scholarly journals Genotyping of ClinicalMycobacterium tuberculosisIsolates Based on IS6110and MIRU-VNTR Polymorphisms

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Anna Żaczek ◽  
Anna Brzostek ◽  
Arkadiusz Wojtasik ◽  
Jarosław Dziadek ◽  
Anna Sajduda
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Variable Number Tandem Repeat ◽  
Pcr Amplification ◽  
Detailed Comparison ◽  
Variable Number ◽  
Apparent Difference ◽  
Typing Method ◽  
Banding Patterns ◽  
Typing Technique ◽  
Discriminatory Index

In this study, 155 clinicalMycobacterium tuberculosisisolates were subject to genotyping with fast ligation-mediated PCR (FLiP). This typing method is a modified mixed-linker PCR, a rapid approach based on the PCR amplification ofHhaI restriction fragments of genomic DNA containing the 3′ end of IS6110and resolving the amplicons by polyacrylamide gel electrophoresis. The results were compared with previous data of the more commonly used methods, 15-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and, to verify combined FLiP/MIRU-VNTR clusters, the reference IS6110restriction fragment length polymorphism (RFLP). FLiP banding patterns were highly reproducible and polymorphic. This method differentiated 119 types among the study set compared to 108 distinct MIRU-VNTR profiles. The discriminatory power of FLiP was slightly higher than that of MIRU-VNTR analysis (Hunter-Gaston Discriminatory Index = 0.991 and 0.990, resp.). Detailed comparison of the clusters defined by each of the methods revealed, however, a more apparent difference in the discriminatory abilities that favored FLiP. Clustering of strains by using combined results of these two PCR-based methods correlated well with IS6110RFLP-defined clusters, further confirming high discriminatory potential of FLiP typing. These results indicate that FLiP could be an attractive and valuable secondary typing technique for verification of MIRU-VNTR clusters ofM. tuberculosisstrains.

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.

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

2010 ◽  
Vol 58 (3) ◽  
pp. 297-308 ◽  
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.

scholarly journals Multi-laboratory validation study of multilocus variable-number tandem repeat analysis (MLVA) for Salmonella enterica serovar Enteritidis, 2015

2017 ◽  
Vol 22 (9) ◽  
Author(s):  
Tansy Peters ◽  
Sophie Bertrand ◽  
Jonas T Björkman ◽  
Lin T Brandal ◽  
Derek J Brown ◽  
...  
Keyword(s):  
North America ◽  
Tandem Repeat ◽  
Validation Study ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
European Economic Area ◽  
The European Union ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Typing Method ◽  
Repeat Analysis

Multilocus variable-number tandem repeat analysis (MLVA) is a rapid and reproducible typing method that is an important tool for investigation, as well as detection, of national and multinational outbreaks of a range of food-borne pathogens. Salmonella enterica serovar Enteritidis is the most common Salmonella serovar associated with human salmonellosis in the European Union/European Economic Area and North America. Fourteen laboratories from 13 countries in Europe and North America participated in a validation study for MLVA of S. Enteritidis targeting five loci. Following normalisation of fragment sizes using a set of reference strains, a blinded set of 24 strains with known allele sizes was analysed by each participant. The S. Enteritidis 5-loci MLVA protocol was shown to produce internationally comparable results as more than 90% of the participants reported less than 5% discrepant MLVA profiles. All 14 participating laboratories performed well, even those where experience with this typing method was limited. The raw fragment length data were consistent throughout, and the inter-laboratory validation helped to standardise the conversion of raw data to repeat numbers with at least two countries updating their internal procedures. However, differences in assigned MLVA profiles remain between well-established protocols and should be taken into account when exchanging data.

scholarly journals Epidemiology of a Salmonella enterica subsp. enterica Serovar Typhimurium Strain Associated with a Songbird Outbreak

2012 ◽  
Vol 78 (20) ◽  
pp. 7290-7298 ◽  
Author(s):  
Sonia M. Hernandez ◽  
Kevin Keel ◽  
Susan Sanchez ◽  
Eija Trees ◽  
Peter Gerner-Smidt ◽  
...  
Keyword(s):  
United States ◽  
Salmonella Enterica ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Pfge Type ◽  
Eastern United States ◽  
Typing Method ◽  
Content Type ◽  
Typhimurium Strain ◽  
Serovar Typhimurium

ABSTRACTSalmonella entericasubsp.entericaserovar Typhimurium is responsible for the majority of salmonellosis cases worldwide. ThisSalmonellaserovar is also responsible for die-offs in songbird populations. In 2009, there was anS. Typhimurium epizootic reported in pine siskins in the eastern United States. At the time, there was also a human outbreak with this serovar that was associated with contaminated peanuts. As peanuts are also used in wild-bird food, it was hypothesized that the pine siskin epizootic was related to this human outbreak. A comparison of songbird and humanS. Typhimurium pulsed-field gel electrophoresis (PFGE) patterns revealed that the epizootic was attributed not to the peanut-associated strain but, rather, to a songbird strain first characterized from an American goldfinch in 1998. This sameS. Typhimurium strain (PFGE type A3) was also identified in the PulseNet USA database, accounting for 137 of 77,941 totalS. Typhimurium PFGE entries. A second molecular typing method, multiple-locus variable-number tandem-repeat analysis (MLVA), confirmed that the same strain was responsible for the pine siskin epizootic in the eastern United States but was distinct from a genetically related strain isolated from pine siskins in Minnesota. The pine siskin A3 strain was first encountered in May 2008 in an American goldfinch and later in a northern cardinal at the start of the pine siskin epizootic. MLVA also confirmed the clonal nature ofS. Typhimurium in songbirds and established that the pine siskin epizootic strain was unique to the finch family. For 2009, the distribution of PFGE type A3 in passerines and humans mirrored the highest population density of pine siskins for the East Coast.

scholarly journals Molecular Typing of Mycoplasma genitalium-Positive Specimens Discriminates between Persistent and Recurrent Infections in Cases of Treatment Failure and Supports Contact Tracing

2019 ◽  
Vol 7 (12) ◽  
pp. 609 ◽  
Author(s):  
Luis Piñeiro ◽  
Pedro Idigoras ◽  
Gustavo Cilla
Keyword(s):  
Molecular Typing ◽  
Variable Number Tandem Repeat ◽  
Mycoplasma Genitalium ◽  
Variable Number ◽  
Contact Tracing ◽  
Polymorphism Analysis ◽  
Recurrent Infections ◽  
Transmitted Infection ◽  
Typing Method ◽  
Sexually Transmitted

Mycoplasma genitalium causes a sexually transmitted infection that sometimes persists or recurs despite adequate antibiotic treatment. Between 2014 and 2018, molecular typing was applied to 75 M. genitalium-positive samples from 48 patients with repeated infection and/or couples/groups of other infected sexual contacts. MG191 adhesin, MG309 lipoprotein, and the rRNA operon were amplified, sequenced, and typed using phylogenetic, variable number tandem repeat, and single-nucleotide polymorphism analysis, respectively. Amplicons were obtained in 74/75 samples, and the combination of locus patterns gave 44 different genetic profiles (discriminatory index of 0.987), with 43 considering only MG191 and MG309. Interestingly, 15/17 patients who presented a first sample sensitive and a second resistant to macrolides had the same genetic variant in the samples (persistence of the same strain). In 2/17 patients, discordant variants (one mixed infection and one recurrence due to incomplete contact tracing) were detected. In 31 additional not related and randomly distributed samples, MG191 typing obtained 23 different genotypes, with no appreciable clustering over time. The typing method allowed persistent and recurrent infections to be distinguished, indicating that macrolide resistance-associated mutations mostly developed during treatment. To detect these secondary resistant strains, prevent reinfections, and improve the control of M. genitalium infections, tests of cure and contact tracing of sexual partners should be mandatory.

A new typing technique for the Rickettsiales Ehrlichia ruminantium: Multiple-locus variable number tandem repeat analysis

2012 ◽  
Vol 88 (2) ◽  
pp. 205-211 ◽  
Author(s):  
Héloïse Pilet ◽  
Nathalie Vachiéry ◽  
Moez Berrich ◽  
Rim Bouchouicha ◽  
Benoît Durand ◽  
...  
Keyword(s):  
Tandem Repeat ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Ehrlichia Ruminantium ◽  
Multiple Locus ◽  
Repeat Analysis ◽  
Typing Technique

scholarly journals Prevalence and Characterization of Heterogeneous Variable-Number Tandem-Repeat Clusters Comprising Drug-Susceptible and/or Variable ResistantMycobacterium tuberculosisComplex Isolates in the Netherlands from 2004 to 2016

2018 ◽  
Vol 56 (11) ◽  
Author(s):  
Inge Roof ◽  
Rana Jajou ◽  
Miranda Kamst ◽  
Arnout Mulder ◽  
Albert de Neeling ◽  
...  
Keyword(s):  
The Netherlands ◽  
Tandem Repeat ◽  
Variable Number Tandem Repeat ◽  
Multivariate Logistic Regression Analysis ◽  
Variable Number ◽  
Linkage Data ◽  
Nucleotide Polymorphisms ◽  
Typing Method ◽  
Content Type ◽  
Heterogeneous Clustering

ABSTRACTThe variable-number tandem-repeat (VNTR) typing method is used to study tuberculosis (TB) transmission. Clustering ofMycobacterium tuberculosisisolates with identical VNTR patterns is assumed to reflect recent transmission. Hence, clusters are thought to be homogeneous regarding antibiotic resistance. In practice, however, heterogeneous clusters are also identified. This study investigates the prevalence and characteristics of heterogeneous VNTR clusters and assesses whether isolates in these clusters remain clustered when subjected to whole-genome sequencing (WGS). In the period from 2004 to 2016, 9,072 isolates were included. Demographic and epidemiological linkage data were obtained from the Netherlands Tuberculosis Register. VNTR clusters were defined as homogeneous when isolates shared identical resistance profiles or as heterogeneous if both susceptible and (variable) resistant isolates were found. Multivariate logistic regression analysis was performed to identify factors associated with heterogeneous clustering. Isolates from 2016 were subjected to WGS, and a genetic distance of 12 single nucleotide polymorphisms (SNPs) was used as the cutoff for WGS clustering. In total, 4,661/9,072 (51%) isolates were clustered into 985 different VNTR clusters, of which 217 (22%) were heterogeneous. Patient characteristics associated with heterogeneous clustering were non-Dutch ethnicity (odds ratio [OR], 1.46 [95% confidence interval {CI}, 1.22 to 1.75]), asylum seeker (OR, 1.51 [95% CI, 1.24 to 1.85]), extrapulmonary TB (OR, 1.26 [95% CI, 1.09 to 1.46]), previous TB diagnosis (OR, 1.38 [95% CI, 1.04 to 1.82]), and not being a contact of a TB patient (OR, 1.35 [95% CI, 1.08 to 1.69]). With WGS, 34% of heterogeneous and 78% of homogeneous isolates from 2016 remained clustered. Heterogeneous VNTR clusters are common but seem to be explained by a substantial degree of false clustering by VNTR typing compared to WGS.

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 Comparison between Salmonella enterica Serotype Enteritidis Genotyping Methods and Phage Type

2015 ◽  
Vol 53 (9) ◽  
pp. 3021-3031 ◽  
Author(s):  
Alessandra De Cesare ◽  
Keshav Krishnamani ◽  
Antonio Parisi ◽  
Antonia Ricci ◽  
Ida Luzzi ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Phage Type ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Quantitative Comparison ◽  
Pulsed Field Gel Electrophoresis ◽  
Typing Method ◽  
Content Type ◽  
Repeat Analysis ◽  
Automated Ribotyping

A quantitative comparison between discriminatory indexes and concordance among multilocus variable-number tandem-repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE), automated ribotyping, and phage typing has been performed, testing 238Salmonella entericaserotype Enteritidis isolates not epidemiologically correlated. The results show that MLVA is the best choice, but each typing method provides a piece of information for establishing clonal relationships between the isolates.

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