Comparing microbiological and molecular diagnostic tools for the surveillance of anthrax

Abstract

AbstractThe diagnosis of anthrax, a zoonotic disease caused byBacillus anthraciscan be complicated by detection of closely related species. Conventional diagnosis of anthrax involves microscopy, culture identification of bacterial colonies and molecular detection. Genetic markers used are often virulence gene targets such asBacillus anthracisprotective antigen (pagA,as also called BAPA, occurring on plasmid pXO1), lethal factor (lef,on pXO1), as well as chromosomal (Ba-1) and plasmid (capsule-encodingcapB/C, located on pXO2). Combinations of genetic markers using real-time/quantitative polymerase chain reaction (qPCR) are used to confirmB. anthracisfrom culture but can also be used directly on diagnostic samples to avoid propagation and its associated biorisks and for faster identification. We investigated how the presence of closely related species could complicate anthrax diagnoses with and without culture to standardise the use of genetic markers using qPCR for accurate anthrax diagnosis. Using blood smears from 2012-2020 from wildlife mortalities (n=1708) in Kruger National Park in South Africa where anthrax is endemic, we contrasted anthrax diagnostic results based on qPCR, microscopy, and culture. From smears, 113/1708 grew bacteria in culture, from which 506 isolates were obtained. Of these isolates, only 24.7% (125 isolates) were positive forB. anthracisbased on genetic markers or microscopy. However, among these, merely 4/125 (3.2%) were confirmedB. anthracisisolates (based on morphology, microscopy, and sensitivity testing to penicillin and gamma-phage) from the blood smear, likely due to poor survival of spores on stored smears. This study identifiedB. cereus sensu lato, which includedB. cereusandB. anthracis, Peribacillus spp., andPriestia spp.clusters usinggyrB gene in selected bacterial isolates positive for BAPA. Using qPCR on blood smears, 52.1% (890 samples) tested positive forB. anthracisbased on one or a combination of genetic markers which included the 25 positive controls. Notably, the standardlefprimer set displayed the lowest specificity and accuracy. Interestingly, various marker combinations, such as Ba-1+capB, BAPA+capB, Ba-1+BAPA+capB+lef, and BAPA+lef+capB, all demonstrated 100.0% specificity and 98.7% accuracy, while maintaining a sensitivity of 96.6%. The BAPA+lef+Ba-1 combination showed 100% specificity, sensitivity, and accuracy. Using Ba-1+BAPA+lef+capB, as well as Ba-1+BAPA+lefwith molecular diagnosis accurately detectsB. anthracisin the absence of bacterial culture. Systematically combining microscopy and molecular markers holds promise for notably reducing false positives, thereby significantly enhancing the detection and surveillance of diseases like anthrax in southern Africa and beyond and reducing the need for propagation of the bacteria in culture.