First Molecular Characterisation of Clinical Strains of Mycobacterium ulcerans in Togo (West Africa)

Background: Buruli ulcer is the third most common mycobacterial disease worldwide. Cases most occur in 30 countries but severe cases occur in West Africa countries such as Benin, Cote d’Ivoire and Togo mainly in rural regions. Early diagnosis may prevent severe disability. The molecular technique seems the best solution and new Mycobacterial Interspersed Repetitive Units (MIRU) and variable number tandem repeats (VNTR) typing method are themost reproducible in this regard. They propose geographical, inter and intraspecies differentiation and can be used as a diagnosis tool. Objective: The objective of this study was to investigate the molecular diversity by using MIRUVNTR typing in clinical samples of BU patients in Togo. Study Design: 64 DNA extracts from clinical samples were collected from BU patients in the two principal endemics districts in Togo (Yoto and Zio) with three less endemic districts (Bas Mono, Lacs and Vo). First, IS2404 and KR real-time PCR plus IS2606 conventional PCR were performed. In a second step, the strains were analysed by PCR typing for five specific and sensitive markers MIRU1, VNTR6, ST1, VNTR19 and VNTR9. Results and Conclusion: 71.11% were positive for IS2404, 3.13% were positives for PCR-KR and 31.11% for IS 2606. By MIRU-VNTR typing, 48.86% positive result was found for MIRU1 and 25.00%, 20.31%, 18.75% and 14.06% for VNTR6, ST1, VNTR19 and VNTR9 respectively. One of the samples was negative for all genotyping markers. Two different genetic profiles were identified by MIRU1, ST1 and VNTR loci by gel-analysed of the amplified products. The VNTR profile B (3,1,1,2) corresponding of 3 copies MIRU1, 1 copy VNTR6, 1 copy ST-1 and two copies of VNTR19 was detected in 15.63% of samples and the VNTR profile A (1,1,1,2) corresponding of 1 copy MIRU1, 1 copy VNTR6, 1 copy ST-1 and 2 copies of VNTR19 was detected in 3.13% of samples and confirms the West African genotype (3,1,1) in Togo. Different genetic strains of Mycobacterium ulcerans (M. ulcerans) were co-circulated in the same endemic region in the country. This study has described first the circulating of different genetic strains of M. ulcerans in Togo.

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Multilocus VNTR analysis of Mycobacterium ulcerans strains isolated in Côte d’Ivoire

The Journal of Infection in Developing Countries ◽

10.3855/jidc.1364 ◽

2010 ◽

Vol 5 (01) ◽

pp. 059-063 ◽

Cited By ~ 3

Author(s):

Grossmann Marie-David Coulibaly-N´Golo ◽

Euloge Ekaza ◽

Bakary Coulibaly ◽

N’guetta Aka ◽

Raymond Kouassi N’Guessan ◽

...

Keyword(s):

Cote D'ivoire ◽

Tandem Repeats ◽

Côte D’Ivoire ◽

Buruli Ulcer ◽

Variable Number ◽

Mycobacterium Ulcerans ◽

Clinical Samples ◽

Bacterial Strains ◽

Cote D’Ivoire ◽

Côte D'ivoire

Introduction: Buruli ulcer, caused by Mycobacterium ulcerans, is endemic in more than 30 countries worldwide, with Côte d'Ivoire being among the most affected countries. Methodology: We used seven variable number of tandem repeats (VNTR) markers and analyzed 114 samples from 11 Ivorian localities consisting of 33 bacterial strains and 81 clinical samples. Complete data sets at loci 1, 6, 9 and 33 were obtained for 18 of these strains (n = 15) and samples (n = 3) collected in each of the localities. Results: All the strains had allelic profile [3113], corresponding to the previously described Atlantic Africa genotype. Conclusion: Sequencing of PCR products at all loci showed no variation in sequence or repeat number, underlining the genetic monomorphism of M. ulcerans in Côte d'Ivoire.

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Community-based geographical distribution of Mycobacterium ulcerans VNTR-genotypes from the environment and humans in the Nyong valley, Cameroon

Tropical Medicine and Health ◽

10.1186/s41182-021-00330-2 ◽

2021 ◽

Vol 49 (1) ◽

Author(s):

Francis Zeukeng ◽

Anthony Ablordey ◽

Solange E. Kakou-Ngazoa ◽

Stephen Mbigha Ghogomu ◽

David N’golo Coulibaly ◽

...

Keyword(s):

Environmental Samples ◽

Tandem Repeats ◽

Buruli Ulcer ◽

Variable Number ◽

Mycobacterium Ulcerans ◽

Community Based ◽

Human Samples ◽

Route Of Transmission ◽

Mode Of Transmission ◽

Animal Reservoirs

Abstract Background Genotyping is a powerful tool for investigating outbreaks of infectious diseases and it can provide useful information such as identifying the source and route of transmission, and circulating strains involved in the outbreak. Genotyping techniques based on variable number of tandem repeats (VNTR) are instrumental in detecting heterogeneity in Mycobacterium ulcerans (MU) and also for discriminating MU from other mycobacteria species. Here, we describe and map the distribution of MU genotypes in Buruli ulcer (BU) endemic communities of the Nyong valley in Cameroon. We also tested the hypothesis of whether the suspected animal reservoirs of BU that share the human microhabitat are shedding contaminated fecal matters and saliva into their surrounding environments. Methods Environmental samples from suspected MU-risk factors and lesion swabs from human patients were sampled in BU-endemic communities and tested for the presence of MU by qPCR targeting three independent sequences (IS2404, IS2606, KR-B). Positive samples to MU were further genotyped by VNTR with confirmation by sequencing of four loci (MIRU1, Locus 6, ST1, Locus 19). Results MU was detected in environmental samples including water bodies (23%), biofilms (14%), detritus (10%), and in human patients (73%). MU genotypes D, W, and C were found both in environmental and human samples. The micro geo-distribution of MU genotypes from communities showed that genotype D is found both in environmental and human samples, while genotypes W and C are specific to environmental samples and human lesions, respectively. No obvious focal grouping of MU genotypes was observed at the community scale. An additional survey in the human microhabitat suggests that domestic and wild animals do not shed MU in their saliva and feces in sampled communities. Conclusions VNTR typing uncovered different MU genotypes circulating in the endemic communities of the Akonolinga district. A MU environmental genotype was found in patients, yet the mechanism of contamination remains to be investigated; and recovering MU in culture from the environment remains key priority to enable a better understanding of the mode of transmission of BU. We also conclude that excretions from suspected animals are unlikely to be major sources of MU in the Nyong Valley in Cameroon.

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First isolation of Mycobacterium ulcerans from Swabs and Fine-Needle-Aspiration Specimens in Togo.

10.21203/rs.2.17920/v1 ◽

2019 ◽

Author(s):

Tchalare Kondi Makagni ◽

Maman Issaka ◽

Piten Ebekalisai ◽

Disse Kodjo ◽

Essossimna A. Kadanga ◽

...

Keyword(s):

Fine Needle Aspiration ◽

Slow Growth ◽

Buruli Ulcer ◽

Needle Aspiration ◽

Mycobacterium Ulcerans ◽

Clinical Samples ◽

Direct Examination ◽

Fine Needle ◽

Pcr Targeting

Abstract Background Buruli ulcer is a skin disease caused by a mycobacterium called Mycobacterium ulcerans . It is prevalent in more than 33 countries on several continents but West Africa is the most affected. The isolation in culture of the bacteria is difficult because of its slow growth and the facilities required. In Togo, studies have been done on the risk factors for Mycobacterium ulcerans infection and the detection of cases by the Ziehl-Neelsen and PCR technique on clinical and environmental samples, but to date no data of isolates from clinical samples are available. The purpose of this study was to perform an in vitro culture of M. ulcerans from swab and fine needle aspiration samples through the confirmation stages of direct examination and IS2404 -PCR. Method A total of 70 clinical samples from Togo and 10 clinical isolates from Benin are analyzed by the three techniques indicated in the diagnosis, in particular the direct examination of acid-fast bacilli (AFB) using the Ziehl-Neelsen staining, the PCR targeting the IS2404 sequence, and the culture after transport of the samples in a transport medium made of Middlebrook 7H9 medium supplemented with a mixture of PANTA and OADC and decontamination by the modified Petroff method. Results The application of the three techniques of diagnosis for clinical samples yielded 44.28% of positivity rates on direct examination of AFB, 35.71% on culture and 77.14% on qPCR IS2404 with a significantly higher rate for qPCR (0.0001). All samples positive for Ziehl-Neelsen staining and culture were also positive for qPCR. Conclusion : Our results show that the culture, despite it difficulty and the slow growth of the bacteria, can be carried out with recommended tools of the mycobacteria culture and a good method of decontamination of the samples can improve the positivity rates. Its realization will allow the assessment of the in vitro sensitivity to the antibiotics used in the treatment and the discovery of new strains of Mycobacterium ulcerans .

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OC 8173 RAPID DETECTION OF MYCOBACTERIUM ULCERANS BY RECOMBINASE POLYMERASE AMPLIFICATION

BMJ Global Health ◽

10.1136/bmjgh-2019-edc.2 ◽

2019 ◽

Vol 4 (Suppl 3) ◽

pp. A2.1-A2

Author(s):

Michael Frimpong ◽

Hubert Ahor ◽

Francisca Sarpong ◽

Ken Laing ◽

Mark Wansbrough-Jones ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Point Of Care ◽

Clinical Signs ◽

Buruli Ulcer ◽

Cross Reactivity ◽

Current Control ◽

Mycobacterium Ulcerans ◽

Recombinase Polymerase Amplification ◽

Clinical Samples ◽

Clinical Sensitivity

BackgroundThere are no primary measures to prevent people from contracting Buruli ulcer, mainly due to poor understanding of its epidemiology. The current control strategy emphasises early diagnosis and prompt treatment, with the goal of avoiding the complications associated with advanced stages of the disease. There is no diagnostic test for the disease appropriate for use at the primary health care level where most cases are detected and treated. Diagnosis based on clinical signs is unreliable in inexperienced hands and complicated by infections that have similar presentations. This study was to develop and evaluate the use of recombinase polymerase amplification (RPA) assay for the detection of Mycobacterium ulcerans at the point of patient care.MethodsA specific fragment of IS2404 of M. ulcerans was amplified in 15 min at a constant temperature of 42°C, using the RPA assay and analysed on a portable fluorometre. Themethod was tested for sensitivity and specificity with molecular standard of IS2404 DNA fragment, various M.ulcerans strains, other mycobacteria and environmentally associated bacteria. Additionally, the assay performance as a diagnostic tool was tested with archived DNA from symptomatic patients. All results were compared with that of a highly sensitive IS2404 PCR.ResultsThe detection limit was 50 copies of IS2404 in 15 min using plasmid standard and 125 fg with genomic Mu DNA equivalent 25 genomic copies. The assay was highly specific in detecting all strains of M. ulcerans with no observed cross reactivity with other mycobacteria and common skin colonising bacteria. The clinical sensitivity and specificity of the BU-RPA assay using clinical samples was 86% and 100% respectively.ConclusionWe have developed a real-time isothermal RPA assay for the detection of M. ulcerans as a cheaper alternative to PCR. Combining this assay with a simple extraction protocol will maximise its use as point-of-care test for Buruli ulcer.

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Whole Genome Comparisons Suggest Random Distribution of Mycobacterium ulcerans Genotypes in a Buruli Ulcer Endemic Region of Ghana

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0003681 ◽

2015 ◽

Vol 9 (3) ◽

pp. e0003681 ◽

Cited By ~ 12

Author(s):

Anthony S. Ablordey ◽

Koen Vandelannoote ◽

Isaac A. Frimpong ◽

Evans K. Ahortor ◽

Nana Ama Amissah ◽

...

Keyword(s):

Random Distribution ◽

Buruli Ulcer ◽

Mycobacterium Ulcerans ◽

Whole Genome ◽

Endemic Region ◽

Genome Comparisons

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Genetic diversity of Mycoplasma hyopneumoniae isolates from conventional farrow-to-finish pig farms in Serbia

Acta Veterinaria Hungarica ◽

10.1556/avet.58.2010.3.3 ◽

2010 ◽

Vol 58 (3) ◽

pp. 297-308 ◽

Cited By ~ 4

Author(s):

Bozidar Savic ◽

Vojin Ivetic ◽

Vesna Milicevic ◽

Ivan Pavlovic ◽

Milenko Zutic ◽

...

Keyword(s):

Tandem Repeats ◽

Genetic Relatedness ◽

Pcr Amplification ◽

Mycoplasma Hyopneumoniae ◽

Variable Number ◽

Repeat Motif ◽

Typing Method ◽

Pig Farms ◽

Different Ages ◽

Swine Population

Mycoplasma hyopneumoniaeis a primary agent associated with mycoplasma pneumonia and the porcine respiratory disease complex (PRDC). Various reports have indicated that different strains ofM. hyopneumoniaeare circulating in the swine population. Lysates from lung swabs from naturally infected pigs of different ages were tested according to a new variable number of tandem repeats (VNTR) genetic typing method based on the polyserine repeat motif of the P146 lipoproteoadhesin, which can be applied directly on clinical material without isolation ofM. hyopneumoniae. The aim was to determine the diversity ofM. hyopneumoniaeisolates from conventional farrow-to-finish pig farms located in different geographical areas of Serbia. PCR amplification was carried out usingM. hyopneumoniae-specific designed, conserved primers (p146MH — L and p146MH — R) flanking the region encoding the repeat motif, followed by sequencing and cluster analysis. Five groups ofM. hyopneumoniaewith thirteen to twenty-four serine repeats were observed. Analysis of three samples from each farm indicated that the specific isolate is ubiquitous in pigs of different ages. Furthermore, seven clusters were observed within 27 tested samples. The results indicated a considerable diversity amongM. hyopneumoniaefield isolates in the swine population from conventional farrow-to-finish farms in Serbia and suggest close genetic relatedness of the corresponding isolates.

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

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0000751 ◽

2010 ◽

Vol 4 (7) ◽

pp. e751 ◽

Cited By ~ 37

Author(s):

Katharina Röltgen ◽

Weihong Qi ◽

Marie-Thérèse Ruf ◽

Ernestina Mensah-Quainoo ◽

Sacha J. Pidot ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Buruli Ulcer ◽

Mycobacterium Ulcerans ◽

Nucleotide Polymorphism ◽

Endemic Region ◽

Single Nucleotide ◽

Single Nucleotide Polymorphism Typing

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Uneven Distribution of Mating Types among Genotypes of Candida glabrata Isolates from Clinical Samples

Eukaryotic Cell ◽

10.1128/ec.00215-08 ◽

2009 ◽

Vol 8 (3) ◽

pp. 287-295 ◽

Cited By ~ 45

Author(s):

Sylvain Brisse ◽

Christine Pannier ◽

Adela Angoulvant ◽

Thierry de Meeus ◽

Laure Diancourt ◽

...

Keyword(s):

Mating Type ◽

Candida Glabrata ◽

Tandem Repeats ◽

Minimum Spanning Tree ◽

Variable Number ◽

Clinical Samples ◽

Population Genetic Analysis ◽

Pathogenic Yeast ◽

Diploid Genotype ◽

Mating Type Switching

ABSTRACT In order to shed light on its basic biology, we initiated a population genetic analysis of Candida glabrata, an emerging pathogenic yeast with no sexual stage yet recognized. A worldwide collection of clinical strains was subjected to analysis using variable number of tandem repeats (VNTR) at nine loci. The clustering of strains obtained with this method was congruent with that obtained using sequence polymorphism of the NMT1 gene, a locus previously proposed for lineage assignment. Linkage disequilibrium supported the hypothesis of a mainly clonal reproduction. No heterozygous diploid genotype was found. Minimum-spanning tree analysis of VNTR data revealed clonal expansions and associated genotypic diversification. Mating type analysis revealed that 80% of the strains examined are MAT a and 20% MATα and that the two alleles are not evenly distributed. The MAT a genotype dominated within large clonal groups that contained only one or a few MATα types. In contrast, two groups were dominated by MATα strains. Our data are consistent with rare independent mating type switching events occurring preferentially from type a to α, although the alternative possibility of selection favoring type a isolates cannot be excluded.

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