Anthrax threatens human and animal health, and people's livelihoods in many rural communities in Africa and Asia. In these areas, anthrax surveillance is challenged by a lack of tools for on-site detection. Furthermore, cultural practices and infrastructure may affect sample availability and quality. Practical yet accurate diagnostic solutions are greatly needed to quantify anthrax impacts. We validated microscopic and molecular methods for the detection of Bacillus anthracis in field-collected blood smears and identified alternative samples suitable for anthrax confirmation in the absence of blood smears. We investigated livestock mortalities suspected to be caused by anthrax in northern Tanzania. Field-prepared blood smears (n = 152) were tested by microscopy using four staining techniques as well as polymerase chain reaction (PCR) followed by Bayesian latent class analysis. Median sensitivity (91%, CI 95% [84-96%]) and specificity (99%, CI 95% [96-100%]) of microscopy using azure B were comparable to those of the recommended standard, polychrome methylene blue, PMB (92%, CI 95% [84-97%] and 98%, CI 95% [95-100%], respectively), but azure B is more available and convenient. Other commonly-used stains performed poorly. Blood smears could be obtained for <50% of suspected anthrax cases due to local customs and conditions. However, PCR on DNA extracts from skin, which was almost always available, had high sensitivity and specificity (95%, CI 95% [90-98%] and 95%, CI 95% [87-99%], respectively), even after extended storage at ambient temperature. Azure B microscopy represents an accurate diagnostic test for animal anthrax that can be performed with basic laboratory infrastructure and in the field. When blood smears are unavailable, PCR using skin tissues provides a valuable alternative for confirmation. Our findings lead to a practical diagnostic approach for anthrax in low-resource settings that can support surveillance and control efforts for anthrax-endemic countries globally.

Genomic sequencing has revolutionized our understanding of bacterial disease epidemiology, but remains underutilized for zoonotic pathogens in remote endemic settings. Anthrax, caused by the spore-forming bacterium Bacillus anthracis , remains a threat to human and animal health and rural livelihoods in low- and middle-income countries. While the global genomic diversity of B. anthracis has been well-characterized, there is limited information on how its populations are genetically structured at the scale at which transmission occurs, critical for understanding the pathogen’s evolution and transmission dynamics. Using a uniquely rich dataset, we quantified genome-wide SNPs among 73 B. anthracis isolates derived from 33 livestock carcasses sampled over 1 year throughout the Ngorongoro Conservation Area, Tanzania, a region hyperendemic for anthrax. Genome-wide SNPs distinguished 22 unique B. anthracis genotypes (i.e. SNP profiles) within the study area. However, phylogeographical structure was lacking, as identical SNP profiles were found throughout the study area, likely the result of the long and variable periods of spore dormancy and long-distance livestock movements. Significantly, divergent genotypes were obtained from spatio-temporally linked cases and even individual carcasses. The high number of SNPs distinguishing isolates from the same host is unlikely to have arisen during infection, as supported by our simulation models. This points to an unexpectedly wide transmission bottleneck for B. anthracis , with an inoculum comprising multiple variants being the norm. Our work highlights that inferring transmission patterns of B. anthracis from genomic data will require analytical approaches that account for extended and variable environmental persistence, as well as co-infection.

23Background: Anthrax threatens human and animal health, and people's livelihoods in 24 many rural communities in Africa and Asia. In these areas, anthrax surveillance is 25 challenged by a lack of tools for on-site detection. Furthermore, cultural practices and 26 infrastructure may affect sample availability and quality. Practical yet accurate diagnostic 27 solutions are greatly needed to quantify anthrax impacts. We validated microscopic and 28 molecular methods for the detection of Bacillus anthracis in field-collected blood smears 29 and identified alternative samples suitable for anthrax confirmation in the absence of blood 30 smears. 31 Methodology/Principal Findings:We investigated livestock mortalities suspected to be 32 caused by anthrax in northern Tanzania. Field-prepared blood smears (n = 152) were tested 33 by microscopy using four staining techniques as well as polymerase chain reaction (PCR) 34 followed by Bayesian latent class analysis. Median sensitivity (91%, CI 95% [84-96%]) and 35 specificity (99%, CI 95% [96-100%]) of microscopy using azure B were comparable to 36 those of the recommended standard, polychrome methylene blue, PMB (92%, CI 95% [84-37 97%] and 98%, CI 95% [95-100%], respectively), but azure B is more available and 38 convenient. Other commonly-used stains performed poorly. Blood smears could be 39 obtained for <50% of suspected anthrax cases due to local customs and conditions. 40 However, PCR on DNA extracts from dried skin, which was almost always available, had 41 high sensitivity and specificity (95%, CI 95% [90-98%] and 95%, CI 95% [87-99%], 42 respectively), even after extended storage at ambient temperature. 43 Conclusions/Significance: Azure B microscopy represents an accurate diagnostic test for 44animal anthrax that can be performed with basic laboratory infrastructure and in the field. 45When blood smears are unavailable, PCR using skin tissues provides a valuable alternative 46 for confirmation. Our findings lead to a practical diagnostic approach for anthrax in low-47 resource settings that can support surveillance and control efforts for anthrax-endemic 48 countries globally. 49 Author summary50 Anthrax, an ancient disease largely controlled in the developed world, is still widespread in 51 remote and rural communities of low-and middle-income countries where it affects human 52 and animal health, and livelihoods. To control anthrax effectively, detection and accurate 53 confirmation are important, but solutions need to be feasible for the most-affected areas 54 3 where resources and infrastructure are typically limited. To achieve this, we assessed a 55 newly proposed stain, azure B, for microscopic confirmation on animal blood smears, as 56 this method can be implemented in low-resource laboratories and in the field. Microscopy 57 using azure B was highly accurate compared to other recommended stains and has the 58 added advantage of being more readily available and convenient. However, blood smear 59 samples were unavailable for more than half of suspected case...

Disease mapping reveals geographical variability in incidence, which can help to prioritise control efforts. However, in areas where this is most needed, resources to generate the required data are often lacking. Participatory mapping, which makes use of indigenous knowledge, is a potential approach to identify risk areas for endemic diseases in low- and middle-income countries. Here we combine this method with Geographical Information System-based analyses of environmental variables as a novel approach to study endemic anthrax, caused by the spore-forming bacterium Bacillus anthracis, in rural Africa. Our aims were to: (1) identify high-risk anthrax areas using community knowledge; (2) enhance our understanding of the environmental characteristics associated with these areas; and (3) make spatial predictions of anthrax risk. Community members from the Ngorongoro Conservation Area (NCA), northern Tanzania, where anthrax is highly prevalent in both animals and humans, were asked to draw areas they perceived to pose anthrax risks to their livestock on geo-referenced maps. After digitisation, random points were generated within and outside the defined areas to represent high- and low-risk areas, respectively. Regression analyses were used to identify environmental variables that may predict anthrax risk. Results were combined to predict how the probability of being a high-risk area for anthrax varies across space. Participatory mapping identified fourteen discrete high-risk areas ranging from 0.2 to 212.9 km2 in size and occupying 8.4% of the NCA. Areas that pose a high risk of anthrax were positively associated with factors that increase contact with Bacillus anthracis spores rather than those associated with the pathogen’s survival: close proximity to inland water bodies, where wildlife and livestock congregate, and low organic carbon content, which may indicate an increased likelihood of animals grazing close to soil surface and ingesting spores. Predicted high-risk areas were located in the centre of the NCA, which is likely to be encountered by most herds during movements in search for resources. We demonstrate that participatory mapping combined with spatial analyses can provide novel insights into the geography of disease risk. This approach can be used to prioritise areas for control in low-resource settings, especially for diseases with environmental transmission.