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Background: Buruli ulcer (BU) is a neglected tropical disease caused by infection of subcutaneous tissue with Mycobacterium ulcerans . BU is commonly reported across rural regions of Central and West Africa but has been increasing dramatically in temperate southeast Australia around the major metropolitan city of Melbourne, with most disease transmission occurring in the summer months. Previous research has shown that Australian native possums are reservoirs of M. ulcerans and that they shed the bacteria in their fecal material (excreta). Field surveys show that locales where possums harbor M. ulcerans overlap with human cases of BU, raising the possibility of using possum excreta surveys to predict the risk of disease occurrence in humans., Methods: We thus established a highly structured 12 month possum excreta surveillance program across an area of 350 km 2 in the Mornington Peninsula area 70 km south of Melbourne, Australia. The primary objective of our study was to assess using statistical modeling if M. ulcerans surveillance of possum excreta provided useful information for predicting future human BU case locations., Results: Over two sampling campaigns in summer and winter, we collected 2,282 possum excreta specimens of which 11% were PCR positive for M. ulcerans -specific DNA. Using the spatial scanning statistical tool SaTScan , we observed non-random, co-correlated clustering of both M. ulcerans positive possum excreta and human BU cases. We next trained a statistical model with the Mornington Peninsula excreta survey data to predict the future likelihood of human BU cases occurring in the region. By observing where human BU cases subsequently occurred, we show that the excreta model performance was superior to a null model trained using the previous year's human BU case incidence data (AUC 0.66 vs 0.55). We then used data unseen by the excreta-informed model from a new survey of 661 possum excreta specimens in Geelong, a geographically separate BU endemic area to the southwest of Melbourne, to prospectively predict the location of human BU cases in that region. As for the Mornington Peninsula, the excreta-based BU prediction model outperformed the null model (AUC 0.75 vs 0.50) and pinpointed specific locations in Geelong where interventions could be deployed to interrupt disease spread., Conclusions: This study highlights the One Health nature of BU by confirming a quantitative relationship between possum excreta shedding of M. ulcerans and humans developing BU. The excreta survey-informed modeling we have described will be a powerful tool for the efficient targeting of public health responses to stop BU., Funding: This research was supported by the National Health and Medical Research Council of Australia and the Victorian Government Department of Health (GNT1152807 and GNT1196396)., Competing Interests: KV, AB, JP, AV, JW, KB, MD, VB, JF, ET, PJ, SW, NG, TS No competing interests declared, (© 2023, Vandelannoote, Buultjens et al.)

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. Identification of environmental reservoirs and agents associated with disease transmission is crucial to understanding the risk factors for this emerging infectious disease. Since culture of M. ulcerans from the environment still proves to be problematic, the direct detection of M. ulcerans in environmental samples via PCR has become increasingly important as the research community seeks to elucidate the mode(s) of transmission and environmental reservoir(s) of this elusive organism., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. Rapid detection of M. ulcerans in clinical specimens is essential for ensuring early diagnosis and prevention of disability. This chapter describes a real-time PCR method for the direct detection of M. ulcerans from a variety of clinical and environmental samples (Fig. 1). Methods for the extraction of DNA from swabs, fresh tissue biopsies, and fixed tissue sections, which are the most common types of specimens used in the diagnosis of Buruli ulcer, are described in Chapter 6 . Chapter 7 describes the appropriate DNA extraction methods for environmental samples including soil, detritus, water, animal feces, and insects, as reliable detection of M. ulcerans in the environment is becoming increasingly important for understanding the ecology and transmission of this elusive pathogen., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer (BU). Rapid detection of M. ulcerans in clinical specimens is essential for early diagnosis so that patients can be treated appropriately as soon as possible. This chapter describes suitable methods for the extraction of M. ulcerans DNA from the most common specimens submitted to the laboratory for confirmation of BU: swabs, fresh tissue biopsies, and fixed tissue sections. The resulting DNA extracts may be used for downstream procedures including standard gel-based PCR and real-time PCR assays. Protocols for direct detection of M. ulcerans DNA by real-time PCR are described in Chapter 8 ., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mycobacterium kansasii can cause serious pulmonary disease. It belongs to a group of closely-related species of non-tuberculous mycobacteria known as the M. kansasii complex (MKC). Here, we report a population genomics analysis of 358 MKC isolates from worldwide water and clinical sources. We find that recombination, likely mediated by distributive conjugative transfer, has contributed to speciation and on-going diversification of the MKC. Our analyses support municipal water as a main source of MKC infections. Furthermore, nearly 80% of the MKC infections are due to closely-related M. kansasii strains, forming a main cluster that apparently originated in the 1900s and subsequently expanded globally. Bioinformatic analyses indicate that several genes involved in metabolism (e.g., maintenance of the methylcitrate cycle), ESX-I secretion, metal ion homeostasis and cell surface remodelling may have contributed to M. kansasii's success and its ongoing adaptation to the human host.

Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics . For L1 we identified signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Menardo F et al.)

Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics. For L1 we identified signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Menardo F et al.)

Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). The MTBC comprises several human-adapted lineages known as M. tuberculosis sensu stricto , as well as two lineages (L5 and L6) traditionally referred to as Mycobacterium africanum . Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known of their genomic diversity, phylogeography and evolution. Here, we analysed the genomes of 350 L5 and 320 L6 strains, isolated from patients from 21 African countries, plus 5 related genomes that had not been classified into any of the known MTBC lineages. Our population genomic and phylogeographical analyses showed that the unclassified genomes belonged to a new group that we propose to name MTBC lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, we found important differences between L5 and L6 strains with respect to their phylogeographical substructure and genetic diversity. Finally, we could not confirm the previous association of drug-resistance markers with lineage and sublineages. Instead, our results indicate that the association of drug resistance with lineage is most likely driven by sample bias or geography. In conclusion, our study sheds new light onto the genomic diversity and evolutionary history of M. africanum , and highlights the need to consider the particularities of each MTBC lineage for understanding the ecology and epidemiology of TB in Africa and globally.

Tuberculosis (TB) affects humans and other animals and is caused by bacteria from the Mycobacterium tuberculosis complex (MTBC). Previous studies have shown that there are at least nine members of the MTBC infecting animals other than humans; these have also been referred to as ecotypes. However, the ecology and the evolution of these animal-adapted MTBC ecotypes are poorly understood. Here we screened 12,886 publicly available MTBC genomes and newly sequenced 17 animal-adapted MTBC strains, gathering a total of 529 genomes of animal-adapted MTBC strains. Phylogenomic and comparative analyses confirm that the animal-adapted MTBC members are paraphyletic with some members more closely related to the human-adapted Mycobacterium africanum Lineage 6 than to other animal-adapted strains. Furthermore, we identified four main animal-adapted MTBC clades that might correspond to four main host shifts; two of these clades are hypothesized to reflect independent cattle domestication events. Contrary to what would be expected from an obligate pathogen, MTBC nucleotide diversity was not positively correlated with host phylogenetic distances, suggesting that host tropism in the animal-adapted MTBC seems to be driven by contact rates and demographic aspects of the host population rather by than host relatedness. By combining phylogenomics with ecological data, we propose an evolutionary scenario in which the ancestor of Lineage 6 and all animal-adapted MTBC ecotypes was a generalist pathogen that subsequently adapted to different host species. This study provides a new phylogenetic framework to better understand the evolution of the different ecotypes of the MTBC and guide future work aimed at elucidating the molecular mechanisms underlying host range.

Buruli ulcer (BU) is a destructive soft-tissue infection caused by the environmental pathogen Mycobacterium ulcerans. In response to rising BU notifications in the state of Victoria, Australia, we reviewed all cases that occurred during 2011-2016 to precisely map the time and likely place of M. ulcerans acquisition. We found that 600 cases of BU had been notified; just over half were in residents and the remainder in visitors to defined BU-endemic areas. During the study period, notifications increased almost 3-fold, from 66 in 2013 to 182 in 2016. We identified 4 BU-endemic areas: Bellarine Peninsula, Mornington Peninsula, Frankston region, and the southeastern Bayside suburbs of Melbourne. We observed a decline in cases on the Bellarine Peninsula but a progressive increase elsewhere. Acquisitions peaked in late summer. The appearance of new BU-endemic areas and the decline in established areas probably correlate with changes in the level of local environmental contamination with M. ulcerans.

Background: Buruli ulcer has been increasing in incidence in southeastern Australia with unclear transmission mechanisms. We aimed to investigate the link between rainfall and case numbers in two endemic areas of the state of Victoria; the Bellarine and Mornington Peninsulas., Methodology: We created yearly and monthly graphs comparing rainfall with local Buruli ulcer incidence for the period 2004-2016 by endemic region and then considered a range of time lag intervals of 0-24 months to investigate patterns of correlation., Conclusions: Optimal positive correlation for the Bellarine Peninsula occurred with a 12-month prior rainfall lag, however, no significant correlation was observed on the Mornington Peninsula for any time lag. These results provide an update in evidence to further explore transmission mechanisms which may differ between these geographically proximate endemic regions., Competing Interests: The authors have declared that no competing interests exist.

Since 2000, cases of the neglected tropical disease Buruli ulcer, caused by infection with Mycobacterium ulcerans , have increased 100-fold around Melbourne (population 4.4 million), the capital of Victoria, in temperate southeastern Australia. The reasons for this increase are unclear. Here, we used whole-genome sequence comparisons of 178 M. ulcerans isolates obtained primarily from human clinical specimens, spanning 70 years, to model the population dynamics of this pathogen from this region. Using phylogeographic and advanced Bayesian phylogenetic approaches, we found that there has been a migration of the pathogen from the east end of the state, beginning in the 1980s, 300 km west to the major human population center around Melbourne. This move was then followed by a significant increase in M. ulcerans population size. These analyses inform our thinking around Buruli ulcer transmission and control, indicating that M. ulcerans is introduced to a new environment and then expands, rather than it being from the awakening of a quiescent pathogen reservoir. IMPORTANCE Buruli ulcer is a destructive skin and soft tissue infection caused by Mycobacterium ulcerans and is characterized by progressive skin ulceration, which can lead to permanent disfigurement and long-term disability. Despite the majority of disease burden occurring in regions of West and central Africa, Buruli ulcer is also becoming increasingly common in southeastern Australia. Major impediments to controlling disease spread are incomplete understandings of the environmental reservoirs and modes of transmission of M. ulcerans The significance of our research is that we used genomics to assess the population structure of this pathogen at the Australian continental scale. We have then reconstructed a historical bacterial spread and modeled demographic dynamics to reveal bacterial population expansion across southeastern Australia. These findings provide explanations for the observed epidemiological trends with Buruli ulcer and suggest possible management to control disease spread., (Copyright © 2018 American Society for Microbiology.)

Background: Buruli ulcer (BU) is a geographically-restricted infection caused by Mycobacterium ulcerans; contact with an endemic region is the primary risk factor for disease acquisition. Globally, efforts to estimate the incubation period of BU are often hindered as most patients reside permanently in endemic areas. However, in the south-eastern Australian state of Victoria, a significant proportion of people who acquire BU are visitors to endemic regions. During a sustained outbreak of BU on the Bellarine peninsula we estimated a mean incubation period of 4.5 months. Since then cases on the Bellarine peninsula have declined but a new endemic area has developed centred on the Mornington peninsula., Method: Retrospective review of 443 cases of BU notified in Victoria between 2013 and 2016. Telephone interviews were performed to identify all cases with a single visit to an endemic region, or multiple visits within a one month period. The incubation period was defined as the time between exposure to an endemic region and symptom onset. Data were subsequently combined with those from our earlier study incorporating cases from 2002 to 2012., Results: Among the 20 new cases identified in short-term visitors, the mean incubation period was 143 days (4.8 months), very similar to the previous estimate of 135 days (4.5 months). This was despite the predominant exposure location shifting from the Bellarine peninsula to the Mornington peninsula. We found no association between incubation period and age, sex, location of exposure, duration of exposure to an endemic region or location of BU lesion., Conclusions: Our study confirms the mean incubation period of BU in Victoria to be between 4 and 5 months. This knowledge can guide clinicians and suggests that the mode of transmission of BU is similar in different geographic regions in Victoria.

Background: The origin and spread of tuberculosis (TB) in Tasmania and the types of strains of Mycobacterium tuberculosis complex (MTBC) present in the population are largely unknown., Objective: The aim of this study was to perform the first genomic analysis of MTBC isolates from Tasmania to better understand the epidemiology of TB in the state., Methods: Whole-genome sequencing was performed on cultured isolates of MTBC collected from 2014-2016. Single-locus variant analysis was applied to determine the phylogeny of the isolates and the presence of drug-resistance mutations. The genomic data were then cross-referenced against public health surveillance records on each of the cases., Results: We determined that 83.3% of TB cases in Tasmania from 2014-2016 occurred in non-Australian born individuals. Two possible TB clusters were identified based on single locus variant analysis, one from November-December 2014 (n = 2), with the second from May-August 2015 (n = 4). We report here the first known isolate of multi-drug resistant (MDR) M. tuberculosis in Tasmania from 2016 for which we established its drug resistance mutations and potential overseas origin. In addition, we characterised a case of M. bovis TB in a Tasmanian-born person who presented in 2014, approximately 40 years after the last confirmed case in the state's bovids., Conclusions: TB in Tasmania is predominantly of overseas origin with genotypically-unique drug-susceptible isolates of M. tuberculosis. However, the state also exhibits features of TB that are observed in other jurisdictions, namely, the clustering of cases, and drug resistance. Early detection of TB and contact tracing, particularly of overseas-born cases, coordinated with rapid laboratory drug-susceptibility testing and molecular typing, will be essential for Tasmania to reach the World Health Organisation's TB eradication goals for low-incidence settings.

To identify potential reservoirs/vectors of Mycobacterium ulcerans in northern Queensland, Australia, we analyzed environmental samples collected from the Daintree River catchment area, to which Buruli ulcer is endemic, and adjacent coastal lowlands by species-specific PCR. We detected M. ulcerans DNA in soil, mosquitoes, and excreta of bandicoots, which are small terrestrial marsupials.

Objectives: This paper, the last in a series of three on 'feline leprosy', provides a detailed description of disease referable to the previously unnamed species, Candidatus 'Mycobacterium lepraefelis', a close relative of the human pathogens Mycobacterium leprae and Mycobacterium lepromatosis., Methods: Cases were sourced retrospectively and prospectively for this observational study, describing clinical, geographical and molecular microbiological data for cats definitively diagnosed with Candidatus 'M lepraefelis' infection., Results: A total of 145 cases of feline leprosy were scrutinised; 114 'new' cases were sourced from the Victorian Infectious Diseases Reference Laboratory (VIDRL) records, veterinary pathology laboratories or veterinarians, and 31 cases were derived from six published studies. Thirty-eight cats were definitively diagnosed with Candidatus 'M lepraefelis' infection. Typically, cats tended to be middle-aged or older when first infected, with a male predilection. Affected cats typically had widespread cutaneous lesions, in some cases after initially localised disease. Advanced cases were often systemically unwell. All cats had outdoor access. The histological picture was lepromatous in the majority of patients, although two cases had tuberculoid disease. In one case that underwent necropsy, lesions were evident in the liver, spleen and lungs. Treatment was varied, although most cats received a combination of oral clarithromycin and rifampicin. Prognosis for recovery was variable, but typically poor., Conclusions and Relevance: Candidatus 'M lepraefelis' typically causes high bacterial index (lepromatous) feline leprosy that in some cases progresses to systemic mycobacteriosis. The disease has a variable clinical course and prognosis. Many cases either died or were euthanased due to the infection. Multilocus sequence analysis reveals a heterogeneous picture and further analysis of draft genome sequencing may give clues to the taxonomy and epidemiology of this organism. Prospective treatment trials and/or additional drug susceptibility testing in specialised systems would further inform treatment recommendations. Comparative aspects: This paper finishes with a discussion of comparative aspects of infection caused by the three feline leproid disease agents that have been the subject of this series: Candidatus 'Mycobacterium tarwinense', Mycobacterium lepraemurium and Candidatus 'M lepraefelis'.

This study aimed to contribute to the understanding of Mycobacterium ulcerans (MU) ecology by analysing both clinical and environmental samples collected from ten communities along two major river basins (Offin and Densu) associated with Buruli ulcer (BU) at different seasons. We collected clinical samples from presumptive BU cases and environmental samples from ten communities. Following DNA extraction, clinical samples were confirmed by IS2404 PCR and environmental samples were confirmed by targeting MU-specific genes, IS2404, IS2606 and the ketoreductase (KR) using real-time PCR. Environmental samples were first analysed for IS2404; after which, IS2404-positive samples were multiplexed for the IS2606 and KR gene. Our findings indicate an overall decline in BU incidence along both river basins, although incidence at Densu outweighs that of Offin. Overall, 1600 environmental samples were screened along Densu (434, 27 %) and Offin (1166, 73 %) and MU was detected in 139 (9 %) of the combined samples. The positivity of MU along the Densu River basin was 89/434 (20.5 %), whilst that of the Offin River basin was 50/1166 (4.3 %). The DNA was detected mainly in snails (5/6, 83 %), moss (8/40, 20 %), soil (55/586, 9 %) and vegetation (55/675, 8 %). The proportion of MU positive samples recorded was higher during the months with higher rainfall levels (126/1175, 11 %) than during the dry season months (13/425, 3 %). This study indicates for the first time that there is a seasonal pattern in the presence of MU in the environment, which may be related to recent rainfall or water in the soil.

Objectives: This paper, the second in a series of three on 'feline leprosy', provides a detailed description of disease referable to Mycobacterium lepraemurium, the most common cause of feline leprosy worldwide., Methods: Cases were sourced retrospectively and prospectively for this observational study, describing clinical, geographical and molecular microbiological data for cats definitively diagnosed with M lepraemurium infection., Results: A total of 145 cases of feline leprosy were scrutinised; 114 'new' cases were sourced from the Victorian Infectious Diseases Reference Laboratory records, veterinary pathology laboratories or veterinarians, and 31 cases were derived from six published studies. Sixty-five cats were definitively diagnosed with M lepraemurium infection. Typically, cats were 1-3 years of age when first infected, with a male gender predilection. Affected cats were generally systemically well. All had outdoor access. Lesions tended to consist of one or more cutaneous/subcutaneous nodules, typically located on the head and/or forelimbs, possibly reflecting the most likely locations for a rodent bite as the site of inoculation for organisms. Nodules had the propensity to ulcerate at some stage in the clinical course. The cytological and histological picture varied from tuberculoid, with relatively low bacterial numbers, to lepromatous with moderate to high bacterial numbers. Treatment was varied, although most cats underwent surgical resection of lesions with adjunctive medical therapy, most often using a combination of oral clarithromycin and rifampicin. Prognosis for recovery was generally good, and in two cases there was spontaneous remission without the requirement for medical intervention. Untreated cats continued to enjoy an acceptable quality of life despite persistence of the disease, which extended locally but had no apparent tendency to disseminate to internal organs., Conclusions and Relevance: M lepraemurium causes high bacterial index (lepromatous) or low bacterial index (tuberculoid) feline leprosy. The infection typically causes nodules of the skin and/or subcutis (which tend towards ulceration) on the head and/or forelimbs. The disease usually has an indolent clinical course and infected cats have a generally favourable response to therapeutic interventions, with rare cases undergoing spontaneous resolution. Genomic analysis may yield clues as to the environmental niche and culture requirements of this elusive organism. Prospective treatment trials and/or additional drug susceptibility testing in specialised systems would further inform treatment recommendations.

Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium - M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico -predicted specificity for M. chimaera Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera ., (Copyright © 2017 American Society for Microbiology.)

Background: The incidence of cutaneous nontuberculous mycobacterial (NTM) infection has increased in recent decades because of widespread use of immunosuppressive therapy and better detection methods. The histopathology of cutaneous NTM infection is not pathognomic and the organisms are slow and difficult to culture, making diagnosis challenging., Methods: We reviewed the clinical and histopathological features of 13 cases of cutaneous NTM infection, and performed panmycobacterial polymerase chain reaction (PCR) on the paraffin blocks., Results: The immunocompetent patients presented with localized lesions on the extremities, whereas the immunocompromised patients presented with disseminated cutaneous lesions. The histopathology in immunocompetent patients was characterized by pseudoepitheliomatous epidermal hyperplasia, intraepithelial abscesses, transepidermal elimination and dermal granulomatous inflammation accompanied by necrosis and suppuration. The immunocompromised patients showed suppurative inflammation with little granuloma formation and numerous acid-fast bacilli. Paraffin block PCR was positive in 4 of 13 cases (31%), whereas culture was positive in 11 of 13 cases (85%)., Conclusion: The aforementioned histological features should help in diagnosing cutaneous NTM infection when combined with clinical and microbiological correlation. In our study, we did not find paraffin block PCR to be superior to conventional culture in detecting cutaneous NTM infection., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: This paper, the first in a series of three on 'feline leprosy', provides a detailed description of disease referable to Candidatus 'Mycobacterium tarwinense', the most common cause of feline leprosy in Victoria, Australia., Methods: Cases were sourced retrospectively and prospectively for this observational study, describing clinical, geographical and molecular microbiological data for cats definitively diagnosed with Candidatus 'M tarwinense' infection., Results: A total of 145 cases of feline leprosy were scrutinised; 114 'new' cases were sourced from the Victorian Infectious Diseases Reference Laboratory records, veterinary pathology laboratories or veterinarians, and 31 cases were derived from six published studies. Forty-two cats were definitively diagnosed with Candidatus 'M tarwinense' infection. Typically, cats were between 3 and 11 years of age, with no gender predilection, and were generally systemically well. All had outdoor access. Most cats underwent surgical resection of lesions with adjunctive medical therapy, often utilising a combination of oral clarithromycin and rifampicin for at least 3 months. Prognosis for recovery was generally good. Resolution of lesions was not observed in the absence of treatment, but a number of untreated cats continued to enjoy an acceptable quality of life despite persistence of the disease, which extended locally but did not appear to disseminate to internal organs. Preliminary results of draft genome sequencing confirmed that the species is a member of the Mycobacterium simiae complex., Conclusions and Relevance: Candidatus 'M tarwinense', a fastidious member of the M simiae complex, is capable of causing feline leprosy with a tendency to produce lesions on the head, particularly involving the eyes and periocular skin. The disease has an indolent clinical course and generally responds favourably to therapy despite lesions often containing large numbers of organisms. Detailed genomic analysis may yield clues as to the environmental niche and culture requirement of this elusive organism. Prospective treatment trials and/or drug susceptibility testing in specialised systems would further inform treatment recommendations.

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration., Competing Interests: Statement The authors have no competing interests as defined by Springer Nature, or other interests that might be perceived to influence the results and/or discussion reported in this paper.

We report a case of chronic pulmonary multi-drug-resistant tuberculosis. Despite 14 years of treatment, Mycobacterium tuberculosis was persistently isolated from sputum. Following treatment cessation the patient remained well, although M. tuberculosis was isolated from sputum for a further 8 years. Genome sequencing of eight serial M. tuberculosis isolates cultured between 1991 and 2011 revealed 17 mutations (0.8 mutations per genome year - 1 ). Eight of these were persisting mutations and only two mutations were detected in the 7 years following cessation of treatment in 2004. In four isolates there were mixed alleles, suggesting the likely presence of bacterial subpopulations. The initial 1991 isolate demonstrated genotypic resistance to isoniazid ( katG W91R), rifampicin ( rpoB S531L), ethambutol ( embB M306V), streptomycin ( gidB L16R), quinolones ( gyrA S95T) and P-aminosalicylic acid ( thyA T202A). Subsequent resistance mutations developed for pyrazinamide ( pncA I31F) and ethionamide ( ethA frameshift). Such information might have been instructive when developing a treatment regimen. In retrospect and with the benefit of high-resolution genomic hindsight we were able to determine that the patient received only one or two active anti-tuberculous agents for most of their treatment. Additionally, mutations in bacA and Rv2326c were detected, which may have contributed to the persistent but mild disease course. BacA is likely to be associated with maintenance of chronic infection and Rv2326c with a decreased bacterial metabolic state. These results expand our understanding of M. tuberculosis evolution during human infection and underline the link between antibiotic resistance and clinical persistence.

Introduction: Mycobacterium ulcerans (M. ulcerans) is a necrotizing skin infection endemic to the Bellarine Peninsula, Australia. Current treatment recommendations include 8 weeks of combination antibiotics, with adjuvant surgery if necessary. However, antibiotic toxicity often results in early treatment cessation and local experience suggests that shorter antibiotic courses may be effective with concurrent surgery. We report the outcomes of patients in the Barwon Health M. ulcerans cohort who received shorter courses of antibiotic therapy than 8 weeks., Methodology / Principal Findings: A retrospective analysis was performed of all M. ulcerans infections treated at Barwon Health from March 1, 1998 to July 31, 2013. Sixty-two patients, with a median age of 65 years, received < 56 days of antibiotics and 51 (82%) of these patients underwent concurrent surgical excision. Most received a two-drug regimen of rifampicin combined with either ciprofloxacin or clarithromycin for a median 29 days (IQR 21-41 days). Cessation rates were 55% for adverse events and 36% based on clinician decision. The overall success rate was 95% (98% with concurrent surgery; 82% with antibiotics alone) with a 50% success rate for those who received < 14 days of antibiotics increasing to 94% if they received 14-27 days and 100% for 28-55 days (p<0.01). A 100% success rate was seen for concurrent surgery and 14-27 days of antibiotics versus 67% for concurrent surgery and < 14 days of antibiotics (p = 0.12). No previously identified risk factors for treatment failure with surgery alone were associated with reduced treatment success rates with < 56 days of antibiotics., Conclusion: In selected patients, antibiotic treatment durations for M. ulcerans shorter than the current WHO recommended 8 weeks duration may be associated with successful outcomes.

This report describes the first case in South Australia, Australia, of Mycobacterium pinnipedii tuberculosis in a free-ranging Australian fur seal (Arctocephalus pusillus doriferus). Severe pyogranulomatous pleuropneumonia with intrahistocytic acid-fast beaded filamentous bacilli was seen on histology. M. pinnipedii was confirmed by full 24-loci mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. Spillover concerns for public health and cattle are discussed.

A case of fever, sepsis, and chest lesions evident on a computed tomography scan of an indigenous man in northern Australia following burns to the feet is described. Sputum PCR testing revealed Mycobacterium leprae, and a fine-needle aspirate of the chest lesions demonstrated Cryptococcus coinfection., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Tuberculosis (TB) represents a serious public health problem in Mozambique, with an estimated incidence rate of 548 cases per 100,000 population in 2011. Information on the molecular epidemiology of Mycobacterium tuberculosis (MTB) strains circulating in Mozambique is limited. This study provides the first description of the genetic diversity of MTB strains circulating in Beira city, the second largest town in Mozambique., Methods: A total of 67 MTB isolates were tested to determine genetic lineages and diversity. The genetic lineages were determined using real-time PCR while genetic diversity was assessed by obtaining Mycobacterial Interspersed Repetitive Unit-Variable Numbers of Tandem Repeat profiles., Results: Only three of the six major lineages were represented, with 41 (61%) strains belonging to lineage 1, 25 (37%) belonging to lineage 4 and the remaining isolate belonging to lineage 3. No lineage 2 strains (containing the Beijing family) were identified. A high degree of diversity amongst the strains from both lineages 1 and 4 were observed. Comparison of the profiles of representative strains with those of reference strains in the MIRU-VNTRplus database revealed that all lineage 1 isolates clustered with the Eastern African Indian (EAI) 5 sub-family. The lineage 4 strains clustered with a variety of different sub-family strains, including the Latin-American-Mediterranean (LAM) 1 sub-family, the Haarlem, Uganda 1 and Cameroon sub-families and the T2-S sub-family., Conclusions: The TB epidemic in Beira city is caused by a diverse group of MTB strains predominantly belonging to lineages 1 and 4., (Copyright © 2014 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.)

Background: Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a necrotizing disease of the skin, soft tissue and bone. PCR is increasingly used in the diagnosis of BU and in research on the mode of transmission and environmental reservoir of M. ulcerans., Methodology/principal Findings: The aim of this study was to evaluate the performance of laboratories in detecting M. ulcerans using molecular tests in clinical and environmental samples by implementing sequential multicenter external quality assessment (EQA) programs. The second round of the clinical EQA program revealed somewhat improved performance., Conclusions/significance: Ongoing EQA programs remain essential and continued participation in future EQA programs by laboratories involved in the molecular testing of clinical and environmental samples for M. ulcerans for diagnostic and research purposes is strongly encouraged. Broad participation in such EQA programs also benefits the harmonization of quality in the BU research community and enhances the credibility of advances made in solving the transmission enigma of M. ulcerans.

Background: Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans, with endemicity predominantly in sub-Saharan Africa and south-eastern Australia. The mode of transmission and the environmental reservoir(s) of the bacterium and remain elusive. Real-time PCR investigations have detected M. ulcerans DNA in a variety of Australian environmental samples, including the faeces of native possums with and without clinical evidence of infection. This report seeks to expand on previously published findings by the authors' investigative group with regards to clinical and subclinical disease in selected wild possum species in BU-endemic areas of Victoria, Australia., Methodology/principal Findings: Twenty-seven clinical cases of M. ulcerans infection in free-ranging possums from southeastern Australia were identified retrospectively and prospectively between 1998-2011. Common ringtail possums (Pseudocheirus peregrinus), a common brushtail possum (Trichosurus vulpecula) and a mountain brushtail possum (Trichosurus cunninghami) were included in the clinically affected cohort. Most clinically apparent cases were adults with solitary or multiple ulcerative cutaneous lesions, generally confined to the face, limbs and/or tail. The disease was minor and self-limiting in the case of both Trichosurus spp. possums. In contrast, many of the common ringtail possums had cutaneous disease involving disparate anatomical sites, and in four cases there was evidence of systemic disease at post mortem examination. Where tested using real-time PCR targeted at IS2404, animals typically had significant levels of M. ulcerans DNA throughout the gut and/or faeces. A further 12 possums without cutaneous lesions were found to have PCR-positive gut contents and/or faeces (subclinical cases), and in one of these the organism was cultured from liver tissue. Comparisons were made between clinically and subclinically affected possums, and 61 PCR-negative, non-affected individuals, with regards to disease category and the categorical variables of species (common ringtail possums v others) and sex. Animals with clinical lesions were significantly more likely to be male common ringtail possums., Conclusions/significance: There is significant disease burden in common ringtail possums (especially males) in some areas of Victoria endemic for M. ulcerans disease. The natural history of the disease generally remains unknown, however it appears that some mildly affected common brushtail and mountain brushtail possums can spontaneously overcome the infection, whereas some severely affected animals, especially common ringtail possums, may become systemically, and potentially fatally affected. Subclinical gut carriage of M. ulcerans DNA in possums is quite common and in some common brushtail and mountain brushtail possums this is transient. Further work is required to determine whether M. ulcerans infection poses a potential threat to possum populations, and whether these animals are acting as environmental reservoirs in certain geographical areas.

The last 20 years has seen a significant series of outbreaks of Buruli/Bairnsdale Ulcer (BU), caused by Mycobacterium ulcerans, in temperate south-eastern Australia (state of Victoria). Here, the prevailing view of M. ulcerans as an aquatic pathogen has been questioned by recent research identifying native wildlife as potential terrestrial reservoirs of infection; specifically, tree-dwelling common ringtail and brushtail possums. In that previous work, sampling of environmental possum faeces detected a high prevalence of M. ulcerans DNA in established endemic areas for human BU on the Bellarine Peninsula, compared with non-endemic areas. Here, we report research from an emergent BU focus recently identified on the Mornington Peninsula, confirming associations between human BU and the presence of the aetiological agent in possum faeces, detected by real-time PCR targeting M. ulcerans IS2404, IS2606 and KR. Mycobacterium ulcerans DNA was detected in 20/216 (9.3%) ground collected ringtail possum faecal samples and 4/6 (66.6%) brushtail possum faecal samples. The distribution of the PCR positive possum faecal samples and human BU cases was highly focal: there was a significant non-random cluster of 16 M. ulcerans positive possum faecal sample points detected by spatial scan statistics (P<0.0001) within a circle of radius 0.42 km, within which were located the addresses of 6/12 human cases reported from the area to date; moreover, the highest sample PCR signal strength (equivalent to ≥10(6) organisms per gram of faeces) was found in a sample point located within this cluster radius. Corresponding faecal samples collected from closely adjacent BU-free areas were predominantly negative. Possums may be useful sentinels to predict endemic spread of human BU in Victoria, for public health planning. Further research is needed to establish whether spatial associations represent evidence of direct or indirect transmission between possums and humans, and the mechanism by which this may occur.

Background: Ten cats with skin lesions characteristic of cutaneous mycobacteriosis were included in this retrospective clinical, pathological and molecular study., Hypothesis/objectives: The aim of this study was to identify the causative agent and to compare the clinicopathological features of these cases with those of previous studies., Methods: Cats were from the south east of France (eight cases), central France (one case) and New Caledonia (South Pacific; one case). Criteria for inclusion were histological evidence of granulomatous dermatitis and/or panniculitis, with acid-fast bacilli within macrophages or extracellularly in regions of tissue necrosis. PCR targeting the 16S-23S internal transcribed spacer region and sequence analysis were performed using DNA extracted from formalin-fixed, paraffin-embedded tissues from all cases., Results: All cats were presented with a history of alopecic to ulcerated nodules. Most cases had limited disease, with one to few nodules, while others (three cats) showed a more aggressive clinical course. Lesions from eight cats yielded a sequence consistent with Mycobacterium lepraemurium, while Mycobacterium microti was identified postmortem from the cutaneous lesion in the cat originating from central France and euthanized for its debilitating condition. No PCR product could be amplified from the remaining specimen., Conclusions and Clinical Importance: Based on this geographically restricted case series, feline leprosy in southern France is most likely to be caused by M. lepraemurium and presents as a generally self-limiting disease. Molecular testing is essential to assess zoonotic potential, because M. microti-induced cutaneous mycobacteriosis can resemble feline leprosy syndrome., (© 2013 ESVD and ACVD.)

Introduction: Buruli Ulcer (BU) is caused by the environmental microbe Mycobacterium ulcerans. Despite unclear transmission, contact with a BU endemic region is the key known risk factor. In Victoria, Australia, where endemic areas have been carefully mapped, we aimed to estimate the Incubation Period (IP) of BU by interviewing patients who reported defined periods of contact with an endemic area prior to BU diagnosis., Method: A retrospective review was undertaken of 408 notifications of BU in Victoria from 2002 to 2012. Telephone interviews using a structured questionnaire and review of notification records were performed. Patients with a single visit exposure to a defined endemic area were included and the period from exposure to disease onset determined (IP)., Results: We identified 111 of 408 notified patients (27%) who had a residential address outside a known endemic area, of whom 23 (6%) reported a single visit exposure within the previous 24 months. The median age of included patients was 30 years (range: 6 to 73) and 65% were male. 61% had visited the Bellarine Peninsula, currently the most active endemic area. The median time from symptom onset to diagnosis was 71 days (range: 34-204 days). The midpoint of the reported IP range was utilized to calculate a point estimate of the IP for each case. Subsequently, the mean IP for the cohort was calculated at 135 days (IQR: 109-160; CI 95%: 113.9-156), corresponding to 4.5 months or 19.2 weeks. The shortest IP recorded was 32 days and longest 264 days (Figure 1 & 2). IP did not vary for variables investigated., Conclusions: The estimated mean IP of BU in Victoria is 135 days (IQR: 109-160 days), 4.5 months. The shortest recorded was IP 34 days and longest 264 days. A greater understanding of BU IP will aid clinical risk assessment and future research.

Background: Mycobacterial granulomas of the skin and subcutis can be caused by one of a number of pathogens. This review concentrates on noncultivable species that cause diseases characterized by focal granuloma(s), namely leproid granuloma (in dogs) and feline leprosy (in cats). Clinically indistinguishable lesions can be caused by tuberculous organisms (Mycobacterium bovis and Mycobacterium microti) and members of the Mycobacterium avium complex. Rapidly growing mycobacterial species that cause infection of the subcutaneous panniculus associated with draining tracts are not discussed. Disease caused by Mycobacterium ulcerans is an important emerging differential diagnosis for ulcerated cutaneous nodules in certain localized regions. CLINICAL LESIONS: Lesions comprise one or multiple nodules in the skin/subcutis. These are generally firm and well circumscribed, and typically become denuded of hair. They may or may not ulcerate, depending on the virulence of the causal organisms and the immune response of the host., Diagnosis: The most inexpensive, noninvasive means of diagnosis is by submission of methanol-fixed, Romanowsky-stained smears to a Mycobacterium Reference Laboratory after detecting negatively stained or acid-fast bacilli on cytological smears. Scrapings of material from slides usually provide sufficient mycobacterial DNA to enable identification of the causal organism using sequence analysis of amplicons after PCR using specific mycobacterial primers., Therapy: Therapy relies upon a combination of marginal resection of easily accessible lesions and treatment using two or three drugs effective against slowly growing mycobacteria, choosing amongst rifampicin, clarithromycin, clofazimine and pradofloxacin/moxifloxacin., (© 2013 The Authors. Veterinary Dermatology © 2013 ESVD and ACVD, Veterinary Dermatology.)

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. Rapid detection of M. ulcerans in clinical specimens is essential to ensure early diagnosis and prevention of disability. This chapter describes a real-time PCR method for the direct detection of M. ulcerans from swabs, fresh tissue biopsies, and fixed tissue sections, which are the most common types of specimens used in the diagnosis of Buruli ulcer. The chapter also briefly describes methods for PCR detection of M. ulcerans in environmental samples, as reliable detection of M. ulcerans in the environment is becoming increasingly important for understanding the ecology and transmission of this important pathogen.

Background: Canine leproid granuloma (CLG) characteristically presents as single to multiple circumscribed dermal to subcutaneous nodules in haired skin. An unidentified mycobacterium is considered be the aetiological agent of this entity., Animals: Several cases of canine leproid granulomas occurred in dogs in New Zealand during 2010 and 2011. Cases appeared in clusters, affecting multiple closely related foxhounds domiciled in the same kennels. All affected hounds recovered after topical and/or systemic antimicrobial therapy. Two similar outbreaks that occurred in foxhounds near Melbourne, Australia are also reported., Methods: Cases were investigated using cytological, histological, microbiological and several molecular techniques. An environmental epidemiological study was also performed., Results: A diagnosis of CLG was established in 11 dogs. Molecular identification of the causative agent confirmed that it was a mycobacterial species with 100% sequence homology within the amplified regions of the 16S rRNA gene and internal transcribed spacer (ITS1) with that found in association with similar infections from the USA, Brazil and Australia., Conclusion and Clinical Importance: This report details the first occurrence of multiple cases of CLG occurring in in-contact dogs and the first proven case of CLG in dogs in New Zealand., (© 2012 The Authors. Veterinary Dermatology © 2012 ESVD and ACVD.)

Buruli ulcer (BU) is a necrotizing infection of skin and soft tissue caused by Mycobacterium ulcerans. In Australia, most cases of BU are linked to temperate, coastal Victoria and tropical, northern Queensland, and strains from these regions are distinguishable by variable-number tandem repeat (VNTR) typing. We present an epidemiological investigation of five patients found to have been infected during interstate travel and describe two nucleotide polymorphisms that differentiate M. ulcerans strains from northern Australia.

Background: Mycobacterium ulcerans is an unusual bacterial pathogen with elusive origins. While closely related to the aquatic dwelling M. marinum, M. ulcerans has evolved the ability to produce the immunosuppressive polyketide toxin mycolactone and cause the neglected tropical disease Buruli ulcer. Other mycolactone-producing mycobacteria (MPM) have been identified in fish and frogs and given distinct species designations (M. pseudoshottsii, M. shinshuense, M. liflandii and M. marinum), however the evolution of M. ulcerans and its relationship to other MPM has not been defined. Here we report the comparative analysis of whole genome sequences from 30 MPM and five M. marinum., Results: A high-resolution phylogeny based on genome-wide single nucleotide polymorphisms (SNPs) showed that M. ulcerans and all other MPM represent a single clonal group that evolved from a common M. marinum progenitor. The emergence of the MPM was driven by the acquisition of the pMUM plasmid encoding genes for the biosynthesis of mycolactones. This change was accompanied by the loss of at least 185 genes, with a significant overrepresentation of genes associated with cell wall functions. Cell wall associated genes also showed evidence of substantial adaptive selection, suggesting cell wall remodeling has been critical for the survival of MPM. Fine-grain analysis of the MPM complex revealed at least three distinct lineages, one of which comprised a highly clonal group, responsible for Buruli ulcer in Africa and Australia. This indicates relatively recent transfer of M. ulcerans between these continents, which represent the vast majority of the global Buruli ulcer burden. Our data provide SNPs and gene sequences that can differentiate M. ulcerans lineages, suitable for use in the diagnosis and surveillance of Buruli ulcer., Conclusions: M. ulcerans and all mycolactone-producing mycobacteria are specialized variants of a common Mycobacterium marinum progenitor that have adapted to live in restricted environments. Examination of genes lost or retained and now under selective pressure suggests these environments might be aerobic, and extracellular, where slow growth, production of an immune suppressor, cell wall remodeling, loss or modification of cell wall antigens, and biofilm-forming ability provide a survival advantage. These insights will guide our efforts to find the elusive reservoir(s) of M. ulcerans and to understand transmission of Buruli ulcer.

Treponema pallidum is the causative agent of syphilis, a sexually transmitted infection of significant public health importance. Since 2000 there has been a marked increase in the number of cases of syphilis infections notified in Victoria, Australia, with the majority of cases occurring in men who have sex with men (MSM) and the highest incidence being in HIV-infected MSM. The molecular subtyping method described by Pillay et al. (A. Pillay et al., Sex. Transm. Dis. 25:408-414, 1998) has been used in this study to determine the diversity of T. pallidum subtypes circulating locally and to look for any relationship between T. pallidum subtypes and HIV status over a 6-year period (2004 to 2009). Treponema pallidum DNA was detected in 303 patient specimens (n = 3,652), and full subtyping profiles were obtained from 90 of these (from 88 patients). A total of 11 T. pallidum subtypes were identified: types 14e (28, 31.1%), 14d (15, 16.7%), 14k (13, 14.4%), 14p (12, 13.3%), 14i (7, 7.8%) 14b (6, 6.7%), 14l (5, 5.6%), and 12i, 13b, 13i, and 13e (1 each, 1.1%). This study showed a similar level of variation among circulating T. pallidum strains compared with that in other studies using the same methodology. A different mix of strains and different predominating strains have been found at each geographical study location, with type 14e emerging as the predominant local strain in Victoria. There was no detectable trend between T. pallidum subtypes and the specimen collection site or stage of syphilis (where known), nor was there any relationship between particular strains and HIV status.

This work reports the design and evaluation of a rapid loop-mediated isothermal amplification test for detecting Mycobacterium ulcerans DNA based on the multicopy insertion sequence IS2404. The test is robust and specific with a detection limit equivalent to 20 copies of the target sequence (0.01 to 0.1 genome). The test has potential for the diagnosis of Buruli ulcer under field conditions.

Background: Buruli ulcer (BU), caused by Mycobacterium ulcerans (M. ulcerans), is a necrotizing skin disease found in more than 30 countries worldwide. BU incidence is highest in West Africa; however, cases have substantially increased in coastal regions of southern Australia over the past 30 years. Although the mode of transmission remains uncertain, the spatial pattern of BU emergence in recent years seems to suggest that there is an environmental niche for M. ulcerans and BU prevalence., Methodology/principal Findings: Network analysis was applied to BU cases in Victoria, Australia, from 1981-2008. Results revealed a non-random spatio-temporal pattern at the regional scale as well as a stable and efficient BU disease network, indicating that deterministic factors influence the occurrence of this disease. Monthly BU incidence reported by locality was analyzed with landscape and climate data using a multilevel Poisson regression approach. The results suggest the highest BU risk areas occur at low elevations with forested land cover, similar to previous studies of BU risk in West Africa. Additionally, climate conditions as far as 1.5 years in advance appear to impact disease incidence. Warmer and wetter conditions 18-19 months prior to case emergence, followed by a dry period approximately 5 months prior to case emergence seem to favor the occurrence of BU., Conclusions/significance: The BU network structure in Victoria, Australia, suggests external environmental factors favor M. ulcerans transmission and, therefore, BU incidence. A unique combination of environmental conditions, including land cover type, temperature and a wet-dry sequence, may produce habitat characteristics that support M. ulcerans transmission and BU prevalence. These findings imply that future BU research efforts on transmission mechanisms should focus on potential vectors/reservoirs found in those environmental niches. Further, this study is the first to quantitatively estimate environmental lag times associated with BU outbreaks, providing insights for future transmission investigations.

Background: Previous analyses of sera from a limited number of Ghanaian Buruli ulcer (BU) patients, their household contacts, individuals living in BU non-endemic regions as well as European controls have indicated that antibody responses to the M. ulcerans 18 kDa small heat shock protein (shsp) reflect exposure to this pathogen. Here, we have investigated to what extent inhabitants of regions in Ghana regarded as non-endemic for BU develop anti-18 kDa shsp antibody titers., Methodology/principal Findings: For this purpose we determined anti-18 kDa shsp IgG titers in sera collected from healthy inhabitants of the BU endemic Densu River Valley and the Volta Region, which was so far regarded as BU non-endemic. Significantly more sera from the Densu River Valley contained anti-18 kDa shsp IgG (32% versus 12%, respectively). However, some sera from the Volta Region also showed high titers. When interviewing these sero-responders, it was revealed that the person with the highest titer had a chronic wound, which was clinically diagnosed and laboratory reconfirmed as active BU. After identification of this BU index case, further BU cases were clinically diagnosed by the Volta Region local health authorities and laboratory reconfirmed. Interestingly, there was neither a difference in sero-prevalence nor in IS2404 PCR positivity of environmental samples between BU endemic and non-endemic communities located in the Densu River Valley., Conclusions: These data indicate that the intensity of exposure to M. ulcerans in endemic and non-endemic communities along the Densu River is comparable and that currently unknown host and/or pathogen factors may determine how frequently exposure is leading to clinical disease. While even high serum titers of anti-18 kDa shsp IgG do not indicate active disease, sero-epidemiological studies can be used to identify new BU endemic areas.

Background: Buruli ulcer (BU) is a destructive skin condition caused by infection with the environmental bacterium, Mycobacterium ulcerans. The mode of transmission of M. ulcerans is not completely understood, but several studies have explored the role of biting insects. In this study, we tested for an association between the detection of M. ulcerans in mosquitoes and the risk of BU disease in humans in an endemic area of southeastern Australia., Methodology/principal Findings: Adult mosquitoes were trapped in seven towns on the Bellarine Peninsula in Victoria, Australia, from December 2004 to December 2009 and screened for M. ulcerans by real-time PCR. The number of laboratory-confirmed cases of BU in permanent residents of these towns diagnosed during the same period was tallied to determine the average cumulative incidence of BU in each location. Pearson's correlation coefficient (r) was calculated for the proportion of M. ulcerans-positive mosquitoes per town correlated with the incidence of BU per town. We found a strong dose-response relationship between the detection of M. ulcerans in mosquitoes and the risk of human disease (r, 0.99; 95% CI, 0.92-0.99; p < 0.001)., Conclusions/significance: The results of this study strengthen the hypothesis that mosquitoes are involved in the transmission of M. ulcerans in southeastern Australia. This has implications for the development of intervention strategies to control and prevent BU.

It has recently been shown that in a Buruli ulcer (BU) endemic region of southeastern Australia, significant numbers of possums (native tree-dwelling marsupials) have clinical BU disease. Furthermore, based on quantitative PCR (qPCR) analysis, animals with BU lesions (and some without) shed M. ulcerans DNA in their faeces, indicative of bacterial loads of up to 10(8) organisms/gram. These findings led us to propose that humans might also harbour M. ulcerans in their gastrointestinal tract and shed the bacterium in their faeces. We conducted a pilot study and collected faecal swabs from 26 patients with confirmed BU and 31 healthy household controls. Faecal samples were also collected from 10 healthy controls from non-endemic regions in Ghana. All 67 specimens were negative when tested by IS2404 PCR. The detection sensitivity of this method was ≥10(4) bacteria per gram (wet-weight) of human faecal material. We conclude that the human gastrointestinal tract is unlikely to be a significant reservoir of M. ulcerans.

Background: Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a destructive skin disease found predominantly in sub-Saharan Africa and south-eastern Australia. The precise mode(s) of transmission and environmental reservoir(s) remain unknown, but several studies have explored the role of aquatic invertebrate species. The purpose of this study was to investigate the environmental distribution of M. ulcerans in south-eastern Australia., Methodology/principal Findings: A range of environmental samples was collected from Point Lonsdale (a small coastal town southwest of Melbourne, Australia, endemic for BU) and from areas with fewer or no reported incident cases of BU. Mycobacterium ulcerans DNA was detected at low levels by real-time PCR in soil, sediment, water residue, aquatic plant biofilm and terrestrial vegetation collected in Point Lonsdale. Higher levels of M. ulcerans DNA were detected in the faeces of common ringtail (Pseudocheirus peregrinus) and common brushtail (Trichosurus vulpecula) possums. Systematic testing of possum faeces revealed that M. ulcerans DNA could be detected in 41% of faecal samples collected in Point Lonsdale compared with less than 1% of faecal samples collected from non-endemic areas (p<0.0001). Capture and clinical examination of live possums in Point Lonsdale validated the accuracy of the predictive value of the faecal surveys by revealing that 38% of ringtail possums and 24% of brushtail possums had laboratory-confirmed M. ulcerans skin lesions and/or M. ulcerans PCR positive faeces. Whole genome sequencing revealed an extremely close genetic relationship between human and possum M. ulcerans isolates., Conclusions/significance: The prevailing wisdom is that M. ulcerans is an aquatic pathogen and that BU is acquired by contact with certain aquatic environments (swamps, slow-flowing water). Now, after 70 years of research, we propose a transmission model for BU in which terrestrial mammals are implicated as reservoirs for M. ulcerans.