Comparing microbiological and molecular diagnostic tools for the surveillance of anthrax

Abstract

The diagnosis of anthrax, a zoonotic disease caused by Bacillus anthracis can 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 as B. anthracis protective antigen (pagA, also called BAPA, occurring on plasmid pXO1), lethal factor (lef, on pXO1), capsule-encoding capB/C (located on pXO2) as well as chromosomal Ba-1. Combinations of genetic markers using real-time/quantitative polymerase chain reaction (qPCR) are used to confirm B. anthracis from 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 for B. anthracis based on genetic markers or microscopy. However, among these, merely 4/125 (3.2%) were confirmed B. anthracis isolates (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 identified B. cereus sensu lato, which included B. cereus and B. anthracis, Peribacillus spp., and Priestia spp. clusters using gyrB gene in selected bacterial isolates positive for pagA region using BAPA probe. Using qPCR on blood smears, 52.1% (890 samples) tested positive for B. anthracis based on one or a combination of genetic markers which included the 25 positive controls. Notably, the standard lef primer set displayed the lowest specificity and accuracy. The Ba-1+BAPA+lef combination showed 100% specificity, sensitivity, and accuracy. 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%. Using Ba-1+BAPA+lef+capB, as well as Ba-1+BAPA+lef with molecular diagnosis accurately detects B. anthracis in the absence of bacterial culture. Systematically combining microscopy and molecular markers holds promise for notably reducing false positives. This significantly enhances the detection and surveillance of diseases like anthrax in southern Africa and beyond and reduces the need for propagation of the bacteria in culture.

Fig 1. The study area, Kruger National Park (KNP), is located in South Africa.

The map of 210 KNP divides the park into three regions, with the distribution of 1708 animal mortalities (S1 Table) investigated in this study are shown as dots; presumptive anthrax positive cases, identified through microscopic examination of blood smears, are marked with red dots, while green dots indicate anthrax-negative mortalities. South Africa provincial and municipal map obtained from africa-latest.osm.pbf. KNP shape files were obtained from from Navteq (2024). The Africa map was obtained from the natural earth data (https://www.naturalearthdata.com/downloads/10m-cultural-vectors/10m-admin-1-states-provinces/)).

Fig 2. Microscopic examination of Gram and polychrome methylene blue-stained cultures from bacterial isolates collected from wildlife blood smears in Kruger National Park, South Africa, identified Bacillus anthracis based on morphology.

Images show square-ended bacilli: (A) Isolate AX2015-1270, (B) AX2015-1277A, (C) AX2015-1152, and (D) AX2015-1136. Encapsulation is visible in (E) AX2015-1270, (F) AX2015-1277A, and (G) AX2015-1152, except in (H) AX2015-1136, which lacked a capsule.

Fig 3. Bar plots displaying the counts of bacterial isolates from blood smears testing positive for Bacillus anthracis using various molecular markers or combinations.

Results are based on qPCR of 80 positive isolates, collected from wildlife mortalities in Kruger National Park, South Africa, between 2012 and 2020. Molecular markers include Bacillus anthracis protective antigen (pagA with BAPA probe), lethal factor (lef), chromosomal marker (Ba-1), and capsule region (capB).

Fig 4. Phylogenetic tree of bacterial isolates from Kruger National Park, South Africa, based on the gyrB gene, constructed using the neighbor-joining method and p-distance model among three closely related genera (formerly Bacillus spp.).

Isolates labeled with AX are from this study and were compared to the closest reference isolates from the National Center for Biotechnology Information (NCBI) (via BLASTn searches) of Bacillus cereus sensu lato, Priestia spp., and Peribacillus spp. The scale bar represents 0.010 substitutions per nucleotide position. Isolates confirmed as B. anthracis through microscopy, culture, molecular diagnosis, and sensitivity to penicillin and gamma phage are marked with an asterisk (*).

Fig 5. Multiple nucleotide sequence alignment of the Bacillus anthracis protective antigen (BAPA) region (targeting the pagA gene of B. anthracis with the BAPA probe) from isolates in this study (starting with AX) compared to the NCBI reference strain B. anthracis DFRL BHE-12.

BAPA_S (Forward) and BAPA_R (Reverse) indicate the BAPA probe targeting sequences. Coloured blocks represent related species clusters, as shown in Fig 4. Sequences were aligned using BioEdit. Two isolates (AX2014-721 and AX2016-1705) lack colour blocks since their species clusters were not included in the previous analysis (Fig 4).