Heterogeneity among Mycobacterium ulcerans isolates from Africa

Mycobacterium ulcerans causes Buruli ulcer, an ulcerative skin disease in tropical and subtropical areas. Despite restricted genetic diversity, mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis on M. ulcerans revealed 3 genotypes from different African countries. It is the first time this typing method succeeded directly on patient samples

Evaluation of different DNA extraction methods and loop-mediated isothermal amplification primers for the detection of Mycobacterium ulcerans in clinical specimens

Background: Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. Methods: In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. Results: Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75-91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. Conclusions: For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50-91.49%) and specificity (89.23-100%), depending on the DNA extraction methods used.This study was supported by the Swiss Agency for Development and Cooperation and the UBS Optimus Foundation through FIND, Geneva-Switzerland. The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.S

Single Nucleotide Polymorphism Typing of Mycobacterium ulcerans Reveals Focal Transmission of Buruli Ulcer in a Highly Endemic Region of Ghana

Buruli ulcer (BU) is an emerging necrotizing disease of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. While proximity to stagnant or slow flowing water bodies is a risk factor for acquiring BU, the epidemiology and mode of M. ulcerans transmission is poorly understood. Here we have used high-throughput DNA sequencing and comparisons of the genomes of seven M. ulcerans isolates that appeared monomorphic by existing typing methods. We identified a limited number of single nucleotide polymorphisms (SNPs) and developed a real-time PCR SNP typing method based on these differences. We then investigated clinical isolates of M. ulcerans on which we had detailed information concerning patient location and time of diagnosis. Within the Densu river basin of Ghana we observed dominance of one clonal complex and local clustering of some of the variants belonging to this complex. These results reveal focal transmission and demonstrate, that micro-epidemiological analyses by SNP typing has great potential to help us understand how M. ulcerans is transmitted

Mycobacterium ulcerans in Mosquitoes Captured during Outbreak of Buruli Ulcer, Southeastern Australia

Mosquitoes positive for M. ulcerans were linked to outbreaks of Buruli ulcer in humans

Virulence potential of Staphylococcus aureus isolates from Buruli ulcer patients

Buruli ulcer (BU) is a necrotizing infection of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. BU wounds may also be colonized with other microorganisms including Staphylococcus aureus. This study aimed to characterize the virulence factors of S. aureus isolated from BU patients. Previously sequenced genomes of 21 S. aureus isolates from BU patients were screened for the presence of virulence genes. The results show that all S. aureus isolates harbored on their core genomes genes for known virulence factors like alpha-hemolysin, and the a and beta-phenol soluble modulins. Besides the core genome virulence genes, mobile genetic elements (MGEs), i.e. prophages, genomic islands, pathogenicity islands and a Staphylococcal cassette chromosome (ECG) were found to carry different combinations of virulence factors, among them genes that are known to encode factors that promote immune evasion, superantigens and Panton-Valentine Leucocidin. The present observations imply That the S. aureus isolates from BU patients harbor a diverse repertoire of virulence genes that may enhance bacterial survival and persistence in the wound environment and potentially contribute to delayed wound healing

Molecular Characterization of Staphylococcus aureus Isolates Transmitted between Patients with Buruli Ulcer

BACKGROUND:Buruli ulcer (BU) is a skin infection caused by Mycobacterium ulcerans. The wounds of most BU patients are colonized with different microorganisms, including Staphylococcus aureus. METHODOLOGY:This study investigated possible patient-to-patient transmission events of S. aureus during wound care in a health care center. S. aureus isolates from different BU patients with overlapping visits to the clinic were whole-genome sequenced and analyzed by a gene-by-gene approach using SeqSphere(+) software. In addition, sequence data were screened for the presence of genes that conferred antibiotic resistance. PRINCIPAL FINDINGS:SeqSphere(+) analysis of whole-genome sequence data confirmed transmission of methicillin resistant S. aureus (MRSA) and methicillin susceptible S. aureus among patients that took place during wound care. Interestingly, our sequence data show that the investigated MRSA isolates carry a novel allele of the fexB gene conferring chloramphenicol resistance, which had thus far not been observed in S. aureus

Genomic Diversity and Evolution of Mycobacterium ulcerans Revealed by Next-Generation Sequencing

Mycobacterium ulcerans is the causative agent of Buruli ulcer, the third most common mycobacterial disease after tuberculosis and leprosy. It is an emerging infectious disease that afflicts mainly children and youths in West Africa. Little is known about the evolution and transmission mode of M. ulcerans, partially due to the lack of known genetic polymorphisms among isolates, limiting the application of genetic epidemiology. To systematically profile single nucleotide polymorphisms (SNPs), we sequenced the genomes of three M. ulcerans strains using 454 and Solexa technologies. Comparison with the reference genome of the Ghanaian classical lineage isolate Agy99 revealed 26,564 SNPs in a Japanese strain representing the ancestral lineage. Only 173 SNPs were found when comparing Agy99 with two other Ghanaian isolates, which belong to the two other types previously distinguished in Ghana by variable number tandem repeat typing. We further analyzed a collection of Ghanaian strains using the SNPs discovered. With 68 SNP loci, we were able to differentiate 54 strains into 13 distinct SNP haplotypes. The average SNP nucleotide diversity was low (average 0.06–0.09 across 68 SNP loci), and 96% of the SNP locus pairs were in complete linkage disequilibrium. We estimated that the divergence of the M. ulcerans Ghanaian clade from the Japanese strain occurred 394 to 529 thousand years ago. The Ghanaian subtypes diverged about 1000 to 3000 years ago, or even much more recently, because we found evidence that they evolved significantly faster than average. Our results offer significant insight into the evolution of M. ulcerans and provide a comprehensive report on genetic diversity within a highly clonal M. ulcerans population from a Buruli ulcer endemic region, which can facilitate further epidemiological studies of this pathogen through the development of high-resolution tools

Detection of Mycobacterium ulcerans by the Loop Mediated Isothermal Amplification Method

In order to develop a simple and rapid test that can be used to diagnose Buruli ulcer under field conditions, we modified the conventional LAMP assay by using a disposable pocket warmer as a heating device for generating a constant temperature for the test reaction and employed the use of crude sample preparations consisting of boiled and unboiled extracts of the clinical specimen instead of using purified DNA as the diagnostic specimen. Thirty clinical specimens from suspected Buruli ulcer patients were investigated by the modified LAMP (or pocket warmer LAMP) and the conventional LAMP, as well as IS2404 PCR, a reference method for the detection of Mycobacterium ulcerans. There was no significant difference in the detection rate (63–70%) in all of the methods when purified samples were used for the tests. On the other hand the use of crude specimen preparation resulted in a drop in detection rate (30–40%). This study demonstrates that the LAMP test can be used for rapid detection of M. ulcerans when purified DNA preparations are used. With further improvements in the sample reaction, as well as in specimen purification, the pocket warmer LAMP may provide a simple and rapid diagnostic test for Buruli ulcer

Domestic animals infected with Mycobacterium ulcerans - implications for transmission to humans

Open Access Journal; Published online: 2 July 2018Background The environmental pathogen, Mycobacterium ulcerans (MU) can infect both humans and animals and cause Buruli ulcer (BU) disease. However, its mode(s) of transmission from the colonized environment to human/animal hosts remain unclear. In Australia, MU can infect both wildlife and domestic mammals. Till date, BU-like lesions have only been reported in wildlife in Africa. This warrants a thorough assessment of possible MU in domestic animals in Africa. Here, we screened roaming domesticated animals that share the human microhabitat in two different BU endemic sites, Sedje-Denou in Benin and Akonolinga in Cameroon, for MU lesions. Methodology/Principal findings We screened roaming mammals and birds across 3 endemic villages of Sedje-Denou in Southern Benin and 6 endemic villages of Akonolinga in Cameroon. After approval from relevant authorities, specimens (wound swabs and tissue fragments) were collected from animals with open or active lesion and systematically screened to detect the presence of MU though the diagnostic DNA targets IS2404, IS2606 and KR-B. Out of 397 animals surveyed in Akonolinga, 44 (11.08%) carried skin lesions and all were negative for MU DNA. For Sedje-Denou, only 25 (6.93%) out of 361 animals surveyed carried external skin lesions of which 2 (8%) were positive for MU DNA targets. These MU infected lesions were found in two different villages on a goat (abdominal part) and on a dog (nape area of the neck). Source-tracking of MU isolates within infected animal lesions was performed using VNTR genotyping and further confirmed with sequencing. One MU VNTR genotype (Z) was successfully typed from the goat lesion. The evolutionary history inferred from sequenced data revealed a clustering of animal MU isolates within isolates from human lesions. Conclusion/Significance This study describes the first report of two MU infected lesions in domestic animals in Africa. Their DNA sequence analyses show close relationship to isolates from human cases. It suggests that MU infection should be suspected in domestic hosts and these could play a role in transmission. The findings further support the hypothesis that MU is a ubiquitous environmental pathogen found in endemic areas, and probably involved in a multiple transmission pathway

Distribution of Mycobacterium ulcerans in Buruli Ulcer Endemic and Non-Endemic Aquatic Sites in Ghana

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is an emerging environmental bacterium in Australia and West Africa. The primary risk factor associated with Buruli ulcer is proximity to slow moving water. Environmental constraints for disease are shown by the absence of infection in arid regions of infected countries. A particularly mysterious aspect of Buruli ulcer is the fact that endemic and non-endemic villages may be only a few kilometers apart within the same watershed. Recent studies suggest that aquatic invertebrate species may serve as reservoirs for M. ulcerans, although transmission pathways remain unknown. Systematic studies of the distribution of M. ulcerans in the environment using standard ecological methods have not been reported. Here we present results from the first study based on random sampling of endemic and non-endemic sites. In this study PCR-based methods, along with biofilm collections, have been used to map the presence of M. ulcerans within 26 aquatic sites in Ghana. Results suggest that M. ulcerans is present in both endemic and non-endemic sites and that variable number tandem repeat (VNTR) profiling can be used to follow chains of transmission from the environment to humans. Our results suggesting that the distribution of M. ulcerans is far broader than the distribution of human disease is characteristic of environmental pathogens. These findings imply that focal demography, along with patterns of human water contact, may play a major role in transmission of Buruli ulcer