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

Abstract

Since 2004, variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex 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.

MIRU-profiler: a rapid tool for determination of 24-loci MIRU-VNTR profiles from assembled genomes ofMycobacterium tuberculosis

“…The microevolution could also result in minor discrepancies between the experimental genotyping and WGS. Indeed, the most discrepant loci in our validation (QUB26 and QUB11b) were reported to be frequently associated with double alleles, a probable consequence of the microevolution ( Jajou et al, 2017 ). The accuracy of the genome assembly, which crucially depends on the read length, is also vital to the digital inference of the MIRU-VNTR profile.…”

Methods for Detecting Mycobacterial Mixed Strain Infections–A Systematic Review

“…Considering that the criteria necessary for identifying different types of M. tuberculosis polyclonal infections using IS6110-RFLP and MIRU-VNTR are well-defined, several studies were also found in the current review that reported on microevolution (Martín et al, 2007;Streit et al, 2015;Cohen et al, 2016;Jajou et al, 2018). The classification was not evident in some other reports that used IS6110-RFLP or MIRU-VNTR, due to results potentially indicating either an MSI or a microevolution event depending on the applied criteria (Al-Hajoj et al, 2010;Alves et al, 2011), or due to WGS not having a well-defined SNP threshold (Liu et al, 2015).…”

Pyrazinamide/Pyrazinoic Acid Resistance in Mycobacterium Tuberculosis: Recent Findings and Implications for Improving the Treatment of Tuberculosis

“…This may be true but caution is warranted as routine culture often misses > 90% of viable cells [18]. Also, the observation that infections with double MIRU-VNTR bands are mixed infections that can be transmitted between patients [33] provides circumstantial evidence that new infections are often the result of larger numbers of mycobacterial cells that preserve some of the genetic diversity present in the source case. This preserved diversity within the infecting bacterial population may ultimately be useful to identify more details of transmission dynamics [8,33,39].…”

“…Also, the observation that infections with double MIRU-VNTR bands are mixed infections that can be transmitted between patients [33] provides circumstantial evidence that new infections are often the result of larger numbers of mycobacterial cells that preserve some of the genetic diversity present in the source case. This preserved diversity within the infecting bacterial population may ultimately be useful to identify more details of transmission dynamics [8,33,39]. Even more interestingly, it was recently observed that patients with a higher proportion of latent bacteria in their sputum appeared more likely to transmit the disease to close contacts [16].…”