Describe the laboratory diagnosis of viral infections including specimens, microscopy, serology and molecular methods.

Approaches to Diagnosis

Specimens

• Collect early — virus shedding is usually greatest in the first few days of illness and falls rapidly, so a late specimen is often negative
• Respiratory — nasopharyngeal swab or aspirate, throat swab, bronchoalveolar lavage; Enteric — stool and rectal swab; Vesicular rash — vesicle fluid and the scraped base of the lesion; Neurological — cerebrospinal fluid, and brain tissue post mortem; Blood — for serology, antigen and nucleic acid
• Viral transport medium is required for swabs — a buffered balanced salt solution with protein (albumin or gelatin) to stabilise the virus and antibiotics to suppress bacterial and fungal overgrowth
• Transport on ice at 4°C for prompt processing, or at −70°C for prolonged storage; never at −20°C, which damages viruses more than either
• Paired sera — acute and convalescent samples 10 to 14 days apart, stored and tested together in the same run

Microscopy and Inclusion Bodies

• Electron microscopy — can visualise the virion directly and identify it by morphology; useful in vesicular rashes and in diarrhoea (rotavirus, norovirus). It requires about 106 particles per mL and is available in few centres
• Immunoelectron microscopy uses antibody to clump the particles, raising sensitivity
• Inclusion bodies — aggregates of virus or of altered cell material, seen by light microscopy and characteristic enough in some infections to be diagnostic

Cultivation, Serology and Molecular Methods

• Cell culture — growth is recognised by cytopathic effect (CPE) (rounding, giant cell formation, inclusion bodies, detachment), by haemadsorption (red cells adhering to infected cells expressing haemagglutinin), by interference, or by immunofluorescence
• Serological tests — ELISA is the workhorse; also haemagglutination inhibition, complement fixation, neutralisation (the most specific) and immunofluorescence
• Interpreting serology — IgM means recent infection; a fourfold rise in IgG titre between paired sera is diagnostic; IgG avidity distinguishes recent from past infection, which is particularly useful in toxoplasma and rubella in pregnancy
• Molecular methods — PCR, reverse-transcriptase PCR for RNA viruses, real-time PCR for viral load (essential in HIV, hepatitis B and C monitoring), multiplex panels, and sequencing for genotyping and resistance
• Limitations of molecular tests — they detect nucleic acid, not viable virus, so they may stay positive after recovery; and they are exquisitely vulnerable to contamination, requiring separated work areas and controls

Matching the Test to the Illness

Applied Aspects

• Choose the test to match the stage of illness — antigen and nucleic acid early, antibody later; testing for IgM on day two of a dengue illness or requesting a culture in convalescence both waste the specimen
• Tell the laboratory which virus is suspected; virology is not a single test, and the specimen, medium and assay differ completely between viruses
• A single positive IgG means past exposure, not current disease, which is a persistent source of misdiagnosis and of unnecessary treatment
• Serology is unreliable in the immunosuppressed, who may not mount an antibody response; antigen or nucleic acid detection is required
• Rapid point-of-care tests have transformed practice in India — dengue NS1 and IgM, HIV, hepatitis B surface antigen and influenza can all be tested without a virology laboratory
• Handle specimens as infectious and observe biosafety; hepatitis B and HIV are transmitted to laboratory and clinical staff by needlestick, and some viral agents require BSL-3 or BSL-4 containment
• Collect the specimen in the first few days; viral shedding falls rapidly, and a swab taken in the second week of illness is frequently negative in a patient who certainly has the infection
• Use viral transport medium and never plain saline for swabs; the protein stabiliser and antibiotics in the medium are what allow the virus to survive transport
• A rising titre requires paired sera tested together in the same run, since inter-assay variation can otherwise create or conceal a fourfold change
• PCR positivity does not prove infectivity; nucleic acid persists after viable virus has gone, which matters when deciding how long to isolate a patient
• Avidity testing distinguishes primary from past infection in pregnancy, which is the distinction that determines fetal risk in toxoplasma, rubella and cytomegalovirus
• Rapid tests have brought virology to the bedside in India, and their availability at district level changes management far more than the marginally higher sensitivity of a reference laboratory assay
• Electron microscopy needs about a million particles per mL, so it is useful in diarrhoeal stool and vesicle fluid where virus is abundant, and useless in blood or cerebrospinal fluid