Comparing microbiological and molecular diagnostic tools for the surveillance of anthrax

dc.contributor.authorOchai, Sunday Ochonu
dc.contributor.authorHassim, Ayesha
dc.contributor.authorDekker, Edgar H.
dc.contributor.authorMagome, Thuto
dc.contributor.authorLekota, Kgaugelo Edward
dc.contributor.authorMakgabo, Sekgota Marcus
dc.contributor.authorDe Klerk-Loris, Lin-Mari
dc.contributor.authorVan Schalkwyk, Louis O.
dc.contributor.authorKamath, Pauline L.
dc.contributor.authorTurner, Wendy C.
dc.contributor.authorVan Heerden, Henriette
dc.date.accessioned2025-05-20T10:40:44Z
dc.date.available2025-05-20T10:40:44Z
dc.date.issued2024-11-21
dc.descriptionSUPPORTING INFORMATION : FIGURE S1. Agarose gel image of Bacillus anthracis protective antigen gene region, pagA (BAPA), for B. anthracis and other bacterial species isolated from cultured blood smears obtained from wildlife mortalities in Kruger National Park, South Africa. The 100 bp (Thermo Scientific, USA) ladder was used. The B. anthracis Sterne and Vollum (labelled as B. anthracis V) strains served as the positive controls. Bacillus cereus ATCC3999 and distilled water (labelled as Negative) were used as negative controls. Sample numbers highlighted with blue rectangles indicate B. anthracis confirmed samples. The assay was repeated three times. TABLE S1. Wildlife species in Kruger National Park, South Africa that tested positive for Bacillus anthracis protective antigen (pagA with BAPA probe sequence), lethal factor (lef), chromosomal marker (Ba-1) and the capsule region (capB) or a combination of these genetic markers and count of animals positive. TABLE S2. Positive results of scraped blood smears using Bacillus anthracis protective antigen (BAPA), lethal factor (lef), chromosomal marker (Ba-1) and the capsule region (capB) molecular markers and marker combinations in probe-based real-time/quantitative polymerase chain reaction (qPCR), with "only" indicating exclusive positivity for the respective marker or combination. TEXT S1. Confirmation of pagA Positivity in Blood Isolates through Conventional PCR. DATASET S1. The S1 dataset encompasses various data used to generate the results in this article, including Bacillus species, Culture, PCR, Morphology, GTBR Report, Probe Smear PCR, Probe-only isolates, Smear Combined Count.
dc.description.abstractThe 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.
dc.description.departmentVeterinary Tropical Diseases
dc.description.librarianam2025
dc.description.sdgSDG-03: Good health and well-being
dc.description.sdgSDG-02: Zero Hunger
dc.description.sponsorshipThe National Science Foundation (NSF) through the NSF- National Institutes of Health (NIH)- United States Department of Agriculture (USDA) Ecology and Evolution of Infectious Diseases program.
dc.description.urihttps://journals.plos.org/plosntds/
dc.identifier.citationOchai, S.O., Hassim, A., Dekker, E.H., Magome, T., Lekota, KE, Makgabo, M.S., et al. (2024) Comparing microbiological and molecular diagnostic tools for the surveillance of anthrax. PLoS Neglected Tropical Diseases 18(11): e0012122. https://DOI.org/10.1371/journal.pntd.0012122.
dc.identifier.issn1935-2727 (print)
dc.identifier.issn1935-2735 (online)
dc.identifier.other10.1371/journal.pntd.0012122
dc.identifier.urihttp://hdl.handle.net/2263/102443
dc.language.isoen
dc.publisherPublic Library of Science
dc.rightsThe work is made available under the Creative Commons CC0 public domain dedication.
dc.subjectAnthrax
dc.subjectDiagnosis
dc.subjectBacteria
dc.subjectCulture
dc.titleComparing microbiological and molecular diagnostic tools for the surveillance of anthrax
dc.typeArticle

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