Heart (myocarditis, valves); joints (arthraligias, arthritis), skin (erythema marginatum), CNS
(Sydenham’s chorea)
o Glomerulonephritis (few days post-infection)
 Specific M-types implicated
 Proliferative disorder of renal glomerulus
 Edema, hypertension, hematuria, proteinuria
 Can have no sequellae or progress to end-stage renal disease

More likely to transmit if high amount, in nose/throat; less likely if you’ve been a carrier for a while.

Str. agalactiae
Regular polysaccharide capsule (inhibits phagocytosis & C’)
 9 serotypes (M-proteins) with different capsule composition
CAMP factor (hemolysin)
Small zone of B-hemolysis
Found in normal GI flora; vaginal tract of 5-30% sexually active women

6
Can be primary or co-pathogen in immunocompromised

Clinical presentation: causes neonatal sepsis and meningitis (early & late forms)
 Associated with prolonged rupture of membranes in colonized mother
 Early onset: 1st 6 days. Septicemia (60%), also pneumonia (30%), meningitis (10%). 10% mortality
 Late onset: 7d-3mo. Majority due to type III; may be fulminant with septic shock & severe meningitis
 Neurologic sequellae: 25-50% children
Prevention:
 Maternal screening
 Prophylactic antibiotics (if colonized mother or any mother with prolonged membrane rupture)
Str. viridians Group
General characteristics:
 Alpha-hemolytic (green pigment)
 Relatively non-virulent
 Normal flora of respiratory tract, vaginal tract, skin, other

Pathogenicity
 Endocarditis: relatively indolent, really needs damaged setting (e.g. IV drugs, then lands on already damaged
heart valve)
 Abcesses(most common cause of liver abscesses. Complications: rupture  hemorrhage, high mortality rate.)
o Str. anginosis, Str. intermedius, Str. constillatus
 Dental caries: Str. mutans (part of gingival flora, clings to enamel)
o To get caries: need right microflora, diet, and host/teeth situation.
o Str. viridians is most common aerobe in oral flora
o Virulence: from high acid production
 Septicimia in immunocompromised patients
 Co-pathogens in mixed flora infections (e.g. Gram + and – together)
 Non-infectious events: chronic gingivitis  atherosclerotic plaques?

Str. pneumonia (“the pneumococcus”)


 Gram positive, in pairs & chains like others (prominent pairs)
 Physiology: fastidious, narrow pH/temp ranges, fermentative Lab diagnosis:
metabolism, catalase negative (like other strep), prefer 5% CO2  GPC in pairs (& chains)
 Alpha hemolytic
Polysaccharide capsule: high MW, complex polysaccharides  inhibited by optochin
 Types defined by capsule antigens  Bile soluble
 Different types have different virulence, C’ and cytokine reactivity,  Quellung reaction (add anti-
Abx resistance, and preferred sites of pathogenicity pneumococcal Ab, see swelling)
 Lots of cross-reactions (other microbial capsules, blood group
antigens, other foreign polysaccharides)
o Can get antibodies very early in life via cross-reactivity
o Capsule very antigenic; repeat infection with same type is rare

Genetic features
 Transfer of DNA via transformation, phages, conjugation
o Transformation ability varies with capsule type, inflammation (cytokine activation), other types of
stress, antibiotics, starvation, chemical exposure. E.g. Abx can induce ability to acquire resistance!

Virulence factors: major one is the capsule (blocks phagocytosis if specific antibody not present).
 Need capsule for virulence (capsule + rest of pneumococcus too)

7
 Cell-surface-associated:
o pili (adherence)
o cell wall polysaccharides (induce inflammation, activate C’)
o surface protein A: inhibits activation of C’,
o autolysin (how pneumococci commit suicide when they don’t like where they are)
 Cytoplasmic (release of these as cells lyse are primary cause of death within 48 hours despite abx):
o Autolysin: releases pnemolysin & cell wall products
o Pneumolysin: cytotoxin, activates C’/cytokines
o Neurominidase: exposes host cell receptor sites
o Peptide permeases: enhance adhesion
o Others (hemolysin, hydrogen peroxide, etc.)

Pathogenicity:#1 cause worldwide of pneumonia, meningitis, otitis, sinusitis

 Normal flora in 20-100% individuals (colonization: normally host makes protective Ab preventing blood-based
spread, from this normal exposure)
o Direct infections (otitis, sinusitis) can still occur
 Facultative pathogen; produces intense inflammatory response without necrosis
 Major cause of death at age extremes

Pneumonia
 Occurs with newly acquired type that patient doesn’t have Ab to
 Typical case: respiratory viral infection, fever recurs 4-7d post viral infection, pneumonia sets in
o Virus damages ability to clear tracheobronchitic tree, destroys ciliary epithelial cells, produces excess
mucus & fluid, blocking normal clearance
 Often begins with aspiration of oropharyngeal contents (alcoholism, neurologic impairment, etc.)
 Purulent nasal discharge, lobar pneumonia, intact alveolar structure, inside of alveoli jammed with
inflammatory cells. Radiology: alveolar infiltrate
 Little residual morbidity – can resolve well (not destroying alveoli)

Meningitis: Meninges jammed with PMNs, not in brain tissue


 3x mortality of N. meningitis; more frequent, more sequelae (CNS, deafness)

Pneumococcal septicemia: especially common in sickle cell children (give PCN prophy until vaccination possible)

Management:
1. Antibiotics (Penicillins, others).
a. Rapidly emerging resistance
i. PBP mutations: “intermediate” resistance, can overcome with ↑ dose sometimes. Increasing
doses doesn’t help for endocarditis or meningitis (just can’t get *drug+ high enough in there)
ii. B-lactamase development
b. Less active: cephalosporins (used to be 2nd line, now resistance emerging too)
c. Varies with location
2. Prevention (vaccines)
a. 23-component vaccines (23 different types / polysaccharides)
b. Different kinds: peds vs adults
c. Vaccine works less well in older patients (although mandated to offer to all pts > 55yo on discharge
from hospital & document reasons for refusal)

8
Enterococci
General features:
 Look like strep (GPC in chains); metabolism like strep (facultative anaerobes, fermentative metabolism)
 Catalase negative (but can make a little bit if they receive DNA from other spp)
 Not fastidious (permissive pH range, no enriched media required, etc.).
o Survive heating, dessication, resistant to disinfectant products (good at health care spread)
o Not damaged by gastric pH
 Part of normal gut flora (E. faecalis & E. faecium are most prevelant)
 Mostly non-hemolytic (some spp alpha-hemolytic; very rare strains beta-hemolytic)
 Grow as small grayish colonies on blood agar

Structure: normal gram+ cell wall, no flagellae, +/- capsule

Infectivity:
 Commensals (below diaphragm – colonic flora, vaginal flora, external genitalia)
 Facultative pathogens

Virulence factors (importance unknown): adherence factors, cytolysins, permeability factor, gelatinase, aggregation
factor (clumping), superoxide production
 LTA: can exhibit endotoxin-like features of septic toxicity (incl. hypotensive shock) in large amounts
 Biofilm formation (especially hearty)

Diseases:
 E. faecalis (60-70%) & E. faecium (10-20%)
 Most commonly: mixed infections (e.g. abscesses below the diaphragm)
o Intra-abdominal abscesses, colonic diverticulitis, urinary tract, gall bladder
 Enterococcal endocarditis
o Classic presentation: elderly male with obstructive uropathy
o Unlike S. aureus, symptoms commonly indolent; low grade fever often ignored
o (especially big abx diffusion problem)
 Septicemia (predominantly in debilitated / immunocompromised; mortality 42-68%)

Resistance:
 VRE: vancomycin-resistant enterococci (esp. E. faecium). Hospitals, nursing homes (where abx are used
commonly); hand-spread; 50%+ mortality
o Some strains have developed resistance to all new alternatives – no effective antimicrobial choices
 Can transmit resistance genes to S. aureus: MAJOR CONCERN

9
Listeria & Other Gram-Positive Rods

Listeria Monocytogenes
Characteristics:
 Short, gram-positive rods, can live intracellularly
 Survives & grows at low temp (fridge), low pH (stomach acid), high [salt]: overcomes food prep barriers
Colonizes: animals, humans, soil, vegetative matter
 Food-borne illness (listeriosis): contaminated meat, dairy, fruits, vegetables (rinse!)

Pathogenesis:
 GI tract  blood  meninges
 Invades, survives in variety of cell types

 Intestine: provokes active endocytosis (“internalins”) to cross mucosal barrier; survives intracellularly
o Escapes death in lysozome: formes pores to get out of phagolysosomes
o Replicates in cytoplasm (molecular mimicry: proteins look like host proteins)
o Promotes actin polymerization, makes “comet’s tail” that pushes organism out into adjacent cell
o Covered by host cell membrane (more molecular mimicry) in
next cell Lab diagnosis of Listeria:
 Spread hematogenously  Culture: grows well (30-37 °C best)
 Virulence factors: many; survival & invasion. Internalin helps  Blood agar:
internalize, listeriolysin O (LLO) helps organism escape from o small white colonies
phagocytic vacuoles (inserts pore), Act A induces actin polymerization o beta-hemolytic
 Catalase positive
Epidemiology: major cause of bacteremia & meningitis
 Characteristic tumbling motility
 Immunocompromised (especially cell-mediated immunity) and
elderly patients
 Colonized mothers (can be asymptomatic)  can pass to fetus
 High mortality (500-600 deaths; 2500 cases in US/yr)

Clinical presentation:
 Febrile gastroenteritis (6-48h incubation) in healthy persons. Self-limited. Many asymptomatic.
o Fever/chills, abdominal pain, diarrhea, nausea/vomiting, myalgias
 Bactermia: (pregnancy or immunocompromise)
o “bactermia without an obvious source”
o Fever/arthalgias, headache, GI symptoms, backache: or asymptomatic.
 Meningitis: 2nd most common cause of bactermia in adults > 50 yo; 5th overall
o Usual presentation of meningitis (stiff neck, headache) but:
 CSF glucose not low
 Mononuclear cells can predominate (intracellular; others = PMNs)
 Pregnancy: somewhat immunocompromised (predisposition)
o Can be infected in last ½ 2nd trimester & 3rd trimester
10
o Listeria can proliferate in placenta (infect child in utero)
o Stillborn / neonatal death: up to 20%
 Neonatal infection
o Disseminated form of disease: granulomatosis infantisepticum
o Late onset up to 2 wks post-partum; meningitis
o Hepatomegaly

Treatment: Ampicillin; TMP+SMX for PCN allergic (but watch out for kernicterus!?)
Bacillius spp.
General Characteristics:
 Aerobic, Gram positive rod
 Spore forming; ubiquitous
 Major pathogens: B. anthracis (anthrax), B. cereus (bacteremia, wound / eye infections, food poisoning)

Bacillus anthracis
Acquisition: Soilherbivoreshumans acquire via inoculation with spores:
 Inhalation*, ingestion*, or trauma (*=more lethal)
 Mostly in agrarian societies
 Category A biological terrorism agent (easy dissemination, high mortality, social disruption)

Pathogenesis
 Spores  skin, GI tract, lung  germinate in Mφ to regional lymph nodes  local production of
toxinsedema, necrosis  bacteremia, toxemia
 Virulence factors:
o Capsule: inhibit phagocytosis
o Protective antigen: binds to cell membranes (better binding/transport of edema factor & lethal factor)
o Edema factor:
 ↑ cellular cAMP; ↑membrane permeability (↑edema)
 ↓PMN function, ↓cytokine pathways (↑ proliferation, bacteremia, systemic infection)
o Lethal factor: Zn-dependent protease. Cleaves MAP kinases, oxygen radicals released
 Leads to Mφ lysis and cell death
Pruritic: itchy
Eschar: scab
Clinical presentations: Papule: circumscribed, solid elevation
 Cutaneous anthrax: of skin with no visible fluid, varying in
o Pruritic papule  enlarges (round ulcer)  painless, black size from a pinhead to 1 cm
eschar (dries, falls off); regional lymphadenitis FYI: Anthracis = “coal” (black eschar)

 Inhalational anthrax: mortality close to 100%


o 1-6d incubation; initial Sx flu-like (non-specific:
Anthrax Dx:
malaise, fever, non-productive cough, chest
 Hx of exposure; appropriate presentation
discomfort)
 Gram stain
o Rapid progression: dyspnea, cyanosis, shock
o Large, gram(+) rods in chains
o Hemorrhagic mediastinitis
 Culture:
o Meningitis can occur
o Blood, CSF, pleural fluid
o Radiography: mediastinal enlargement (hilar,
o 6-24h growth on 5% sheep’s blood
mediastinal lymphadenopathy  hemorrhage)
o Medium gray irregular colonies, swirling
o Pathology: tons / sheets of purple anthrax bacilli, tons
projections
of hemorrhage
o Non-hemolytic & non-motile
o PCN susceptible
 Gastrointestinal anthrax: 25-60% mortality

11
o Ingestion of vegetative bacteria (not spores) from poorly cooked, contaminated meat  ulcers in all
areas of GI tract  regional lymphadenopathy  nausea, vomiting, bloody diarrhea, sepsis
(excruciating pain)
o Pathology: intense submucosal hemorrhage

Treatment:
 EARLY administration of antibiotics is crucial
 Penicillin, doxycycline, ciprofloxacin
 Vaccine: military use only unless exposed to spores (lots of side effects; anthrax is rare)

Bacillus cereus
Soil organism; broad range of clinical syndromes
B. cereus Dx:
 Gram’s stain: GPR in chains
Clinical presentations:
 Culture
 Food-borne illness (especially rice that was left sitting out)
o Grows well on 5% sheep’s blood
o Diarrheal type: heat-labile enterotoxin; 8-16h post exposure
o Large, feathery, spreading colonies
o Emetic type: heat-stable enterotoxin; 1-5h post-exposure
o Beta-hemolytic (not anthrax)
 Ocular disease (penetrating trauma: bacteria in soil; almost always
o Organisms are motile, lecthinase
lose eye) & Wound infections (healthy persons): extensive damage,
(+) (not anthrax)
liquefactive necrosis
 Bacteremia, endocarditis, abscesses in immunocompromised pts & IV drug users

Treatment:
 Food poisoning: usually self-limited
 Produces broad-spectrum β-lactamase: resistant to PCNs & cephalosporins
 Vancomycin, clindamycin are active

Cornyebacterium diptheriae
General characteristics of Cornyebacterium spp.
 Aerobic bacteria, non-spore forming, Gram-positive bacilli
 Curved or club-shaped (corney = club)
 Catalase positive
 Normal flora of skin, mucous membranes of mammals; hard to distinguish colonization/contamination/infection
 Need to ID to species level when isolated from normally sterile sites, urine (if lots), clinical material

Cornyebacterium diptheriae: most important pathogen


 Humans = only known reservoir; Asx carriers = important for epidemics
 Spread: airborne respiratory droplets, direct contact with resp. secretions or exudates from skin lesions,
contaminated milk
 Most cases: colder months
 N. America: mostly disadvantaged groups (skin infections)
 Vaccine-preventable

Pathogenesis
 Non-invasive
 Exotoxin: major virulence factor
o 2-segment polypeptide
 B-segment (binding): bind to receptors on susceptible cells
 A-segment (active): inhibits protein synthesis in mammalian cells

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o Can affect all cells in body (heart, nerves, kidney most common)
o Also contributes to pseudomembrane production

Clinical presentation:
 Respiratory tract disease: 2-4d incubation period; can have local inflammation at various sites
o Pharyngeal (most common)
1. Abrupt onset (fever, malaise, sore throat)
2. Pseudomembrane forms & spreads
 one or both tonsils  oropharynx, nasopharynx, soft palate
 white then dirty gray with black necrosis
 Can compromise & distort lower airway: “bull’s neck”
o Systemic complications from toxin: myocarditis, neurotoxicity (cranial neuropathies)
 Cutaneous:
o Non-healing ulcers, dirty gray membranes, C. diptheriae Dx:
superinfected with other bacteria  Presumptive dx: clinical
o Outbreaks among alcoholic homeless, impoverished
 Definitive dx: isolate, ID organism
(no boosters, poor sanitation)
 Notify state lab (reportable) & send
o Rarely associated with toxigenic illness
material
 Others: can have invasive disease (e.g. endocarditis) with
 Lab dx: sheep’s blood & selective medium
non-toxigenic C. diptheriae

Treatment & prevention


 Strict isolation
 Antibiotics: PCN or erythromycin for 14 days
 Supportive care
 Vaccine: part of childhood DTaP series (4 doses total); booster at school entry, every 10 years with tetanus

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Introduction to Gram-negative Bacilli
Gram negative bacilli: rods that stain red by Gram’s stain;
 large, diverse group
 Classification: growth requirements, phenotypic or genotypic characteristics, disease associations

Major categories of GNRs:


 Enterobacteriaceae – fermenters. Enteric pathogens & non-enteric infections
 Glucose non-fermenting GNRs
 Oxidase positive fermenters (cause diarrhea)
 Fastidious GNRs: require specialized media / growth conditions
 Anaerobic GNRs
 GNRs associated with zoonoses

Can use selective material which is inhibitory to Gram (+), e.g. MacConkey agar, to isolate Gram (-)
 Red/pink: acid produced, lowers pH, lactose fermenter
 Clear: non-lactose fermenter; diarrheal pathogens
 No growth: not Gram (-)

Enterobacteriaceae (this lecture)


General characteristics:
 Tons of diversity, found in GI tracts of humans, animals, fish, etc. Some Enterobacteriaceae:
also in environment: soil, water, plants. Certain kinds (Salmonella,  Ferment glucose (often with
Shigella, Yersinia) shouldn’t be in humans ever. gas production)
 Facultative anaerobes (rapid growth with or without O2)  Reduce nitrates to nitrites
 Grow readily on simple material (12-8h incubation).  Catalase: positive
 Diseases: all major categories except STDs  Cytochrome oxidase: negative
Morphology: large, parallel sides, rounded ends
 Some strains: peritrichous flagella (go around whole circumference)
 Some: encapsulated
 Many: surface pili for adherence
 Do NOT form spores

Antigenic structure (serotyping):


 O (somatic) antigen (= LPS = endotoxin): cell-wall LPS, heat-stable (IgM response)
 K or Vi (capsular) antigens: cell surface polysaccharide (Vi = virulence, for salmonella)
 H antigens: externally located flagellar proteins (IgG response)

Remember the gram (-) cell wall: has porins in OM; antimicrobial resistance: mutate porin so drugs can’t get in

Urinary tract infections


Community-acquired:
Types of UTIs
 80% from uropathogenic E. coli, then Klebsiella and Proteus
 Urethritis (urethra)
 Predisposition: anatomy, density of mucosal receptors, sexual activity
 Cystitis (bladder)
Hospital-acquired  Pyelonephritis (kidney)
 Frequently in patients with indwelling bladder catheters
Usually ascending; can be
 E. coli still main cause; also other resistant Gram (-)s
descending (from kidneys)
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E. coli: Need uropathogenic strains: low pH in urine, lots of antimicrobial compounds in urine
 Specific fimbriae (type 1, good for colon/vagina colonization), pili (P pili:
E. coli: Lab Dx
adherence to urinary tract)
 β-hemolytic on sheep’s blood
 Toxins: endotoxin and α-hemolysin (pore forming cytotoxin; explains
 Lactose fermenter
hemolysis, causes toxicity to uroepithelial cells too)
 Rapid indole positive
 Aerobactin – siderophore (chelates iron)

Proteus spp.
 Associated with UTIs, urolithiasis (stones) Proteus: Lab Dx
 Proteus mirabilis, Proteus vulgaris  Non-lactose fermenter
 Cycle: Proteus produces urease, hydrolyzes urea to CO2 + NH3   Very motile (swarming over
neutralize/alkalinize urine  inorganic compounds fall out of solution & agar plate)
crystallize  stones  become embedded & reinfected

Klebsiella pneumoniae
 UTIs, pneumonia; hospital-acquired infections with multidrug resistance Klebsiella pneumoniae: Lab Dx
 Mucoid capsule (important for virulence)  Non-lactose fermenter
 Get in lungs/urine; huge inflammatory response  Non-motile
 Disgusting mucoid colonies

Gram-Negative Pneumonia
Predisposing factors: Gram (-) pneumonia Pneumonia
NON-HOSPITALIZED HOSPITALIZED 1. Rapid growth
 Underlying cirrhosis (↑ encapsulated infections)  Diminished cough reflex, anesthesia 2. Inflammation (intense,
 Loss of consciousness, alcoholism, drug abuse  Mechanical ventilation PMNs)
 Elderly, immunocompromised  Immunocompromised 3. Inefficient killing of organism
(varies with host)
Klebsiella pneumoniae 4. Obstruction, obliteration of
 Pneumonia: necrotizing, develop cavitation in areas of consolidation lung tissue
o If you survive: chronic lung disease (pulmonary fibrosis) 5. Death (tissue / host)

Aerobic Gram-Negative Rod Meningitis


0-4 wks Grp B strep, E. coli, L. monocytogenes, Salmonella
Aerobic GNR meningitis
4-12 wks Grp B strep, E. coli, H. influenza, S. pneumoniae, N. meningitidis
3mo – 50 yrs S. pneumoniae, N. meningitides (H. influenza in adolescents)  E. coli (75% capsular type K1)
> 50 yrs S. pneumoniae, N. meningitides, L. monocytogenes, aerobic GNR  Klebsiella sp
 Predominantly neonates, elderly, neurosurgery pts  Salmonella sp
 Don’t let your babies play with snakes & iguanas! (salmonella)  Pseudomonas aeruginosa
 Other factors: immunocompromise, head trauma/neurosurgery, CNS
shunt (↑ aerobic GNR meningitis)
Bacterial meningitis: Lab Dx:
Neonatal meningitis: Exposure/colonization (Asx?) during birth + poor immune
LP: response  bactermia  meningitis
Other agents:  ↑: pressure, WBC, PMNs, protein, lactate
 Citrobacter koseri (sporadic cases, utilize citrate);  ↓: glucose
 Enterobacter sp (yellow pigmented, highly virulent, MDR) Gram stain & culture: GNRs & PMNs
Gross path: cloudy, pus in meninges
High mortality with GNR meningitis

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Intestinal infections
Salmonella (S. enterica = most pathogens; also S. bongori)
 Various virulence factors (Vi = salmonella typhi) Salmonella: Lab Dx
 Exposure: poultry or products (eggs)  Gram (-), non-motile
 Stays in vacuoles inside cells; enters submucosa, basal membrane; goes to  Produces H2S (black colonies)
mesenteric lymphocytes  blood stream  Use selective media to
 Clinical presentations: recover from stool
o Gastroenteritis (S. typhimurium mostly)
 6-24h post-exposure: nausea, vomiting, abd. pain, diarrhea; 50% have fever
o Bacteremia (non-typhoidal): much more frequently found in blood than Shigella
o Typhoid fever (S. typhi, S. paratyphi)
 1-4wk incubation; fever, multi-organ system infection, may or may not have diarrhea
 Multiplication in spleen, liver  gall bladder during infection

Shigella
 Four serotypes: S. sonnei (major in US), S. flexneri, S. boydii, S. dysenteriae (epidemic dysentery, produces
Shiga toxin),
 Presentation: fever, severe, cramping abd. pain, bloody diarrhea Shigella: Lab Dx
 Virulence factors plasmid associated; LPS in all  Gram (-), non-motile
 More severe pain than Salmonella, lower abd. (colon)
 Pathogenesis:
o Through stomach acid & small bowel  terminal ileum/colon
o Engineers own phagocytosis
o Escapes from vacuole! Stays in mucosa(fecal WBC confined to mucosal layer)
o Non-motile; cause invasive infection by spreading cell-cell via actin polymerization.
o Destroys colonic epithelial cells in process (blood, pus in stool); self-limiting 2-5d

E. coli
 EnteroToxigenicEC (traveler’s diarrhea), EnteroInvasiveEC
(uncommon, invasive) EnteroPathogenicEC (infantile, ST E. coli: Lab Dx
childhood diarrhea), EnteroAggregativeEC (traveler’s  Gram (-)
diarrhea): from previous lecture  Hand-deliver quickly!
 Direct detection of SLT in STEC pts’ stool
 Shiga-toxin-producing E. coli: STEC  Culture: grows well on standard lab material;
o Hemorrhagic Colitis selective material can be usd
o Hemolytic-Uremic Syndrome (thrombocytopenia,  PMNs + GNRs: think this family!
renal failure, hemolytic uremia) – associated with production of Stx-2 (Stx-1 only: EPEC)

Yersinia can cause diarrhea too


Antibiotics & GNRs

 Order susceptibility testing on organisms from


clinical material (disk diffusion / microbroth
dilution)
o Look for MIC
 MDR control: hand hygiene, contact
precautions, antimicrobial stewardship
 Klebsiella can be especially bad (resistant to
almost everything!)

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Fastidious Gram Negative Rods
Haemophilus sp
Characteristics: normal resp flora of humans & animals
 Pleomorphic, GNRs
 Invasive strains = encapsulated
 Requires special growth factors in vitro: X factor, V factor. Optimal growth on chocolate agar

Lots of different kinds. No capsule = not invasive (H. influenza non-typeable for instance)
 H. influenzae type b (a-f are other types).
o Type b causes epiglottitis, sinusitis, meningitis, pneumonia, septic arthritis, etc.
 H. influenzae non-typeable (unencapsulated): now #1 cause pediatric acute otitis media; sinusitis too
o Replaced S. pneumoniae

H. influenzae type B (HiB)


 Fallen 99% post-vaccine area (22 cases 2007, unvaccinated/incompletely vaccinated children)
Haemophilus: Lab Dx
Epidemiology/pathogenesis
 Gram Stain (pleomorphic GNR)
 Nasopharynx of 3-5% healthy people colonized with Hib
 Culture (chocolate agar)
 Pathogenesis: penetrates submucosa of nasopharynx  o Look for X, V factor requirements
bloodstream  CNS  meningitis o Hemolysis?
 Virulence factors: capsular polysaccharide, fimbriae, IgA protease,
OM proteins, ciliostatic glycopeptides (paralyzes cilia to make colonization easier)

Treatment/prevention:
 33% produce plasmid-mediated beta-lactamase (PCN resistant)
 Invasive disease: 3rd gen cephalosporin
 Non-invasive disease: amoxicillin + clavulanate
 HiB Vaccine x 4: 2,4,6,12-15 mo of age

Bordetella pertussis
 Gram (-) coccobacilli (single or pairs; faintly-staining); strict aerobe; needs special media (have to ask for it)

Epidemiology: B. pertussis: Lab Dx


 Can’t survive in environment: requires direct, person-person  Gram Stain (single/pairs, Gram (-)
spread via airborne droplets coccobacillus)
 Adults can be reservoir for infection  Strict aerobe
o (unexplained cough = avoid babies!)  Best specimens: NP aspirates/swabs
 Children < 1yo: most severe disease (immune system not at peak)  *Culture (special media)
 Vaccine preventable! o Can require up to 10d to recover
(way too long for Dx)
Pathogenesis: Non-invasive (secretes toxins)  *Nucleic acid detection: faster &
1. Adheres to non-ciliated resp. epithelium (fimbrae) most sensitive
2. Produces local damage; disrupts host defenses
 Serology = only retrospective
3. Systemic disease
Key virulence factor: pertussis toxin (all stages)
* = recommended by CDC
Clinical presentation: Pertussis syndrome (more atypical presentation in adult)
0. Incubation period (1-3 weeks)
1. Catarrhal stage (1-2 weeks): Most contagious; rhinorrhea, malaise, sneezing, low-grade fever
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2. Paroxysmal stage (2-4 weeks): paroxysmal coughing with inspiratory whoops; post-cough vomiting, air hunger /
apnea / cyanosis, peripheral lymphocytosis (hallmark: tens of thousands!)
a. Basically can’t get air! Whooping = trying to inspire against closed glottis. Really sucks to have.
b. Radiography: Peri-hilar infiltrates, distal airways spared,
3. Complications: secondary bacterial pneumonia (damaged resp. epithelium), pulmonary hemorrhage (exertion
of coughing), encephalopathy (toxin to brain), seizures (hypoxemia), coma/sudden infant death

Treatment / Prevention
 Supportive care (vent, ICU, fluids, etc.)
 Erythromycin: doesn’t alter course but decreases bacterial load (less infective)
 Vaccine:
o used to use whole cell (DTP); high reactogenicity, 50-90% efficacy, wanes in adolescents/adults
o Discontinued in some countries  pertussis outbreaks
o Acellular vaccine now in use: DTaP (diphtheria, tetanus, aceullular pertussis)
 Immunogens of B. pertussis, no endotoxins (less side effects) with equal efficacy
 5 doses (2mo-5yr); one-time booster for adults (Tdap)

Legionella
General characteristics:
 thin, poorly-staining GNRs
 Require L-cysteine for growth; use AA for growth (non-fermenters)
 Biochemically inert (weak oxidase rxn; catalse positive, liquefy gelatin)
 Motility: polar flagella

Epidemiology: tons of species; L. pneumophila is responsible for >90% disease (rest named by where they’re from)
 Ubiquitous, widely distributed in environment (aquatic settings: natural or man-made)
 Wide temperature range (0-63C)
 Parasitize & survive in free-living amoebae!
 Form biofilms in water systems
Transmission: inhalation of droplets; aspiration of water; wound infection (more rare) by contaminated water

Pathogenesis:
1. Adhere to resp epithelium Risk factors for Legionella infections
2. Phagocytosed  Cigarette smoking, chronic lung
3. Intracellular survival / multiplication disease, alcoholism
a. Inhibits acidification of lysosome  Immunosuppresion (corticosteroids,
b. No fusion of lysosome with phagosome malignancies, HIV, transplantations)
c. Lysosome associates with rER (molecular mimicry; lined  Diabetes, end-stage renal dz, CV dz,
with ribosomes); bacteria replicates here advanced age
4. Cell rupture & release
24 kD protein macrophage infectivity potentiator (mip): target of many Legionella: Lab Dx
molecular tests  Culture: need special medium
(request!) to inhibit normal flora (abx,
Clinical presentations: etc). Can take up to 7 days (slow
 Legionella pneumonia: 2-15% cases of CAP growing)
 Legionnaire’s disease: systemic illness with pulmonary and  Legionella urinary antigen: only
extrapulmonary manifestations (rare to have extrapulmonary serotype 1, but that causes most dz
alone)  Nucleic acid amplification (no FDA but
o Radiologically indistinguishable from other some in-house depending on lab)
pneumonias
 Direct fluorescent antibody
 unilateral, lower-lobe alveolar infiltrates; some

18
pleural effusion
 cavities/abscesses can occur if immunocompromised
o Slight, non-productive cough (no PMNs because intracellular)
o GI: watery diarrhea; abd. pain
o CNS: mental confusion, headache
o Bradycardia; hyponatremia, hypophosphatemia, elevated CK, increased transaminases
o Doesn’t respond to beta-lactams
Getting a sample: no special transport, room temp OK, refrigeration if delayed.
 Send sputum, aspirates, BAL fluids, pleural, lung tissue.

Legionella urinary antigen detection


 Detects LPS of cell wall
 Serotype 1 only (80% of dz though)
 Antigen shows up day 1-3 of Sx; can persist for weeks/months

Treatment: 10-14d
 Newer macrolides (azithromycin, clarithromycin); quinolones (levofloxacin)
 Can also use tetracyclines or TMP+SMX

Prevention:
 Routine culture surveillance of hospital water distribution systems
 Treat If pathogenic legionella found (hard to get biofilm out). Chemicals, heating, etc.

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Non-fermentative Gram Negative Bacteria
General features:
 White on MacConkey agar = non-fermenter (pink=fermenter)
 Complex mix (opportunistic pathogens of plants, animals, (most) Non-Fermentive Gram (-)s:
humans)  Aerobic, Non-spore forming, Bacilli
 75% in clinical specimens are one of these four  Cytochrome oxidase positive
1. Psudomonas aeruginosa  Catalase positive
2. Acinetobacter baumanii  Don’t ferment CHOs; may oxidatively
3. Burkholderi cepecia metabolize some sugars
4. Stenotrophomonas maltophilia  Motile, nutritionally versatile
 Opportunistic pathogens: most often hospital-acquired  Grown on MacConkey Aga

Pseudomonas spp

 Ubiquitious (soil, water, decaying organic matter, vegetation)


 Found throughout hospital environment (moist reservoirs, incl. dialysis equipment, mops, toilets, etc.)
 Simple growth requirements: wide temp range; can use lots of nutrients for C&N source
o Can even grow in distilled water and disinfectant solutions!
 Lab classification: fluorescent (make pyoverdin green under UV) & non-fluorescent
o Fluorescent group includes P. aeruginosa and P. fluorescens, most important for clinical
o P. aeruginosa also makes pyocyanin
 Important for invasiveness & characteristic of aeruginosa on plate

Pseudomonas aeruginosa
 Aerobic, straight/slightly curved, non-spore forming, Gram(-) rod
 Motile (1+ polar flagella)
 Grows well on SBA, chocolate, MacConkey; wide temp range; Pseudomonas aeruginosa: definitive Lab Dx
 Catalase positive  Gram (-) rod
 Makes both pyoverdin & pyocyanin  Oxidase positive (rapid oxidase test)
 Grape-like or corn-tortilla odor
Virulence: multifactorial (structural components, toxins, enzymes)  Recognizable colony morphology
 Structural: o SBA/chocolate: large colonies, metallic
sheen, mucoid/rough/pigmented
o LPS (endotoxin), Pili (adhesion; neuraminidase to remove
o MacConkey: lactose negative; green
sialic acid from pili receptor); capsule (adhesion &
pigmentation or metallic sheen
suppression of phagocytosis / immune response);
pyocyanin (tissue damage: hydroxyl radical products; IL-8 stimulus)
 Toxins/enzymes:
o Exotoxin A & S(inhibits protein synthesis); cytotoxin (leukocydin) (cytotoxic for eukaryotic membranes;
microvascular injury); Elastase (disrupts elastin-containing tissues, collagen)

Clinical presentation:
 Bacteremia, pneumonia top 2
 Others: pretty much all sites of body but particularly adapted to respiratory tract
o CF patients; chronic colonizer of pts with chronic lung P. aeruginosa: Predisposing factors
disease
 Chronic debilitating illness (lots of hosp)
o #1-2 cause of Ventilator-Associated Pneumonia (VAP)
 Prior therapy with broad-spectrum abx
 Can produce disease far away from initial site of tropism
 Breach of airway (tracheostomy,
 Intact host defenses: not at much risk (opportunist) endotracheal tube)
 Imparied host immunity (primary disease
Pulmonary infections in CF patients: colonization  tracheobronchitis or iatrogenic)

20
 necrotizing bronchopneumonia
 Tropism for CF epithelial cells; actually switches to a CF phenotype (rough LPS, mucoid, less motility)
o Increased Abx resistance because of prior broad-spectrum abx in CF pts

Skin infections from P. aeruginosa:


 Ecthyma gangrenosum secondary to P. aeruginosa bacteremia (pretty classic for P.aerug)
 Folliculitis (e.g. after hot-tub)
 Burn wounds: P. aeruginosa very important!
o Colonization  local vascular damage / necrosis  bacteremia
o Risk correlates to extent of burned surface
o Commonly <1wk from burn injury

“Community-acquired” P. aeruginosa infections: can happen in outpatient setting


 Corneal ulcers, keratitis (extended wear contact lenses / abrasions / scratches)
 Endopthalmitis (deeper eye infection; after eye trauma or surgery)
 Exposure to moist environment (hot tub, whirlpool, swimming pool)
 Otitis externa : “swimmer’s ear”; malignant external otitis can extend to cartilage & bones (pts with diabetes,
elderly, etc.)
 Puncture wounds (through tennis shoes, etc.)  osteochondritis
 Endocarditis (IV drug users)

Treatment, prevention, control: recognize high level antimicrobial resistance worldwide; carbapenems may be driving
emerging resistance; lots of MDR Pseudomonas.
 Might be able to use old drug (colistin) despite bad side effect profile
 Can’t eliminate from hospital environment: need infection control (safeguard patients)
o Keep equipment sterile; prevent health care worker  pt transfer; responsible ABx use

Acinetobacter spp
 Widely distributed (nature & hospital)
 2nd most common non-fermenter found in humans after P. aeruginosa Acinetobacter spp
 Can survive on moist & dry surfaces; present in food & on skin  Gram(-) coccobacillary rods
 Increased frequency: immunocompromised, debilitated pts  Strictly aerobic
 Grow on MacConkey (colorless)
Acinteobacter baumanii:  Non-motile, non-fermentative
 Most frequently isolated from human specimens  >> Oxidase negative << (key)
 Most often responsible: hospital-acquired infections  Usually nitrate negative

Clinical presentations: Risk factors: Acinetobacter


 Hospital acquired:  Stay in ICU
o Respiratory tract infection  Abx treatment
o UTI, Wound infection, Bacteremia
 Surgery
o Nosocomial pneumonia / VAP
 Mechanical ventilation
o Digestive tract of ICU patients can be reservoir for MDR strains
 Community-acquired:
o CA-Pneumonia with fatal outcome (initially misdiagnosed; wrong Tx)
o Wound infections & osteomyelitis (e.g. battlefield infections)

Tx & prevention: Need Abx susceptibility testing for every clinically significant isolate!
 Intrinsic resistance to cephalosporins; carbapenem resistance ~ 20%; high frequency of MDR
 Ampicillin-sulbactim, ticarillin-clavulanate or imipenem are most effective
 Also: TMP-SMX, quinolones, doxycycline. Can add aminoglycoside if severe infection; colistin possible if MDR
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Stenotrophomonas maltophilia
 Similar profile to Burkholderia cepacia
 Widely distributed: nature & hospital S. maltophilia
 Can colonize resp tract in pts. with prolonged hospitalization (e.g.  Gram(-) rod
immunocompromised)  Motile, non-fermentative
 Virulence factors unknown, opportunistic human pathogen  Grows well on MacConkey
 Oxidase negative
Clinical presentation:
 Nosocomial; high morbidity/mortality
o Bacteremia, pneumonia, UTI, wound infctions
o Increasing incidence: resp tract infections in CF patients
Treatment:
 Intrinsic resistance to almost every antimicrobial commonly used (B-lactams, AGs, others)
 TMP+SMX is primary choice for treatment

Burkholderia cepacia complex


B. cepacia: most commonly isolated Burkholderia sp. in clinical specimens; consists of 9 complex genovars
 Plants, soil, water as reservoir
 NOT part of normal human flora; can colonize resp tract
 Nosocomial: associated with contaminated hosp equipment, medications, and disinfectants
o (even iodine & chloride solutions! Albuterol! Sterile blood culture systems!)
Clinical presentation: opportunistic pathogen, variety of infections
 Bacteremia, UTI, septic arthritis, peritonitis, pneumonia
 Risk factor: chronic granulomatous disease or CF pts

B. cepacia and cystic fibrosis


 Emerged in 1980s; require special infection control practices
 Approx 3% CF pts in US infected with B. cepacia, 6-7% adults
 Most strains inherently resistant (broad spectrum abx)
 May be absolute contraindication for lung transplant
 20% develop cepacia syndrome: bacteremia, fatal outcome

Treatment: resistant to most antimicrobials (B-lactams, AGs, others)


 Susceptible: TMP+SMX, ceftazime, imipenem, meropenem, some fluroquinolones

Burkholderia pseudomalli
 Found in soil, water, vegetation: SE Asia, Northern Australia (watch out for travelers)
 Wrinkled colonies on plate
 Acquisition: inhalation or inoculation (trauma/wounds); Potential bioterrorism agent

Clinical presentation:Causes melioidosis


 Can be acute, subacute, or chronic (chronic can mimic TB)
 Pneumonia = most common clinical presentation (abscess formation, cavitation)
 Protean manifestation (assumes many forms)
o Asx, cutaneous, pulmonary dz
o High propensity for bacteremia
o Diabetes, renal dz, cirrhosis, thalassemia, alcoholism (& immunocompromise) predispose

Treatment: TMP+SMX and broad-spectrum cephalosporin


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Neisseria Species
General characteristics:
 Gram (-) diplococci, inhabit mucous membrane surfaces Features of Neisseria
 Group contains:  Aerobic
o Non-pathogens (upper resp tract dwellers)
 Non-motile, non-spore forming
o Strict pathogens:
 Oxidase, catalse positive
 N. gonorrhoeae (STI: gonorrhea);
 Chocolate agar (best)
 N. meningitidis (epidemic/endemic meningitis)
 Use CHO oxidatively

Neisseria gonorrhoeae
Same characteristics as other Neisseria
Nutritionally: more fastidious
 Requires cysteine for growth; other requirements too

Epidemiology: STI except in newborns


 humans are only host; huge incidence (355,000/yr), mostly in sexually active teens & young adults
 Women (men too) can be asymptomatic and transmit infection
 Risk factors:
o Social: lower SES, urban, lack of education, unmarried, STD history)
o Behavioral: unprotected intercourse, multiple / high risk partners, drug use, MSM

Pathogenesis: adherence  cell entry/transport  evade  stimulate PMN host response (pyogenic)
 Special features of surface structure:
o Oligosaccharide endotoxin (smaller than LPS)
o Pili (attachment), peptidoglycan (toxic to fallopian tubes), membrane proteins (survival & invasion)
 Adheres to non-ciliated cells (e.g. fallopian tubes); adjacent ciliated cells damaged, slough off, more can attach
o Loss of ciliated cells  obstruction, infertility, ectopic pregnancy, etc.

Clinical presentation N. gonorrhoeae: Lab Dx


 Women: Mucopurulent cervicitis is typical, also urethral  Gram stain (better in male urethral samples)
syndrome (distal urethra), pelvic inflammatory disease  Culture-based: sexual abuse, treatment
(ascending disease; scarring, etc.). failures, test of cure
 Neonates: perinatal transmission; Ophthalmia neonatorum o Multiple methods (need 2nd to confirm:
(purulent conjunctivitis); also gonococcal scalp abscesses or important to be right, esp. child abuse)
systemic infections.  Nucleic acid amplification (PCR) – very
 Men: Urethritis, epididymitis, rectal infections (if MSM) sensitive (can’t use for test of cure)
 Common features: purulent exudates, inflammation,
erythema (penis/os). Burning on urination (men)
o Unusual adult manifestations: conjunctivitis, disseminated gonococcal infection (vesicle  purulent papule,
erythema, central necrosis)

Culture: selective media; supplemented with growth factors; 72h, small, glistening, raised.
 Glucose metabolizer(not maltose or sucrose)

Treatment:
 Uncomplicated: Ceftriaxone IM or Cefixime PO
o Treat for CHLAMYDIA TRACHOMATIS (lots of coinfection): AZI PO x1 dose or doxycycline PO bid x 7d)
 Report & contact-trace infected persons
Prevention: sex ed, abstinence, condoms, no vaccine
 use 1% silver nitrate or macrolide antibiotic prophylax for newborns
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Neisseria meningitidis (“meningococcus”)
All age groups, both individual (sporadic) & epidemic
Multiple serotypes: B,C,Y most severe in US
Immunization possible (esp. closed populations)

Epidemiology
 All age groups, both individual (sporadic) & epidemic
 Multiple serotypes: B,C,Y most severe in US
 Asymptomatic nasopharyngeal carriage (8-25%), precedes infection
o Colonization ↑ in closed populations (college, military)  need vaccination
 Terminal C’ deficiency (C5-9): recurrent, severe infections
 Worldwide: Epidemics in some countries (at end of rainy season)
 USA: Vast majority sporadic with localized outbreaks
o Case fatality: 10-14% !!

Pathogenesis
 Virulence factors: Pili, engulfment, transport in host cells; Polysaccharide capsule (↓ phagocytosis, C’); Lipo-
oligosaccharide endotoxin (very toxic!)
 Process: HAPPENS REALLY FAST
1. Pili adhere to non-ciliated resp epithelium invades submucosa
2. Capsule prevents phagocytosis
3. Replicates in submucosa; evades
4. Spreads into bloodstream
5. Endotoxin in blood: cytokine & alternative C’ activation

Clinical presentations: N. meningitidis Lab Dx:


1. Bacteremia without sepsis (rare)  Direct exam of CSF (presumptive, quick)
2. meningitis with neurologic sequelae  Culture: CSF, blood, other
3. meningococcemia (overwhelming sepsis & DIC) o Chocolate agar
a. with adrenal insufficiency: Waterhouse-Frederichsen  Definitive ID: glucose & maltose use
Syndrome

Meningitis  Meningococcemia
 Like other meningitis but faster.
 Sudden onset: Fever, chills, myalgias, arthralgias, headache, confusion, nuchal rigidity
 CSF: 1200 WBC/mL, Glc↓, protein ↑
 Meningococcemia
o Rash: palpable purpura (infection of endothelial cells  DIC, small hemorrhages)
o Can get peripheral gangrene; lose digits
 Adrenal hemorrhage can cause insufficiency (W-F syndrome)

Treatment:
 Meningitis/meningococcemia: Penicillin (alternatives: ceftriaxone, chloramphenicol in other countries)
o Not PCN resistant!
 Prophylaxis (close contacts) – trace! Rifampin, cipro, ceftriaxone
Vaccines:
 Old: polysaccharide tetravalent (poor immunogenicity, not long-lasting, no reduction in NP carriage)
 New: tetravalent conjugate vaccine (Menactra) for 2-55yo (give 11-12 or entry to HS; should have for college)
o Capsular polysaccharide conjugated to diphtheria toxin; induces T-cell dep response (good for infants)
o Reduces Asx carriage
o Indications: asplenia, C’ deficient, traveling to endemic areas, closed pops, prevention, lab workers
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Anaerobic Gram Positive Bacteria
General characteristics:
 Don’t grow in presence of oxygen
Anaerobic infections (Clinical Dx)
 Two groups:
 Foul-smelling discharge
o Obligate anaerobes (strict or moderate)
 Proximity to mucosal surface
o Aerotolerant anaerobes
 Gas in tissues
 Tons of different kinds (many part of normal microflora, protective)
 Negative aerobic cultures
 Classification (pathogenic): shape, gram, spore formation, toxin
production
 Widespread: environment (soil, sewage, foods, etc.)
o animals & humans: oral cavity around teeth, GI tract, skin (some), G/U tract
o habitats with low oxygen tension & reduced oxidation reduction potential

Pathogenesis:
 Disruption of normal mucosal barriers
 Anaerobic infections often mixed (synergy with aerobes, other anaerobes)
 Virulence factors: capsules, adhesions, enzyme production, toxin production (some very potent toxins)

Presentations:
 brain abscess (usually anaerobes or microaerophilic strep)
 bacteremia (need virulence factors to get into bloodstream)
 lots of others (mixed) – e.g. empyema (pus in pleural cavity)

Lab Dx:
 Get a good specimen (not anything where anaerobes would be in normal flora; Exception: C. diff & feces)
o gastric washings, urine, vagina/cervix, feces, upper resp. secretions, etc. are bad
 Swabs are bad specimens, aspirates & tissue biopsies are best (transport immediately)
o Inject into anaerobe transport media
 Semi-solid, 5% CO2, reducing agent  shows anaerobiosis
o Culture in complex media: supplemented; chopped meat glucose; use anaerobe chamber

The following are GRAM POSITIVE ANAEROBES


Peptostreptococcus
 Anaerobic, Gram (+) cocci; variable shape & size
 Found on skin, mucous membranes
 Importance: Causes bacteremia & mixed infections

Propionibacterium spp
(Major metabolic product = propionic acid)
 Anaerobic, Gram (+) rods, highly pleomorphic (curved, clubbed, pointed ends)
 Normal flora of skin, mucous membranes; major contaminant of blood cultures
 Non-spore forming
 Importance: acne, also opportunist with medical devices(shunts, caths, prosthetic valves)

Actinomyces
Anaerobic, Gram (+) rods, pleomorphic (short/club shaped, long/thin/beaded rods, branching)
Slow-growing – makes a “ molar tooth” colony. Reason why labs hold AnO2 cultures for up to 1 wk

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Actinomycosis
 Indolent, progressive infection that progresses across tissue boundaries
 Purulent foci with dense fibrotic tissue around
 Can look like neoplasia/tumor (mass)
 Later: sinus tract; drainage with “sulfur granules” (yellowish)
 E.g. cervical actinomycosis (cervicofacial is most common)

Clostridium species: overview


 Obligate anaerobes, Gram (+) bacilli
 spore-forming, pleomorphic, most motile
 Large group; ubiquitous in nature (GI tract of humans/animals; soil)

Clostridium perfringens
Clostridial myonecrosis (“gas gangrene”)
 Pathogenesis: production of phospholipase C (very potent alpha toxin)tissue destruction
 Diagnosis: clinical: septic appearance, gas in tissues (palpable bubbles),
o Necrosis with gas & no inflammatory cells: phospholipase C kills them too quickly!
 Treatment: need extensive surgical debridement! (can’t contain with abx & host immune system)
o Supportive care
o Abx: penicillin G + clindamycin
o Hyperbaric oxygen for some locations
Food poisoning: toxin made after food ingested
Necrotizing enterocolitis: beta-toxin production after poorly cooked pork, rare in US

Clostridium difficile
Pseudomembranous colitis & antibiotic associated diarrhea;
 Pathogenesis: Toxin A (enterotoxin), Toxin B (cytotoxin)
o Extra toxins: epidemic strain
 Clinical presentation: bloody diarrhea; can include toxic megacolon (distended)
 Diagnosis: DIRECT DETECTION OF TOXIN IN STOOL is major way to diagnose
 Treatment:
o Withdraw offending antibiotic
o Metronidazole PO or vancomycin
o Surgery if toxic megacolon, intestinal perforation, severe illness

Clostridium botulinum
Botulinum toxin: most potent neurotoxin known; bioterrorism agent
Botulism: three types
1. Foodborne: typically adults; from ingestion of preformed toxin in contaminated food (poorly-made jams, etc)
2. Infant: organism in GI tract; toxin produced in vivo, most common form of disease
3. (Wound) – rare

Pathogenesis: toxin production; potent neurotoxin that blocks Ach release at neuromuscular junctions

Clinical presentation:
1. Incubation: 18-36h, dose dependent
2. Afebrile, alert, oriented, normal sensory exam; early nausea/vomiting, diarrhea
3. Cranial nerve symptoms (ptosis, blurry/double vision, trouble swallowing/talking, ↓ salivation)
4. Progressive motor symptoms: BILATERAL DESCENDING FLACCID PARALYSIS  resp paralysis
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5. Death in 60% (untreated; 5% with Tx)

Lab Dx: toxin in serum, gastric fluid, stool

Treatment:
1. Supportive care (airway support)
2. Administer antitoxin (equine serum for adults – 9-20% hypersensitivity – or human botulism Ig for infants)
3. (if wound: debride too)

Clostridium tetani
VACCINE PREVENTABLE!
Widespread distribution of spores in soil & aquatic environments
Pathogenesis:
1. Spores contaminate puncture wounds, etc.
2. Spores germinate (low oxidation-reduction from poor vascular flow)
3. Vegetative cells multiply; release tetanospasmin (attaches to peripheral nerve endings; travels up to CNS)
4. Toxin: binds gangliosides in CNS and blocks inhibitory impulses (PROLONGED MUSCLE SPASMS)

Clinical presentation
 Spastic muscle contractions
 Difficulty opening the jaw (“lock-jaw”)
 Characteristic smile (“risus sardonicus”)
 Contractions of back muscles can result in arching – can actually snap spine!

Treatment:
1. Supportive care (airway maintenance, antispasmodics)
2. Antitoxins: human tetanus Ig, tetanus toxoid
3. Antibiotics: metronidazole

Prevention: vaccine!

Clostridium septicum
Bacteremia associated with malignancy
 Pathogenesis: high dose chemo damages GI; organisms translocate at site of mucosal damage
 Blood cultures positive
 Treatment: Penicillin G, supportive care, sometimes surgery

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Emerging and Re-emerging Bacterial Zoonoses
 Zoonosis: any infectious disease that may be transmitted from other animals (wild & domestic) to humans or
from humans to animals
 Vector: any animal that transmits an agent of human disease or plays an essential role in the agent’s life cycle
o E.g. mosquitos / malaria; snail hosts / schistosomiasis; rodents / leishmaniasis
o Typically arthropod vectors (mosquitos, ticks, flies, lice, fleas)
 Emerging infections: diseases that have recently appeared or are growing in incidence. Lots!
o Zoonotic, vector-borne, bacterial  high RR for disease to emerge

Transmission of bacterial zoonoses


Direct contact: animals/infected materials Leptospirosis (Leptospira); Brucellosis (Brucella)
Animal bites / scratches Cat scratch disease / bacillary angiomatosis (Bartonella henselae)
Arthropod vectors Plague (Yersinia pestis)
Contaminated food Lyme disease (Borrelia burgdorferi), Rocky Mountain Spotted Fever
(Rickettsia rickettsi)

Leptospirosis
Leptospira: spirochete (spiral-shaped); Enormous taxonomic group

Leptospirosis:
 Estimated >10M cases worldwide, uncommon in US
(Hawaii, Wisconsin?), more common in tropics
 Acquisition: contaminated animal or rodent urine: lives in
bladder, urinary tract of asymptomatic animals
o Water/soil
o Skin abrasions; conjunctivae
o Domestic pets (dogs) / livestock too
Clinical presentation:
 Fever, headache, myalgia, abdominal pain, conjunctival suffusion (inflammation / red eyes)
 5-10%: Icteric form (Weil’s disease)
o Can precede worse outcomes: renal failure, pulmonary hemorrhage, cardiac arryhmias
 Uveitis (late manifestation, can lead to blindness)
 Death in 5-15% (esp old age)
Histopathology:
 Cholestasis: brown pigment in liver (jaundice too)
 Pulmonary hemorrhage(mechanism not known)
 Interstitial nephritis: chronic inflammatory cells in interstitial around tubules

Pathogenesis:
 urine contaminated water  mucous membrane / skin abrasion  liver, CNS, kidney, all organs  localized in
kidneys  patient sheds Leptospira in urine
 Virulence factors: motility, hemolysins, adhesions / invasions (bind & localize), hemostasis & coagulation genes
 Immunologic pathogenesis:
o Proinflammatory response to LPS (via TLR2); activates host inflammation
o Multi organ-system failure due to DIC
o Pulmonary hemorrhage (Ig, C’ fixation on host cells; more common with high Ab titers)
o Ocular disease: uveitis during “immune phase” – Ig & C’? uvea = iris, choroid, ciliary body

Dx: can culture during acute phase (1st week); IHC / microscopy / PCR not great.

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 Microagglutination test (MAT): detect Abs as early as 5-7d post-onset;
o single high titer acceptable; seroconversion preffered
Tx: doxycycline, penicillin, cefotaxime.
 Jarisch-Herxheimer reaction (proinflammatory cytokine cascade; like sepsis, should anticipate possibility)

Brucellosis (Brucella spp)


α-2 proteobacteria
Gram (-) coccobacillus; facultative intracellular
 B. melitensis is key organism. Not sure if 1 or 3 species

Epidemiology: worldwide distribution (Eurasia, Africa, S. America most) ;


domestic wild populations coexisting
 Human transmission:
o Oral contamination (domestic farm animals)
o Consumption of unpasteurized dairy products
o Inhalation of aerosolized contaminated animal products

Pathogenesis:
1. Enters via mucosa, endocytosed by phagocytes
2. Survives in acidified phagolysosome; enters into ER (abx resistant), delays apoptosis
3. Regulates TNFα by translocating a protein into cell’s cytoplasm

Granulomas everywhere = characteristic of intracellulars like brucella

Clinical presentations

Classic febrile brucellosis: acute infection


 insidious onset (fever, sweating, malaise, headache, weight loss; aches/pains  frank arthritis)

Relapsing / undulant brucellosis (Malta fever)


 > 2mo after classical febrile brucellosis if un/der treated
 Liver involvement, arthritis, uveitis, orchiepididymitis
 Can be chronic; > 1 yr

Complications:
 Osteoarticular disease
o Peripheral arthritis (acute infection, non-erosive, knees, hips, ankles, wrists)
o Sacroilitis (acute infection)
o Spondylitis (lumbar spine; often irreparable damage)
 Epididymoorchitis (granulomatous inflammation with lots of lymphocytes in epididymis; enlarged testicles)
 Abortion in pregnant females, liver, CNS (meningitis, etc  grave prognosis)
 ENDOCARDITIS: main cause of mortality, usually aortic valve, generally requires surgery
 Relapse: inadequate treatment; usually 1st year

Diagnosis:
 Blood or bone marrow culture: need Biosafety level 3 (easily aerosolized)
 Detect Abs (serum agglutination test: draw pt serum, add antigens for Brucella). Safer, for presumptive Dx

Treatment:
 Doxycycline + rifampin (or streptomycin/netilomycin) for 4-6 weeks

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Bartonella spp
 Small, Gram (-) rods; facultative intracellular bacterium; α-2 proteobacteria, related to Brucella
 Mammalian, arthropod reservoirs (rodents, felids, lice, fleas, ticks)
 Infects erythrocytes & endothelial cells

Bartonella henselae: Cat-scratch disease

Epidemiology:
 ↑risk: kitten exposure, ownership, bites/scratches
 Cats: often seropositive; persistently infected with B.
henselae (“normal blood flora”-ish)
 Fleas: vector between cats, not humans (except maybe
immunocompromised?)

Clinical diagnosis:
 Regional lymphadeopathy with or without fever
 Cat/kitten scratch/bite
 Papule at inoculation site (small, red, elevated lesion)
 Characteristic histopathological features
o Stellate microabscess in granuloma (in lymph nodes)
 Apoptotic cells, macrophages, PMNs, epitheliod histiocytes, etc.
o Clusters of bartonella inside lesions

Pathogenesis:
 Fleas on infected cats defecate; cats clean, flea feces under claws
 Inoculation of bacteria in cat scratch
 Spread: draining lymph nodes  infection
 Occasional: systemic spread but resolves spontaneously in most cases

Clinical manifestations:
 Cat-scratch episcleritis (Parinaud’s oculoglandular syndrome)
o Inoculation in/around eye; drains to post-auricular node
o Neuroretinitis: CNS involved; fluffy infiltrates (bacteria in retina)
 Usually in immunocompromised pts:
o Bacillary angiomatosis / peliosis
 Proliferation of blood vessels & capillaries in skin (angiomatosis) or liver/spleen (peliosis)
 Inflammatory cells in interstitium; bacteria cluster there
o Endocarditis – very important
 Thrombus-like material forms; most of necrotic lesion filled with Bartonella

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Pathology of Mycobacteria Infection
M. leprae: Leprosy
 Caused by M. leprae (acid-fast; obligate aerobe)
 Prefers cooler parts of body (lesions on skin prominences, nose, URT, peripheral nerves, testes)
o Neuropathy secondary damage to fingers, etc – secondary infection; loss of digits.
 2 types: tuberculoid (nothing to do with TB) & lepromatous
LEPROMATOUS TUBERCULOID
 Weak host immune response  Strong host immune response
 Numerous bacilli (MB: multibacillary)  Few bacilli (PB: paucibacillary)
 Sheets of macrophages, not granulomas  Granulomatous reaction
 Non-reactive lepromin (skin) test  Reactive lepromin (skin) test
o Note how immune reaction is responsible for other differences
 Extremely rare in USA; more common in other countries (esp. Africa, Nepal) – usually travelers if in US

M. tuberculosis: TB
 Epidemic waves of morbidity/mortality: sharp rise; peak, gradual decline over 100s of years
 Worldwide: #2 ID killer (HIV, 2.7M; TB: 2.2M; Malaria: 1.1M)
o 2 billion infected with M. tb; 8M new cases/yr, 1 death every 10 seconds; 500k infected with HIV too
o One untreated pt infects 10-15 new pts / yr
 Epidemiology: poverty, overcrowded housing, undernourishment (airborne)
o Slums of US, etc. where poor, elderly together

Mycobacterium tuberculosis:
 Acid-fast bacteria (high lipid content of cell walls; Ziehl-Neelsen stain)
o Red dye, washed away from other cells by acid alcohol, counterstain blue
 Obligate aerobes, hard to detect in tissue, slow to grow in culture
 Can survive intracellularly

Transmission:
 Person-person (small airborne droplet nuclei)
o Have to be small to avoid mucociliary apparatus
o Produced when coughing/sneezing/speaking/singing
o Remain airborne; disperse uniformly throughout enclosed space (can’t use regular surgical mask)
 Expt: guinea pigs in cages on roof infected via aerosolization; distance didn’t matter
 MDR-TB is more easily spread!

Primary TB:
Initial spread & development of Ghon focus / complex
1. Goes to middle, lower lung fields (in periphery) – greatest
volume of air goes there
2. Nuclei implant on respiratory bronchioles/ alveoli (past
mucociliary system, way out into lungs)
3. Initially: non-specific PMN response (usually not observed)
4. Alveolar Mφ engulf mycobacteria  multiply within Mφ
5. Some mycobacteria are killed by Mφ  process/present
antigen to T-helper lymphocytes  release lymphokines to
attract more Mφ & activate  monocytes infiltrate;
activated histiocytes  granuloma forms
o Caseous center, etc. If you hear caseous or AFB,
think TB!
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6. Some Mφ with M. TB transported to regional lymph nodes, then throughout body
o Ghon focus: initial site of implantation
o Ghon complex: Ghon focus + lymph node (classic for primary TB)
Simon foci: some bacteria  lung apacies (higher oxygenation, lower blood flow so lymphostasis)
 Apical scars: common in tuberculin + pts; harbor more bacilli (dormant) than other areas of lung
 1st part driven by anatomy/physiology; this is driven by metabolic character of the organism

Outcomes of this part:


 >90% heal; have positive PPD; viable bacilli remain! (calcified granule in lung can harbor dormant M. TB)
 5% go on to develop progressive primary TB

Progressive Primary TB
 5-10% of patients (especially lowered immunity, kids, elderly) progress to primary progressive TB
 If granulomata erode, can discharge into different spaces
1. Miliary TB: erode into vessel
 Characteristics:
 tons of evenly-sized, tiny foci throughout lung (bacteria well mixed in blood)
 larger foci towards apex (better oxygenation)
 Worst prognosis
 Pulmonary vein: left heart; to rest of body: new lesions in both lungs
 Pulmonary artery: back into that lung; new lesions in one lung
2. TB Bronchopneumonia: erode into airway
 Characteristics: larger pieces of granuloma breaking off  unevenly sized, clustered nodules
3. TB empyema: erode into pleural space
 Characteristics: can see granulomatous change, caseation in former pleural space

Post-primary TB
Infections which develop in individuals with immunity to bacillus.
1. Reactivation of previously healed TB (Simon foci)
a. Begins in posterior segment of upper lobe
b. About 1-2% untreated PPD + pts will reactivate <
5yrs
2. Reinfection of previously infected individual (with
different strain)
a. Not totally prevented; suggests BCG vaccine not
effective

CAVITATION IS HALLMARK OF POST-PRIMARY TB:


 Granulomata eroding, eventually leave cavitation where
they were before (especially near apex)
 Can manifest as TB bronchopneumonia (airway), Miliary
TB (vein), TB empyema (pleura) like before or…
o Massive hemorrhage (erode into artery)  specific for post-primary TB
o Leads to hemoptysis
 Cavity can also heal (risk of aspergilloma – fungal - infection)

Preventing post-primary TB: treat PPD+ even if asymptomatic!


 Cuts risk of developing active disease; can treat while still healthy!

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Non-Tuberculous Mycobacteria
 A.k.a. “atypical” mycobacteria, “anonymous” mycobacteria, “pseudotubercule bacilli”
 Classified into 4 groups, don’t produce disease in guinea pigs
 Acquired from ENVIRONMENT (soil, water) – not person-person like M. tb
 Opportunists: usually infect people with underlying lung dz or decreased immunity

HIV & Mycobacterium tuberculosis


 HIV: depletes CD4+ cells; decreases monocyte/macrophage function
 HIV+ & +PPD: very high active disease rate
 Highest AIDS demographic groups: blacks/Hispanics 22-44 = greatest increase in TB
 Reversal of downward TB trend (1985-6: AIDS)

HIV patients & TB: TB usually diagnosed 6mo before opportunistic infections (more virulent); now an AIDS-defining dz
 More often extrapulmonary TB
 TB in lungs often non-apical, non-cavitary; poorly-developed granuloma (no good immune response)
 Drug-resistance higher; PPDs more often negative
 Complications: crushing spine (Pott’s disease); ocular involvement
 Rest of world: TB is leading cause of death in HIV+ individuals

Worrisome: TB form in Africa resistant all known drugs; usually fatal.

HIV & NTM


 NTM (e.g. mycobacterium avium complex) common in pts with AIDS (need low CD4 count)
 Enter via GI tract; occur late (less virulent), no person-person transmission
 No granulomas, histiocytes packed with bacilli (look pale blue & striated)

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Vector-Borne Zoonoses
Vector: any animal that transmits an agent of human disease or plays an essential role in the agent’s life cycle
 typically refers to arthropod vectors (mosquitos, ticks, fleas, flies, lice, etc.)
 Vector transmission only: Lyme disease, RMSF
 Direct-contact and vector transmission: plague
Yersinia pestis (Plague)
Gram-negative coccobacillus; stains in a bipolar pattern; facultative intracellular bacterium
Ecology: endemic foci maintained; persistant rodent hosts; flea vector active year round
 Epizootic hosts; low resistance to infection (squirrels / chipmunks), high mortality/population density
 Rural rats  fleas  urban rats  fleas  humans(bubonic)  humans(pneumonic)
Potential bioterrorism agent (pneumonic)

Transmission:
 Flea bite: bubonic plague
1. Rat flea bites patient
2. Inoculate flea material into wound  drain to local lymph nodes  bubo (hyperplasia & necrosis of LN)
3. Fever, headache, chills, malaise, painful lymphadenopathy (2-6d later)
4. With Tx: cure in 3-5 days
 Aerosol: pneumonic plague
 Pulmonary infection  rapid spread & septicemia
 Either: septicemic plague
 Invade blood without buboes; high frequency of death
 Endotoxin / cytokine release / Gram (-) sepsis; resp. distress syndrome (ARDS); SIRS
 Complications: endophthalmitis, mmeningitis, tissue abscesses

Epidemiology: endemic in Africa, Asia, N/S America; >2,000 cases / yr in world, high case fatality rate
 Human transmission: depends on environment, host mammals, arthropods

Pathophysiology: rapid spread & growth in tissue & blood


 PMN: engulf & kill Yersinia
 Mφ: just get infected; Yersinia propogates inside, kills Mφ
 Bacteria suppresses leukocyte function
o Yops: suppress inflammation
o LcrV: stimulates TLR-CD14 on MφIL-10(anti-inflammatory )
 LPS activates systemic inflammatory responses, coagulation, fibrinolytic pathways (SEPSIS)
 Pla protease (plasmid)  tissue destruction & C’degradation (required for bubo)

Pathology: Extensive tissue necrosis without extensive inflammation

Diagnosis: clinical suspicion, blood culture, bipolar staining


Treatment: streptomycin, doxycycline, sulfonamides

Borrelia burgdorferi (Lyme Disease)


Borrelia burgdorferi:
 Spiral-shaped (corkscrew / twisting motility); small, obligate parasite found with mammals, birds, ticks
 Transmitted exclusively through tick bites
 Maintained in small mammal via Ixodes species tick cycle (mammal reservoir, tick vector varies with location)
o Tick: larvae  nymph  adult; nymphs do most of the biting (small tick = immature)
o Larvae needs to acquire borrellia from mammal reservoir (last season’s nymphs did the infecting!)
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Tick-bite (ixodes scapularis, “deer tick”) transmitted
 Tick life cycle:
 Tick bite: inserts through epidermis into dermis; both insert/secrete saliva & aspirate tissue (infects & can be
infected); spirochete to tick midgut
o Mild inflammatory lesion where bite occurs

Epidemiology: N. America (northeast, Wisconsin/Michigan area, etc.), Europe, Asia


 Seasonality: bimodal distribution (most May-July: nymphal deer ticks; secondary peak in late fall: adult ticks)
 Incidence increasing

Clinical presentation:
 Early localized infection: erythema migrans (“bull’s eye”)
o EM +/- draining lymphadenopathy; usually 1st week post-tick bite. EM not always present!
o Nonspecific inflammation; perivascular lymphocytes: looks like chronic inflammation
 Early disseminated infection: systemic manifestation (disseminates to lymphatics, blood vessels, capillaries)
o Fever (infrequent in early-localized)
o Multiple erythema migrans
o CN VII palsy, Carditis (arrhythmias), Oligoarticular arthritis, Meningitis
 Late infection: oligoarticular, chronic arthritis, encephalopathy (very rare), chronic pain/memory loss/etc.
 Post-Lyme disease syndrome: no response to antibiotics, not present in vaccine placebo group from trial,
independent of serological results, no resistance to Abx: NO EVIDENCE FOR LONG TERM ABX and this cluster of
symptoms really seems unrelated to Borrelia!

Pathogenesis: inflammatory response to spirochetal lipoproteins in specific anatomical sites


 OpsA (adhesion for borreliae in tick midgut)
 OpsC (transmission from tick salivary gland; infection in mammal)
 Dissemination: spirochetes blind plaminogen; converted to plasmin, helps in interstitial / cell invasion
o Host plasmin + spirochete: better movement through ECM (degrade); activate matrix metalloproteases
to cleave ECM more
 Lots of lipoproteins (interact with TLRs host proinflammatory cytokine & chemokine production)
o Joints: arthritis; heart: carditis; neural structures: neuritis

Diagnosis: Clinical (Hx exposure; endemic region, erythema migrans), culture, SEROLOGY
 Serology: enyzme immunoassay (sensitive, not specific) first, then western blot to confirm
Treatment: amoxicillin, doxycycline, ceftriaxone (CNS infections)
Prevention:
 Vaccine no longer available
 Prophylatic doxycycline for tick bites in highly endemic areas (85-90% effective < 24 hrs)
 Remove attached ticks, avoid areas, etc.

Rickettsia rickettsii (Rocky Mountain Spotted Fever)


Gram (-), Obligate intracellular bacteria (Rickettsia = intracytoplasmic; Erlichia & Anaplasma = intravacuolar)
True bacteria (debated at first: DNA, RNA, ribosomes, binary fission)
Close relative to mitochondria (ATP transporter to steal ATP from cell: opposite of mito, etc.)
Tick-bite transmitted

Epidemiology: SE USA, emerging in Southwest. North, Central, South America


 Case-fatality rate: up to 8% overall, higher in children & elderly.
 Lyme disease: non-fatal; RSMF: can be deadly!
35
Pathogenesis:
 attach & enter endothelial cells  escape from phagosome & inhibit phagolysosome fusion  escape, cell dies
 Causes lymphocytic vasculitis

Clinical presentation: SUDDEN ONSET Rash progression in RSMF
 High fever, severe headache, severe myalgias  Macular: flat, pink-red
 Maculopapular-petechial rash after 3-5d  Maculopapular: raised; blanches
 Normal WBC with left shift; thrombocytopenia  Petichiae: blood extravasated
 Lymphocytic vasculitis  loss of intravascular fluid (won’t blanch)
o Hypotension; shock, end-organ ischemic injury
 GI system, renal system (acute tubular necrosis secondary to hypotension)
o Cerebral edema; meningoencephalitis
 E.g. herniated brainstem through foramen magnum, crush respiratory centers
o Non-cardiogenic pulmonary edema
 Pathology: inflammation, damage to endothelium, damage to dermis, not epidermis (around endothelial cells)

Diagnosis: clinical (h/o tick bite, fever, headache); SEROLOGY


 5x risk death after 5d of illness
 Most not treated until after 5d: “hallmark” rash comes in late!
o Presentation during non-peak tick activity, early presentation (1st 3 days) higher risk for missed Dx
 Culture not advocated: VERY HAZARDOUS (use PCR or skin biopsy with antigen)
o Serology not useful in first 7-14 days

Treatment: doxycycline (or tetracycline; doxy has better outcome than chloramphenicol; tooth-staining uncommon
with these doses in kids)

36
Pathophysiology: ID & Micro
Introduction to Infectious Diseases ........................................................................................................................................ 2
Prokaryotic Structure & Physiology ........................................................................................................................................ 5
Bacterial Toxins ....................................................................................................................................................................... 9
Fever & Sepsis ....................................................................................................................................................................... 12
Diarrheal Disease Caused by Bacteria................................................................................................................................... 15
Staphylococci ........................................................................................................................................................................ 22
Prion Disease......................................................................................................................................................................... 25
Academy Awards of Infectious Diseases .............................................................................................................................. 27
Anaerobic Infections ............................................................................................................................................................. 28
Chlamydia & Mycoplasma .................................................................................................................................................... 30
Non-Tuberculous Mycobacteria (NTM) & Nocardia ............................................................................................................. 32
Antimicrobial Stewardship .................................................................................................................................................... 36
Syphilis (& other STIs) ........................................................................................................................................................... 39

1
Introduction to Infectious Diseases
Infectious Disease: When an interaction with a microbe causes damage to the host, Steps in microbial
resulting in clinical signs and symptoms of disease pathogenesis
1. Contact
Pathogen: Any organism that has the capacity to cause disease 2. Attachment
3. Invasion
Factors that affect pathogenicity: Host, Agent, and Environment 4. Evasion
5. Cell Damage
I. Host: WHO IS THE PATIENT? (must develop a specific plan for that pt) 6. Spread

a. Age: According to P. Murphy, all organ functions decline at about 1% per year.

Immunologic parameters which decline with age Immunologic parameters which increase with age
 Thymus atrophies (by age 40)  Variability of response to a new antigen ↑
 Primary antibody responses ↓  Incidence of monoclonal immunoglobulins ↑
 Skin reactivity ↓  Incidence of auto-Ab against DNA (anti-nuclear),
 T cell proliferation to mitogens ↓ immunoglobulins (rheumatoid factor), and organs
 # naïve T cells ↓ (thyroid) ↑
 T cells make less IL-12 (immunostimulatory)  # memory T cells ↑
 T cells make more IL-10 (immunosuppressive)

b. Nutritional status
 Need mixed protein (40g/day, all 20 amino acids in correct proportions) for protein synthesis
 Can’t store amino acids – unused ones used as energy
 Protein deficiency & immunity: T-cell function ↓ (main problem); PMN production also ↓ but less obvious.
Liver makes less albumin
 Lots of hospital pts. malnourished (esp in ICU, etc.) Can be a big predictor of outcome

c. Host genetics
 E.g. breed susceptible/resistant animals, N. Europe may have been selected for TB, measles, smallpox
resistance, certain human mutations lower resistance to certain organisms, genetic basis for some HIV
resistance in certain patients.

d. Host co-morbidities
 Most clinical infections are situational: reason exists for why pt. became infected
o Chronic medical illnesses (diabetes, etc); immunosuppressive
drugs (steroids, transplants, chemo, rheumatoid arthritis tx); Types of Pathogens:
IV access for many reasons  Prions (single protein molecule,
II. Pathogen PrP)
a. Endogenous vs. Exogenous Flora  Viruses (Nucleic acid –RNA or DNA
 Endogenous flora: organisms which are long-term residents of - & proteins)
body surfaces (“commensals”)  Bacteria (prokaryotes; nucleic acid
o Colonization by endogenous flora happens right after birth - DNA & RNA – not separated from
o Good for nutrient acquisition, differentiation of mucosal sites, rest of cell by membrane)
stimulates immune system, provides accessory growth factors  Fungi (eukaryotes)
o Harder for pathogenic organisms to establish residence on  Parasites (eukaryotes)
body surface or multiply & cause problems
 Exogenous flora: organisms which can’t survive indefinitely in a single individual but depend on transmission
from one person to the next

2
b. Better classification scheme
 Commensals: Very normal to have; rarely cause disease
 Facultative Pathogens: some patients get sick, others don’t (who’s the patient?)
 Obligate pathogens: never should be there; is essentially always causing disease if present

c. Human microbiome: effort to categorize what kinds of flora live where in / on humans

Probability of disease equation:

inoculum size × growth rate × virulence


𝑷𝒅𝒊𝒔𝒆𝒂𝒔𝒆 =
host resistance

Inoculum size:
 If you brush teeth, some bacterial always entering your blood, but you’re ok (small size so neutrophils take care
of it).
 If you have an abscess burst of the same bacteria, you’re in trouble (tons entering blood stream at once)
 About 1011 Gram-negative bacilli (1 mL stool), 1012 Gram-positive bacilli can kill you

Growth rate:
 Doubling time varies among organisms
 Bacteria = minutes (exceptions: mycobacteria, chlamydia, etc. are slower)
 Virus = hours (poliovirus @ 5hrs is fastest)
 Fungi, parasites = slower

Virulence factors: properties that enable a microorganism to establish itself on or within a host & enhance its potential
to cause disease (resist host defenses, multiply from small inoculum to concentration that causes disease)
 Growth @ 37 C, for instance; toxicity, etc).

Host resistance
 Non-immunological: HOST RESISTANCE
o Skin impermeable to most bacteria Immunological
Nonimmunological
o Mucous membranes allow small #s bacteria Innate (“Natural”) Acquired
to pass through (induce immunity)  Skin  Macrophages  B-cells
o Constant flow in body tubes washes out  Mucous membranes  Neutrophils (PMNs)  T-cells
bacteria (saliva, bile, urine)  Constant flow in  NK cells
o Lungs protected by cilia and cough reflex body tubes  Complement
o Breaking these barriers (e.g. central lines, IV  Clilia, cough in lungs  Interferon
drugs, Foley cath) is a great way to cause infection

 Innate (“Natural”)
o Macrophages & Dendritic cells
 Roles: Phagocytosis, antigen presentation, secrete cytokines to drive acquired immune
response differentiation,
 use pattern recognition receptors (CD14, Toll-like Receptors) to recognize molecules from
pathogens (e.g. LPS in Gram (-), peptidoglycan in Gram (+))
o Polymophonuclear cells (PMNs = neutrophils)
 Roles: phagocytosis (opsonic, PAMPs, etc.)
 Microbes can escape phagocytosis:
o have a capsule to protect
o inhibit fusion of phagolysosome
o escape into cytoplasm & replicate there
3
o resist killing (e.g. catalase)
 Reach infection site via chemotaxis (chemicals from initial host-pathogen interaction) and
adhesion interactions (sticking & migration from vessel)

o Complement: series of plasma proteins, cell receptors that mediate inflammation


 Liver produces complement (starvation: protein ↓, complement ↓)
 Most important effects: activated C3 and C5-9
 Pathways:
 Classical pathway (Ag-Ab complexes)
 Alternative pathway (polysaccharides via C3)
 Lectin-binding pathway
 Mechanisms of action:
 opsonization (C3b) – increase attachment & phagocytosis (Mφ & PMN)
 chemotaxis (C5a) - attracts PMNs
 cell destruction (membrane attack complex)

o NK Cells: large, low-density, granular cells without T-cell receptors or surface IgG
 Don’t require activation before function
 Triggered by cells that do not display self class I MHC
 Mostly antiviral activity

o Interferons: cytokine (glycoprotein) cell-signaling molecules produced by


immune system cells in response to challenge (immunuloregulatory Responses to infection
signals) (will discuss in detail later)
 Interferon α – “leukocyte”interferon; from all cells in response to  Fever
viral infection or dsRNA, upregulate MHC class I  Anorexia
 Interferon β – “fibroblast”interferon; from all cells in response to  Lethargy
viral infection or dsRNA, upregulate MHC class I  Myalgia
 Interferon γ - “immune” interferon, from T-cells in response to
antigen/mitogen, upregulate macrophages & MHC classes I & II

 Acquired immunity
o T-cells (cell-mediated immunity)
 Roles: directly kill infected cells, generate DTH responses, promote Ab formation
 TH (CD4+) cells: work with MHC class II molecules
 facilitate immune response by T-cells & B-cells
 secrete products that promote antiphagocytic activity
 TCTL (CD8+) cells: work with MHC class I molecules
 Lyse infected cells
o B-cells (humoral immunity)
 Produce immunoglobulins which recognize unique antigenic structures

NB: Good review slides for the end of the block at the end of this “Healthy people who are well-fed,
lecture: defect in ____, increased susceptibility to_____. reasonably separated from each other, and have
access to pure water
seldom have serious infectious illnesses”
– P. Murphy

4
Prokaryotic Structure & Physiology
Prokaryotes vs. Eukaryotes PROKARYOTE EUKARYOTE
SIZE OF CELL Small Bigger
NUCLEUS No nuclear membrane / nucleoli Nuclear membrane / nucleoli
Important things: differences between MEMBRANE-ENCLOSED Nope Yep
prokaryotes and eukaryotes are good ORGANELLES
targets for antibiotics (cell wall, ribosomes, FLAGELLA Simple (2 building blocks) Complex (microtubules)
etc.) GLYCOCALYX Capsule / slime layer Present in some cells (if no
cell wall)
CELL WALL Usually present; complex When present, simple
Bacterial shape & size PLASMA MEMBRANE No CHO; mostly lacks sterols Sterols & CHO as receptors
CYTOPLASM No cytoskeleton Cytoskeleton
 Cocci (round) RIBOSOMES Smaller (70S) Bigger (80S); smaller (70S) in
 Bacilli (rods) organelles
CHROMOSOME (DNA) Single circular chromosome Multiple linear chromosomes
 Pleomorphic (shape is variable or shape No histones Histones
is “in-between”, e.g. “coccobacilli”) CELL DIVISION Binary fission Mitosis
 Also vibrio (=commas), spirochetes SEXUAL REPRODUCTION Transfer of DNA fragments only Meiosis
(=spirals), etc.
 Size: about 0.2-5 μm

Gram’s Stain
Process:
 Get sample, fix by air drying (don’t heat – could hurt cell wall)
 Crystal violet (all purple) iodine (stabilizes)  alcohol (decolorizes G- ) Safranin (counterstains G- red)
 End result: Gram (+) = purple, Gram (-) = red
 Gram stain adheres to gram+ peptidoglycan layer & resists decolorization

Exception:
 Acid-fast bacteria cannot be Gram-stained
 Resist decolorziation with alchol because of a high lipid concentration in cell walls)
 Mycobacteria, etc: have to use an acid-fast stain (show up red)

Uses:
1. Bacterial identification (especially in low resource areas, or quick ID)
2. Early identification of an appropriate antibiotic (e.g. could start empiric treatment to cover Gram (+) )

Tips:
 Try to minimize epithelial cells (would take up safranin, so would be red)
 Neutrophils usually show up but are much bigger than your bacteria
o Can use neutrophils to judge the decoloration (pale red = too declored, too blue = not declored enough)
o If you leave alcohol on too long, could decolor even Gram (-) bacteria

Structural differences: Gram Positives vs Gram Negatives

Cell membrane in prokaryotes:


 no sterols (unlike eukaryotes)
 Important: active transport, energy generation, cell wall precursor synthesis, secretion of enzymes/toxins
Gram Positive Gram Negative
1. Phospholipid cell membrane, then 1. Inner phospholipid membrane, then a
2. large peptidoglycan layer anchored to cell 2. small peptidoglycan layer in the periplasmic space, covered by
membrane by lipoteichoic acids (LTA) 3. outer phospholipid membrane with lipopolysaccharide (LPS)

5
Important features of bacterial cell walls:

Teichoic Acids: (Gram + only)


 antigenic (glycerol or ribitol phosphate)
 anchor to cytoplasmic membrane or NAM of peptidoglycan
 useful for anchoring cell wall & adherence to cell membrane

Peptidoglycan (Gram + and -, but more & more exposed in Gram +)


 Cross linked chains of monomers (NAG-NAM pentapeptide)
 Transglycosidase enzymes join monomers to make chains
 Transpeptidase enzymes make peptide cross-links between
chains (strength & enable bacterium to resist lysis)
o Penicillin blocks this transpeptidase
o Note the peptide side chains coming out at right angle

Gram + (A) and Gram - (B) Cell Walls

Lipopolysaccharide (LPS): (Gram – only)


 Lipid A: “endotoxin”
o Conserved among Gram (-)
o Highly immunostimulatory
o Causes unforgettable sepsis
o Made of sugars & fatty acids
 Core polysaccharide somewhat conserved among species
 O-specific polysaccharide different even within species

Gram Negative Sepsis


 Gram (-) sepsis is really bad – death not due to organism but rather immune system going crazy
 Tx: give fluid (will accumulate in extremities after leaking out: puffy appearance)
 Short-term Tx: Abx expose lipid A, cause worse short-term consequences
o Why can’t we get mortality in gram (-) sepsis to 0% (around 30% for years)?
o Maybe because Abx precipitate some LPS/Lipid A release & toxicity results
 Lipid A is key player: actions include
o Febrile response
o Activation of complement, granulocytes, macrophages
o ↑ interferon production
o ↑TNF (↓ capillary endothelial cell permeability; development of shock)
o ↑ colony-stimulating-factor production
o B-cell mitogen

Bacterial capsule:
 Gelatinous layer, covers bacterium, found in some spp, usually polysaccharide
o Sugars vary spp to spp; can use to do serological typing
6
o Virulence: prevents phagocytosis
o Swells if homologous Ab around: QUELLUNG REACTION
 Example: think you have strep; mix with an anti-strep-capsule Ab, get Quellung reaction to
confirm that you’re right (organism has that capsule)
o Can be used as antigen with some vaccines

Spore formation:
 Clostridium and Bacillus produce them (e.g. recurrence of C. diff, etc.)
 Response to adverse conditions
 Form inside the cell (DNA, cytoplasm, cell membrane, peptidoglycan, thick keratin-like structure around it)
 No metabolic activity (can be dormant for years)
 When environment more favorable, enzyme degradation of coat, germination into bacterium
 Highly resistant to heat and chemicals

Bacterial division (Binary Fission)


 1 parent cell  2 identical progeny cells
 Exponential growth (2n where n = # generations)
 Doubling time: 20 minutes to days

Bacterial growth cycle


1. Lag phase: get used to environment, get ready to
start dividing (A)
2. Log growth phase (C)
3. Run out of space & food; equal # die & divide (E)
4. No more food, etc. death (F)

NB: Many Abx work best in log growth phase

Aerobic & Anaerobic Growth


Using oxygen generates two toxic molecules: H2O2 and O2•
(superoxide). Bacteria need two enzymes to use oxygen.
1. Superoxide dismutase (2 O2• + 2 H+  H2O2 + O2)
2. Catalase (2 H2O2  2 H2O + O2)

 Obligate aerobes cannot grow without oxygen (ATP-generating system needs oxygen as hydrogen acceptor)
 Faculative anaerobes can use oxygen for respiration if it’s around, but they can also go anaerobic
 Obligate anaerobes lack one or both of these enzymes so they can’t generate ATP via respiratory pathway

Bacterial genetics: bacteria are haploid with a single chromosome (usually circular, ~2000 proteins; can transfer to
other bacteria, e.g. as plasmid); fission results in identical progeny.

Mutations are changes in base sequence of DNA that results in altered phenotype
 Substitutions (missense, nonsense); frame shift, transposons / insertion sequences.
 Can occur randomly & may be caused by chemicals, radiation, viruses – but genetic diversity not generated
during reproduction like in eukaryotes

Transfer of DNA within bacterial cells


 Transposons: DNA from one site on chromosome to another site or to plasmid. Can lead to antimicrobial drug
resistance
 Programmed reagrangements: movement of gene from silent site to site where it’s transcribed & translated.
Can lead to antigenic variation

7
 These are two ways that inducible resistance can arise, or production of a toxin which wasn’t previously
produced, for example.

Transfer of DNA between bacterial cells


 Conjugation: mating of bacterial cells, DNA transferred via sex pilus, etc.
 Transduction: transfer of genetic material via a bacteriophage (bacterial virus)
 Transformation: transfer of DNA (extracellular) itself from one cell to another

Bacteria & Iron


 All organisms need exogenous iron source; free-living bacteria commonly use siderophores to chelate iron from
environment for their use
 Human host: iron not found in unbound form (maybe a way to control bacterial growth?). Instead bound to
proteins (transferring, lactoferrin, ferritin, myoglobin, hemoglobin, etc.)
 How do bacteria get iron?
o Produce special siderophores (compete with iron-binding proteins)
o Direct uptake by stealing from tranferrin / lactoferrin
o Use of heme from breakdown of tissues
 Clinical importance: host sequestration
o chronic infections can lead to anemia
o not iron-deficiency anemia – instead, host tries to hold onto iron more
o iron stores are actually increased in the red cells that are present).

8
Bacterial Toxins
Toxins: molecules produced by microbes that can produce disease
Toxoids: detoxified toxins that retain antigenicity / immunogenicity)  vaccinations (diphtheria, tetanus)

Categorization: chemical composition (protein/lipid/LPS), cell/tissue target (enterotoxin/neurotoxin), mechanism of


action (proteolytic/adenylate cyclase toxin) biologic effect(lethal factor / edema factor), organism (pertussis toxin,
cholera toxin), intracellular target, specificity (broad/targeted)

EXOTOXIN ENDOTOXIN
 molecule (usually protein) produced and released by a  intracellular / cell-associated structural component
microorganism to affect target cells at a distance of Gram (-) bacteria (e.g. LPS)
 Act enzymatically or directly with host cells; stimulate  Most located in cell envelope & act locally
variety of host responses

Exotoxins
Toxin genes:
 chromosomes (cholera),  bacteriophage (diphtheria),
 plasmids (E. coli heat-labile),  combination (Staph enterotoxin)

Control & regulation of exotoxin synthesis & release:


 Diptheria toxin: production↓when iron around; Cholera toxin / virulence factors: environmental osmolarity, temperature
 Toxin often required for virulence & pathogenicity
 Distinct pathways for secretion; details have significant impact on toxin/pathogen virulence
o Direct injection, series of pores, assembly in periplasmic space, etc.

Structure: A-B toxins


 Most exotoxins: A (active, enzymatic  toxin effects) and B (binding) domains
 Need both for effects to be exerted on cells (binding & activity)
 Permutations: A:5B, A-B, A+B, etc.
 Attachment & entry: direct entry (B-subunit  pore formation), receptor-mediated endocytosis, both

Mechanisms of action of A-B exotoxins (organism can have more than 1 toxin too)
1. ADP-ribosylating toxins. Remove ADP ribosyl group from NAD & stick it on host-protein, inactivating or
modifying function
2. Adenylate cyclase toxins. Synthesize cAMP after binding host cell calmodulin
3. RNA glycosidase toxins. Cleave host cell rRNA, stopping protein synthesis  cell death
4. Metalloprotease toxins. Proteolytic  disrupt cell function

Attatchment & entry: different types


 Non-selective (e.g. cholera toxin) so clinical manifestation is from localization of organisms / toxin
 Coupling between toxin-receptor complex; lipid rafts in host cell plasma membrane (anthrax LF/EF)
 Some could act against most cells, but only can bind to certain surface receptors on certain cells (pertussis)
 2 toxins may catalyze the same intracellular reaction (e.g. diphtheria toxin, pseudomonas exotoxin A) but have
different clinical manifestations (different receptors, different cell distributions)

Membrane-damaging toxins: release of host cell nutrients (cell death) & better direct injection of bacterial components
 Pore-forming toxins: trans-membrane pores into phospholipid bilayer; disrupt selective ion movement.

9
o B. anthracis edema factor, S. aureus α-toxin (abscesses), streptolysin O of S. pyogenes (strep throat)
 Cytotoxins: hydrolyze / solubilize phosopholipid bilayer
o (α-toxin of C. perfringens)

Exotoxin examples:

Anthrax: uncommon, risk factors: animals & hides, industrial activities, bioterrorism. Cutaneous/inhalational/GI; any
can be associated with hemorrhagic meningitis. Radiography: widened mediastium (hemorrhagic lymphadenitis).
Substantial edema (around lesions on skin).

Produces three toxin components (need all 3 together)


 A: Edema factor (EF): an adenylate cyclase (↑cAMP in phagocytes, forms ion-permeable pores). Edema,
phagocytosis inhibition, impaired host defenses.
 A: Lethal factor (LF): a protease (cytokine release, cytotoxicity: lysis of Mφ, ↓PMN activity)
 B: Protective antigen. Binds glycoprotein receptor; cleaved by host protease, then binds EF or LF; complex
endocytosed, increased cAMP.

Diptheria: uncommon (universal vaccination with diphtheria toxoid); endemic in Latin America / Carribean, immunity
does not prevent carriage, adult immunity wanes without booster.

Diphtheria toxin: produced as single polypeptide; cleaved by trypsin


 A: catalytic domain
 B: binding domain (receptor for cell attachment)
 T: hydrophobic domain (insertion into endosome membrane, secure A release)
 Process: B binds, RME  endosome; acidification  unfolding of A&B to expose T  T inserts into endosome 
A translocated to cytoplasm  enzymatic activity (ADP-ribosylates)
 Causes systemic toxicity proportional to the burden of pharyngeal exudates
 Most severe manifestations:
o Cardiac toxicity (myocarditis): arrhythmia, can lead to heart failure & hemodynamic collapse
o Neurotoxicity: cranial neuropathies, paralysis of pharyngeal wall / soft palate, delayed peripheral
neuritis

Endotoxins
Endotoxin: LPS, located in outer membrane of Gram (-) bacteria.
 Released from lysed bacteria (host defense and/or Abx)
 Generally acts locally.
 Structure:
o O-antigen: variable among Gram (-) bacilli. Facilitates tissue adherence, carrier for lipid A, antigenic
variation, phagocyte resistance, protection from Ab & C’
o Core (R) polysaccharide: conserved within / distinct between genera
o Lipid A: highly conserved
 Generates febrile response (direct: hypothalamus; induces endogenous pyrogens like IL-1,
prostaglandins)
 Directly activates Mφ, coagulation cascade
 Activates C’: histamine release PMN chemotaxis
 Induces interferon production, TNFα (↑capillary endothelial cell permeability  shock)
 ↑colony stimulating factor production; polyclonal B-cell production, immunoglobulin secretion
 Inject LPS: fever, leukocytosis, disseminated intravascular coagulation (DIC), hypotension, shock, death.

Mechanism of action:

10
1. Lysis  release of LPS  binds to circulating LPS-binding protein; complex then binds to CD14 on Mφ cell
membranes (associates with other proteins)
2. Triggers secretion of pro-inflammatory cytokines (IL-1, IL-6, IL-8, TNFα, PAF) from Mφ.
3. Cytokines bind cytokine receptors on target cells (inflammation, C’ activation, coagulation pathway)
4. Leads to endotoxic shock, multi-organ systemic failure
 High fevers, severe back pain, pain on urination; CVA tenderness (costovertebral angle  kidneys);
PMNs in urine, Gram (-) on gram stain
 Hypotension, tachycardia, dyspnea

Summary: Endotoxin vs. Exotoxin


PROPERTY ENDOTOXIN EXOTOXIN
Chemical nature Lipopolysaccharide Protein
Relationship to cell Part of outer membrane Extracellular, diffusible
Antigenic Yes Yes
Form toxoid No Yes
Potency Relatively low Relatively high
Enzymatic activity No Often
Pyrogenicity Yes Occasionally

Superantigens
Biological activities: overstimulation of immune system, pyrogenicity, shock
Mechanism:
 Superantigen mediates non-specific interaction between class II MHC on APCs and specific Vβ chains of TCR on
T-lymphocytes
 Massive stimulation of T-cells (20% activated  massive cytokine release)
 Toxic shock syndrome: Hypotension, fever, diffuse erythematous rash

Examples: pyrogenic exotoxins of Group A strep and Staph enterotoxins

Scarlet fever:
 Pharyngitis from Group A Streptococcus usually self-limited (2-5d); Tx: prevent complications
 Scarlet fever: pyrogenic exotoxin (Group A strep): complication from pharyngitis
 “Sandpaper rash”: 1-2d after onset; upper chest  rest of body, texture key in Dx
o Rash fades  desquamation
 “Strawberry tongue”: bright red tongue

Toxins as friends:
1. Immunogens in vaccines directed against toxin (toxoid)
2. Direct targeting of cells with receptor can exploit toxin B subunits (e.g. fusion proteins in cancer chemo)
3. Botulinum toxin (Botox ®)
a. Control of disorders with uncontrolled muscle spasms
i. Blepharospasm (uncontrollable blinking)
ii. Torticollis (relentless turning of neck to one side)
b. Chronic anal fissures
c. Temporary reduction of skin wrinkles

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Fever & Sepsis
Fever: “a state of elevated core temperature which is often, but not necessarily, part of the defensive response of a
multicellular organism (host) to the invasion of live or inanimate matter recognized as pathogenic or alien by the host.”
 Drugs, cancers, etc. can also cause fever – not just infection

Variability of temperature:
1. observer (calibration, etc.)
2. anatomic (oral is best – easily accessible & responds promptly to core changes; also rectal & tympanic)
3. physiological: ↓(age, in morning), ↑(ovulation, exercise, at night)
a. No set “normal body temperature”: normally distributed among individuals
b. Varies : 96°F 101.3°F (> 101.3 usually defined as fever)

Thermoregulation:
 Heat derived from internal work (peristalsis, myocardial contraction), biochemical reactions, external work
(exercise, shivering)
 Core heat distributed via circulatory system (e.g. ↑core temp, ↑cutaneous blood flow to dissipate via skin)
o Turn red with fever (dilating vessels)
 Pre-optic area controls body temp
o If over set point: activate heat loss responses (lower body temp)
 Pyrogens affect temperature regulation (drugs too!)
o Endogenous: PGE2 (from COX-2, PGE2 synthase via arachadonic acid pathway)  to preoptic area
o Pyrogenic cytokines: IL-1 (most commonly associated, also TNF, IL-6, IFN)
 Source: Mφ (response to endotoxin, peptidoglycan, fungal cell walls, bacterial toxins, drugs)
 Example: LPS + LPS-binding protein  Mφ CD14 reeptor  cytokines  PGE2 production from
endothelial cells  pre-optic area  increase body temperature (feeds back on cytokine
expression)
o Non-infectious pyrogens: non-infectious febrile disease from pyrogens produced in immune response
 Fever in malignancy: generally infection or body’s attempts to reject tumor
 Certain tumors (e.g. Hodgkin’s disease): malignant cells can produce endogenous pyrogens

FEVER GOOD! FEVER BAD!


 Closely regulated, purposeful response (evolved)  Basal metabolic rate ↑10% for 1°C rise in temp
 Stimulates the immune response  3rd world countries: chronic infection & fever in children;
 1°C rise in temp = 10x the stimulatory effect of IL-1 on T- food supply already marginal, metabolic needs can’t be
cells  more B-cell activity, more Mφ activity, more met, promotes more infection (cycle)
cytotoxic lymphocyte output  Hospitals: gross malnutrition common in chronically
 Most organisms have growth optima ~ 37C (want to infected adults; don’t heal surgical wounds, ↑risk more
make environment inhospitable) infections

Treatment:
 Antipyretics: if you’re going to give, GIVE CONSISTENTLY AND CONTINUOUSLY
o nontoxic, analgesic effects, reduces metabolic demands & fever-induced alterations in mentation
 People feel worse when temperatures are changing (e.g. chills & sweats)
 No definitive studies to show benefits or harmful effects for moderate fever
 Temperature > 106°F: enzymatic processes start to break down (big trouble)

Acute-phase response (APR):


 various physiological reactions mediated by same group of pyrogenic cytokines that activate the thermal
response of fever (mostly IL-6)

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 Stimuli: infections, trauma, cancer, burns, exercise, childbirth
 Body trying to limit actions of what it perceives to be invader:
o IL-6: changes in hepatic protein synthesis
 ↓ albumin
 ↑ fibrinogen, haptoglobins (increased amounts);
 ↑ C reactive protein and serum amyloid A associated protein (not present in normal plasma)
o IL-1: ↓ serum iron/zinc (withhold from bacteria), ↑ PMNs & bands
 C-reactive protein:
o binds phosphocholine on microorganism, damaged host cells;
o activates C’ & ↑phagocyte adherence (better clearance)
o Good marker for following Tx of chronic infection (e.g. osteomyelitis). (Can also look at anemia of
chronic disease: Iron increased)
 Serum amyloid A: ↑adhesiveness, chemotaxis of phagocytic cells & lymphocytes

SEPSIS
Definitions:
 SIRS: systemic inflammatory response syndrome:
o “abnormal, generalized inflammatory reaction in organs remote from the initial insult
o (fever, tachycardia, leukocytosis / PMNs)
o Could also be from drug rxn, tumor, etc.
 Sepsis: when SIRS occurs in pt with suspected or proven infection, then SIRS = sepsis
 Severe sepsis: sepsis + hypotension
 Septic shock: severe sepsis + organ dysfunction that cannot be reversed by fluids (usually liver, kidney)

Sepsis: (85% SIRS have bacterial causes = sepsis): Caused by host reaction to agents (all good things but too magnified)

Syndrome of injury to small vessels


1. Mφ secrete cytokines (TNF, IL-1,etc.) producing inflammation in capillaries all over body
a. Endothelial cells lose contact with each other; express proteins to bind PMNs
b. Secretion of:
i. Prostaglandins (vasodilation, low BP, vascular permeability ↑)
ii. Leukotrines (permeability ↑)
iii. Thromboxanes
iv. Platelet activating factor (↓BP, ↑aggregation)
v. Nitric oxide (vasodilation) Clinical Features of Sepsis
2. Vessels: dilated & leaky (obstruction of vessels / plugs of platelets &  High cardiac output
PMN)  Low systemic vascular resistance
a. Lightly bound PMN: emigrate into tissue, cause damage there  Tachycardia
b. Tightly bound PMN: degranulate directly onto vessel walls  A-V shunting with poor tissue
c. Vessel walls destroyed: lipid peroxidation, proteolysis, extraction of O2
induction of apoptosis  Acidosis / elevated blood lactate
d. ↑ thrombin production, ↑ clotting  Increased gut permeability
3. Blood shunted around tissues, fluid & protein lost from blood, tissues
waterlogged, tissue ischemia DDx: conditions where BP decreases
4. Organ dysfunction:  MI, bleeding: SVR↑, CO↓
a. Lungs fill with fluid, oxygen uptake fails (further ischemia)  Sepsis: SVR↓, CO↑ (HR↑)
b. Heart: acidotic, ischemic: fails
Treatment:
Clinical problems:  support vital functions (FLUIDS) &
 Antibiotics can control infection oxygenation if needed
 give ANTIBIOTICS,
 hope for the best
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 Fluid infusions restore volume & BP, but fluid doesn’t stay intravascular (tissue edema gets worse)
 Ventilator support: restores oxygenation temporarily but can damage lungs (O2 toxicity, high pressures)
 Often survive a few days, then die of progressive multi-organ failure (MOF)

Role of activated Protein C (not CRP)


 Activated by thrombin bound to thrombomodulin
 Normally inhibits clotting cascade (↓thrombosis, ↑fibinolysis)
 Body runs out of protein C during sepsis
 Protein C levels predict mortality
 Replete protein C as Tx (inhibit thrombosis, promote fibrinolysis): cut mortality from 30% to 25%
o (other interventions post-antibiotics didn’t affect long-term mortality)

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Diarrheal Disease Caused by Bacteria
Diarrhea: #2 cause child death worldwide (#1 = acute respiratory disease)
 5 million /yr in 1980  1.5-1/7 million today because of ORS)
 Most cases: 1st 2 years of life; mortality highest in < 1yo.

Frequency of bowel movements: most 1-2 per day (varies in general population, not impossible for 3/day to be normal)

Diarrhea defined by frequency, consistency, volume, change from normal habits


 Acute: Secretory (mostly viral & bacterial) or invasive (mostly bacterial)
 Chronic (2-3wks+): mostly parasitic, less commonly bacterial; can be non-infectious (eg. inflammatory bowel dz)
 Traveler’s diarrhea: mostly bacterial, reflecting organisms that cause diarrhea in local children

Diagnosis: clinical symptoms, microscope exam, culturing stool (primary), ELISA, PCR.
 Looking at Ab can be used only retrospectively (takes too long)

Normal GI function: 2L fluid in, 200mL out in stool (98% absorbed, most in small bowel).
 Villus cells absorb, crypt cells secrete.
 Continuous removal of intestinal epithelial cells (shed in stool ~2-3d)
o Divide @ crypt, travel up & sloughed at top. Normal: # cells entering villus = # cells dying
 Normal microbial flora: few in small intestine; abundant in large bowel (also in mouth)
o Mostly anaerobes (99%); facultative anaerobes (e.g. E. coli) ~1%
o Provide protection against enteric pathogen colonization (Salmonella, C. difficile)
o Non-immunologic control: gastric acid, normal peristalsis, bile
o Immunologic control: sIgA from mucosal immune system, cell-mediated immunity in gut
 FYI: lactating mammary gland makes these Ab too; important for newborns

Pathophys: Mechanisms for Diarrhea


1. Decreased absorption
a. inhibited/defective absorption from villous cells Oral vaccines for diarrheal dz prevention
b. luminal presence of osmotically active agents  Typhoid
c. decreased contact time (rapid transit time)  Cholera
2. Increased secretion  ETEC, Shigella, Campylobacter in
a. Stimulated anion secretion from crypt cells development

Bacterial agents of acute diarrhea: mostly Gram (-) rods


 E. coli: Enterotoxigenic (ETEC), Enteropathogenic (EPEC),
Enterohemorrhagic (EHEC)
 Vibrio: V. cholerae O1 & O139, V. parahemolyticus, V. vulnificus
 Shigella: S. dysenteriae, S. flexneri, S. sonnei, S. boydii
 Salmonella: Salmonella (non-typhoid), S. typhi
 Campylobacter jejuni
 Yersinia entercolitica
 Anaerobes: C. difficile, C. perfringens, B. fragilis (enterotoxigenic
strains)
 Others: aeromonas, plesiomonas, L. monocytogenes, more

Fecal-oral transmission
 Developing countries: contaminated water supply, inadequate latrines, poor sanitation
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 Developed countries: contamination of processed foods
Virulence factors
 Inoculum size: variable (Shigella, EHEC: 10-100 organisms for; cholera, ETEC: 105-8)
o Person-person requires small inoculum size; food/water requires large inoculum size
 Enterotoxigenic vs Invasive
o Enterotoxigenic: V. cholerae, ETEC. Attach to mucosal
cells in intestine by pili, other mechanism; sit on border
& secrete toxins
o Enteropathogenic: attatch & efface microvilli; cause
some damage to the border (EPEC, EHEC)
o Invasive: Shigella, Campylobacter, Salmonella, Yersinia,
Listeria. Enter cells & replicate there

Enterotoxins:
 Cholera toxin (CT); E. coli heat-labile toxin (LT): large MW toxins,
5B:1A, arranged like donut
 Heat stable (ST) E.coli toxin is Enterotoxigenic Invasive
smaller (E. coli, Cholera) (Campylobacter, Shigella)
 Shiga and Shiga-like toxins (S. Diarrhea Severe Moderate
dysenteriae, EHEC) also large MW , Major site of disease Small bowel Colon
different action Major defect Increased secretion Decreased absorption,
 Bacteriophage control (horizontal Increased secretion
gene transfer): CT & Shiga-like Character of fecal loss Isotonic electrolyte Same + mucus, ±blood,
 Plasmid control: LT & ST E. coli solution ± pus
toxins Primary Rx Fluid-electrolyte Fluid-electrolyte + Abx

Incubation period generally 1-3 days, rarely 4-5 days; very rare 30d+

Secretory Diarrhea: Vibrios & ETEC


Both Gram (-) rods

V. cholerae:
 O1, O139 only serogroups that cause epidemic cholera
 Most severe of all diarrheas (60-70% mortality untreated; 12-24h  death, > 1L/hr)
 Result of the cholera toxin
 Clinical course: exposed, 1d incubation, vomiting at first / “rice water” diarrhea, low BP, tachycardic
 Findings: “washerwoman’s hands”, skin turgor, sunken eyes (severe dehydration), no real histological changes
 May see metabolic acidosis (losing bicarbonate): huffing & puffing
 Potassium lost may lead to hypotension & even renal failure
 Stool: NO RBC, NO PROTEIN (similar to serum: isoelectric!)

ETEC: most common bacterial cause of diarrhea in developing world; most frequent cause of traveler’s diarrhea. Very
similar to V. cholerae.

Both V. cholerae & ETEC: similar mechanisms of pathogenesis (CT & ETEC LT are very similar)
 Large inoculum size (105-8 organisms); achlorhydria can predispose
 Colonize small bowel via fimbriae attaching to mucosal receptors
 Produce enterotoxins while sitting on surface
o B subunits of CT/LT attach to GM1 ganglioside receptors; ST attaches to different one)
o A subunits of CT/LT activate adenylate cyclase: ↑cAMP
 ↑ Cl secretion from crypt cells
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 ↓ NaCl absorption from villus cells
o ST: activates guanylate cyclase; ↑cGMP, same changes
o Coupled Na/glucose absorption NOT affected: important for ORS
Lab diagnosis:
Treatment: FLUID REPLACEMENT (IV or oral)
 If cholera, antibiotics help decrease stool output & shorten disease. Tetracycline, Cholera: use special
erythromycin, cipro. media to culture
 Oral rehydration therapy (ORS): Na, Cl, K, Citrate + carbohydrate. Does not (TCBS agar)
decrease severity/duration of diarrhea. Uses coupled Na/glucose transport to
efficiently absorb sodium. Universal therapy for all dehydrating diarrhea. ETEC: need
o Keep osmolarity lower than normal serum (don’t want to suck fluid out) molecular methods
o Use citrate because it’s more stable than bicarbonate (no easy culture)
 With good treatment: mortality <1%

Vibrio parahaemolyticus
Gram (-) rod, halophilic (thrives on high salt); normal inhabitant of costal waters
 Common cause of diarrheal disease after eating undercooked shellfish (especially oysters)
 Major cause of diarrheal illness in Asia (esp. Japan)  eating raw fish
 Mechanism of action not well understood (heat-stable toxin?)
 One serotype “pandemic”: O5:K6 (Asia Europe, US)

Clinical presentation:
 Seasonal: summer months predominate (warmer water)
 24h incubation, mild diarrhea (±nausea, vomiting, low-grade fever)

Diagnosis: difficult, need special media (TCBS agar)


Treatment: Abx usually NOT required (self-limiting; 2-3d)

Vibrio vulnificus
Gram(-) rod, lives in sea water (mostly in summer along Gulf Coast)
 Blood stream infections (ingestion in seafood like raw oysters, wounds after exposure to salt water)
 Pts with liver disease or severe immunocompromise almost exclusively affected (very rare in normal pts)
 Sepsis results

Treatment: infections very difficult to treat; mortality very high in spite of Abx & surgery

Invasive diarrheas: Shigella & more


Shigella (prototype of invasive diarrhea): Gram (-), non-spore-forming rod
 Low inoculum size (101-2): person-person spread is usual The 4 Shigellas
 Pass through small bowel  invade large bowel; move laterally causing  S. dystereria (Shiga bacillis) =
cell death. most severe
o inflammatory changes of mucosa (huge changes on histology  S. flexneri: most prevalent in
o diarrhea with blood & pus developing countries
o fever & abdominal pain  S. sonnei: most prevalent in
 Dx: stool culture (non-lactose fermenting, non-pigmented colonies) developed countries &
 Tx: antimicrobials. Increasing resistance in developing countries travelers to developing
o Cipro when resistance is high countries
o Loss of fluid isn’t huge, but ORS can be used when necessary  S. boydii: relatively uncomon

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o Nutritional therapy can be important (not absorbing nutrients from damaged bowel)

Campylobacter jejuni
Gram (-), slightly curved rod
 Common in small children of developing world, young adults of developed world
 Present in >50% supermarket chicken packages (prevalent in poultry and other mammals too)
o Transmission: food preparation; on outside of meat (easily cooked away)
 Mechanism for diarrhea production not known
 Requires 42C incubation, microaerophilic conditions to grow

Clinical presentation: invasive diarrhea, much like Shigella


 Stool: pus, RBC
 Fever, abdominal pain; can be Asx in yong children in tropics
 Association with Guillain-Barré syndrome (autoimmune, ascending paralysis): serotype O19 may have
immunologic cross-rxns with GM1 ganglioside in nerve tissue

Treatment: antimicrobial therapy useful (azithromycin is drug of choice; erythromycin, fluroquinolones too).
 Often cipro-resistant
 Dx can take several days, pt may recover by then.
 Oral therapy not too helpful

Enterohemorrhagic E. coli (EHEC)


Gram (-) rod
 Most common E. coli in USA
 Can cause bloody diarrhea with possible life-threatening sequelae: hemolytic uremic syndrome (HUS)

Source: food industry failures. Healthy young cows are reservoir; contamination from stool
 Low inoculum size so big mixtures of hamburger meat can still be infectious
 Irradiation can control but not widely used

Pathogenesis: colonize large bowel mucosa; attach & efface microvilli of mucosal cells
 Produce two shiga-like enterotoxins (SLT-1, SLT-2) controlled by bacteriophages
o 5B:1A toxins; A subunit inhibits 60S ribosomal subunit (kills cells)

Clinical presentation: 1-2d incubation  hemorrhagic colitis (blood in stool); HUS can develop in 10% pts after 6 days
(diarrhea has already disappeared)
 HUS: hemolysis, renal failure, thrombocytopenia. 3-5% HUS pts die, 3-5% get chronic renal dz

Dx: stool cultures on special media (Sorbitol MacConkey agar); otherwise look like normal E. coli
 ID toxin in stool; look for serological responses to toxin & O antigens too

Tx: supportive
 ANTIMICROBIALS ARE CONTRAINDICATED (can increase the production/release of the toxins!) 

Prevention:
 Avoid undercooked hamburger, unpasteurized milk / apple cider (steaks=OK because not a big mixture).
 Wash hands after petting zoos
 Food industry: improve methods, culture all hamburger prior to shipping, irradiate to sterilize meat

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Enteropathogenic E. coli (EPEC)
Gram (-) rod
 Limited to children < 2yo; previously big cause of nursery diarrhea
 Mild illness; can use ORS as tx (no evidence for Abx)
 Not as frequent as ETEC; particularly common in Brazil

Pathophys: attach to small intestine (attach & efface microvilli), do not produce enterotoxins
 doesn’t cause too much damage
Dx: serotype E. coli in stool

Non-typhoid Salmonella
Gram (-), non-lactose-fermenting rods Non-typhoid Salmonella:
 Fecal-oral transmission; generally through prepared foods in developed world  S. typhimurium
 Chickens thought to be primary reservoirs (eating contaminated chicken or  S. enteritidis
infected eggs via shell contamination or transovarial passage)
 S. Heidelberg
o Also: amphibians, reptiles (turtles!)
 S. Newport
 One of the most common causes of large food-borne illnesses in USA

Clinical presentation:
 Inflammatory changes in small bowel
 Generally mild disease except in children, elderly, immunocompromised (sepsis, mortality↑)
 Strains can differ in virulence (some produce enterotoxins)

Dx: stool culture, blood culture if bacteremia suspected


Tx: Abx optional for mild cases, essential in severe cases. Fluoroquinolones/ceftriaxone/cipro

Typhoid Fever (S. enterica, serotype typhi)


Gram (-), non-lactose fermenting rod, single clone worldwide
 Only infect humans (chronic carriers important: food handlers, etc.)
o Chronic carriers: ID via stool cultures, Ab to Vi antigen
 Can treat with prolonged Abx, may require removal of gall bladder (previously more common)

Typhoid fever: a.k.a. enteric fever, also caused by Salmonella paratyphi A & B
 Primarily in the developing world, rare in developed world except for travelers
 Found in all age groups, normally mild in young children

Fecal-oral transmission (from chronic carriers); carried primarily in gall bladder


 Requires high inoculum size (104-7)

Pathogenesis: Incubation: 7-14 days


 Invade small intestine through M-cells (in Peyer’s patches, take up antigens & present to T-cells)
 Transported to lymphatics  blood stream  all parts of body
 Persist in mononuclear cells

Clinical presentation: prolonged fever, constipation, hepatomegally, occasionally rose spots on abdomen/chest
 Mortality 10-20% w/o tx; 1% with good tx
 Complications: intestinal hemorrhage, intestinal perforation, cholecystitis (can affect any organ system)

Dx: cultures: blood, bone marrow, stool, duodenal fluid


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 (string test: swallow string to obtain sample of upper small intestine)
 Some serological tests but not diagnostic acutely

Tx:
 Antimicrobials (fluoroquinolones, AZI, ceftriaxone = 1st line; chloramphical, ampicillin, TMP+SMX = 2nd line if
susceptible). Increasing resistance.
 Vaccines: two effective ones now
o Live vaccine: oral, 70% protection for 5 yrs
o Injectible vaccine: 70% protection, 2 years, not for kids

Clostridium difficile
Gram (+), spore-forming, anaerobic rod
 Part of normal flora of large bowel (3% normal stools, 25% hosp pts); cause disease infrequently
 Seen after use of broad-spectrum Abx (grow to high concentrations)
 Produce toxins leading to illness
o TxA, TxB – mechanism not known

Risk factors: use of broad-spectrum Abx, intestinal surgery, age

Clinical presentation:
 Diarrhea, colitis (fever, pain, cramping), ↑ WBC, hypoalbuminemia
 Advanced form: pseudomembranous enterocolitis
 Mostly hospitals & medical institutions; responsible for ~25% antibiotic-related diarrheal illness

Dx: toxins A/A+B in stool (enzyme immunoassay or tissue culture), also endoscopy for pseudomembranes
 Stool culture not useful (high frequency of normal colonization)

Tx: Stopping/changing antimicrobial therapy, metronidazole (250 mg po qid x 10d) or vancomycin (po) if failure
 Relapses are common (spore-forming) – can treat with same drugs, longer duration
 Also: cholestyramine, probiotics

Prevention: infection control procedures; controlling use of antimicrobials


 Doxycycline, macrolides, aminoglycosides, fuloroquinolones less likely to result in C.diff overgrowth
 Isolation, use disposable materials, careful hand washing
 Bacterial spores survive on inanimate objects (wash & decontaminate hospital rooms)

Listeria monocytogenes
Small, Gram (+), intracellular rods
 Can be part of normal bowel flora in adults
 Soil / animals
Transmission: contaminated foods (cheese, pork, milk)  can be outbreaks (esp. soft-cheese related)

Epidemiology:
 Long incubation period (~30d)
 Can be asymptomatic
 Can cause disseminated infections (e.g. acute meningitis) especially in immunocompromised
 Pregnant women: especially susceptible (spontaneous abortion)

Diagnosis: culture of normally sterile body fluids (CSF, blood). Difficult to culture from stool; not diagnostic if found
 Serology can be useful (outbreaks)

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Treatment: ampicillin +/- gentamicin (gentamicin accumulates in Mφ, Listeria killed when they escape cytoplasm)
Prevention: avoid contaminated foods (soft cheese, pork products, esp. for immunocompromised / pregnant)
 TMP+SMX prophylaxis for AIDs patients

Yersinia enterocolitica
Gram (-) coccobacillus; intracellular

Reservoir: environment, animals (especially pigs)


 Grows at refrigerator temperature (fairly unique for food-borne); found in most colder temperate areas
(Canada, not tropics)
Transmission: ingesting contaminated foods (raw milk, contaminated water, pig tongues)

Clinical presentation:
 self-limited, mild gastroenteritis;
 can provoke mesenteric adenitis which can present as “pseudo-appendicitis”

Dx: culture of stool, blood


Tx: not needed except in severe cases (gentamicin, fluoroquinolones used)
 Avoid unnecessary surgery
Prevention: avoid undercooked meat (e.g. pigs) & unpasteurized milk

Helicobacter pylori
Small microaerophilic Gram (-) rods; colonize stomach
 Don’t cause diarrhea (dyspepsia)
 Can be Asx for life, very common in developing countries
 Major cause of peptic ulcer disease; major contributor to gastric cancer

Dx: serology or hydrogen breath test


Tx: combination of 2+ antibiotics and proton pump inhibitor to reduce gastric acid; reinfection is common

Organisms not covered in this lecture


 Clostridium perfringens: anaerobic Gram (+) spore-forming rod; normal inhabitants of GI tract; produce lots of
toxins, responsible for multiple diarrheal diseases (enteritis necroticans) & “Pig Bel”
 Bacteroides fragiles: anaerobic Gram(-) rod; produces enterotoxin; responsible for mild diarrhea (developed &
developing countries)
 Enteroadherent E. coli (EAEC): diarrheal dz in children (developing & developed countries); relatively common
cause of traveler’s diarrhea
 Enteroinvasive E. coli (EIEC): dz resembles shigellosis; closely related to Shigella spp.

Summary of treatment:
1. Rehydration (IV/oral) most important in secretory diarrheas, where stool output is great
2. Antimicrobials critical for tx of shigellosis & invasive diarrheas but should be withheld if EHEC is suspected
3. Nutritional therapy important in children in developing countries (Zn therapy if zinc deficiency common)

Control by vaccines:
1. Typhoid: two vaccines on market
2. V. cholerae & ETEC: live, killed oral vaccines being tested
3. Shigella/Campylobacter: live, killed oral vaccines being tested in military
4. EHEC, Helicobacter: vaccines being developed

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Staphylococci
General characteristics:
 Staphyle = “a bunch of grapes”, kokkus = “berry” Medically important Staphylococci
 Gram (+) cocci 1. Staph. aureus (most virulent)
 Divide randomly in 3 planes; daughter cells don’t  Most important
separate completely: grape-like clusters  Healthy & hospitalized (“staph infections”)
2. Staph. epidermidis (less virulent)
Distinguishing staph:  Opportunist, oncology pts, implanted
 Blood agar: medical equipment (biofilm), neonates
o S. epidermidis: white colonies, no hemolysis 3. Staph. saphrophyticus (less virulent)
o S. aureus: golden yellow colonies, beta-  UTI in young women
hemolysis
 Coagulase test (rabbit plasma + staph: does it coagulate?)
o S. aureus: coagulase positive
o All other staph: coagulase negative

Virulence factors:
 Polysaccharide capsule (some species)
o Most S. aureus infections caused by 2 types (5+8)
 Peptidoglycan: minor differences between Staph species (major scaffold anchoring surface adhesions)
 Teichoic acids: water soluble polymers, linked covalently to peptidoglycan backbone, site of attachment of cell-
wall active enzymes / proteins (not virulence factor; may be important in adhesion to nasal epithelium)
 NO LPS (Gram positive!)

Coagulase negative Staphylococci


Examples: S. epidermidis, et al.
 No virulent exotoxins
 Form biofilms (secreted polymeric carbohydrate gel; surrounds micro-colonies of bacteria)
o Acts like capsule (keeps out Ab, C’, PMN, Abx)
o Adheres to plastic (catheters, pacemakers!) Rule of thumb: less virulent =
o Hard to eradicate more abx resistant

Pathogenesis: e.g. attach during insertion and/or migrate down cath (sterile technique important!)
 Virtually non-pathogenic in normal people
 Resistant to many antibiotics

Clinical presentation: variety of clinical disease; usually indolent, rarely fulminant or life-threatening
 Risk factors: hardware/foreign material (IV cath, dialysis access, implants/implanted devices)
 Pre-term infants at risk (fragile skin, low integrity)
 Can lead to:
o neonatal septicemia (NEC = neonatal necrotizing enterocolitis, bowel wall injury / necrotizing infection)
o endocarditis

S. aureus
Extremely common in N. America
 Can acquire/integrate accessory genetic elements (↑pathogenicity)
 Evolves to elude antimicrobials

Epidemiology: pretty much everyone infected between birth & death


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 NOSE, skin/appendages (hair follicles, sweat glands), GI tract, vagina common colonization sites
 Minor skin infections: pretty much everybody (intact immunity prevents spread)
 Major disease: immunosuppressed; normal people after initial insult (wound/injury, IV drugs, influenza)

Virulence factors most associated with S. aureus:


 Surface proteins
o Protein A: binds Fc of Ab; exposes Fab to phagocytes (don’t recognize; so antiphagocytic)
o Coagulase: binds prothrombin; forms staphylothrombin, which catelyzes fibrinogen  fibrin
o A & B clumping factors: bind fibrinogen, with coagulase lead to localized clotting (other spp mostly)
 Why promote clotting? Wall off from immune system & response; leads to ischemic injury
 Toxins
o Exoenzymes (proteases, lipases, allow spread through tissues)
o Hemolysins (4 types; lyse RBC & other eukaryotic cells)
o Leucocidins: e.g. Panton-Valentine Leukoidin, pore-forming hemolysin; results in degranulation/lysis of
PMNs, associated with aggressive infections (furunculosis, necrotizing pneumonia)
o Superantigens: stimulate T-cells nonspecifically (cytokine release, clinical shock)
 Enterotoxins (food poisoning), exfolatins (scalded skin syndrome), TSST-1 (toxic-shock-syndrome
toxin)

Transmission: person-person (direct or indirect: intermediary


person’s hands)
 Hospitals: patient-patient or via healthcare worker’s
hands
 Community-based: household contact
 Environmental surfaces commonly contaminated (role in
MRSA spread controversial)

Immunity: conditions with increased susceptibility


 PMNS are primary defense against S. aureus
 Deficient neutrophil number
o Congenital: congenital neutropenia; bone marrow aplasia/failure
o Acquired: leukemia, chemotherapy
 Defective neutrophil function
o Chronic granulomatous disease (CGD): defect in oxidative burst, still opsonize/internalize
 Path: see PMNs packed with bacteria (chronic infection; can’t kill)
o Leukocyte adhesion deficiency (LAD): make normal PMNs but can’t extravasate
 Path: see tons of bacteria, no PMNs in infected area
o Chediak Higashi syndrome (# & function), Hyper immunoglobin E syndrome (Job’s syndrome)

Clinical presentation: TONS of clinical presentations (see below) Toxic shock syndrome
1. Impetigo (infected eczema, etc.)  E.g. following introduction of high-
2. Cellulitis (deeper than impetigo) absorbency tampons (overgrowth of
3. Cutaneous abscesses (MRSA most common cause of staph)
skin/soft tissue infection in US EDs)  Fever, profound hypotension,
o Boils (= furuncle; skin infection involving entire hair erythematous rash
follicle & nearby skin tissue)  Vomiting/diarrhea common
o Carbuncles (involves group of hair follicles)  Multisystem organ dysfunction
4. Wound infections  Usually no bacteremia
5. Deep abscesses  TSST-1 and other superantigen toxins to
o Abdominal, neck/sinus blame (Ab against TSST-1 = protective)
o E.g. pyomyositis: bacterial infection of skeletal
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muscle leading to pus-filled abcess
6. Osteomyelitis (bone infection)
7. Septic arthritis
8. Septicemia
9. Endocarditis
10. Toxin-mediated
o Toxic-shock syndrome
o Scalded skin syndrome (esp. children & neonates)
 Basement membrane destroyed, skin sloughing off, follows cellulitis
o Food poisoning
o Necrotizing fasciitis
NOTE: S. aureus usually causes non-severe infection (celulitis, boils, etc.) but fulminant infections make the headlines

Antibiotic resistance:
 Abx pressure  acquisition / transfer of resistance genes (between and within species)
 Penicillinase: plasmid-encoded; opens beta-lactam penicillin ring, no cephalosporin action
o Inhibited by most beta-lactamase inhibitors
o Present in 95%+ of S.aureus isolates: never choose PCN for empiric S.aureus treatment!

 MRSA: Methacillin-resistant S. aureus


o Chromosomal (mecA): on a mobile genetic element (SCCmec, staphylococcal cassette chromosome)
o Results in resistance to all beta-lactam antibiotics (includes cephalosporins)
o 2 types of MRSA: different SCCmec types, different abx resistance genes/toxins

 Hospital-acquired MRSA (HA-MRSA)- “USA 100-200”


 Hosp. pts, often very old/young, major illness, surgical incisions, IV cath, trach tubes,
dialysis, etc.; more likely to have implanted foreign bodies
 Handled frequently & all over by other people
 2008: 60% HA-S. aureus = MRSA!
 Multidrug resistant (type I,II,III SCCmec); no PVL

 Community-acquired MRSA (CA-MRSA) - “USA 300-400”


 Up to 75% SSTI now CA-MRSA in most areas of country
 Now also causing hospital outbreaks/infections
 Nosocomialcommunitynosocomial
 Not multidrug resistant (type IV SCCmec); produces PVL (cytolytic toxin)
 CA-MRSA (PVL-producing) clinical manifestations
o Skin infections (Children, athletes, prisons, previously healthy persons)
o CA-pneumonia (esp. after influenza; necrotizing; associated bone infection,
thrombi; unusually severe/high mortality, previously healthy persons!)

 Vancomycin-resistant S. aureus
o Has been mainstay of treatment for serious MRSA
o MIC levels for vancomycin keep rising (some isolates have popped up with high MICs); now MIC of 4 is
no longer “susceptible” (4-8mcg/mL from “susceptible” to “intermediate”)
o Limited right now but will likely change
o Worry: Staph are good at horizontal gene transfer – could pick up VanA from VRE?

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Prion Disease
Prion: a proteinaceous infectious particle which is resistant to inactivation by most procedures that modify nucleic acids

Transmissible spongiform encephalopathies: Prion Diseases


 Animals:
1. Scrapie (sheep)
2. Bovine spongiform encephalopathy (BSE)
3. Wasting disease of deer & elk
 Humans:
1. Kuru (no longer around)
2. Creutzfeldt-Jakob disease (most common TSE in USA)
a. Incidence: 1 per million per year
b. Incubation: up to 50 years
c. Mortality: 100%
3. Variant CJD (what people mean when they say a human has “mad cow disease”)

Scrapie: ataxic sheep; wasting, cerebellar dysfunction, commonest fatal genetic disease of sheep
 Studied agent:
o Resistant to things that inactivate nucleic acids: formaldehyde, ethanol, proteases, nucleases, UV/ionizing radiation, etc.
o Inactivated by things that inactivate proteins: autoclaving, pH extremes, inorganic salts, detergents
o Isolated the protein: had same sequence as naturally occurring proteins
 PrPC is normal form (α-helical, monomeric, on cell surface, soluble)
 PrPSC is changed form (β-sheets, big aggregates, insoluble amyloid fibrils/rods, found in
intracellular vesicles / extracellular space)
 Misfolded: nobody knows how it starts

Kuru: Fore people group in Papua New Guinea; very isolated.


 Big epidemic of Kuru (= “trembling”) especially among women and children (7 of 8)
o Cerebellar dysfunction, then rapid decline
 Autopsy: no inflammation, no characteristic cellular changes, just loss of Purkinje cells
 Similarities to scrapie identified; inoculated primates with Kuru pt. brain, 2-4 yrs later had kuru (1st transmission
of human infection to subhuman primates, nobel prize, etc.)
 Ritual cannibalism was the cause: started in 1900s, suppressed in 1960s by Australians
o Men would eat more choice cuts
o Women & children would eat brains & other parts
o 50+ year incubation: cases from 50s  today (last few)

CJD
 Transmitted, rare (1:1,000,000/yr), no regional pattern, varied presentation
 Vacuolization in neurons; like pathology of scrapie & kuru
 Presentation: myoclonic jerking  dementia  death
o No recovery, rapid progressive decline (~5 months on average, no good days), 90% dead < 1yr
NATURAL SPREAD OF CJD
LACK OF COMMUNICABILITY EVIDENCE OF TRANSMISSIBILITY
 Absence of temporal/geographical  Transmission to experimental host (brain, viscera, CSF inoculation)
clustering  Transmission by physician (corneal transplantations, cerebral
 Lack of conjugal cases corticography, dural grafts)
 Lack of predominance by occupation  Transmission to children (growth hormone injection from human
pituitary glands)

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 Growth hormone: 1985: from human pituitary, combined many sources, some children infected. Resulted in
development of recombinant GH
 Lack of transmission by blood products: case control studies (transfusion ≠ higher vCJD risk), no CJD in 342
recipients from donors who developed CJD, 12,000 hemophiliacs & no CJD

Mad cow disease (BSE)


 Started 1985, England
 Looks like scrapie in cow; didn’t do anything about it, big explosion of cases
 Pattern: throughout England, Scotland, Wales; one case pretty much everywhere (opposite of kuru/scrapie)
o Point-source epidemic
 Bone meal: from all kinds of sources. Cook “greaves” (random animal parts), extract tallow (candles/fat/etc),
get bone meal (for cattle feed; helps calves grow faster)
o 1980s: Iranian gas crisis: short-cuts on cooking of various bone meal prep steps
o 1979: tallow market crash: people decided animal fats were bad for you, switched to vegetable fats
 Stopped processing tallow & left fat (with infectivity) in
 1988: banned cattle carcasses in cattle feed; 1989: banned cattle offal in human food
o After 5-year lag (incubation period), cases started to drop

vCJD
 Colloquially “mad cow” but not technically BSE; only 2 cases in US
 Different from CJD: younger patients, psychiatric abnormalities (not myoclonic movements & dementia)
o CAN be transmitted by transfusion: 2 cases in UK

US food chain concerns:


 imported BSE cattle
 spontaneous BSE in cattle/other animals
 chronic wasting disease in deer and elk (clearly spreading among captive and live deer)

New: BSE diagnosed in cow in Washington, transfusion cases of vCJD, reports of possible BSE strains

Response to BSE in US: increased cattle testing, prohibited non-ambulatory cattle for human consumption, SRM &
mechanically separated meat prohibited from human food, carcasses of tested animals not passed until negative test

Deterrents to risk assessment: unknown mode of natural transmission, species barriers, dose/route of entry, strains,
differences in pathogenesis

Needs:
 Blood test for humans & animals
 Better understanding of pathogenesis (transmission, species barriers, strains, etc)
 Neuropathological exams on all degenerative diseases
 Worldwide surveillance of TSEs (humans & animals)

Current situation:
 Kuru gone (incubation > 50 yr)
 Iatrogenic CJD (hGH & dural graphs) ↓ but will continue for decades
 BSE / vCJD outbreaks ↓ in UK but isolated cases worldwide
 Chronic wasting disease: no spread to cattle or humans but spreading in N. America; wide host range in lab

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Academy Awards of Infectious Diseases
New diseases: 58% from animals (zoonosis)
Most important ID events in 20th century (survey):
 smallpox eradication, penicillin, HIV, 1918 influenza, childhood immunization, clean water

Most likely to be eradicated: probably guinea worm (Jimmy Carter’s campaign!), but talked about polio.
 Poliovaccine: 1000 children paralyzed per day (1988)  herd immunity in US (early 90s)
 Down to 6 countries / 575 cases/yr

New vaccine: Hepatitis B. 60% hepatocellular carcinoma; 300,00 cases / 5,000 deaths/yr
 1st: anti-STD vaccine, anti-cancer vaccine. Can’t cultivate virus in artificial media (impressive)
 Big drop in HBV in USA, Taiwain (decided to vaccinate in 80s). HBV endemic in Asia (lots of perinatal trans)

New bacterium: H. pylori. Role in Type B gastritis, achlorhydria, peptic ulcers, gastric carcinoma & lymphoma, others?
 Some crazy guy drank a glass of it & got sick (achlorhydria, etc.) Non-invasive; tons of polys.
 Abx shown to have better prevention of recurrence than ranitidine
 More and more chronic diseases shown to have microbial components

New viral agent: HIV.


 14,000 new infections/day, 42M living with HIV, 4.3M newly infected 2006, 2.9M deaths/year, 2.2M on ART
 Huge reduction in life expectancy in some African countries (negated all health gains since 1950s)
 PEPFAR continuing to give aid to developing countries; widely expanded in ’08 & focused on across-the-board
health care systemic improvements (and monitoring outcomes, not just # treated or prevented anymore)

New antiviral agents: HAART (HIV) INTERVENTION PER-PERSON SURVIVAL GAIN


 1996 AIDS conference, Vancouver: viral dynamics / # copies Cancer chemo 10 months
explored; clinical trials of triple therapy reported Coronary bypass 20 months
 Protease inhibitors: great example of rational drug design HAART-HIV 43 years
 Now 26 drugs across 6 classes; huge improvements in outcome & survival
 New focuses: Cure HIV (2009 case; empty latent pool); eradicate HIV (test & treat)
 (FYI: HCV antiviral agents represent 28% new drug apps for antimicrobials)

New antibacterial agent: the bacteria


 Great achievement; abused  microbes win!
 MRSA: USA-300, community-acquired; USA-100: hospital-acquired; necrotizing fasciitis & pneumonia
 Pipeline of Abx drying up (fewer approved & just one new class in 20 years): pharma wants to make drugs you
have to take every day!

Infectious disease epidemic: Influenza


 H1N1 (1918): 25-30% world’s population ill; >40 million deaths worldwide (60% in 20-45yo)
o Influenza usually kills old (85+) but avg age in 1918: 28 years old (same with avian influenza today)
 Bird flu today: controlled in birds (vaccines / culling), by isolating patients, and because virus hasn’t gotten good
at human-human transmission yet
H1N1 challenges
 H1N1 today: people born before 1957 not getting this one – H1N1
circulating until 1957.  Vaccine production (solved – 15 μg,
no adjuvant, single dose – but late)
 Seasonal = 65+yo, swine flu = young people
 Antivirals (OK for now)
 Currently at pandemic alert level: 4 influenza pandemic, totally
th

unpredicted, projected US toll: 50% infected, 1.8 mil  Surge capacity (biggest US challenge)
hospitalized, 30-90,000 deaths; will be tracked in real time!  Global sharing unlikely

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Anaerobic Infections
Anaerobes: dominant form of life on/in people
“Organisms of neglect”- important but not much known about them Most anaerobic infections:
 Mixed (polymicrobial)
History: Pasteur (Clostridum butyricum); Veillon & Zuber: intraabdominal sepsis  Endogenous (host flora)
(IAS) & B. fragilis, “classical studies”(1889 – 1938) looking at genitourinary tract  Microbial dx rare
flora, lung abscesses, IAS; “renaissance” (1965-1980) – clinical studies, culture,  Empiric tx with abx
taxonomy, antibiotic development
Classification of anaerobes
Growing anaerobes: commonly use a chopped meat glucose  EOS (extremely O2 sensitive): tolerates
broth seconds/minutes of O2 exposure
 Strict anaerobe: tolerates > 0.4% O2
Much less anaerobic bacteremia now than in past (people
 Moderate anaerobe: tolerates 0.8-2.5% O2
learned how / when to treat anaerobic infections)
 Microaerophilic: tolerates >2.5% O2
 Facultative anaerobes: tolerates anaerobic
Major pathogens:
condition but also grows in air or 10% CO2
 Gram negative bacilli (Bacteroides, Prevotella)
 Gram positive cocci (Peptostreptococci, a.k.a. “peptococci”)
 Gram positive bacilli (Clostridia) – pretty much the only anaerobe transmitted human-human because it’s
spore-forming; the spores can exist aerobically)
SITE RATIO ANO2 : O2
 Hopless (Lactobacillus, Bifidobacteria, Veillonella)
Saliva, tooth 1:1
How to diagnose anaerobes: Stomach, Ileum
Gingiva 1000:1
 Microbiologic diagnosis: get specimen from a normally sterile site;
Colon
transport while protecting from too much O2, think if it makes sense / from
an anaerobic site Vagina 5:1
 Clinical diagnosis: think of site of infection (not pharyngitis, etc. but maybe a peritonsilar abscess); putrid
discharge, polymicrobial flora Gram Stain.

Growing anaerobes: get anaerobic transport media; often grow in anaerobic chamber, etc.
 Is it pratical?
o Specimens are hard to obtain; microbiology is polymicrobial, tedious; the process is expensive &
prolonged, treatment is empiric anyway because susceptibilities are high; patients are often discharged
before report comes in
o Antibiotic sensitivities are of poor quality and not recommended, clinical clues are good
o One exception: blood infections (usually take a sample & put in both aerobic and anaerobic bottles)
 If E. coli, would grow in both; if Bacteroides Fragilis, would grow just in AnO2

Recognizing anaerobes:
 Often see polymicrobial mix
 AnO2 GNRs have an unique morphology (e.g. long & fusiform); AnO2 GPCs just look like cocci

Infection site: ANO2: FREQUENT ANO2: RARE


 Why not UTIs? Urine isn’t Brain abscess, dental
Meningitis, pharyngitis,
appropriate for replication of HEAD & NECK infections, space infections,
acute sinusitis, acute otitis
anaerobes (reduction potential) chronic sinusitis, chronic otitis
 Almost all abdominal abscesses Aspiration pneumonia, lung Bronchitis
CHEST
(except those in table) usually abscess, empyema Non-aspiration pneumonia
have anaerobic involvement Cholecystitis, spontaneous
ABDOMEN Peritonitis, phlegmon/abscess
 Dental infections: 1000:1 bacterial peritonitis
anaerobes:others in gingival GU TRACT Female genital tract UTIs, STDs
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crease; during extraction or other damage, anaerobes can pass to perimandibular space (through mandible &
underneath) and set up infection there.
 “Clenched fist injury” – punch somebody in the mouth & their anaerobes get you back with a nasty infection
 When you see abscess or aspiration pneumonia, think ANAEROBE

Abscesses & IAS (Intra-abdominal sepsis): often E. coli & B. fragilis (if polymicrobial, probably anaerobes involved!)
 If you perforate your colon, tons of anaerobes headed inside
 Flow is slower in colon so anaerobes can grow; by the time stool gets out, it’s almost purely anaerobes (just
about as much as could fit in the space that the stool occupies!)
 Often E. coli early (peritonitis stage; recovery); B. fragilis late (abscess stage, higher mortality).
o Different roles: E. coli causes bacteremia & shock; B. fragilis causes abscess
o Not synergestic: each has its own role
 Remember that other organisms (e.g. S. aureus) can cause abscesses commonly too!

B. fragilis
 bacteremia without septic shock (no active endotoxins)
 capsular polysaccharide: need capsule for sepsis (capsule itself can actually cause an abscess!)
 In vitro resistance to abx: very little
o Metronidazole is best against B. fragilis
o Imepenem or Pip-Tazo work too

Clostridial syndromes
Clostridium sp. Syndrome
C. botulinum Botulism
C. tetanus Tetanus
C. perfringens Gas gangrene
C. perfringens enterotoxigenis Food poisoning
C. difficile Antibiotic-associated colitis
C. sordellii Septic absorption
C. septicum Neutropenic colitis

Anaerobes & Anaerobic Infections: Summary


 Important, neglected, predominant flora, 1 quadrillion per person
 Endogenous infections, abscessogenic or clostridial toxins
 Dx: clinical clues; Rx: empiric

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Chlamydia & Mycoplasma
Chlamydia
 Obligate intracellular organism; tiny
 Thought to be a virus for a time: DNA, RNA, ribosomes; make own proteins & nucleic acid (true bacteria)
 Inner & Outer membrane (like gram neg) but no peptidoglycan layer (don’t Gram stain at all: too small)
 Energy parasite (can’t make own ATP)

Biphasic life cycle:


1. Elementary body (like spore) attaches to, ingested by cell
2. Phagosome fusion; EB  reticulate body (RB)
3. RB multiplies, condenses (RB  EB), forms inclusion body
4. Inclusion body releases EB (infectious particle)
(EB: infectious, not metabolically active; RB: metabolically active, not infectious)

Chlamydiaceae:
 Chlamydia trachomatis: trachoma, oculogenital, LGV: STIs & conjunctivitis
 Chlamydia pneumoniae: atypical pneumonias
 Chlamydia psittaci: psittacosis (from birds)

Chlamydia trachomatis
 Infects non-ciliated, columnar epithelial cells; infection doesn’t confer much resistance to reinfection
 Doesn’t grow on normal lab media: need to use tissue culture (like a virus)
 Culture isn’t great for Dx: use PCR for LGV & D-K (more sensitive)
o Trachoma: clinical diagnosis

Multiple serotypes with different diseases (note conjunctivitis ≠ trachoma)

Serovars D-K:
Common genital infections, conjunctivitis (neonates)
 Presentations
o Men: urethritis  epididymitis (70% due to CT!)
 Serosanguinous penile discharge (gonorrhea more purulent)
o Women: urethritis, cervicitis, (PID, ectopic pregnancy, chronic pelvic pain if untreated)
 Majority asymptomatic
 Cervicitis: mucopurulent discharge (can use swab to test)
o Both: pharyngitis, pneumonia, proctitis (anal sex), conjunctivitis
 Proctitis: direct inoculation from anal sex; rectal bleeding, pain, mucous discharge, diarrhea
o Neonatal conjunctivitis (30-50% exposed babies)
 Highest in women, blacks, ages 15-25; most frequently reported STD & ID in US (2-4M new cases/yr)
 high partner co-infection rate

Remember: notifiable disease; must treat all sex partners from previous 60 days or reinfection is likely
 High reinfection rate
 Screen: all women <25yo yearly; re-screen 2-4 months after Tx

Serovars L1-L3:
Lymphogranuloma venereum (LGV)
 Worldwide; higher in tropical/subtropical; MSM mostly in US
 More invasive strains; cause thrombolymphangitis
30
 Stages:
1. Primary: genital lesion (painless) – transient ulcer
 Ulcer most often undetected; 30d incubation
 Heal without scarring; can get anal/rectal reinfection with anal intercourse/contaminations
2. Secondary: regional lymphadenopathy; systemic Sx
 2-6wks later: buboes (swelling of lymph node); painful!
 “Groove sign” – groove between two LNs
 Rupture or harden, then resolve
 Inguinal LAD is most common (less in Treating Chlamydia
women so go undiagnosed)  Tetracyclines (doxycycline)
 Rectal involvement: MSM, anal sex  macrolides (azithromycin: only need one dose)
3. Tertiary: genital elephantiasis; strictures,  Resistance: extremely uncommon
fistulas, abscesses, frozen pelvis
 More common in women (lack of Sx in 1st 2 stages)
 Elephantiasis! Nasty!

Serovars A-C:
Trachoma: leading cause of preventable (infectious) blindness worldwide
 A chronic keratoconjunctivitis endemic to Africa, Asia, Middle East, Australia (aboriginal groups)
 Transmission: children & women who care for them; Via hand-eye, fomites, flies
 Developing world only
 Pathogenesis:
o repeated reinfection  chronic follicular conjunctival inflammation (active trachoma) 
o tarsal conjunctival scarring distorts tarsal plate 
o entropion (turning in of edges of eyelid so that lashes rub against eye surface) & trichiasis (cicatricial
trachoma) 
o corneal abrasions, scarring, opacification: blindness

Chlamydia pneumoniae

 Same life cycle as C. trachomatis; spread via respiratory route


 Common cause of upper & lower resp. infections
o 7-10% of CAP
o “Atypical pneumonia” – interstitial, not lobar like S. pneumoniae, etc.

Chlamydia psittaci

 Birds (parrots pigeons, hens, turkeys  pet owners, pet shop employees, poultry farmers)
 Severe Atypical Pneumonia, also typhoidal-form fevers, splenomegaly, malaise possible
 Culture is DANGEROUS – use serology or PCR

Mycoplasma
Tiny prokaryotes, lack a cell wall; Cell membrane bound: contain sterols; has RNA, DNA; hard to grow (special agar)

Mycoplasma pneumoniae: URTI & atypical pneumonia (respiratory aerosols)


 Can have complications (derm, cardiac arrhythmias/CHF, neuro: meningitis, encephalitis)
Mycoplasma genitalium: urethritis & cervicitis
 No cell wall; unknown prevalence, not reportable; sequelae unknown
o Smallest prokaryote bacteria capable of self-replication; culture often missed, PCR?
Treatment: Doxycycline, macrolides, fluoroquinolones

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Non-Tuberculous Mycobacteria (NTM) & Nocardia
 NTM & Nocardia: both in same order (Actinomycetales); both found in environment and only cause disease if
immune system compromised somehow
NTM vs Nocardia
Mycobacteria Nocardia
Route of Infection Inhaled Inhaled, direct inoculation
Source/Reservoir NTM-soil,water; leprosy-human Soil
Host range MOTT-large; leprosy-narrow Large
Clinical 1o lung or skin (GI?), can 1o lung or skin, can disseminate
disseminate
Cell Wall PG, AG, mycolates (C60-C90: long!) PG, AG, mycolates (C44-C66: shorter!)
Microbiology Acid fast, aerobic, rod Weakly acid fast, aerobic,
filamentous rod
Host Defense Cell mediated immunity Cell mediated immunity
Evasion Phagosome-lysosome non-fusion Phagosome-lysosome non-fusion

 NTM: large; diverse group


o M. tuberculosis complex
 M. TB; others including M. bovis (attenuated to vaccine strain, M. bovis BCG)
 Mycobacterial cell wall: like a Gram (+) at heart but covered with an outer lipid layer
o Mycolic acid & certain glycolipids are unique to mycobacteriae & nocardia
Diagnosing Mycobacteria
AFB Test
 Rapid diagnostic test (real time!); specific for mycobacteria, but don’t know which species!
 Sensitivity only 40-70%: need high amount of AFB to visualize. CAN’T EXCLUDE TB WITH NEGATIVE SMEAR
 Fluorescent acid-fast stains make visualization quicker & easier to read (e.g. auramine-rhodamine fluorescence)

Decontaminating specimens:
 Mycobacteria grow slowly (1x/day vs. E. coli ~20m); can be overgrown by other bacteria/fungi
o Sterile specimens: blood, CSF  inoculate directly onto media
o Non-sterile specimens: sputum chemical decontamination to remove normal flora/contaminants
 Abx to inhibit other bacteria; decontaminate with pH
 Suppresses both mycobacteria & others but less for myco (suppression can be problem)

Culture: more sensitive than smear, TAKES WEEKS to grow, may require special conditions (low temp, etc.)
 Importance: species ID, drug susceptibility, monitoring response to Tx
 Need low temp (environmental organism needs environmental temperatures): need to notify lab for NTM
 Liquid media faster (1-3wks vs 4-6 for solid)
 Can’t grow M. leprae!

Speciation: traditionally growth characteristics; biochemical tests (slow)  DNA probes, other methods
 M. tuberculosis, M. avium complex, M. kansasii = important pathogens
 M. gordonae: not a pathogen but common lab contaminant

Rapid Dx: Nucleic acid amplification (like PCR)


 More sensitive than smear; less sensitive than culture (90% sputum+ samples; 50% sputum- )
 Positive test: supports TB Dx; NEGATIVE TEST DOES NOT EXCLUDE TB
 Good for specimens other than sputum

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Mycobacterium leprae & Leprosy
 A.k.a. Hanson’s disease, big historical significance (& >500K new cases/yr), mostly Brazil & India
M. leprae
 Obligate intracellular parasite; cannot be cultivated in lab
 50% genes “pseudogenes” (evolutionary reduction: from environment to humans, had excess genes left over)
 Armadillos are a natural reservoir (SE US) & tool for research

Pathogenesis:
 URT transmission (inefficient)  multiples in tissue Mφ & Schwann cells around nerves  mostly skin,
peripheral nerves disease: local effects of multiplication / host cell-mediated immune response; lose sensory
input around lesions, etc,
 Neuropathic effects: neuropathy  trauma/burns  autoamputation of digits
 Sunken nose: bacillary multiplication (low temp)  destroys bone & cartilage

Leprosy spectrum:
Tuberculoid (TT) BB: Lepromatous (LL)
Paucibacillary (few bacteria) borderline Multibacillary (lots of bacteria)
BT: BL:
↑ peripheral nerve thickness Unstable:
borderline borderline
Flat granules, well-defined usually go
tuberculoid lepromatous Leonine facies (lesions heaped up wth
granulomatous lesions one way skin cells; filled with mycobacteriae)
Good TH-1 mediated immunity or other Mostly TH-2 mediated response

Immunologic reactions: (can occur after Tx: unleash immune response)


 Reversal reaction
o Treatment of BB (borderline) disease can cause Leprosy Dx
immune reconstitution  DTH reaction Examination:
o Inflammation of existing lesions  erythematous / hypopigmented skin lesions
o Requires corticosteroid Tx (prevent further  + sensory loss
motor/sensory loss)  ± enlarged peripheral nerves
 Erythema nodosum leprosum
o BL/LL disease: can develop fever, eruption of red Lab: skin smear / biopsy; PCR-based if available
nodules, other inflammatory manifestations
o Already have high burden of organism; may be due to immune complexes
o Can require corticosteroids
Non-tuberculosis Mycobacteria (NTM)

 Free-living, environmental, opportunistic pathogens Main clinical scenarios of NTMs


o Need to distinguish contamination from actual infection 1. Lymphadenitis
2. Inhalational pulmonary disease
Slow-growers: M. avium complex; M. kansasii, M. marinum, M. ulcerans 3. Disseminated disease
Rapid-growers: M. abscessus, M. chelonae, M. fortuitum 4. Skin/skin structure infection
(following inoculation)

Mycobacterium Avium Complex (MAC/MAI)


= Mycobacterium avium-intracellularae: really two organisms
 Environmental organism (soil/water)

Main clinical syndromes

33
 Lymphadenitis (cervical lymphadenitis: MAC is most common entity; need to excise LN)
 Pulmonary MAC: underlying lung disease is common (e.g. COPD,
emphysema, CF: prevent clearance) Diagnostic criteria: NTM lung dz
o Patterns: Solitary pulmonary nodule, fibrocavitary, nodular Clinical (need 2)
bronciectatic (small nodules, esp. in periphery  larger 1. Typical Sx & radiology
bronchi) 2. Exclusion of other Dx
o Hot tub lung: hypersensitivity pneumonitis caused by reaction
to MAC in hot tub water Microbiologic (need 1)
 Subacute SOB, cough, fever 1. + culture x2 (sputa)
 + MAC culture in hot tub & lung 2. + culture x1 (bronchoscopy)
 May not require Abx 3. Biopsy evidence of granulomas
 Disseminated (AIDS pts) + culture x1
o CD4 VERY LOW (<50), Incidence way down (HAART)
o Entry: GI tract; Subactute presentation: fever/abd pain/diarrhea/wt loss; hepatosplenomegaly,
pancytopenia, adenopathy
o Dx: blood culture (can grow from blood = tons of organism)/biopsy
o Path: phagocytes jammed with MAC

M. kansasii
 TB mimic: most likely to present like TB (upper lobe cavitary dz)
 Strictly transmitted via water supply (midwest, SE)
 Risk factor: underlying lung disease (COPD, smoker)

M. marinum
 Aquatic/marine environments; natural pathogen of fish
 Human dz: inoculated into skin post-trauma (fishermen, watermen, aquarium hobbyists)
 Chronic ulceronodular skin disease: “Fish tank granuloma”
o Chronic: dx often after weeks/months
 Grows best at low temp: 28-30 C (notify lab)

M. ulcerans
 Close relative of M. marinum; presumed aquatic
 ONLY TOXIN PRODUCING MYCOBACTERIUM (has a huge PLASMID that encodes)
 Buruli ulcer: emerging dz of sub-saharan Africa; chronic painless cutaneous ulcer
 Major cause of disability (esp. children – aquatic environment) because of scarring, fibrosis (e.g. over joint!)
 Tx: streptomycin + rifampin x 8wks

Rapidly-growing Mycobacteria
 Colonies in ≤ 7days (relatively fast next to others; still pretty
slow) – can be longer for primary isolate RGM: Diagnosis
 M. abscessus, M. chelonae, M. fortuitum: 90% clinical  Grow on mycobacterial media (may also
isolates grow on normal blood agar/culture systems)
 Ubiquitous in home & hospital; common contaminants of  Need to notify lab if suspect RGM:
fluids & devices 1. More readily decolorized in AFB stain
 Pseudo-outbreak of M. fortuitum pulmonary dz at JHH: from 2. More susceptible to decontamination
ice machine! 3. Require cooler temperatures for growth

Opportunistic pathogens: surgical site infections, implant-associated, pulmonary infection of diseased lungs (M.
abscesses), disseminated in immunocompromised pts.
 Example: surgeon’s dye to mark incision sites contaminated; inoculated pt when cuts made
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Nocardia
 Aerobic, Gram (+), filamentous rod
o Require modified acid-fast stain (weaker decolorization)
o Various spp with varying Abx susceptibility
 Ubiquitous in soil, decaying vegetation
 Infection: inoculation or inhalation
o NO PERSON TO PERSON TRANSMISSION

Cutaneous Nocardosis: immunocompentent host


 Manifestations: Celulitis / abscess / lymphocutaneous (can track up lymph vessels)
1. Mycetoma (N. brasiliensis only)
 Common where shoes aren’t worn
 Host & microbe interact, chronic inflammation
 Huge exophytic (growing outward) ulcerative masses; ooze & pus)

Pulmonary & Disseminated Nocardosis


 Host: reduced cell-mediated immunity
 Nodular/cavitary infiltrates; disseminates to brain / skin
 Diagnosis: difficult: no pathognomonic features; often not suspected; may delay Dx procedure (pt
immunocomporomised); may be partially treated / difficult to isolate after empiric Tx
 THINK NOCARDIA IF:
1. Pulmonary AND brain disease
2. Nodular / cavitary
3. Immunosuppresed

M. TUBERCULOSIS NOCARDIA
Order: Actinomycetales
SIMILARITIES

Cavitary lung disease;


risk for dissemination if immunocompromised
Risk: impaired cell-mediated immunity
Acid-fast (mycolates in cell wall)

Can be cultured on blood agar


Filamentous rods
DIFFERENCES

Ubiquitous in environment
Latent infection; gives +
tuberculin skin test
Slower generation time
Longer mycolates
(more resistant to
decolorization; more acid-fast)

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Antimicrobial Stewardship
Why is this important?
 200-300M abx annually, 45% outpatient
 25-40% hosp pts get abx; 10-70% unnecessary or suboptimal; 5% adverse reaction
 Abx unlike other drugs: use in one pt can compromise use in another!
 Resistance (up), really expensive ($30B annually)
 # new abx (down), and mostly just modifications of existing classes
o Timeline for development:9 -10yrs, really hard

Definitions
 Prophylaxis: use of antimicrobial agents to prevent the development of an infection
o Pre-exposure: e.g. surgical prophylaxis
o Post-exposure: e.g N. meningitidis prophylaxis
 Empiric treatment: use of antimicrobial agents when infection is suspected and patient is ill enough to require
treatment
o e.g. tx of pt with possible sepsis
 Pathogen-directed treatment: use of antimicrobial agents to treat a proven infection
o e.g. tx of pt with blood cultures growing S. aureus

Principles of antibiotic treatment


1. Develop differential diagnosis
o Most likely disease states based on history and Principles of Antibiotic Treatment (overview)
physical exam 1. Develop differential diagnosis
 Demographics: age, race, geography, 2. Determine if antibiotics are necessary
speed of onset, rapidity of progression, 3. Choose an antibiotic
course 4. Refine antibiotic choice
 Organ systems affected, 5. Have a plan for length of therapy
signs/symptoms, prior Dx & therapies
o Is there an infectious process on the short list?

2. Determine if antibiotics are necessary right now


o Does antibiotic therapy alter the course of disease?
 No effect on some mild bacterial infections: especially those with primary viral problem (acute
bronchitis, acute sinusitis, acute otitis media) or natural mechanism to clear (infectious
diarrheas)
o Are antibiotics required immediately?
 Risk/benefit: risk of deferring therapy vs benefit of waiting to confirm dx
GIVE ABX! DON’T GIVE ABX (WAIT!)
 known focus of infection that requires tx with  Stable patient with subacute process in
abx to prevent the patient from getting sicker whom culture data may be hard to obtain but
(acute meningitis, pneumonia, acute is critical to management
endocarditis, epidural abscess with cord
compromise, etc.) (pt with fever of unknown origin: get origin
first, suspected vertebral osteomyelitis w/o
 no known focus of infection but other neurological sx, pt with wt loss & mass-like
important feature lesion in right middle lobe of lung:
(neutropenia/cancer + fever, asplenia + fever, malignancy?)
high immunosuppression + fever, toxic-
appearing patients with unstable vital signs)

36
o Avoid "antibiotics for every fever"
 Fever is not an illness: it's a sign of illness
 Non-specific, host response to inflammation, not always an infectious cause, don't
always need abx even if infectious
 Same goes for "antibiotics for increased WBC"

3. Choose an antibiotic
o Empiric therapy: when we don’t know what’s causing the disease, but we need to start something
 What bacteria are likely to be involved & what antibiotics cover these bacteria?
 Common error: try to cover every possible organism
 Cover most likely & tailor to sickness of patient
o Pathogen-directed therapy: ID’d organism & have antibiotic susceptibilities
 Choose antibiotic with narrowest coverage that will treat organism.

4. Refine antibiotic choice


o Keep an open mind. Follow progress over time; reassess DDx if not progressing as expected
o Watch for & anticipate side effects:
 Allergies: PCNs, other drugs
 Drug-drug interactions (know current meds)
 Rifampin (oral contraceptives), voriconazole/posaconazole, erythromycin
 Look out for pts on warfarin, oral contraceptives
 Drug-food interactions: antacids inactivate oral fluoroquinolones

5. Have a plan for length of therapy


When to stop antibiotics?
o Empiric treatment:
 trust cultures & stop if from sputum/blood/urine before antibiotics started aren’t growing
organisms (not pneumonia, bacteremia, UTI)
 MRSA and pseudomonas grow easily: if they’re not isolated in culture, they’re most likely not
there and you don’t have to cover them
 NO REQUIREMENT TO “COMPLETE A COURSE” just because you started empirically
o Pathogen-directed treatment
 Length of most courses of therapy: arbitrary multiples of 7 days!
 Some have been clinically investigated: S. aureus bacteremia w/o endocarditis (14-28d); N.
meningitidis meningitis (7d), uncomplicated UTI in woman (3d)
 Try to give shortest course possible

Cultures & Antibiotics


Common error: antibiotics for every possible culture. Make sure to evaluate the culture!
Blood cultures
EXAMPLE ORGANISMS CLASSIFICATION WHAT TO DO
S. aureus (coag +)
Gram (-) rods NEVER A CONTAMINANT ALWAYS treat
Yeast
Usually don’t treat
Coag (-) Staph Usually a contaminant Exceptions:
Gram (+) rods (skin organisms)  patients with indwelling hardware
 signs of infection & >1 positive culture
Evaluate clinical picture for possible source (e.g.
Strep viridans
50/50 oral, GI, endocarditis)
Enterococcus
 signs of infection & >1 positive culture

37
Urine cultures
4 questions
1. What is source of urine? (clean catch good; cath/foley bag not good)
2. Does patient have symptoms of a UTI? ( dysuria, frequency, suprapubic pain, fever: don’t culture if none!)
3. What does urinalysis show? (should be sterile & uncontaminated. Suggestive of UTI: > 5-10 WBC/µL, no
epithelial cells – don’t rely on dipstick only)
4. What does the culture show? (positive ≥ 105 colonies; common UTI pathogens = E. coli, K. pneumoniae)

Wound cultures
 Superficial cultures of chronic ulcers & cultures from drains: usually polymocribial; contaminated/colonized
 More reliable: cultures from newly incised / drained abscesses
o Can be polymicrobial too; significant pathogens = heavy growth (S. aureus, GNR)
o Organisms like coag neg staph, enterococcus, strep viridans: if light growth, don’t need abx coverage
unless they’re only organism present
o Think of anaerobes in abdominal infections (might not grow in culture)

Sputum cultures
 Lab classification of quality of sputum sample: PMNs = good, epithelial cells = bad (want lower resp secretions)
o Type 1: discarded; lots of squamous cells, problem in specimen collection
o Type 2: adequate; PMNs=epithelial
o Type 3: good! PMNs > epithelial (rare or none)
o Type 4: just spit (bad)
 From endotracheal tubes: often colonized (look out) – should have clinical evidence of pneumonia to start abx

Cases:
1. 68 yo diabetic male; osteomyelitis, no systemic sx – don’t start abx right away (wait & check it out – stable pt)
2. 35 yo male, no comorbidity, picture of CA-pneumonia: want to start abx right away (pneumonia), thinking of S.
pneumoniae, HiB, (M. catarralis)+ atypical (legionella, Chlamydia, etc.) as common, give ceftriaxone (GPC) + azithromycin
(atypical) as empiric coverage. Find out susceptible to PCN! Give PCN (actually amoxicillin – easier PO because you don’t
have to give it as often) as pathogen-directed therapy.
3. 45 yo woman, severe Crohn’s disease, on TPN via central venous cath, fever 101 F & fatigue. Start abx (has cath!); probably
want to cover staph (vanco empirically for MRSA possibility), get a blood culture. Comes back MSSA; give oxicillin for
definitive Tx.
4. 50 yo man; central venous cath, took blood culture for no clinical reason, doing fine at home, culture comes back GPCC
(don’t start abx! Asymptomatic)
5
5. 55yo woman HT/high cholesterol; has acute MI; U/A & Cx show >10 colonies of E. coli. Find out if she has Sx, check U/A
results, think about source before starting abx.

Explaining antibiotic control to patients:


 educate physicians + patients + office staff (not effective to focus on one group, have office-based materials,
reinforce education during well-patient visits)
 Patients: explain, share facts (not for viral infections), build cooperation & trust, don’t say “just a viral infection”,
get pt involved, give alternative Tx for symptoms when possible, be confident

Pharmaceutical representatives: varying perception about appropriateness of gifts; people think that it only affects
other doctors & not me, etc.

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Syphilis (& other STIs)
NB: Most STIs are ASYMPTOMATIC  ask about BEHAVIOR, not Genital ulcer diseases:
just symptoms!  Syphilis (Treponema pallidum)
 Herpes (HSV 1&2)
Syndromes & causes:
 Chancroid (Haemophilus ducreyi) – mostly in
 Genital ulcer diseases: syphilis & herpes are the big
the south; uncommon
ones
 Lymphogranuloma venereum (Chlamydia
 “Drips” (discharges): gonorrhea, chlamydia,
trachomatis L1-L3)
trichonomiasis are main ones
o Possible presentation esp in developing
o Pt presents with a drip: treat for gonorrhea &
world; usually causes proctitis (MSM, etc.)
chlamydia empirically & then trich if sx don’t
 Granuloma inguinale or donovanosis
resolve
(Klebsiella granulomatiosis)
 Urethral/vaginal/cervical inflammation; proctitis (receptive anal sex), pediculosis pubis too.

Syphilis (Treponema pallidum)


“The great imitator / imposter” – can cause disease in pretty much any internal organ

Treponema pallidum
 Spirochete (slender, tightly coiled, unicellular, helical)
 Can’t culture in vitro (hard to study – inject into bunny testicles)
 Has very few proteins on outer membrane – relatively inert; has very little genetic diversity

Transmission: primarily sexual contact; also kissing, in utero, blood transfusions

Pathogenesis:
1. Penetrates skin through little microabscesses common with intercourse 
disseminates quickly (hours/days) via lymphatics/blood to any organ Stages of syphilis:
(especially CNS: in 1st few days!) divides ~30h
2. T. palladium gets to deep tissues (induces matrix metalloprotease-1 “early syphilis”
production) quickly, induces endothelial cells to express ICAM-1/VCAM- 1. Primary syphilis
1/E-selectin  inflammatory cells migrate to tissues 2. Secondary syphilis
a. PMNs respond first 3. Latent syphilis
b. Dendritic cells stimulated (TLR2 recognizes T. pallidum PAMPs)  a. Early latent
phagocytosis  taken to regional LNs  T-cells activated (slow
process b/c T. pallidum outer membrane is relatively inert) “late syphilis”
3. [CD4] & [CD8] peak 13-18d post-exposure (ulcers resolve); T. pallidum b. Late latent
survives in an unknown reservoir 4. Late syphilis
4. Incubation period: ~3wks 5. (Neurosyphilis: early &
5. Very low inoculum size (~10 spirochetes = infection!) late)

Stages (natural history: assume no treatment)


1. Primary syphilis Early neurosyphilis
a. Chancre: single papule at site of inoculation  ulcer;  CNS involvement can be
painless, well demarcated, heaped-up edges, smooth base, symptomatic during primary or
highly vascularized. secondary syphilis
i. Heals in 3-6wks without therapy  Presentation like meningitis (stiff
ii. If grouped, painful ulcer neck, headache, photophobia)
iii. Harder to recognize in women (more likely to have  Usually self-resolving ( immune
worse outcomes with syphilis) system can control)
 More common in HIV patients
39
2. Secondary syphilis
a. Lymph nodes  blood, multiplication & dissemination
b. 2-8 wks after chancre if untreated: most common = skin manifestations (rash on palms & soles is classic
for secondary syphilis, but can look like anything! Macular/popular/ulcers/pustules/psoriasis/etc)
c. Skin lesions: filled with tons of spirochetes, very infectious (only if both people have abrasions on skin)
i. Condyloma lata: gray raised lesions; look papillomatous, almost like genital/anal warts
ii. Mucous patches in mouth, etc.
d. Other manifestations: all organ systems possible or even asymptomatic (even without rash)
i. Fever, lymphadenopathy, meningitis, optic neuritis, gastritis, hepatitis, glomerulonephritis, arthritis

3. Latent syphilis: no manifestations at all!


a. Immune system controls secondary manifestation; patient becomes Asx (latency)
b. Early latency: 1st year, pt. can have relapses of secondary syphilis; still infectious
c. Late latency: > 1 yr, pt is immune to relapse or reinfection
i. CAN STILL TRANSMIT (BLOOD TRANSFUSION or IN UTERO)
ii. 60% will stay in late-latent forever! (even without treatment)
iii. 30% will develop late syphilis & its manifestations if untreated

4. Late syphilis: a.k.a. “tertiary syphilis”; occurs decades later


a. Cardiovascular syphilis
i. Endarteritis obliterans of vasa vasorum of the aorta  aortitis  sacular aneuyrisms
1. FYI: Endarteritis obliterans = “inflammatory condition of the lining of the arterial walls in which the intima
proliferates, narrowing the lumen of the vessels and occluding the smaller vessels”
2. Usually ascending aorta; used to be most common cause of aortic aneuyrisms
b. Gummatous syphilis: almost like TB
i. Benign, granulomatous-like lesions usually affecting skin / bones but can occur in any organ;
causes local destruction via immune response
ii. Called “Gummas”, especially common in face & nasal cartilage (path: look like TB granulomata)
iii. “Benign” unless they’re in a bad place (heart, etc).
c. Late neurosyphilis: can be symptomatic or asymptomatic
i. Symptomatic: >10yr after primary infection; 2 main groups (meningovascular/parenchymatous)

ii. Meningovascular: endarteritis of small blood vessels (meninges, brain, spinal cord)
1. Strokes, seizures
2. MCA STROKE is especially common site

iii. Parenchymatous: actual destruction of nerve cells “Argyll Robertson (AR) pupil”
1. Tabes dorsalis: affects spinal cord  a.k.a. “Prostitute’s Pupil”, although
(shooting pains down leg, ataxia, cranial that’s probably not too PC these days
nerve abnormalities)  small pupils, accommodate (to near
2. General Paresis: affects brain (dementia, objects) but don’t react (to bright light)
psychosis, slurring speech, “Argyll  Wikipedia notes this fun mnemonic:
Robertson” pupil). Schizophrenia-type “like a prostitute, they ‘accommodate
but do not react.’”
symptoms of general paresis are big in
literature, Law & Order: SVU
Transmissibility
 Sexual transmission: only possible prior to late-latent syphilis
 In utero transmission: possible at any time
o All pregnant women need a syphilis test at their 1st visit
Congenital syphilis
 In utero infection can occur at any stage of syphilis; tends to happen after 4th month of gestation
 Baltimore has a number of cases each year (marker of public health quality)

40
 Perinatal manifestations: rhinitis (“snuffles”) followed by diffuse rash (esp. soles of feet), splenomegaly,
anemia, jaundice, thrombocytopenia; osteochondritis not uncommon (predilection for long bones & cartilage in
nasal area; causes deformity)
 Later manifestations: neurosyphilis, deafness, keratitis, recurrent arthropathy, Hutchinson’s teeth (widely-
spread incisors; look kind of like Dracula)

Diagnosis
 Dark-field microscopy: gold standard test for primary syphilis (serology negative in ~ 30% cases); not often
used (unavailable), can’t use for oral /GI lesions (lots of oral/GI nonpathogenic spirochetes)
 Serology: primary tests used, not sensitive in primary syphilis (prior to Ab formation); pt can become non-
reactive in secondary, early latent, early-late-latent syphilis

1. Non-treponemal tests: RPR & VRDL


 RPR = rapid plasmid reagent; VRDL = venereal disease research laboratory
 Nonspecific but very sensitive (almost 100%); quantitative
 testing for anticardiolpin IgM & IgG (marker of host cell – treponeme interaction: looking for Ab
against mitochondrial self-antigens; treponemes make cells explode & immune response against
these Ag follows)
 VERY CHEAP: 1st test to get if you suspect syphilis
 Result negative & don’t suspect primary syphilis: unlikely to have syphilis, no more tests
 Result positive: confirm by ordering treponemal test
 Provide titer that can be followed (1:1 = low, 1:2048 = very high; want 4-fold reduction in 12
months to indicate a cure)
 Re-test to follow treatment & check for re-infection

2. Treponemal tests: test for treponemal-specific antibodies;


 Tests are specific but expensive (use RPR/VRDL first)
 Don’t provide a titer that can be followed after therapy; once-positive = always positive

Treatment: need both a good immune system and penicillin to cure


 PENICILLIN; dose depends on stage of disease
o Early: 2.4M units PCN x1 dose
o Late-latent (gummas, etc.): 2.4M units PCN x3 doses over 3 weeks
o If patient is PCN-allergic: desensitize & use PCN anyway
 Treat patient and ALL SEX PARTNERS (track down & treat, even if you have to use the police to do it)
 ALWAYS TEST FOR OTHER STIs (including HIV)

Other diseases (DDx)


STI DDx: Genital ulcers
 Granuloma inguinale donovanosis:
o From Klebsiella granulomatis, rare in US (more SE Asia, Africa)
o Painless, progressive, ulcerative lesions without regional lymphadenopathy
o “beefy red” & highly vascular
Painless ulcers Painful ulcers
o Dx: tissue biopsy (no culture; PCR not FDA approved)
Syphilis, LGV, Herpes,
o Rx: doxycycline (100 mg po BID x 3wks)
granuloma inguinale chancroid
 Chancroid
o From Haemophilus ducreyi, endemic in some parts of southern US
o Painful genital ulcer & tender supperative inguinal adenopathy; often co-infected (syphilis / HSV)
o Dx: culture
o Rx: azithromycin 1g po x1 or ceftriaxone 250mg IM x1

41
STI DDx: urethral / vaginal / cervical inflammation
 N. gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis
 Symptoms: dysuria (pain with urination), increased urinary frequency, urethral/cervical/vaginal discharge,
occasionally epididymitis / orchitis (testicular pain) in men

STI DDx: Pelvic inflammatory disease


 Infection spreading to upper genital tract in women (uterus/fallopian tubes/ovaries) (STIs in general)
 Symptoms: abdominal pain, fever
 Signs: uterine tenderness and cervical motion tenderness
 Complications: infertility, chronic pelvic pain, ectopic pregnancy

STI DDx: Proctitis


 N. gonorrhoeae, Chlamydia trachomatis, HSV 1&2
 Can be infected but asymptomatic
 Generally via receptive anal sex
 Symptoms: pain on defecation, rectal discharge with blood & mucus

General principles of STI management:


 NO SEX (even with a condom) until patient and partners are treated
 Find one STI  test for ALL THE OTHERS
 Most STIs are reportable infections: report to health department
 When possible, use a treatment that you can watch them take(e.g. PCN injection)
 Don’t judge patient (better to get partners treated, etc)

42
Pharmacology: ID & Micro

Introduction to Antibiotics ...................................................................................................................................................... 2


Cell Wall Active Antibiotics (I & II) .......................................................................................................................................... 5
Sulfonamides and Antimicrobial Antifolates ........................................................................................................................ 11
Ribosomal Inhibitors ............................................................................................................................................................. 14
Drugs for Mycobacterial Infections....................................................................................................................................... 21
Quinolones ............................................................................................................................................................................ 25
Antibiotic Resistance ............................................................................................................................................................. 27

1
Introduction to Antibiotics
Antibiotic Definition:
 Classic: substances produced by one microorganism to inhibit growth or destroy another
 Functional: Drugs that kill “bugs” (mostly bacteria but also others)
Therapeutic objectives:
Example: for a given clinical syndrome, figure out what the most common organism is, 1. Right drug
then choose an appropriate class & drug and individualize therapy (all depends on who 2. Right place
& where pt is). After empiric therapy, get lab results (if applicable) & give definitive tx. 3. Right time

Empiric therapy: begin broad


 “What’s likely?” - depends on probability of organism in given clinical syndrome (pt.,geography, hospital vs
community, etc.)
 Worst-case scenario – what’s the worst that could happen even if less probable
 Local antibiotic sensitivity patterns (community, esp. hospital)

Definitive therapy: go narrow


 Get culture & know sensitivity
 Side effects, co-morbidities, other patient characteristics inform choice
 Not probability-dependent

Narrowest spectrum good: ↓ resistance, adverse effects, cost; ↑ adherence, simplicity


Obstacles: tons of organisms, tons of abx with different characteristics; agents can cause many syndromes & vice versa,
many causes still unknown, causes for a syndrome can vary with population, geography, age

RIGHT DRUG
Selective toxicity: less sensitivity to toxicity in humans than bacteria
host efficacy bacterial toxicity
 Therapeutic window = Selective toxicity =
host toxicity host toxicity
 Relies on the distant genetic relationship between humans & bacteria (as opposed to fungi, parasites, viruses)
 Various mechanisms (see table)

Adverse effects
 Often unrelated to antimicrobial effects
 Large therapeutic index usually (can give
a high molar quantity)

Coverage: individualize for organism, local


epidemiology / resistance, patient
 E.g. antibiotic guidelines / susceptibility grids for each hospital.

Bactericidal vs. Bacteriostatic


 Microbiologic definition = MBC:MIC > 4 for ‘cidal
o Minimum bactericidal concentration : minimum bacteriostatic concentration > 4
 Clinical definition:
o ‘Cidal = Dead bugs
o ‘Static + good immune system & good penetration = Dead bugs
o ‘Static drug alone (immune dysfunction or sanctuary) = Sleeping bugs

2
 Table has good summary but not hard & fast rules.
 Bacteriostatic are mainly ribosome inhibitors.
 Can be variable even for a given drug (‘static against
some, ‘cidal against others)
 Clinical relevance: sometimes ‘static drugs penetrate
better, or ‘cidal drugs are needed (see table)
o Endocarditis: poor penetration, slow growth
o Meningitis: poor penetration, immune
sanctuary
o Osteomyelitis: poor penetration
o Neutropenia: immunosuppresion

Combinations of antibiotics: save for special situations


 Mixed infection: at least 2 microorganisms growing and
causing disease, e.g. intra-abdominal infection
 Resistance prevention: e.g. anti-TB therapy
 Initial empiric therapy with serious infection
 Synergy: a few situations where 1+1 > 2 in the clinic
RIGHT PLACE
Antibiotic Tissue Penetration
 Recall first unit: []blood, size, protein binding in plasma, lipid
solubility, ionic charge, tissue binding, active transport,
excretion pathways
 Inflammation: some drugs can penetrate better in places of
inflammation (e.g. get into sanctuary like past BBB if
inflammation present).
o Example: penicillin <1% CSF penetration without
inflammation, 5% with inflamed meninges
o Penicillins usually pumped out across BBB;
inflammation reduces this efflux

Extravascular penetration of drugs


 Interstitial fluid: rapid equilibrium with vascular space
 Large reservoir (e.g. pleural fluid): equilibrium occurs slowly, smaller fluctuations (akin to oral dosing)
 Specialized site with barrier (e.g. CSF): much lower concentrations overall

 Loading dose can rapidly achieve target concentration


o Especially relevant if reservoir situation
o How fast you can get it up to 95% still depends on half-life!

Bacterial penetration:
 Outer membrane of Gram (-) bacteria, periplasmic β-lactamases around cell wall,
 Need to get past cytoplasmic membrane & cytoplasmic pumps
 Think: how does drug work? Where? How does it get there? How could resistance block its arrival?

RIGHT TIME
Pharmacokinetics / pharmacodynamics:
 time course of drug concentration often doesn’t parallel time course of drug effect

Time vs. Concentration-dependent killing


3
 Concentration-dependent killing (aminoglycosides, quinolones)
o Increased killing rate as concentration rises above MIC
o Therapeutics: get concentration up high & fast
 Time-dependent killing (β-lactams)
o Killing rate doesn’t rise past a point (~4x MIC)
o No correlation between drug peak/MIC or AUC/MIC
o Instead, killing increases with % time spent above MIC
o Therapeutics: get concentration to effective level &
keep it there

Post-Antibiotic Effect (PAE)


 Suppression of bacterial growth that persists after short
exposure of organisms to antibiotic agents
 Means effect-time relationship might be different from drug-time relationship
 So something with a short half-life but with a PAE might be able to be dosed less frequently than you’d expect

PAE against…
PAE
Gram (+) Gram (-)
β-lactam Abx YES No
Aminiglycosides (yes,but not clinically YES
Quinolones relevant)

4
Cell Wall Active Antibiotics (I & II)
Cell Wall Basics
Gram (+): large pepditoglycan cell wall on outside, β-lactamases on surface of cell wall
Gram (-): small cell wall in periplasmic space between OM & IM; β-lactamases in periplasmic space

Cell wall: NAM/NAG make long CHO strands; tied together by peptide cross links
 Cross-links differ in AAs, # between GPC (L-lysine, pentaGlycine) & GNR (m-DAP)
 Both have terminal D-Ala-D-Ala that is reactive for cross-linking

E. coli division: elongation; incorporation of new material, formation of septum, splitting into daughter cells

Peptidoglycan “3-for-1” Growth model


1. Synthesize 3 new strands
2. Chew up an existing strand
3. Insert 3 new strands for existing strand (“3 for 1”)
4. Cross-link via transpeptidase at D-ala-D-ala
a. D-alanine not present in vertebrates
b. Terminal D-ala cleaved off
c. Cross-linkage formed by acylation at CO-N bond (m-DAP in GNR, Gly in GPC)
5. Expand

 Involves big multienzyme complex that assembles in cell wall area to form new cell wall
 Just behind this cassette is a point of transition and vulnerability: where the cross-links have been broken but
not yet reformed (so inhibiting cross-linking = multienzyme complex just breaking apart the wall)

Penicillin & general β-lactam mechanisms


β-lactams inhibit cross-linking of peptidoglycan strands
 Suicide substrate for transpetidase
 Forms covalent linkage: leaves acylated, inactivated enzyme
 Structural analogs of D-ala-D-ala with β-lactam ring in place of peptide
bond
o Ring constrains the structure to keep homology
o Cleave ring = lose structure
 Autolysins (peptidoglycan hydrolases) usually break apart cell wall during
synthesis; here simply degrading wall
 Selective toxicity: humans don’t have cell walls (no D-ala / transpeptidase)

Penicillin binding proteins:


 Multiple kinds, located in cell membrane of both GNR and GPC
 Transpeptidases, transglycosylases
 Localization depends on function (elongation, shape maintenance, septa formation, cell divison)
 Inhibiting some can be lethal to cell, others won’t be lethal

β-lactam effects: leads to dramatic morphologic changes (failure to divide, failure to form septa, etc.)

Penicillin G
Classic penicillin: as development continues, pick up more gram (-) and more anaerobes
Key points:
 Inflammation inhibits organic anion transport pump, so better CSF penetration (not pumped out as much)

5
 Time-dependent killing (no need to go above 4x MIC)
 Post-antibiotic effect in GPC, NOT GNR – can dose less frequently than 45m half life suggests

Allergic reactions:
 Most dermatologic, idiopathic, drug fever, etc. – less severe and have longer onset (~72h)
 Most worrisome: anaphylaxis – very severe & quick onset (~1 h)
 Management:
o No Hx allergy: no skin test (2% positive skin tests, much rarer anaphylaxis)
o Hx penicillin allergy: order skin test
 Positive skin test: avoid penicillins or desensitize first. 50-70% will have anaphylaxis &
accelerated urticaria!
 Negative skin test: can use (3% will have cutaneous reactions)
 Up to 30% cross-reactivity with cephalosporins (if positive skin test, avoid cephalosporins or desensitize!)

β-lactamase inhibitors
Resistance:
 β-lactamases (can use inhibitors)
o Hydrolyze β-lactam ring; no longer has constrained D-ala-D-ala steric homology
 Also alteration in PBP site, ↓ permeability too

Clavulanate:
 competes with penicillin for β-lactamase active site access
 β-lactamase hydrolyzes clavulanate & forms irreversible complex (suicide substrate)

Augmentin: Amoxicillin + Clavulanate.


 Much lower MIC than just amoxicillin if β-lactamase producing bug

Other β-lactamase inhibitors: Sulbactam, Tazobactam


Limitations of β-lactamase inhibitors
 Only active against one class (A) of serine hydrolases (exception: Tazobactam on class C&D too)
 Degraded by β-lactamase after binding
 Clavulanate induces expression of β-lactamase
 Emergence of inhibitor-resistance Class A enzymes; multiple classes produced by one organism; multiple-
organism infections can have different β-lactamases
Development of more penicillins
Problems with Penicillin G:
 short half-life
 unstable to gastric acidity (hard to give PO for lower GI stuff)
 inactivated by β-lactamase
 poor Gram(-) spectrum
 allergenic

Goals of penicillin’s spawn:


 Improve activity against GNR (anti-pseudomonals like
piperacillin, etc. and extended GNR spectrum of
amox/ampicillin)
 PCN-ase resistance (anti-staph like oxacillin, methicillin, etc.)
 Pharmokinetic advantage (prolonged half-life like penicillin G
benzathine/procaine, etc.)

6
Goals of modifying penicillin side chains:
 ↑ β-lactamase stability; ↑PBP affinity, better PK, more acid stable
 better permeability across outer membrane (↑ Gram (-) coverage)

Dorothy Crowfoot Hodgkin: x-ray crystal structure of penicillin (β-lactam) and pretty much everything else.

MODIFICATIONS OF PENICILLIN
Improved Pharmacokinetics
Longer acting: good because of time-dependent killing (more efficient than high peaks)
 Procaine penicillin: 1:1 salt (penicillin G:procaine). Given IM, near painless
 Penzathine penicillin: 2:1 salt (penicillin G: dibenzylethylene diamene). VERY slowly absorbed IM
 Both of these: penicillin molecule has same t½ but absorption from IM slow: almost like continuous infusion

Orally available: lower peak, a few hours of action (along with PAE, makes for acceptable po dosing)
 Penicillin V: acid stable

Improved Gram (-) Spectrum (Aminopenicillins)


In general: cover penicillin’s spectrum (GPC, above-the-belt anaerobes) + enterococcus & some “dumb GNR”
Ampicillin (can add sulbactam to cover β-lactamase producers) – often IV
ampicillin Like penicillin but with improved Gram (-) spectrum
Indications: Penicillin's spectrum + enterococcus and "dumb gram negatives". GPC: pneumococcus,
streptococcus, enterococcus (faecalis, not faecium); GNR: H. influenzae, E. coli, P. mirabilis, Salmonella.
Administration: often IV; can be paired with β-lactamase inhibitor (amp + sulbactam = Unasyn, given IV) to
improve spectrum to include β-lactamase producing S. aureus, H. influenzae, M. catarrhalis, many GNR
Resistance: β-lactamase, PBP mutations, etc.
Toxicity: Diarrhea, skin rash (macular, evanescent). Not allergy.
Other: 50% orally bioavailable. PAE against GPC, not GNR.

Amoxicillin (can add clavulanate to cover β-lactamase producers) – often PO


amoxicillin Like penicillin but with improved Gram (-) spectrum; like ampicillin but with 100% bioavailability
Indications: Penicillin's spectrum + enterococcus and "dumb gram negatives". GPC: pneumococcus,
streptococcus, enterococcus (faecalis, not faecium); GNR: H. influenzae, E. coli, P. mirabilis, Salmonella.
Administration: often PO, can give 3 times a day (q8h); can be paired with β-lactamase inhibitor (amoxicillin +
clavulanate = Augmentin, given po) to improve spectrum to include β-lactamase producing S. aureus, H.
influenzae, M. catarrhalis, many GNR
Resistance: β-lactamase, PBP mutations, etc.
Toxicity: Diarrhea, skin rash (macular, evanescent). Not allergy.
Other: 100% orally bioavailable. Just ampicillin with one OH group added. PAE against GPC, not GNR.

Amoxicillin = Ampicillin + one hydroxyl (bioavailability 50% for amp 100% for amox)

Antipseudomonal (aminoacylpenicillins)
Piperacillin (can add tazobactam to cover β-lactamsae producers)

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piperacillin Like ampicillin but with coverage of more serious GNRs (e.g. pseudomonas)
Indications: Ampicillin's spectrum + more nosocomial GNRs ("more serious GNR"). Adds pseudomonas
aeruginosa & bacteroides fragilis
Administration: Can pair with tazobactam to increase coverage (β-lactamase producing staphylococci and
many GNR). Doesn't help against pseudomonas! In combination for serious Gram (-) infections (e.g.
nosocomial / empirical)
Resistance: β-lactamase, PBP mutations, etc.
Other: PAE against GPC, not GNR. (Ampicillin coverage = GPC: pneumococcus, streptococcus, enterococcus
(faecalis, not faecium); GNR: H. influenzae, E. coli, P. mirabilis, Salmonella.)

Penicillinase-resistant
Methicillin: historic interest only; not hydrolyzed by β-lactamase
 “Methicillin resistant S. aureus” (MRSA) or S. epidermidis (MRSE): means it’s not resistant because of β-
lactamase
o Resistance due to altered PBP
o Resistance extends to ALL OTHER β-LACTAMS! Includes cephalosporins, imipenem

Oxacillin:
oxacillin Like penicillin but pencillinase resistant
Indications: Penicillin's spectrum + penicillinase-producing staph, pneumococcus, Group A streptococci
Resistance: PBP mutations, like MRSA or MRSE. (resistance to one of these extends to ALL β-lactams)
Other: PAE against GPC, not GNR.

Dosing penicillins Things to remember


 Half-life 0.5-1.5 hrs but PAE allows dosing q4h about all these penicillins:
 Not good for PO dosing 1. PAE against GPC, not GNR
 Exceptions: 2. B-lactamase inhibitors only help if B-
o Meningitis: dose q2h lactamase is mechanism of inhibition
o Amoxicillin: 3x/day (q8h) 3. Time-dependent killing! (continuous
o Benzathine & procaine: very long half life infusion increases % time > MIC, so
better killing. E.g. piperacillin study)
4. Renal clearance predominates
5. Low penetration (CSF, eye, brain,
prostate) unless inflammation

8
Other β-lactam classes: Cephalosporins (cephalosporin ring instead of azothine); Monobactams (one ring only),
carbapenems (different 5-membered ring) – but all have β-lactam ring

Cephalosporins
General characteristics:
 β-lactam ring, resistant to β-lactamases  Time-dependent killing (continuous infusion
 Spectrum: broad (GPC, GNR incl. pseudomonas) more efficient)
 Safety: betterthan penicillins  PAE vs. Gram (+)
 Four “generations”  Allergy: Up to 1/3 cross-reactivity with PCNs!

GENERATION SPECTRUM EXAMPLES


1st Strep, S. aureus. No activity against Enterococci, Listeria Cephalexin (Keflex)
2nd E. coli, Klebsiella, Proteus, H. influenza, M. catarrhalis Cefuroxime
Less activity against Gram (+) than 1st gen Cefotetan (also B. fragilis)
3rd Enterobacteria, Serratia, N. gonorrhea. Ceftriaxone*(Rocephin)
GPC: S. aureus & Str. pyogenes covered as well as 1st gen
4th Comparable to 3rd generation but more β-lactamase Cefepime
resistant
Used for pseudomonas too

*Dose all q8h except ceftriaxone (once daily)


Special ones: cefotetan (anti-anaerobe) & cefepime (anti-pseudomonal)

Carbapenems
General characteristics:
 Actively transported into bacteria: imipenem-specific porin
o Helps reach periplasmic space & cell wall in Gram (-) like pseudomonas
 PAE FOR GRAM (-): only one in class
 Renal toxicity: filtered by kidney;
o β-lactam ring hydrolyzed by proximal tubular dehydropeptidase I, producing renal tubular toxin
o Coadministration with cilastatin fixes the problem by blocking renal dehydropeptidase I
o ↑ *active drug]urine, so more efficacy for UTI treatment
o ↓ *toxic metabolite], so less renal tubule toxicity

Imipenem – Cilastatin
imipenem Indications: Very broad spectum: Gram (+) incl. enterococcus, Gram (-) incl. pseudomonas, anaerobes including
bacteroides
Administration: q8h (half life short but PAE for all)
Toxicity: Seizures (incidence not defined). Renal clearance; urinary carbopenem hydrolyzed by proximal tubular
dehydropeptidase I; results in metabolite: renal tubular toxin. Co-administration with cilastatin (inhibits renal
dehydropeptidase I) both 1) increases efficiency for UTI tx and 2) decreases renal tubular toxicity
Resistance: Rare (so far). Imipenem + cilistatin = Primaxin
Other: PAE against Gram (+) AND GRAM (-)! Renal metabolism.

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Monobactams
Aztreonam: like a non-aminoglycoside “aminoglycoside”
 Totally different spectrum: good Gram (-) coverage
 No cross-allergenicity with penicillins
aztreonam Indications: "Non-aminoglycoside aminoglycoside". Gram (-) coverage
Toxicity: No cross-allergenicity with penicillins
Resistance: β-lactamase, etc.

Vancomycin
Key features:
 Not β-lactam, so β-lactamase doesn’t hurt it.
 VRE: multiple genes mutated (D-ala-D-Lac instead of D-ala-D-ala)
 Allergenicity: skin rash, eosinophilia, drug fever; Phlebitis, “Red Man” flushing if IV dose too rapid
 Time-dependent killing (continuous IV best)
 No absorption (actually good for C.diff colitis – want it all to go on surface of GI; otherwise IV)

vancomycin Mechanism of Action: H-bonds to D-Ala-D-Ala so transpeptidase can't access (not β-lactam)
Effects: Inhibits cross-linking of peptidoglycan layer of bacterial cell wall
Selective Toxicity: Humans don't have bacterial cell wall
Indications: Great against S. aureus (e.g. MRSA); C. difficile colitis, Enterococci if susceptible
Administration: No absorption (good for C. diff). Enters CSF poorly without inflammation. Usually dose IV a
few times per day.
Toxicity: Allergenicity: skin rash, eosinophilia, drug fever. Phlebitis, "Red Man" (flushing if dose IV too fast),
ototoxicity & nephrotoxicity are doubtful
Resistance: NOT β-LACTAM so not affected by β-lactamase. Vancomycin resistance emerging (e.g. in E
faecium. - VRE). Mechanism: changing D-ala-D-ala to D-ala-D-lac (9 genes required)
Other: Renally excreted. Half life 6 hrs; 9 days if anuric. Time-dependent killing (continuous dose best)

10
Sulfonamides and Antimicrobial Antifolates
Paul Ehrlich = Mr. Magic Bullet (“die Zauberkugel”) & the father of Uber Modern Chemotherapy.
 Things he came up with: new principles for discovering anti-infectives (synthesized, not just natural, structure-activity relationships, used
standardized animal infectious models, came up with the idea of chemotherapeutic index, had a “receptor theory” – selective toxicity to
pathogen; drug resistance is characteristic of the infecting organism, not the host.
 Worked with organic arsenicals & azo dyes; voted “historical figure I’d most like to have a baby with” by pharmacology lecturers

Gerhard Domagk came up with prontosil around 1932-5, a red dye with azo link and sulfonamide group
 First big time antibacterial agent (highly effective, relatively
nontoxic) SA: active form of prontosil
 Turns out that it’s really a prodrug; cleavage @ azo linkage 1. SA: active in vitro & in vivo; prontosil
in metabolism yields sulfanilamide (active part) only in vivo
2. Equally active on molar basis
Sulfanilamide 3. SA produced from prontosil in tissues
 Active part of prontosil 4. Patients / lab animals given prontosil or
 Looks like PABA; reacts with 7,8-dihydropterin SA excrete SA
pyrophosphate via DHP synthase but leads to a metabolic
dead end (further steps inhibited – product isn’t a substrate for dihydrofolate reductase)

Sulfonamides: how they work in general


Folate metabolism in bacteria

1. 7,8-Dihydropterin pyrophosphate + para-amino benzoic acid (PABA) 7,8-DHP (via DHP synthase)
2. 7,8-DHP + several glutamates  dihydrofolate (FH2)
3. FH2 + NADPH tetrahydrofolate (THF or FH4) + NADP+ (via dihydrofolate reductase)
4. TH4 needed for TMP synthesis from dUMP; DNA synthesis down the line

KEY to SELECTIVE TOXICITY:


 HUMANS TRANSPORT DIHYDROFOLATE INTO CELLS; BACTERIA CAN’T (impermeable membrane)
 BACTERIA SYNTHESIZE DIHYDROFOLATE IN CELLS; HUMANS CAN’T (no DHP synthase)

Mechanisms of resistance
 ↓ bacterial permeability to sulfonamides
 Mutations in DHP synthase – so the sulfonamides don’t fit as well
 ↑ DHP synthase activity (constant % inhibition, so increase in # of enzymes increases overall activity)
 ↑ PABA to outcompete for enzyme spots

Structure / Activity Relationships


 Sulfanilamide crystallizes in urine (not cool)
 New versions: better solubility in water & longer half-life by substituting R and R’ groups

Clinical uses
 Cheap, easy to administer, orally bioavailable, narrow spectrum, well tolerated
 Rarely monotherapy
 Prophylaxis of simple UTI from Gram (-) bacteria
 Drug of choice for Nocardia
 Occasionally: H. ducrei, Chlamydia, Lymphogranuloma venereum (not so much anymore)

Pharmokinetics
 Shorter half lives (sulfamethoxazole = 11h, sulfisoxazole = 6h) are more manageable
11
 Longer half lives (sulformethoxine = 150h) aren’t really used anymore: can give infrequently (good) but also side
effects take a long time to go away (big problem)

Toxicity of sulfonamides
 Most hypersensitivity or idiosyncratic (only partially dose-dependent)
 Flu-like symptoms, skin rashes, drug fever, joint pain, lymphadenopathy
 Rare: Stevens-Johnson syndrome (danger with long-acting sulfonamides: can’t reverse)

 Higher rate in patients with AIDS (nobody knows why)

 Mechanism of idiosyncratic toxicity


o Sulfonamides can be broken down by two pathways
1. N-acetyl transferase (yields non-toxic metabolite)
2. Cytochrome P450 (yields reactive metabolite)
 Reactive metabolite can be detoxified or bind proteins (becomes hapten; generates
immunologic response & cytotoxicity)
o Slow acetylators might be at a higher risk for side effects

Toxicity in premature infants


 Premature rupture of membranes = ↑ risk infections
 Trying to find abx to treat empirically: sulfisoxazole & penicillin lead
to high mortality
 Hyperbilirubinemia
o Physiologic in all newborns (physiologic jaundice) – peaks @ Normal bilirubin: mostly bound to albumin
7-10d, then falls
o Bilirubin lower in sulfonamide treated pts
o But kernicterus present (yellow staining of basal ganglia from
bilirubin)
 Normal situation: bilirubin mostly bound to albumin, etc. – only
unbound crosses membranes
 Sulfonamide bumps bilirubin off of albumin: higher free bilirubin, so
more can cross into the cells (e.g. brain) Kernicterus in newborn: bilirubin bumped off
o Explains findings: [bilirubin]blood ↓, [bilirubin]cells ↑ albumin by sulfonamide; accumulates in cells
Moral of the story: don’t give sulfonamides to a nursing mother or her child

Structural analogues of sulfonamides


For mycobacterial infections
 DDS (Dapsone, 4,4’-diaminodiphenylsulfone): M. leprae (leprosy)
o Usually with Rifamipicin; ineffective vs. other mycobacteriae (unique dihydropteroate synthase)
 PAS (p-aminosalicylic acid): M. tuberculosis (TB)
o Inactive against most other bacteria; remarkably specific for M. tuberculosis
o Other sulfonamides ineffective against TB
For non-infectious diseases: antithyroid, antihyperglycemic, blocker of penicillin secretion & stimulate uric acid
secretion for gout (probenecid), diuretics, others.

Trimethoprim
Diaminopyrimidines (e.g. methotrexate, folic acid, etc.) – two amino groups on a pyrimidine moiety

Trimethoprim: a diaminopyrimidine
 Blocks dihydrofolate reductase (inhibits FH2  THF & limits TMP pool for DNA synthesis)
 Selective toxicity: doesn’t fit into human dihydrofolate reductase enzyme
12
 Huge difference: IC50 300,000:5 (humans:E. coli)
 Resistance:
1. Mutation in dihydrofolate reductase (so trimethoprim inhibits less)
2. ↑ dihydrofolate reductase expression (constant % effect)
 Toxicity
 Rare: rash, nausea +/- vomiting (3-5%)
 Folate deficiency in pregnant, malnourished, alcoholic patients (who are already low in folate)
 Result: neutropenia, thrombocytopenia, megaloblastic anemia

Cotrimoxazole
 Trimethoprim + Sulfamethoxazole (“Bactrim”, “Septa”)
 Sulfamethoxazole inhibits DHP synthase; Trimethoprim inhibits DHFR (later step in same pathway)
 Advantages: synergism, broader spectrum, ↓ resistance, ↓ dosage = ↓ toxicity
o ‘Cidal instead of ‘Static (complicated reasons)
 Half-lives well matched (11-10 hrs)

Similar combo: Fansidar


 Pyrmethamine (diaminopyramidine DHFR blocker like TMP); sulfadoxine (like SMX)
 For malaria

13
Ribosomal Inhibitors
Basics of Bacterial Ribosome
 70S ribosome
o 50S subunit (peptide exit tunnel, peptidyl
transferase cavity, etc.)
o 30S subunit (A - acceptor, P - protein, E – exit sites)
 Process of elongation: A to P to E; tRNA/mRNA moves,
polypeptide stays still
o Movement to hybrid states is spontaneous, 30S
subunit movement requires energy

Ribosomal inhibitors
 Site specific; work on different subunits. Only
aminoglycosides are ‘cidal in all situations
 See slide for good summary

Aminoglycosides
Structure: 3 hexose sugars, O-glycosidic linkage, 1+ amino
group/sugar

Gentamicin, tobramycin, amikacin

Pharmacokinetics:
 Poor oral bioavailability (IV)
 Distribution: good into interstitial,
poor into cells (except PCT & ear),
poor into CSF (give intrathecal if
needed)
 Not metabolized
 Glomerular filtration; PCT
accumulation
 2-3h half-life but dose once daily (PAE
& concentration-dep killing)

Once-daily dosing
 Get high peak ([]-dep killing)
 Drug-free interval
o Reverse adaptive post-exposure resistance by bacteria (not genetic; will revert & bacteria become more
susceptible)
o Minimize toxicity (need 3-5h drug-free)
 Rationale: []-dep killing, PAE, saturation of PCT & inner ear cells at low [], adaptive post-exposure resistance
 Meta-analysis: reduces / delays nephrotoxicity, no change in clinical efficacy, no change in ototoxicity

Therapeutic drug monitoring: further reduces nephrotoxicity (give dose, wait 30m, draw level, adjust dose)
 Wide PK variability; Cmax related to efficacy, trough related to nephrotoxicity: important to hit targets
 Easy, rapid AG assays exist; can improve outcome
 Hit targets quicker, higher peaks & lower troughs, less nephrotoxicity & costs
14
Which AG to use:
 Check local pattern of resistance first
o Gentamicin: standard (cheap)
o Tobramycin: more expensive but not worth the difference; JHH: a little better vs pseudomonas
o Amikacin: much more expensive but much more expensive (save for when you need it!)
o Streptomycin for TB; Neomycin for topical; Neomycin or kanamycin for oral in hepatic coma
 Ototoxicity similar for all

gentamicin Mechanism of Action: Aminoglycoside antimicrobial agents. Bind 30S ribosomal A site rRNA
Effects: Inhibit protein synthesis, misreading, freezing initiation complex. Actively imported into bacteria via
tobramycin polyamine transporter (inhibited by chloramphenicol, calcium, anaerobic or acidic environment, mutations).
Causes lysis & death of bacteria
amikacin
Selective Toxicity: human 80S ribosomes don't bind aminoglycosides well (exception: cells with megalin
membrane transporter: PCT, inner ear, pigmented retina epithelia; mitochondrial rRNA)
Indications: Gram negative rods (good against E. coli, Klebsiella, proteus, even some "bad" gram negatives.
Gentamicin not great against Pseudomonas or Acinetobacter - TOB or AMI better. TOB not worth the cost).
Use for severe gram(-) infections & for synergy (S. aureus, enterococci, PCN-resistant S. pneumoniae!) with
PCNs.

Administration: IV, once-daily dosing (high peak for concentration-dep killing, drug-free interval to reverse
adaptive post-exposure resistance by bacteria & minimize toxicity). Takes advantage of PAE. Therapeutic
drug monitoring useful too

Toxicity: Nephrotoxicity (10-20%, PCT changes in all, glomerular changes in few, rarely severe, reversible).
Ototoxicity (uncommon, cochlear & vestibular 3-15%ish, irreversible); Neuromuscular paralysis (exceedingly
rare, increased with fast infusion, myasthenia, succinyl choline anesthesia). Ethacrynic acid (loop diuretic)
potentiates nephro/ototoxicity!

Resistance: very slow development of resistance (ribosomal & transport mutations rare). Enzymatic
modifications most common (acetylation, phosphorylation, adenylation). One enzyme may inactivate all
AGs; most that inactivate GEN also inactivate TOB. Very few mutations inactivate AMI

Other: Synergistic with beta-lactams. Antagonistic with chloramphenicol. Has activity against GPC but not
used clinically except in synergistic combinations. PAE against Gram (-) and (+)

streptomycin Like other AGs but used for TB

neomycin Used topically (triple antibiotic cream, etc.)

Toxicity in depth:
 cells with megaline membrane transporters: PCT, inner ear, pigmented retina epithelia

Nephrotoxicity:
 binds brush border phospholipid (MMT) in PCT between microvillus projections; accumulates in renal cortex
15
 gets endocytosed; toxicity from lysozome processing products

Risk factors for nephrotoxicity: Pros & Cons of Aminoglycosides


 Increasing age (PK issue only?)
 Volume depletion (concentrating effect)
 Normal renal function (can’t cause toxicity if
can’t get to PCT!)
 Hepatic dysfunction
 Duration > 72hrs (3-7d on average for toxicity) –
can use eperically
 Concomitant medications:
o loop diuretics, especially ethacrynic acid (1 dose does it!)
o Antibiotics (vancomycin, ampho B, clindamycin, cephalosporins)

Ototoxicity: perilymph concentration sustained over time; total destruction of hair cells
Clinical features of ototoxicity
 High frequency lost first; clinically notable rarely; tinnitus; high low frequency hearing loss
 Imbalance, vertigo, nausea/vomiting if vestibular involvement
 50% irreversible; cumulative (don’t give AG at all if history of ototoxicity); progresses after cessation of drug

Risk factors for ototoxicity:


 ↑age, underlying renal dz, previous AG tx or auditory damage, duration of treatment
 Ethacrynic acid (loop diuretic)
 Genetic mutation (1555AG in mitochondrial rRNA; G mutation binds AGs to mito rRNA!)

Tetracyclines
Tetracycline, doxycycline, minocycline: 4-rings

Allow binding in 30S A site but not transition step;


transported into bacteria; passive absorption to eukaryotes
(doesn’t accumulate)

Originally: broad spectrum; lots of resistance now


(mutations in bacterial active transport system; confers
resistance to all tetracyclines). Good for treatment of atypicals (Chlamydia, borrelia, H. pylori)

Pharmacokinetics: orally bioavailable formulations (doxycycline ~100%)


 Advantages of doxy, minocycline:
o Available IV;
o Can be dosed 1-2x daily; (tetracycline 3-4x daily): longer half-lives and better bioavailability
 Adverse effects: chelated with calcium & deposited in teeth, bones
o Sunlight: darkened bands appear on teeth (important from last few days of pregnancy  6 yo)
o Don’t give with antacids, milk (Ca), Maalox (Mg)
 Doxycycline: least GI side effects

16
tetracycline Mechanism of Action: Ribosomal inhibitor, antimicrobial agent. Inhibits bacterial ribosomal 30S subunit
at A site
doxycycline Effects: Blocks A/T > A/A alignment (aminoacyl-tRNA can't "stand up")
Selective Toxicity: Will inhibit eukaryotic protein synthesis, but actively transported into bacteria &
minocycline accumulates. Doesn't accumulate in eukaryotic cells

Indications: Atypicals: Chlamydial infections (e.g. Chlamydia pneumonia, most common young adult
CAP, STIs, PID, etc.). Borrelia burgdorferi (Lyme dz). H. pylori (along with other abx & bismuth
subsalicylate).

Administration: q3-4h (less half-life, bioavailability than doxy or mino). Can't administer with antacids,
Maalox, milk (Ca & Mg, see below)
Toxicity: Chelates with calcium (teeth/bones; darkened bands on teeth with sunlight exposure,
important from few days before birth to 6yo)

Resistance: Genetically altered active transport system (shared by all tetracyclines)


Other: Originally broad spectrum, now lots of resistance

“Glycylcycline” class: tigecycline

tigecycline Mechanism of Action: Ribosomal inhibitor, antimicrobial agent. Inhibits bacterial ribosomal 30S subunit
at A site. New "class"- glycylcycline - minocycline with a large sterically limiting side chain
Effects: Blocks A/T > A/A alignment (aminoacyl-tRNA can't "stand up") Overcomes 2 resistance
mechanisms: (1) active efflux from bacteria; (2) protection of ribosomes

Spectrum: broad, ‘Static


 GPC (incl. MRSA & VRE!)
 GNR (incl. Acinetobacter, NOT pseudomonas),
 AnO2 (incl. Bacteroides)

Good efficacy: intra-abdominal, skin/skinstructure, pneumonia


Good tissue penetration, 36h half life! no drug-drug interactions or food effect

Chloramphenicol
From here on out, talking about the 50S subunit (here, peptidyl
transferase cavity adjacent to A & P sites)
 Common / overlapping binding sites for linezolid,
chloramphenicol, clindamycin, macrolides

Chloramphenicol: inhibits peptidyl transferase step; forms a


number of H-bonds with the molecules in the site. Does bind our
mitochondrial ribosome (like AGs a bit)

17
Cool things about chloramphenicol:
 can start IV, finish po or do a whole course of a powerful antimicrobial PO in developing countries!
 Great CSF penetration! Up to 50% plasma concentrations!

chloramphenicol Mechanism of Action: Ribosomal inhibitor, antimicrobial agent. Inhibits bacterial ribosomal 50S
peptidyl transferase at A site.
Effects: Blocks peptide elongation by blocking peptidyl transferase step
Selective Toxicity: Doesn't bind human large ribosomal subunit, does bind mitochondrial peptidyl
transferase
Indications: 'Cidal against S. pneumoniae, N. meningitidis, H. influenzae. 'Static against many Gram
(-) & anaerobes. Used for CNS infections of these.
Administration: insoluble. palmitate ester used for PO (children syrup, hydrolyzed by gut).
succinate ester used for IV (hydrolyzed by liver).

Toxicity:
 Bone marrow suppression: transient, reversible (dose-related, plasma-level related,
marrow vacuolated; dec. reticulocytes, serum iron incr. with RBC decrease, from inhibition
of mito protein synth).
 Aplastic anemia: irreversible > 50% (not dose/plasma/time related; often fatal, 1:40,000,
just takes 1 dose).
 Gray baby syndrome: don't glucuronidate well, blood levels > 50mcg/mL, e- transport
inhibited (ashen gray color, vomiting, refusal to suck, rapid/irreg breathing, abd distension,
cyanosis, diarrhea, flacidity, hypothermia, death).

Resistance: Enzymatic modification (acetylation)


Other: Metabolism: glucuronidated by liver; mostly glucuronidated product cleared by kidney.
Distribution: wide, intracellular, excellent CSF penetration (>50% plasma concentrations). Not often
used in US (afraid of aplastic anemia) although risk ~ PCNs, <NSAID/yr.

Clindamycin
Inhibits 50S peptidyl transferase @ A&P site (peptidyl transferase cavity)

clindamycin Mechanism of Action: Ribosomal inhibitor, antimicrobial agent. Inhibits bacterial ribosomal 50S peptidyl
transferase (A & P site)
Effects: Peptide bond formation blocked
Selective Toxicity: Doesn't bind human ribosomal subunit or inhibit eukaryotic peptidyl transferase
Indications: GPC (S. pneumoniae, Group A Strep, Staph aureus), most anaerobes (important)
Administration: q6-8h, orally absorbed
Toxicity: Pseudomembranous colitis (alters gut flora, C. diff, nursing homes, etc. Use metronidazole (and
vancomycin if failure) for C.diff tx. Associated with clindamycin most commonly but also other broad
abx)
Resistance: Seldom a clinical problem

18
Linezolid
Blocks movement of fMet-tRNA into 50S P site (inhibits formation of 70S initiation complex) - doesn't block peptidyl
transferase step like clinda, chloramphenicol

Activity for clindamycin, linezolid, chloramphenicol is additive although their binding sites overlap.

linezolid Mechanism of Action: Ribosomal inhibitor, antimicrobial agent. Blocks formation of 70S initiation
complex (blocks movement of fMet-tRNA into bacterial ribosomal 50S P site)
Effects: No formation of 70S ribosome, no protein synthesis in bacteria
Selective Toxicity: Doesn't bind human 80S ribosomal subunit
Indications: WORST OF THE WORST (VRE, MRSA, PCN-resistant S. pneumoniae). 'Static except against S.
pneumoniae.
Other: PAE of 1-2 hrs; time-dependent killing

Macrolides
14-15 member ring with 2 monosaccharide moieties
Erythromycin, azithromycin, clarithromycin (old new)
 Erythromycin used in military recruits to eradicate strep pharyngeal carriage: PCN allergy will pop up in huge
population of recruits.
 Common strategy: start with cheapest (erythromycin); move up if it doesn’t work.
 AZI, clarithromycin q24h: more convenient & better adherence; erythromycin: cheap but GI side effects

erythromycin Mechanism of Action: Ribosomal inhibitor, antimicrobial agent. Binds in bacterial ribosomal peptidyl
transferase cavity (50S), blocks peptide exit tunnel via H-bonding.
azithromycin Effects: Occupies only exit tunnel; allows up to 6-8 peptide bond formation, then termination
Selective Toxicity: Don't bind to human larger subunit; don't inhibit human protein synthesis
clarithromycin Indications: Atypical organisms/pneumonias (Mycoplasma pneumoniae, Chlamydia pneumoniae,
Legionella pneumophila). Can use instead of PCN if allergic (S. pneumoniae, Group A Streptococcus)
Administration: q6h (ERY), q24h (AZI + clarithromycin)
Toxicity: Safest of all antimicrobials, some nausea & vomiting (more ERY)
Resistance: Becoming a problem (very frequently used)
Other: Great cellular uptake (AZI > ERY for macrophage penetration)

19
Ribosomal Inhibitors: Mechanisms of Action

20
Drugs for Mycobacterial Infections
Special features of Mycobacteria
Cell walls:
 Unique, highly lipophilic with mycolic acid  confers acid-fastness; target for selective chemotherapy
 Many of these drugs are only active against mycobacteria for this reason

Metabolic features:
 Can replicate intracellularly or extracellularly
 Can undergo prolonged periods of metabolic inactivity / dormancy
o Dormant = less susceptible to killing by bactericidal agents
o Need prolonged therapy (months) to completely eradicate infections

Drug resistance:
1. Primary resistance: spontaneous acquisition of resistance
a. 1 out of every 106-8 M. tuberculosis organisms are resistant
to at least one of the standard anti-TB drugs (based on
mutation rate; point-mutations)
b. Not consequential for small, non-cavitary lesions (e.g.
asymptomatic PPD converters) who have less than 1 million
organisms (104-5 organisms means ~0 are resistant)
c. TB pneumonia; TB meningitis: huge organism load, very
likely resistant to 1-2 drugs. (109-11 organisms means 103-
105 are resistant)
d. Single drug (isoniazid) is therefore adequate to treat a recent PPD converter
e. Multiple drugs must be used to treat an individual with clinically apparent disease

2. Secondary resistance: acquisition of resistance during treatment


a. Pts. asymptomatic before all organisms eradicated
i. Slow mycobacterial growth/metabolism, especially in
caseating / cavitary lesions
b. Noncompliance is common with prolonged treatment, esp.
after pt. starts to feel better (only take some pills, skip doses, etc.)
i. Provides selective pressure for the emergence of drug-resistant organisms
ii. Directly-observed therapy (DOT) is how this is handled in USA (Baltimore: down to ~7 cases/yr)
1. Expensive: other countries can’t afford it!

3. MDR & XDR TB


a. MDR TB (multi-drug resistant): resistance to isoniazid & rifampin (two mainstays of bactericidal tx)
within a single organism
i. Can develop MDR TB via secondary resistance, or can acquire as primary infection
b. XDR TB (exceptionally drug-resistant): combined resistance to all first line drugs
i. Most dangerous bacterial infection on planet: lethal, highly communicable, incurable
ii. Treat with 5 drugs, all second-line, not fun!

Molecular mechanisms of resistance:


 Always through chromosomal mutaitons (M. TB not permissive for plasmids or transposable elements)
 MDR resistance is therefore a staged process facilitated by noncompliance
o Can’t get an “MDR plasmid” or something like that; doesn’t occur precipitously

21
Drugs for Tuberculosis
General principles:
 Slow growth, characteristics of ‘cidal drugs = intermitant therapy (2-3x/week)
o Makes directly observed therapy (DOT) possible
 Duration of tx: reduced from 126 months (4,3 month tx under investigation; 1mo would be best)

Isoniazid (INH):
 Developed as isonicotinic acid hydrazine (INH): nicatinomide analog; nervous system agent; isopropyl
metabolite was more potent than isoniazide but more toxic (lead to MAOI development though)
 Mycobacteria specific

isoniazid (INH) Mechanism of Action: antituberculosis agent; inhibits synthesis of mycolic acids
Effects: 'Cidal. Blocks cell wall component (mycolic acid) production; accumulates inside mycobacteria &
forms oxygen free radicals, killing the cell.
Selective Toxicity: just passes in and out of human cells (doesn't accumulate, no free radicals)
Indications: tuberculosis (as single therapy for PPD converters or part of multi-drug scheme for active
disease). Can be used for post-exposure prophylaxis in pts under 35 (>35yo = risk of hepatotoxicity
outweighs benefit)
Administration: if normal TB: 4 drugs x 2 months, then 2 active drugs x 4 months. Coadminister with
vitamin B6 to prevent neurotoxicity
Toxicity:
 hepatitis (old age, slow acetylators, esp. in combo with rifampin; need to monitor monthly and
stop drug as soon as hepatic transaminases increase significantly).
 neurotoxicity (peripheral neuropathy; INH competes with nicatinomide & leads to relative
vitamin B6 deficiency; coadminister vitamin B6 to prevent)
Resistance:
 katG: catalase-peroxidase enzyme that probably "activates" drug through oxygen free radical
formation; mutation in katG means the drug doesn't accumulate inside the cell and INH isn't toxic.
 inhA: encodes mycolic acid synthesis enzyme; contains binding pocket for nicotinamide, confers
cross-resistance to ethionamide (INH analog), probably INH target; mutation is less common cause
of resistance that katG.
 MDR-TB is by definition resistant to rifampin and isoniazid; XDR resistant to all 1st line agents.
Other: metabolized by hepatic N-acetyltransferase (classic example of variable halflife because of gene
polymorphisms). Slow acetylators (83% egyptians, 50% caucasian americans) half-life = 6hrs, fast
acetylators half-life = 1 hr

22
Rifampin
 Unlike INH, is a broad-spectrum antibiotic.
 Also active against Gram (+) & some Gram (-) bacteria (Neisseria, H. influenzae, S. aureus).
rifampin Mechanism of Action: antituberculosis agent. Macrocyclic antibiotic, broad-spectrum inhibitor of bacterial
DNA-dependent RNApol
Effects: 'Cidal
Indications: 1st line anti-TB agent, part of multi-drug scheme for active disease.
Administration: if normal TB: 4 drugs x 2 months, then 2 active drugs x 4 months.
Toxicity:
 Orange discoloration (urine, sweat, tears, soft contact lenses).
 Hepatitis (can be more common in children; opposite of INH hepatitis).
 Hypersensitivity reactions (flu-like syndrome; more common in healthy patients).
 Light-chain proteinuria in > 50% pts ("unexplained" proteinuria in labs).
 Note: pharmacokinetics are non-linear! drug can accumulate if high doses (saturating clearance
mechanisms).
 Potent inducer of cyt P450s, including CYP3A4 (oral contraceptives, cyclosporin, coumarin, etc!)
Resistance: Via RNApol gene mutations. Emerges quickly if used as single agent. MDR-TB is by definition
resistant to rifampin and isoniazid; XDR resistant to all 1st line agents.
Other:
 Also highly bactericidal against most Gram (+) and some Gram (-) bacteria (H. influenzae, S. aureus)
 Neisseria - commonly used as PEP for N. meningitidis exposure or to eradicate nasal carriage.
 Metabolized by deacetylation (biliary excretion, enterohepatic recirculation, makes elimination half-
life longer than would be expected & unpredictable).

pyrazinamide Mechanism of Action: antituberculosis agent.


Effects: 'Cidal. Structural analog of nicatinomide, like INH
Indications: Tuberculosis
Toxicity:
 Hepatotoxicity (now rare; common with previous higher doses).
 Hyperuricemia (gout-like Sx possible);
 Photosensitivity dermatitis (rare, give at night to prevent).
Other: active at acidic pH; may be especially useful for killing intracellular mycobacteria. Not effective
against dormant organisms. Metabolized to pyrazinoic acid (renally excreted); half-life 12-24 hrs.

ethambutol Mechanism of Action: antituberculosis agent; mechanism of action unknown


Effects: inhibits both RNA synthesis & mycolic acid metabolism
Indications: Tuberculosis
Toxicity: Affects optic nerve: peripheral neuropathy (esp. retrobulbar optic neuritis) with color blindness &
eventual loss of peripheral vision. PERMANENT! Need to screen at baseline / every few months via
opthalmologist; stop as soon as color blindness occurs (esp important in children)
Aminoglycosides; quinolones can also be used against TB
 AGs:Streptomycin: less nephrotoxic, more vestibulotoxic than other AGs
o Others (e.g. amikacin) are active & have been tested; not widely used (toxicities & costs)
 Fluoroquinolones (esp. moxifloxacin) highly active & increasingly used (MDR/XDR-TB)

23
Dapsone (Leprosy)
dapsone Mechanism of Action: Anti-leprosy agent (sulfone; sulfonilamide analog); inhibits folate synthesis
Effects: Inhibits folate synthesis; bacteria can't produce nucleotides for DNA synthesis
Indications: Leprosy
Toxicity:
 Hemolytic anemia (esp. pts with severe G6PD deficiency - southern Mediterranean);
 Methemoglobinemia and subclinical hemolysis common;
 Hypersensitivity reactions (rash/fever) like sulfonamides;
 Agranulosis & fatal infectious mononucleosis-like syndrome (rarely);
 Reversal reactions and erythema nodosum leprosum can occur during initiation of therapy (kills bacteria
so fast that all bacteria lyse & cause reaction: severe fevers, big thick skin lesions, etc.).
Resistance: Increasingly common (previous extensive use as "monotherapy for life" for leprosy)
Other:
 Also active against Pneumocystis carinii/jiroveci (used as prophy in HIV pts); broad spectrum activity.
 Pharmacokinetics:n-acetylation (like INH, slow & fast acetylators genetic polymorphism).Half life 10-50h

Historically: Dapsone monotherapy for life


Now: 2-3 drugs for ≤ 6 months
Pauci-bacillary (PB) Leprosy Multi-bacillary (MB) Leprosy
(1-5 skin lesions) (>5 skin lesions)
2 drug regimen: 3-drug regimen:
Rifampin + Dapsone x 6 months Rifampin + Clofazimine + Dapsone x 12 months

Drugs for other mycobacterial infections:


 Rifabutin
rifabutin Similar to rifampin; more potent in vitro & longer half-life

Mechanism of Action: antituberculosis agent. macrocyclic antibiotic, broad-spectrum inhibitor of bacterial


DNA-dependent RNApol
Effects: 'Cidal
Indications:
 prophylaxis of MAI in AIDS patients with CD4 < 100 (only FDA use);
 also used for M. avium treatment and active against M. TB (but more expensive)
Toxicity:
 Orange discoloration (urine, sweat, tears, soft contact lenses).
 Uveitis (inflammation of anterior chamber of eye: dose-dependent; rare with regular doses, more
common if pts. on Rx slowing hepatic clearance - clarithromycin, fluconazole, etc.)
 Rare: granulocytopenia, rash.
 Potent inducer of cyt P450s, including CYP3A4 (oral contraceptives, cyclosporin, coumarin, etc!)
Resistance: Via RNApol gene mutations. Cross-resistance with rifampin.
 Macrolides (clarithromycin / azithromycin)
 Fluoroquinolones
Future directions for antimycobacterial therapy
New targets: ATP synthase; FA synthetases; 5-10 new drugs by 2020 but challenging under current pharma model

24
Quinolones

Gram (-):
First Generation  Nalidixic Acid UTIs only
Tissues
 Norfloxacin  Ofloxacin Gram (-)
“Fluoroquinolones” Second Generation some (+)
 Ciprofloxacin  Levofloxacin
 
Gram (+)
Third Generation  Gatifloxacin

AnO2
Fourth Generation
 Moxifloxacin  Besifloxacin
 Gemifloxacin

(First gen: concentrated in urine; only place that they reach therapeutic levels)

Discovery: chloroquine synthesis; thousands of analogs, rely on fluroquinolone pharmacophore for activity. 9 on
market; 4 “generations” based on activity/coverage, F introduced at C6 = “fluoro”quinolone

Mechanism of action: Inhibit prokaryotic type II topoisomerases (DNA Gyrase & Topoisomerase IV)

DNA topoisomerase review: ENZYME DRUG


Type I (single-strand break)
 Used to relieve supercoiling & get DNA in correct
IA prokaryotes None
topology for all aspects of its metabolism (RNA synth,
IB eukaryotes Camptothecins
DNA synth, recombination, higher order structure). Type II (double-strand break)
 Gyrase: introduces negative supercoils (relieves positive II prokaryotes (gyrase, topo IV) Quinolones
supercoils); operates ahead of the replication fork II eukaryotes Etoposide
 Topo IV: segregates replicating chromosomes
 Both are A2B2 tetramers; (2 DNA-binding and 2 ATPase subunits (also contact DNA)

DNA topo catalytic reaction (ATP dependent)


1. Tetrameric enzyme binds circular DNA chromosome
2. DNA cleaved
a. A “four base stagger” is introduced (see picture): four unpaired bases on
each cleaved side of the strand
b. Two tyrosines on the enzyme bind the 5’ phosphate on each cleaved strand piece, preventing DSB
c. This is a transient covalent intermediate
3. Strand passage through the cleaved strand
4. DNA ligated back together

Mechanism of action
 Form tetramers and base-pair with 4-base stagger + enzyme
 Stabilize DNA-topoisomerase catalytic intermediate (the “cleavable complex”).
o Enzyme can’t ligate DNA substrate; gets stuck in catalytic cycle
o This itself would be reversible (‘STATIC): if [drug] falls, FQ dissociates & enzyme can proceed
 Cleavable complex  irreversible DNA breaks (‘CIDAL)
o Replication fork collision  DNA strand breakage  SOS repair response

25
Target:
 Gram (-): Gyrase is primary target
 Gram (+): Topo IV is primary target

Selective toxicity:
 FQs bind selectively to DNA-gyrase / DNA-topo IV complexes,
not their human equivalents (topo II / gyrase)

Resistance:
 Mutations in gyrase/topo IV most common
1. DNA-binding subunit (first)
2. Secondary mutations: any other subunit
3. Gram (-): gyrase mutates first, Gram (+): Topo IV first
 Can also have mutations in membrane protein transporters (less common)
 Plasmid-mediated resistance is rare (encodes a protein mimic of DNA substrate; FQ binds this, won’t bind
enzyme complex instead)
 Increasing resistance = big worry! (up over 80% in some countries)

Spectrum of activity: 1st gen = gram (-), UTI only; more recent  gram (+), even anaerobes

Pharmacokinetics:
 Absorption: rapid & complete, great bioavailability (>85%)
o Magnesium/aluminum (antacids!) or iron reduces absorption!
 Distribution: wide; intracellular (up to 24x those of serum)
o CSF: 10-25% serum distribution (without inflammation)
 Metabolism:
o Ciprofloxacin: ~15% metabolized, mostly Phase I enzymes (oxidation); interferes with theophyline metabolism
 Mostly renal elimination
o Moxifloxacin: ~35% metabolized, mostly Phase II enzymes (conjugation / glucuronidation)
 1/3 renal; 2/3 biliary (conjugation) elimination
 Elimination: predominantly renal; some biliary/transintestinal
o In RENAL FAILURE, adjust for all EXCEPT moxifloxacin
o NO adjustment for hepatic failure (phase II enzymes sufficiently active even in advanced hep failure)

Toxicities
Class adverse effects
 Gastrointestinal (2-11%): nausea, vomiting, diarrhea
 CNS (1-7%): headache, dizziness, fatigue, sleep disorder; <0.5%: hallucinations, depression, seizures!
 Skin: up to 20% phototoxicity; 0.4-2% hypersensitivity (rash, pruritis)
 Arthropathy in juvenile animals  not FDA approved for children < 18yrs
 Tendon rupture: bizarre & rare but FQs are used a lot! > 200 cases reported; black box warning

Idiosyncratic / rare side effects: illustrate the importance of postmarketing surveillance for practicing docs
 Temafloxacin withdrawn because 1/3500 developed HUS; 2 deaths
 Grepafloxacin withdrawn: small number  torsade de oints
 Trovafloxicin withdrawn: serious liver toxicity, 1/25000

Future: looking to develop a “wundafloxacin”: this is a class that is broad spectrum, safe, excellent bioavailability, good
tissue penetration, ongoing subject of research; growing bacterial resistance & expense are downsides!

26
Antibiotic Resistance
Antibiotic drug resistance model: "every infection is a mini-epidemic"
 Assumption: microorganisms must reproduce in a manner that maintains or increases the body burden of the
microbe (otherwise it'll become extinct)
 Reproductive number (R): ratio new infectious organisms / original number (or infected cells produced) after
some arbitrary time period during which the organism's replicating.
o E.g. 1 organism --> 5 organisms, R=5
o R < 1: organism continues to grow & reproduce; R > 1: organism becomes extinct
 Effective antibiotic therapy: R < 1 in presence of antimicrobial drug
 Drug resistant organism: R > 1 in presence of antimicrobial drug

How do antibiotics promote drug resistance?


 Genetic mutations occurring continually in bacteria; use of abx reveals resistant organisms (selective pressure;
doesn't facilitate resistance-conferring mutations)

 Primary drug resistance: pre-dates drug therapy.


o When patient acquires the infection, the organism already has R > 1 for the drug.
o Exists because of improper use / overuse of antibiotics.
o Need to treat with drug that is known to be sensitive or combination, where R < 1 for at least one of
drugs. e.g. HA-MRSA.

 Secondary drug resistance: occurs during drug therapy.


o If therapy is effective, R < 1 & secondary resistance not likely to occur.
o If effective therapy taken incorrectly or irregularly, drug concentrations decrease & resistant organisms
can begin to survive & replicate.
o Selective pressure still applied by inadequate doses, so resistant organisms emerge (R>1)
o Non-adherence to prescribed drug regimen is cause of most infections exhibiting secondary resistance

Mechanisms of resistance:
1. Inactivation of antibiotic
o Beta-lactamase & beta lactams,
o aminoglycosides (acetylation, adenylation, p-ation), chloramphelicol (actylation)
2. Modification of antibiotic target
o D-ala-D-ala to D-ala-D-lac & vancomycin,
o alteration of PBPs & beta-lactams, methylation of rRNA to block macrolides, point mutations (RNApol:
rifampin, DNA gyrase: quinolones)
3. Efflux of antibiotic from cell
o Resistance to tetracyclines: inducible expression of efflux pump in response to tetracycline presence
o similar for macrolides, quinolones

Where do these come from?


 Pathogens' resistance mechanisms: similar to self-resistance mechanisms in organisms that produce antibiotics
(prevent self from destruction as they're killing their neighbors to compete for niche)
o Inactivation of oleandomycin via glycosylation, then efflux of inactive antibiotic (S. antibioticus)
o Modification of rRNA (erythromycin target) by methylation in S. erythraeus, which produces it

 Resistance determinants: clustered with biosynthetic genes to produce antibiotics in organism. Can be
transferred horizontally or acquired by neighboring microbes in several ways:
1. Transduction: transfer of DNA inside bacteriophage
2. Transformation: uptake of free DNA from environment, usually post-cell-lysis.

27
 Haemophilus, Neisseria, S. pneumonia are most common
3. Conjugation: Transfer of DNA that occurs during contact between bacterial cells (sex pili, etc).
 Most efficient than transduction/transformation
 Both plasmid and chromosome transfer can occur.
 Most common mechanism.

Practical implications & antibiotic use: bacteria are good at this, so abx resistance will always be around
1. Modification of existing scaffolds: might not work for much longer
2. Combination therapy: combine abx with different modes of action; lower chance that dual-resistance-
conferring organisms can emerge
3. Selective removal or restriction of antibiotic classes to reduce likelihood of resistance to particular classes of
antibiotics (don't prescribe it if you don't really need it)

New directions:
 New targets: unique/conserved in bacteria & essential for viability. DNA replication, metabolism, cell walls, etc.
 New molecules: keep screening (new places: ocean, etc.); combinatorial methods, traditional methods

28
Viruses

RNA DNA
(all listed are linear genomes)

SS (-) DS
DS Parvovirus (B19) Circular Linear
Rotavirus
(a reovirus)
Segmented Hepatitis B
SS Herpesviruses
 HSV 1,2 (oral/gen lesions,
keratoconjunctivitis, viral
(+) (-) Papillomavirus encephalitis)
 VSV
(chickenpox/shingles/zoster)

Picornaviruses Hepatitis E  EBV (mono, Burkitt’s, etc)


(PERCH) Influenza  CMV (immunosuppressed /
(an orthomyxovirus) transplant; congenital defects)
 Poliovirus Norovirus Segmented  HHV-6 (roseola/exanthema
 Echovirus (a calcivirus) subitum)
 Rhinovirus  HHV-8 (Kaposi’s sarcoma)
 Coxsackievirus Paramyxoviruses
 Hep A virus Flaviviruses (PaRaMMyxo)
 HCV  Parainfluenza Adenovirus
 Yellow fever  RSV  Febrile pharyngitis (sore throat)
 Pneumonia
 Dengue  Measles (rubeola)
Togaviruses  Conjunctivitis (pink eye)
 St. Louis Encephalitis  Mumps
 Rubella
 West Nile
 Eastern / Western
Equine Enceph.
Rabies
Coronaviruses (a rhabdovirus)
 Common cold
Retroviruses  SARS
 HIV HDV
(a deltavirus)
Pathology: ID & Micro (Viruses)
Pathology of Viral Infection .................................................................................................................................................... 2
Negative Strand Viruses .......................................................................................................................................................... 5
The Herpesviruses ................................................................................................................................................................. 10

1
Pathology of Viral Infection
Pathogenesis: how viruses cause disease in the host VIRAL STRATEGIES HOST DEFENSES
 Viruses: too small to examine with a light microscope like  Rapid replication  Barriers to viral entry
bacteria. Have to look for patterns in path.  Mutation  Innate immunity
 Virulence genes  Adaptive immunity
Viruses are obligate intracellular parasites
 Genome: RNA or DNA
 Must enter intact host cell; use host to synthesize components
 Progeny virus = virions are assembled in cell, can spread to another cell
 Each class of virus has specific host cells (species and often tissue specific)

Many similarities in viruses that have similar classification; similar symptoms Taxonomy:
even across species (good for animal models) 1. nucleic acid (DNA/RNA; +/-, ds/ss)
2. capsid (symmetry of protein shell:
Typical life cycle: entrygenome exposure  genome replicationmRNA icosahedral/helical)
synthesis  protein synthesis  assembly (viral proteins/virions) release 3. envelope (lipid membrane,
infection of new cell naked/enveloped)
4. dimensions of virion / capsid
Budding: enveloped viruses take part of the host cell membrane with ‘em.

Patterns of disease: vast majority of infections are subclinical. See chart (dark sections = viremia; can detect in blood )
 Acute (rhino, rota, influenza)
 Persistent (lymphocytic choriomeningitis v.)
 Latent/reactivating (herpes)
 Slow (HIV, measles)

Virulence: ability to cause disease (=pathogenicity)


 Polygenic control: different genes control
binding/entry/replication/effects on cells
 How does it enter/spread? Why in certain
cells/tissues? How does it cause damage?
How does immune system cause indirect
damage? How does it go to a new host?
 Can measure as: mean time to death (animal
models), viral load, T-cell counts, other
measures specific to pathogen.
Virulence classification
 Virulent: causes disease;
 Attenuated: no/reduced disease
 Avirulent: no disease

Types of viral virulence genes: studied with tissue culture & animal models + mutations
1. Viral replication: herpesviruses’ DNApol brain only; poliovirus 5’ NCR mutated so not in brain
2. Defeat host defense:
o virokines (viral equivalent of chemokines, subvert immune response),
o viroceptors (tie up host chemo/cytokines)
o not required for growth in vitro but help out in vivo
3. Promote virus spread within/among hosts: gD protein in HSV1 recognizes cell receptors; pt mutation blocks CNS spread
4. Toxic gene products: cause cell injury directly (virotoxins), cause Cl secretion (osmotic diarrhea), etc.

2
Tropism: virus has to enter the cell (susceptibility) and then replicate inside it (permissivity)
 Neurotropism, Pneumotropism, Enterotropism: or all (pantropism)

Viral receptors: required for viral entry; determines tropism (host & tissue), some also need co-receptor, active process
 E.g. HIV-1: two tropic strains depending on co-receptors
o T-cell-line-tropic strain (CD4 + CXCr4 co-receptor)
o Macrophage tropic strain (CD4 + CCr5 co-receptor)
 Receptors can be integrins, Ig-like molecules, GAGs, CHOs
 target for treatment & protection (e.g. CCR5 antagonists in HIV Rx)

Spread: direction of release determines infection pattern (superficial or down deep?)


 local replication (influenza through respiratory epithelium; papilloma through skin)
 systemic spread (must cross basement membrane, etc.)

Viremia: viruses can be carried to blood & disseminated


 often within cells: monocytes (measles, HIV, CMV); lymphocytes (HIV, EBV, HHV), neutrophils (influenza) or free in
plasma (poliovirus, HBV). In cells is a good way to circumvent BBB
 Can disseminate through lymphatics too

TRANSMISSION HOST DEFENSES EXAMPLES


Hand-shaking Mucociliary apparatus URT: Rhinovirus, coronavirus,
RESPIRATORY parainfluenza virus, RSV, influenza
Coughing Alveolar Mφ LRT: adenovirus, parainfluenza, RSV,
SYSTEM
Sneezing Adaptive immune response influenza virus
Eating Stomach pH, Digestive enzymes
Rotavirus, reovirus, measles,
GI TRACT Drinking Flow of ingesta poliovirus, adenovirus
Poor hygiene Adaptive immune response
Urine flow
Sexual activity Thick epithelial layer HIV, HSV (lifelong persistent/latent)
UROGENITAL TRACT HPV: cervical cancer
Fecal contamination Acid pH
Adaptive immune response
Conjunctiva Tears
EYES Abrasions Thick epithelial layer HSV: lifelong persistent/latent
Direct inoculation Adaptive immune response
Cuts, abrasions Poxviruses, papillomaviruses, rabies,
Epidermis Insects: togaviruses/alphavirues
SKIN Insect bites Emerge from below: systemic
Skin oils
Needles infection: measles, chicken pox
Cuts, animal bites; Inhalation
Rabies, herpes simplex, HIV,
NERVOUS SYSTEM Cell trafficking (retrograde transport Blood-brain barrier measles, alphaviruses
up axons to DRG/CNS)

Types of Viral Damage to Tissues

Cytopathic effects (cyto=cell, pathic = abnormal): can be in vivo or in vitro; NOTE: not all viruses produce CPE
1. Cell swelling: bloating of cells
2. Necrosis:
a. ballooning degeneration from membrane injury Cytopathic effects
b. host protein/nucleic acid synthesis shuts down 1. Cell swelling
c. Cell death (pyknosis, hypereosinophilia) 2. Necrosis
i. Single cell necrosis 3. Apoptosis
ii. more widespread (depending on virulence of pathogen) 4. Inclusion bodies
d. Lysis / detachment allows virion release 5. Syncitia/multinucleated
e. Tissue architecture disrupted (caseous or coagulation) giant cells
f. Vesicles can form (necrotic cells, fluid-filled space under epithelium) 6. Cellular hyperplasia /
proliferation 3
g. Can cause malformations during fetal development
3. Apoptosis:
a. Some viral genes promote apoptosis (aid in virus dissemination)
b. Some inhibit apoptosis (longer replication, establish latency)
c. Some do both (HIV’s “Tat”) depending on context.
4. Inclusion bodies: arrays/aggregates of viral/cellular products
a. Often present only very early in infection
b. Intranuclear and/or intracytoplasmic
c. Can be eosinophilic, basophilic, or amphophilic
d. Usually > ½ diameter of cell
e. Can see peripheralization of chromatin in big inclusions; some look like owl’s eyes
f. Not pathognomonic but a signature microscopic finding, good for aiding in Dx
i. Not all viral: e.g. bismuth inclusions in liver
5. Syncitia/multinucleated giant cells
a. Viral fusion proteins expressed on cell surface  cells fuse together (in vivo/vitro)
b. Allows virus transmission without exposure to host defenses
c. Differentiate from: foreign body giant cells, osteoclasts, megakaryocytes
6. Cellular hyperplasia/proliferation
a. Self-limited & transient usually but may be PRE-NEOPLASTIC
b. May be due to atypical differentiation or accumulation of viral products
c. E.g. molluscum contagiosum, pox virus, EBV burkitt’s lymphoma, HPV cervical carcinoma

Alteration of host cell functions leads to the visible cytopathic effects – can also alter other functions (cytoskeletal
depolymerization, for instance)

Host Responses

Classic: MONONUCLEAR CELL INFILTRATES (LYMPHOCYTES, lymphocytes, lymphocytes… and macrophages)


 Exception: arbovirus can produce PMN response

HIV encephalitis (somewhat general features of viral incephalitis)


 Microglial nodules (collection of macrophages)
 Perivascular cuff of lymphocytes & macrophages
 Multinucleated giant cells (macrophages fuse; full of virions)

Virus-induced immunopathology: can be CD8 or CD4 (Th1 or TH2) T-cell mediated, antibody mediated, etc.
 Can result in immune deposits in glomeruli & cause pathology there

Host susceptibility can vary:


1. Genetic:
o MHC class I diversity: different ability to present peptides;
o Chemokine receptor tropism (HIV elite suppressors); other genetic polymorphisms for different viruses
2. Non-genetic:
o Age (infants/elderly usually)
o Gender (males, pregnant women more susceptible)
o Malnutrition (measles: protein deficiency)
o Other: corticosteroids, cig smoking, stress, etc.

Why study this stuff? Viruses are constantly emerging and re-emerging; classes tend to cause similar diseases, so if we
study something we’ve seen before, we might be better prepared when something new emerges.

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Negative Strand Viruses
Positive vs Negative strand viruses
 Negative strand: antisense genome, polymerase included with Key features of Negative Strand Viruses
incoming virion; first step is to make a + strand (full length  RNA is not infectious
antigenome)
 Virion contains RNA-dep-RNApol
 Postive strand: sense genome, no polymerase included with
 Encapsidation: Genomic RNA
incoming virion; polymerase synthesized as first step via packaged in protein (“nucleocapsid”)
genomic RNA translation
 Nucleocapsids have helical structure
 Enveloped virions
Influenza Viruses  Entry: virion fusion or cell-cell fusion

3 types: A, B, C
 A/B antigentically distinct, structurally similar
 Both cause dz in adults and children
 A more prevalent than B
 Influenza A: ducks, chickens, horses, swine
o Birds = largest reservoir

Transmission:
 Birds  pigs, other non-humans (rarely humans
with some exceptions)
 Pigs  Humans is most common

Virion: structural features


 Major genes: HA, NA, M2
 Segmented genomes (1-3 genes/segment; HA & transcription complex on separate segments)
 Lipid envelope with viral glycoproteins (HA/NA)
o Envelope fuses with host lysosome membrane to allow genome to enter cell

Reassortment: Antigenic shift: possible mechanism


1. 2 strains infect same cell (e.g. two swine strains in a pig cell) 1. chicken strain / human strain
2. All genome segments replicate infect a pig,
3. When virus assembles, mixing of segments happens  reassortant 2. reassortant virus results with:
progeny viruses a. chicken strain antigens
(evades human defense) and
b.human strain machinery
(replicates in humans)
Hemagglutinin
3. pandemic strain can result
 16 antigenic types (flu A, H1-14); trimer with globular head on stalk
 Functions:
o binds sialic acid
o binds and agglutinates RBCs (have sialic acid)
o mediates fusion of viral envelope with cell membrane
 Targeted by neutralizing antibodies: keep virus from binding to host cells
 Minor mutations result in antigenic drift: year/year variation, causes seasonal epidemics
 Replacement with gene from alternate hosts results in antigenic shift (causes pandemics)

Mechanism of entry:
1. virus binding (HA/sialic acid)  endocytosed 
2. low pH induces conformational change in HA in endosome  hydrophobic AAs in HA exposed 
3. fusion of envelope with endosome membrane  release genomes into cytoplasm
5
HA – needs cleavage for activity:
 synthesized in pro-form; needs cleavage for conformational change/activation of fusion function
 cleaved by tryptase Clara, serine protease secreted by nonciliated Clara cells in bronchial/bronchiolar
epithelium (lumen of respiratory tract; may account for restricted location of virus replication)
 happens at defined site (R/K); both HA fragments remain bound together (dipeptide linkage)

Neuraminidase
 Tetramer; 9 recognized subtypes in influenza A (N1-9)
 Cleaves sialic acid residues on cell surface during virus exit; if mutated, can’t exit cell surface

Nomenclature: Type / # of isolate / year of first isolation / HA&NA subtypes


 Example: A/Hong Kong/156/97
 H1N1, H3N2 currently circulating (usually 1-2 for seasonal epidemics)

M2 protein
 Tetramer, spans viral envelope, activated via acidity of endosome
 Pumps protons into virion: loosens protein-protein contacts, facilitates virus uncoating
 Target of amantadine

Replication
 Genome replication happens in nucleus
o No mRNA capping/methylating enzymes, so steals caps (+10-13nts) to prime mRNA synthesis
 Viral mRNAs translated on sER & rER
 HA/NA transported through Golgi to cell membrane,
 Glycoproteins (HA/NA) aggregate on surface of cell membrane, viral proteins & genomes aggregate underneath,
and the virus buds off, coated in an envelope

Location: ciliated columnar epithelial cells in respiratory tract; causes tracheobronchitis


 Lots of virions  shed into respiratory tract (better transmission)
 Virus-induced apoptosis of infected cells, damages respiratory tract
o Protective mucus layer disrupted
o Respiratory epithelium denuded
o Transudates / exudates (inflammatory cells, dead epithelial cells)

Clinical features: Respiratory, seasonal (winter) transmission


 1-4d post-infection: H/A, fever, myalgias, non-productive cough, sore throat, no rinorrhea (3-7d)
o Sx: local production of IFN & IL-1 (localized cell destruction b/c of immune response)

Immunity
 Innate resistance: mucus barrier, clearance by cilia, alveolar Mφ
o Impairment in any of these: ↑ risk infection (elderly, smokers, COPD, immunocomp, pregnant)
 Adaptive immunity:
o Protection: IgA (mucosal), IgG (serum)
o Clearance: IgG + complement, CTL

Complications:
 Primary virus infection: Interstitial pneumonitis
o Cardiovascular dz; pregnancy predispose
o Progression from classical 3d sx  bilateral findings, no consolidation

6
o CXR: bilateral infiltrates
o Non-bacterial: normal flora in sputum, no Abx response (high mortality)
 Secondary bacterial pneumonia:
o from damage to innate immune system, destruction of ciliated epithelial cells, abnormal Mφ function
o Age > 65yo, pulmonary dz predispose
o Improve, then worsen; consolidation
o CXR: consolidation
o Bacterial: sputum shows S. pneumo, S. aureus, H. flu, Abx response (low mortality)

Immunization
1. Killed/inactivated vaccine (mostly HA/NA)
a. Reformulated annually (WHO isolate/IDs viruses, reports strains to reference lab, panel makes rec)
b. Health care workers, populations at high risk of morbidity & mortality
c. Partial protection: incidence ↓ 30-70%, morbidity/mortality ↓ 60-90%
2. Live attenuated intranasal (FluMist)
a. Replication restricted to nasopharynx: cold-adapted (grows best @ intranasal temp); restricted
replication at 37C)
b. Reformulated annually; approved for use in healthy people 5-49yo

Diagnosis
 Direct detection (stain NP aspirates with flu-specific mAb), culture

Paramyxoviruses
General characteristics:
 No epidemiologically important antigenic change Paramyxoviruses: medically important
 No natural reservoir: constant person-person spread 1. Parainfluenza 1-4
2. Respiratory Syncitial Virus
 Spread: respiratory route
3. Human metapneumovirus
 Various proteins: H (receptor binding), F (fusion), M (assembly),
4. Measles
others too.
5. Mumps
 Genome nonsegmented, mRNA generated by polymerase
reinitiation at different promoter regions.

Surface glycoproteins: CHO attached during ER/Golgi transit; usually 2 proteins


1. Cell attachment: binds cellular receptor, elicits neutralizing Ab
2. Fusion protein (F): must be cleaved to F1/F2 by intracellular proteases to be active; required for virion
infectivity (happens only at neutral pH)

Replication cycle:
1. fuse @ neutral pH  intracellular replication (all RNA in cytoplasm, H/F  ER/Golgi  PM)
2. exit: nucleocapsids assemble underneath H/F; virion assembly mediated by matrix protein
a. Virion budding from cell membrane
b. Fusion with adjacent cell (surface proteins fusogenic @ neutral pH)
i. Leads to GIANT CELLS & syncitia formation

Respiratory Syncytial Virus (RSV)


 Outbreaks of respiratory disease in winter
 Transmission: direct contact with respiratory secretions (not aerosolized)
Clinical presentation:
 Otitis media, bronchitis, bronchiolitis, croup (inspiratory stridor), pneumonia
o Most severe in young infants; partial immunity after primary infection (less severe disease)
 Can progress: cough, wheezing, dyspnea, ↑ RR, hypoxemia
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 1% infants require hosp, 1% of those die
Diagnosis:direct staining of NPAs with fluoro mAB, culture
Vaccine: under development

Human Metapneumovirus
ID’d Netherlands 2001 from respiratory specimens from 20+ yrs; cytopathic effect similar to RSV (syncytia),
 Most infections: childhood (<5yr)
 Causes 7-40% ped resp infections
 Parallels to RSV:
o seasonal (winter), initial exposure in childhood, severe dz in infants/elderly
o range of clinical sx: mild respiratory symptoms to severe cough, bronchiolitis, pneumonia
o repeated infections occur but less severe dz (URI only)
Dx: RT-PCR, Ab for direct-detect

Parainfluenza
 Common cause of URIs
 Most common cause of croup (laryngotracheo-bronchitis) in young children
 Most children: infected by 5 yo, can be re-infected by less severe
 Diagnosis: culture; vaccine not yet available

Measles
 Worldwide distribution; incidence varies with vaccination rates
o Epidemic if high vaccine coverage; Endemic if low
 Age: changed with countries with high vax rate
 Transmission: Respiratory & Aerosol
o Attack rate: 99.9% (only 1:1000 escapes infection if exposed!)
o Mortality rate: developing countries, 30% in infants 6-9mos

Pathogenesis: Systemic replication


1. Respiratory epithelium  local lymph nodes  dissemination via infected monocytes from resp LNs
a. Primary viremia (LOW) from this first dissemination
2. Epithelial / endothelial cells infected throughout body, release virus into blood
a. Secondary viremia (HIGH) from this second dissemination

Clinical Symptoms: happen during viremia


 Prodrome: fever, cough, coryza (sx of head cold), conjunctivitis, Koplik spots (in oral mucosa: red dot & white
clearing around it, virtually pathognomonic)
 Maculopapular rash with immune response (virtually diagnostic): cellular response
o Immune responses: CD4 help, CD8 clearance, IgM, neutralizing IgG

Measles-induced immune deficiency


 Generalized immunosuppression; increased susceptibility to secondary infections
 Multiple mechanisms:
o Early: lymphopenia (activation-induced cell death, apoptosis of infected cells)
o Middle: delayed DTH, decreased lymphoproliferation
o Late: thymus effects?
Diagnosis: clinical picture, direct mAb stain of resp secretions, culture, serology (IgM)
Prevention: live attenuated vaccine (infants 2-15mo)
 has changed age distribution (now very young infants and older children/young adults 10-22yo)

8
Mumps
Infection of glandular epithelial cells
 Parotitis & orchitis most commonly recognized (big swollen jowls or testicles; mumps gives ya bumps)
 Pancreatitis/ovarian infection occur but infrequently recognized
 Meningitis 10% all cases
Diagnosis: Culture (saliva, urine, CSF); serology, molecular methods now
Prevention: live attenuated vaccine

Rhabdoviruses
Rabies is only important human pathogen
 Incidence: depends on control of domestic animals (better in US than developing world)
 Endemic in wildlife: bats, raccoons, skunks, coyotes, foxes
 < 10 cases / yr in US; mostly imported or contact with rabid bats

Pathogenesis:
1. in saliva of bite of infected animal  limited replication in muscle, subepithelial tissue
2. uptake by sensory/motor neurons; retrograde transport to cell body & major replication there
3. trans-synaptic transmission: early avoidance of immune reponse

Prodrome: fever, malaise, parasthesias at bite site


Later (in CNS now): anxiety, aggressive behavior, seizures, hypertonia, paralysis

Diagnosis: clinical Hx of exposure, biopsy, immunohistochemical staining, PCR


Prevention: inactivated vaccine
 Long incubation period allows for post-exposure use
 Pre-exposure if in vocation with high risk of exposure: vet, wildlife managers, etc.

9
The Herpesviruses
Note that these are in the same family as EBV, HHV8, etc. which cause cancer (previous lecture)

Common features: similar morphology, ubiquitous, asymptomatic infection, common modes of replication / life cycles
 ALL establish LATENT infections ≪ notorious feature of herpesviruses; Reactivation can produce disease
 Seroprevalence: extremely high in young adults (latent & lifelong)
o HHV8 is the only rare one

Physical characteristics:
 Enveloped, have nucleocapsid with genome woven into protein coat
 Gargantuan genome (>100k, 50+ genes)
1. Enzymes & structural genes
2. Non-structural genes too: modulate host cell gene expression, host immune responses

Infection: 2 “modes”
1. Productive (“lytic”) infection: release of progeny virions
2. Latent infection: no virions produced, reservoir for recurrent disease
a. Recurrent disease results from:
renewed replication or induced cell proliferation (tumor-inducing γ-herpesviruses only)
Lytic infection:
1. Viral entry (envelope fusion  nucleocapsid transported to nucleus 
2. Gene tx, genome rep, progeny nucleocapsid assembly in nucleus 
3. Nucleocapsids bud from nucleus – viral envelope is formed from nuclear membrane (unusual)! 
4. Release via exocytosis
TEMPORAL CASCADE OF LYTIC INFECTION GENE EXPRESSION
Genes Functions
Although latent infection has restricted cell tropism (see
α (immediate early) regulators of viral gene expression
comparison table), knowing where the virus “hides out”
β (early) proteins for genome replication
during latency is important (see slide on right)
γ (late) viral structural proteins
Transmission:
LYTIC INFECTION LATENT INFECTION
Natural modes: “mixing & matching of skin & mucous
membranes” Lots, temporal
 skin, genital tract: HSV-1/2 GENE EXPRESSION cascade (see Restricted
 oral secretions: HSV-1/2, CMV, HHV-6, EBV table)
INFECTED CELL TYPES
 respiratory tract: VSV (weird) Many (≥2) Few (1-2)
(TROPISM)
Iatrogenic modes
VIRION PRODUCED? Yep Nope
 transfusion (e.g. CMV, hiding in monocytes)
 transplants

Disease manifestations:
 Low severity: Recurrent infections, immune competent
 High severity: Primary infections, immune impairment
o Populations with severe infections:
immunodeficient (HIV, etc.),
immunosuppressed (transplant pts, cancer pts),
fetuses/newborns, malnourished pts, burn
victums
o Use prophylactic antibiotics when indicated; high index of suspicion to dx/treat

10
Herpes Simplex Virus Diseases
Most common presentations: herpes labialis & genital herpes

Pathogenesis
1. Transmission: skin/skin or mucous
membrane/mucous membrane contact
2. Primary infection: epithelial cells (productive)
3. Retrograde transport up axon
4. Secondary infection: sensory neuron cell body
(latent)
a. Orolabial: trigeminal ganglia
b. Genital: sacral ganglia
5. Reactivated(stress, illness, UV light)
6. Anterograde transport down axon
7. Recurrent infection: epithelial cells (productive)

Genital herpes infections: mostly HSV-2 (20-50% HSV-1)


Oral herpes infections: mostly HSV-1 (5-20% HSV-2)
(Note that it’s not “1 above the waist, 2 below” like people think)

Reactivation can be symptomatic or asymptomatic (1-5% Asx in orolabial, 3% Asx in genital)


 Still shedding during Asx reactivation! Good way to spread

Clinical presentations (non-genital herpes)


1. Primary gingivostomatitis (lesions on gums, oral cavity, lips)

2. Herpes Whitlow (from sucking thumb – lesion on end of finger)

3. HSV keratitis: #1 cause of infectious blindness in developing world; dendritic ulcers in eye
2 mechanisms of pathogenesis
o Autoinoculation (orolabial, spread to eye)
o Trigeminal nerve ophthalmic root infection (after ganglion reactivation)

4. Neonatal herpes: 1/3500-5000 deliveries, Neonatal Herpes: 3 syndromes


o transmission through infected birth canal (rarely placental) 1. Skin, eye, mouth (SEM): 40%.
o Most women asymptomatic during labor Usually not fatal but recurs;
o Presentation: first 1-2 wks life; 3 syndromes (see box) 30% w/ neuro sequelae
o Vesicles: can’t use to rule in/out (SEM>CNS>disseminated) 2. Encephalitis:35%
3. Disseminated: 25%
5. Herpes encephalitis:
o most common acute, sporadic encephalitis (10-20% cases)
o Primarily HSV-1
o Classic presentation: fever + focal neuro defects (temporal lobe: memory, mental status)

Genital Herpes
TRANSMISSION IS COMMONLY UNRECOGNIZED
 Asymptomatic shedding is common Epidemiology of genital herpes
(70% acquired from asymptomatic partner)  500K cases/yr in US; 40-60M prevalence
 Primary infections are often asymptomatic  Correlated to number of sexual partners
(80-90% infections unrecognized!)  Women > Men for susceptibility
 Factors unknown (shedding rates don’t impact transmission!) (unknown why, 8% vs 2% /yr)
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Recurrence
 Recurrence w/ Sx can occur after years of “silent” infection; don’t assume infidelity!
 Symptoms less severe than primary (shorter shedding duration, fewer lesions)
 Frequency: 90% have >1 recurrence / yr, 40% >6, 20% >10
Acyclovir & Genital Herpes
 Factors that affect recurrence:
 Reduces recurrences
o time since acquisition (shedding declines 70% over 10 yrs) (w/Sx by 75%, Asx too)
o virus type (HSV-2: more, more severe recurrences than HSV-1)  Reduces transmission
o immune status (immunocompromised = more frequent (50%)
recurrence)

Varicella-zoster Virus (VSV)


Primary infection: Varicella (chicken pox) Recurrent infection: Herpes zoster (shingles)
“dew drop on rose petal” lesions Reactivation in sensory ganglion
(glistening vesicle, red base) Lesions localize to innervated dermatome

Causes severe disease in: Immunocompromised pts:


1. Teens/adults  can disseminate (bad) & spread epidermally
o At risk for varicella pneumonia  ≥ 2 dermatomes
2. Immunocompromised/newborns  Across midline
o life-threatening pneumonia
o encephalitis Disseminated zoster infection: everywhere
o progressive/disseminated varicella

Cytomegalovirus
Usually asymptomatic but can cause disease:
CMV Mononucleosis 80-20 rule (wards)
 20% of mono (EBV = 80%) If common: say 80%
 Frequent manifestation of primary CMV infection in young adults If uncommon: say 20%
 Fever, lymphadenopathy, lymphocytosis without exudative pharyngitis
o EBV = sore throat, CMV = usually not
 HETEROPHIL ANTIBODY NEGATIVE: monospot test will come up negative! (unlike EBV)

Congenital infection
 Most common congenital viral infection
 Severity depends on maternal serostatus in pregnancy
o Primary maternal infection: severe symptoms ~25% births
 Jaundice, hepatosplenomegaly, petichial rash, cerebral calcifications, chorioretinitis, motor disability
 20%: late onset hearing loss
o Reactivation during pregnancy: usually asymptomatic at birth
 but 15% with late onset hearing loss!

Special populations (solid-organ/bone marrow transplants; leukemia/lymphoma pts, AIDs pts with low CD4)
 HIV: reactivation/disease when CD4 < 50 (uncommon with HAART)
o Retinitis, encephalitis, colitis with ulcers
 Bone marrow transplant: commonly pneumonia (prophy with ganciclovir)
 SOLID ORGAN TRANSPLANT: huge problem!
o Usually manifests as disease in allograft
 liver = CMV hepatitis, lung = CMV pneumonitis (renal ≠ nephritis though)
o Highest risk: CMV seronegative recipient and seropositive donor

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HHV-6 and -7
Roseola infantum (exanthum subitum): rash-like illness in young kids & transplant patients
 Can see febrile seizures, other sx possible, may contribute to HIV disease progression & exacerbate other viral dz
 Erythematous rash

Herpesvirus diagnostics
 Viral culture (HSV from all sites except CSF, makes it hard to culture for HSV meningitis!)
 Rapid antigen detection from lesions
 PCR for nucleic acids (including CSF)
 Antibody detection: limited utility except mono & to identify high risk pts for transplants

13
Pathophysiology: ID & Micro (Viruses)
Introduction to Virology.......................................................................................................................................................... 2
(+) RNA Viruses ....................................................................................................................................................................... 5
HIV (and retroviruses) ............................................................................................................................................................. 8
Small DNA Viruses: Parvoviruses & Papillomaviruses .......................................................................................................... 12
Influenza: Epidemics, Pandemics, and Prevention Strategies .............................................................................................. 15
Viral gastroenteritis............................................................................................................................................................... 18
Gammaherpesviruses: EBV / KSHV ....................................................................................................................................... 20
Viral Hepatitis........................................................................................................................................................................ 23

1
Introduction to Virology
History: “filterable agent” (not like bacteria); nucleic acid infectious, no binary 3 basic types of virus
fission, requires host, first described in 19th century (agriculture (TMV)  1. Bacteriophage
animals (foot/mouth) humans (yellow fever) 2. Animal/plant (DNA or RNA)
3. Retrovirius (RNADNARNA)
Virion: virus particle (viral nucleic acid + structural proteins)
 Structural proteins = payload vehicle to deliver nucleic acids Properties of viruses
 Example: alphavirus is enveloped, icosahedral
 Small, infectious, obligate
Structural proteins: encoded by viral genome; packaged into virion (protective intracellular parasite
coat for nucleic acid)  Genome: DNA or RNA
 Protein capsid ± lipoprotein envelope (cell membrane of host cell)  In host cell: genome
replicated, synthesis of other
Nonstructural proteins: encoded by viral genome, not packaged into virion virion components via host
 Enzymes (polymerases, helicases, etc) or transcription factors systems, progeny assembled
 Needed for viral replication in cell

Usually nonstructural proteins encoded 1st on genome (5’ end) because they’re needed for translation/transcription

Basic structures: icosahedral / helical; enveloped or non-enveloped Taxonomy:


 Example: picornavirus (common cold). Icosahedral (20 triangular faces, 1. nucleic acid (DNA/RNA; +/-, ds/ss)
12 verteces). 2. capsid (symmetry of protein shell:
 Common motif for icosahedral: 8-stranded antiparallel β-barrel icosahedral/helical)
3. envelope (lipid membrane,
Basic viral genome structures naked/enveloped)
4. dimensions of virion / capsid
(+) strand (sense) Non-segmented
Single stranded Non-segmented
RNA

(-) strand (antisense)


Segmented
Double stranded Segmented
Single stranded
DNA

Linear
Double stranded
Circular

Viruses evolve rapidly; produce large #s progeny; RNApol has no proofreading function (population = “quasispecies”)
 Mutation
 Recombination (two viruses in same cell, recombine)
 Reassortment (2 viruses with segmented genomes in same cell, e.g. flu)

One step growth curves: takes a day or two, then kicks into gear. Many fold higher # organisms than bacteria

Viral replication cycle: attachment, penetration, uncoating, transcription of early mRNA/translation early proteins,
replication of viral DNA, transcription of late mRNA, translation of late proteins, assembly, release.

Cell surface molecules for virus attachment:


 CHOs: linked to proteins/lipids (sialic acid, GAGs)
 Lipids (glycolipids, proteolipids)
 Proteins (immunoglobulin superfamily, C’ –regulatory proteins, integrins, TNF receptor superfamily)

Receptor binding sites: can be depressions (picornavirus “canyons”) or projections (rotavirus “fibers”)
2
 Neutralizing antibodies can bind to these receptor sites; block ability to interact with receptor (e.g. bind to
rhinovirus canyon)

Viral genome: all viruses need a strategy to make RNA


 (+) strand RNA viruses: make (-) strand as template to copy, or can undergo direct translation
 (-) strand RNA: need to make (+) strand to copy (have polymerase)
 DNA viruses: need to get into host nucleus for transcription; then mRNA translated in cytoplasm

 Early mRNA: products used to help with transcription, etc.


 Late mRNA: products for structural organization, assembly, etc.

Problems for viruses to overcome


1. Package info for replication into small genome
o Big variety of sizes of genomes
o Strategies: overlapping reading frames, code from both strands, splice RNA, frame-shift, RNA editing

2. Maintain in population without dying out


o Transmission
 Humans: respiratory/salivary, fecal-oral, or sexual contact
 Animals: vector (arthropod), vertebrate reservoir, vector+vertebrate reservoir
o Types of infection: acute, persistent, latent, relapsing
o Persistence: can’t kill host, kill cells in which virus replicates, or be eliminated by immune response

3. Need both stability (transmission) and instability (infection)


o Entry & uncoating strategies
 Endocytosis: both enveloped/non-enveloped,use clathrin-coated pits, enter cytoplasm; fuse
with endosome with acidification (often needed for viral protein conf. changes)
 Fusion: enveloped only, fuse directly with cell
Cytopathic effects:
membrane, discharge virus into cytoplasm
o Outcomes of infection  Rounding / swelling
1. Lysis  lysis
2. Transformation (e.g. pre-neoplastic)  syncytia formation (fusion, esp.
3. No pathological effects enveloped viruses)
 Chronic dysfunction can still result  hemadsorption (absorption on RBC)

Pathogenesis of viral infection

Disease : can be at site of entry (e.g. HSV) or at distant target organs(Coxsackie virus, enters via GI tract  myocarditis)
Time course of symptoms: due to local & systemic infection
 Local: earlier onset of Sx, due to infection of body surface (e.g. cold)
 Systemic: later onset of Sx, from immune response (e.g. measles)
 Rabies, hepatitis can be weeks, others are pretty short

Immune response
 Interferon:
1. dsRNA intermediate presence triggers Mϕ to synthesize & release IFN
2. IFN signals other cells via JAK-STAT pathway to induce antiviral protein genes (inhibit viral release /
products; ↑ MHC CLASS I EXPRESSION
3. Actually appears to control spread of virus before acquired immune response (acquired mops up,
allows for long-lasting immunity)
 Antiviral Antibodies:

3
1. Serological tests: Dx (ELISA, radioimmunoassay, Westerns)
2. Biological activity: function of Ab?
 Neutralizing (can’t cause productive infection). Effective immune response
1. Eliminate virus from blood/other
Can block attachment, endocytosis, uncoating.
 C’ fixation (causes cell death) fluids (prevent further spread)
 Hemagglutination inhibition (binds viruses 2. Eliminate virus-infected cells
together; can’t productively infect) from tissues (“cure infection”)
3. Roles of antibodies: protect against reinfection, clear 3. Immunity to re-infection
virus from fluids, downregulate intracellular virus replication (not completely understood)
 MHC Class I:
1. Mouse experiment: cytotoxic t-cells only kill MHC-I matched virus-infected target cells
 Cell-mediated immunity: focus immune response (target), clear infected cells, recruit other effector cells,
activate Mϕ , provide help for production of Ab by B cells

Basic immune response scheme:


1. Virus enters
2. Mϕ are 1st responders, pick it up
3. If dsRNA: IFN made
a. T-helpers activated to recruit B cells, Ab made against Mϕ and other infected cells
b. ↑ Mϕ activation (more MHC class I, etc.)
4. Infection cleared
a. T-suppressor cells help tone down immune response
b. Memory B-cells produced (longer-lasting immunity)

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(+) RNA Viruses
Picornaviruses
Picornaviruses: Pico (small) RNA Viruses
 Icosahedral Human picornaviruses
 Receptor binds into canyon; neutralizing antibodies bind canyon too  Rhinoviruses
 Entry: endocytosis uncoating  conformational change in acidified  “Enteroviruses”
endosome  extrusion of RNA into cytoplasm (injected) o Polioviruses
 Plus-strand viruses: produce (-) strand intermediate in cytoplasm of cell o ECHO viruses
 Replication: (+) RNA translated as single polyprotein; viral proteases o Enteroviruses 68-71
cleave into individual proteins o Coxsackie viruses A&B
 Translation: have internal ribosomal entry site (IRES) in 5’UTR of RNA  Hepatitis A virus
o IRES: RNA can bind directly to ribosome w/o 5’ 7-methyl cap or
cap-binding protein

Clinical presentation
 Rhinoviruses: cause local upper respiratory disease (stay in resp. tract)
o Generally pediatric problem and nuisance
o Exception: asthma patients
 Enteroviruses: systemic infection
o Fecal-oral transmission  GI tract  viremia (in blood)
o Can go to
o Skin (hand-foot-mouth disease): Rash: pustules on skin
o Muscle (echovirus, coxsackie A/B): myocarditis, pericarditis
o CNS: Brain (polio, coxsackie A&B), meninges (echo, polio, coxsackie)
 Example: paralytic sequelae of poliovirus: limb atrophy

Poliovirus
Transmission: fecal-oral (land runoff, sewage, solid waste landfills)

Pathogenesis: infects throat, feces, blood, CNS (major disease effects


 Replicates in motor neurons of spinal cord
 Poliomyelitis: inflammation, death of motor neurons
o Phrenic nerve involvement especially bad: needed iron lung to support respiration

 Can shut off host protein synthesis


o IRES allows viral mRNA to bind / assemble
o 2A is a viral protease that cleaves elements of cap-binding protein assembly (initiation factor)
o Cellular RNA production stops but viral mRNA is fine!
o Cell death results (very little replication of own proteins)
Clinical manifestations of
Epidemiology: poliovirus infection
 summertime (virus not good in the cold! Seasonality: NE > south, etc.)  90-95% asymptomatic
 age dependence: was early in developing countries, late in industrialized  4-8% flu-like symptoms
countries  1-2% major disease
History: Unclear why some people get
 epidemics started early 20 c. (more leisure time, more time in common
th severe dz, others asx: virus/host
factors?
swimming pools, etc.)
 early attempts to control: quarantine  vaccines

5
Vaccines:
 Sabin’s live virus vaccine helped reduce polio SALK VACCINE SABIN VACCINE
incidence big-time; wild polio eradicated (inactivated) (live virus)
o Advantages: spread immunity via shedding, Use Currently in US Not used in US
mucosal immunity, etc. Revertants to wt? No Yes (rare)
 Problem: tendency to revert to virulence (rapid Administration Injected Sugar cube
emergence of mutations) Mucosal immunity? No Yes
o Vaccine-associated paralytic polio: couldn’t completely get rid of polio as a disease with Sabin’s vaccine
(all new polio cases due to live virus vaccine)
 Switched to Salk’s inactivated virus vaccine (no more revertants)

Current problems:
1. importation of polio from endemic to polio-free areas
2. circulation of virulent vaccine-derived/recombinant viruses
3. prolonged excretion of vaccine viruses by immunodeficient individuals (e.g. AIDS pts)

Togavirus (rubella & alphaviruses)


 enveloped, (+)-strand RNA, icosahedral virus
o 2 types: rubella virus & alphaviruses
 Genome: RNA
o Genome is mRNA for nonstructural proteins (needed to synthesize RNA)
o Second subgenomic RNA is synthesized from part of genome for translation of structural proteins

Rubella
Respiratory transmission, worldwide distribution

Clinical presentation:
 Children / adults: mild maculopapular rash
 Congenital rubella syndrome (CRS):
o requires: maternal exposure, maternal blood invasion, placental Features of CRS:
infection, entry to baby’s blood, fetal infection 1. mental retardation
 lack of any of these means the baby will be healthy. 2. heart defects
o Don’t see CRS if mom gets rubella after 17-18 wks gestation 3. cataracts

Arbovirus encephalitis (caused by alphaviruses & flaviviruses)

Examples: alphaviruses, eastern equine encephalitis, western equine encephalitis


Remember: encephalitis occurs in a minority of cases (most flu-like if even have Sx)

Flaviviruses
 Mosquito-borne viruses (yellow fever, dengue, Japanese encephalitis, West Nile)
 Tick-borne viruses
 Hepatitis C too!

Transmission: birds are animal reservoir; humans infected incidentally via mosquito
West nile virus: spread really fast
 appeared in 1999, across USA over 5 years, caused lots of human disease
 now seems like more American birds have acquired immunity, human cases more sporadic

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Coronaviruses
 (+)-strand RNA virus, transcribed and then translated
o Uses subgenomic RNA (along with genomic RNA) as mRNA, like togaviruses
 Morphology: looks like a crown
 Cause common cold and severe acute respiratory syndrome (SARS), which has pretty much disappeared

Summary of (+)-strand RNA viruses

Transmission Presentation
Picornaviruses Human (resp, fecal/oral) Variety: colds, polio, rashes
Togaviruses Human (resp) for rubella Rash, CRS
Mosquitos for alphaviruses encephalitis
Flaviviruses Mosquitos/ticks Fever, encephalitis
Coronaviruses Humans, ?animal for SARS Colds, SARS

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HIV (and retroviruses)
History
AIDS: originally described as opportunistic infections in young adults: PCP pneumonia / oral candidiasis (1981)
Thought to be transmissible (epidemiology, hemophiliacs, epidemic in NYC & SF); HIV-1 discovered in 1983
 HIV-1: 3 groups, from SIV (simian), cross-species transmission responsible (SIV doesn’t often cause disease in
natural hosts but does in humans  animal model, use Asian macaques, which aren’t usual host  causes dz)
o M group causing AIDS epidemic currently
o (33M+ living with AIDS, 2.7M new each year, 2.0M deaths each year)
 HIV-2: SIV from West Africa, more slowly progressive, not as widespread as HIV-1

Found RNA-containing virus with reverse transcriptase activity; retrovirus morphology by EM


 immunologically distinct from human T-cell leukemia virus, only other significant retrovirus in humans
 much more like lentivirus (slow disease)
 Can’t really fulfill Koch’s postulates (hemophiliacs kind of?)  can’t put back into humans

Retroviruses
Enveloped, small genome (10kb), (+) ssRNA
 ssRNA capped, polyadenylated like host mRNA Retrovirus genes:
 Has reverse transcriptase & can integrate into host cell genome  gag: structural proteins
 RNA virus benefit: high mutation rate; DNA virus benefit: latent form in  pol: enzymes (protease,
host genome RT, integrase)
 env: coat protein
Complex viruses (also have accessory genes – regulatory gene expression)

HIV Structure
 gp120: surface glycoprotein, trimers, mediates interaction between virus & cell receptor
o Target of neutralizing & cytotoxic AB
 gp41: transmembrane glycoprotein: causes fusion of cell membrane, anchors gp120
 Core:
o 2 copies of viral RNA (needed for the RT step
o Protease, integrase, reverse transcriptase already packaged inside
Cell targets of HIV
CD4+ lymphocytes are targeted and killed by HIV
 Lose CD4+ lymphocytes in: peripheral blood, lymphoid/gut-associated lymphoid tissues
o (normal: 46%, decreased to 3%, etc). CD8 stays the same, so CD8/CD4 ratio increases
 CD4 < 200 is AIDS-defining (normal > 1000); blood level gives good indication of whole compartment
o Onset of opportunistic infections
 Normal jobs: Central in immune response (all arms)
o Mature in thymus into blood
o Recognize antigenic peptides (MHC class II), activate Mϕ, activate B-cells to produce antibodies

HIV also infects CD4+ monocytes/macrophages


 CD4+ monocytes in blood, bone-marrow-derived, migrate into tissues and take virus with them (brain, etc)
o Spread all over body in first few weeks of infection
 Normal jobs: antigen presentation, host defense, repair  differentiate into Mϕ
 Express MHC Class II molecules; chemokine receptors (CCR5/CCR2)

Natural History
 Initial viremia (virus up, CD4 down)
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 Innate, adaptive immune can control at first, CD4 rebounds but not to normal
 Virus keeps replicating (lymphoid tissues, dumped into blood), goes to set point (for longer period of time)
o The lower the set point, the better the prognosis
(immune system doing better)
o Therapy: keep viral load low

HIV Life cycle


Note: targets for antiretrovirals (except gene expression)
1. Attachment-fusion
a. CD4 (host) and gp120 (virus) interact; CD4 gp120
conf change
b. gp120 (virus) can then interact with CCR5 or CXCR4
chemokine receptor (host) (CD4 not sufficient)
i. CCR5-tropic HIV: on Mϕ (and T-cells
too)(primary infection, most infection)
ii. CXCR4-tropic HIV: T-cells only express
iii. Tropism can shift (R4 is much more pathogenic & cytolytic)
c. Conformational change of gp41 after chemokine
receptor/gp120 interaction Summary of HIV Life Cycle
d. gp41 mediates fusion of membranes & viral entry (core: 1. Attachment-fusion
RT, genome, etc) 2. Reverse transcription (RNA  DNA)
i. some viral particles still left on cell 3. Integration of viral DNA
4. Virus gene expression
2. Reverse transcription (RNA  DNA) 5. Assembly & budding
a. Very complex process 6. Maturation
b. Primer (tRNA from cell) bind primer binding site of viral
RNA in virion (near 5’ end)
c. RT uses primer to start making (-) DNA from RNA (RNADNA)
d. Rapidly runs out of RNA: goes to 5’ end of RNA, then jumps to either 3’ end of RNA or to the second
copy of RNA
e. Duplication results in DNA copy with LTRs (long-terminal repeats) on each end:
i. Allows you to transcribe another RNA without loss of genetic material
f. RNase H chews up the RNA as you go along (no editing capacity)
i. Higher mutation rate than in normal DNA replication

3. Integration of viral DNA


a. Random cut into non-histone coated DNA (endonucleolytic, sticky ends)
b. Insert viral genome
c. Host proteins repair the cuts: looks the same!
d. Until cell dies, can’t clear! – can sit here latently, carry to other parts of body, etc. (e.g. monocytes)
e. Latent virus is a reservoir

4. Virus gene expression


a. Activation state of cell determines either latency or productive replication (e.g. activated T-cell,
maturation of monocyte to Mϕ, etc. triggers replication)
b. Cellular transcription factors; cellular RNA pol II complex used to transcribe HIV DNA
i. Note switch to cellular machinery now (before was viral RT) – not drug targets!
ii. Cellular transcription factors, RNA pol II and HIV’s Tat assemble to achieve high levels of HIV
DNA transcription
iii. TAT also turns on some genes that are toxic to cell (would be good target)
c. Long terminal repeat is a cellular promoter region
d. Full length mRNA produced (whole viral genome), spliced (host cell proteins)
9
e. Translated to make structural proteins (full length mRNAGag polyprotein) and a longer protein (via
read-through of Gag’s stop protein to make Gag-pro-pol polyprotein, encoding protease/enzymatic
activity)
f. Cleaved via protease (viral)

5. Assembly & budding


a. Structural proteins (gag, gag-pol) myristolated (targeted for cell membrane), RNA targeted to nuclear
capsid & associates, then budding of the whole complex happens
b. This forms an immature virion which is non-infectious

6. Maturation
a. Protease gets bundled along; cleaves itself out of Gag-pol precursor protein
b. After budding: cleaves gag & gag-pol to form mature virion (infectious)
c. Maturation is essential to be infectious  PROTEASE INHIBITOR TARGET

Pathogenesis
Not that virus itself kills all CD4 cells: 2 accepted theories
 Immune activation: so much activation of immune system  exhausted (high level of activation)
 Bystander killing: activated T-cells more prone to apoptosis (more dying)

Transmission
Not a tough virus: fragile (not on surfaces, aerosol, etc)
 Sexual transmission (incl oral)
 Contaminated needles (IV drug use mainly; P=0.3% for needle stick, use antiretroviral PEP, call 5-STIX)
 Mother-child (in utero, at delivery, breastfeeding: all preventable with antiretroviral Rx)
 Probability of transmission depends on viral load (highest in acute infection & during AIDS)

Genetic polymorphisms: CCR5


 CCR5 is primary tropism for HIV transmitted sexually
 Some have 32 base pair deletions (Δ32/ Δ32) in CCR5 and are resistant to infection (1% Caucasians)

Spread:
1. Transmission  dendritic cells / infected Mϕ 
2. local LN  CD4 lymphocytes, Mϕ  viremia in blood 
3. spread to tissues  viremia in CSF (brain infected)
4. Long-lived reservoirs: resting lymphocytes (blood, tissues), Mϕ (tissues)

1st sign of infection: local LN involvement (make tons of viruses; viremia)

Stages of infection
1. Primary (acute) HIV infection: rapid replication (first few weeks),
a. Ab tests initially negative, viral load varies (104-106/mL), CD4 depletion (esp. GALT).
b. Acute retroviral syndrome: fever, lymphadenopathy, pharyngitis, rash
c. Viremia falls: innate, adaptive (CTL) immune response develops
d. Levels off to set point (different in different pts; prognostic)
e. LN full of virus; dendritic cells trapping virus inside LN, adaptive immune response clears
f. Viral load lowers, CD4 counts rebound

2. Asymptomatic (8-10 yrs usually, 20+ in long-term progressors)


a. Persistent infection, rise in viral load, decrease in CD4
b. Current guidelines: start Rx at CD4 > 350 (now being reconsidered)
10
i. Sooner is better for prognosis! Getting immune response the whole time (continual activation
of immune system, damage being done the whole time)
3. AIDS
a. CD4 < 200
b. Opportunistic infections (fungal, bacterial, parasitic, CNS, lungs, etc.)
c. Use prophylactic Rx to prevent opportunists

Treatment
Multiple drugs: mutation rate high (1 error/genome per 3 replication cycles)
 No editing function (single strand)
 “every base pair mutates every day”
 Partial suppression: rapid production of mutant viruses
 “Do it right or don’t do it”  sequential monotherapy = develop resistance to all!
o Never treat with one drug
o Never add 1 drug to a failing regimen
 3 drugs: likelihood of getting resistance to 3 drugs on same viral genome is low!

Latency & HAART


 Eradication was predicted (1st, 2nd phases showed you’d get done) – but latency was 3rd phase
 Latency: reversibly non-productive state of infection
o Resting CD4 T-cells & Mϕ in sites like CNS
o Normal T-cells: some activated T-cells return to resting state (1 in million) for ready response to future
infections, re-activate when activated
o Stable reservoir of latent cells throughout HAART (would need ~73 yrs to eliminate)

Vaccine?
 6 yr trial in Thailand: guarded possibility of vaccine? 30% reduction in those who receive vaccine; no reduction in
HIV load in vaccinees with HIV (?)
 Why so hard? All current viral vaccines prevent development of disease, don’t stop infection; HIV vaccine would
need to induce “sterilizing immunity” to prevent infection/latency; HIV infection doesn’t induce natural
immune response to prevent progression; would need vaccine against many variable clades of HIV-1/2, diverse
antigenicity among HIV in population

Tests for HIV

Serology: remember: HIV antibodies take 2-4wks to develop (can’t use right away!)
1. ELISA used as first test
ELISA + WESTERN PCR
o Pt. serum + HIV proteins in well; look for binding of
 Inexpensive  Expensive
pt. antibodies
 Rapid  Requires sample prep
o False-positive: 0.4%
2. Western Blot: Blood test (1985)  Requires Ab against  Detect early infection
o Used after ELISA to confirm (combined false- virus (2-4wks post- (3d post infection)
positive 0.005%) infection)  Can quantify viral load
o Purified virions lysed, run on SDS-PAGE Almost no difference in sensitivity
o Western-blot with patient sera to look for anti-HIV ab
RT-PCR
 Amplify RNA in virus (detects infection earlier: 1st week!)
 Gives you viral load: how much virus do you actually have in blood?
 CD4 and viral levels are most important clinical measures

Viral load, CD4 count (via flow cytometry) are the two best prognostic indicators
11
Small DNA Viruses: Parvoviruses & Papillomaviruses
DNA viruses: unlike RNA viruses, can use host cell nuclear enzymes to transcribe DNARNA & replicate DNADNA
Must either:
1. infect a dividing cell (parvoviruses)
2. induce host cell DNA synthesis (papillomaviruses, polyomaviruses, adenoviruses)

Parvoviruses
 Among smallest of DNA viruses; icosahedral virion (3 proteins + linear ssDNA, ~5000nt)
 Replicate in host cell nucleus
 Don’t have enough room to code for DNA synthesis enzymes: can only replicate in:
1. dividing cells that have necessary DNA synthesis enzymes
 autonomous parvoviruses can replicate alone
2. cells co-infected with a “helper” virus (that provides the enzymes)
 dependoviruses need a helper virus like adeno/herpes

PARVOVIRUSES THAT CAUSE HUMAN INFECTION: QUICK LOOK


Parvovirus B19 Autonomous Erythema infectiosum (“fifth disease”, childhood rash dz)
Acute/recurrent arthritis (adults)
Aplastic anemia/crisis (pts with chronic hemolytic anemia)
Chronic anemia (immunocomp / hydrops fetalis)
Bocavirus Autonomous Respiratory disease (infants)
Adeno-associated virus (AAV) Dependovirus No dz: gene vector (integrates into cellular DNA)

Virions: non-enveloped, icosahedral, linear + or – sense ssDNA, no enzymes, very resistant to inactivation

Genome: 2 reading frames


1. structural coat proteins (overlapping in-frame sequences) Parvoviruses
2. nonstructural proteins for transcription/DNA replication 1. Respiratory transmission 
2. Respiratory epithelium
Replication: nucleus 3. Viremia
1. cellular DNApol makes dsDNA  4. Skin, bone marrow
2. cellular RNApol makes mRNA (erythroblasts), fetus, GI tract
3. If autonomous: need host cell in S phase
a. (can’t stimulate S phase like papilloma viruses)
b. Predilection for bone marrow, GI tract, developing fetus (dividing cells!)

Parvovirus B19
Pathogenesis:
 B19 cellular receptor = globoside (P antigen), found
primarily on erythroid cells
 Virus replicates primarily in erythroid precursor cells
 Cytopathic effect: giant pronormoblasts with nuclear
inclusions, cytoplasmic vacuolization in bone marrow
 Toxicity: express B19 nonstructural protein (NSP) 
apoptosis induction
o Megakaryocytes: nonproductively infected
(no transcription of mRNA for structural
proteins) but NSP compromises & kills

Normal child/adult
12
0. 5-6d incubation
1. Viremic phase (108-1014/mL): fever, malaise, myalgias
2. “slapped face” rash (erythematous, strikingly flushed) afterwards ( immune response )
a. extends to extremities (lacy, evanescent, maculopapular)
b. Adults: develop arthritis during immune response

Destruction of erythroid precursors during acute phase  absence of reticulocytes in blood (transient mild anemia)
 Not clinically important usually, unless:

1. If patient has chronic hemolytic anemia (sickle cell, thalassemia, hereditary spherocytosis)
 More virus made & released (more bone marrow cells being produced & turned over more quickly)
 Already have shortened life for circulating erythrocytes, add on more anemia
 Result: APLASTIC CRISIS (“Transient Aplastic Crisis = TAC”, life threatening!)

2. If patient is immunodeficient:
 Can’t clear virus  chronic anemia (“pure red cell aplasia”)

3. If fetus
 Can cause severe anemia  hydrops fetalis (abnormal fluid in at least 2 compartments), infant death
 Greatest risk: first 2 trimesters,
 Treat: transfuse in utero, but baby might become tolerant to virus & have persistent infection / red cell aplasia

Epidemiology:
 Humans only (esp. school age kids & parents), respiratory transmission (also possible by transfusion)
 Dx: serology later, PCR during acute phase
 If immunocompetent: clear w/o tx, immunity is life-long
 Tx for immunocompromised: immune globulins; no vaccine

Papillomaviruses
HPVs: human papillomaviruses
 Icosahedral, covalently closed supercoiled circular dsDNA molecule, 8kb with histones (“minichromosome”)
 Cause warts & squamous carcinomas (e.g. cervical carcinoma)
 Culture: difficulty; typed via PCR usually

Replication: nucleus of squamous epithelial cells


 2 phases (overlapping reading frames on single strand)
o Early (E) genes: regulatory proteins for replication, transcription, transformation. E2,E6,E7 important
o Late (L) genes: capsid structural proteins (L1, L2)
o Long control region: origin of replication, control elements for transcription/replication

Disease/pathogenesis: species-specific, restricted tissue tropism


1. Cutaneous types: warts
o Virus enters skin via abrasion  basal layer of epidermis  express early genes
 E7: induces DNA synthesis
 Cellular proliferation (hyperplasia)  wart forms eventually
o As infected cells differentiate to keratinocytes, late genes expressed (L1/2), producing infectious virions
o Incubation period: months/years, crops of warts clear at same time (immune mechanism?)
2. Mucosal types: genital/oral/respiratory mucosa
o Worldwide issue
o Women: target proliferating cells at border of squamous/columnar epithelium of cervix
 Several months later: flat condyloma (asymptomatic)
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 Clearance of virus: 1-2 years, longer if HIV infected (↑ risk)
 Associated with ↑ cytologic abnormality in Pap smear
HPV & Cervical Carcinoma
Essentially all cervical carcinoma worldwide is initiated by HPV infection
 HPV-16, HPV-18 = MOST ONCOGENIC (70%)
 MORE RISK with longer persistent infection (e.g. HIV pts)

Oncogenicity: function of E6/E7 oncoproteins (both required for immortalization of keratinocytes)


 E7: induces DNA synthesis in resting cells (want basal cells to proliferate more)
o Binds retinoblastoma tumor suppressor pRB
 normally regulates growth by binding E2F, keeping G1/S checkpoint in check
 If RB bound by E7: E2F can go do its thing & release G1/S checkpoint (progress to S)
 E6: activates telomerase in epithelial cells; can complex with p53 in high-risk HPVs
o Targets p53 for ubiquitin-mediated degradation (no checkpoint control)
 E2: usually controls E6/7 expression
o HPV usually exists as unintegrated autonomously-replicating episome in nucleus
o In malignant cells: viral genome integrated in a way that disrupts E2 (cut circular genome in middle of
E2 to insert)  no control of E6/E7

Epidemiology:
 Common worldwide in men & women; also linked to penile squamous carcinoma, some head/neck tumors
 Most infected women are asymptomatic, clear infection, and do NOT develop malignant disease
 If developing disease
o Histopathologic progression: cervical intraepithelial neoplasia (CIN)  invasive disease
o Papanicolau smear: screening device; detects cellular changes
o PCR can be used to detect type
o Tx: removal of involved tissue

Immunization:
 Virus-like particles (VLPs) from L1 capsid protein (antigenically different between strains)
o Immunogenic: assemble into empty aggregate
 VLPs for HPVs 16, 18 (high-risk) & 6 ,8 (low risk, cause condyloma, prevent warts = good for marketing) in
current vaccine: prevents against both cancer & genital warts
 Possible therapeutic vaccine: E6/E7; for HPV-induced tumors

14
Influenza: Epidemics, Pandemics, and Prevention Strategies
Influenza quick review:
 wild waterfowl = natural reservoir; many strains circulate in birds
 influenza A & B = major cause of human disease (A is vast majority)
 Subtypes: classified by Hemagglutinin (H x 16), Neuraminidiase (N x 9)
o H1N1, H1N2, H3N2, novel (Swine) H1N1 circulating recently
 Mutations: antigenic drift (variations within same H&N classes) vs antigenic shift (complete H/N change)
o Shift: H, N, or both H & N: e.g. bird strain & human strain re-assort
 pigs are good facilitators (resp epithelium have both human-like & bird-like receptors)
 high association of shift with pandemics
 Pandemics of 20th c: 1918-19 (Spanish, H1N1, 20M dead Steps to cause an epidemic
worldwide), 1957-8 (asian, H2N2), 1968-9 (“hong kong”) 1. Susceptible population
2. Animalhuman transmission
Novel H1N1 3. Human  human transmission
 Very rapid progression, in viral spread & response (says good 4. Sustained humanhuman
things about current collaborative epidemiological efforts)
 Several steps removed phylogenetically from seasonal flu
 Now counting deaths instead of cases, expect combinations of infections with seasonal flu in winter
 Unusual features:
o Summer outbreak (seasonal=winter)
o High mortality in young adults without comorbidities (seasonal = elderly, infants, comorbidities)
 Symptoms = usually the same, just in young, healthy people too! 5-24yo unusually affected
 Asthma, COPD, CVD, diabetes, immunosuppresed seem to be important comorbidities

Seasonal influenza
 Annual epidemic spread like clockwork: late fall, winter, early spring (peak = Jan, Feb).
 All ages affected, highest rates among children, most serious in >65 and <2 years old + high risk conditions
 Annually 36k deaths, 226k hospitalizations in US; most deaths in >65yo
 50% peds deaths: no underlying high risk condition (secondary bacterial pneumonia is #1 cause)

Signs/symptoms: malaise, myalgias, headache, fever, non-productive cough, rhinitis, sore throat, otitis (peds)
 Normally a non-specific viral constellation; together = influenza-like illness (ILI)
 Uncomplicated: resolves in 3-7d with cough/malaise up to 2 wks (self-limited)

Complications
 Primary viral pneumonia
 Can exacerbate underlying medical diseases
 Secondary bacterial pneumonia / sinusitis / otitis
 Co-infection with viral/bacterial pathogens
 Uncommon: encephalopathy, transverse myelitis, myocarditis, pericarditis, Reye’s syndrome

Dx: difficult clinically to distinguish from other resp viruses; absence of ILI Sx doesn’t rule out flu
 Need lab Dx + high level of suspicion
 Lab dx:
o Nasopharyngeal aspirate: suction catheter, mucous trap, aspirate from posterior nasopharynx, add to
transport media, process < 1 hr
o Nasopharyngeal swab: have to get back to NP, better because won’t aerosolize (esp H1N1)
o After you get the sample: viral culture, immunofluorescente DFA antibody, RT-PCR, Serology

15
Transmission of influenza
 Person-Person via large particle respiratory droplets, coughs/sneezes, 3 foot radius – can use surgical mask
 Close contact, contaminated surfaces
 Some evidence of airborne spread (small particle residue evaporated/suspended like TB – would indicate more
than just a surgical mask!)
 Observational studies in healthcare settings: contact/droplet are primary means; anecdotal airborne spread
 Incubation: 1-4d
o Adults: infectious from 1d prior to Sx through 5d post sx
o Children: infectious from several days prior to 10+d post sx
o Immunocompromised: can shed virus for months
o Shedding prior to Sx: more transmission (less precautions taken)

Vaccines
 Most effective way to prevent infection/complications
 Annually (antigenic drift)
 Two types
o Trivalent inactivate vaccine (TIV)
 Injected, grown in eggs, 3 strains (A/H3N2, A/H1N1, B)
 Inactivated/killed; subunit/subvirion/purified surface protein
 Cannot cause influenza (killed!) ACIP Recommendations
o Live, attenuated influenza vaccine (LAIV) for seasonal flu vaccine
 Intra—nasal administration; grown in eggs, 3 strains
 Children (6mo-19yr)
(A/H3N2, A/H1N1, B)
 Pregnant women
 Live attenuated virus; can cause mild signs / sx of
 >50yo
attenuated influenza
 Chronic med conditions
o Cold-adapted, LAIV (FluMist)
 2-50yo (FDA) and also 50-64; efficacy comparable to  Nursing homes / long-term
injected (85% healthy adults) care
 Well tolerated (rhinorrhea, nasal congestion)  Live with / care for high risk
 Don’t give to pregnant/immunosuppressed for complications
 Safe in healthcare setting (shedding short duration,  Healthcare personnel
less than dose to vaccinate, doesn’t replicate well at  Household contacts of
37F, genotypically stable, etc.) persons of high risk for
 Efficacy: prevention of illness among vaccinated subjects in complications; out of home
controlled trials caregivers of children < 6mo
 Effectiveness: prevelance of illness among vaccinated populations
o Depends on age, immunocompetence, match between ACIP Recommendations
vaccine/strains, outcome measured (death, hosp., etc) for H1N1 flu vaccine
o Good (80-87% kids, 77-90% working adults, less in elderly in  Pregnant women
community / long-term-care)  Household contacts of
persons of high risk for
Medical conditions with ↑ risk of complications complications; out of home
 COPD + asthma caregivers of children < 6mo
 CVD (not HTN)  Healthcare / EMS
 Renal, hepatic, hematological, metabolic disorders (incl. DM)  6mo – 24yo (ALL)
 Immunosuppresion (meds/disease like HIV)  25-64 with higher risk
 Cognitive/neuro dysfunction that compromises resp function or conditions
increases risk of aspiration
*note: no prioritization of
elderly!
Hard to make vaccine: WHO decides in feb which strains to include; 6-8 mo

16
of production, 10s of millions of hand-picked 11-day-old chicken eggs to inject with strain, incubate for several days,
extract/purify egg white: LABORIOUS
Vaccination “season”: people stop getting vaccinated after Thanksgiving although most influenza is in Jan/Feb
 Need to keep up vaccination efforts!

Influenza in health care workers: common (23%, most can’t remember flu or resp symptoms)
 Vaccinate: ↓ patient mortality, ↓ lost hours, ↑ normal function of institution in flu season
 Doesn’t make you sick (large double-blind placebo study)

Antiviral meds: adjunct to vaccination, not substitute


 Adamantanes (amantadine, rimantadine)
o Single point mutation confers resistance; was common in H3N2 circulating strains, was recommended
against for a while, now active against H1N1?
 Neurominidase inhibitors (oseltamivir, zanamivir)
o Resistance mostly in seasonal influenza, increasing over last few seasons
o Active against flu A & B; 83-4% active for prevention, bad in pregnancy?
o Can use for chemoprophylaxis
 NOT vaccine substitute; adjunct
 ~85% effective in household exposure, use in institutional settings (prevent spread in
outbreak), protect high risk when flu circulating (vaccine takes 2 wks to make Abs), protect
immunocompromised, protect those with contraindication for vccine
 Can use for therapy or prophylaxis, both have similar effects in decreasing length of illness

Other prevention
 Hand-washing, respiratory etiquette, community mitigation (close schools, avoid mass gatherings wear masks)
 Respiratory etiquette:
o Cover nose/mouth, use tissues, use hand-hygiene after resp secretions / contaminated objects,
healthcare facilities need to make tissues / hand sanitizer available in waiting rooms!
o Provide no-touch receptacles, tissues; dispensers of alcohol, use masking/separation if resp. symptoms
o Droplet precautions: use mask if sx of resp infection, esp in setting of fever

17
Viral gastroenteritis
Some viruses replicate in GI tract but don’t cause GI disease: (Enteroviruses: polio, coxsackie, echo, HAV, reoviruses, adenoviruses)

To infect GI tract: need resistance to low pH, detergents (bile), proteases (small intestine)
 Some viruses even co-opt these features as part of life cycle!
Review: anatomy of small intestine
Some viruses replicate in GI tract and cause gastroenteritis  Crypt cells (dividing, secretory)
 Norovirus: (+) RNA no envelope  Villus cells (tip = mature, non-dividing, absorptive)
 Rotavirus: segmented dsRNA no envelope  M-cells (Peyer’s patches, like LNs)

Pathogenesis of viral gastroenteritis: Different viruses infect different sites in small intestine
ingestion  mucosal infection  diarrhea  more transmission

Rotavirus
Rotavirus diarrhea: most common cause of severe dehydrating diarrhea in young children

Rotavirus: segmented, dsRNA (1 segment = 1 protein), no envelope


 In Reovirus family
 RNA alone not infectious
 Different segments = different viruses; can distinvirionguish rotaviruses based on electrophoresis
o Can reassort during dual infection of cells
Structure
 Outer capsid: VP4, VP7 (role: attachment & entry; neutralization / protective immunity target)
o VP7: viral surface glycoprotein, major part of virion
o VP4: much smaller component
 Trypsins from small intestine: cleave VP4  VP8* + VP5*
 Required for infectivity: once cleaved, can exposes fusion domain & allows fusion/entry
 VP5* selectively permeabilizes membranes
 Core:dsRNA genome woven into capsule structure on inside

Steps in infection:
1. Ingest virion  to small intestine  trypsin cleaves VP4  entry mediated
2. Intermediate sub-viral particle formed (ISVP)  enters lysosome, cytoplasm, etc.
 STAYS as ISVP (intact) – doesn’t fully uncoat like other viruses to show genome
3. ISVP has its own VIRAL RNApol – makes (+)-strand RNA & extrudes into cell
4. New RNA can be used as mRNA to make proteins, assemble virus, etc.

Cypopathic effects:
 see blunted, vacuolated villi
 MATURE enterocytes infected, not dividing cells at base of crypts
o In one week: everything restored (more being made at base, infected cells turned over)

Pathogenesis:
 If you have neutralizing antibodies (anti-VP4/7), halts infection (VACCINE target)
 Rotavirus infects mature absorptive enterocytes in small intestine; produce & release NSP4
o Cellular disruption leads to ↑Ca+2  malabsorption & osmotic diarrhea
 Productive infection  production of NSP4: viral enterotoxin
 NSP4:
1. Stimulates Cl- secretion from crypt cells (causes osmotic diarrhea)

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2. May also stimulate enteric nervous system (more diarrhea)
Diarrhea: good for virus: more transmission Rotavirus Dx
 Often hard to
Clinical features: (vs other virus causes): high prevalence of vomiting & dehydration culture viruses
 Esp. important in infants – can’t tolerate huge volume depletion  Use antigen-
specific enzyme
Epidemiology: immunoassay
 younger kids (6mo-1yr) get more rotavirus gastroenteritis, diarrhea (stool specimen)
 Biggest single cause of infant diarrhea in both developing & developed countries
o (US: million cases/yr, 150 deaths, $350M in costs; developing countries: 150M cases, 900K deaths/yr)
 Seasonality: more in winter (opposite of enteroviruses)

Vaccine: made by reassortants (rhesus monkey/bovine + human – less virulent but still antigenic)
 One was pulled (linked to intussusception – one part of bowel slides into another like telescope – in infants) in
1999 (rare cases)
 Two live, oral, attenuated vaccines are FDA approved now (bovine reassortment, no intussusception risk)
o Now routine in US

Norovirus
Outbreak of gastroenteritis (1972, Norwalk, OH) – “winter vomiting disease” but no true seasonality
Found viral source: related to small rounded structure viruses; all termed Calciviruses

Norovirus:
 (+) ssRNA, no envelope
 Cup-shaped indentations on surface (β-parallel sheets)
 Only infects some people: depends on receptor status in host & blood type
o FUT2 encodes a carbohydrate that’s part of receptor
o If receptor present: “secretor” (secretor ≫ non-secretor for susceptibility)
o O > A/B for susceptibility (blood types)

Clinical features: high level of variability (some vomit w/o diarrhea, others vice-versa, some both)
 Delayed gastric emptying might be involved (asx infected = no delay)

Epidemiology: all ages, all groups, across the board


 (% with serum Ab increases with age, esp. post 6yrs, depends on country)
 Acute gastroenteritis outbreaks (e.g. banquets,day care, cruise ships, nursing homes, etc.)
o Most common etiology of foodborne illness outbreaks (& foodborne illness overall!)
o Easy to spread (hand-hand, surfaces, etc.)
o Also: wells, water supply, nursing
homes/hospitals, etc. SUMMARY OF GASTROENTERITIS VIRUSES

Infectivity: stool, vomit are infectious Rotavirus


 Many strains with little durable immunity  dsRNA virus that synthesizes RNA inside a
transcriptionally active particle
Dx: no routine tests (usually just for investigations)  Most common cause of dehydrating diarrhea in
Rx: supportive (usually self-limiting 2-3d) children <2yo worldwide

Calicivirus (e.g. Norovirus)


 (+) ssRNA virus with genome similar to
picornaviruses
 Outbreaks of gastroenteritis in all ages
 Most common cause of infectious GI illness
(food-borne, cruise ships, water-related, etc).
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Gammaherpesviruses: EBV / KSHV
Epstein-Barr Virus (EBV)
 dsDNA, large genome
 Immortalized B-cells: after infection with EBV: enter cell cycle, proliferate, grow indefinitely in tissue culture,
tumorigenic in mouse models

Epidemiology:
 Everybody, everywhere (>95% adults worldwide)
 Primary infection: transmitted in saliva
o Asymptomatic if infected as a kid (most exposed in early childhood)
o Infectious mononucleosis if infected later in life (25-70% of adults infected develop Sx)
 Most important diseases are associated with latency (tumors) – very uncommon

Virology:
 Has a lytic and a latent phase of infection (Burkitt’s cells are slightly different because they have less protein
expression and are therefore less immunogenic)
o Lytic phase: spread via infectious virions like normal virus
o Latent phase: hangs out in epitopes
Lytic infection of B-cells Latent infection of B-cells Burkitt’s B-cells
(“immortalized”)
Genome Linear Circular epitopes
Viral (acyclovir susceptible), Host (not acyclovir susceptible)
DNApol used
Viral enzymes expressed Viral enzymes not expressed
Lots of proteins expressed Lots (antigenic) Only one (invisible to CD8+
Gene expression T-cells because of lack of
MHC-1 presentation)
Immune response Big response Big T-cell response No T-cell response
Infectious virions Host cellular proliferation; no virions made
Spread
(epitopes partitioneddaughter cells when replicating)

Keeping virus under control:


 T and NK Cell response (atypical lymphocytosis)
o Kills many infected B-cells
o > 1% T cells in most seropositive healthy people target EBV (huge response, surveillance)
o T cells rapidly kill off immortalized B-cells, not Burkitt’s cells

Infectious Mononucleosis
 Especially prevalent if primary EBV infection occurs as adult
Pathogenesis
1. Transmission: Saliva (“kissing disease”)
2. Immortalization of lymphocytes in vivo
3. T cell response, most immortalized B-cells are killed
4. A small number of EBV-infected resting B-cells have minimal antigen expression (like Burkitt’s cells)  escape
5. Reactivation of these infected, resting B-cells occurs sporadically (unknown why)
6. Intermittent in everyone (reactivation): Production of virus, shedding in saliva, infectivity
Clinical features
 Sore throat, fever, generalized lymphadenopathy (esp. cervical)
 Atypical lymphocytosis (activated T & NK cells): “ mononucleosis” is really a lymphocytosis
Diagnosis
 (+) heterophilic monophile test
20
o Turns out that Abs generated during infectious mononucleosis will agglutinate horse RBCs (weird &
accidental cross-rxn)
o “ Monospot” test used currently based on this
o Disappears with resolution of acute illness
 Serology: IgM to viral capsid antigen (VCA) for current infection; IgG for post-infection

Burkitt’s Lymphoma
 Young males, maxillary / periorbital tumor
EBV Tumor associations
 Equitorial Africa only (malarial distribution): not high
Lymphomas Other
altitudes or deserts
Endemic Burkitt’s Nasopharyngeal carcinoma
 Escape immune detection (makes few viral proteins) B-cell in immunodeficient Gastric carcinoma
 Exact EBV – BL relationship unknown Hodgkin’s disease

B-cell lymphoma (immunodeficient pts)


Basic idea:
 T-cells suppressed, pretty much everybody has EBV B-cells
 can’t keep them in check anymore and end up with B-cell lymphoma (uncontrolled growth)

Patients:
 Transplant patients on cyclosporine, etc – if stop suppression, tumor regresses
 Severe combined immunodeficiency (SCID), X-linked immunodeficiency: often die of EBV B-cell lymphoma
 AIDS lymphoma: 50% increased risk
 (X-linked agammaglobulinemia, XLA): no risk (no B-cells = no EBV, no B-cell lymphoma)

Hodgkin’s lymphoma
 EBV in tumor cells in 30% of cases (associated)
o Find EBV DNA/RNA/Ag at each tumor site, during presentation & during relapse

Nasopharyngeal carcinoma
 Especially prevalent in Southern China (genetic & environmental)
 Virtually ALWAYS EBV-associated (not well understood)

Kaposi’s Sarcoma Herpesvirus (KSHV)


KSHV
 Unlike EBV, doesn’t infect most people (rare except in HIV, MSM, special pops)
 Found via PCR in B-cells of seropositive individuals, DOESN’T IMMORTALIZE like EBV
 Several genes closely mimic human genes (e.g. viral IL-6)
 Transmission: early childhood in endemic regions (saliva?), ? sexual trans in MSM? Rare in transfusion, IV

Kaposi’s sarcoma
 KHSV infection is required
 Geographic: Children in Africa (hands/legs); old men in Mediterranean (Italy, Greece, etc.),
 Immunosuppresion:
o Organ transplant recipients (regress with withdrawal of immunosuppresion
o AIDS patients: especially MSM in North America & Western Europe
 Presentation: tumor, most commonly on skin, may also be GI/lungs
o Neovascular proliferation  purplish color

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Primary Effusion Lymphoma
 In AIDS patients, B-cells float in pleural/peritoneal fluid (no solid component)
 Exceedingly rare
 Pts DUALLY INFECTED (EBV+KSHV)

Main Concepts (review)


Latent infection:
 How can a virus establish latency in a dividing cell? How can a virus spread inside a host while latent?
o Need to be able to make DNA that can go into daughter cells
o EBV/KSHV have episomes that are replicated using host machinery & partitioned to daughter cells
 Are viral genes expressed in latency?
o Yes: can be one, a few, many – and virus can take over cell, make it grow out of control
 Can disease processes be associated with latent infections?
o Yes: tumors for example
Lytic infection/reactivation GAMMAHERPESVIRUS TUMOR ASSOCIATIONS
 Is it always harmful to the host? Nasopharyngeal carcinoma
Always EBV associated, regardless
o Not usually – don’t usually want to of geography
measure EBV in blood (diseases aren’t EBV associated in malarial areas of
Burkitt’s lymphoma Africa but not in North
virions but are latent virus)
America/Europe
Tumor associations Hodgkin’s lymphoma Sometimes EBV associated
 What does tumor association mean? Kaposi’s sarcoma Always KHSV associated
o Not just that colon cancer has EBV – Primary effusion lymphoma KSHV and EBV associated
everybody is EBV positive!
o Need to find virus in cancer cells

22
Viral Hepatitis
Hepatitis = inflammation in the liver
 Nonspecific: alcohol use, acetaminophen, etc. can cause too
 Infectious causes: nonviral (syphilis, TB, histo, etc.) and viral (CMV, EBV, HIV, H[A-C]V, etc.)

Hepatitis viruses: Certain viruses only cause hepatitis clinically


 A to E, really a clinical grouping, not biological (some DNA, some RNA, etc.)

Clinical course of Hepatitis: exposure  incubation (3-4wks)  symptoms (jaundice)  recovery or persistence
 Acute viral hepatitis (USA): A>B>C for frequency
 Chronic viral hepatitis: B & C (A can’t cause chronic hepatitis)

Transmission:
 A (& E) is nonenveloped, not killed by bile, so can be Exposure HAV & HEV HBV & HDV HCV
transmitted fecal-oral (acute) Fecal-oral +4 0 0
 Note that the chronic ones can be transmitted via blood Sexual +1 +4 +1
(makes sense) Blood +1 +4 +4
 Sexual: oral or vaginal Perinatal +1 +4 +2

Clinical features of acute viral hepatitis


 HAV+HEV: provokes a stronger immune response than B/D, C
o Shorter incubation HAV+HEV HBV+HDV HCV
o Higher % with jaundice Incubation (weeks) 2-6 6-24 6-300
o No persistence % Jaundice 30-70 20-40 15-25
 HCV: big one for persistence % Persist 0 5 80

Consequences:
 Liver: largest organ in body, stores vitamins A, B12, D, E, K; metabolizes lipids, makes cholesterol, stores
glycogen
 Fibrosis: scarring (overgrowth of connective tissue), restricts function  bridging (bands of fibrosis)
o Cirrhosis: widespread fibrosis with nodule formation macronodular cirrhosis
 Hepatocellular carcinoma (primary cancer of the liver; one of most common in world, cirrhosis is risk)

Lab Dx:
 Elevated transaminases (ALT, AST > 10x normal): liver-specific enzymes, spilled out in ongoing damage
 Antibodies
o IgM antibodies: markers of recent infection (6 mo)
o IgG: markers of any past infection
o Neutralizing Ab: recovery process under way
 Viral particles(protein/nucleic acid, “antigen”): ongoing infection and infectivity

Prevention: Vaccines (HAV, HBV) or immunoglobulin administration (HAV, HBV)


Treatment: none for acute hepatitis; there are treatments for chronic HBV/HCV

Hepatitis A virus
 Picornavirus, RNA virus
 NO ENVELOPE  bile stable (can be transmitted fecal-oral)
 Capsid proteins elicit a universal neutralizing antibody (one serotype  vaccine possible)

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Hepatitis B virus
 S-gene: surface antigen, makes surface antigen in outer envelope;
o first recombinant vaccine (yeast) produced against it (1st anti-cancer vaccine)
 Genome: tiny (3200nt), uses overlapping reading frames
 Replication: entry  uncoating  genome incompletely closed (opened from circle) to be imported to nucleus
o Completly Closed Circular DNA (cccDNA): genome closed & repaired inside nucleus
 Makes a bunch of transcript for viral replication
 Can be INTEGRATED into host genome (stable, reservoir) – hard to eliminate
 Transmission: makes TONS of virus and extra surface antigens (serum packed); environmentally stable (can
hang out on tables, equipment, etc). Very transmissible

Hepatitis D virus
 Has Dependency issues: needs Hepatitis B (either via co-infection or prior chronic infection)
o Uses HBV to put on its capsule (has HbsAg) but has its own RNA

Hepatitis C virus
 Tons of genomic diversity
o Error rate: 1x10-4; turnover is really high (1010-12 per day)
o Mutations: every base, every day, every person (like HIV)
 Forms quasispecies
 Even more genetic diversity than HIV
o Explains failure of vaccine & immune response to clear (some variants can evade & persist)
 Abs don’t neutralize (too much diversity)
 Steady progression of chronic disease, often cirrhosis  end-stage liver disease (ESLD)
 ALT at constant elevated rate; RNA present the whole time
 Clinical correlations of genetic diversity
o 80% persistence, resistance to treatment
o HCV is hard vaccine target, hard target for antiviral drugs
o Reservoir: infections last for decades

Hepatitis E
 40d average incubation; 1-3% CFR
 Pregnancy: often fulminant (15-25% CFR!)
 Higher severity with age; no chronic sequelae

Summary/Review

5 hepatotropic viruses
TRANSMISSION COURSE KEY FEATURE
HAV Fecal/oral Self-limited No envelope = bile stability
HBV Surface antigen in vaccine
HCV Blood/sex/etc Chronic Viral diversity
HDV Needs HBV
HEV Fecal/oral Self-limited Fatal in pregnant women

 Viral particles: ongoing infection


 Anti-viral Abs: IgMs are recent

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Pharmacology: ID & Micro (Viruses)
Anti-HIV Drugs......................................................................................................................................................................... 2
Vaccines I ................................................................................................................................................................................ 5
Vaccines II ............................................................................................................................................................................... 7
Antivirals ................................................................................................................................................................................. 9

1
Anti-HIV Drugs
Goals: how does chronic HIV cause disease? CD4+ T-cell depletion  immune suppression; direct consequence of HIV
replication, so inhibit HIV replication!
 Decrease HIV replication by as much as possible for as long as possible in every patient

HIV dynamics
 HIV replicates very rapidly (can be good: can shut off consequences of infection very quickly)
 Can only suppress chronically, not eradicate (latent reservoir)
 Need to get into brain, LN, genital tract, etc where infection is
 Can develop resistance extremely rapidly (esp. on monotherapy)

Nucleoside analog reverse transcriptase inhibitors


(NRTIs)

Zidovudine (azidothymidine, AZT)


 Also: ddI, ddC, d4T, 3TC, ABC, FTC, TDF
 thymidine analog; 5’ OH replaced with N3
 Synthesized in 1964 as anti-cancer; known
antiretroviral in ‘ 74, anti-HIV in ‘ 85, approved in ‘87
really quickly (single small trial)
 Still one of most potent anti-HIV drugs

zidovudine Mechanism of Action: NRTI, Anti-HIV antiretroviral agent. Thymidine analog. Triphosphate form inhibits
(azidothymidine, HIV reverse transcriptase, acts like chain terminator
AZT) Effects: Incorporated but not substrate for elongation in RNA-dep-DNA-pol activity of HIV RT
Selective Toxicity: poor, also inhibits mitochondrial DNApol
Indications: HIV
Administration: Short plasma half-life (1h) but much longer intracellular AZT-TP half-life (allows more
infrequent dosing, q12h).
Toxicity:
 Bone marrow suppression (common, mostly anemia, less commonly granulocytopenia).
 Rare: Myopathy, lactic acidosis/steatosis
 (steatosis = accumulation of fat in liver cells, fatal and class-wide for NRTIs albeit rare)
Resistance: Need 5+ AA changes. Slow to develop (only 1/3 on monotherapy resistant in 1 year), limited
cross-resistance with other NRTIs
Other:
 Phosphorylated by cellular enzymes to triphosphate (active form). Rapidly converted to AZT-MP,
accumulates in cell (-DP, -TP formation more slow)
 Well absorbed, eliminated by glucuronidation (Phase II).
 Commonly used in other countries (cheap generic) and sometimes for needle-stick prophy here,
although others probably work as well (only one studied)
Tenofovir (TDF) is most common in US now
tenofovir (TDF) NRTI antiretroviral; unlike AZT is a broad-spectrum antiviral (anti-HBV too),
more commonly used than AZT in USA

2
Non-nucleoside reverse transcriptase inhibitors (NNRTIs)
 Not lots of structural homology in this group (unlike NRTIs) – binding to flexible binding pocket
Nevirapine (NVP)
 Also: delavirdine, etravirine, efavirenz
 Only one that doesn’t inhibit HIV-2, resistance develops rapidly (makes sense)

nevirapine Mechanism of Action: NNRTI Anti-HIV antiretroviral. Non-competitive reverse transcriptase inhibitor
(NVP) Effects: Binds to HIV RT distant from active site, causes conf. change to make RT less efficient
Selective Toxicity: No effect on human DNApols (incl. mitochondrial)
Indications: HIV
Administration: bid but could be qd (long half life)
Toxicity: mostly immune-mediated
 Rash, hypersensitivity (common), hepatitis (rare)
 Stevens-Johnson syndrome (rare, systemic attack of immune system against epithelium: full body
burn, slough off mucosa/epithelium)
 IMPORTANT: CYP450 3A4 INDUCER (drug interactions - like rifampin).
Resistance: FAST (very poor monotherapy) - needs single AA change (1000x resistance), days to weeks,
Cross-resistance to other NNRTIs (exception = etravirine)
Other: no intracellular activation required. Doesn't work against HIV-2 (doesn't bind RT). Well-absorbed,
eliminated by CYP450 3A4

Efavirenz is currently most used in US


efavirenz NNRTI like nevirapine
 Less toxicity, longer half life most common NNRTI in use in USA
 part of Atripla (1 pill qd);

Protease inhibitors (PIs)


HIV Protease: usually cleaves immature surface proteins (immature virion mature core/capsid structure, infectious)\
 Doesn’t prevent virion formation or release
 Inhibits maturation

3
Ritonavir
Ritonavir Mechanism of Action: PI, anti-HIV antiretroviral.
(r)  Competitive inhibitor of HIV protease (mimics transition state)
Effects: Inhibits HIV protease; prevents viral maturation (can make immature virus, but can't make it
infectious). 2-3 log reduction in VL (can't keep replicating + fast turnover of HIV = quick drop!), partially
restores CD4 count even on own
Selective Toxicity: No known human aspartyl proteases inhibited
Indications: HIV: PI but mostly used now as booster (increase concentrations of other HIV drugs)
Administration: bid (3-5h half life)
Toxicity:
 Inhibits CYP450 3A4 (also induces hepatic enzymes but net block). Drug interactions (but also used
for boosting).
 GI intolerance (nausea, vomiting, diarrhea), hyperlipidemia (elevated cholesterol & TGs, reversing
metabolic disturbance created by virus),
 First few weeks: common circumoral & extremity parasthesias (important for adherence)
 Rare: glucose intolerance, hepatic transaminitis (inceased AST/ALT).
Resistance: Weeks to months. Not necessarily in all subjects (unlike NRTIs)
 Primary resistance mutation: 1AA, 3-5x resistance.
 Secondary resistance changes accumulate, resistance keeps increasing, cross-resistance increases.
 If you can keep at high levels (e.g. boosting, drugs with high therapeutic index), 1st mutation will still
be suppressed (dose-response curve).
Other: no intracellular activation required. 99% protein bound. Variable bioavailability (first-pass metabolism,
autoinduction). Eliminated by CYP450 3A4 (oxidative)

Others
 Integrase inhibitors (e.g. raltegravir), inhibit HIV integrase (no chromosomal integration), very non-toxic (like
placebo!)
 Entry inhibitors
o Fusion inhibitors (e.g. enfuvirtide = T-20), interferes with membrane protein bundle formation needed
for fusion, only injectible BID & really expensive (salvage pts only)
o CCR5 antagonists (e.g. maraviroc) – inhibit host cell CCR5! Only effective if CCR5-trophic HIV, approved
for salvage pts only

HAART (highly active antiretroviral therapy)


 Combination therapy ONLY and for EVERY PATIENT
 Potent combinations possible for pretty much everybody
 Popular starting regimens:
o Efavirenz + 2 NRTIs (e.g. Atripla, 1 pill qd, 80% new HIV Rx in US
Atripla 1 pill qd for HIV! efavirenz (NNRTI) + emtricitabine (NRTI) + tenofovir (NRTI)
o Potent PI + 2 NRTIs (usually a boosted PI with ritonavir)

 Rationale: prevent drug resistance (probably need ~3 agents to prevent resistance emergence)
o Probably not synergy (3 NRTIs nearly as active as regimens with 2-3 different targets)
 Trends: less pills, co-formulated drugs, qd regimens, better tolerability, better resistance testing
o Make it easier since it’s for life

4
Vaccines I
Vaccine: any formulation able to elicit antigen specific protective immunological memory

Classic vaccine principle: vaccine protection based on exposure of host to immunogenic agent followed by the natural
development of immunity (failed in HIV, malaria, cancer, etc)
 Adaptive immune response: only one that has memory, against certain antigens
 Primary response smaller,
 Secondary response faster & bigger to prevent infection
o Can prevent clinical disease!

Main types of vaccines


Live Vaccines Inactive Vaccines
antigens encoded by a genetic material and synthesized in protein/polysaccharide antigen is directly injected into
host (e.g.live attenuated) host (inactivated vaccines, recombinant proteins, purified
polysaccharides, etc)

presented on MHC class I (made inside cells) and MHC II; injected antigens presented on MHC class II (internalized)
make CD8 (cytotoxic) T-cells and helper T-cells too! make mostly helper T-cells

infectious agent, locally deposited, distributed to regional lymphnodes


makes strong innate inflammatory responses, weak, innate inflammatory response
strong induction of all adaptive responses (B and T cells) (requires addition of adjuvant)
longer memory mainly induce antibody responses (weak CD8 responses)

Essential components of vaccine formation


1. Route of administration: Appropriate presentation to immune system (B cells, CD4/CD8 T-cells)
2. Adjuvant(danger signal): immune stimulatory signals
o Start innate immune responses, shape adaptive effector mechanism
o Only really need for inactive vaccines – live vaccines have enough “danger” on their own
3. Active principle: Antigenic epitopes correlated with protection!
o Exact region of molecule & pathogen recognized by T-cell / antibodies

Route of administration (oral, subcutaneous, intradermal, intramuscular)


 Changes how antigen is processed (which cells?), how it modulates immune system
 Want mucosal immunity? Have to expose to mucosa!
 Want B-cell epitope? Polysaccharides (IgM, CD4-independent Ab) or protein (IgG, CD4-dep Ab)
 Want T-cell epitope? CD4: needs class II presentation, CD8: needs to be intracellular, class I presented

5
Adjuvant: Immunology mini-review:
 Live vaccines naturally activate innate immune responses (retain PAMPs,
ability to have C’ fixed, opsonization, etc.) so they don’t need adjuvant T-cells: immune system
 Inactive vaccines: include some stuff that binds to innate immune receptors degrades pathogen protein,
(adjuvant) processes via Class I or Class II
o Aluminum compounds, liposomes, virosomes, viruslike-particles, etc. MHC, presented as small
o Can also conjugate your antigen to an immunogenic protein (older) fragments

Mechanism of vaccine action  T-cells recombine to


1. Activate B-cell response  make Ag-specific antibodies generate molecules;
some can perfectly bind
a. Neutralization: block biological function of antigen
to MHC-epitope
b. Opsonization: faster clearance of antigen combination

2. Activate T-cell response  CD4 helper & CD8 cytotoxic B-cells: antibody’s exact
a. CD4+: cytokine secretion, supports B-cell/CD8+ cytolytic cell conformation is important
activation, proliferation, maturation, memory differentiation (needs to bind actually virus,
b. CD8+: cytolytic (kill infected cells) not virus+MHC)

Vaccines against viruses


 neutralizing antibodies (target surface envelope glycoproteins / proteins)
o Can use plaque reduction neutralization assay (mix virus & ab on culture, if neutralizing no plaques form
in culture cause virus can’t get in; otherwise plaques form where cells infected)
 CD8+ cytotoxic action (target cytoplasmic non-structural proteins)

Vaccines against bacteria (if extracellular)


 Opsonizing antibodies for ↑phagocytosis (target surface polysaccharides, envelope glycoproteins/proteins)
 Antitoxin antibodies (bind & neutralize toxins) – may not even need to kill bacteria to neutralize disease

T-cell independent response (for vaccines against sugars, for instance)


 make mostly IgM because they don’t stimulate CD4+
 Go away relatively quickly – conjugate to protein to induce helper T-cells & get better response!

T-cell dependent response (vaccines against proteins)


 Have antigen and stimulate T-helper cells (make IgG after IgM, differentiate into memory phenotypes)

Vaccines require very high standards


 High safety: Giving to large numbers of healthy people / especially babies!
 High benefit / efficacy:
o High individual protection levels (reduces risk greatly in individual)
o Herd immunity (reduce contagion in community, disease in unvaccinated population)

Herd Immunity

6
Vaccines II
Lots of vaccine preventable diseases; these are hard to make though! US health care is expensive! Yada yada yada…

Main classes of antigen formation in clinical use


Host attenuated (mutant)
Whole organism
“Live” e.g. BCG; adapt pathogen to non-human host & back into person
(weakened agent)
Recombinant mutant
Purified fraction from organism (part of toxin, antigen, etc)
Sub-unit
“Inactivated” Recombinant antigen
“Whole organism”

Vaccines with live-replicating organisms


 Need to attenuate pathogenicity but preserve immunogenicity
Immune response:
 activate innate immune system, don’t need adjuvant
 presented on MHC I/II, whole shebang: T-cell/CD4-Th/CD8-CTL/B-cells/memory/neutralizing Ab
Can sometimes cause disease if immunocompromised (risk/benefit; depends on disease burden, etc.)

Examples:
1. Attenuated virus: e.g. varicella virus vaccine (varivax)
 Changing hosts can cause virus to adapt in ways that make it less pathogenic to humans
 This vaccine: from child with varicella to human lung cell cx  guinea pig cell cx (gets adapted to different host)  back to
human cell cx (ensure immunogenicity); given sub-q
Oral vaccines:
2. Recombinant/Reassortment virus: e.g. rotavirus vaccine (RotaTeq) can provide mucosal
 Reassorted with different animal strain, using reverse genetics, to immunity (IgA)
attenuate pathogenicity
 Cover multiple strains if there are various pathogenic strains of virus
 This vaccine: human + bovine rotavirus reassorted, covers 5 rotavirus strains, given orally

Vaccines that are inactive/non-replicating


Immune response
 presented on MHC class II (CD4, not CTL CD8s, good memory response)
 often need to give adjuvant to better stimulate innate immune system (often Aluminum)

Examples:
1. Whole organism e.g. hepatitis A vaccine
o Need inactivation of pathogenic properties but preservation of immunogenicity
o Need to give with adjuvant (aluminum)
o This vaccine: given IM; formalin-inactivated whole virus vaccine from attenuated HAV in cell culture (fibroblasts);

2. Subunit: Polysaccharide e.g. meningococcal polysaccharide vaccine (Menomune)


o Need to purify antigen & inactivate toxicity (if applicable); no epitope mapping needed
o Stimulates innate immune system via TLR receptors
o Produces CD4-independent B-cell response (IgMs produced, short-lived)
o This vaccine: sub-q, from several groups of N. meningitides polysaccharides

3. Subunit: Protein-conjugated polysaccharide e.g. conjugated meningococcal polysaccharide vaccine (Menactra)


o Same basic idea as above; still stimulates innate immune system, need to purify / remove toxicity, etc.
o Conjugate polysaccharide to something immunogenic (inactivated diphtheria protein in this case)
7
o CD4-dependent B-cell response (IgGs, better memory response)

4. Subunit: Recombinant protein e.g. Hepatitis B vaccine


o Express antigen in genetically modified heterologous organism
o Requires adjuvant; produces CD4-dep B-cell response, IgGs, etc. (longer-lived)
o This vaccine: included in childhood to prevent perinatal transmission & cause it’s hard to diagnose

Vaccine uses:
 Active immunity: protection from person’s own immune system after vaccine, long-lasting
 Passive immunity: transferred from another person or animal as antibody; temporary, wanes with time
o Transplacental IgGs passed from mother to child: need to schedule childhood vaccinations accordingly
(or else mom’s IgGs will neutralize the vaccine antigen!)
o Results from: all blood/blood products, homologous pooled human Abs / Igs, antitoxins (pooled serum)

Immunizing during pregnancy


 Protects both mother and infant; transplacental Abs are cheaper & safer than Ig therapy
 Factors that influence Ab transfer in pregnancy
o Time from vax to delivery
o IgG level/subclass (IgM, IgA, IgE: DO NOT CROSS PLACENTA)
o Gestational age: @ 33wks IgGmaternal = IgGfetal, then fetal Abs more prevalent
 Influence on neonatal immunization
o Protect the infant
o Neutralize live attenuated vaccines; influence immunization schedule

Neonates’ Immunological Responses: immunize as early as possible; boost as needed


 Immature lymphoid organs; need 8+ wks post-natal age, adjust for premies
 Limited responses: CD8, innate system
 Immature dendritic cells (less CD4 activation, fewer germinal centers formed)
 Maternal Abs: don’t affect T-cell priming; epitope specific

The Elderly
 Thymus regresses
 Less naïve T-cells, mostly memory population: if you introduce antigen, it might be half-recognized by some cells
that are already around, and a less specific/effective immune response is mounted
 CD4 impaired (TRC/MHC signaling impaired)
 CD8 cells  senescence; Mϕ impaired

Immunocompromised: don’t give most of these vaccines!


 HIV patients can often mount immune responses if CD4+ are OK, check recs for vaccine

8
Antivirals
Background
Viruses: obligate intracellular parasites.
 Initial thoughts: kill viruses, kill the cell (bad), so develop ways to Need for therapeutic antivirals:
stimulate the immune system (vaccines).  more immunosuppression (chemo, transplants)
 Amantadine: selectively target influenza without killing cells, made  pathological / genetic immunodeficiency (AIDS)
antivirals seem plausible  greater potential for rapid spread of infection
 Current strategies: use basic science discoveries about viruses, (higher population density, greater global
biological screening / high throughput screening, rational drug mobility, emergence of new antivirals)
design
Antivirals against HBV
HBV: 300M+ worldwide, major cause of chronic hepatitis, cirrhosis, hepatocellular carcinoma
 Tiny genome (dsDNA, circular, single-stranded region) copied from RNA template by viral reverse transcriptase
after incorporation into virion

α-IFN Mechanism of Action: Anti-HBV agent. Stimulates Jak/STAT pathways leading to transcription of genes with
"interferon-specific response element (ISRE)"
Effects: ISRE genes interfere with pretty much all aspects of viral life cycle (especially protein synthesis)
Selective Toxicity: IFN is part of normal human antiviral response
Indications: HBV (chronic active HBV)
Administration: Subcutaneous or IM (poor oral bioavailability)
Toxicity: flu-like symptoms and sometimes neuropsychiatric problems
Resistance: tolerance develops in most patients; HBV terminal protein blocks signal transduction
Other: very short-lived effects

lamivudine Mechanism of Action: nucleoside analog (NRTI), inhibits both HIV and HBV reverse transcriptase
(3TC) (similarities in enzymes)
Effects: converted to triphosphate by cellular enzymes; competitive inhibitors / chain terminator of HBV
DNApol (no 3' OH)
Selective Toxicity: humans don't have RT
Indications: HIV, HBV
Administration: po (good oral bioavailability)
Toxicity: negligible
Resistance: mutations in viral RT, some mutants less fit in vitro, others 3TC-dependent. Discontinuing
leads to rebound of viremia.

Antivirals against Influenza (A, B, avian)


Influenza: (-) sense ssRNA genome, segmented, synthesis relies on viral RNA-dep-RNA-pol

M2 inhibitors
amantadine Mechanism of Action: Anti-influenza agent. Inhibits M2 protein
Effects: M2 protein = ion channel used to pump protons into virion compartment & reduce pH, which is
rimantadine required for uncoating.
1. Primary effect: Drug binds inside M2 channel & blocks.
2. Secondary effect: decreases pH in Golgi, causing premature HA conf change, decreasing release.
Selective Toxicity: Humans don't have M2 protein
Indications: Influenza; not used as much anymore
Administration: Oral (rimantadine is methylated deriviative of amantadine, better oral bioavailability)
Toxicity: CNS side effects (rimantadine can't cross BBB as well; less side effects)
Resistance: Rapid (30% in 5 days) via mutations in M2 AA's lining channel. Same mutations overcome
early & late effects. Mutants retain fitness.

9
Neuraminidase inhibitors
zanamivir Mechanism of Action: Anti-influenza agent. Competitive, reversible inhibitor of viral neuraminindase (NA)
Effects: NA cleaves terminal sialic acids from glycoproteins, glycolipids, proteoglycans, promoting effective
oseltamivir spread of virus throughout respiratory epithelium. Drug is sialic acid analog with larger, positively-charged
(Tamiflu) guanadine to interact more strongly with negative AA in active-site cleft. Oseltamivir also has a
hydrophobic region to bind to enzyme hydrophobic pocket
Selective Toxicity: humans don't have NA
Indications: prophylaxis and treatment of influenza
Administration:
 Zanamivir: poor oral bioavailability (IV or aerosol spray) - CAN'T USE IN PTS WITH RESP PROBS
 Oseltamivir: prodrug, better oral bioavailability, can give PO
Toxicity: Minimal (some nausea)
Resistance: inefficient in vitro; no cross-reactivity between zanamivir/oseltamivir, mutants have reduced
fitness

Antivirals against herpes viruses


Herpes: large, complex virus, linear, dsDNA genome, famous for latent infection
 Tons of interesting targets (tons of proteins) but worst choice - viral DNApol – is most common target

acyclovir Mechanism of Action: Nucleoside analog antiviral agent. Inhibits DNA synthesis
Effects: Chain terminator (no 3' OH) and competitive inhibitor of viral DNApol
valacyclovir Selective Toxicity: Two mechanisms:
1. For initial phosphorylation (ACV to ACV-MP) viral TK >cellular TK in affinity, so drug accumulates
in infected cells. (Next two P-lations via cellular TKs.)
2. ACV-3P inhibits viral DNApol much more than cellular DNApol.
Indications: HSV-1 (facial), half as active against HSV-2 (genital), not useful against CMV or HHV6
Administration:
 Acyclovir: 10-30% orally bioavailable
 Valcyclovir: prodrug with more bioavailability (substrate for intestinal/renal peptide
transporters; rapidly converted to ACV by intestinal/hepatic enzymes after oral administration)
Toxicity: Well tolerated: some nausea, diarrhea, rash, headache; rare renal/neural toxicity
Resistance: Mutation in viral TK (can't P-late ACV); causes cross-resistance to analog. Less frequent:
mutations in viral DNApol (less incorporation)

ganciclovir Similar to ACV but active against CMV


Similarities: converted to monophosphate by viral kinase, then -2P, -3P by cellular enzymes; competitive
inhibitor of viral DNApol
Differences:
 single hydroxymethyl group on sugar side chain
 no viral TK (p-lated by UL97, a protein kinase)
 not an absolute chain terminator (competitive inhibitor)
 accumulates to higher concentrations in CMV-infected cells (although ACV is better viral DNApol
substrate)
Toxicity: SERIOUS. affects bone marrow progenitor cells (low therapeutic index); inhibits lymphocyte
blastogenic responses
Resistance: mostly kinase mutations (like ACV)

10
foscarnet Mechanism of Action: antiviral (anti-herpes) agent. Pyrophosphate analog (inhibits all herpesviruses)
Effects: reversibly blocks pyrophosphate binding site on DNApol, inhibiting cleavage of pyrophosphate from
nucleoside-3P during elongation (pushing reaction backwards)
Selective Toxicity: viral DNApol is 100x more sensitive than cellular DNApol
Indications: only for life-threatening infections with no other treatment available (mechanism of action
different, so often works against ACV/GCV -resistant mutants of HSV/HZV/CMV)
Administration: Oral
Toxicity: SERIOUS. accumulates in bone, causes kidney toxicity
Resistance: Mutations in viral DNApol

fomvirisen Mechanism of Action: anti-CMV agent. Anti-sense RNA complementary to a viral mRNA
Effects: Binds, inhibits translation of a CMV mRNA encoding a protein essential for viral replication
Selective Toxicity: Doesn't bind human mRNAs
Indications: ganciclovir-resistant or -contraindicated CMV retinitis
Administration: injected into eye (completely unstable)
Toxicity: to your wallet
Resistance: has been reported
Other: VERY EXPENSIVE, only FDA-approved antisense drug

11
Pathology: ID & Micro (Fungi & Parasites)
Characteristics & Concepts of Medically Important Fungi ..................................................................................................... 2
Superficial / Cutaneous Fungal Infections .............................................................................................................................. 5
Opportunistic Mycoses ........................................................................................................................................................... 8
Pathogenic Mycoses ............................................................................................................................................................. 12
Introduction to Parasitology ................................................................................................................................................. 14

1
Characteristics & Concepts of Medically Important Fungi
 Candida: 3 -4 most common cause of blood
rd th
What is a fungus?
 Eukaryotic (hard to treat; close relationship to other euk) stream infection
 Heterotrophic: feed off of other sources  Aspergillis: most common cause of infectious
pneumonic mortality in BMT recipients
 Polymorphic: different shapes/forms
 PCP/PJP, crytococcus: among most common
 Cell wall: complex, heteropolysaccharides/peptides, target of AIDS-defining infections in HIV pts
antimicrobial therapy
 Cell membrane: contains sterols, commonly ergosterol (target of ampho B)
 Reproduction: all reproduce asexually, 75% have sexual cycle

Fungi contain chitin but not cellulose (plants have both)

Taxonomy based on characteristics of sexual reproduction; 4 classes cause human infections:


 Zygomycetes (mucoralis is order): lower fungi, reproduce sexually
o Rhizopus
 Ascomycetes: reproduce sexually
 Bacidiomycetes: reproduce sexually, basically mushrooms, one exception (Cryptococcus neoformans)
 Deuteromycetes: (deutero = “other”), sexual function has been lost (Candida spp.)

Morphologic Forms
 Yeast: unicellular fungus, reproduces by asexual budding (generation time = hours)
o Budding: create daughter cell, leave mother cell
 Filamentous: fungus whose vegetative form is a mass of individual hyphae (mold)
o Hyphae: characteristics used for dx in laboratory
Branching Septation
 dichotomous = “Y-shaped”  septate, e.g. Apergillus,
 right-angled = “T-shaped”  non-septate, zygomycetes, e.g. rhizopus)
 If NON-SEPTATE, think ZYGOMYCOSIS  AMPHOTERICIN is immediate response
 BRANCHING SEPTATE hyphae in immuncompromised with PNEUMONIA  Aspergillus
 Pseudohyphae: look like hyphae but not filamentous (yeast elongating)
 If HYPHAE, PSEUDOHYPHAE, and YEAST forms present: CANDIDA

Dimorphism: ability of some fungi to exist in two different morphological forms

Classic dimorphism: e.g. Histoplasmosis


 MOLD in ENVIRONMENT (room temp), YEAST in US
(tissue/lab)
 Taken up by Mϕ, cell-mediated immunity critical
Candida: opposite of classic dimorphism
 YEAST in environment, MOLD in us

Structure of a fungus
Encapsulated: only CRYPTOCOCCUS NEOFORMANS!
 Protects against host response
 Cryptococcal antigen: capsular antigen can be detected from LP in CSF via latex agglutination assay or ELISA.
Extremely sensitive test, targets glucuronxylomannan, produced in huge amounts in cryptococcal infections
Cell Wall:
 Rigid, heteropolysaccharide wall, very resistant to hydrolysis, strength & stability

2
 NOT a barrier to environment (cell membrane): like a chain link fence
 Multi-layered: glucans: inner fibrillar/inner matrix of cell wall; glycopeptides: inner/outer layers.
o 90% polysaccharide, 10% peptides
o 1,3-β-glucans: enchinocandins target this specific component of cell wall (Candida, Aspergillus)
o Also mannans, chitin, 1,6-β-glucans
 Can monitor mannans or glucans as markers in detection of invasive fungal infections
 Composition varies between different forms of fungi; target of cell/humoral immune response
 Important receptors for cells, intracellular matrices, and HARDWARE (catheters!)

Septum / septae FUNGUS SEPTAE


 Ingrowth of cell wall; appears to divide hyphae into individual cells; different Zygomycetes Few/none
septae for different organisms Ascomycetes Simple
Basidiomycetes Elaborate
Cell membrane
 Typical bilayer membrane; this is the real barrier between fungal cell/environment
 Sterols incorporated into lipid portion; most common is ergosterol, help maintain fluidity
 TARGET for drugs (ampho B = targets ergosterol in membrane directly, alloamines/azoles target biosynthesis)

Other structural features: ER/ribosomes, unstacked Golgi, simple mitochondria, membrane bound vacuoles,
 most haploid in vegetative form

Reproduction
Sexual reproduction: via fusion of hyphae (see picture on right)
Asexual reproduction:
 asexual spores, germinate  colony with identical genetic composition to parent
strain
 more resistant to organism, better dispersion
 can be infective respiratory inoculum in patients (esp. immunocompromised e.g.
AIDS pt, raking leaves  aspergillosis)
 Sporangiospores: asexual spores, produced in sac-like cell called
sporangium by zygomycetes
 Condida: asexual spores (not sporangium) by all other major
groups (e.g. Aspergillus)

Mycoses & Humans


 Virulence: varies widely between fungi; depends on host status
 Cause wide spectrum of infection ( -osis = disease)
 Transmission: endogenous flora, natural environment; most not
person-person
 Pathogenic morphologic forms can be varied; different than those in vitro

Fungal structure Examples


Budding yeast forms only Crypto, histo, blasto, sporo
Budding yeast + hyphae Candida, tinea versicolor
Hyphae Aspergillosis, zycomycosis, dermatophytosis
Spherule Coccidiomycosis
(Large spherical structure with internal spores)

Virulence factors:
 Cell surface receptors (epithelial cells, endothelial cells, caths, etc.)
3
 Hydrolytic enzymes, host mimicry
 Polysaccharide capsule (Cryptococcus)
 Melanin production: inhibits oxidative response (dampens host response)

Stains:
STAIN FEATURES
H&E Differentiate host response, not sensitive for fungi detection
PAS (periodic acid-Schiff) Stains acid polysaccharide cell wall of fungi
GMS (Gomorri’s methenamine silver) Deposits silver on fungal cell wall, better sensitivity of detection
Mucicarmine / Alcican blue Specific for Cryptococcus capsule
(Fontana Masson) Melanin in cell wall of some fungi

Classification by Host Response / Disease

1. Superficial: no inflammation, cosmetic


 E.g. tinea versicolor: very superficial blanching, “spaghetti & meatballs” (hyphae & clusters of yeast)

2. Mucocutaneous: inflammation occurs but no invasion of deeply viable tissue


 E.g. dermatophytosis: tinea corporis, ringed, papulosquamous (silvery, raised) eruption
o Tinea: initially thought was worm (“ringworm”)

3. Subcutaneous: localized infection following trauma


 Chromoblastomycosis (“copper pennies” + sheets of PMNs)
 Mycetoma (highly destructive, muscles/tendons/bones)
 SPOROTRICHOSIS: much more common than the other two
o Traumatic inoculation  single lesion  lymphocutaneous propogation (up lymphatic)
o Working with hay, prick from ROSE PETAL

4. Deep mycoses: life-threatening


TYPE OF FUNGI HOST IMMUNE STATUS MAJOR IMMUNE RESPONSE EXAMPLES
OPPORTUNISTIC Common fungi Compromised PMN Candida, aspergilla,
zygomycetes
PATHOGENIC More virulent fungi Can be CMI (cell-mediated Histoplasmosis, other
immunocompetent immunity) endemic mycoses

IMPORTANT TO REMEMBER:
Diagnosis: key features of certain organisms
FEATURE ORGANISM
Zygomycosis / Rhizopus
Non-septate hyphae
(give ampho B)
Branching septate hyphae
Aspergillus
(pneumonia in immunocomp pt)
Hyphae + pseudohyphae + yeast form Candida
Capsule Cryptococcus

4
Superficial / Cutaneous Fungal Infections
1. Dermatophytosis
2. Onchomycosis (nail infection)
3. Tinea versicolor (superficial, variable color, nape of shoulder  across chest)

Superficial mycosis Cutaneous mycosis


Layer affected Superficial straum corneum Epidermis/dermis
Host response No host response Inflammatory response
Example Tinea versicolor Dermatophytoses

Cutaneous fungal infections

Dermatophytes cause most cutaneous fungal infections Dermatophytes


Infections often named by region of body that they inhabit (see table)  Microsporum spp
 Tinea capitis: only children (adults- change in cebaceous glands  stop  Epidermophyton floccosum
getting tinea capitis infections)  Trichophyton spp
 Tinea = “ringworm” (not actually worm) – NOT name of species! Tinea… Dermatophyte
infection of…
Also classified by environmental reservoir Pedis Foot
 Anthropophilic(humans) – e.g. T. tonsurans Capitis Head
 Zoophilic (animals) – e.g. M. canis Corporis Body
 Geophilic (soil) – e.g. M. gypseum Barbae Beard
Cruris Groin
What causes what? Unguium Nail
 Trichophyton (most in US): EVERYTHING (more or less) Manuum hand
o capitis, barbae, corporis, cruris, pedis, unguium
 Microsporum (more common worldwide): capidtis, corporis, cruris, pedis,
o NOT UNGUIUM (no nails)
 Epidermophyton floccosum (only spp of this genus): TINEA CRURIS (groin)

Pathogenesis / Host Defense


 Dermatophytes use keratin as nutrient source
 Inflammatory host responses responsible for involvement of surrounding tissues
 Cellular immunity is key factor in host defense (iron metabolism too)

Hair invasion (arthrocondia = asexual spores)


Type of invasion Athrocondia form… Cuticle Clinically Examples
M. canis
More inflammation
Ectothrix Outside of hair shaft Destroyed M. gypseum
More likely to grow back
T. equinum
Can pull out hair with bulb, without pain.
Endothrix Within hair shaft Intact T. tonsurans
Less likely to grow back

Infections: if possible, can use skin scraping + wet mount to dx

1. Tinea pedis:
a. most common (70% adults worldwide); often Trichophyton rubrum
b. 3 clinical forms: interdigital, moccasin, vesiculobullous (can treat topically)
c. Can have 1 hand + 2 feet: tinea pedis et manuum
d. Can have onychomycosis along with tinea pedis (need to treat systemically)
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2. Tinea corporis:
a. non-glabrous skin (trunk, extremities)
b. “Ringworm” – erythematous, round, scaly patch; red, raised, advancing border +/- papules/pustules
c. Itchy (pruritic)
3. Tinea cruris:
a. Invasion of hair follicles (can confuse with cutaneous candida)
b. Predisposition: tinea pedis/onchyomycosis at same time (transfer?)
4. Tinea capitus:
a. Infants, children, young adolescents, in US mostly urban (AA/Hispanic preschoolers)
b. Can transmit child-child or animals/humans
c. Usually Trichophyton (T. tonsurans especially) in US; Microsporum canis most common worldwide
d. Variety of manifestations (pustles/papules/etc) on scalp
i. Inflammation  scaling, alopecia, erythema/exudate/edema
ii. Ectothrix: “black dot alopecia” (some patches preserved)
iii. Endothrix: total hair loss
iv. Kerion (scalp condition; thickened raised area with set of spongy lesions) forms
1. increased cell-mediated immune response; all Mϕ & mono not PMNs)
2. Severe inflammation, hair loss, cervical lymphadenopathy
e. Need to hit hair follicles: systemic + cutaneous treatment

Lab Dx of Dermatophyte Infections


 KOH of scale scraped from leading edge of lesion (destroys most cellular debris)
 Culture to confirm; special agar (Sabouraud dextrose) for up to 4 weeks
o ANY GROWTH OF DERMATOPHYTES IS SIGNIFICANT (not a contaminant)
 Wood’s light: UV light, M. canis will fluoresce blue-green

Treatment of Dermatophyte Infections:


Tinea Capitis: Topical + Systemic Tx
1. Oral antifungals (griseofulvin, others)
2. Ketaconozale shampoo (reduce fungal shedding)
3. Prevent spread (clean contaminated brushes, pillows; selenium sulfide for other family members)
4. Kid is OK to go to school as soon as he’s on treatment

Tinea pedis, corporis, cruris, manuum: Topical Tx


1. Miconazole, clotrimazole, etc.
2. Oral if extensive/severe/recalcitrant infection

Onchyomycosis
Onchyomycosis: infection of nail plate and/or nail bed that interferes with normal nail function
Epidemiology: mostly dermatophytes (T. rubrum, others)

Presentation: pain, dysfunction, paronychia (skin infection around nails)


 Increased risk: diabetes (bad!), HIV/AIDS, compromised hosts, elderly

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Clinical classifications: PSO/DSO/WSO
DISTAL SUBUNGUAL (DSO) PROXIMAL SUBUNGUAL (PSO) WHITE SUPERFICIAL (WSO)
Immunocompromised hosts
PREVALENCE Most common (90%) 10%
(early HIV infection indicator)
Distalproximal
Proximaldistal Dorsal surface of nail
(hyphae under nail plate, spread
INVASION (starts at cuticle, spreads to
proximally, digest stratum plate attacked
entire nail bed)
corneum of nail bed & nail plate)
Whole nail involved / Minimal inflammation
PRESENTATION Proximal parts relatively intact
obliterated (not attacking viable tissue)
HOST RESPONSE Cell-mediated immunity
T. rubrum (most common)
SPECIES T. tonsurans, T. mentagrophytes, E. T. rubrum T. mentagrophytes
floccosum)

Diagnosis: KOH + culture of nail


Treatment/prevention: need ORAL THERAPY (get into nail bed, e.g. griseofulvin)

Tinea Versicolor
Superficial mycotic infection
 Young, middle-aged adults
 Upper trunk/neck/arms; often manifests as depigmentation (“ sun spots” because they don’t tan)

Malassezia furfur is causative agent


 Lipophilic yeast (needs olive oil or something in the agar to grow)
 May also cause fungemia with parenteral lipid solutions (e.g. in babies)
 might be involved in other conditions too

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Opportunistic Mycoses
Candida, Aspergillus, Zygomycetes, Cryptococcus, Pneumocystis

Mycoses: 2 groups based on ability of host’s non-immune cells to phagocytose & kill the challenging fungal spore/yeast
Mucocutaneous candidiasis
Altered T-cell function
Cryptococcus
(e.g. AIDS)
Opportunistic compromised Pneumocystosis
mycoses hosts only Altered phagocytic Invasive candidiasis
activity (quantitative or Aspergillosis
qualitative defects) Zygomycosis
Histoplasmosis (Histoplasma capsulatum)
Blastomycosis (Blastomyces dermatitidis)
Pathogenic,
Coccidiomycosis (Coccidioides immitis)
deep, systemic normal hosts Cellular/T=cell function
Paracoccidiomycosis (Paracoccidoides brazilensis)
mycoses – Latin America, N. Brazil
Penicilliosis (Penicillium marneffei)

Candidiasis
 Opportunist (causes wide range of infection)
 Candida is genus, albicans is most common member
o Albicans is Germ tube POSITIVE, others aren’t
 Hyphae + pseudohyphae + yeast
 Components of normal flora on mucosal surfaces (skin/oral/GI tract/female GU)
 Causes infection only in compromised hosts
Infections
Mucocutaneous Deeply invasive
THINK NORMAL MUCOSAL DISTRIBUTION.  Candidemia: #3-4 for blood infections overall
oropharyngeal (thrush), esophageal  Endocarditis, hepatosplenic candidiasis
Clinical
candidiasis, candida epiglottis (chronic/disseminated),
presentation cutaneous, onchyomycosis, keratitis,  Acute disseminated candidiasis (high burden  septic shock)
vulvovaginal.  Renal candidiasis (filtering out candida sets up shop)
 Altered barriers (vascular/urinary cath, peritoneal dialysis,
 Underlying disease (HIV/diabetes)
trauma, burns, cytotoxic drugs)
 Corticosteroids
Risk factors  Neutropenia, BMT/solid transplants, surgery
 Pregnancy, elderly (↓ immune)
 Broad spectrum Abx
 Antibacterial Abx (kill normal flora)
 Hyperalimentation, hemodialysis
Topical if not serious
 Clotrimazole, etc. Need SYSTEMIC Tx (Fluconazole, etc.)
Treatment
Systemic if serious (e.g. if esophageal) (Ampho B as salvage b/c of toxicity)
IV if needed

Mucocutaneous candidiasis
Smear / scrape:
 Mucosal: mucosal surfaces; white pseudomembranous placque
hyphae + pseudohyphae
 Cutaneous: intertriginous (where 2 areas of skin rub together) areas: scalded
+ budding yeast
lesions, punctuate satellite lesions
o Diaper dermatitis, paronychial/onchyomycosis, moist areas
o Diabetes
 Chronic mucocutaneous candidiasis: genetic inherited disorder, big scarring; disfiguring
o ↓ cellular immunity to Candida + polyendocrinopathies
 (DM I, adrenal insufficiency, hypothyroid/gonad/parathyroid/etc.)
o Intractable candida: mucocutaneous surfaces (oropharynx, face, toes, fingers, intertriginous areas)
o “Autoimmune-polyendocrinopathy-candidosis-ectodermal dystrophy” (APCED)

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o Tx: fluconazole but worry about resistance in long term use
Invasive candidiasis
 Pathogenesis:
1. Adherence/colonization 
2. Penetration through mucosa  angioinvasion / access to venous caths 
3. Hematogenous dissemination 
4. Replication in tissues (necrosis +/- abscess formation) Dx in Tissues:
 Host response: hyphae + pseudohyphae
1. Immune competent: acute + chronic inflammatory cells + budding yeast
2. Neutropenic: no abscesses form, lots of hyphae
If you suspect invasive candidiasis:
GET A FULL OPHTHALMIC EXAM
Candida Albicans (to check for involvement of vitriol –
 GERM TUBE POSITIVE species (form hyphae; others are negative) candida endophthalmitis)
 Virulence factors:
o surface receptors (epithelial/endothelial cells; extracellular matrices, hardware)
 can act as immunomodulator
 sticky for cardiac valves, caths, etc.
o Hydrolytic enzymes, host mimicry
o dimorphic (yeast in environment  colonizes  sets up shop as hyphae)

Aspergillus spp (Aspergillosis)


 Filamentous; common in environment
 All infections OPPORTUNISTIC
Treatment of aspergillosis
 Voriconizole / ampho B
Clinical presentations
 Need host immune
1. Toxin-mediated:
response: reverse immune
o aflatoxins (extremely carcinogenic; cause hepatocellular carcinoma) suppression!
–on stored grains/peanuts; not elaborated inside a patient
2. Allergic syndromes (atopic pts)
3. Colonizations / saphrophytic
o fungal ball = “aspergilloma” in old TB cavity or impacted paranasal sinuses
4. Infections (deep infections)
o Keratitis (post corneal trauma)
o Invasive disease: pulmonary +/- dissemination

Invasive Aspergillosis
 Risk factor: PMN FUNCTION depression Conidia = asexual spores
o Quantitiative (neutropenia)
o Qualitiative (function: CGD, post-BMT, high dose corticosteroids, HIV)

 Pathogenesis: Invasive Aspergillosis:


1. Inhaled (conidia)  alveoli   “Angular dichotomously
 Normal host: phagocytosis (Mϕ, killing) branching septate hyphae”
 Compromised host: may or may not phagocytose; don’t kill o = Y-shaped with
2. germinate  hyphal invasion of lung parenchyma  septae
3. Angioinvasion (thrombosis, ischemia, infarction)   Angioinvasion, thrombosis
4. Hematogenous dissemination (sometimes)  areas of necrosis

 Role of host defenses


1. Neutropenia: angioinvasion, infarction, dissemination, hemorrhage

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2. Immunosuppresion: inflammatory necrosis, local invasion

 Radiology: (not specific for aspergillus – anything that invades a large blood vessel - but commonly present)
1. Halo sign (dense nodule = infarction; delicate structure of local ischemia around it)
2. Air crescent sign (e.g. in recovery; infarcted area
where aspergillus was, separated by a crescent of air
from surrounding parenchyma)

Aspergillus fumigates

Pathology: has characteristic structure:


 Culture: condiophores, vesicles, phialides, conidia
o Powdery surface colonization on plate
 Tissue: septate hyphae (filamentous)
o Note: culture is diagnostic form, tissue just
highly suggestive
Virulence factors
 Adherence receptors, hydrolytic enzymes, complement inhibition; etc.
 Toxins: not afalotoxin in vivo but others

Aspergillus niger
 Commonly saprophytic (in fungus balls; lives off of dead tissue)
 Black color; commonly found in environment

Zygomycosis (mucormycosis)
 Opportunistic; caused by several zygomycetous fungi (Rizopus is most
common species)
 Pathology: wide, non-septate hyphae that branch at right angles
o Key: NON-SEPTATE; T-SHAPED
o Invasion of blood vessel walls/nerves; extensive necrosis in
advance of fungus
 Rapid-growing “LID-LIFTERS” (both in lab and in vivo!)
 Sporangiophores have large “sporangia” sacs filled with sporangiospores
(asexual spores)

Disease Description At risk patient


Saphrophytic / Old TB or other lung cavity; no invasion or
Post-TB, bronchiectasis, etc
colonization dissemination.
FAST course – need to diagnose quickly and get biopsy
1. Inhalation (asexual spores from environment) 
Rhinocerebral paranasal sinuses 
Diabetes mellitus + ketoacidosis
Invasive

zygomycosis 2. tissue invasion (nerves, blood vessels)  cranial nerve


palsies, thrombosis, necrosis 
3. invasion of orbit & eye  extension to brain.
Pulmonary +/-
dissemination
Marked by angioinvasion Neutropenia

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Cryptococcus neoformans
At risk: T-cell-compromised (corticosteroids, transplants, HIV with CD4 < 100)

Pathogenesis:
1. inhale yeast (environment)  lung replication  CD4/CD8 recruited  usually cleared (specific response)
2. If immunocompromised (T-cells)
a. Progressive pulmonary infection
b. Hematogenous dissemination (cross BBB to BRAIN)

Pathology:
 Normal host: chronic inflammation +/- granulomatous response; resolve w/o calcification
 Compromised: mild to non-inflammatory response
 Gelatinous lesion (ENCAPSULATED) Diagnosis of cryptococcus
 Spherical yeast cells with:  Antigen test or direct
o clear area (capsule), obs. in CSF
o narrow/pinched mother-daughter attachment
 Need PAS/GMS (H&E doesn’t really work) Clinically:
 confusion, decreased
Radiography: disseminated infection concentration, headache
(increased ICP)
Virulence factor: CAPSULE
 Glucuronxylomannan with different side chains (different serotypes) Treatment:
o Produced in excess: detectable as ANTIGEN FOR RAPID Dx  AMPHO B + 5FC
 Inhibits phagocytosis; poor in vivo antigen
 Others: phenoloxidase (produces melanin, which inhibits oxygen-dep killing & is stainable)

Pneumocystis carinii/jiroveci
 Opportunistic
o CELL-MEDIATED IMMUNITY is key (not neutropenia) Diagnosis of PCP
 Alveolar-interstitial pneumonia (fever, dyspnea, non-productive cough)  Bronchioalveolar lavage:
o Extrapulmonary dz is uncommon cysts of trophozoites
o Tachypnea + hypoxia  DFA (mAb available)
 Risk factors: immunosuppresion, corticosteroids, HIV infection, elderly  PCR

Radiography Treatment:
 No nodules or infarcts  TMP+SMX
 Interstitial / alveolar involvement, multilobar
 Delicate proteinaceous debris in alveoli, blocks oxygen exchange (alveolar / interstitial disease)
o Trophozoites from cyst damage & create interstitial rxn / debris

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Pathogenic Mycoses
Histoplasma, Coccidiodes, Blastomyces, Paracoccidiodes

 Can cause infection in normal host; all are endemic dimorphic fungi
o Contact with organism in well-defined ecological niches
Organism Niche Geography
Histoplasma capsulatum Soil, caves (bird/bat feces) Ohio/Mississippi Valley regions
Coccidiodes immitus Desert soil SW USA (Sonora desert)
Blastomyces dermatitidis Water North/Central, SE USA
Paracoccidiodes brasiliensis unknown South America (Venezuela, N. Brazil)

General features:
 Entry = inhalation (asexual spores from environment); NOT PERSON-PERSON (even if mimics TB)
 Asymptomatic or mild in most hosts
 Disseminated progressive infection: 1/1000 infections Dermatophytes are
o More frequent in T-CELL COMPROMISED transmissible, others
 Pathology: chronic inflammation, granuloma formation generally aren’t

Histoplasmosis (Histoplasma capsulatum)


 Range: Asx to life-threatening
 Histoplasma capsulatum: dimorphic fungus Histo radiography
 Mostly soil/caves in Central USA  acute = infiltrate
o Nature/room temp: filamentous;  chronic = cavitary
 makes micro/macroconidia (microconidia = infectious)
o In vivo/37C: yeast

Disease:
 Pulmonary entry
o Acute: (90-95% have asx or mild resp sx; 5%: moderate mild to severe resp dz)
o 1/1000: disseminated infections (more common in T-cell compromised);
 Severity/progression: related to host status
 Disease of the reticuloendothelial system: Mϕ lining lung, spleen, LN, bone marrow

Pathology
 Early / active infection: intracellular budding yeast cells (in Mϕ & monocytes)
 Normal hosts: Granulomas (fibrosis, calcification in old lesions); Few intracellular yeasts
 Immunocompromised (e.g. HIV+): poorly formed granulomas; Many intracellular yeasts
In vivo yeasts
Diagnosis: DNA probes Cell response Location Disease
Histoplasmosis Monocytes Intracellular Lung dz
Culture: SLOW; takes weeks. Molecular Candida glabrata Lymphocytes Extracellular UG / bloodstream opportunist
probes are faster.
 See conversion to yeast at 37C (reverse of candida); macroconidia + hyphae
 Organism is HIGHLY TRANSMISSIBLE in this form (careful! Advise!)

Virulence factors: evades killing by phagocytes; replicates in phagolysosome (neutralize acid environment?)

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Blastomycosis (Blastomyces dermatitidis)
 Pneumonia + other presentations
 Water in NE/central USA
 Less propensity for reticuloendothelial system than histoplasmosis
 Hyphae (25C)  large yeast, double wall, broad-based budding (in vivo, 37C)

Coccidiomycosis (Coccidiodes immitis)


 Soil of Sonoran desert of SW USA

Characteristic structure
 Hyphal form at room temperature
o Arthrocondia: little boxcar units
 hydrophobic & easily transmitted (room temp)
 Dispersed throughout environment
 Spherules in tissues (very characteristic); invasive

Clinical presentation: infects lungs, usually mild,


 sometimes can disseminate (brain, joints, other organs)
Tx: antifungal drugs (sometimes for life)

Virtual Rounds
PATIENT DIAGNOSIS TREATMENT PLAN
Little boy with itching scalp; hair falling out. Exam: Tinea capitis: High temp cycle for clothes, systemic Tx +
small areas of inflammation/erythema/scratching; pull Trichophyton spp ketoconazole shampoo, selenium sulfide
hair out including bulb. shampoo for other kids
Migratory farm worker, was working with moss. sporotrichosis Azole
Multiple cutaneous lesions, draining.
sICU pt: came back from surgery starting to spike high Candida (if germ tube +, Fluconazole. If liver enzymes elevated,
temperatures, came back as yeast. albicans) can’t use (use echinocandins) Think of
eyes (call ophthalmologist); think of cath
(make sure it’s clean)
Oncology: AML pt in high dose chemo; cough/high Aspergillus Voriconazole; if liver enzymes elevated,
spiking temperature / pleuritic pain. See halo sign on maybe ampho B
radiography. See branching septate hyphae from
bronchioalveolar lavage
14 year old diabetic girl. Acidotic, sinus infiltration that Zygomycosis Ampho B. Debride, correct underlying
shows black, darkened, necrotic nasal turbinate on immune deficit
biopsy. Broad, non-septate hyphae
24 yo IV drug user; minimal access to medical care. Cryptomycosis Ampho + 5 FC. Worried about
Headache. Get LP with antigen test, positive meningitis, increased ICP
Pt with high risk for HIV. Shortness of breath, 85% Pneumocystis (PCP/PJP) TMP+SMX
O2Sat, diffuse interstitial infiltrate with no nodules.

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Introduction to Parasitology
Parasite: organism living in complete dependency in or on another living organism (host)
 Host shields parasite from outer world; provides food (parasite’s “restaurant”) Major themes of
 Generally protozoa, worms, arthropods parasitology
Host:  Attachment/invasion
 Host cell invasion
 Definitive: host where sexual reproduction of parasite occurs
 Host-parasite
 Intermediate: host for immature parasite stage / asexually-reproducing stage interaction
Vector: disease-causing parasite is conveyed from this host to another host  Obtaining nutrients
 Immune evasion
Parasitism: most common way of life (>50% all spp);  Encystation/eggs
 all living creatures have parasites (tiny viruses to big tapeworms)  Behavioral changes
 Humans: found in variety of tissues/organs
 man-made ecological changes responsible for perpetuating/intensifying most infectious/parasitic diseases

Epidemiology: huge burden; most morbidity from chronic infection; mortality figures high (malaria in Africa > CVD in US)

Attachment/invasion of host
Attatchment: Parasite needs mechanism to interact with host & prevent its expulsion
 Molecular (receptor-ligand)
o e.g. Plasmodium & RBC molecules; falciparum/vivax use different molecules
 Physical interaction:
o e.g. hookworm, attach with sharp teeth/hooklets
o Feces eggs in soilstepped on  footlungaspirate (weird!)  GI tract

Invasion: Obligate intracellular parasites need host cell to survive & replicate Mechanisms of invasion
 Helminths: different modes of invasion 1. receptor/ligand interaction
1. Direct from environment– worms penetrate skin directly, go to 2. subvert host cell transmembrane
blood (shistosomes, hookworm) signaling pathway
2. Along vector bite path – bite  bloodstream (brugia) 3. modify host cytoskeleton
3. Dispersed from vector bite – enter skin, then go all around through tissue (onchocerca)
Host cell invasion
 Apicomplexan invasion (Toxoplasma gondii)
o glides along surface  apical tip (rhoptry neck) invades; forms
moving junction  rest of parasite pulled in behind (like boat in
Panama canal) – see picture to right
o Result: parisotophorous vacuole. Parasite proteins not
expressed in vacuole, but later help it survive

 Attraction of lysosomes (Trypanosoma cruzi / Chagas dz)


o Secretes molecules to attract lysosome in endothelial/cardiac
cells, gets inside (can also enter via parisotophorous vacuole & fuse)
Host-parasite interaction
Host: Dynamic interaction; host tries to reject, can release cytokines (e.g. IL-8)
Parasite:
 can de-differentiate cells to suite needs
o e.g. Trichinella worm; de-differentiates host muscle by shutting off muscle-specific genes – better for parasite,
huge inflammatory response for host)
 can redecorate host cells to suit needs
o e.g. Plasmodium falciparum: inserts protein into RBC PM; bind to host endothelium so they don’t get destroyed by
spleen (sequestered)

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Obtaining nutrients
By definition, parasites obtain nutrients from their hosts
 E.g. plasmodium digests Hb from RBC; some protozoa (Toxoplasma) can’t synthesize purines on own

Immune evasion
1. Interfere with host immune system to co-exist
 Block Ag processing by inhibiting protease cleavage in APC; induce suppressor Mϕ & Treg cells, induce tolerance,
use superantigens, inhibit T/B activation
Clearing: Ab / CMI can be important, or innate/Mϕ can be important (Depends on parasite; intracellular = more innate)
Worms: EOSINOPHILS & IgE RESPONSE 

2. Some protozoa can multiply within Mϕ (have to escape lysosome digestion)


Activated Mϕ (T-cell help)  kills parasite; fusion of phagosome with lysosome
Toxoplasma gondii
Naïve T-cells  parasite lives in endosome; doesn’t fuse with lysosome
Trypanosoma cruzi Killed or escapes phagosome to divide in cytoplasm
Leishmania Doesn’t care: resists lysosome enzymes; lives in phagosome
3. Antigenic variation: African trypanosomes express hundreds of VSG (surface proteins); can’t make vaccine
 Waves of infection: one VSG type cleared; another takes over; cleared, etc.

Encystation: eggs/cysts
 Environmentally stable forms (good for transitioning between hosts) Good for diagnosis:
o Thick walled cysts (protozoa, esp. intestinal) stool ova & parasite exams
o Eggs (worms) (“stool O&P”)
 Can be signature forms:
o oocysts in cryptosporidium
o Schistosome eggs
 lateral spur = mansoni; end-spur = haematobium, round = hepatica)

Host behavior
Parasites can alter host behavior
 E.g. Toxoplasma gondii: from cat feces; rats eat it, stop being afraid of other animal
scents (makes it easier for cat to catch them!)

Mechanisms of Pathogenesis
1. Direct cellular damage
 Need to balance host cell damage & needs from host cell
 Direct damage from lysing cell during egress; secreting pore-forming How do parasites cause disease?
peptides, secreting proteolytic enzymes 1. Direct cellular damage
 E.g. Toxoplasma lyses cells during egress – necrotic cell death; invades 2. Mechanical
adjacent cells during the process obstruction/compression
3. Host immunological response
2. Mechanical obstruction/compression 4. Other disease mechanisms
 Helminths are prototype: obstruct GI tract or lymphatics
o e.g. Ascaris  intestinal obstruction in kids in developing countries
o e.g. lymphatic filariasis: block LN, backup of lymph  elephantiasis
 Parasite-filled abscesses/cysts compress vital organs
o e.g. pork tapeworm: brainmass effect seizures
o Encephalitis/brain abscesses in HIV  cerebral Toxo

3. Host immune response

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 Eosinophilia (helminths) irritate GI lining, increase bowel permeability, produce more eosinophils
 Granulomas (around destroyed larvae or eggs)  colon/rectum walls, elsewhere (back up fluids, damage)
 Cytokines: IL-8, TNF-α, etc.
 Damage from parasite itself can be minimal; host immune reaction  extreme & harmful
o Schistosomiasis: eggs in bladder  granulomatous rxn  fibrosis  obstruction; carcinoma!

Other
 Anemia, fever, organomegaly, malnutrition, diarrhea, rash, etc.

Intestinal protozoa
 Fecal-oral route; cyst-tryphozite stages (Giardiasis, amebiasis, crytptosporidiois)
o Cyst: resistant wall (infective, found in feces)
o Trophozoite: metabolically active & mobile (non-
infective)
Diarrhea:
 Secretory: small intestine, ↑Cl- secretion from crypt cells
o E.g. giardia (cyst ingested, releases trophozites, differentiates
into cyst again in gut lumen in response to bile  shed in
watery diarrhea  infective).
 Villus blunting, infiltrating lymphocytes, secretory diarrhea
 Invasive / malabsorptive: esp. colon
o Normally need brush border, good epithelium
o Damage to brush border (break junctions/ulcerate)  malabsorption
o Dysentery: diarrhea + blood/mucus in stool
 E.g. Entamoeba histiolytica: protozoa; common cause of dysentery in developing countries;
 Trophozoite / cyst life cycle
 invades host intestinal mucosa; can spread to liver to make abscesses (lung, brain too)
 Can cause colitis (flask-shaped ulcers – spread laterally)

Trypanosoma brucei: African Sleeping Sickness


 Parasitic protozoa; 2 subspecies
T.b. rhodesiense T.b. gambiense
Geography East africa West africa
Chancre: Inflammatory reaction at site Winterbottom’s sign: Enlarged lymph nodes
of tsetse fly bite in neck
Early Sx/ Signs
Rashes with fever if fair-skinned
Kerandel’s sign: press hard on hands; severe pain shortly after release
Cross BBB to CNS Quickly More slowly

 Transmitted by tsetse flies; prefer to bite humans


o disseminate everywhere post infection & multiply wherever blood is
 Epi: 100% FATAL IF UNTREATED; 500k+ infected; thousands permanently disabled from treatment

 Sx: confused with malaria (high, spiking fever)


o CNS involvement:
 Neuro problems: conflicting psych Sx(agitation, indifference, irritability, uncontrolled sex
impulses, violence)  motor system distrubrances (paralysis, tremors, twitching, slurring), pain,
itching (leads to suicide in some!)
 Sleep disturbances (reverse sleep cycle: insomnia + irresistible urge to sleep)
 Seizures, incontinence, total body cachexia (CNS-mediated apoptosis), coma, death

16
 Tx: need early treatment (otherwise could cross BBB  CNS involvement; white matter encephalitis
o Early stage: without CNS involvement
 Suramin (rhodesiense) or Pentamidine (gambiense), good prognosis
o Late stage: CNS involvement
 Melarsoprol – arsenical; HIGH TOXICITY: 4-12% MORTALITY
 Eflornithine – expensive, injections x14d; phase III trials for oral underway (good for preventing
unwanted hair growth in women too!)

17
Pathophysiology: ID & Micro (Fungi & Parasites)
Malaria .................................................................................................................................................................................... 2
Helminth Parasitism ................................................................................................................................................................ 6
Other Protozoa...................................................................................................................................................................... 10

1
Malaria
Global Burden
 Was formerly prevalent in US; eradicated via infection controls & social improvement
 1st global push (‘50s) to eradicate based on DDT+chloroquine; success in some areas, partial in others
o No serious attempt in Africa
o Failed: unrealistic expectations, no integration with existing infrastructure
 Chloroquine-resistant P. falciparum & DDT-resistant Anapholes
 Global distribution today: Africa biggest, SE Asia drug resistant, also other parts of world
o Very different distribution in different countries within Africa – some much higher than others

Epidemiology
 Parasitic, mosquitos; 247M cases/yr, 891K deaths, 85% in sub-saharan Africa (YOUNG KIDS & PREGNANT)
o Resurgence: drug resistance, other factors, no vaccine
 Four species of malaria:
o Plasmodium falciparum: 90% infection; almost all death in Africa, MDR, vaccine efforts
o P. vivax: big contributor in SE Asia morbidity (& mortality)
o P. ovale (Africa only), P. malariae too

 Highly variable around world & within countries with different presentation
o Related to intensity of burden, duration of transmission
o Classic definitions: Spleen rate: hypoendemic < meso < hyper < holo
 Acquired immunity:
o Stable malaria: heavy, perennial transmission; endemic
 Generally protected from severe dz after age 5 (except for in pregnancy
o Unstable malaria: less intense transmission; epidemics / outbreaks
 Protective immunity: later age or not at all; all ages vulnerable

Immunity
 Humoral & Cellular; Initially: innate + spleen
 Maternal Ab last 3-6 mo (don’t see severe dz in children < 6mo)

Protection: slow, need prolonged, repeated exposure; protection from infection is not achieved
 Immunity lost if exposure stops: very common to see expat visit old country & get malaria
 Diminished immunity in pregnancy: increased risk of disease & complications, incl. still birth/miscarriage/low birth wt)
 Limited interaction with HIV: co-infection, not opportunist
o Viral load increases in acute phase; lost protection against malaria
o Biggest interactions in HIV+ pregnant women

Innate immunity:
 Malaria hypothesis: red cell polymorphisms distributed geographically because of selective pressure of malaria
o Hb structure, thalassemias (Hb synth), G6P deficiency (RBC enzyme), Duffy negative blood (PM of cell)
o HLA types? May protect against severe malaria
Duffy receptor and vivax malaria
 Chemoine receptor; spans PM, present in endothelial cells, only P. vivax binds for entry to RBC
 Duffy negative: primarily present in Africans (no vivax)

2
Life Cycle
A. Mosquito bite (female
anopheles mosquito at
night)  Sporozoites
injected; clinically Asx
B. Hepatic stage: multiple
stages, 6d-weeks of
“incubation”, results in
hepatic schizont filled with
merozoites (still Asx)

(P. vivax & P. ovale can


arrest here as hypnozoites in
liver & relapse months to
year after primary infection)

C. Schizont ruptures and


releases merozoites into
blood stream, which infect erythrocytes.
If patient has Sx post-Tx, what’s up?
D. Erythrocytic schizonts filled with merozoites rupture; more red cells
released: periodicity (via asexual reproduction) Recrudesence: P. falciparum & P.
E. Some merozoites differentiate into gametocytes malariae, reappearance of parasites in
F. Gametocytes taken up by female anapholes mosquito; sexual the blood (e.g. after being pushed down
reproduction takes place in her, infects other host below detectable threshold.
Relapse: P. vivax & P. ovale, revival of
Note: SEXUAL forms responsible for transmission
hypnozoites in the liver.
ASEXUAL for periodicity of symptoms
Re-infection: new infection for patient
Invasion of erythrocytes leads to knobs forming (“sticky” RBC)

Species-specific characteristics:

P. falciparum: P. vivax
 ~5.5d incubation in liver  8 day incubation
 48 hr erythrocytic cycle (fever periodicity)  48h periodicity
 Tons of merozoites per schizont  Fewer merozoites /schizont
 Infects ALL KINDS of RBC (HIGH parasitemia)  Invades mostly RETICULOCYTES (LOW parasitemia)
 Need HIGH burden for fever (even more if immune)  Need lower burden for fever
 Can form hypnozoites (dormancy!)
P. malariae: P. ovale:
 72h periodicity  Similar to P. vivax
 Can form hypnozoites (dormancy!)

Clinical presentation & Diagnosis


 Complex; many vital systems involved; Asx in hepatic & sporozoite stages
 Disease from red blood cell stage: stimulates host immune response
 Periodic fever (chillrigorshigh feversweatingrelease), non-specific
o Can also have cough, H/A, body ache, malaise, weakness, diarrhea
o Signs: fever, anemia, jaundice, enlarged spleen/liver

3
LAB FINDINGS KEY: DIAGNOSIS: no
1. low platelets (first) later than 1 hour
2. low WBC, low RBC (second, third) after malaria first
suspected
Take blood when FEVER is present (higher burden of organisms)
TREATMENT: no
Suspect in US if: later than 1 hour
1. Any fever in exposed person (cough, diarrhea don’t rule out; think 7-25d incubation; after smear read
can relapse (vivax/ovale))
Tx based on:
2. Fever of unknown origin in “unexposed” (P. vivax/ovale ~3-5yr relapse; P. malariae
1. speciation
up to 50yr recrudescence!) 2. quantification
3. geography (drug
Clinical spectrum resistance?)
Mostly uncomplicated malaria if dz present in patients 4. assessment of
 See above symptoms severe malaria
 Tx: oral antimalarial drugs; confirm drug susceptibility by region
 Follow decline of parasitemia post-Tx initiation

Severe malaria = complicated malaria; set of overlapping problems. UP TO 50% MORTALTITY WITH TX
 Can lead to profound anemia, seizures, coma, death
 CAN BE VERY RAPID (esp. if non-immune, immunocompromised)
 Tx: IV drugs & intensive care

Types of severe malaria


A. Acidosis: final common pathway
a. Oxygen delivery impaired (lack of RBC)  metabolic acidosis
b. Sequestration of infected cells in brain/kidneys/lungs: can be organ specific
c. Proinflammatory cytokines, nitric oxide involved,
organ dysfunction leads to coma Severe malaria manifestation
B. Cerebral malaria: altered consciousness, seizures, rapid onset depends on endemicity
but rapid recovery if not fatal; immune-mediated Holoendemic Young patients; mostly
cerebral malaria
C. Severe anemia: hemolysis
Hyper/ Young patients; cerebral early
a. destroy uninfected RBC in spleen; malaria suppresses mesoendemic then severe anemia later
bone marrow (erythropoieses ineffective) Hypoendemic All ages; mostly severe anemia
b. Making less & destroying more
c. Associated with secondary bacterial infections;
d. Tx: transfusion if blood supply is safe

Pathological features
P. falciparum: cytoadherence important for sequestration (knobs with receptors for endothelial cells)
 Ring stage: circulates freely
 Schizont stage: generally sequestered in capillaries & venules (see more in other forms of malaria)

Sequestration & rosetting (P. falciparum / malariae)


 Sequestration: binding of infected RBC to capillary endothelium (keep away from spleen!)
 Rosetting: binding of uninfected RBC to infected RBC (responsible for pathophysiology): see “rosette” of healthy
RBC around infected RBC

Placental malaria: cytoadherance to placental endothelium; placental sequestration & exudates


 LOW BIRTHWEIGHT IS THE SINGLE MOST IMPORTANT PREDICTOR OF INFANT MORTALITY

4
If you suspect malaria
 Ideal: Giemsa stain of thick and thin smears;
o Can quantify (determine risk of severe dz, drug susceptibility)
 Based on RBC (thin) or WBC (thick) count
o Thick: more sensitive, hard to read / speciate, use for quick dx
o Thin: helps with speciation to determine Tx
o quick dry some to read fast: delay can be fatal!
P. falciparum: P. vivax P. malariae
 Normal RBC size; preserved  Fewer merozoites in schizont,  Band-form schizonts
morphology RBCs dysfigured
 Fine delicate rings  Large, irregular rings
 Gametocytes: sickle shaped (but rare)  Round gametocytes
 Rare trophozoites & schizonts  Amoeboid trophozoites present

 Also: dipstick antigen (no quantification or speciation but no microscope needed), PCR

Chemotherapy
 Chemoprophylaxis for travelers
 No prophylaxis generally in endemic countries
o Specific indications are exception sometimes: pregnant women, infants, children
 If it fails: think drug resistance, PK failure, fake drug?

Control
 ITN: insecticide-treated nets
 Indoor house spraying, vector control (limited utility), personal barriers
 Integrate with local systems when present; give effective/prompt treatment
o Currently: Tx without definitive Dx in endemic regions (but drug resistance)?
 Monitor drug resistance!
 Vaccine problems: natural protective immunity is present but restricted; immune response contributes to
pathology; antigenic variation + efficient parasite; lack of good outcome measures

Clinical significance review


P. falciparum infection is MEDICAL EMERGENCY: can infect RBC of all ages, severe anemia, high multiplication rate
 sequesters (microvascular obstruction, tissue hypoxia, capillary leakage, end organ failure)
 Almost always cause of severe malaria
o Cerebral: seizures, obtundation, coma
o Severe anemia
o Hyperparasitemia; severe prostration, end organ failure, acidosis, diffuse bleeding, more

P. vivax: Anemia & ruptured spleens


P. malariae: can cause nephrotic syndrome in African kids

5
Helminth Parasitism
Cause more disability than death; neglected tropical diseases
100+ spp of helminthes (vs 40 protozoa)
Nematodes (roundworms ) Flatworms:
 hookworm, Ascaris, Strongyloides,  trematodes (flukes/Schistososma)
pinworm/whip-worm, filaria  cestodes (tapeworms)

General principles of helminth dz


 Don’t multiply within definitive host (reproduce sexually & produce transmission stage but not more adults)
o Exceptions: Strongyloidiasis / capillariasis
 Low worm burdens (minority has high & is important for severe dz/high transmission)
o High worm burden = high exposure, not that they’re reproducing inside you
 Disease correlates with worm burden
Endemic regions Expatriates
 Heavily parasitized  Big inflammatory response
 High worm burden  Severe disease
 Little disease (little inflammation)  Low worm burden
 No TX = long term infection
o can live for years [nematodes] to decades [river blindness] to host’s lifetime [strongyloides stercoralis]
 Most produce eosinophilia + elevated IgE response
o Mast cell proliferation, too; all T-cell dependent & down-regulated with continued exposure
o Can cause pathology

Helminth pathogenesis
Mechanical attachment/damage
 Block internal organs (Ascaris, tapeworms, flukes, filiaria, schistosomes)
 Pressure atrophy (echinococcus, cysticercus) Deficiency Organism
 Tissue migration (helminthic larvae) Iron Hookworm
Nutritional depletion: see table Vitamin B12 Tapeworm
Metaplastic changes Macronutrients Ascariasis, Strongyloides
 Hepatoma = liver flukes; bladder cancer = schistosomes
Immunopathology
 Anaphylactic response (IgE/histamine)
 Immune complexes (Ag+Ab deposition in brain,kidney, etc)
 Cell-mediated reaction (monos & Mϕ)

Just because this will almost certainly be on the exam:

6
Intestinal Roundworms

Organism Mug shot Pathology Transmission Life Cycle Clinical presentation Other
Low worm burden is Asx
Think: irritable kid and
Mechanical GI  Lung High worm burden: abd. pain & intestinal obstruction
Ascaris Ingest egg then these come out after
blockage  GI Migrating larvae/adults: Pulmonary eosinophilia syndrome (Loeffler’s
Tx!
syndrome); biliary/liver inflammation, intestinal obstruction

SKIN: Larva Skin 


Hookworms Blood loss Lose lots of blood; ANEMIA Make anticoagulant
penetrate Lung  GI

Whipworms GI (local Mostly Asx


Think: bloody stools &
(Trichuris damage/rectal Ingest egg All in gut Heavy infection in children: GI problems (abd. pain, bloody diarrhea,
rectal prolapse!
trichiura) prolapse) prolapse; growth retardation)

Hyperinfection into tissues in transplant patients


Strongyloi- Local GI SKIN: Larva Skin  Think: Vietnam vet getting
Initial infection  migration to brain, muscle, other organs with gut
diasis damage penetrate Lung  GI a transplant
flora sepsis (after immune suppression)

Pinworm
Perianal Scotch tape test to see
(Enterobious Ingest egg All in gut Itchy butt at night; adults migrate to anus to lay eggs (E.g. kids) st
pruritus eggs! (1 thing in morning)
vermicularis)

7
Tissue Roundworms: Filaria
Insect vectors
blood = microfilia; tissues = adult worms

Organism Mug shot Pathology Transmission Life Cycle Clinical presentation Other
Spectrum of disease:
Filiarasis Damage Microfiliare circulate at
Mosquito/ 1. Asymptomatic
(Wucheria & lymph vessels Mosquitos night when mosquitos
human 2. Night fevers (when microfiliare circulate)
Brugia spp) (elephantiasis) feed!
3. Chronic: elephantiasis

Migrate to,
Calabar swelling; can migrate to eye!
Loa loa across sclera Flies Fly /human
Doesn’t cause blindness!
of eye

Inflammatory reaction due


Chronic
Oncheo- RIVER BLINDNESS (whole towns sometimes in Africa) to bacterial co-infection
inflammation Flies Fly/human
cerciasis Subcutaneous nodules (LPS) brought in by
of eye
parasite

Flatworms

Organism Mug shot Pathology Transmission Life Cycle Clinical presentation Other
Liver/bladder fibrosis; cancer
Cercariae
Granuloma S. mansoni: GI disease (in portal veins): cirrhosis, etc. Ingest RBC to eat Hb
Schisto- penetrate skin Snail /
reaction to S. haematobium: (in bladder)  ureter obstruction, bladder cancer Eggs have characteristic
somiasis after release human
eggs Can go to CNS, inflame  paralysis! shapes / spines : see slide
from snail
Swimmer’s itch in Great Lakes: from bird schisto (penetrates only)
Nutritional
Pigs or
deprivation; Ingest larvae Taenia solium: pork/pigs
cows / Make & excrete adults
big worm in (via raw meat) Taenia saginata: beef/cows
humans
intestine
Cestodes Cysticercosis (T. solium ONLY) – can go to all kinds of tissues
(Tapeworms) Larval forms Neurocysticercosis is most serious Note EGG not larva
Pigs or
in tissues Ingest egg  3-5y incubation ingested
cows /
(cysticercus in (fecal/oral)  Psychiatric syndromes; epilepsy, cysts, rarely SC involved/eye Make larvae; can go all
humans
brain, etc.) Dx: CT+ELISA or Western around!
Tx: steroids/albendazole +/- surgery

8
Eosinophilia:
Worms, wheezes & weird diseases Eosinophilia & Helminths
 Asthma, IBD, cancer, rheum stuff, drugs, etc.
 Not caused by protozoa
 Higher in short-term visitors
Helminth eosinophilia: Usually higher in acute infection
 Often highest before eggs form
o Chronic, high eosinophilia – think helminth!
 Infections with eosinophilia often
o Differs among species (often absent or lower in adult forms)
Asx (sx months to years later)
 Ascariasis: often absent with adult worms
 Hookworm can be low too in adult worms  Absence doesn’t exclude helminth
 Malaria, other bacterial infections
can suppress eosinophilia
Life cycle vocabulary for eukaryotic parasites  Chronic: can cause endomycardial
fibrosis
Malaria: ring trophozoite / trophozoite / schizont containe merozoites
Toxoplasma: tachyzoite divides rapidly, infectious; bradyzoite slowly
Cryptosporidium: sporozoites shed infective eggs;
Leishmania: amastigote in reticuloendothelial cells is infective
Trypanosoma: trypomastigote is infective (fly human); amastigote is intracellular
Giardia: trophozoite is active & replicating
Entoamoeba: cyst; no replication for transmission
Trichomonas: trophozoite only

Helminths

Roundworm
 Adult in intestine, eggs shed in feces, larva (freeliving/parasitic) can go to various tissues, encyst, etc.
Filarial roundworm
 Adult in bloodstream, microfilariae cause disease in tissues; are infective for insect
Fluke flatworm
 Adult in portal/bladder veins, shed eggs in bloodstream
Cestode flatworm
 Adult in intestine, release proglottid with eggs, form cysterci / hydatid cysts in mm/brain

GUINEA WORM is almost eradicated (dracunculiasis) , a roundworm


 99% eradicated
 Roll up on stick!
 If you put your foot in water to cool, larvae burst out

DDx of fever in endemic area: Malaria, malaria, malaria – then other parasites/virus/bacteria, other causes of fever
MALARIA DOESN’T HAVE EOSINOPHILIA

N. meningitides & malaria are two infectious diseases that can kill you in 24h

9
Other Protozoa
Organism Mug shot Pathology Transmission Life Cycle Clinical presentation Other

1. Cutaneous ulcer: worldwide (esp. Middle East, Central Asia, N.


Africa; Argentina  TX); at sandfly bite site. 2wks-years of
incubation. Non- or slow-healing ulcer on exposed skin, heaped up
edges
Leishmania Lives in Mϕ Dogs / Cutaneous: think vet from
and other RES Sandfly bites; 2. Mucosal: (Central/South America); metastatic from skin; extensive
Sandfly / Iraq or tourist from /to
(Leishman- cells IV drug use non-healing ulcers on mucosa (nose, oral cavity, pharynx, larynx)
Humans South/Central America
iasis) (promastigote)
3. Visceral: (Asia, southern Europe, Brazil): disseminates within RES
cells; 3-8mo incubation; EXTENSIVE NONTENDER
HEPATOSPLENOMEGALY, fever, weakness, weight loss, GRAY
DISCOLORATION of EXTREMITIES (kala-azar; “black fever”)

Think: Primary in healthy


1. Immune-competent: primary infection usually subclinical; can
Forms cysts patient = mono
produce mononucleosis-like syndrome with painless lyphadenitis
(latent;
(esp. cervical)
bradyzoites) if HIV patient reactivates &
Toxoplasma Undercooked
immune Cat/rat, gets brain lesions / neuro
beef/pork; 2. Immunocompromised: reactivation of dormant infection
reaction; Cat-feces- problems
(Toxoplasm- eggs in cat (encephalitis, brain lesions, chorioretinitis, myocarditis,
otherswise human
osis) feces pneumonitis)
proliferates in Pregnant woman changes
lots of tissues litterbox for the first time
3. Pregnant: Primary infection  transinfection of fetus  CNS
as tachyzoites (primary) & fetus gets birth
sequelae, chorioretinitis, severe disease.
defects
Leading curable STI in US
Trichomonas
(7.3M new cases/yr)
vaginalis Pear-shaped Human / Women: 50% Asx  PID & severe complications
Sexual
Motile Sex /
intercourse Theoretically survives up to
(Trichomon- Flagella Human Men: 75% Asx  severe infection, epidiymitis / prostatitis
45m on clothes,
iasis)
washcloths, bath water

10
Diarrheal Protozoa
Organism Mug shot Pathology Transmission Life Cycle Clinical presentation Other
Cyst active
trophozoite in
Developing countries mostly (also immigrants, travelers, MSM in US) Cyst outside host;
Entamoeba GI tract; Human:
Ingest cyst trophozite (active) inside –
histolytica ingestGI
(water, soil, 1. Diarrhea (severe & bloody – dysentery) see pictures
can invade liver /
food) 2. Liver abscesses
(Amebiasis) (flask abcess) brain
3. Brain abscesses Dx: stool o+p (about 50%)
& spread to
liver, brain
Worldwide: epidemic diarrhea (contaminated water)
AIDS pts: severe diarrhea if low CD4 ct
Oocyst Contaminated
Cryptospor- Sporadic: day care, child care, travelers, backpacker/hiker/swimmer Need special stains (Stool
outside / water (shallow GI only
idium O+P with AFB)
troph inside wells, other)
Large volume secretory diarrhea with nausea/cramps/vomiting/wt loss
Self limited (2-3wks; >2mo in AIDS)
#1 fecal parasite for diarrhea in USA
Think: hiker who drank the
Contaminated Day care, travel to endemic areas, ingestion of unfiltered water while water; smelly stool
Giardia Cyst outside water camping; fecal-oral sex contact (esp. MSM), well water on farms
GI only
lamblia Troph inside (mountain Both trophs & cysts shed in
streams) Acute diarrhea, abdominal cramping, bloating, flatulence, stool; only cyst survives
Stools become NASTY SMELLING & GREASY over time (malabsorptive) Dx: Stool O&P; antigen
No blood/pus/mucous

11
Pharmacology: ID & Micro (Fungi & Parasites)
Antifungal Drugs ..................................................................................................................................................................... 2
Chemotherapy of Parasitic Infections ..................................................................................................................................... 5

1
Antifungal Drugs
The big picture:
DRUG INFECTION ROUTE MECHANISM CLASS
Amphotericin B
(deoxycholate)
Deep IV Ergosterol binding
Amphotericin B Polyene
(lipid formulation)
Nystatin Derm/yeast PO/topical/vaginal
Ketoconazole Deep/derm PO/topical
Fluconazole
Deep IV/PO
Itraconazole Ergosterol synthesis Azole
Clotrimazole
Derm/yeast Topical/vaginal
Miconazole
Flucytosine Deep PO DNA/protein synthesis Pyrimidine
Caspofungin
Micafungin Deep IV Cell wall synthesis Echinocandin
Anidulafungin
Gresofulvin Derm PO Microtubule formation Griseofulvin
Terbinafine Derm PO/topical Squalene synthesis Allylamine

General principles: need to be highly specific for fungal target without affecting human counterparts (tough because
both are eukaryotes)

Sterol biosynthesis:
 First part (common to both animals & fungi)
1. Squalene  2,3-oxidosqualene (via squalene 2,3-epoxidase)
2.  lanosterol (via 14-α-demethylase)
3.     zymosterol
 Second part
1. Humans: zymosterol  cholesterol
2. Fungi: zymosterol  ergosterol
 Key point: fungi use ergosterol (more hydrophobic & rigid) instead of cholesterol in their cell membranes

Target: ergosterol in cell membrane (polyenes)


 Note: these act directly on ergosterol (others interfere with biosynthesis)
amphotericin B Mechanism of Action: polyene antifungal agent. Big macrolide ring; half hydrophobic, half hydrophilic, forms a
channel or pore in fungal membranes

Effects: forms cylindrical channel (hydrophobic sides outside, against cell membrane) when bound to sterols &
allows leakage of small molecules resulting in fungal death
Selective Toxicity: Binds more avidly to ergosterol (fungi) than cholesterol; selective toxicity not great

Indications: potentially fatal fungal infections(think of toxicity)


1. invasive aspergillosis,
2. disseminated candidiasis Administration: IV
Toxicity: A lot. NEPHROTOXICITY is dose limiting. Fever, chills, hypotension ("shake 'n bake")
Other: deoxycholate is usual form; also lipid formulations available: same efficacy, less toxicity, 30-40x more $$

Nystatin Very similar to amphotericin, but in topical preparation


Indications: treatment of oral, vulvovaginal, cutaneous candidiasis
Administration: Topical

2
Target: ergosterol biosynthesis
 Note: none of our current drugs target the fungi-specific part of ergosterol biosynthesis!

Azoles (ergosterol biosynthesis)


 imidazole (2 nitrogen atoms) or triazole (3 nitrogen atoms)
Azoles (in general) Mechanism of Action: azole antifungal agent.
 Low doses: inhibits ergestol biosynthesis.
 High doses: may directly damage fungal cell membrane
Effects:
 Low dose: blocks 14-alpha-demethylase, a CYP450 enzyme (lanosterol  ergosterol).
 High dose: direct damage
Selective Toxicity: Binds fungal demethylase more than human (although both have this enzyme)
Toxicity: GI distress, rash, hepatotoxicity, drug interactions (CYP3A4 inhibitor)

DRUG ORAL ABSORPTION METABOLISM EXCRETION INDICATIONS


Alternative to amphotericin B
Ketoconazole Effective Hepatic Urine
(systemic/mucocutaneous fungal infections)
Good; Urine (80% dose No postantifungal effect; fungistatic.
Mostly non-
Fluconazole independent of excreted in urine 1. Maintenance cryptococcal meningitis
hepatic
gastric acidity unchanged) 2. Prophy for Candida (transplant, etc.)
Erratic, better Systemic; fewer side-effects than ketoconazole
Itraconazole Hepatic only Urine + bile
with acid/food (but still hepatotoxic)
80-90%, better on 1. Invasive aspergillosis
Voriconazole Hepatic only Urine + bile
empty stomach 2. Candidemia
Ketaconazole has shortest half-life (~12h); others in 20-50h range

Allylamines (ergosterol biosynthesis)


naftifine Mechanism of Action: Allylamine antifungal drug. Inhibits ergosterol biosynthesis.
Effects: Inhibit squaline-2-3-epoxidase (earlier step in ergosterol biosynthesis than azoles); lead to membrane
terbinafine disruption and leakage of small molecules.
Selective Toxicity: Highly selective for fungal squalene epoxidase over human (no effect in vivo on cholesterol
biosynthesis
Indications: Candida, etc.
Administration: Topical (+oral for terbinafine)
Other: terbinafine is active ingredient in Lamisil®

Target: nucleotide metabolism (pyrimidine analogs)


Flucytosine Mechanism of Action: Pyrimidine analog antifungal agent. Active form interferes with nucleotide metabolism
(5-FC) Effects: Converted to 5-FU in fungi by cytosine deaminase. 5-FU has two roles:
1. metabolized to 5-FUMP, incorporated into fungal mRNA, interferes with protein synthesis
2. 5-FUMP ; 5-dUMP via ribonucleotide reductase; inhibits thymidylate synthase
Selective Toxicity: Human cytosine deaminase can't deaminate 5-FC (note that fungi and GI FLORA can!)

Indications: systemic Candida/Cryptococcus infections


Administration: co-administered with amphotericin B to combat resistance
Toxicity: generally well tolerated but:
 bone marrow depression,
 GI distress (gut flora killed);
 reversible hepatotoxicity
Resistance: MAJOR PROBLEM - develops very quickly with monotherapy

3
Target: microtubule formation
Griseofulvin Mechanism of Action: Antifungal agent; Interferes with microtubule formation
Effects: actively transported into fungal cells; disrupts microtubules (mitotic & cytoplasmic), cell cycle arrest at
mitosis, formation of multinucleate cells.
Selective Toxicity: Humans don't actively transport into our cells

Indications: Severe infection of hair, nails, palm, soles (concentrates highly in keratin layers)
Administration: Almost complete distribution; goes to keratin layers
Toxicity: Relatively safe (GI distress, temporary headache)

Target: cell wall biosynthesis


Caspofungin Mechanism of Action: Antifungal agent. Inhibits cell wall biosynthesis
Effects: irreversible inhibitor of 1,3-beta-D-glucan synthase (makes glucan polymers for fungal cell wall); fungicidal
Micafungin Selective Toxicity: Humans don't have cell walls; fungi need glucan polymers for structure & viability
Indications:
Anidulafungin  Caspofungin: Salvage therapy for invasive aspergillosis; esophogeal candidiasis, candidemia. Used when
ampho B, others don't work.
 Micafungin: prophylaxis of candidiasis in BMT recipients
 Anidulafungin: Tx of candidemia
Toxicity: Important (limits usage, 14% of recipients). fever, nausea, vomiting, infusion site complications
Resistance: No cross-resistance with other classes

4
Chemotherapy of Parasitic Infections
1/3 of world’s pop has parasites. 1:5 Americans! NO VACCINES so you have to use drugs for prophylaxis & treatment
Can classify as protozoa or helminths, or (more useful for pharm): Gastrointestinal vs Tissue/blood

Selective toxicity of antiparasitics (4 mechanisms)


1. Parasite location
A good antiparasitic drug:
a. if in the lumen of the bowel only, use a luminal agent that’s not
 Safe
absorbed (won’t hurt host cells!) (widespread use / prophy)
2. Differences in host/parasite metabolic pathways  Orally effective
3. Differences in isofunctional enzymes  Cure in 1 dose
4. Concentration of drug by parasite  Cheap

Mechanisms of actions for antihelminthics (2)


Note: adult worms don’t multiply in humans
 if you can get them to stop moving & hanging on, your body can flush ‘em out
 Worms have complex nervous systems; need active motility to resist expulsion by peristalsis

2 Targets: motility & energy generation

1. Parasite motility
pyrantel Mechanism of Action: Antihelminthic agent. Ach analog; neuromuscular blocking agent & Ach inhibitor
(causes spastic paralysis & constant muscle contraction of worm)
Effects:Worms can't resist bowel peristalsis; get swept out
Selective Toxicity: Luminal agent (poorly absorbed) - only affects parasites

Indications: Ascaris infection (helminths)


Administration: PO

praziquantel Mechanism of Action: Antihelminthic agent. Causes tetanic contraction of schistotomes (alters Ca
transport) & alters membrane integrity
Effects: Worms can't resist bowel peristalsis; get swept out to liver/lungs (portal circulation from gut or
systemic from bladder area). Surface of worm disrupted then, leading to death.
Selective Toxicity: unknown (NOT LUMINAL)

Toxicity: frequent GI/CNS but mild


Indications: Cestodes / trematodes (schistosomiasis & tapeworms - taenia solium, etc.)

2. Parasite energy generation

Enteric helminths live in anaerobic environment, so they have a special, different way to get energy:
 Transport glucose across membrane; different end of glycolysis (malate) & Krebs-cycle type thing
 Uses succinate dehydrogenase in respiratory chain (reverse direction from humans: fumaratesuccinate)
 Both the transporter & the succinate DH enzyme are different isoforms in parasites

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Benzimidazoles:
albendazole Mechanism of Action: Antihelminthic agent.
 Block glucose transport
mebendazole  inhibit succinate dehydrogenase activity; an enzyme used in parasite's respiratory chain.
 (also disrupt microtubules selectively)
Effects: No energy for parasites; die & get swept out

Selective Toxicity: Helminths have different metabolism because they're anaerobic; parasitic isoforms are
different for these enzymes than humans'.
 Albendazole: Variable absorption (good to get more coverage; bad because of human
interactions - not a luminal agent)
 Mebendazole: Luminal agent (good absorption
Indications:
 Both: Gut organisms (enterobius, ascaris, trichuris, hookworm)
 Albendazole: tissue too (strongyloides, tapeworm which have tissue parts of life cycle)
Toxicity: Potent teratogen in animals. Don't give to pregnant women. Minimal otherwise
Administration: PO

Gut-dwelling Can go to Tissue


Trematode Cestode
Enterobius Trichuris Luminal?
Ascaris Hookworm (Flukes) (Taenia sp.)
(pinworm) (whipworm)
(Strongyloides) (Tapeworm)
Albendazole +/- absorption
Mebendazole Luminal
Pyrantel Luminal
Praziquantel NO

Classes of antimalarial drugs (3)


Malaria: 100+ countries where P. falciparum (most important) is chloroquine resistant
 all through Africa, Asia, S. America
 Important in USA too: 73 cases in MD last year (more than meningococcus!)
Remember life cycle: sporozoites enter; go to liver, can be hypnozoites in vivax or ovale, form schizonts which burst & release
merozoites, which attack RBC, forming erythrocytic schizonts, which burst in a periodic manner, with more merozoites leaving
(periodic fever); some change to gametocytes, which can be transmitted via new mosquito

 Important: ASYMPTOMATIC until RBC START BURSTING

Note that no drugs have Sporozoite Liver  RBC


activity against all 4 phases  Initial Hypnozoite Asexual Gametes
 Chloroquine
Class I: Asexual RBC form ONLY  Mefloquine
 can’t truly prophylax (need  Quinine
infection to RBC stage to  Fansidar
Class I

start killing) (pyrimethamine +


sulfadoxine)
 Need to start before,
 Coartem
continue through, and
(armether +
continue 3wks after lumefantrine)
exposure for prophylaxis  Tetracyclines
(organisms would have to Class II Primaquine
reach RBC stage to be Atovaquone +
killed by drug) Class III
Proguanil

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Class II: opposite of Class I
 Prophylaxis only: can’t use in symptomatic patient (no effect on RBC stage)
 Can use primaquine to eradicate latent hypnozoites (use if worried about vivax/ovale exposure)

Class III: treatment & prophylaxis (liver stage & asexual stage)

Molecular mechanisms of action: chloroquine, atovaquone/proguanil


4-subsituted quinolones (chloroquine, mefloquine, quinine)

Heme biosynthesis: When Hb is broken down; heme is formed (toxic). Humans & plasmodia (feed on Hb) both have
mechanisms to detoxify
 Humans: break down via heme oxygenase to make bilirubin & excrete
 Plasmodium: no heme oxygenase: form heme polymers (visible as “malaria pigment” or “hemozoin” in
plasmodia).
 Chloroquine blocks this breakdown; heme accumulates; plasmodia die
o Resistance: enhanced efflux mechanisms

chloroquine Mechanism of Action: Antimalarial drug. Inhibits heme detoxification in plasmodia


Effects: Inhibits formation of heme polymers in plasmodia, leading to buildup of toxic heme
Selective Toxicity: Humans use heme deoxygenase to make bilirubin & excrete (different pathway).
Concentrated 100-200fold in infected RBC

Indications: First line for ovale & malariae


Toxicity: Retinopathy (&gt;100g cumulative dose, permanent)
Resistance: Widespread (more common than not: Sub-saharan Africa, South America, SE Asia).
Mutations in transporter proteins (enhance efflux)
Other: Cheap & available

Atovaquone/proguanil: synergistic against malaria parasites in vivo


1. Atovaquone: Binds cytochrome b in P. falciparum; inhibits mitochondrial electron transport
a. Pyrimidine synthesis inhibited
b. Mitochondrial membrane potential collapses
c. Resistance is rapid if monotherapy
2. Proguanil: metabolized to form cycloguanil, which selectively inhibits dihydrofolate reductase
3. Synergy: Not totally known; probably based on collapse of mitochondrial transmembrane potential (not
pyrimidine synth in experiments although that would be logical)

Antimalarial chemotherapeutic regimens


General principles to keep in mind:
Class Drug Use Notes
Cheap, available
I Chloroquine Ovale/malariae malaria
Widespread resistance for falciparum
Prophy against chloroquine-
I Mefloquine CNS toxicity prevents tx of established infection
resistant falciparum
Old; Quinidine is stereoisomer (can use in a pinch)
I Quinine Tx of chloroquine-resistant
IV for serious infection
falciparum malaria
I Coartem Two-drug combo; non-synergistic
Active against liver hypnozoites (only one)
II Primaquine Add for Vivax/ovale
Hemolytic anemia in G6PD-deficient pts!
Atovaquone + Proguanil
III Tx of MDR falciparum More expensive; synergistic
(Malarone)

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Situation Treatment
Prophylaxis Before, during, 3wks after
 Chloroquine if sensitive; malarone, doxycycline, mefloquine if resistant
 Add primaquine if exposure to vivax/ovale (test for G6PD 1st)
Mild/moderate infection ORAL
 Chlorquine if sensitive
 Malarone [or Coartem or quinine (+doxy/tetra/clinda)] if resistant
 Add primaquine when recovered if exposed to vivax/ovale (test for G6PD 1st)
Severe illness IV
 Quinidine or quinine + (doxy/tetra/clinda)
 Add primaquine when recovered if exposed to vivax/ovale (test for G6PD 1st)
Note: only 15-30% US travelers take malaria prophy, docs usually get it wrong

Other protozoal infections (metronidazole)


Metronidazole:
metronidazole Mechanism of Action: Anti-protozoal & anti-anaerobe antibiotic.
 When two nitro-radical anion forms collide, they create a reactive complex that causes
alkylation & strand breakage of DNA
Selective Toxicity: Only works against anaerobes:
 has aromatic NO2 group that accepts an electron from reductive metabolism process
(production of ferredoxin), generating nitro-radical anion only in anaerobes
Indications: Protozoa + anaerobes.
 Trichomonas vaginalis Entamoeba histolytica, anaerobes; off-label for Giardia & H. pylori.
Toxicity:
 Genetic toxicity (? - controversial).
 GI common (nausea, metallic taste, disulfiram-like rxn with alcohol),
 Neuro (headache, ataxia, peripheral neuropathy, seizures - can be irreversible but rare).
 Avoid in pregnant/lactating women
Other: Prefers organisms with high A/T content in their genomes. Penetrates well into abscess cavities,
CSF, bile, bone, placenta, milk. Excreted by kidneys (modify for renal failure)

Anti-amebics
3 things amoebae can do:
1. Hang out as cysts (infective form)
2. Turn into trophozoites but hang out in lumen (commensal)
3. Invade as trophozoites (flask shaped ulcers, etc.)

Infection Treatment
Asymptomatic (luminal carrier) Luminal agent only Paromycin, diloxanide fuorate
Symptomatic (tissue invasion) Luminal agent + tissue agent Metronidazole, tinidazole

Pentamidine
pentamidine Mechanism of action: Active transport & accumulation in parasites.
 Activity is multifactorial: disorganize mitoDNA, inhibit mito Topo, bind ribosomes, inhibit
phospholipid synth, etc.
Indications: T. brucei, Leishmania, Blastomycosis, Babesia, P. jiroveci.
 Aerosolized form recommended for PCP prophylaxis in HIV patients (second line because of
toxicity for Tx behind TMP+SMX)
Toxicity: severe in 55% AIDS pts with PCP. leukopenia, azotemia, hepatitis, unpredictable hypoglycemia
(insulin similarities), others.
Other: Structural analog of synthalin (synthetic insulin)

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Pathophysiology: Skin
The Dermatologic Vocabulary ................................................................................................................................................. 2
Histopathology of the Skin ...................................................................................................................................................... 4
Acne & Rosacea....................................................................................................................................................................... 6
Cutaneous Autoimmune Bullous Diseases: Pemphigus & Bullous Pemphigoid ..................................................................... 9
Psoriasis & Atopic Dermatitis ................................................................................................................................................ 11
Pigmented Lesions & Melanoma .......................................................................................................................................... 14
Non-Melanoma Skin Cancer ................................................................................................................................................. 17
Dermatology of Pigmented Skin ........................................................................................................................................... 19
Birthmarks in Babies ............................................................................................................................................................. 20
Drug Eruptions ...................................................................................................................................................................... 22
Cutaneous Manifestations of Internal Diseases ................................................................................................................... 25
Common Infections of the Skin ............................................................................................................................................. 27

1
The Dermatologic Vocabulary
Lesion morphology: shape and relative size of the lesion(s)
non-palpable, circumscribed, color change <1 cm; (“macular”
1. MACULE or “patch” are used to describe larger areas of the color junctional nevus
change)
molluscum contagiosum,
2. PAPULE palpable, circumscribed lesion, < 1 cm
intradermal nevus
palpable, circumscribed, relatively flat topped lesion, greater
3. PLAQUE psoriasis, lichen simplex chronicus
in surface area than in thickness, > 1 cm;
melanoma, squamous cell
4. NODULE palpable, circumscribed lesion, ≤ 1 cm and < 2 cm;
carcinoma
squamous cell carcinoma, basal cell
5. TUMOR large nodular lesion,≥ 2 cm
carcinoma
herpes simplex and zoster infections,
6. VESICLE clear fluid –filled lesion (blister), < 0.5 cm
vesicular foot dermatitis
bullous impetigo, toxic epidermal
7. BULLA clear fluid-filled lesion (blister), > 0.5 cm
necrolysis, bullous pemphigoid
8. PUSTULE turbid fluid-filled lesion folliculitis, acne
9. CYST nodule filled with a semisolid or liquid substance epidermal inclusion cyst
transient palpable lesion (hive) caused by an interstitial serous
10. W