Occurrence and Nature of Double Alleles in Variable-Number Tandem-Repeat Patterns of More than 8,000 Mycobacterium tuberculosis Complex Isolates in The Netherlands

ABSTRACTSince 2004, variable-number tandem-repeat (VNTR) typing ofMycobacterium tuberculosiscomplex isolates has been applied on a structural basis in The Netherlands to study the epidemiology of tuberculosis (TB). Although this technique is faster and technically less demanding than the previously used restriction fragment length polymorphism (RFLP) typing, reproducibility remains a concern. In the period from 2004 to 2015, 8,532 isolates were subjected to VNTR typing in The Netherlands, with 186 (2.2%) of these exhibiting double alleles at one locus. Double alleles were most common in loci 4052 and 2163b. The variables significantly associated with double alleles were urban living (odds ratio [OR], 1.503; 95% confidence interval [CI], 1.084 to 2.084;P= 0.014) and pulmonary TB (OR, 1.703; 95% CI, 1.216 to 2.386;P= 0.002). Single-colony cultures of double-allele strains were produced and revealed single-allele profiles; a maximum of five single nucleotide polymorphisms (SNPs) was observed between the single- and double-allele isolates from the same patient when whole-genome sequencing (WGS) was applied. This indicates the presence of two bacterial populations with slightly different VNTR profiles in the parental population, related to genetic drift. This observation is confirmed by the fact that secondary cases from TB source cases with double-allele isolates sometimes display only one of the two alleles present in the source case. Double alleles occur at a frequency of 2.2% in VNTR patterns in The Netherlands. They are caused by biological variation rather than by technical aberrations and can be transmitted either as single- or double-allele variants.

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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

Journal of Clinical Microbiology ◽

10.1128/jcm.00887-18 ◽

2018 ◽

Vol 56 (11) ◽

Cited By ~ 3

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.

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A Predominant Variable-Number Tandem-Repeat Cluster of Mycobacterium tuberculosis Isolates among Asylum Seekers in the Netherlands and Denmark, Deciphered by Whole-Genome Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.01100-17 ◽

2017 ◽

Vol 56 (2) ◽

Cited By ~ 5

Author(s):

Rana Jajou ◽

Albert de Neeling ◽

Erik Michael Rasmussen ◽

Anders Norman ◽

Arnout Mulder ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

The Netherlands ◽

Genetic Distance ◽

Genome Sequencing ◽

Tandem Repeat ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Whole Genome ◽

Average Genetic Distance ◽

Content Type

ABSTRACT In many countries, Mycobacterium tuberculosis isolates are routinely subjected to variable-number tandem-repeat (VNTR) typing to investigate M. tuberculosis transmission. Unexpectedly, cross-border clusters were identified among African refugees in the Netherlands and Denmark, although transmission in those countries was unlikely. Whole-genome sequencing (WGS) was applied to analyze transmission in depth and to assess the precision of VNTR typing. WGS was applied to 40 M. tuberculosis isolates from refugees in the Netherlands and Denmark (most of whom were from the Horn of Africa) that shared the exact same VNTR profile. Cluster investigations were undertaken to identify in-country epidemiological links. Combining WGS results for the isolates (all members of the central Asian strain [CAS]/Delhi genotype), from both European countries, an average genetic distance of 80 single-nucleotide polymorphisms (SNPs) (maximum, 153 SNPs) was observed. The few pairs of isolates with confirmed epidemiological links, except for one pair, had a maximum distance of 12 SNPs. WGS divided this refugee cluster into several subclusters of patients from the same country of origin. Although the M. tuberculosis cases, mainly originating from African countries, shared the exact same VNTR profile, most were clearly distinguished by WGS. The average genetic distance in this specific VNTR cluster was 2 times greater than that in other VNTR clusters. Thus, identical VNTR profiles did not represent recent direct M. tuberculosis transmission for this group of patients. It appears that either these strains from Africa are extremely conserved genetically or there is ongoing transmission of this genotype among refugees on their long migration routes from Africa to Europe.

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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

Journal of Clinical Microbiology ◽

10.1128/jcm.01016-17 ◽

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.

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Clustering of Tuberculosis Cases Based on Variable-Number Tandem-Repeat Typing in Relation to the Population Structure of Mycobacterium tuberculosis in the Netherlands

Journal of Clinical Microbiology ◽

10.1128/jcm.00489-13 ◽

2013 ◽

Vol 51 (7) ◽

pp. 2427-2431 ◽

Cited By ~ 19

Author(s):

R. Sloot ◽

M. W. Borgdorff ◽

J. L. de Beer ◽

J. van Ingen ◽

P. Supply ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Population Structure ◽

The Netherlands ◽

Tandem Repeat ◽

Variable Number Tandem Repeat ◽

Variable Number

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Standard Genotyping Overestimates Transmission of Mycobacterium tuberculosis among Immigrants in a Low-Incidence Country

Journal of Clinical Microbiology ◽

10.1128/jcm.00126-16 ◽

2016 ◽

Vol 54 (7) ◽

pp. 1862-1870 ◽

Cited By ~ 58

Author(s):

David Stucki ◽

Marie Ballif ◽

Matthias Egger ◽

Hansjakob Furrer ◽

Ekkehardt Altpeter ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Nucleotide Polymorphisms ◽

Foreign Born ◽

Single Nucleotide ◽

Content Type ◽

Weak Evidence ◽

High Incidence ◽

Low Incidence

Immigrants from regions with a high incidence of tuberculosis (TB) are a risk group for TB in low-incidence countries such as Switzerland. In a previous analysis of a nationwide collection of 520Mycobacterium tuberculosisisolates from 2000 to 2008, we identified 35 clusters comprising 90 patients based on standard genotyping (24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat [MIRU-VNTR] typing and spoligotyping). Here, we used whole-genome sequencing (WGS) to revisit these transmission clusters. Genome-based transmission clusters were defined as isolate pairs separated by ≤12 single nucleotide polymorphisms (SNPs). WGS confirmed 17/35 (49%) MIRU-VNTR typing clusters; the other 18 clusters contained pairs separated by >12 SNPs. Most transmission clusters (3/4) of Swiss-born patients were confirmed by WGS, as opposed to 25% (4/16) of the clusters involving only foreign-born patients. The overall clustering proportion was 17% (90 patients; 95% confidence interval [CI], 14 to 21%) by standard genotyping but only 8% (43 patients; 95% CI, 6 to 11%) by WGS. The clustering proportion was 17% (67/401; 95% CI, 13 to 21%) by standard genotyping and 7% (26/401; 95% CI, 4 to 9%) by WGS among foreign-born patients and 19% (23/119; 95% CI, 13 to 28%) and 14% (17/119; 95% CI, 9 to 22%), respectively, among Swiss-born patients. Using weighted logistic regression, we found weak evidence of an association between birth origin and transmission (adjusted odds ratio of 2.2 and 95% CI of 0.9 to 5.5 comparing Swiss-born patients to others). In conclusion, standard genotyping overestimated recent TB transmission in Switzerland compared to WGS, particularly among immigrants from regions with a high TB incidence, where genetically closely related strains often predominate. We recommend the use of WGS to identify transmission clusters in settings with a low incidence of TB.

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Mycobacterium tuberculosis in Ontario, Canada: Insights from IS6110 Restriction Fragment Length Polymorphism and Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Genotyping

Journal of Clinical Microbiology ◽

10.1128/jcm.01946-08 ◽

2009 ◽

Vol 47 (8) ◽

pp. 2651-2654 ◽

Cited By ~ 6

Author(s):

D. C. Alexander ◽

J. L. Guthrie ◽

D. Pyskir ◽

A. Maki ◽

N. Kurepina ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Restriction Fragment Length Polymorphism ◽

Restriction Fragment ◽

Length Polymorphism ◽

Tandem Repeat ◽

Fragment Length ◽

Fragment Length Polymorphism ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Restriction Fragment Length

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Multiple-Locus Variable-Number Tandem Repeat Analysis of Dutch Bordetella pertussis Strains Reveals Rapid Genetic Changes with Clonal Expansion during the Late 1990s

Journal of Bacteriology ◽

10.1128/jb.186.16.5496-5505.2004 ◽

2004 ◽

Vol 186 (16) ◽

pp. 5496-5505 ◽

Cited By ~ 99

Author(s):

Leo M. Schouls ◽

Han G. J. van der Heide ◽

Luc Vauterin ◽

Paul Vauterin ◽

Frits R. Mooi

Keyword(s):

The Netherlands ◽

Tandem Repeat ◽

Bordetella Pertussis ◽

Minimum Spanning Tree ◽

Whooping Cough ◽

Variable Number Tandem Repeat ◽

Clonal Expansion ◽

Variable Number ◽

Multiple Locus ◽

Repeat Analysis

ABSTRACT Bordetella pertussis, the causative agent of whooping cough, has remained endemic in The Netherlands despite extensive nationwide vaccination since 1953. In the 1990s, several epidemic periods have resulted in many cases of pertussis. We have proposed that strain variation has played a major role in the upsurges of this disease in The Netherlands. Therefore, molecular characterization of strains is important in identifying the causes of pertussis epidemiology. For this reason, we have developed a multiple-locus variable-number tandem repeat analysis (MLVA) typing system for B. pertussis. By combining the MLVA profile with the allelic profile based on multiple-antigen sequence typing, we were able to further differentiate strains. The relationships between the various genotypes were visualized by constructing a minimum spanning tree. MLVA of Dutch strains of B. pertussis revealed that the genotypes of the strains isolated in the prevaccination period were diverse and clearly distinct from the strains isolated in the 1990s. Furthermore, there was a decrease in diversity in the strains from the late 1990s, with a remarkable clonal expansion that coincided with the epidemic periods. Using this genotyping, we have been able to show that B. pertussis is much more dynamic than expected.

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