Can cross‑reactivity between hepatitis B surface antigen (HBsAg) and syphilis cause a false‑positive HBsAg result, and how should it be confirmed?

Cross-Reactivity Between HBsAg and Syphilis Testing

False-positive HBsAg results do not occur due to cross-reactivity with syphilis antibodies, but syphilis-positive patients have higher rates of true HBV coinfection that must be distinguished from false positives through confirmatory neutralization testing.

Understanding the Relationship

The evidence does not support immunologic cross-reactivity between HBsAg assays and syphilis antibodies. However, there is an important epidemiologic association:

• Syphilis-positive patients have significantly higher rates of true HBV infection (5.2% vs 1.4% in blood donors) 1, with occult HBV coinfection rates as high as 5.1% in syphilis-positive blood donors 2
• This reflects shared sexual transmission routes, not laboratory cross-reactivity
• Among females with syphilis, HBsAg positivity reaches 7.2% compared to 3.5% in controls 3

Causes of False-Positive HBsAg Results

False-positive HBsAg results occur from specific technical and clinical factors, not syphilis:

According to the most recent EASL guidelines 4, false-positive HBsAg results can occur in:

• Patients on hemodialysis
• Post-mortem organ donors
• Individuals with heterophilic antibodies
• Patients receiving G-CSF
• Technical issues (e.g., dislodged separator gel from collection tubes) 5

Notably absent from this list is any mention of syphilis or treponemal antibodies causing false-positive HBsAg results.

Confirmatory Testing Algorithm

When you encounter a positive HBsAg result, especially with low signal strength (COI <6.0), perform neutralization testing with anti-HBs:

1. For COI values between 0.9-6.0: Mandatory confirmatory neutralization test 4, 6

   • Use manufacturer-recommended anti-HBs neutralization
   • Sample is confirmed positive if COI after neutralization ≤60% of control-treated COI
   • Specificity reaches 100% at COI ≥6.0 6

2. For equivocal neutralization results: Add supplementary testing 4

   • Anti-HBc testing (most reliable marker)
   • HBV DNA quantification by NAT
   • Sequential HBsAg measurements over time

3. Second-step neutralization is superior to first-step for high-titer samples, as it doesn't require dilution 7

Clinical Pitfalls to Avoid

Do not assume a positive HBsAg in a syphilis patient is automatically false-positive:

• The higher prevalence of true HBV coinfection in syphilis patients (5.1-7.2%) means most positive results are genuine 3, 1, 2
• Occult HBV infections are particularly common in this population, with anti-HBc positivity reaching 56.5% in syphilis-positive donors 2
• Some coinfected patients may show unusual serologic patterns, including HBsAg/anti-HBs coexistence (89% confirmed true coexistence) 8

Watch for these specific scenarios:

• Low COI values (1.0-6.0) warrant immediate confirmatory testing 6
• Visual inspection of serum is essential—unusual appearance may indicate preanalytical errors 5
• In sexually transmitted infection clinics, assume higher pretest probability of true HBV infection

Biological False Positives in Syphilis Testing

The syphilis literature addresses biological false positives (BFPs) in nontreponemal tests (RPR/VDRL), occurring in <0.85% of tested individuals 9. However, there is no reciprocal documentation of syphilis antibodies causing false-positive HBsAg results.

The prozone phenomenon in syphilis testing (false-negative due to antibody excess) is unrelated to HBsAg testing 9.