Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets

Microbiology ◽  
2002 ◽  
Vol 148 (2) ◽  
pp. 519-528 ◽  
Author(s):  
Robin A Skuce ◽  
Thomas P McCorry ◽  
Julie F McCarroll ◽  
Solvig M. M Roring ◽  
Alistair N Scott ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Epidemiology ◽  
Tandem Repeats ◽  
Antigenic Variation ◽  
Variable Number ◽  
Strain Typing ◽  
The Novel ◽  
Typing Method ◽  
Intergenic Regions ◽  
Typing Methods

The lack of a convenient high-resolution strain-typing method has hampered the application of molecular epidemiology to the surveillance of bacteria of the Mycobacterium tuberculosis complex, particularly the monitoring of strains of Mycobacterium bovis. With the recent availability of genome sequences for strains of the M. tuberculosis complex, novel PCR-based M. tuberculosis-typing methods have been developed, which target the variable-number tandem repeats (VNTRs) of minisatellite-like mycobacterial interspersed repetitive units (MIRUs), or exact tandem repeats (ETRs). This paper describes the identification of seven VNTR loci in M. tuberculosis H37Rv, the copy number of which varies in other strains of the M. tuberculosis complex. Six of these VNTRs were applied to a panel of 100 different M. bovis isolates, and their discrimination and correlation with spoligotyping and an established set of ETRs were assessed. The number of alleles varied from three to seven at the novel VNTR loci, which differed markedly in their discrimination index. There was positive correlation between spoligotyping, ETR- and VNTR-typing. VNTR-PCR discriminates well between M. bovis strains. Thirty-three allele profiles were identified by the novel VNTRs, 22 for the ETRs and 29 for spoligotyping. When VNTR- and ETR-typing results were combined, a total of 51 different profiles were identified. Digital nomenclature and databasing were intuitive. VNTRs were located both in intergenic regions and annotated ORFs, including PPE (novel glycine-asparigine-rich) proteins, a proposed source of antigenic variation, where VNTRs potentially code repeating amino acid motifs. VNTR-PCR is a valuable tool for strain typing and for the study of the global molecular epidemiology of the M. tuberculosis complex. The novel VNTR targets identified in this study should additionally increase the power of this approach.

Molecular epidemiology of Mycobacterium tuberculosis in adult Filipino TB-HIV co-infected patients

2021 ◽  
Vol 25 (4) ◽  
pp. 285-291
Author(s):  
J. C. M. Malabad ◽  
C. F. Ang ◽  
F. A. R. Palabrica ◽  
M. A. M. Cajucom ◽  
N. G. Gloriani ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Molecular Epidemiology ◽  
Disease Transmission ◽  
Tandem Repeats ◽  
The Philippines ◽  
Variable Number ◽  
People Living With Hiv ◽  
East African ◽  
Living With Hiv

BACKGROUND: TB is the leading cause of death from a single infectious disease, particularly among people living with HIV (PLHIV). Molecular epidemiology provides information on prevalent genotypes of Mycobacterium tuberculosis and disease transmission dynamics, which aid in TB control. Identification of mutations that confer drug resistance is essential for the rapid diagnosis of drug-resistant TB, especially in high TB burden settings, like the Philippines.METHODS: This study aimed to determine mutations in M. tuberculosis drug resistance-conferring genes and circulating genotypes in PLHIV. MIRU-VNTR (mycobacterial interspersed repetitive unit-variable number of tandem repeats) typing using a set of 24-loci and sequencing of drug resistance-conferring genes were performed in 22 M. tuberculosis isolates from TB-HIV co-infected patients.RESULTS: The prevalence of resistance to any drug was 31.8%, 18.2% for isoniazid monoresistance, 4.5% for streptomycin monoresistance and 9.1% for multidrug resistance. The identified mutations in the katG, rpoB, pncA, rpsL and gyrA genes have been reported in the literature; none was found in the inhA and embB genes. All isolates belonged to the EAI2-Manila family and were grouped into four clusters based on their phenotypic drug resistance and mutation profiles.CONCLUSION: The use of 24-loci set may be used as a more discriminatory MIRU-VNTR typing in settings where the East African-Indian lineage is predominant, like the Philippines.

scholarly journals Typing Method for the QUB11a Locus ofMycobacterium tuberculosis: IS6110Insertions and Tandem Repeat Analysis

2016 ◽  
Vol 2016 ◽  
pp. 1-6
Author(s):  
Eriko Maeda-Mitani ◽  
Koichi Murakami ◽  
Akira Oishi ◽  
Yoshiki Etoh ◽  
Nobuyuki Sera ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Mycobacterium Tuberculosis ◽  
Tandem Repeat ◽  
Tandem Repeats ◽  
Variable Number ◽  
Typing Method ◽  
Repeat Analysis ◽  
Ofmycobacterium Tuberculosis ◽  
High Discrimination ◽  
Vntr Analysis

QUB11a is used as a locus for variable number of tandem repeats (VNTR) analysis ofMycobacterium tuberculosisBeijing lineage. However, amplification of QUB11a occasionally produces large fragments (>1,400 bp) that are not easily measured by capillary electrophoresis because of a lack of the typical stutter peak patterns that are used for counting repeat numbers. IS6110insertion may complicate VNTR analysis of large QUB11a fragments inM. tuberculosis. We established a method for determining both tandem repeat numbers and IS6110insertion in the QUB11a locus ofM. tuberculosisusing capillary electrophoresis analysis andBsmBI digestion. All 29 large QUB11a fragments (>1,200 bp) investigated contained IS6110insertions and varied in the number of repeats (18 patterns) and location of IS6110insertions. This method allows VNTR analysis with high discrimination.

Strain Typing of Mycobacterium tuberculosis Isolates from Korea by Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats

2009 ◽  
Vol 29 (4) ◽  
pp. 314-319 ◽  
Author(s):  
Kyung Won Yun ◽  
Eun Ju Song ◽  
Go Eun Choi ◽  
In Kyung Hwang ◽  
Eun Yup Lee ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Tandem Repeats ◽  
Variable Number ◽  
Strain Typing

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 Population structure of drug-resistant Mycobacterium tuberculosis in Central Asia

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Anna Engström ◽  
Uladzimir Antonenka ◽  
Abdylat Kadyrov ◽  
Gulmira Kalmambetova ◽  
Katharina Kranzer ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Population Structure ◽  
Central Asia ◽  
Tandem Repeats ◽  
Relative Size ◽  
Multidrug Resistant ◽  
Variable Number ◽  
Health Concern ◽  
Drug Resistant ◽  
Recent Emergence

Abstract Background Drug-resistant tuberculosis (TB) is a major public health concern threathing the success of TB control efforts, and this is particularily problematic in Central Asia. Here, we present the first analysis of the population structure of Mycobacterium tuberculosis complex isolates in the Central Asian republics Uzbekistan, Tajikistan, and Kyrgyzstan. Methods The study set consisted of 607 isolates with 235 from Uzbekistan, 206 from Tajikistan, and 166 from Kyrgyzstan. 24-loci MIRU-VNTR (Mycobacterial Interspersed Repetitive Units - Variable Number of Tandem Repeats) typing and spoligotyping were combined for genotyping. In addition, phenotypic drug suceptibility was performed. Results The population structure mainly comprises strains of the Beijing lineage (411/607). 349 of the 411 Beijing isolates formed clusters, compared to only 33 of the 196 isolates from other clades. Beijing 94–32 (n = 145) and 100–32 (n = 70) formed the largest clusters. Beijing isolates were more frequently multidrug-resistant, pre-extensively resistant (pre-XDR)- or XDR-TB than other genotypes. Conclusions Beijing clusters 94–32 and 100–32 are the dominant MTB genotypes in Central Asia. The relative size of 100–32 compared to previous studies in Kazakhstan and its unequal geographic distribution support the hypothesis of its more recent emergence in Central Asia. The data also demonstrate that clonal spread of resistant TB strains, particularly of the Beijing lineage, is a root of the so far uncontroled MDR-TB epidemic in Central Asia.

scholarly journals Evaluation of variable numbers of tandem repeat as molecular epidemiological markers of Mycobacterium tuberculosis in Japan

2007 ◽  
Vol 56 (8) ◽  
pp. 1052-1057 ◽  
Author(s):  
Takayuki Wada ◽  
Shinji Maeda ◽  
Atsushi Hase ◽  
Kazuo Kobayashi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Tandem Repeat ◽  
Tandem Repeats ◽  
Allelic Diversity ◽  
Rflp Analysis ◽  
Strain Typing ◽  
Beijing Family ◽  
Discrimination Power ◽  
Family Based ◽  
Vntr Analysis

Using 243 Mycobacterium tuberculosis isolates obtained in 2001 in Osaka City, Japan, the discriminatory power of variable numbers of tandem repeats (VNTRs) of 12 standard mycobacterial interspersed repetitive units (MIRUs) was assessed. The biggest cluster defined by MIRU-VNTRs consisted of 57 (23.5 %) isolates and they belonged to the Beijing family based on spoligotyping. When additional VNTR loci were included in the MIRU-VNTR analysis, the 57 originally clustered strains were further differentiated by the addition of Queen's University Belfast (QUB)-VNTRs, but not exact tandem repeat-VNTR. The allelic diversity of additional VNTR loci such as VNTR 3232 (QUB-3232), VNTR 2163a (QUB-11a), VNTR 2163b (QUB-11b) and VNTR 1982 (QUB-18) was high in the 57 strains. When the 243 M. tuberculosis isolates were analysed using 16-locus VNTR (the 12 standard MIRUs and the 4 QUB loci) and IS6110 RFLP, the respective Hunter–Gaston discriminatory indexes were 0.9966 and 0.9971. The discrimination power of 16-locus VNTR was equal to that of IS6110 RFLP analysis. If appropriate loci are added to the standard MIRU analysis, VNTR genotyping could be a valuable tool for strain typing and epidemiological research of M. tuberculosis in Japan.

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 Can 15-Locus Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Analysis Provide Insight into the Evolution of Mycobacterium tuberculosis?

2005 ◽  
Vol 71 (12) ◽  
pp. 8207-8213 ◽  
Author(s):  
Andrea Gibson ◽  
Timothy Brown ◽  
Lucy Baker ◽  
Francis Drobniewski
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Tandem Repeats ◽  
Variable Number Tandem Repeat ◽  
Epidemiological Studies ◽  
Variable Number ◽  
Molecular Techniques ◽  
Nucleotide Polymorphisms ◽  
East African ◽  
Phylogeny And Evolution ◽  
Phylogenetic Lineages

ABSTRACT The phylogeny and evolution of the bacterium Mycobacterium tuberculosis is still poorly understood despite the application of a variety of molecular techniques. We analyzed 469 M. tuberculosis and 49 Mycobacterium bovis isolates to evaluate if the mycobacterial interspersed repetitive units-variable-number tandem repeats (MIRU-VNTR) commonly used for epidemiological studies can define the phylogeny of the M. tuberculosis complex. This population was characterized by previously identified silent single-nucleotide polymorphisms (sSNPs) or by a macroarray based on these sSNPs that was developed in this study. MIRU-VNTR phylogenetic codes capable of differentiating between phylogenetic lineages were identified. Overall, there was 90.9% concordance between the lineages of isolates as defined by the MIRU-VNTR and sSNP analyses. The MIRU-VNTR phylogenetic code was unique to M. bovis and was not observed in any M. tuberculosis isolates. The codes were able to differentiate between different M. tuberculosis strain families such as Beijing, Delhi, and East African-Indian. Discrepant isolates with similar but not identical MIRU-VNTR codes often displayed a stepwise trend suggestive of bidirectional evolution. A lineage-specific panel of MIRU-VNTR can be used to subdivide each lineage for epidemiological purposes. MIRU-VNTR is a valuable tool for phylogenetic studies and could define an evolutionarily uncharacterized population of M. tuberculosis complex organisms.

scholarly journals Strain Typing Methods and Molecular Epidemiology ofPneumocystisPneumonia

2004 ◽  
Vol 10 (10) ◽  
pp. 1729-1735 ◽  
Author(s):  
Charles Ben Beard ◽  
Patricia Roux ◽  
Gilles Nevez ◽  
Philippe M. Hauser ◽  
Joseph A. Kovacs ◽  
...  
Keyword(s):  
Molecular Epidemiology ◽  
Strain Typing ◽  
Typing Methods

scholarly journals Severe Clinical Outcomes of Tuberculosis in Kharkiv Region, Ukraine, Are Associated with Beijing Strains of Mycobacterium tuberculosis

Pathogens ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 75 ◽  
Author(s):  
Olha Konstantynovska ◽  
Mariia Rekrotchuk ◽  
Ivan Hrek ◽  
Anton Rohozhyn ◽  
Nataliia Rudova ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Outcomes ◽  
Tandem Repeats ◽  
Genotypic Variation ◽  
Variable Number Tandem Repeat ◽  
Severe Infection ◽  
Variable Number ◽  
Beijing Genotype ◽  
Multiple Drug ◽  
Public Health Response

Genotypic variation in Beijing lineages of Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), has been associated with hyper virulence and the spread of extensively and multiple drug (X/MDR) resistant MTB strains in Eastern Europe, Central Asia, and East Asia. The clinical outcomes of 215 new cases of TB among the population of the Kharkiv region of Eastern Ukraine were analyzed to uncover factors associated with severe infection. Infecting MTB strains were profiled by 5 locus exact tandem repeats (ETRs) and 15 locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) genotyping. Among diverse MTB genotypes discovered in Ukraine, the Beijing genotype (MIRU-VNTR 42425) was significantly associated with risk factors for severe outcomes of disease in the study population, including TB/HIV co-infection and treatment failure. Strain replacement (superinfection) was observed in 10 patients, suggesting repeated exposure to novel MTB strains in hospital or community settings. Inclusion of MTB genotyping data may identify at-risk patients and improve treatment adherence to prevent X/MDR development for effective public health response against tuberculosis in Ukraine.

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