scholarly journals Development and Application of Two Multiplex Real-Time PCR Assays for the Detection of Mycobacterium ulcerans in Clinical and Environmental Samples

2007 ◽  
Vol 73 (15) ◽  
pp. 4733-4740 ◽  
Author(s):  
Janet A. M. Fyfe ◽  
Caroline J. Lavender ◽  
Paul D. R. Johnson ◽  
Maria Globan ◽  
Aina Sievers ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Polyketide Synthase ◽  
Variable Number Tandem Repeat ◽  
Buruli Ulcer ◽  
Turnaround Time ◽  
Positive Control ◽  
Variable Number ◽  
Mycobacterium Ulcerans ◽  
Taqman Assay ◽  
Vntr Locus

ABSTRACT Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. PCR has become a reliable and rapid method for the diagnosis of M. ulcerans infection in humans and has been used for the detection of M. ulcerans in the environment. This paper describes the development of a TaqMan assay targeting IS2404 multiplexed with an internal positive control to monitor inhibition with a detection limit of less than 1 genome equivalent of DNA. The assay improves the turnaround time for diagnosis and replaces conventional gel-based PCR as the routine method for laboratory confirmation of M. ulcerans infection in Victoria, Australia. Following analysis of 415 clinical specimens, the new test demonstrated 100% sensitivity and specificity compared with culture. Another multiplex TaqMan assay targeting IS2606 and the ketoreductase-B domain of the M. ulcerans mycolactone polyketide synthase genes was designed to augment the specificity of the IS2404 PCR for the analysis of a variety of environmental samples. Assaying for these three targets enabled the detection of M. ulcerans DNA in soil, sediment, and mosquito extracts collected from an area of endemicity for Buruli ulcer in Victoria with a high degree of confidence. Final confirmation was obtained by the detection and sequencing of variable-number tandem repeat (VNTR) locus 9, which matched the VNTR locus 9 sequence obtained from the clinical isolates in this region. This suite of new methods is enabling rapid progress in the understanding of the ecology of this important human pathogen.

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

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.

scholarly journals Subtractive Hybridization Reveals a Type I Polyketide Synthase Locus Specific to Mycobacterium ulcerans

2003 ◽  
Vol 185 (23) ◽  
pp. 6870-6882 ◽  
Author(s):  
Grant A. Jenkin ◽  
Timothy P. Stinear ◽  
Paul D. R. Johnson ◽  
John K. Davies
Keyword(s):  
Genetic Basis ◽  
Polyketide Synthase ◽  
Buruli Ulcer ◽  
Subtractive Hybridization ◽  
Mycobacterial Infection ◽  
Mycobacterium Ulcerans ◽  
Suppressive Subtractive Hybridization ◽  
Type I ◽  
The Third ◽  
Close Genetic Relationship

ABSTRACT Mycobacterium ulcerans causes Buruli ulcer, the third most prevalent mycobacterial infection of immunocompetent humans after tuberculosis and leprosy. Recent work has shown that the production by M. ulcerans of mycolactone, a novel polyketide, may partly explain the pathogenesis of Buruli ulcer. To search for the genetic basis of virulence in M. ulcerans, we took advantage of the close genetic relationship between M. ulcerans and Mycobacterium marinum by performing genomic suppressive subtractive hybridization of M. ulcerans with M. marinum. We identified several DNA fragments specific to M. ulcerans, in particular, a type I polyketide synthase locus with a highly repetitive modular arrangement. We postulate that this locus is responsible for the synthesis of mycolactone in M. ulcerans.

scholarly journals Improvement of Multiple-Locus VNTR Analysis Typing Scheme for Helicobacter pylori

2019 ◽  
pp. 1-7
Author(s):  
Vladimir M. Sorokin ◽  
Ruslan V. Pisanov ◽  
Aleksej S. Vodop'janov
Keyword(s):  
Helicobacter Pylori ◽  
Genetic Relatedness ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Genome Database ◽  
Vntr Locus ◽  
Multiple Locus ◽  
H Pylori ◽  
Mlva Typing ◽  
Vntr Analysis

Aims: To improve a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) assay for Helicobacter pylori typing. Materials and Methods: Polymorphic VNTRs were searched by Gene Expert. The distribution and polymorphism of each VNTR locus were analyzed in 18 H. pylori genomes from the NCBI genome database by BLAST and were compared with a collection of 15 clinical H. pylori strains. The MLVA assay was compared with MLST-typing for discriminating H. pylori isolates. Results: Twelve VNTR loci were identified by bioinformatic screening of H. pylori genomes, and five of them were highly polymorphic. Therefore, an MLVA assay composed of five VNTR loci was developed with greatest discriminatory power. Conclusion: MLVA typing is a faster and more standardized method for studying the genetic relatedness of H. pylori isolates. At preliminary stage it is sufficient to use only 3 VNTR loci for the differentiation of H. pylori strains.

scholarly journals Evaluation of multiple-locus variable-number tandem repeat analysis (MLVA) for genotyping of Escherichia coli isolated from Karaj River

2017 ◽  
Vol 18 (1) ◽  
pp. 160-166
Author(s):  
Reza Ranjbar ◽  
Samaneh Shokouhi Mostafavi ◽  
Hamed Memariani
Keyword(s):  
Escherichia Coli ◽  
Water Quality ◽  
Tandem Repeat ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Coliform Bacteria ◽  
Vntr Locus ◽  
Multiple Locus ◽  
E Coli ◽  
Genotyping Method

Abstract Most microbiological water quality regulations rely upon the detection of indicators of fecal pollution, such as coliform bacteria, or more specifically Escherichia coli. In order to further understand the source, fate, and implications for water quality regulation, environmental E. coli isolates should be assessed genetically to observe various levels of genotypic diversity. Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) is a novel, simple and inexpensive polymerase chain reaction (PCR) based genotyping method which relies on the detection of different copy numbers inside each VNTR locus. In this study, we evaluated MLVA as a tool for the genotyping of E. coli strains of water samples collected from the Karaj River, Iran. Overall, high genetic diversity was observed among environmental E. coli isolates. We also proved the feasibility of MLVA as a complementary or even future replacement genotyping method for the average routine laboratory.

scholarly journals Isolation of Nontuberculous Mycobacteria from the Environment of Ghanian Communities Where Buruli Ulcer Is Endemic

2016 ◽  
Vol 82 (14) ◽  
pp. 4320-4329 ◽  
Author(s):  
Samuel Yaw Aboagye ◽  
Emelia Danso ◽  
Kobina Assan Ampah ◽  
Zuliehatu Nakobu ◽  
Prince Asare ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Nontuberculous Mycobacteria ◽  
Buruli Ulcer ◽  
Mycobacterium Ulcerans ◽  
Mycobacterium Fortuitum ◽  
Mycobacterial Species ◽  
Mycobacterium Chelonae ◽  
Content Type ◽  
Rate Of Recovery ◽  
Soil And Water

ABSTRACTThis study aimed to isolate nontuberculous mycobacterial species from environmental samples obtained from some selected communities in Ghana. To optimize decontamination, spiked environmental samples were used to evaluate four decontamination solutions and supplemented media, after which the best decontamination solution and media were used for the actual analysis. The isolates obtained were identified on the basis of specific genetic sequences, including heat shock protein 65, IS2404, IS2606,rpoB, and the ketoreductase gene, as needed. Among the methods evaluated, decontamination with 1 M NaOH followed by 5% oxalic acid gave the highest rate of recovery of mycobacteria (50.0%) and the lowest rate of contamination (15.6%). The cultivation medium that supported the highest rate of recovery of mycobacteria was polymyxin B-amphotericin B-nalidixic acid-trimethoprim-azlocillin–supplemented medium (34.4%), followed by isoniazid-supplemented medium (28.1%). Among the 139 samples cultivated in the main analysis, 58 (41.7%) yielded mycobacterial growth, 70 (50.4%) had no growth, and 11 (7.9%) had all inoculated tubes contaminated. A total of 25 different mycobacterial species were identified. Fifteen species (60%) were slowly growing (e.g.,Mycobacterium ulcerans,Mycobacterium avium,Mycobacterium mantenii, andMycobacterium malmoense), and 10 (40%) were rapidly growing (e.g.,Mycobacterium chelonae,Mycobacterium fortuitum, andMycobacterium abscessus). The occurrence of mycobacterial species in the various environmental samples analyzed was as follows: soil, 16 species (43.2%); vegetation, 14 species (38.0%); water, 3 species (8.0%); moss, 2 species (5.4%); snail, 1 species (2.7%); fungi, 1 species (2.7%). This study is the first to report on the isolation ofM. ulceransand other medically relevant nontuberculous mycobacteria from different environmental sources in Ghana.IMPORTANCEDiseases caused by mycobacterial species other than those that cause tuberculosis and leprosy are increasing. Control is difficult because the current understanding of how the organisms are spread and where they live in the environment is limited, although this information is needed to design preventive measures. Growing these organisms from the environment is also difficult, because the culture medium becomes overgrown with other bacteria that also live in the environment, such as in soil and water. We aimed to improve the methods for growing these organisms from environmental sources, such as soil and water samples, for better understanding of important mycobacterial ecology.

scholarly journals High-Throughput Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat Genotyping for Mycobacterium tuberculosis Epidemiological Studies

2014 ◽  
Vol 53 (2) ◽  
pp. 498-503 ◽  
Author(s):  
Marie Gauthier ◽  
Floriane Bidault ◽  
Amandine Mosnier ◽  
Nino Bablishvili ◽  
Nestani Tukvadze ◽  
...  
Keyword(s):  
High Throughput ◽  
Tandem Repeat ◽  
Variable Number Tandem Repeat ◽  
Epidemiological Studies ◽  
Turnaround Time ◽  
Variable Number ◽  
Automated System ◽  
Health Concern ◽  
Automated Method ◽  
Allele Calling

The emergence of drug-resistant forms of tuberculosis (TB) represents a major public health concern. Understanding the transmission routes of the disease is a key factor for its control and for the implementation of efficient interventions. Mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) marker typing is a well-described method for lineage identification and transmission tracking. However, the conventional manual genotyping technique is cumbersome and time-consuming and entails many risks for errors, thus hindering its implementation and dissemination. We describe here a new approach using the QIAxcel system, an automated high-throughput capillary electrophoresis system that also carries out allele calling. This automated method was assessed on 1,824 amplicons from 82 TB isolates and tested with sets of markers of 15 or 24 loci. Overall allele-calling concordance between the methods from 140 to 1,317 bp was 98.9%. DNA concentrations and repeatability and reproducibility performances showed no biases in allele calling. Furthermore, turnaround time using this automated system was reduced by 81% compared to the conventional manual agarose gel method. In sum, this new automated method facilitates MIRU-VNTR genotyping and provides reliable results. Therefore, it is well suited for field genotyping. The implementation of this method will help to achieve accurate and cost-effective epidemiological studies, especially in countries with a high prevalence of TB, where the high number of strains complicates the surveillance of circulating lineages and requires efficient interventions to be carried out in an urgent manner.

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

2019 ◽  
pp. 1-14
Author(s):  
Menssah Teko ◽  
Mounerou Salou ◽  
Solange E. Kakou Ngazoa ◽  
Issaka Maman ◽  
Kodjovi Agbodeka ◽  
...  
Keyword(s):  
West Africa ◽  
Tandem Repeats ◽  
Buruli Ulcer ◽  
Variable Number ◽  
Mycobacterium Ulcerans ◽  
Clinical Samples ◽  
Molecular Technique ◽  
Endemic Region ◽  
Typing Method ◽  
Genetic Strains

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.

scholarly journals MIRUReader: MIRU-VNTR typing directly from long sequencing reads

2019 ◽  
Author(s):  
Cheng Yee Tang ◽  
Rick Twee-Hee Ong
Keyword(s):  
High Throughput Sequencing ◽  
Variable Number Tandem Repeat ◽  
Epidemiological Studies ◽  
Variable Number ◽  
Supplementary Information ◽  
Vntr Locus ◽  
Tuberculosis Transmission ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read

Abstract Summary Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing is widely used to genotype Mycobacterium tuberculosis complex in epidemiological studies for tracking tuberculosis transmission. Recent long-read sequencing technologies from Pacific Biosciences and Oxford Nanopore Technologies can produce reads that are long enough to cover the entire repeat regions in each MIRU-VNTR locus which was previously not possible using the short reads from Illumina high-throughput sequencing technologies. We thus developed MIRUReader for MIRU-VNTR typing directly from long sequence reads. Availability and implementation Source code and documentation for MIRUReader program is freely available at https://github.com/phglab/MIRUReader. Supplementary information Supplementary data are available at Bioinformatics online.

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