One Health investigation of anthrax outbreaks in Uganda, 2022–2024: real-time PCR detection of Bacillus anthracis virulence-associated targets

Anthrax remains an important zoonotic disease in Uganda, where recurrent outbreaks affect livestock-dependent communities and require coordinated human, animal, environmental, and laboratory response. Although real-time polymerase chain reaction (PCR) is increasingly used for outbreak confirmation, multi-year evidence on the distribution and concordance of virulence-associated molecular targets among outbreak-investigation specimens remains limited. This study characterised detection of the pagA and capC targets among specimens generated during One Health anthrax outbreak investigations in Uganda between 2022 and 2024. A retrospective descriptive analysis of secondary epidemiological and laboratory data from 194 specimens collected opportunistically during anthrax outbreak investigations was conducted. Real-time PCR assays targeted pagA, associated with plasmid pXO1, and capC, associated with plasmid pXO2. The primary outcome was dual-target positivity, defined as detection of both targets in the same specimen. Descriptive analyses characterised target detection and positivity patterns. Exploratory univariable modified Poisson regression with robust standard errors estimated crude prevalence ratios (CPRs) and 95% confidence intervals. Of the 194 specimens tested, 31 (16.0%) were positive for pagA and 29 (14.9%) for capC. Twenty-nine specimens (14.9%) were positive for both targets and met the dual-target positivity criterion. Two specimens were pagA-positive/capC-negative, while none were pagA-negative/capC-positive. Annual dual-target positivity was 33.3% in 2022, 11.8% in 2023, and 13.4% in 2024. No statistically significant differences were observed by species, specimen type, region, or selected district. Compared with 2022, dual-target positivity was lower in 2024 (CPR = 0.40; p = 0.021), whereas the difference observed in 2023 was not statistically significant (CPR = 0.35; p = 0.071). Dual-target real-time PCR identified specimens containing both pagA and capC during anthrax outbreak investigations in Uganda, with limited single-target discordance. Findings reflect patterns among opportunistically collected outbreak-investigation specimens and should not be interpreted as population-level prevalence or evidence of geographic hotspots. Strengthening quality-assured molecular confirmation, specimen referral, standardised outbreak documentation, and cross-sector One Health coordination remains important for anthrax surveillance and response.