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lab diagnosis of viral infection

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viral infections AND diagnosis

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CDC laboratory testing viral infections PCR antigen antibody diagnosis

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Laboratory diagnosis of viral infections

Diagnosis is based on the appropriate clinical specimen, collected at the right time, followed by direct detection of the virus or indirect detection of the host immune response.

1. Specimen collection

The sample depends on the site of infection:
Clinical syndromeCommon specimens
Respiratory infectionNasopharyngeal/throat swab, nasal aspirate, sputum
CNS infectionCSF, sometimes serum
GastroenteritisStool
HepatitisSerum/plasma
Herpes lesionsVesicle fluid, lesion swab, scraping
Congenital infectionAmniotic fluid, neonatal blood/urine/saliva
Systemic or blood-borne infectionBlood, serum, plasma
Tissue disease or fatal infectionBiopsy/autopsy tissue
Collect samples as early as possible, ideally during the acute phase when viral load is highest. Use proper viral transport medium and cold-chain transport where needed.

2. Direct detection of virus

A. Nucleic acid amplification tests

PCR, RT-PCR, real-time PCR, multiplex PCR detect viral DNA or RNA.
  • Most important and commonly used method
  • Highly sensitive, specific, and rapid
  • Can identify and quantify viral load
  • Useful for viruses that are difficult or slow to culture
  • Examples: SARS-CoV-2, influenza, HIV, hepatitis B/C, HSV in CSF, CMV
A positive PCR generally supports current infection, but results must be interpreted with symptoms, specimen quality, and timing. Molecular methods have largely replaced viral culture for many viruses. Henry's Clinical Diagnosis and Management by Laboratory Methods, p. 1463.

B. Antigen detection

Detects viral proteins in the specimen by:
  • ELISA
  • Immunofluorescence assay
  • Rapid immunochromatographic tests
  • Immunohistochemistry in tissue
Examples:
  • Influenza and SARS-CoV-2 rapid antigen tests
  • Rotavirus antigen in stool
  • Hepatitis B surface antigen (HBsAg)
  • RSV antigen in respiratory samples
These tests are rapid and useful at the point of care, but are generally less sensitive than NAATs. The CDC respiratory-testing guidance notes that antigen-negative results can occur despite infection, particularly when viral circulation is high.

C. Virus isolation in cell culture

The specimen is inoculated into susceptible cell lines and observed for cytopathic effect (CPE), such as cell rounding, syncytium formation, or inclusion bodies.
  • Confirms viable virus
  • Useful for epidemiology, antiviral susceptibility testing, and research
  • Slow, costly, and requires specialized facilities
  • Now used less often for routine diagnosis

D. Electron microscopy

Can demonstrate virus particles directly in stool, vesicle fluid, or tissue.
  • Rapid but insensitive
  • Requires a high viral load and expensive equipment
  • Mainly used in specialist/reference laboratories

E. Cytology and histopathology

Characteristic inclusions or cellular changes may suggest viral infection:
  • Tzanck smear: multinucleated giant cells in HSV or VZV lesions
  • Negri bodies: rabies
  • Owl-eye inclusions: CMV
  • Koilocytes: HPV
  • Immunohistochemistry can detect viral antigen in tissue.

3. Indirect diagnosis: serology

Serology detects virus-specific antibodies in serum.
MarkerInterpretation
IgM antibodyUsually indicates recent or acute infection
IgG antibodyPast infection, immunity, or later phase of current infection
Fourfold rise in IgG titre in paired seraEvidence of recent infection
Neutralizing antibody testMay confirm specific infection, especially when cross-reactivity is an issue
Paired sera are collected during:
  • Acute phase: early in illness
  • Convalescent phase: about 2 to 4 weeks later
A significant rise in IgG titre or detection of virus-specific IgM supports acute infection. Serology is especially helpful when viremia is brief, when the patient presents late, or when molecular testing is unavailable. Henry's Clinical Diagnosis and Management by Laboratory Methods, p. 1463.
Limitations of serology
  • Antibodies may not yet be present early in infection.
  • IgM can persist after recovery.
  • Cross-reactivity may occur, especially among related viruses such as flaviviruses.
  • Immunocompromised patients may not mount an adequate antibody response.
For example, CDC notes that West Nile virus IgM may persist for months and can cross-react with other flaviviruses, so confirmatory neutralization testing may be needed in selected cases (CDC West Nile diagnosis guidance).

4. Interpretation and reporting

Laboratory results should always be correlated with:
  • Clinical presentation
  • Day of illness
  • Type and quality of specimen
  • Vaccination history
  • Immune status
  • Epidemiologic exposure
A negative antigen test does not necessarily exclude infection. A negative PCR may occur if the specimen is taken too late, collected poorly, or tested from the wrong anatomical site.

Short summary

The main diagnostic methods are:
  1. NAAT/PCR - preferred method for most acute viral infections
  2. Antigen detection - rapid but less sensitive
  3. Viral culture - slow, specialized, now limited in routine use
  4. Microscopy/cytology/histopathology - supportive or specialized
  5. Serology - detects IgM, IgG, or rising antibody titres, especially useful for late presentation or retrospective diagnosis

Mechanism of antimicrobial resistance

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antimicrobial resistance mechanisms

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WHO mechanisms antimicrobial resistance bacteria enzyme inactivation target modification efflux reduced permeability

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Mechanisms of antimicrobial resistance

Antimicrobial resistance (AMR) occurs when a microorganism survives or grows despite an antimicrobial concentration that would normally inhibit or kill it.
Resistance may be:
  • Intrinsic (natural): an inherent property of a species. Example: Gram-negative bacteria are naturally resistant to vancomycin because the drug cannot adequately cross the outer membrane.
  • Acquired: develops through mutation or acquisition of resistance genes from other organisms.

How resistance genes arise and spread

  1. Spontaneous chromosomal mutation
    Random mutations may alter a drug target, reduce drug uptake, or increase efflux-pump activity. Antibiotic exposure selects the resistant mutants.
  2. Horizontal gene transfer
    Resistance genes spread between bacteria by:
    • Conjugation: plasmid transfer through cell-to-cell contact
    • Transformation: uptake of naked DNA from the environment
    • Transduction: bacteriophage-mediated transfer
    • Transposons/integrons: mobile genetic elements that carry and assemble resistance genes

Major biochemical mechanisms

1. Enzymatic inactivation or modification of the drug

Bacteria produce enzymes that destroy the drug or chemically modify it so it no longer works.
Examples
  • Beta-lactamases hydrolyse the beta-lactam ring of penicillins, cephalosporins, and sometimes carbapenems.
    • ESBLs inactivate many third-generation cephalosporins.
    • Carbapenemases inactivate carbapenems.
  • Aminoglycoside-modifying enzymes acetylate, phosphorylate, or adenylate aminoglycosides.
  • Chloramphenicol acetyltransferase inactivates chloramphenicol.

2. Alteration of the drug target

The microbial target is changed, so the antimicrobial cannot bind effectively.
Examples
  • Altered penicillin-binding proteins (PBPs) cause methicillin resistance in Staphylococcus aureus (MRSA), via PBP2a encoded by mecA.
  • Mutation in DNA gyrase or topoisomerase IV causes fluoroquinolone resistance.
  • Methylation of 23S rRNA causes resistance to macrolides, lincosamides, and streptogramin B antibiotics.
  • Replacement of D-Ala-D-Ala with D-Ala-D-Lac in peptidoglycan precursors causes vancomycin resistance in enterococci.
  • Altered dihydropteroate synthase or dihydrofolate reductase can cause sulfonamide or trimethoprim resistance.

3. Reduced permeability or reduced drug uptake

The organism prevents enough drug from reaching its intracellular target.
Examples
  • Loss or alteration of porins in Gram-negative bacteria reduces entry of beta-lactams, quinolones, and carbapenems.
  • The outer membrane of Gram-negative bacteria itself limits entry of several drugs.
  • Altered transport systems can reduce uptake of fosfomycin or aminoglycosides.

4. Active efflux of the drug

Efflux pumps actively transport the antimicrobial out of the cell, keeping intracellular drug levels below effective concentrations.
Examples
  • Tetracycline resistance through efflux pumps
  • Fluoroquinolone resistance through multidrug efflux pumps
  • Macrolide resistance through active efflux
An efflux system may remove more than one class of drug and therefore contributes to multidrug resistance.

5. Bypass of the inhibited metabolic pathway

The organism bypasses the drug-blocked step by using an alternative enzyme or pathway.
Examples
  • Acquisition of an alternative dihydrofolate reductase causes trimethoprim resistance.
  • Acquisition of an alternative dihydropteroate synthase causes sulfonamide resistance.
  • Some bacteria can use preformed folate, bypassing the pathway blocked by sulfonamides.

6. Overproduction of the target or enzyme

The bacterium produces excess target molecule or metabolic enzyme, so normal drug concentrations cannot inhibit all of it.
Example: increased production of dihydrofolate reductase may reduce trimethoprim effectiveness.

7. Biofilm formation and persister cells

Bacteria in a biofilm are embedded in an extracellular matrix and may grow slowly. Drug penetration is reduced and cells can become metabolically inactive, making them tolerant to antibiotics.
Common examples include infections involving:
  • Urinary catheters
  • Prosthetic valves or joints
  • Endotracheal tubes
  • Chronic wounds
This often represents antimicrobial tolerance rather than stable genetic resistance, but it is clinically important.

Quick mnemonic: DART-E

  • Drug destruction or modification
  • Altered target
  • Reduced permeability
  • Transport out by efflux pumps
  • Evasion or bypass of metabolic pathway
The four central molecular mechanisms are reduced uptake, active efflux, target modification, and enzymatic drug inactivation. GOLDMAN-CECIL Medicine, mechanisms of multidrug resistance section. Lippincott also describes target alteration, reduced penetration, efflux, and antimicrobial-inactivating enzymes as the main mechanisms. Lippincott Illustrated Reviews: Pharmacology, p. 926.
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