Community-Based Double Reflex Screening Reveals Widespread Hepatitis D Viremia Among Hepatitis B Surface Antigen Carriers in Pakistan.

Background Hepatitis D, caused by the hepatitis delta virus (HDV), poses a significant public health concern in many parts of the world. However, HDV screening remains suboptimal in most regions, leading to underdiagnosis of the infection. This study aimed to determine the prevalence of HDV infection through double reflex testing of hepatitis B surface antigen (HBsAg)-positive individuals at screening camps in two high-prevalence towns in the eastern part of Balochistan province, Pakistan. Methods A total of 1,643 individuals were screened for HBsAg and antibodies to the hepatitis C virus (anti-HCV). Those who tested positive for HBsAg were further tested for antibodies to HDV (anti-HDV). Samples positive for anti-HDV were subsequently analyzed for HDV RNA using real-time PCR. Results Of the 1,643 individuals screened, 277 (16.9%) tested positive for HBsAg and 227 (13.8%) for anti-HCV. Among the HBsAg-positive individuals, 186 (67.1%) were positive for anti-HDV antibodies, including 133 men and 53 women, with a mean age of 32.2 ± 11.3 years. HDV RNA was detectable in 108 (58.1%) of the anti-HDV-positive individuals, with mean HDV RNA levels of log₁₀ 6.69 ± 1.35 IU/mL. Of the 277 HBsAg-positive individuals, five also tested positive for anti-HCV; one of these was positive for anti-HDV but negative for HDV RNA. Conclusions Reflex testing of HBsAg-positive individuals in this HDV-endemic area showed that 67% had been exposed to HDV, and 58.1% of those with anti-HDV antibodies had active viremia. These findings underscore the importance of implementing double reflex testing - screening HBsAg-positive individuals for anti-HDV antibodies, followed by HDV RNA testing in those who are anti-HDV positive - in regions with high HDV prevalence.