12 - VIROLOGY LABORATORY METHODS

Antiviral chemotherapy

Blood donor screening

Prevent outbreaks

Seroepidemiological studies

Viral disease process

Importance of Laboratory Diagnosis (5)

Antiviral chemotherapy

Importance of Laboratory Diagnosis

to confirm viral etiology of diseases for which antiviral chemotherapeutic agents are available

Blood donor screening

Importance of Laboratory Diagnosis

for bloodborne viral pathogens such as Hepatitis B, C, and HIV to prevent transmission of infection by transfusion of blood and blood formed products contaminated with viruses

Prevent outbreaks

Importance of Laboratory Diagnosis

to monitor and detect epidemics of serious viral infections such as influenza, encephalitis, and poliomyelitis earlier in order to initiate appropriate control measures to prevent further spread of diseases

Seroepidemiological studies

Importance of Laboratory Diagnosis

to perform seroepidemiological studies on viral infections

Viral disease process

Importance of Laboratory Diagnosis

define the viral disease process and understanding it in order to make plans on how to manage, prevent, and treat viral diseases

Vesicle fluid, skin scrapings

Specimen Consideration

Vesicular rash (2)

Throat, stool, and rectal swabs

Specimen Consideration

Maculopapular rash (3)

Stool and rectal swabs

Specimen Consideration

Gastrointestinal tract infections (2)

Conjunctival swab/scraping

Specimen Consideration

Eye infections

Nasal and bronchial washings, throat and nasal swabs, sputum

Specimen Consideration

Respiratory tract infections (5)

Stool, tissue, saliva, brain biopsy, cerebrospinal fluid

Specimen Consideration

CNS (encephalitis and meningitis cases) (5)

Blood

Specimen Consideration

Bloodborne infections

Urine

Specimen Consideration

Urinary tract infections

Vesicle fluid or swab

Specimen Consideration

Genital infections

Enteroviruses, adenoviruses, and HSV

Throat swabs

Viruses (3)

inflamed

vesiculated

purulent

posterior pharynx

Throat swabs

Collected with a dry, sterile swab by passing the swab over the _, _, or areas on the _ _

tongue

buccal mucosa

teeth

gums

Throat swabs

Swab should not be touched by the _, _ _, _, or the _

RSV, influenza and parainfluenza viruses

Nasopharyngeal swab or aspirate specimens

Viruses (3)

nasopharynx

3-7

buffered saline

Nasopharyngeal swab or aspirate specimens

Collected by:

Inserting a swab in a flexible shaft through the nostril into the _

By washing and collecting the secretions by rinsing the bulb syringe at _-_ mL of _ _

Rhinovirus

Nasal specimen

Viruses (1)

respiratory

All _ specimens are acceptable for culture of most viruses

bronchoscopy

lower, influenza, adenoviruses

Bronchial and Bronchoalveolar Washes

Washings and lavage fluid collected during _

Excellent specimens for detecting viruses that infect the _ respiratory tract, especially _ viruses and _

PCR

electron microscopy

Rectal Swabs and Stool Specimens

Many agents of viral gastroenteritis do not grow in cell culture and require _ or _ _ for detection

Stool specimens

Rectal Swabs and Stool Specimens

More sensitive for viral gastroenteritis (stool specimens or rectal swabs)

Stool specimens

Rectal Swabs and Stool Specimens

provide a larger sample volume, are more likely to contain a higher concentration of virus, and offer better sensitivity and reliability for detecting viruses that are shed in the feces

Rotavirus and enteric adenovirus (types 40 & 41)

Stool Specimens

Viruses (2) and type

5-10

Stool Specimens

_-_ mL stool sample

Enterovirus

Rectal Swab

Viruses (1)

3-5

Viral Transport Medium (VTM)

Rectal Swab

Swab is inserted _-_ cm in the mucosa to ensure adequate sampling and placed in _ _ _ to maintain the viability of the virus during transport

CMV, mumps, rubella and measles viruses, polyomaviruses, and adenoviruses

Urine

Viruses (6)

2-3

Urine

Multiple specimens (_-_ vials)

10

first-morning clean-catch

Urine

_ mL _-_ _-_ urine

7.5%, sodium bicarbonate

pH, bacteria

Urine

Neutralized with _% solution of _ _ before testing

Urine _ and contaminating _ may interfere in viral replication

Enteroviruses, HSV, VZV, and in rare cases CMV or poxviruses

Skin and Mucous Membrane Lesions

Viruses (5)

Tzanck Smear

Skin and Mucous Membrane Lesions

Prepared by carefully unroofing the vesicle and making an “impression smear”

Tuberculin Syringe

Skin and Mucous Membrane Lesions

Aspirate a drop of vesicle fluid and place in VTM

For further use in the event a viral or bacterial culture

Viruses are difficult to detect in areas with ulcerations because it may hide the virus behind the pus/additional WBCs

Scalpel

Skin and Mucous Membrane Lesions

Direct examination

physician

Sterile Body Fluids (CSF, Pericardial and Pleural Fluid)

Collected aseptically by the _ (aspiration)

Enterovirus, HSV, VZV, influenza viruses, or CMV

Sterile Body Fluids (CSF, Pericardial and Pleural Fluid)

Viruses (5)

Blood and Bone Marrow

Used primarily to detect CMV

5-10

Blood and Bone Marrow

_-_ mL whole blood

Heparin

Citrate

EDTA

Blood and Bone Marrow

Anticoagulants used (3)

Citrate

Blood and Bone Marrow

Anticoagulants used

other virus/general viral detection

EDTA

Blood and Bone Marrow

Anticoagulants used

nucleic acid testing

Serum

Blood and Bone Marrow

serological assays

Acute

Blood and Bone Marrow

serological assays

immediately

convalescent

Blood and Bone Marrow

serological assays

2-3 weeks

Tissue

Useful for detecting viruses that commonly infect the lungs, brain, and GI tract

CMV, influenza virus, adenovirus, sin nombre virus

Tissue

Lungs viruses (4)

HSV

Tissue

Brain viruses (1)

CMV

Tissue

GI tract viruses (1)

surgical procedures

Tissue

Specimens are collected during _ _

Fresh tissue

Tissue

_ _ is preferred especially for nucleic acid assays to ensure accurate viral detection

HSV

human papillomavirus (HPV)

Genital Specimen

Detection of _ and _ _

immediately

Specimen Consideration, Transport, and Storage

Ideally should be processed _

4

Specimen Consideration, Transport, and Storage

Short-term transportation storage: Keep samples at _ C

4 C

-20 C or -70 C

Specimen Consideration, Transport, and Storage

Storage

≤5 days: ?

≥6 days: ?

freezing

VTM

Specimen Consideration, Transport, and Storage

Specimen for _ must be diluted or emulsified in _

Dacron

Rayon

Specimen Consideration, Transport, and Storage

Synthetic swabs used in specimen collection (2)

calcium alginate

Specimen Consideration, Transport, and Storage

Cotton swabs are avoided because they may contain _ _ which can interfere with viral recovery, specifically the inactivation of HSV

Viral Transport Media (VTM or UTM)

Specimen Consideration, Transport, and Storage

Useful for maintaining viral stability

Viral Transport Media (VTM or UTM)

Specimen Consideration, Transport, and Storage

Used to transport small volumes of fluid specimens, small tissues, and scrapings, and swab specimens

Protein

Antimicrobials

Specimen Consideration, Transport, and Storage

Composition of VTM (2)

Protein

Specimen Consideration, Transport, and Storage

Composition of VTM

• Stabilizes viral agents  

• Ex: Serum, albumin, or gelatin

Antimicrobials

Specimen Consideration, Transport, and Storage

Composition of VTM

• To prevent overgrowth of bacteria and fungi

• Ex: Penicillin, streptomycin (traditionally used)

Vancomycin, gentamicin, amphotericin

Specimen Consideration, Transport, and Storage

Composition of VTM

• More potent mixture of antimicrobials

Stuart’s medium

Amie’s medium

Leibovitz-Emory medium

Hanks Balanced Salt Solution (HBSS)

Eagle’s Tissue Culture medium

Specimen Consideration, Transport, and Storage

Examples of VTM (5)

Blood Viral Culture

Specimen Consideration, Transport, and Storage

Sterile anticoagulated tube (4 C)

Blood Viral Serology

Specimen Consideration, Transport, and Storage

Serum in sterile tube (4 C for a few days)

Serum in sterile tube (-20 C for weeks/long term preservation)

IgM

Specimen Consideration, Transport, and Storage

Testing for virus-specific _ should be completed before freezing whenever possible because _ may form insoluble aggregates upon thawing, producing a false negative result

direct

indirect

Viral Testing

Classified into _ and _ methods

Direct

Viral Testing

Detects virus itself through:

Microscopy

Antigen detection

Molecular testing

Indirect

Viral Testing

Detects the body’s response to the virus typically through serology

Viral Isolation

Indirect Examination

plays a vital role in establishing the cause of viral diseases

Animal inoculation

Embryonated eggs

Tissue culture

Indirect Examination

Methods/Gold standards for confirming viral etiology (3)

Animal Inoculation

Viral Isolation

Experimental animals are used for cultivation of viruses

Plays an essential role in the study of pathogenesis of viral infections and that of viral oncogenesis

Mouse, rabbits, hamsters, newborns or suckling rodents

Viral Isolation – Animal Inoculation

Animals used (4)

Embryonated Eggs

Viral Isolation

Initially used for the growth of viruses

Classic and widely used system especially in research and vaccine production

Goodpasture (embryonated chick egg)

Viral Isolation – Embryonated Eggs

among the first to demonstrate that embryonated eggs can be used to grow viruses

Burnet (different sites)

Viral Isolation – Embryonated Eggs

later refined the technique by identifying different sites within the egg, where specific viruses can be grown most effectively

8-11

Viral Isolation – Embryonated Eggs

_-_ days old chick egg

8-11

Viral Isolation – Embryonated Eggs

_-_ days old chick egg

Yolk sac

Amniotic cavity

Allantoic cavity

Chorioallantoic membrane

Viral Isolation – Embryonated Eggs

Sites (4)

HSV

Viral Isolation – Embryonated Eggs

Yolk sac (1)

influenza virus, mumps virus

Viral Isolation – Embryonated Eggs

Amniotic cavity (2)

influenza virus, mumps virus, newcastle disease virus, avian adenovirus

Viral Isolation – Embryonated Eggs

Allantoic cavity (4)

herpes simplex virus, poxvirus, rous sarcoma virus

Viral Isolation – Embryonated Eggs

Chorioallantoic membrane (3)

Embryonated egg

Viral Isolation

provides sterile and self-contained system, cost effective and relatively easy to handle, and the different sites would offer versatility for cultivating various viruses

Tissue Culture

Viral Isolation

Demonstration of virus in appropriate clinical specimens by culture establishes diagnosis of viral diseases

Acute stage of infection

Viral Isolation – Tissue Culture

Time of collection

Enterovirus

Viral Isolation – Tissue Culture

present in CSF for 2-3 days after onset of CNS manifestations

HSV & VZV

Viral Isolation – Tissue Culture

found in lesions within the first 5 days of the onset of symptoms

Respiratory viruses

Viral Isolation – Tissue Culture

present in respiratory secretions during the first 3-7 days of onset of symptoms

In ice

Viral Isolation – Tissue Culture

Respiratory viruses transport conditions

Organ Culture

Viral Isolation – Tissue Culture

Gold standard for establishing viral etiology of a disease

Organ Culture

Viral Isolation – Tissue Culture

Small bits of the organs are maintained in vitro for days and weeks preserving their original morphology and function

Ideal for respiratory viruses

Ex: Tracheal ring organ culture

Tracheal ring organ culture

Viral Isolation – Tissue Culture

Example of organ culture

Explant Culture

Viral Isolation – Tissue Culture

Components of minced tissue are grown as explants embedded in plasma clots

More stable environment

Compared to individual cells

Ex: Adenoid tissue explant cultures

Adenoid tissue explant cultures

Viral Isolation – Tissue Culture

Example of explant culture