Virology Laboratory Diagnosis

8/3/2019 Virology Laboratory Diagnosis

1/31

delfin, rmt

Virology

Laboratory Diagnosis


2/31

Three Groups of Diagnostic Test

y Direct detection

y the clinical specimen is examined directly for the

presence of virus particles, virus antigen or viral

nucleic acids

y

y Indirect examination (virus isolation)

y the specimen is subjected into cell culture, eggsor animals in an attempt to grow the virus.

y


3/31

y Serology.

y constitutes by far the bulk of the work of any

virology laboratory.

y serological diagnosis can be made by the

detection of rising titres of antibody between

acute and convalescent stages of infection, or the

detection of IgM.y In general, the majority of common viral

infections can be diagnosed by serology.


4/31

Specimen Collection

y Viral antibody studies

y

y For immune status testing, a single serum sample is sufficient.

y

y To detect acute infection

y

y Both acute and convalescent sera are generally required to

detect an antibody rise.

y Virus infections whose clinical symptoms are immune-mediated

are exceptions (like EBV, HBV, parvovirus B19).

y Specimen collection for viral culture

y Specimen containers that are not labeled will be rejected.

y Collect specimens for culture early in illness when viral

shedding is maximal.


5/31

Sample Collection device* Holding

Temperature

Comments

Virus isolation or antigen test:

Swabs Use viral transport

medium Large swab for

throat, lesion, etc. Small

wire shaft swab for

nasopharynx in young

children or urethral

samples

Refrigerate Viral transport

medium with swabs

should be capped

tightly

Body fluids,

BAL, stool

Use sterile leakproof

containers

Refrigerate Do not dilute body

fluids or BAL in

transport medium

Tissues Place in tubes containing

liquid viral transportmedia to keep tissue

moist

Refrigerate .

Blood

(Leukocytes or

plasma)

Collect 2 lavender top

tubes. Collection time

required

Room temperature Sample must be

processed within 4-6

hours of collection.


6/31

Specimen collection instructions for

selected specimen

yNasopharynx swaby Insert swab into nasopharynx, just past point of

resistance. Leave in place for 1 min or rotate to

dislodge respiratory epithelial cells;

y remove and place in transport medium. For small

children, thin, flexible wire shaft can be used.


7/31

y Nasopharynx aspirate

yUse suction pump connected to a catheter through a

mucus trap;

y Insert catheter as far into nose as possible.y Specimen should be taken from posterior part of nasal

mucosa, which is lined with respiratory epithelium,

and not from anterior part, which is lined with

squamous epithelium.yCollect as much ofNP secretions as possible; do not

dilute sample with saline unless necessary.


8/31

y Throat swab

y Swab posterior pharyngeal wall, not buccal mucosa,

tonsils, tongue or palate.

y Swab firmly and thoroughly.y Rectal swab

y Stool specimen or Rectal swab is required for enteric

pathogens


9/31

y Lesion swab

yClean lesion with sterile saline soaked gauze pad.

yUnroof vesicles or remove crusts. Firmly swab base

and margins of the lesion, obtaining fluid and cells.

yAfter sample collection, clean lesion thoroughly

with betadine. Do not use disinfectant prior to

sample collection or virus may be inactivated.


10/31

Methods of Examination:

y Direct Examination

ycalled rapid diagnostic methods because they

can usually give a result either within the same

or the next day.

y

yextremely useful in cases when the clinical

management of the patient depends greatly onthe rapid availability of laboratory results.


11/31

1. Antigen Detection

y Examples of antigen detection include

immunofluorescence testing of nasopharyngeal

aspirates for respiratory viruses e.g.. RSV, flu A, fluB, and adenoviruses.

y The quality of the specimen obtained is of utmost

importance in order for the test to work properly.


12/31

2. Electron Microscopy (EM)

yVirus particles are detected and identified on the basis

of morphology. A magnification of around 50,000 isnormally used.

ymainly used for the diagnosis of viral gastroenteritis by

detecting viruses in faeces e.g. rotavirus, adenovirus,

astrovirus, calicivirus and Norwalk-like viruses.yOccasionally it may be used for the detection of

viruses in vesicles and other skin lesions, such as

herpesviruses and papillomaviruses.

y The sensitivity and specificity of EM may be enhanced

by immune electron microscopy, whereby virus

specific antibody is used to agglutinate virus particles

together and thus making them easier to recognize.


13/31

3. Light Microscopy

yReplicating virus often produce histological changes

in infected cells.These changes may be characteristic

or non-specific. Viral inclusion bodies are basically

collections of replicating virus particles either in the

nucleus or cytoplasm.

y Examples of inclusion bodies include the negri

bodies and cytomegalic inclusion bodies found in

rabies and CMV infections respectively.

y

Although not sensitive or specific, histologynevertheless serves as a useful adjunct in the

diagnosis of certain viral infections


14/31

4. Viral Genome Detection

yMethods based on the detection of viral genome are

also commonly known as molecular methods.

y It is certain though that the role of molecular methods

will increase rapidly in the near future. Classical

molecular techniques such as dot-blot andS

outhern-blot depend on the use of specific DNA/RNA probes

for hybridization.

y These techniques may allow for the quantification ofDNA/RNA present in the specimen. However, it is

often found that the sensitivity of these techniques is

not better than conventional viral diagnostic methods.


15/31

Virus Isolation

y Cell cultures, eggs, and animals may be used for

isolation.

y However eggs and animals are difficult to handle

and most viral diagnostic laboratories depend oncell culture only.


16/31

There are 3 types of cell cultures:

y Primary cells - e.g. Monkey Kidney.

y These are essentially normal cells obtained from

freshly killed adult animals.These cells can only be useonce or twice.

y Primary cell cultures are widely acknowledged as the

best cell culture systems available since they support

the widest range of viruses.y However, they are very expensive and it is often

difficult to obtain a reliable supply.


17/31

y Semi-continuous cells - e.g. Human embryonic

kidney and skin fibroblasts.

yThese are cells taken from embryonic tissue, and

may be use up to 50 times.


18/31

y Continuous cells - e.g. HeLa, Vero, Hep2, LLC-

MK2, BGM.These are immortalized cells i.e.

tumour cell lines and may be passaged indefinitely.

yContinuous cells are the most easy to handle butthe range of viruses supported is often limited.


19/31

Identification of growing virus

y The presence of growing virus is usually detected by:

y Cytopathic Effect (CPE) - may be specific or non-specific

e.g. HSV and CMV produce a specific CPE, whereas

enteroviruses do not.

y Haemadsorption - cells acquire the ability to stick to

mammalian red blood cells. Haemadsorption is mainly used for

the detection of influenza and parainfluenzaviruses.

y Confirmation of the identity of the virus may be carried out

using neutralization, haemadsorption- inhibition,

immunofluorescence, or molecular tests


20/31

Problems with cell culture

y The main problem with cell culture is the long period

(up to 4 weeks) required for a result to be available.

y Also, the sensitivity is often poor and depends on many

factors, such as the condition of the specimen, and the

condition of the cell sheet.

y Cell cultures are also very susceptible to bacterial

contamination and toxic substances in the specimen.y Lastly, many viruses will not grow in cell culture at all

e.g. Hepatitis B and C, Diarrhoeal viruses, parvovirus

etc.


21/31

Serology

y Serology forms the mainstay of viral diagnosis.y This is what happens in a primary humoral immune

response to antigen.

y Following exposure, the first antibody to appear is IgM,

which is followed by a much higher titre of IgG.

y In cases of reinfection, the level of specific IgM either

remain the same or rises slightly.

yBut IgG shoots up rapidly and far more earlier than in aprimary infection.


22/31

y Many different types of serological tests are

available.

yWith some assays such as EIA and RIA, one can

look specifically for IgM or IgG, whereas withother assays such as CFT and HAI, one can only

detect total antibody, which comprises mainly

IgG.


23/31

y Some of these tests are much more sensitive than

others:

yEIAs and radioimmunoassays are the most sensitive

tests available, whereas CFT and HAI tests are not sosensitive.

yNewer techniques such as EIAs offer better

sensitivity, specificity and reproducibility than

classical techniques such as CFT and HAI.


24/31

Criteria fordiagnosing Primary

Infection

yA significant rise in titre of IgG/total antibody

between acute and convalescent sera

yhowever, a significant rise is very difficult to

define and depends greatly on the assay used.


25/31

y Presence of IgM offers a rapid means of diagnosis.

y However, there are many problems with IgM assays,

such as

yinterference by rheumatoid factor

y re-infection by the virus,

y unexplained persistence of IgM years after the

primary infection.

y

y Seroconversion - this is defined as changing from a

previously antibody negative state to a positive state.


26/31

y A single high titre of IgG (or total antibody)

y is a very unreliable means of serological diagnosis since

the cut-off value is very difficult to define.


27/31

Criteria fordiagnosingre-infection/re-

activationy It is often very difficult to differentiate re-infection/re-

activation from a primary infection.

y

y Under most circumstances, it is not important to differentiate b

primary infection and re-infection.y However useful in the diagnosis of rubella infection in the first

trimester of pregnancy

y primary infection is associated with a high risk of fetal

damage whereas re-infection is not.y

y In general, a sharp large rise in antibody titres is found in re-

infection whereas IgM is usually low or absent in cases of re-

infection/re-activation.


28/31


29/31

Limitations of serological diagnosis

yMany viruses often produce clinical disease before the

appearance of antibodies such as respiratory and

diarrhoeal viruses. So in this case, any serological

diagnosis would be retrospective and therefore willnot be that useful.

y

y There are also viruses which produce clinical disease

months or years after seroconversion e.g. HIV andrabies. In the case of these viruses, the mere presence

of antibody is sufficient to make a definitive diagnosis.


30/31


31/31

y Immunocompromised patients often give a reduced

or absent humoral immune response.

y

y Patients with infectious mononucleosis and those

with connective tissue diseases such as SLE may

react non-specifically giving a false positive result

y

y Patients given blood or blood products may give a

false positive result due to the transfer of antibody.

Documents

Virology Laboratory Diagnosis