Intestinal Protozoa

Amoebae and ciliates

Dr. Devika Iddawela Department of Parasitology 2008/2009 Batch

OBJECTIVES
1. Name the common intestinal amoebae &ciliates that infect humans 2. Of the intestinal amoebae, name the organisms that are pathogenic to human 3. Outline the life cycle of Entamoeba histolytica /Balantidium coli indicating the stages that cause pathogenic effects and are of diagnostic importance in the above 4. Identify points in Life cycle where preventive measures are applicable 5. Describe the mechanism of pathogenesis 6. Describe the pathogenesis and clinical features of these stages

7. Describe the mode(s) of transmission, prevention and control of amoebiasis

8. Describe the laboratory methods of diagnosis of these organisms

Intestinal protozoan
Phylum protozoa is classified into 4 subdivisions based on methods of locomotion

1.Amoebae moves by means of pseudopodia 2. Ciliates are propelled by rows of cilia that beat with a wave like motion

3. Flagellates- move by long whip like flagellae 4

. Coccidia: lack the specialized organelles of

motility

Amoebae
Unicellular organisms
Characterized by possessing pseudopodia by which these organisms move and engulf food particles such as bacteria, red blood cells

 Asexual reproduction binary fission

Most are free living

can exist as trophozoite (growing stage) or cyst

( dormant stage)

Differentiate on morphological features of either trophozoite or cyst

Differentiating features of trophozoite: Size,

Type of motility directional or non- directional

fast or sluggish character of pseudopodia ,Cytoplasmic inclusion bodies : Red blood cells, food vacuoles containing bacteria, yeast

Nuclear structure:

number of nuclei, arrangement of peripheral chromatin, position of the karyosome

Chromatin ; Nuclear DNA present as peripheral chromatin Peripheral chromatin chromatin adhering to nuclear membrane Karyosome: small condensed mass of chromatin within the nuclear space

Differentiating features of cyst : size shape

number of nuclei, structure of nuclei

presence of glycogen mass Chromatoid body or bar - coalesced RNA within the cytoplasm

Genus : Entamoeba Parasites of alimentary tract - man, monkeys vertebrates and invertebrates

Characteristics of this genus :

Nucleus more or less spherical Nuclear membrane line with chromatin granules Small karyosome situated at or near the centre Trophozoite has single nucleus

Genus: Entamoeba
karyosome

peripheral chromatin

Genus:

Endolimax and Iodamoeba

Large karyozome

Grouped according to the number of nuclei in the mature cyst (1,4,8)

Amoebae that parasitize humans Intestinal amoebae: ( inhabit the large intestine) Entamoeba histolytica

E.dispar
E.coli E.hartmani

Endolimax nana
Iodamoeba butschlii Dientamoeba fragillis Oral cavity : Entamoeba gingivalis

There are two stages in the life cycle of these amoebae.

1.Trophozoite:mortile and feeding stage. Multiply by binary fission 2. Cyst : Inactive, non motile and infective stage No cyst stages in D.fragilis & E.gingivalis

Of several species of amoebae live in the alimentary tract of human MAJORITY are commensals ONLY Entamoeba histolytica is pathogenic D.fragilis and I.butschlii,
may cause intestinal infection

ENTAMOEBA HISTOLYTICA

Entamoeba histolytica

cosmopolitan distribution worldwide incidence: 0.2-50% highest prevalence in areas with poor sanitation no animal reservoirs estimated 50 million cases/year 100,000 deaths/year

Pathogenic organism parasitize large intestine of man Disease: amoebiasis

Blood and mucous diarrhoea
E. dispar identical morphology but not Invasive ( non-pathogenic)

Trophozoite

RBCs

20-40 m, motility-active, progressive, directional Pseudopodia- finger like, hyaline, very rapidly extruded Inclusions- red blood cells (invasive forms) Nucleus- single, fine central kayosome, regular peripheral chromatin Nucleus

Cyst spherical, 10-20 m (E. hartmanni <10 m) Nuclei: 1-4, structure like in trophozoite Chromatoid bodies: thick, 1-2 stain like chromatin, disappear as cyst matures (does not stain with Iodine)

E. dispar identical morphology

Life cycle
Ingestion of mature cysts Excysts in small intestine Each cyst give rise immature trophozoites Maturation takes place in caecum Trophozoites feed grow and divide causing pathological effects

Infective stage

Amoebiasis
Pathogenesis - Infection with E.histolytica does not necessarily lead to disease. The outcome depends on :

Host factors Parasite factors

Host Factor Contributions

Physico-chemical environment of the gut influenced by bacterial flora, mucus secretion & gut motility Degree of immunological resistance

Parasitic factors Important virulence factors of E.histolytica

Adhesion molecules ( N- acetyl-D-

galactosamine inhibitable lectine Gal/GalNac) adhesion to colonic mucine and host

cells induce contact dependent cytolysis,

Channel-forming peptides(Amoebapores): Stored in cytoplasmic granules & release following target cell contact, forms iron exchanging channels in plasma membrane lysing the target cells

3. Cystein protinases Aid in penetration of host tissue by digesting extracellular matrix, cleaving collagen, elastin,fibrinoge in extracellular matrix by stimulating host cell proteolytic cascade

Resistance to host response

complement

resistance-inactivates the complement factors and are thus resistant to Complement mediated lysis. Limit the effectiveness of humoral response by degrading both IgA and IgG

4. Species/strain

differences; E. dispar non

invasive, Pathogenic zymodemes =E.histolytica

Pathology
Intestinal Amoebiasis LARGE INTESTINE

Adhere to colonic mucin and host clls

Penetration of mucus layer contact-dependent killing of epithelium breakdown of tissues (extracellular matrix) contact-dependent killing of neutrophils, leukocytes, etc. initially produce focal and superficial erosions in large intestine with unaffected mucosa in between

Amoebic ulceration with unaffected mucosa in between

penetrates the intestinal epithelium and then the muscularis mucosa & enter in to submucosa Trophozoites advance laterally and downward into the submucosa producing a 'flask-shaped' ulcer ( typical appearance of intestinal amoebiasis)

Trophozoite

Flask shaped ulcers -Base in submucosa and small opening on the mucosal surface

Trophozoites penetrate the muscle

and serous layers leading to intestinal perforations ,peritonitis
Rarely involvement of blood vessels at the base of the ulcer may produce profuse bleeding

Amoeboma - Amoebic granuloma

An inflammatory thickening of the intestinal wall, due to repeated invasion of colon by E histolytica

common sites- ascending colon & caecum

Haematogenous spread to other organs

Tissue invasion:
Initial lesion large intestine, caecum, ascending colon, sigmoidorectal region.

Blood stream and lymphatic spread cause extra-intestinal amoebiasis(liver, skin, brain, heart)

Extraintestinal Amebiasis
primarily liver (portal vein) other sites less frequent

Hepatic amoebiasis
Single abscess- Rt. Lobe (commonest) predisposing factors: alcohol

Spread to other sites- direct

-blood stream

Hepatic abscess ( common site is right lobe)

Pulmonary Amoebiasis
rarely primary rupture of liver abscess through diaphragm fever, cough, dyspnea, pain,

Cutaneous Amoebiasis
intestinal or hepatic fistula perianal ulcers urogenital (eg, labia, vagina, penis)

Clinical features
Intestinal disease
Majority of infections are asymptomatic [cyst passers are infective carriers]

asymptomatic cyst passer

Amoebic colitis Gradual onset ( symptoms presenting over 1-2 weeks) abdominal pain, tenesmus , watery or bloody diarrhoea, anorexia, loss of weight. Fever only 10- 30% Rectal bleeding without diarrhea can occur, especially in children

fulminant colitis- Rare complication abrupt onset of profuse bloody diarrhoea, high fever,dehydration ,wide spread abdominal pain + perforation (peritonitis)

amoeboma (amoebic granuloma)- painful abdominal mass perianal ulceration

Extraintestinal Disease _ sings & symptoms depend on the organ affected liver abscess
Frequently affect adults than children, Male>female 60-70% of patients with amebic liver abscess do not have concomitant colitis, a history of dysentery within the previous year hepatomegaly, liver tenderness, pain in the upper abdomen, High fever and anorexia, Weight loss, vomiting, fatigue

Diagnosis of Amoebiasis

Trophozoites
Direct wet faecal smears in saline can demonstrate motile trophozoite. Fresh sample of faeces ( preferably with in 30 min) should be examine to visualize live trophozoite. confirmed on a permanently stained smear to identify morphological features of nucleus Eg; Trichrome or Iron haematoxylin Biochemical Methods: Culture and Isoenzyme analysis to differentiate E.dispar from E.histolytica

Entamoeba histolytica

Sigmoidoscopy:
Visualize characteristic ulcers Look for trophozoites in mucosal aspirate Biopsy can be taken from the edge of ulcer stained with H &E

Cyst

Wet faecal smear ( saline or iodine)

Faecal concentration methods

If cysts are few to be present in direct smear, cysts can be concentrated either by floatation ( Zinc sulphate centrifugal floatation) or by sedimentation ( FormalEther )

Immature cyst

Immature cyst

Trophozoite

4.Mature cyst with 4 nuclei

Differentiation of E.Hislolytica from other nonpathogenic intestinal protozoa is very important E.Coli cyst

Size 10 -20 m, >4nuclei Nucleus ; eccentric karyosome with irregular coarse chromatin Chromatoid bodies infrequent ,needle shape when present

Iodamoeba butshclii cyst

7 -15 m, , glycogen mass is large, dark brown with iodine

Acute dysentery- predominant form trophozoites

saline, stained smear, culture Colitis cysts - saline, iodine, concentration methods

Faecal examination: minimum of 3 samples in 7 days wet/permanent/culture

Diagnosis Intestinal amoebiasis

Definitive diagnosis
[GOLD STANDARD]

demonstrate parasite in stools/rectal smears STOOL FULL REPORT = SFR Trophozoites with ingested red blood cells indicate invasive amoebiasis Presence of cysts does not indicate active disease but infective carriers (cysts are infective)

Without the specific presence of ingested RBCs in the cytoplasm the pathogen, E. histolytica & the non pathogen, E. dispar Are morphologicaly identical BUT Biochemically different

Immunodiagnosis
Detection of E.histolytic specific antibodies
By Enzyme linked immunosorbent assy(ELISA) Useful in non-endemic areas where E. histolytica infection is not common

Antigen Detection in stool

Antigen-based ELISA s Advantages Differentiate E. histolytica from E. dispar; (ii) they have excellent sensitivity and specificity;

Emerging methods in Diagnosis

These are considered the most useful tests for detecting E. histolytica. They test directly for the parasite itself by exposing some stool to a strip of paper coated with antibodies. The parasites will stick to the antibodies on the paper. The test distinguishes E. histolytica from other parasites. Disadvantage : costly

Molecular Biology-Based Diagnostic Tests - PCR

Detection of parasite DNA in faeces by PCR Provide high sensitivity and specificity for the diagnosis of intestinal amoebiasis

Extraintestinal -Hepatic WBC/DC leucocytosis >10,000/mm3 immunodiagnosis : serology - Serum antibody detection ELISA Serum antigen detection by ELISA

abscess aspiration
only selected cases
reddish brown liquid trophozoites at the abscess wall Abscess fluid Ag detection (ELISA)

imaging X ray, CT, MRI, ultrasound

Typical aspirate- chocolate syrup Trophozoites are found on marginal wall Commonly found in the last portions of aspirated material

CT scan of abscess in R lobe

X ray showing fluid level

Peters & Gilles. Atlas of Tropical Medicine and Parasitology- 4th Ed. Mosby-Wolfe 1995

amoebic hepatic abscess causing a raised right diaphragm with pleural effusion

Normal chest X ray

sensitivity and specificity of tests of diagnosis for amoebiasisa

Test Colitis Sensitivity <60% Specificity 10-50% NA Liver abscess Sensitivity <10% <25%

Microscopy (stool) Microscopy NAb (abscess fluid)

Stool antigen >95% detection (ELISA) Serum antigen 65% (early) detection (ELISA) Abscess NA antigen detection (ELISA)

>95%

Usually negative

>90%

NA

75% (late), 100% (first 3 days) 100% (before treatment)

PCR (stool) Serum antibody detection (ELISA)

>70% >90%

>90% >85%

Not done 70-80% (acute), >90% (convalescent)

 Direct hand to mouth

Transmission Through cysts

Indirect- contamination of food/water

Sources of infection: Food and water contaminated with infected faeces. Food handlers excreting cysts are an important source of contamination of foods

Houseflies also act as a mechanical vectors contaminating food

Sexual transmission

Man is only reservoir host

Because of the protection conferred by cyst wall , cyst can survive days and weeks in external environment
Cyst Can be killed: Boiling- Above 68 C Iodine (200 ppm)/acetic acid 5-10% Remove from water by sand filtration Ordinary chlorination does not kill cysts

Epidemiology Amoebiasis is cosmopolitan but no correlation between infection and disease

Generally in developed countries asymptomatic In tropics/low socio-economic standards High pathogenicity

High risk groups: travelers, institutional inmates

homosexuals, immunocompromised individuals, children in day care centers

Prevention
Reduce environmental contamination: detecting and treating infected persons

Improve environmental sanitation Avoid ingestion of infected cyst by personal protection

Food safety
Thoroughly cook all raw foods. * Thoroughly wash raw vegetables and fruits before eating. * Reheat food until the internal temperature of the food reaches at least 167. Wash your hands before preparing food, before eating, after going to the toilet or changing diapers

Ciliates

What are ciliates ?

Protozoa with cilia

Cilia -

Hair like structures used for locomotion and feeding.

Shorter than flagella and more in number

Ciliophora ciliates
Use cilia for movement or feeding Can have more than one nucleus (macronucleus, micronucleus) Feed through a mouth like structure (oral groove,

Generally larger than other protozoa

Reproduce by binary fission

ONLY ciliate that is known to parasitize man is Balantidium coli

Balantidium coli
Largest protozoan parasite of man
It is a zoonotic infection

A common parasite of pigs Pig the main reservoir Human infection is less frequent

Parasitize distal ileum and colon

Invade the mucosa and causes blood and mucous diarrheoa

Pathogenic to man as it invade the intestinal tissue

C/f similar to amoebic dysentery but no extra-intestinal spread

Two morphological forms

Trophozoite Cyst

Trophozoit

Cyst

EM view

Cilia

Life cycle

Diagnosis

Detection of cysts and trophozoits in faecal smears.

Regarding E. histolytica A. Cyst is the infective stage B. Does not attach to intestinal mucosa

C. Inhabits the human large intestine

D. Extaintestinal spread is possible E. Nucleus has a central karyosome

Regarding amoeba A. E. gingivalis has cyst stage in their life cycle B. Can differentiated by their characteristic movements C. E. dispar is a human pathogen D. E. coli and E. histolytica are morphologically identical

Regarding Balantidium coli

A. It is not pathogenic to human B. Trophozoite has only one nucleus C. It is a zoonotic parasite

D. Cyst is covered with cilia

E. Trophozoite is the infective stage to human True /false E.histolytica Inhabits human large intestine E. Histolytica cyst is a infective stage to human Transmitted by faeco-oral route

E.Histolytica trophozoite is morphologically identical to E. dispar

True or false

Genus Entamoeba has large katyosome in side the nucleus

E. Histolytica trophozoite moves sluggishly E. Histolytica trophozoite has single nucleus with centrally placed karyosome E. Gingivalis has trophozoite and cyst in their life cycle Acute amoebic dysentery, predominant form is cyst in stools Flask shaped ulcers are typical lesion in intestinal amoebiasis Trophozoites in faecal samples is a commonly associated with hepatic amoebiasis

In amoebic colitis, predominant form in the faeces is trophozoite

E histolytica and E dispar cysts cannot differentiate microscopically Fever is a common clinical feature of amoebic colitis. Abscess fluid microscopy is useful in the diagnosis of amoebic liver abscess