Double reflex testing for anti-HDV antibody following an HBsAg-positive test result and HDV RNA in those anti-HDV positive: a global systematic review and meta-analysis.

Background: Chronic hepatitis D (CHD) coinfection leads to accelerated progression to liver cirrhosis, hepatocellular carcinoma, and increased mortality. Currently, only a small proportion of individuals who are HBsAg positive are tested for anti-HDV antibodies, and among those who test positive, few receive confirmatory HDV RNA testing. Reflex testing, whereby laboratory-based anti-HDV testing is automatically triggered with a positive HBsAg test result in the lab, and also HDV RNA testing in those with a positive anti-HDV result, is one approach to promote the uptake and earlier diagnosis of HDV coinfection. We undertook a systematic review and meta-analysis to evaluate the effectiveness of a laboratory-based reflex testing strategy for both anti-HDV and HDV RNA on uptake of testing and linkage to care and turnaround times across the care cascade. Methods: We searched five databases (PubMed, Scopus, Embase, Cochrane, and Global Health (EBSCOhost)) and conference abstracts for relevant studies from database inception through June 2025, with or without a non-reflex comparator arm, and that had data on at least one step in the care cascade (uptake of HDV antibody and HDV RNA testing, linkage to care, and treatment initiation). Summary estimates were calculated using random-effects meta-analyses with 95% confidence intervals (CIs). The strength of evidence was assessed using the GRADE framework. The protocol was registered with PROSPERO (CRD42023397577). Findings: Of 3160 studies identified in the search, 28 were eligible for inclusion, of which 9 also had a non-reflex comparison arm. The majority (82.1%) were conducted in high-income countries. Among the 28 studies that used a reflex testing, 97.1% (95% CI: 93.9-98.7) of HBsAg-positive individuals had anti-HDV testing, and 7.2% (95% CI: 5.8-8.9) were anti-HDV positive. Of these, 95.3% (95% CI: 90.1-97.8) had HDV RNA testing, and 43.2% (95% CI: 33.2-53.8) were RNA positive. In the nine studies with a non-reflex comparison arm, reflex testing significantly increased anti-HDV testing uptake (98.0% versus 39.7%; RR 2.55, 95% CI: 1.62-4.05). And in the six studies with a comparator arm that also examined HDV RNA testing, uptake was higher with reflex testing but this was not statistically significant (94.7% versus 79.9%; RR 1.02, 95% CI: 0.86-1.20). Nine studies reported turnaround times for reflex testing from HBsAg testing to anti-HDV reflex testing, and all were completed within the same day. Four studies also reported a same-day turnaround for HDV RNA testing. Linkage to care was reported in only three of the reflex testing studies, and all 6 HDV RNA-positive individuals were referred for care. Interpretation: Laboratory-based reflex testing significantly increased the uptake of anti-HDV antibody and HDV RNA testing across diverse populations and settings, with same day turnaround times. The 2024 World Health Organization guidelines for hepatitis B care and treatment now recommend reflex HDV testing as a scalable approach to improve HDV testing uptake, and linkage to care for surveillance for liver cancer, and access to new treatments for Delta coinfection. Funding: The Guangzhou Science and Technology Project; The National Key R& D Program; National Institutes of Health; The University of North Carolina Center for AIDS Research; World Health Organization.