What is the most appropriate combination of NAT, IgG, and IgM tests for diagnosing HHV6 infection?

HHV-6 Diagnostic Testing: NAT vs IgG and IgM

Quantitative PCR (NAT) is the mainstay and recommended test for diagnosing HHV-6 infection, while antibody tests (IgG and IgM) are not indicated in immunocompromised patients and have limited utility in immunocompetent individuals. 1

Primary Recommendation: Use NAT (PCR) Testing

Quantitative PCR that distinguishes between HHV-6A and HHV-6B DNA is the recommended diagnostic approach for HHV-6 infection. 1 This represents the European Conference on Infections in Leukaemia consensus guideline from 2019.

Why NAT is Superior:

• PCR is the mainstay of HHV-6 diagnosis and directly detects viral DNA in peripheral blood, providing objective evidence of viral presence 1
• NAT can quantify viral load, allowing for longitudinal monitoring and assessment of disease activity 1
• PCR can differentiate between HHV-6A and HHV-6B, which antibody tests cannot 1
• A WHO standard for HHV-6B DNA is now available, improving standardization across laboratories 1

Limitations of Antibody Testing (IgG and IgM)

Antibody tests cannot distinguish between HHV-6A and HHV-6B and are not indicated in HSCT patients. 1

Specific Problems with Serologic Testing:

• Severely immunocompromised patients may not develop antibodies despite active infection, making serology unreliable in transplant recipients 1
• IgM has low accuracy (76.1% sensitivity, 87.5% specificity) for detecting primary HHV-6 infection in children 2
• IgM can be present in both primary infection AND reactivation, limiting its ability to distinguish between these scenarios 3
• IgM responses may be detected at very low titers and can be transient or absent even in confirmed infections 4
• Viral isolation remains the only truly reliable method for diagnosing active infection when serologic and PCR results are ambiguous 5

Clinical Algorithm for HHV-6 Testing

Step 1: Initial Testing

• Order quantitative PCR for HHV-6 DNA on peripheral blood as the first-line test 1
• Ensure the assay differentiates HHV-6A from HHV-6B 1

Step 2: Serial Monitoring

• Use the same DNA extraction method, quantitative PCR assay, and specimen type for all repeat testing in a given patient 1
• This ensures accurate comparison of viral load trends over time 1

Step 3: Rule Out Chromosomally Integrated HHV-6 (CIHHV-6)

• Suspect CIHHV-6 if persistently high HHV-6 DNA levels (>5.5 log10 copies/mL) are detected in whole blood 1
• CIHHV-6 shows characteristic patterns: serum levels are 100-fold lower than whole blood in the same patient 1
• If CIHHV-6 is suspected, confirm with droplet digital PCR showing a ratio of one copy HHV-6 DNA per cellular genome 1
• This distinction is critical to avoid unnecessary antiviral therapy 1

Step 4: Consider Additional Testing Only in Specific Scenarios

• Viral culture remains the gold standard but is specialized and labor-intensive 1
• RT-PCR for viral mRNA can confirm active virus replication but lacks standardization 1
• IgG avidity testing may help distinguish primary from recurrent infection in immunocompetent patients, with low avidity indicating primary infection and high avidity indicating past infection 6

Critical Pitfalls to Avoid

Do Not Rely on Serology Alone

• Never use IgM or IgG alone to diagnose HHV-6 infection, especially in immunocompromised patients 1
• Serology may be misleading as it cannot distinguish latent from actively replicating virus 5

Do Not Misinterpret Positive PCR in CIHHV-6

• High-level HHV-6 DNA detection does not always indicate active infection—it may represent chromosomally integrated virus 1
• CIHHV-6 occurs in approximately 1% of the population and will show persistently elevated DNA levels that do not respond to antivirals 1

Do Not Use Different Assays for Serial Monitoring

• Switching PCR assays or specimen types between tests makes viral load trends uninterpretable 1
• Agreement between laboratories for HHV-6 DNA levels is poor, so consistency is essential 1

Special Population Considerations

In Hematopoietic Stem Cell Transplant Recipients:

• PCR is essential because these patients may not mount antibody responses 1
• Donor-derived CIHHV-6 must be excluded by testing pre-transplant samples 1
• HHV-6 DNA will increase post-HSCT in parallel with leukocyte engraftment if the donor has CIHHV-6 1

In Immunocompetent Adults and Children:

• IgG avidity testing may add value when combined with PCR to distinguish primary from recurrent infection 6
• However, PCR remains the primary diagnostic modality 1