What conditions can cause false viral positivities?

Conditions Causing False Viral Positivities

False-positive viral test results are primarily caused by sample cross-contamination in molecular testing and cross-reactivity with other pathogens in serological testing, with specific triggers including recent vaccinations, other viral infections, and structural similarities between viral proteins. 1

RT-PCR/Molecular Testing False Positives

Primary Cause: Sample Cross-Contamination

• Sample cross-contamination is the most common cause of false-positive RT-PCR results, occurring during specimen collection, laboratory processing, or from amplicon carryover from previous testing runs 1
• Environmental contamination at specimen collection sites can introduce viral RNA, particularly when influenza vaccines are administered in the same location where respiratory samples are collected 2
• Vaccine RNA can remain detectable in the environment for at least 66 days after administration, potentially contaminating clinical specimens 2

Vaccine-Related Contamination

• Administration of influenza vaccines in the same room as clinical sampling has resulted in false-positive PCR results showing multiple simultaneous viral strains (H1N1, H3N2, and influenza B) 2
• Trivalent influenza vaccines contain high loads of detectable influenza A and B RNA that can contaminate specimens 2

Important Caveats for Molecular Testing

• Detection of viral RNA does not prove viral viability—only viral culture can confirm infectious virus 1
• Timing of sampling, viral load quantification, and detection method all affect interpretation of positive results 1
• Patients recovered from COVID-19 may continue testing positive by RT-PCR despite no longer being infectious 1

Serological Testing False Positives

Cross-Reactivity Between Pathogens

Flavivirus Cross-Reactivity:

• False-positive dengue antibody results occur due to cross-reactivity with other flaviviruses including West Nile virus, Zika virus, St. Louis encephalitis virus, yellow fever virus, and Japanese encephalitis virus 1, 3, 4
• Prior flavivirus infection or vaccination (particularly yellow fever vaccine) causes false-positive results for dengue IgM and IgG antibodies 1
• Plaque reduction neutralization tests (PRNTs) are required to definitively distinguish between cross-reactive flavivirus antibodies 1, 3

HIV False Positives:

• SARS-CoV-2 infection can cause false-positive HIV screening tests due to structural similarities between SARS-CoV-2 spike protein and HIV-1 gp41 protein 5, 6
• Epstein-Barr virus (EBV) infection causes false-positive HIV immunoassay results 1, 5
• Recent Tdap vaccination (within 7 days) can trigger false-positive HIV-1/2 antigen-antibody screening and HIV-1 antibody differentiation immunoassays, with rapid seroreversion to negative within days 7
• Influenza vaccination has been documented to cause false-positive HIV results 5

EBV and Dengue Cross-Reactivity:

• EBV infection can cause false-positive dengue IgM antibody results through nonspecific reactivity 4
• With decreased disease prevalence, a higher proportion of positive IgM tests may represent cross-reactivity with other pathogens like EBV rather than true infection 4

Other Serological False Positives

EBV Heterophile Antibody Testing:

• False-positive heterophile antibody (Monospot) tests occur in patients with leukemia, pancreatic carcinoma, viral hepatitis, and CMV infection 1
• False-negative heterophile tests occur in approximately 10% of patients, especially children younger than 10 years 1

West Nile Virus:

• False-positive anti-WNV IgM and IgG antibodies occur following yellow fever vaccination or natural infection with other flaviviruses (dengue, St. Louis encephalitis viruses) 1
• Traumatic lumbar puncture or defective blood-brain barrier permeability can lead to falsely elevated IgM levels in CSF 1

Hepatitis A:

• False-positive HAV IgM antibody results occur due to low positive predictive value in populations with low prevalence of acute hepatitis A 1

Clinical Algorithm for Managing Suspected False Positives

When to Suspect False-Positive Results:

1. Multiple simultaneous viral strains detected on single PCR test (suggests environmental contamination) 2
2. Positive screening test with negative confirmatory molecular test (HIV RNA PCR, viral load) 5, 7, 6
3. Recent vaccination within 7-66 days of testing (influenza, Tdap, yellow fever) 7, 2
4. Positive antibody test in low-prevalence population 1
5. Clinical presentation inconsistent with positive test result 5, 6

Confirmatory Testing Strategy:

For Molecular (PCR) False Positives:

• Repeat testing with fresh specimen collected at different location 2
• Review specimen collection procedures and environmental controls 1, 2
• Consider viral culture to confirm viability if clinically indicated 1

For Serological False Positives:

• Perform more specific confirmatory testing: HIV RNA PCR for HIV, PRNT for flaviviruses 1, 4, 6
• Collect convalescent serum 7-10 days after initial specimen to assess for seroconversion 1
• Use comprehensive antibody panels (e.g., VCA IgM, VCA IgG, EBNA for EBV) rather than single tests 1, 4
• For dengue: use NAAT during first 7 days of symptoms rather than relying on serology 3, 4

Critical Pitfalls to Avoid:

• Never rely on single serological test for definitive diagnosis when cross-reactivity is possible 4
• Do not assume acute infection based on positive IgM alone, as IgM can persist for months after initial infection 1, 3, 4
• Do not initiate treatment based solely on screening test without confirmatory testing when clinical suspicion is low 7, 6
• Consider timing of recent vaccinations before interpreting positive results 7, 2