+

Centro Scholar University – Mendiola

College of Medical Technology

Lea Necitas G. Apostol, PhD, MPH

Lab Manager/Supervising Science Research Specilaist
Department of Virology
Research Institute for Tropical Medicine
OUTLINE
I. VIROLOGY
Basics

II. The need to do laboratory investigation

Surveillance/Diagnostic
Vaccine Preventable diseases (Polio, Measles, JE)
Emerging and Reemerging viral Infections (MERS-CoV, Ebola)

III. Current diagnostic tests in virology

Virus Isolation
Immunofluorescence Test
Serology
Molecular Test (PCR, Sequencing)
Biosafety in the lab
• Virology
• Basics
• The need to do laboratory investigation
• Surveillance/Diagnostic
• Vaccine Preventable diseases (Polio,
Measles, JE)
• Emerging and Reemerging viral Infections
(MERS-CoV, Ebola)
• Current diagnostic tests in virology
• Virus Isolation
• Immunofluorescence Test
• Serology
• Molecular Test (PCR, Sequencing)
• Biosafety in the lab
+
VIRUSES
 Non-cellular form of life

 Obligate intracellular parasites

 absolute dependence on a living host organism for their reproduction

 Virions harbor viral genome protected by protein shell

 Can infect ALL life forms (human, plants, fungi, bacteria, insects etc)
+
Biological diversity
between viruses
 Host range (human, zoonoses, arbo)

 Type of disease (CNS, GI etc)

 Virus genome type (DNA or RNA)

 Virion size / structure

 Virus particles (virions) are built of a nucleic acid and a protein shell
 Icosahedral (spherical) virions (protein subunits are arranged with cubic
symmetry)
 Elongated (helical) virions

 Virus genome replication strategy

+ LIFE CYCLE

 Invasion /Entry via host receptors

 Genome uncoating, expression
and replication
 Uncoating
 Modify host cell functions for
replication
 Particle (virion) assembly forming
the capsid
 Virus is released and infects new
cells
+ SOURCES OF TRANSMISSION
Source of virus is called reservoir (e.g., infected animal, cell
culture etc)

Agent or means by which an infection is spread or

transmitted from one individual to another is called a vector
(e.g., aerosol)

Zoonoses - animals

*sometimes the reservoir = the vector like some insect-

transmitted viruses (ARBOVIRUSES)
+
2 WAYS of disease transmission

 Horizontal disease transmission

 from one individual to another in the same generation (peers in
the same age group)

 can occur by either direct contact (licking, touching, biting), or

indirect contact air – cough or sneeze, vectors or fomites that
allow the transmission of disease without physical contact

 Vertical disease transmission

 passing a disease causing agent vertically from parent to
offspring, such as perinatal transmission.
+
ROUTES OF TRANSMISSION
 Airborne contact - if the microorganism can remain in the air for
long periods (chickenpox, influ, mumps/rubella/measles, SARS)
 Droplet transmission – coughing or sneezing on another person;
large and are not suspended in air  dispersed over short
distances (respiratory viruses)
 Fecal-oral transmission - – usually from contaminated food or water
sources due to lack of sanitation and hygiene, (GI, Polio and
enteroviruses)
 Transmission by direct contact - touching an infected person,
including sexual contact; oral (HIV, HSV)
 indirect physical contact – usually by touching soil contamination or a
contaminated surface (fomit
 Vector-borne
+
CLASSIFICATION
 1. Virus genome
 DNA or RNA
 single-stranded (ss) or
 double-stranded (ds)

 2. Virion structure
 icosahedral or helical capsid
 enveloped or non-enveloped
(naked capsid)

 3. Baltimore scheme
 based on how virus produces
mRNAs
- mostly used to distinguish
different types of RNA viruses
(+ sense, - sense)
• Virology
• Basics
• The need to do laboratory investigation
• Surveillance/Diagnostic
• Vaccine Preventable diseases (Polio,
Measles, JE)
• Emerging and Reemerging viral
Infections (MERS-CoV, Ebola)
• Current diagnostic tests in virology
• Virus Isolation
• Immunofluorescence Test
• Serology
• Molecular Test (PCR, Sequencing)
• Biosafety in the lab
Why the need to do lab
investigation?
Why the need to do lab
investigation?

Virus is “everywhere”.
Viruses can infect all life forms
Viral infection can be lethal
+
Viral Disease Surveillance

 Vaccine-Preventable Disease Surveillance

 Polio, Measles/Rubella, Jap encephalitis,
Rotavirus, Influenza
 Emerging and Reemerging Infectious Diseases
 Ebola, MERS-CoV, SARS, pH1N1, Nipah
+ Rationale for Priority
VPD
Global Polio Eradication Initiative(AFP Surveillance)
- No case of polio caused by wild poliovirus worldwide
despite intensive surveillance
 any child under 15 years of age with acute onset of floppy
paralysis, OR a person of any age in whom poliomyelitis is
suspected by a physician

 Measles Elimination
 Absence of endemic measles transmission in a defined
geographical area for a period of >12 months in the
presence of a well performing surveillance system (<1
confirmed / 1,000,000 population)
 any individual, regardless of age, with the following signs and
symptoms: fever (38°C or more) or hot to touch; and
maculopapular rash (non-vesicular); and at least one of the
following: cough, coryza (runny nose), or conjunctivitis
+ What are vaccine preventable diseases?
 Vaccine Preventable Diseases are those illness that can be prevented through
immunization.
 EPI SURVEILLANCE NETWORK
Regional
Reference Labs WHO
Japan, Australia
National Epidemiology
Center

RITM
Immunization
Virology Laboratory Program

Regional Center for Health Development

EPISO / RESU / PESU

RHU
Hospital

Privat
EPI Surv.
e Coordinator
Clinic
+
Responsibilities of Medical Technicians

 Atthe Health facility

 Facilitate specimen collection
 Provide facility for specimen storage in the
facility
 Support disease surveillance officer in the
transport of specimens to RITM
+
So, what are the VPD Surveillance
programs of DOH?
 Acute Flaccid Paralysis (AFP) - Polio

 Measles/Rubella

 Japanese Encephalitis

 Rotavirus

 All “SYNDROMIC” APPROACH

+
Poliovirus
 Genus Enterovirus (Genogroup C)

 3 types: Polio 1, 2, 3

 Vaccines: Oral Polio Vaccine (OPV), Inactivated Polio Vaccine

(IPV)
+
Why is it feasible to eradicate
polio?
 The virus is relatively stable (only 3 serotypes)

 Transmission : faecal-oral route (hygiene)

 Available vaccines

 Herd immunity
 a form of immunity that occurs when the vaccination of a
significant portion of a population (or herd) provides a measure of
protection for individuals who have not developed immunity.

Only 3 countries remain endemic for polio!

+
Poliomyelitis
+
POLIO Laboratory Confirmation

Laboratory Procedures done for detecting

polioviruses:

1. Virus Isolation
2. Intratypic differentiation (ITD) of poliovirus
3. Sequencing

* Typing not required based on revised algorithm

+
MEASLES

one of the leading causes of death

among young children even
though a safe and cost-effective
vaccine is available.

In 2013, there were 145 700

measles deaths globally – about
400 deaths every day or 16 deaths
every hour.
+
MEASLES
• single-stranded, negative-sense,
enveloped RNA virus
• genus Morbillivirus
• family Paramyxoviridae

• Infection of the respiratory system

• Humans are the natural hosts of

the virus; no animal reservoirs are
known to exist.

• WHO and DOH:For elimination

+ Specimen

Serum sample collection remains the

GOLD STANDARD for confirming suspect cases
under surveillance.

Serum should be collected 4-28 days from rash onset

+
Quick Guide for Specimen Collection
+
Measles Laboratory Confirmation

Laboratory Procedures done for detecting measles/rubella:

1. Serological Diagnosis (EIA/ELISA)

2. Virus Isolation*

3. Molecular PCR
* Typing not required based on revised algorithm
+
What is Japanese Encephalitis Virus?

 Most common cause of encephalitis in Asia

 Genus Flavivirus
 Transmitted to humans by bite of Culex mosquito; wading
birds are primary reservoir
 Children <15 years old are
more susceptible
 <2% of cases develops neurological symptoms
 80-90% develops neurological sequelae
+
JE: The most common cause of
encephalitis in Asia
+
Laboratory analysis for JE:

 serological investigation

 Panbio
JE/Dengue IgM
Combo ELISA kit used
+
Possible causal agents of CNS
infections Japanese
Hemophilus Encephalitis Virus
influenzae type b
(Hib),
Polio, Entero 71
Coxsackie A

CNS Infections:
Meningitis
Neiserria
Encephalitis
meningitidis
Myelitis Measles,
Neuritis Mumps, HSV,
VZV, EBV, CMV

Streptococcus Dengue, Malaria,

pneumoniae Other and other arthropod-
borne diseases
bacterial
pathogens
+
Emerging and Reemerging
Infections

MERS CoV

Ebola
+
Family Coronaviridae
 Alphacoronavirus

 Betacoronavirus
 MERS coronavirus

 Human coronavirus HKU1

> BATS

 SARS-related coronavirus

 Human Coronavirus-EMC


Gammacoronavirus

Deltacoronavirus > AVIAN

+
MERS-CoV

 Middle East Respiratory Syndrome Coronavirus

 Causes viral severe acute respiratory illness

 Can affect anyone (1-99 y.o.)

 First case in Jordan in April 2012

 First reported in Saudi Arabia in Sept 2012

 So far, all
cases of MERS have been linked to
countries in/near the Arabian Peninsula
+
The virion
 Enveloped /Capped

 spherical (120 nm)

The genome
 The ssRNA(+) genome is associated
with the N protein = nucleocapsid.

 Monopartite, linear

 27-32kb in size (the largest of

all RNA virus genomes)

 Polyadenylated

 The leader RNA (65-89 bp) at the 5’

end is also present at the end of each
subgenomic RNAs.
+
MERS-CoV
 Host
 Human, cattle, pig, mouse, rat, and bats

 Reservoirs
 SARSV: Bats MERSV:Camel

 Tropism
 Primary site of infection: epithelial cells
of respiratory or enteric tracts.
Neurological tissues
 INTERACTIONS (Cell receptors)
 MHV: Ceacam1 ,
 SARS-CoV: ACE2 ,
 BCoV,HCoV-OC43: sialic acid
+ Countries with Lab-Confirmed
MERS Cases

 in or near the Arabian Peninsula

 ( KSA, UAE, Qatar, Oman, Jordan, Kuwait, Yemen, Lebanon,
Iran)

 with Travel-associated Cases

 (UK, France, Tunisia, Italy, Malaysia, Philippines, Greece,
Egypt, USA, Netherlands, Algeria, Austria, Turkey Two
patients were transferred to Germany for care.
+
The disease: MERS
 Transmission
 close contact

 Caring for/living with an infected person

 Travel

 Incubation Period
 2-14 days

 Mortality
 About 3-4 out of every 10 people reported have died (58% mortality at
day 90)
 Immunocompromised & with pre-existing medical conditions
(comorbidities: diabetes, cancer, kidney/heart/lung dss) => more likely to
be infected with MERS-CoV
+
MERS Clinical Features
 Symptoms & Complications
 SARS: fever, cough, shortness of breath
 Gastrointestinal symptoms including diarrhea and
nausea/vomiting.
 severe complications: pneumonia and kidney failure.

 from asymptomatic  acute upper respiratory illness, 

rapidly progressive pneumonitis, respiratory failure,
septic shock / multi-organ failure  death
+
The disease: MERS
 Prevention
 no vaccine

 Wash your hands often with soap and water for 20 seconds. (OR
use an alcohol-based hand sanitizer)
 Cover your nose and mouth with a tissue when you cough or
sneeze
 Avoid touching your eyes, nose and mouth with unwashed hands.
 Avoid personal contact, (kissing, or sharing cups or eating
utensils) with sick people.

 Treatment
 No specific antiviral treatment is recommended
 For severe cases, current treatment includes care to support vital
organ functions.
+
MERS – CoV Lab Diagnosis

 PCR

 Serology
 Screening (ELISA)
 Confirmatory (IF/NAT)
+
Ebola
+
A disaster!
“Our people are dying, children are
being orphaned, most of the dead
are women and over two-thirds of
those infected belong to the most
economically active age category
of 15 to 50. Children are not going
to school; doctors and nurses are
dying, and non-Ebola illnesses are
adding to the toll of death and
suffering due to further strains and
weakening of the healthcare
delivery system in the country.”

DR ERNEST BAI KOROMA, PRESIDENT OF SIERRA LEONE

 +
Filovirus Cycle of Transmission

Reservoir hosts

Spillover

P Formenty, World Health

Organization, April 2014
+
Ebolavirus
 Filovirus family (Filoviridae)

 Five species
 Zaire Ebolavirus (EBOV)
 Sudan Ebolavirus (SUDV)
 Bundibugyo Ebolavirus (BDBV)
 Tai Forest Ebolavirus (TAFV)
 Reston Ebolavirus (RESTV)

Wikipedia Commons
+ 48

Reston Ebolavirus (REBOV)

The Good Cousin
 Found in Philippines and China

 Causes respiratory disease in pigs

 Infects humans, but no disease

 Pig farmers, abattoir workers, others have antibodies

 Some cross-reaction with Zaire EBOV

 Are people with REBOV antibodies immune?

 Can REBOV antibodies be used for therapy?

 Will REBOV antibodies from natural infections confuse diagnosis

of Ebola virus disease?
Miranda & Miranda 2011; Pan et al 2014
+
Ebolavirus
 Long narrow virus, length 1400 nm &
width 80 nm

 Filamentous

 RNA with protein as inner core; matrix

layer next; then membrane from
host cell outside

 RNA codes for 8 proteins

 Virus takes over the cell

http://visualscience.ru/en/projects/ebola/poster/
+
Natural history
 Virus enters the cell – any cell, but particularly uses
macrophages, dendritic cells and monocytes

 Spreads to lymph nodes via lymphatics and then to liver and

spleen via blood

 Secondary spread to all organs

 Exits the body in faeces, saliva, sweat, tears, sputum, skin cells,
breast milk, semen, urine, vomitus
+ No virus before
symptoms

Martines et al. J Pathol 2014

Detection of Ebola Virus in Different
Human Body Fluids over Time

52
+
Pathogenesis of Ebolavirus
 Destroys cells – focal necrosis in many
organs

 Suppresses inflammation

 Causes cytokine storm

 Induces clotting
Liver with Ebolavirus (red)
 Multi-organ focal necrosis and Martines et al J Pathol 2014
disseminated intravascular coagulation
with focal haemorrhage and minimal
inflammation
+
Survival outside host
 Dried – 24 hr at 25°C; 14 days at 4°C

 In fluids – up to 46 days at 25°C

 Ebolavirus is killed by:

 Heat 60ºC for 1 hr
 Hypochlorite (Chlorine solution)
 Alcohols
 3% acetic acid
 1% glutaraldehyde

Piercy et al J Appl Microbiol 2010;109:1531; Sagripanti et al Arch Virol 2010;

155:2035; Health Canada – PDSS - http://www.phac-aspc.gc.ca/lab-
bio/res/psds-ftss/ebola-eng.php
+ Environmental contamination in
isolation ward
 Sudan Ebolavirus outbreak – Uganda 2000

 2 positives from 33 environmental specimens

 Ebolavirus was detected on a bloody glove and a bloody IV insertion

site

 Not isolated on bedframes, chairs, stethoscopes, clean gloves, food

bowl, spit bowl, body bag cleaned with bleach, body louse

 Suggests that environmental contamination and fomites are possible

modes of transmission in an ETC

.
Bausch et al. J Inf Dis 2007
+
Ebola in West Africa
 Three countries with widespread transmission

 Guinea, Sierra Leone, Liberia

+ 57

Country Classifications (current)

 Countries with Widespread Transmission

 Guinea, Liberia, Sierra Leone

 Countries with Travel Associated Cases

 Mali

 Countries with Travel Associated Cases and Localized

Transmission
 USA, Spain

 Countries with Localized Transmission

 Democratic Republic of Congo (separate outbreak)
+ 58

Is EVD close to Philippines?

+
Diagnosis Assays

 Antigen Detection ELISA (Direct ELISA, Sandwich ELISA)

 IgM ELISA and IgG ELISA (Indirect ELISA)

 Polymerase Chain Reaction

 Virus Isolation
II. The need to do laboratory investigation
Surveillance/Diagnostic
Vaccine Preventable diseases (Polio,
Measles, JE)
Emerging and Reemerging viral
Infections (MERS-CoV, Ebola)
III. Current diagnostic tests in virology
Virus Isolation
Immunofluorescence Test
Serology
Molecular Test (PCR, Sequencing)
Biosafety in the lab
+
Outline

 Virus Isolation

 IF

 Serology

 Molecular
 PCR
 Sequencing
+
CURRENT DIAGNOSTIC TESTS IN
VIROLOGY

SEROLOGIC VIRUS MOLECULAR ELECTRON IF Sequencing

TESTS ISOLATION DETECTION MICROSCOPY Microscopy
CELL CULTURE
Dengue ALL
Dengue, Respiratory Respiratory
Measles ALL
Chikung Viruses (Influ A Viruses (Influ
& B, RSV, Enteroviruses
unya & Parainfluenza,
A & B, RSV,
Parainfluenza,
Influenza,
JEV Adenovirus,
etc) HSV, CMV, JE Adenovirus)
Measles Entero (Polio,
Coxsackie,
MERS-CoV Herpes
Viruses 1 & 2
& Rubella ECHO, etc) SARS
CMV
Rotavirus Dengue and JEV
Ebola
Herpes Viruses
MERS- 1 & 2; CMV
CoV, Measles
 63

Virus Isolation
Minimum Requirement:

Biosafety Laboratory Level 2 – VPD and other viruses (not droplet/airborne)

Biosafety Laboratory Level 3 – EBOLA, MERS-CoV, SARS
+ 64

Learning objectives

 To know the different techniques in virus isolation

 To identify the types of cell culture

 To define cell culture and know its requirements

 To identify the different cell lines and the viruses it can

isolate

 To learn how to perform cell culture

 To know the different techniques for virus identification

 To distinguish or be familiarize with different cytopathic

effect (CPE) of viruses in cell culture
+ 65

The basis….

 VIRUSES

– obligate intracellular parasites

 In the lab:
 Cultured cells
 Primary – seed virus
 Secondary – portions of the seed virus propagated in another
flask
 Eggs
 Laboratory animals
+ 66

Cell Culture/Maintenance
 culturing of cells derived from multicellular eukaryotes under controlled
conditions

 2 systems:
a. adherent culture – as monolayers on an artificial substrate
b. suspension culture – free-floating in the culture medium

 Cell lines:
 Primary – directly prepared from animal cells (monkey kidney cells)
 Semi-continuous – derived from human fetal tissue (HFF – human
fibroblast; MRC-5 cells)
 Continuous – tumors of human or animal (Vero, HEp2)
 Established or immortalized cell line has acquired the ability to
proliferate indefinitely either through random mutation or deliberate
modification

 Vary in their susceptibility to different viruses

+ 67

Cell Lines
Cell Derivative Viruses
line
HEp2 Human epidermoid Entero, HSV, Respiratory viruses

RD rhabdomyosarcoma Enteroviruses

L20B Genetically modified mouse cell Polioviruses and some coxsackieviruses

MDCK (Madin-Darby) Canine kidney Influenza

C6/36 Asian tiger mosquito larva Dengue

Vero African green monkey kidney epithelial Enterovirus, Respiratory viruses

cells
Vero- w/ human signaling lymphocyte activation Measles and Rubella
hSLAM marker
MRC-5 Human fetal lung fibroblast CMV
HFF Human fibroblast CMV
HeLa Human cervical carcinoma Parvoviruses
+ 68

Requirements

 Equipment
 BSC Class II
 Pipettor + tips
 Vortex
 Freezer
 Water bath
 Incubator

 Materials
 Freezing container
 Serological Pipettes
 2% Maintenance Media (10% Growth Media)
 Confluent cell line
 Samples
+ 69

Requirements

• Successful virus isolation depends on:

– Type of specimen (appropriateness)
– Timing of collection
– Amount of virus present
– Cultivability in cell culture/susceptibility of cells
– Handling and transport
– Aseptic techniques

• Growing the virus in susceptible cell lines

• Cultures are incubated for at least 10-14 days; 21 days for
CMV
+ 70

Procedure
 Specimen is inoculated onto confluent cell lines using 2%
Maintenance Medium

 Inoculated tubes/plates are incubated at 34-37°C

 Tubes should be read under an inverted microscope for CPE

(cytopathic effect) for 5-10 days or 14 days

 Repassage (contamination/toxicity/CPE) as necessary

 Confirm the virus (IF, Neutralization test)

+ 71

(Dis)Advantages
 Relative ease

 Broad spectrum

 Sensitivity

 Whole virions can be isolated

 Difficulty in maintaining cell cultures

 Variability of cell cultures

 Expensive materials and supplies

 Strict compliance to GLP

+ 72

Virus Identification in Cell Culture

1. Plaque purification - assumes “clone” from single plaque

2. Microscopy - cytopathic effects (CPE)

Syncytia – RSV, Measles, Mumps
Cell rounding, pyknotic – Enterovirus
Vacuolization, Ballooning - HSV
Inclusion bodies - Rabies
Focus (foci) - CMV
Grapelike clusters – Adenovirus

3. IF

4. PCR
+ 73

Plaque Assay

 used to purify a clonal population of virus or to determine viral

titer as plaque-forming units per ml (pfu/ml) so that known
amounts of virus can be used to infect cells during subsequent
work.

 cell monolayers are infected with a low ratio of virus

 An overlay of agarose keeps the cells stable and limits the spread
of virus.

 Each group of infected cells is referred to as a plaque.

+ 74

Cytopathic Effect

 Morphological changes that happen when viral infection and

replication occur

CMV - ballooning RSV - syncitia

CMV Measles/Rubella
+ 75

CPE

Adenovirus Enterovirus
Grape-like clusters Rounding necrosis
+ 76

CPE
HSV ballooning HSV-1
+ 77

CPE
RSV syncitia Influenza
+ 78

Immunofluorescence Assay

 is a technique used for light microscopy with a fluorescence

microscope and is used primarily
on microbiological samples.

 uses the specificity of antibodies to their antigen to

target fluorescent dyes to specific biomolecule targets within
a cell, and therefore allows visualization of the distribution of
the target molecule through the sample.

 2 types:
 Direct
 Indirect
+ 79

IFA
If the viral Ags are present in the cells,
monoclonal antibodies (MAb) specific
to that Ag will bind to the Ags.

secondary Abs containing FITC

(fluorescence substrate) conjugate will
bind to MAbs.

Under the IF microscope, FITC

substrate is excited by UV light and
gives an apple-green colored
fluorescence.

Conjugate also contains a counter stain,

Evan's Blue, which stains the cells dull
red. Hence, positive test is seen as dull-
red background with green
fluorescence while negative test is seen
as dull-red without any fluorescence.
+ 80

Direct/Indirect IF Assay (IFA)

 Scrape cells grown in culture vessels, transfer into a sterile microcentrifuge tube then
centrifuge for 5 mins @ 5,000 rpm
 Remove supernatant (ITCF), place it in a sterile cryotube with label of the sample/lab. ID.
 Pellet of cells are resuspended in PBS(-), 15-20 drops, to wash cells, then centrifuge for 5
mins @ 5,000 rpm. Wash up to 2x.
 Resuspend pellet in PBS(-), 7 drops, then smear on the wells of an IF slide. 2 wells for each
specimen.
 Air dry slide. Fix in cold acetone for 10 mins, air dry.
 Add monoclonal antibodies (MAb) specific to viral agent to respective wells
 Put slide inside the humidity chamber and incubate at 37°C for 30 minutes.
 Rinse slide with PBS(-) and then wash with PBS(-) for 10 minutes (immerse in shaker with
PBS-). *to remove excess and unbound monoclonal antibodies in the wells
 Add conjugate specific to viral agent to respective wells and the slide is then
put inside the humidity chamber and incubated at 37°C for 30 minutes.
 Rinse slide with PBS and then wash with PBS(-) for 10 minutes (immerse in
shaker with PBS). *to remove excess and unbound conjugate in the wells
 Air dry slide, mount and observe under the fluorescence microscope.
+
SEROLOGY TESTS
Are performed to demonstrate Ags in the serum, or the response
of the human body to these infectious agents (Abs) to establish
its contact with the immune system

Stems from demonstration of a rising titer of Abs to the agent

which among other things indicates a progressive infection.

Serological tests are of importance in epidemiological studies and

ascertain the response of the population to vaccines.

 based on the property of proteins to readily bind to a plastic

surface.
+ Antigen ELISA (Sandwich, Direct ELISA) 82

 Principle of the test

a capture antibody, directed against the protein, is linked to a

solid support such as a plastic plate
If Ags are present, they will be captured by the bound antibody.
The bound Ag is then detected by a 2nd Ab linked to an enzyme.
A chromogenic molecule is then added
+ 83

Indirect ELISA (antibody

detection)
+ 84

Laboratory Diagnosis (e.g. EVD)

Example of a positive microtiter plate

Microtiter washer
+
Diagnosis tests used according to stage
of the Disease

• Acute phase

1. Antigen ELISA

2. PCR

3. IgM ELISA

Timing of sample collection is crucial

• Convalescent Phase

1. IgG ELISA
+
Advantages and Limitations of the
Assays
Assay Advantages Limitations
Antigen ELISA Can detect all species Can not be used to
Has allowance for speciate the virus since
genetic changes it is a cocktail of
Can be set up in the different antibodies,
field may not detect small
viral copies
PCR Sensitive, can detect Can not accommodate
small viral copies, fast changes in sequence
turn around time for (mutation) and difficult
real time PCR to set up in the field
IgM ELISA Useful in the acute IgM does not persist
phase, can be used as a long the immune
complimentary test for system
antigen ELISA and PCR
+
Advantages and Limitations of the
Assays
Assay Advantages Limitations
IgG ELISA Can be used to monitor Not a point of care test
the progress
(convalescence of the
patient), IgG persist
longer in the immune
system, has allowance
for viral mutations
Virus Isolation High sensitivity, “gold Needs specialized
standard” especially in facility (BSL4)
novel outbreak of Needs specialized
pathogens training
Needs strict biosafety
and biosecurity
measures
+
Interpretation of Results

Assay Results
Antigen ELISA Positive Negative
Viral antigen No viral antigen
detected present
PCR Viral RNA No Viral RNA
detected present
IgM ELISA IgM detected No IgM detected
(Acute phase)
IgG ELISA IgG detected No IgG detected
(Exposure to
disease,
convalescent
stage
+
Quality Assurance Measures
Manpower

Training
Quality Control
◦ Internal Kit Control
◦ In-house Control
◦ Standard reagents and cell lines
Quality Assessment
◦ Sample Validation
◦ Proficiency Testing Panel
Equipment Monitoring
◦ Pipette Calibration (every 6 months)
◦ Temperature Monitoring (Incubator, Storage, Room)
◦ Incubator Calibration (once a year)
+
Serology Testing Algorithm for
Serum (Measles, Rota, Dengue, Chik, JE)

MEASLES IgM

Equivocal Positive Negative

Retest in Report as Report as Test for

Duplicate POSITIVE NEGATIVE Rubella IgM

If 2 Equivocals, If 2 Positives, If 2 Negatives, If 1 Positive & 1 If 1 Negative & 1 If 1 Negative & 1 Positive,
Report as Report as Report as Equivocal, Report Equivocal, Report Retest in Duplicate and
EQUIVOCAL POSITIVE NEGATIVE as EQUIVOCAL as EQUIVOCAL use 3 out of 5 consensus
+
Monitoring the Testing Process
Quality Assurance encompasses all measures, from receipt of
specimens through final reporting, to ensure that the final results
are as accurate as the assays allow.
1. Specimens should be inspected upon arrival for suitability.
2. Also included are organized record keeping system,
3. SOPs that acts as references
4. Supervisory review of results
5. System of evaluation of lab personnel
6. Use of the most appropriate tests/strategies
7. Mechanism for timely reporting
8. Storage of specimens for follow-up testing
9. Appropriate reporting forms
10. Good quality control and quality assessment program
+
Monitoring the Testing Process
Quality control refers to those specific measures that ensure the test
is performing as expected. Such measures include

1. Careful inspection of internal (kit) control values that validate the

test

2. Monitoring of physical parameters (temperatures, functioning of

equipment)

3. Validation of new reagents

+
Monitoring the Testing Process
Quality assessment is a means to challenge the overall performance
of the laboratory this process usually consists of the testing of a panel
of samples with known reactivity provided by an external source.

The National Reference Laboratories participate in the Proficiency

Testing panel from the following:

• National Rotavirus Laboratory- Murdock’s Childrens Research

Institute, Melbourne

• National Measles Laboratory- Center for Health Protection, HK

• National Dengue Laboratory- National Institute for Infectious

Diseases, Tokyo and National Environmental Agency, Singapore

• National JE Laboratory- US Centers for Disease Control

+
Good Laboratory Practices
Equipment Maintenance and Calibration. The maintenance
and calibration of your laboratory equipment is extremely important in
obtaining accurate and reproducible results. Ideally, pipettes are
calibrated every six months; incubators, annually. Options for
calibrating pipettes:
• Perform the gravimetric method
• Use a commercial automated calibration system
• Send pipette to the manufacturer
• Send the pipette to a calibration service
It is recommended that the laboratory sets specifications about
micropipettes performance based on their own or accreditation
standards.
As a guideline, micropipettes and multi-dispensing
micropipettes with precision of ± 10% for volumes <10 µl and ± 5%
for larger volumes can be used.
+
Good Laboratory Practices

Sample Handling. Sample quality can have a significant impact on final

assay results. Be sure samples are properly stored.

In general serum samples should be refrigerated at 2-8°C for up to 5

days. For longer period, serum should be removed from the clot and
frozen to a minimum of -20°C.

Frozen samples can be thawed at room or refrigerator temperature and

mixed thoroughly prior to dilution to ensure that proteins are dispersed
throughout the sample. Mix by gentle vortexing or inverting at least five
times. Frothing or over-mixing will cause denaturation of serum proteins.
Limit freeze-thaw cycles to 3.

Gross fungal or bacterial contamination can have adverse effect on the

antibody or other protein components of a sample and may have an
undesirable effect on test results. If sample quality is questionable,
obtaining a fresh sample is strongly advised, when possible.
+
Good Laboratory Practices

Pipetting Methods. Accurate and precise pipetting performance is

critical in laboratory analysis, particularly in highly sensitive tests
where a small mistake in pipetting can cause a large error in the final
result. Therefore it is of great importance to evaluate and to reduce,
wherever possible, both random and systematic errors in liquid
sample handling.

To achieve both good accuracy and precision you require a precision

instrument with quality tips, but you must also follow good
laboratory practice -cleanliness and consistent correct handling.

Regular maintenance/calibration guarantees that your pipettor

performs according to specifications.
+
Good Laboratory Practices
ELISA PLATE TIMING
Adding Samples and Control. During sample dispensing
to the plate, keep the time difference between the first and
the last sample to a minimum (<10 minutes), otherwise
use a multichannel pipette.
If the interval is too long, put a positive control and/or your
in-house control at the end of the plate and compare
results with the control at the beginning of the plate
Multiple Plate Runs. Keep your batch size small enough
so your processes do not overlap or your plates wait in
line to be washed. If possible, use a timer for every plate.
+
Good Laboratory Practices
ELISA PLATE WASHING. In general an automated or
semi automated wash system in proper working order will
provide more consistent washing than manual methods.
Check that all dispensing needles are dispensing with a
smooth, steady stream and that all aspiration ports
aspirate uniformly.
Prepare wash solution according to the package insert.
Use only the wash solution included with or recommended
for your test.
Do not allow plates to dry between plate washings and prior
to addition of reagents.
+
Good Laboratory Practices
ELISA PLATE READING.
Plates should be read as soon as possible following
addition of stop solution. Absorbance reading may drift if
excessive time elapses between stopping the reaction
and reading the plates.
+
Good Laboratory Practices
Reagent Handling and Preparation.
Receiving Tests: When you receive your ELISA kits, record the date on
your Inventory Control Tracking Chart and on the boxes. Use the
first-in-first-out method when using your tests.
If you do not use an entire test, mark the date it is opened and each
time it is used thereafter. This way, you can track of how many times
it moves or cycles from the refrigerator to room temperature. Keep
the number of cycles to the minimum by batching whenever possible.
General Reagent Handing
Be sure to check your package insert for guidelines on handling and
preparing reagents. Allow components to equilibrate to room
temperature (18-26°C) before beginning the assay. Use disposable
pipette tips and reagent reservoir when handling reagents to minimize
the risk of contamination.
+
Good Laboratory Practices
Test Component Handling and Preparation.
If a partial plate is used, aspirate all the liquid from the used well and cover with
sealing tape. Store unused portion of plates with several desiccants in a
resealable bag.
Make sure sample diluent and wash concentrate have come to room
temperature before use.
Controls should be added to the plate in the same manner and at the same time
as the samples.
If the test requires you to prepare a “working” conjugate solution, be sure to
follow the instructions closely. Prepare only what you immediately need. Do
not save leftover for future use. Contaminated or improperly stored
conjugates may lose enzymatic activity or may have apparent increase in
background.
Stop solution should be at room temperature before use, no crystallization and
appears clear.
+
Good Laboratory Practices
Quality Control
In-House Control. Incorporate an in-house control together with the kit
internal control. Aliqout in small quantities and freeze at -20°C. Do
not refreeze leftover. Plot controls and analyze for variations or
trends and review technique and quality control measures .
2.500
OD/CO Ratio
2.000
Mean
1.500
+1SD
1.000
-1SD
0.500 NORMAL TREND SHIFT
+2SD
0.000
-2SD
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Monitoring Temperature. ELISAs are sensitive to temperature

extremes. Try to maintain a laboratory temperature of 18-25°C. Cold
bench tops may affect assay and should be avoided by placing
several layers of paper towels under the assay plate.
+
ELISA TROUBLESHOOTING
High background or excessive Color Development
(High OD Readings)
Possible Causes:
o Poor water quality was used to wash plates or to prepare wash
solution.
o Substrate solution has deteriorated.
o Insufficient washing or poor washer performance.
o Wash system contained an alternate wash formulation.
o Reader was malfunctioning or not blanked properly.
o Laboratory temperature was too high.
o Reagents were intermixed, contaminated or prepared incorrectly.
+
ELISA TROUBLESHOOTING
Insufficient color development (Low Optical Density
Readings)
Possible Causes:
o Laboratory temperature was too low.
o Wash solution incorrectly prepared or wrong wash solution was used.
o Washer system had microbial contamination or contained an alternate
wash formulation.
o Too many wash cycles were used.
o Incubation periods were too short.
o Reagents and plates were too cold.
o Reagents were expired or intermixed from a different lot number.
o Wrong conjugate was used, deteriorated or prepared incorrectly.
+
ELISA TROUBLESHOOTING
Insufficient color development (Low Optical Density
Readings)
Possible Causes:
o Assay plate was read at wrong wavelength, or reader was
malfunctioning.
o Excessive test stress occurred (Plates cycled several time from ref to
room temp or plate was exposed to temperature extremes)
o Assay plates were compromised.
+
ELISA TROUBLESHOOTING
Replicates within a plate show poor reproducibility
Possible Causes:
o Error in sample pipetting.
o Excessive time was taken to add samples, controls or reagents to the
assay plate.
o Multichannel pipette was not functioning properly.
o There was inconsistent washing or washer system malfunctioning.
o There was poor distribution of antibody in the sample.
+
ELISA TROUBLESHOOTING
No color development
Possible Causes:
o Reagents were used in the wrong order or an assay step
was omitted.
o Samples were not added.
o Wrong conjugate was used, has deteriorated or prepared
incorrectly.
+
ELISA TROUBLESHOOTING
Poor reproducibility plate to plate
Possible Causes:
o Inconsistent incubation times occurred from plate to plate.
o Inconsistent washing occurred from plate to plate.
o Pipette was working improperly.
o Controls and samples were at different temperatures.
o Reagents being used were from different lots.
+4. Molecular Testing
+
+
Polymerase Chain Reaction (PCR)

A process used to artificially multiply a chosen piece of genetic material

Also known as DNA amplification
+
Electrophoresis
 Gel Electrophoresis Area
PCR Hood

+
Mupid, Bio-Rad
Sink BioSan
BSC II
ESCO
PCR Hood Minicentrifug
BioSan e
Gel
Documentation Centrifuge Centrifuge Vortex
System Eppendorf Eppendorf Scientific
BioRad 2 Door Ind.
PCR Room Fridge
Panasonic
Post-PCR Sample Preparation Dry Bath
BSC II Bio-Rad
Computer
ESCO
Pre-PCR

Freezer
Fujidenzo
Compartmentalized Computer
Waste
Bins
Table
Unidirectional flow
qPCR Machine
Kits, MCTs

Mini
Centrifuge

Aseptic technique
VWR
Plate Multi-tier
Thermal Table Rack Freezer Autoclave
Centrifuge Cyclers Rack Log Book Supplies, Sanyo Zealway
Labnet
consumables

Wash Area
MCB-IBD VIRO
Stock Stock
Sink
Cabinet Cabinet

BSC II
ESCO
Reagent
Reagent Preparation
Weighing
PrepPre-PCR Balance
Stock Area OHAUS

Gel
PCR Hood Making Area
2 Door
BioSan
Ref
LG
Standing
Cabinet Microwave
Whirlpool
+ Sample • Nucleic

WORKFLOW
Acid
Prep Extraction

Reagent • Reagent
Prep Preparation

Sample • Template
Prep Addition

• Gel
Wash Area Preparation

PCR/Post- • PCR/RT-
PCR Room PCR/qPCR
+
EQUIPMENT
&
MACHINES
IN PCR LAB
+
Reagent Preparation
 Preparation of Master Mixes

 Should be amplicon-free
X
 Materials should be nuclease-free

Equipment available

• BSC II

• PCR Work Station

• Vortex

• Mini-centrifuge

• -30oC Freezer

• Refrigerator
+
Sample Preparation
 Nucleic acid extraction

 Should be amplicon-free

 Materials should be nuclease-free

Equipment available

• BSC II (DNA/RNA)

• PCR Work Station

• Vortex

• Centrifuge

• Mini-centrifuge

• -20oC Freezer

• Dry Bath
+
PCR/Post-PCR
 PCR

 Should be clutter-free

Equipment available

• PCR Work Station

• Vortex

• Mini-centrifuge

• Refrigerator

• Thermal Cyclers

• qPCR machine

• Gel Documentation System

• Gel Electrophoresis Tanks

+ Laboratory Diagnosis and Surveillance of Ebola
Reston Virus

Real time-Polymerase Chain Reaction

+
SEQUENCING

Fragments pass thru a laser beam, excites the flourescent molecule and
recognized the dye (each base has assigned color)….
A- green, C-blue, T-red G-black
 121

BIOSAFETY
+ Laboratory Acquired Infections 122

 Infections, either symptomatic

or asymptomatic, that are
acquired through laboratory or
laboratory-related activities,
as a result of working with
infectious agents.

 Transmission of infection
occurs when the 6 elements of
the “Chain of Infection” are
present.

 Chain of infection is a way of

describing how disease is
transmitted from one living
thing to another.
 123
Actual Reports of LAIs…..
+ 124

Understanding How LAIs Occur

+ 125

Elements of LAIs…

 Infectious Agent

 Laboratory Reservoir

 Host

 Exposure

 Routes of Exposure

 Portal of Entry
+ Infectious agent 126

 An organism that causes

diseases:

 Bacteria
 Viruses
 Fungi
 Protozoa
 Prions
+ Infectious agent 127

Characteristics: Sources of infection:

 Pathogenicity  Endogenous/Self infection

- the ability to cause
disease
 Virulence
- agent factors that  Exogenous/Cross infection
determine disease severity
 Infectious dose
- the approximate number
of organisms required to
cause infection in a host
- Organism which are
harmless in one site cause
infection when transferred
to another.
- Organism are
transferred from one source
e.g.: nurse, doctor, other
patient, environment
Infectious Dose of Selected Agents 128
+ 129

Laboratory Reservoir

 Experimental animals

 Cell cultures

 Growth vessels

 Laboratory workers

 Laboratory stocks
+ 130

Host

Modifiers of Host Susceptibility

 Immune status of host
 Pregnancy
 Immunosuppressant therapies
or medications
 Nutrition
 Genetic make up

 Immunization
+ 131

Exposure

 Accidental releases and spills

 Incidental releases
 Manipulation of samples
 Aerosolization during sonication or centrifugation
 Excretion of agents by animals
+ 132

Types of Exposure:

 Direct
 Close contact between laboratory reservoir and laboratory host
 e.g.: Mouth pipetting, animal bites

 Indirect
 Contact with the agent on surfaces or in air of the laboratory
environment
+ 133

Routes of Exposure:

 Mucous membrane

 Ingestion

 Inhalation

 Direct inoculation
+ 134

Portal of Entry:

 Any body opening that allows the infectious agent to enter:

 Nose
 Mouth
 Eyes
 Mucous membrane
 Break in the skin
 Device inserted into the skin
 135

Understanding the Elements of LAIs Break the Chain of Infection

Exposure

Virulence
+ 136

Breaking the Chain of Infection

+ Laboratory
Biosafety
 Containment principles,
technologies, and practices
implemented to prevent
unintentional exposure to
pathogens and toxins, or their
unintentional release
Laboratory
137
Biosecurity
• Protection, control
and accountability for
valuable biological
materials within
laboratories, in order
to prevent their
unauthorized access,
loss, theft, misuse,
diversion or
intentional release
+ 138

Biorisk Management Process

 Biorisk = biosafety + biosecurity

Assessment Mitigation Performance

Elimination or
Risk identification Control
substitution
Hazard/threat Engineering
Assurance
identification controls
Likelihood Administrative
Improvement
evaluation controls
Consequences Practices and
evaluation procedures
Personal protective
equipment
+ 141

What to Look For?

 Hazard = object with the potential to cause harm

 Risk = likelihood + consequences of a harmful event

 Threat = person with the potential/intention to cause harm

+ 142

Exercise - What to Look For?

Heating a test tube containing Hot drink in

Ebola-suspected blood on mug
burner, but not to the point of
disinfection
+ 143

Exercise - What to Look For?

Some biosafety risks: Some biosafety hazards:

• Infectious material falling onto • Hair not tied back
mug • Infectious material in
• Ingestion of infectious material test tube
through bare hands • Hot drink in mug
• Ingestion of infectious material • Lack of PPE (gown,
through clothing gloves, goggles)
• Infection through eyes Other safety hazards:
• Infection through hair • Open burner
Other safety risks: • Liquid in mug
• Hair getting burned by burner Some threats:
• Limbs getting burned by burner • Person not following
• Burns from hot drink spill protocol
• Slippage and blunt force due to • Supervisor not routinely
drink spill checking on staff
+ 145

Equipment, Procedures, and

Chemicals
HAZARDS
Centrifug (aerosol)
e

Glassware

Ethanol

Guanidiniu
m chloride
+ 148

PPE Hazards

↓ ↓
↓ ↓ ↓
↓ ↓
↓
↓
+ 149

Personnel
 Training and practice

 Workload and fatigue

 Infection control

 Health (incl. mental) and immunity

status

 Professional relationships (buddy,

biosafety officer)
+ 150

Exercise - Facility
 Heating, ventilation, and air
conditioning (HVAC)

 Filtered air

 Windows

 Access control

 Work surfaces

 Pest control
+ 156

Pathogen Safety Data Sheet

+ 158

Biosafety Cabinet Use

+