Multiplex Nucleic Acid Testing Uncovers Hidden Hepatitis B Infections in High-Risk Groups

Screening for HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) has long depended on serological assays that detect antibodies or viral antigens rather than the pathogens themselves. While these tests are inexpensive and widely deployed, they carry a structural blind spot: they cannot reliably identify occult HBV infection, in which the viral surface antigen is absent but viral DNA persists in the blood, nor can they distinguish current, actively replicating HCV infection from antibodies left behind after viral clearance. A new cross-sectional study published in Frontiers in Cellular and Infection Microbiology suggests that a single multiplex nucleic acid test capable of detecting all three viruses simultaneously could close part of that diagnostic gap in the populations that need it most.

The research team, led by investigators from the Chinese Center for Disease Control and Prevention and partner institutions across China, enrolled 1,202 participants: 802 people who use drugs recruited from sentinel surveillance sites in Yunnan Province, and 400 men who have sex with men recruited from a sexually transmitted disease clinic in Beijing. Each participant provided a blood sample that was split for parallel testing. One portion underwent conventional serological screening for HIV antibody, hepatitis B surface antigen, and HCV antibody. The other portion was screened with a commercially approved HIV/HBV/HCV multiplex nucleic acid amplification test that simultaneously detects HIV-1 and HIV-2 RNA, HCV RNA, and HBV DNA in a single reaction.

The analytical logic of the study was deliberately layered. Any sample reactive on initial screening, whether serological or nucleic acid based, was retested independently on orthogonal single-pathogen platforms: the cobas 5800 system for quantitative HBV DNA and HCV RNA, and the Abbott m2000 system for HIV RNA quantification. Samples yielding borderline signals, defined as cycle threshold values above 35, were subjected to an enhanced verification protocol involving repeat extraction, repeat amplification, and orthogonal confirmation. Final infection status was adjudicated by integrating screening results, confirmatory test results, epidemiological history, and clinical symptoms, creating a three-step framework designed to separate true infections from screening artifacts.

The headline finding concerns occult HBV infection. Among the 58 participants ultimately confirmed as HBV-infected, conventional surface antigen screening flagged 46. Adding multiplex NAT to the screening phase surfaced 14 additional reactive samples, of which 12 were confirmed as genuine occult infections on orthogonal retesting; the remaining two proved to be false positives. Critically, these 12 cases would have been missed not only by surface antigen testing alone but also by the reflex strategy in which nucleic acid testing is triggered only after a positive serological result, because the reflex pathway never reaches surface antigen-negative individuals. The incremental yield translates to a number needed to test of approximately 100 to find one additional occult infection.

Characterization of the 12 occult cases revealed a consistent serological signature. All 12 were positive for antibody to the HBV core antigen, a marker of prior exposure that is not part of routine Chinese screening panels. Eight showed isolated core antibody positivity, while four also carried surface antibody, a pattern suggesting resolved infection with residual low-level viremia. Eleven of the 12 had HBV DNA levels below the lower limit of quantification on confirmatory testing, and one measured 60.2 IU/mL. These low-level DNA signals, detectable only by highly sensitive amplification, illustrate why occult infection has historically evaded clinical detection and why it remains a recognized transmission risk in transfusion settings.

For HCV, the value of nucleic acid testing lay in classification rather than in initial detection. Antibody screening identified 307 reactive individuals, but confirmatory RNA testing showed that only 199 carried current, actively viremic infection; 108 were antibody-positive but RNA-negative, indicating past exposure that had either resolved spontaneously or been cured with antiviral therapy. Three additional antibody-reactive samples failed orthogonal confirmation entirely. Because anti-HCV antibodies persist indefinitely after viral clearance, RNA testing is the only way to determine who currently needs treatment, and the study confirmed that this benefit accrues from any RNA testing strategy rather than from the multiplex format specifically.

HIV results told a different story. All 170 confirmed HIV cases were antibody-reactive on initial screening, so multiplex NAT added no incremental diagnostic yield. Yet the combined approach delivered clinically meaningful ancillary information: 91 of the antibody-positive individuals were RNA-positive, indicating incomplete viral suppression despite documented antiretroviral therapy records, while 79 were RNA-negative and confirmed to have successfully suppressed viral loads under treatment. Within a single clinical encounter, clinicians could therefore both rule out active viremia and assess treatment adherence, a capability the authors highlight as an underappreciated advantage of combined screening.

The study also probed whether the technology would be acceptable to the communities it targets. Acceptance was high: 777 of 802 people who use drugs and 368 of 400 men who have sex with men said they were willing to undergo multiplex NAT, citing convenience and a desire to know their health status as primary motivations. Treatment willingness was similarly strong, with more than 96 percent of both cohorts expressing readiness to receive HBV or HCV therapy after diagnosis. The barriers reported were practical rather than clinical: cost, privacy concerns, doubts about accuracy, and limited awareness of combined testing. The authors argue that public health efforts should therefore prioritize subsidized pricing and targeted health education alongside the analytical rollout.

The authors are careful to frame the results as proof of concept rather than a mandate for immediate policy change. Because the reference standard incorporated screening NAT results and confirmatory testing was restricted to screening-reactive samples, the measured sensitivity and negative predictive values are design-dependent and may be overestimated. Infections missed by every assay in the algorithm could not be quantified, and low confirmatory viral loads left open the possibility of transient or assay-edge reactivity in some occult cases. The study also compared two cohorts with different sampling frames and baseline prevalences, and the authors caution that pooled prevalence estimates should not be extrapolated directly to other regions.

Even so, the central insight stands out clearly: the benefit of upfront multiplex NAT derives less from superior analytical sensitivity than from the structure of the screening pathway itself. Routine HBV screening in China relies on surface antigen and does not include core antibody, creating a systematic gap through which occult infections fall. By routing every participant through nucleic acid testing regardless of serological status, the multiplex strategy redirects individuals who would otherwise be invisible to the diagnostic system. The five false-positive nucleic acid screening results observed, two for HBV and three for HCV, were all caught by the standardized retesting protocol and did not lead to misdiagnosis, suggesting that the trade-off in specificity is manageable when orthogonal confirmation is enforced. The authors conclude that multicenter prospective implementation studies and formal cost-effectiveness analyses are needed before the approach can be integrated into routine clinical practice, but the direction of travel is evident: for populations bearing the heaviest co-infection burden, the screening algorithm itself may be the most modifiable target for improving case detection.

Subject of Research: Evaluation of multiplex HIV, HBV, and HCV nucleic acid testing for detecting occult and active infections in high-risk populations in China

Article Title: Enhanced detection of occult and active infections using multiplex HIV/HBV/HCV nucleic acid testing in high-risk populations: a cross-sectional study

Article References: Jin, C., Wang, Y., Zhang, Z., Niu, M., Wang, J., Chen, H., Yuan, H., Liu, N., Yao, E., Li, M., Zhang, Y., Feng, X., Shi, Y., & Lu, K. (2026). Enhanced detection of occult and active infections using multiplex HIV/HBV/HCV nucleic acid testing in high-risk populations: a cross-sectional study. Frontiers in Cellular and Infection Microbiology, 16, Article 1870687. https://doi.org/10.3389/fcimb.2026.1870687

Image Credits: AI Generated

DOI: 10.3389/fcimb.2026.1870687

Keywords: HIV, hepatitis B virus, hepatitis C virus, occult HBV infection, nucleic acid testing, multiplex assay, serological screening, co-infection, people who use drugs, men who have sex with men, diagnostic yield, China