Mycobacterium ulcerans challenge strain selection for a Buruli ulcer controlled human infection model.

Critical scientific questions remain regarding infection with Mycobacterium ulcerans, the organism responsible for the neglected tropical disease, Buruli ulcer (BU). A controlled human infection model has the potential to accelerate our knowledge of the immunological correlates of disease, to test prophylactic interventions and novel therapeutics. Here we present microbiological evidence supporting M. ulcerans JKD8049 as a suitable human challenge strain. This non-genetically modified Australian isolate is susceptible to clinically relevant antibiotics, can be cultured in animal-free and surfactant-free media, can be enumerated for precise dosing, and has stable viability following cryopreservation. Infectious challenge of humans with JKD8049 is anticipated to imitate natural infection, as M. ulcerans JKD8049 is genetically stable following in vitro passage and produces the key virulence factor, mycolactone. Also reported are considerations for the manufacture, storage, and administration of M. ulcerans JKD8049 for controlled human infection.

Pre-extensively Drug-Resistant Congenital Tuberculosis in an Extremely Premature Baby.

We describe a case of congenital tuberculosis in an extremely premature baby, with rapid molecular detection of a pre-extensively drug-resistant (XDR) pattern of drug resistance. The baby was treated successfully with a regimen including bedaquline and delamanid, drugs not previously described in the treatment of congenital tuberculosis (TB).

Whole genome sequencing of drug-resistant Mycobacterium tuberculosis isolates in Victoria, Australia.

OBJECTIVES: Whole genome sequencing (WGS) can identify clusters, transmission patterns, and drug resistance mutations. This is important in low-burden settings such as Australia, as it can assist in efficient contact tracing and surveillance. METHODS: We conducted a retrospective cohort study using WGS from 155 genomically defined drug-resistant Mycobacterium tuberculosis (DR-TB) isolates collected between 2018-2021 in Victoria, Australia. Bioinformatic analysis was performed to identify resistance-conferring mutations, lineages, clusters and understand how local sequences compared with international context. RESULTS: Of the 155 sequences, 42% were identified as lineage 2 and 35% as lineage 1; 65.8% (102/155) were isoniazid mono-resistant, 8.4% were multi-drug resistant TB and 5.8% were pre-extensively drug-resistant / extensively drug-resistant TB. The most common mutations were observed in katG and fabG1 genes, especially at Ser315Thr and fabG1 -15 C>T for first-line drugs. Ser450Leu was the most frequent mutation in rpoB gene. Phylogenetic analysis confirmed that Victorian DR-TB were associated with importation events. There was little evidence of local transmission with only five isolate pairs. CONCLUSION: Isoniazid-resistant TB is the commonest DR-TB in Victoria, and the mutation profile is similar to global circulating DR-TB. Most cases are diagnosed among migrants with limited transmission. This study highlights the value of WGS in identification of clusters and resistance-conferring mutations. This information is crucial in supporting disease mitigation and treatment strategies.

Mycobacterium tuberculosis Genotypes and Drug Susceptibility Test Results from Timor-Leste: A Pilot Study.

Tuberculosis (TB) is prevalent and a major public health problem in Timor-Leste. The government of Timor-Leste is prioritising the surveillance of TB and drug-susceptibility testing (DST) to understand the burden of TB and TB drug resistance in the country. Moreover, little is known about the origin of Mycobacterium tuberculosis (MTB) in Timor-Leste. This study reports MTB DST and sequencing for Timor-Leste. A pilot study was carried out in which a convenience sample of TB isolates from mucopurulent sputum collected from presumptive TB patients in the capital Dili between July and December 2016 was tested for phenotypic and genotypic evidence of drug resistance. Standard MTB culture was performed at the Timor-Leste National Health Laboratory (NHL). The MTB isolates were sent to the Victorian Infectious Diseases Reference Laboratory (VIDRL) in Australia for DST and sequencing. Overall, 36 MTB isolates were detected at the NHL; 20 isolates were recovered during sub-culturing at VIDRL. All 20 isolates were susceptible to rifampicin, isoniazid, pyrazinamide, and ethambutol, with no genotypic markers of resistance identified. On sequencing, lineage 4 was the most common. The results of this study provide a small snapshot of MTB diversity and resistance in an under-sampled region with very high TB incidence. Future investment in whole-genome sequencing capacity in Timor-Leste will make it possible to undertake further, more representative analyses that may be used to evaluate transmission dynamics and epidemiology of genotypic markers of resistance.

Identification and antimicrobial susceptibility of referred Nocardia isolates in Victoria, Australia 2009-2019.

Introduction. Nocardia is an opportunistic pathogen that can cause significant morbidity and mortality, particularly in the immunocompromised host. Antimicrobial susceptibility profiles vary across Nocardia spp. and vary within Australia as well as worldwide. Knowledge of local susceptibility patterns is important in informing appropriate empiric antimicrobial therapy.Gap Statement. This is the largest study to date in Australia that correlates antimicrobial susceptibility profiles with molecular identification of Nocardia species. It is the first study that examines isolates from multiple institutions across the state of Victoria, Australia.Aim. To investigate the species distribution and antibiotic susceptibility of Nocardia spp. isolates referred to the Mycobacterial Reference Laboratory (MRL) in Victoria, Australia from 2009 to 2019.Methodology. We conducted a retrospective review of Nocardia spp. isolates which were identified using molecular sequencing. Antimicrobial susceptibility testing was performed using standardized broth microdilution method with Sensititre RAPMYCO1 plates. Species distribution and antibiotic susceptibility profiles were analysed.Results. In total, 414 Nocardia isolates were identified to 27 species levels, the majority originating from the respiratory tract (n=336, 81.2 %). N. nova (n=147, 35.5 %) was the most frequently isolated, followed by N. cyriacigeorgica (n=75, 18.1 %). Species distribution varied by isolate source, with N. farcinica and N. paucivorans found more commonly from sterile sites. Linezolid and amikacin had the highest proportion of susceptible isolates (100 and 99% respectively), while low susceptibility rates were detected for ceftriaxone (59 %) and imipenem (41 %). Susceptibility to trimethoprim sulfamethoxazole varied by species (0-100 %).Conclusion. This is the largest study to date in Australia of Nocardia species distribution and antimicrobial susceptibility patterns. N. farcinica and N. paucivorans were more likely to be isolated from sterile sites, while N. brasiliensis and N. otitidiscvarium were more likely to be isolated from skin and soft tissue. First line therapeutic antimicrobial recommendations by local guidelines were not necessarily reflective of the in vitro susceptibility of Nocardia isolates in this study, with high susceptibility detected for linezolid and amikacin, but poor susceptibility demonstrated for ceftriaxone and imipenem. Profiles for trimethoprim-sulfamethoxazole varied across different Nocardia species, warranting ongoing susceptibility testing for targeted clinical use.

Whole genome sequencing for tuberculosis in Victoria, Australia: A genomic implementation study from 2017 to 2020.

Background: Whole genome sequencing (WGS) is increasingly used by tuberculosis (TB) programs to monitor Mycobacterium tuberculosis (Mtb) transmission. We aimed to characterise the molecular epidemiology of TB and Mtb transmission in the low-incidence setting of Victoria, Australia, and assess the utility of WGS. Methods: WGS was performed on all first Mtb isolates from TB cases from 2017 to 2020. Potential clusters (≤12 single nucleotide polymorphisms [SNPs]) were investigated for epidemiological links. Transmission events in highly-related (≤5 SNPs) clusters were classified as likely or possible, based on the presence or absence of an epidemiological link, respectively. Case characteristics and transmission settings (as defined by case relationship) were summarised. Poisson regression was used to examine associations with secondary case number. Findings: Of 1844 TB cases, 1276 (69.2%) had sequenced isolates, with 182 (14.2%) in 54 highly-related clusters, 2-40 cases in size. Following investigation, 140 cases (11.0% of sequenced) were classified as resulting from likely/possible local-transmission, including 82 (6.4%) for which transmission was likely. Common identified transmission settings were social/religious (26.4%), household (22.9%) and family living in different households (7.1%), but many were uncertain (41.4%). While household transmission featured in many clusters (n = 24), clusters were generally smaller (median = 3 cases) than the fewer that included transmission in social/religious settings (n = 12, median = 7.5 cases). Sputum-smear-positivity was associated with higher secondary case numbers. Interpretation: WGS results suggest Mtb transmission commonly occurs outside the household in our low-incidence setting. Further work is required to optimise the use of WGS in public health management of TB. Funding: The Victorian Tuberculosis Program receives block funding for activities including case management and contact tracing from the Victorian Department of Health. No specific funding for this report was received by manuscript authors or the Victorian Tuberculosis Program, and the funders had no role in the study design, data collection, data analysis, interpretation or report writing.

Tuberculosis in Australia's tropical north: a population-based genomic epidemiological study.

BACKGROUND: The Northern Territory (NT) has the highest tuberculosis (TB) rate of all Australian jurisdictions. We combined TB public health surveillance data with genomic sequencing of Mycobacterium tuberculosis isolates in the tropical 'Top End' of the NT to investigate trends in TB incidence and transmission. METHODS: This retrospective observational study included all 741 culture-confirmed cases of TB in the Top End over three decades from 1989-2020. All 497 available M. tuberculosis isolates were sequenced. We used contact tracing data to define a threshold pairwise SNP distance for hierarchical single linkage clustering, and examined putative transmission clusters in the context of epidemiologic information. FINDINGS: There were 359 (48%) cases born overseas, 329 (44%) cases among Australian First Nations peoples, and 52 (7%) cases were Australian-born and non-Indigenous. The annual incidence in First Nations peoples from 1989-2019 fell from average 50.4 to 11.0 per 100,000 (P<0·001). First Nations cases were more likely to die from TB (41/329, 12·5%) than overseas-born cases (11/359, 3·1%; P<0·001). Using a threshold of ≤12 SNPs, 28 clusters of between 2-64 individuals were identified, totalling 250 cases; 214 (86%) were First Nations cases and 189 (76%) were from a remote region. The time between cases and past epidemiologically- and genomically-linked contacts ranged from 4·5 months to 24 years. INTERPRETATION: Our findings support prioritisation of timely case detection, contact tracing augmented by genomic sequencing, and latent TB treatment to break transmission chains in Top End remote hotspot regions.

Population genomics provides insights into the evolution and adaptation to humans of the waterborne pathogen Mycobacterium kansasii.

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.

Buruli ulcer: a new case definition for Victoria.

ABSTRACT: Laboratory-confirmed infection with Mycobacterium ulcerans is currently notifiable to health departments in several jurisdictions. Accurate surveillance is imperative to understanding current and emerging areas of endemicity and to facilitate research into a neglected tropical disease with poorly-understood transmission dynamics. The state of Victoria currently reports some of the highest numbers of M. ulcerans cases in the world each year, with 340 cases notified in 2018 (an incidence of 5.5 per 100,000 population). In May 2019, a group of clinical, laboratory and public health experts met to discuss a new case definition for the surveillance of M. ulcerans disease in Victoria, incorporating clinical and epidemiological elements. The new case definition supports important public health messaging and actions for residents and visitors to popular tourist areas in Victoria.

Mycobacterium abscessus bloodstream infection: Unexpected catheter tunnel infection localized by PET/CT.

Mycobacterium abscessus is an emerging cause of invasive infection in the immunosuppressed population. We report a case of M. abscessus bloodstream and catheter tunnel infection localized by positron emission tomography/computer tomography (PET/CT) in an allogeneic haematopoietic stem cell transplant recipient. This case highlights the difficulties in treating invasive M. abscessus infection and the potential role of PET/CT in localizing infection and guiding therapy in this population.

Epidemiology of Buruli Ulcer Infections, Victoria, Australia, 2011-2016.

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.

The incubation period of Buruli ulcer (Mycobacterium ulcerans infection) in Victoria, Australia - Remains similar despite changing geographic distribution of disease.

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.

Comparative Genomics Shows That Mycobacterium ulcerans Migration and Expansion Preceded the Rise of Buruli Ulcer in Southeastern Australia.

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.

Molecular epidemiology of tuberculosis in Tasmania and genomic characterisation of its first known multi-drug resistant case.

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.

Feline leprosy due to Candidatus 'Mycobacterium lepraefelis': Further clinical and molecular characterisation of eight previously reported cases and an additional 30 cases.

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'.

Feline leprosy due to Mycobacterium lepraemurium.

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.