Laboratory Diagnosis Of Viral Infections

Lab Diagnosis of Viral Diseases

S. Saengamnatdej, PhD. for 499305 (2009/2) April 25, 2009

Goals Specimen Processing Collection of Specimens Methods

Goals 1. To confirm the diagnosis (acute or chronic infection). 2. To determine appropriate antiviral therapy. 3. To define the course of disease. 4. To determine the immune status. 5. To establish public health measures to prevent the spread. 6. To study the evolution & epidemiology of viral infections.

Specimen Flow Diagram Request

Report

Transport

Collect

Macroscopic & Microscopic Examination

Antigen detection; nucleic acid probes

Cultivation, isolation

Initial (via phone)

Preliminary (same day) Preliminary Identification (day after receipt) viruses, fungi &some bacteria may take longer time.

Identification; antimicrobial sensitivity testing

Slack, R.C.B. (2006)

Further identification; typing; toxin production; further sensitivities

Final (2nd day)

Supplementary

Collection of Specimens - Avoid contamination (commensals or external sources)

- Use appropriate methods. - In a transport-durable, leak-proof, sterile container. - Use viral transport medium containing albumin and suitable antibiotics.

Specimens - Sent with a request card. (providing sufficient information.)

- Labeled correctly. - Kept separately from food and drug. - Transported on ice (4 ºC) without delay. (frozen, if undelivered for weeks.)

- Follow safety regulation. (each step with a responsible personnel and minimize handling by untrained people.)

Methods of Collection Eye

Conjunctival swab

Respiratory secretions

Nasopharyngeal swab, Throat (pharyngeal) swab, Nasal washing,

Adenovirus, Influenza, Enterovirus, Rhinovirus, Paramyxovirus, Rubella, HSV Nasal swab (ex: meningitis during summer—enterovirus)

GI specimens Reovirus, Rotavirus, Adenovirus, Norwalk, Calicivirus

Vomitus, Stool, Rectal swab (meningitis during summer— enterovirus)

Methods of Specimen Collection (Cont.) CNS

CSF (ex: CNS disease after parotitis—mumps or meningitis during summer—enterovirus)

Brain biopsy: rabies Genital

Ulcer scrape

Blood

Serum antigen, Serum antibody

HIV, HTLV, HBV, HCV, HDV

Urine Rubella, Measles, Adenovirus, CMV, Mumps (ex: CNS disease after parotitis, Maculopapular rash)

Timing : Viral shedding For examples, Respiratory viruses shed for only 3-7 days HSV & VZV from lesions: more than 5 days after the onset. Enteroviruses from CSF , only 2-3 days after the onset.

1.Detect viral components Proteins Nucleic acids

2.Serology

Methods

for hard-to-isolate&grow, slow-disease causing v.

EBV, Rubella, Hepatitis A-E, HIV, HTLV, Arbovirus

3.Cytology CPEs

4.Cultivation & Isolation Animals/ Embryonated eggs/ Organ culture / Tissue culture (primary, diploid, continuous cell lines) CPEs, plaques, interference, hemadsorption

5.Electron Microscopy

Notes on Serology Methods.

To indicate seroconversion, the antibody titre should increase at least fourfold.

Why fourfold?

Because twofold increase in the result may be not correct due to twofold serial dilutions in the serologic assays have the imprecision nature. Let's take these figures as example, “samples with 512 and 1023 units of antibody would both give a signal on a 512-fold dilution but not on a 1024-fold dilution, and the titers of both would be reported as 512. On the other hand, samples with 1020 and 1030 units are not significantly different but would be reported as titers of 512 and 1024, respectively.” (Murray, P.K. et al.,2005)

Notes on Serology Methods.(2)

How does serology help to define the stage of diseases? The antibody against the intracellular viral component indicates the late stage of the infection. For example, the antibody to EBV-nuclear antigen is detected late in the infection.

(Murray, P.K. et al.,2005)