scholarly journals Use of GeneXpert Remnants for Drug Resistance Profiling and Molecular Epidemiology of Tuberculosis in Libreville, Gabon

2017 ◽  
Vol 55 (7) ◽  
pp. 2105-2115 ◽  
Author(s):  
Amel Kévin Alame-Emane ◽  
Catherine Pierre-Audigier ◽  
Oriane Cordelia Aboumegone-Biyogo ◽  
Amandine Nzoghe-Mveang ◽  
Véronique Cadet-Daniel ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Molecular Epidemiology ◽  
Variable Number Tandem Repeat ◽  
Pcr Amplification ◽  
Variable Number ◽  
Line Probe Assay ◽  
Content Type ◽  
Mdr Tb ◽  
Probe Assay ◽  
Xpert Assay

ABSTRACTMultidrug-resistant (MDR) and extensively drug resistant (XDR) strains ofMycobacterium tuberculosispose major problems for global health. The GeneXpert MTB/RIF (Xpert) assay rapidly detects resistance to rifampin (RIFr), but for detection of the additional resistance that defines MDR-TB (MDR tuberculosis) and XDR-TB, and for molecular epidemiology, specimen cultures and a biosafe infrastructure are generally required. We sought to determine whether the remnants of sputa prepared for the Xpert assay could be used directly to find mutations associated with drug resistance and to study molecular epidemiology, thus providing precise characterization of MDR-TB cases in countries lacking biosafety level 3 (BSL3) facilities forM. tuberculosiscultures. After sputa were processed and run on the Xpert instrument, the leftovers of the samples prepared for the Xpert assay were used for PCR amplification and sequencing or for a line probe assay to detect mutations associated with resistance to additional drugs, as well as for molecular epidemiology with spoligotyping and selective mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing. Of 130 sputum samples from Gabon tested with the Xpert assay, 124 yielded interpretable results; 21 (17%) of these were determined to be RIFr. Amplification and sequencing or a line probe assay of the Xpert remnants confirmed 18/21 samples as MDR, corresponding to 12/116 (9.5%) new and 6/8 (75%) previously treated TB patients. Spoligotyping and MIRU typing with hypervariable loci identified an MDR Beijing strain present in five samples. We conclude that the remnants of samples processed for the Xpert assay can be used in PCRs to find mutations associated with the resistance to the additional drugs that defines MDR and XDR-TB and to study molecular epidemiology without the need for culturing or a biosafe infrastructure.

scholarly journals Drug-resistant Mycobacterium tuberculosis and its genotypes isolated from an outbreak in western Thailand

2020 ◽  
Author(s):  
Janisara Rudeeaneksin ◽  
Benjawan Phetsuksiri ◽  
Chie Nakajima ◽  
Supranee Bunchoo ◽  
Krairerk Suthum ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Variable Number Tandem Repeat ◽  
Multidrug Resistant ◽  
Variable Number ◽  
Drug Resistant ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Extensively Drug Resistant ◽  
Mdr Tb ◽  
Vntr Analysis

Abstract Background Multidrug-resistant TB (MDR-TB) outbreaks have occurred in the Thamaka district, Kanchanaburi province in Thailand. Methods Seventy-two isolates, which included 7% mono-, 30.6% MDR and extensively drug-resistant TB (XDR-TB), were genotyped by spoligotyping, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) and single nucleotide polymorphism genotyping, and their drug resistance was analysed. Results The spoligotyping results showed that Beijing spoligo-international type (SIT)1 was predominant (n=38; 52.8%) while the remaining were non-Beijing sublineages (n=34). The MIRU-VNTR analysis showed that Beijing isolates, most of which belonged to the modern type (n=37), formed 5 clusters and 13 individual patterns. In katG, only mutation Ser315Thr was identified. In rpoB, Ser531Leu was predominant, except for His526Arg and Leu533Pro, which were found in two isolates. A cluster of 14 Beijing strains contained these common mutations and shared the MIRU-VNTR genotype with isolates in the Thamaka district that had spread previously. Two U SIT523 isolates contained the mutations A1400G in rrs and Asp94Gly in gyrA genes, indicating a spread of XDR-TB. Conclusions Most mutations were associated with drug resistance and the specific MDR Beijing and XDR-TB in U SIT523 isolates remain. This genotyping is a key tool for tracking TB transmission in the Thamaka district of Thailand.

scholarly journals Transmission of Dapsone-Resistant Leprosy Detected by Molecular Epidemiological Approaches

2011 ◽  
Vol 55 (11) ◽  
pp. 5384-5387 ◽  
Author(s):  
Wei Li ◽  
Rama M. Sakamuri ◽  
Danielle E. Lyons ◽  
Florenda M. Orcullo ◽  
Vidyagouri Shinde ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Target Gene ◽  
Variable Number Tandem Repeat ◽  
Mycobacterium Leprae ◽  
The Philippines ◽  
Variable Number ◽  
Content Type ◽  
Leprosy Patients ◽  
Content Variable ◽  
History Of

ABSTRACTDrug resistance surveillance identified six untreated leprosy patients in the Philippines withMycobacterium lepraefolP1mutations which confer dapsone resistance. Five patients share a village of residence; four who carried the mutation, Thr53Val, were also linked byM. lepraevariable-number tandem repeat (VNTR) strain types. In India,folP1mutations were detected in two relapse patients with a history of dapsone treatment. Mutations were not found in the rifampin target generpoB. These findings indicate that dapsone resistance is being transmitted.

scholarly journals Potentially Human-Pathogenic Escherichia coli O26 in Norwegian Sheep Flocks

2011 ◽  
Vol 77 (14) ◽  
pp. 4949-4958 ◽  
Author(s):  
C. Sekse ◽  
M. Sunde ◽  
B.-A. Lindstedt ◽  
P. Hopp ◽  
T. Bruheim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Human Pathogens ◽  
Content Type ◽  
Representative Sampling ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Close Relationship ◽  
Large Survey

ABSTRACTA national survey ofEscherichia coliO26 in Norwegian sheep flocks was conducted, using fecal samples to determine the prevalence. In total, 491 flocks were tested, andE. coliO26 was detected in 17.9% of the flocks. One hundred forty-twoE. coliO26 isolates were examined for flagellar antigens (H typing) and four virulence genes, includingstxandeae, to identify possible Shiga toxin-producingE. coli(STEC) and enteropathogenicE. coli(EPEC). Most isolates (129 out of 142) were identified asE. coliO26:H11. They possessedeaeand may have potential as human pathogens, although only a small fraction were identified as STEC O26:H11, giving a prevalence in sheep flocks of only 0.8%. Correspondingly, the sheep flock prevalence of atypical EPEC (aEPEC) O26:H11 was surprisingly high (15.9%). The genetic relationship between theE. coliO26:H11 isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA), identifying 63 distinct PFGE profiles and 22 MLVA profiles. Although the MLVA protocol was less discriminatory than PFGE and a few cases of disagreement were observed, comparison by partition mapping showed an overall good accordance between the two methods. A close relationship between a few isolates of aEPEC O26:H11 and STEC O26:H11 was identified, but all theE. coliO26:H11 isolates should be considered potentially pathogenic to humans. The present study consisted of a representative sampling of sheep flocks from all parts of Norway. This is the first large survey of sheep flocks focusing onE. coliO26 in general, including results of STEC, aEPEC, and nonpathogenic isolates.

scholarly journals Prediction of Local Transmission of Mycobacterium tuberculosis Isolates of a Predominantly Beijing Lineage by Use of a Variable-Number Tandem-Repeat Typing Method Incorporating a Consensus Set of Hypervariable Loci

2017 ◽  
Vol 56 (1) ◽  
Author(s):  
Yoshiro Murase ◽  
Kiyohiko Izumi ◽  
Akihiro Ohkado ◽  
Akio Aono ◽  
Kinuyo Chikamatsu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Tandem Repeat ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Trend Test ◽  
Typing Method ◽  
Content Type ◽  
Tuberculosis Transmission ◽  
High Prevalence ◽  
Hypervariable Loci

ABSTRACT Strain genotyping based on the variable-number tandem repeat (VNTR) is widely applied for identifying the transmission of Mycobacterium tuberculosis. A consensus set of four hypervariable loci (1982, 3232, 3820, and 4120) has been proposed to improve the discrimination of Beijing lineage strains. Herein, we evaluated the utility of these four hypervariable loci for tracing local tuberculosis transmission in 981 cases over a 14-month period in Japan (2010 to 2011). We used six different VNTR systems, with or without the four hypervariable loci. Patient ages and weighted standard distances (a measure of the dispersion of genotype-clustered cases) were used as proxies for estimating local tuberculosis transmission. The highest levels of isolate discrimination were achieved with VNTR systems that incorporated the four hypervariable loci (i.e., the Japan Anti-Tuberculosis Association [JATA]18-VNTR, mycobacterial interspersed repetitive unit [MIRU]28-VNTR, and 24Beijing-VNTR). The clustering rates by JATA12-VNTR, MIRU15-VNTR, JATA15-VNTR, JATA18-VNTR, MIRU28-VNTR, and 24Beijing-VNTR systems were 52.2%, 51.0%, 39.0%, 24.1%, 23.1%, and 22.0%, respectively. As the discriminative power increased, the median weighted standard distances of the clusters tended to decrease (from 311 to 80 km, P < 0.001, Jonckheere-Terpstra trend test). Concurrently, the median ages of patients in the clusters tended to decrease (from 68 to 60 years, P < 0.001, Jonckheere-Terpstra trend test). These findings suggest that strain typing using the four hypervariable loci improves the prediction of active local tuberculosis transmission. The four-locus set can therefore contribute to the targeted control of tuberculosis in settings with high prevalence of Beijing lineage strains.

scholarly journals Examination of Taxonomic Uncertainties Surrounding Brucella abortus bv. 7 by Phenotypic and Molecular Approaches

2013 ◽  
Vol 80 (5) ◽  
pp. 1570-1579 ◽  
Author(s):  
Bruno Garin-Bastuji ◽  
Virginie Mick ◽  
Gilles Le Carrou ◽  
Sebastien Allix ◽  
Lorraine L. Perrett ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Epidemiological Methods ◽  
Content Type ◽  
Phenotypic Profile ◽  
Molecular Approaches ◽  
Content Classification ◽  
Species Specific ◽  
Kenyan Strain

ABSTRACTBrucellataxonomy is perpetually being reshuffled, at both the species and intraspecies levels. Biovar 7 ofBrucella abortuswas suspended from theApproved Lists of Bacterial NamesBrucellaclassification in 1988, because of unpublished evidence that the reference strain 63/75 was a mixture ofB. abortusbiovars 3 and 5. To formally clarify the situation, all isolates previously identified asB. abortusbv. 7 in the AHVLA and ANSES strain collections were characterized by classical microbiological and multiple molecular approaches. Among the 14 investigated strains, including strain 63/75, only four strains, isolated in Kenya, Turkey, and Mongolia, were pure and showed a phenotypic profile in agreement with the former biovar 7, particularly agglutination with both anti-A/anti-M monospecific sera. These results were strengthened by molecular strategies. Indeed, genus- and species-specific methods allowed confirmation that the four pure strains belonged to theB. abortusspecies. The combination of most approaches excluded their affiliation with the recognized biovars (biovars 1 to 6 and 9), while some suggested that they were close to biovar 3.These assays were complemented by phylogenetic and/or epidemiological methods, such as multilocus sequence analysis (MLSA) and variable-number tandem repeat (VNTR) analysis. The results of this polyphasic investigation allow us to propose the reintroduction of biovar 7 into theBrucellaclassification, with at least three representative strains. Interestingly, the Kenyan strain, sharing the same biovar 7 phenotype, was genetically divergent from other three isolates. These discrepancies illustrate the complexity ofBrucellataxonomy. This study suggests that worldwide collections could include strains misidentified asB. abortusbv. 7, and it highlights the need to verify their real taxonomic position.

scholarly journals Mycobaterial interspersed repetitive units-variable number of tandem repeats analysis and pattern of drug resistance in extended drug resistant (XDR) TB isolates from pulmonary tuberculosis patients in Bangladesh

2020 ◽  
Vol 46 (1) ◽  
pp. 22-28
Author(s):  
Shirin Tarafder ◽  
Md Bayzid Bin Monir
Keyword(s):  
Drug Resistance ◽  
Molecular Typing ◽  
Tandem Repeats ◽  
Variable Number ◽  
Drug Resistant ◽  
Gyra Gene ◽  
Drug Resistant Tuberculosis ◽  
Tree Analysis ◽  
Resistant Tuberculosis ◽  
Mdr Tb

Background: To investigate the spread of specific genotypes in a defined geographical area and to determine any relationship of these genotypes with drug resistance the most essential method is molecular typing. It allows a rapid and precise species differentiation. Objective: This study was intended to observe the genotypes of XDR mycobacterium tuberculosis by determining 24 loci MIRU-VNTR analysis. Methods: To gain an insight about molecular typing of MTB and drug resistance-associated mutations in XDR-TB isolates a total of 98 multi drug resistant tuberculosis (MDR-TB) isolates collected through Xpert MTB/RIF assay. They were subjected to 2nd line (Fluoroquinolones, kanamycin, capreomycin and amikacin) drug susceptibility testing through line probe assay (LPA) in a view to detect extensively drug resistant tuberculosis (XDR-TB). Genotyping was done for XDR-TB isolates using 24 loci Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) using the online tool at http://www.MIRU-VNTRplus.org.. Out of 98 MDR-TB isolates 11(11.23%)  XDR-TB isolates were typed and analysed. Results: Twenty four loci MIRU-VNTR genotyping involving similarity searching and phylogenetic tree analysis revealed that six (54.60%) XDR-TB isolates belonged to Beijing strain, Other MTB strain also detected were Delhi/CAS two(18.20%), Haarlem two(18.20%) and New-1, one (9.10%) in number. Minimum spanning tree analysis showed two strain of Beijing family form a clonal complex. Beijing strains were more common among younger age group and within urban population. Beijing strains were also predominant in treatment failure patient. Only one new case of XDR-TB belongs to Delhi/CAS family. Second line mycobacterial drug resistance (MTBDRsl) detected by LPA showed the most prevalent mutations involved in Fluoroquinolones drug resistance (FQ) was Asp94Gly in gyrA gene (54.55%) in quinolone resistance determining region (QRDR) and for Injectable 2nd line Drug resistance (ISL) was A1401G, C1402T in 16S rrs gene (100%)..  All XDR-TB isolates showed resistance to Levofloxacin in 2nd line LPA but Moxifloxacin showed low level resistance to some cases. Conclusion: Molecular typing of XDR- TB isolates and pattern of drug resistance associated mutations in XDR-TB isolates in Bangladesh have not been reported previously. The result of this study highlights the need to reinforce the TB policy in Bangladesh with regard to control the spread and transmission as well as detection and treatment strategies regarding XDR-TB. Bangladesh Med Res Counc Bull 2020; 46(1): 22-28

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