Prevalence and Characterization of Heterogeneous Variable-Number Tandem-Repeat Clusters Comprising Drug-Susceptible and/or Variable Resistant Mycobacterium tuberculosis Complex Isolates in the Netherlands from 2004 to 2016.

The variable-number tandem-repeat (VNTR) typing method is used to study tuberculosis (TB) transmission. Clustering of Mycobacterium tuberculosis isolates 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.

Correction: Epidemiological links between tuberculosis cases identified twice as efficiently by whole genome sequencing than conventional molecular typing: A population-based study.

[This corrects the article DOI: 10.1371/journal.pone.0195413.].

Epidemiological links between tuberculosis cases identified twice as efficiently by whole genome sequencing than conventional molecular typing: A population-based study.

BACKGROUND: Patients with Mycobacterium tuberculosis isolates sharing identical DNA fingerprint patterns can be epidemiologically linked. However, municipal health services in the Netherlands are able to confirm an epidemiological link in only around 23% of the patients with isolates clustered by the conventional variable number of tandem repeat (VNTR) genotyping. This research aims to investigate whether whole genome sequencing (WGS) is a more reliable predictor of epidemiological links between tuberculosis patients than VNTR genotyping. METHODS: VNTR genotyping and WGS were performed in parallel on all Mycobacterium tuberculosis complex isolates received at the Netherlands National Institute for Public Health and the Environment in 2016. Isolates were clustered by VNTR when they shared identical 24-loci VNTR patterns; isolates were assigned to a WGS cluster when the pair-wise genetic distance was ≤ 12 single nucleotide polymorphisms (SNPs). Cluster investigation was performed by municipal health services on all isolates clustered by VNTR in 2016. The proportion of epidemiological links identified among patients clustered by either method was calculated. RESULTS: In total, 535 isolates were genotyped, of which 25% (134/535) were clustered by VNTR and 14% (76/535) by WGS; the concordance between both typing methods was 86%. The proportion of epidemiological links among WGS clustered cases (57%) was twice as common than among VNTR clustered cases (31%). CONCLUSION: When WGS was applied, the number of clustered isolates was halved, while all epidemiologically linked cases remained clustered. WGS is therefore a more reliable tool to predict epidemiological links between tuberculosis cases than VNTR genotyping and will allow more efficient transmission tracing, as epidemiological investigations based on false clustering can be avoided.

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

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.

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

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.

Analysing Tuberculosis Cases Among Healthcare Workers to Inform Infection Control Policy and Practices.

OBJECTIVE To determine the number and proportion of healthcare worker (HCW) tuberculosis (TB) cases infected while working in healthcare institutions in the Netherlands and to learn from circumstances that led to these infections. DESIGN Cohort analysis. METHODS We included all HCW TB patients reported to the Netherlands TB Register from 2000 to 2015. Using data from this register, including DNA fingerprints of the bacteria profile and additional information from public health clinics, HCW TB cases were classified into 4 categories: (1) infected during work in the Netherlands, (2) infected in the community, (3) infected outside the Netherlands, or (4) outside these 3 categories. An in-depth analysis of category 1 cases was performed to identify factors contributing to patient-to-HCW transmission. RESULTS In total, 131 HCW TB cases were identified: 32 cases (24%) in category 1; 13 cases (10%) in category 2; 42 cases (32%) in category 3; and 44 cases (34%) in category 4. The annual number of HCW TB cases (P<.05), the proportion among reported cases (P<.01), and the number of category 1 HCW TB cases (P=.12) all declined over the study period. Delayed diagnosis in a TB patient was the predominant underlying factor of nosocomial transmission in 47% of category 1 HCW TB patients, most of whom were subsequently identified in a contact investigation. Performing high-risk procedures was the main contributing factor in the other 53% of cases. CONCLUSION In low-incidence countries, every HCW TB case should warrant timely and thorough investigation to help further define and fine-tune the HCW screening policy and to monitor its proper implementation. Infect Control Hosp Epidemiol 2017;38:976-982.

Underdiagnosis of foodborne hepatitis A, The Netherlands, 2008-2010(1.).

Outbreaks of foodborne hepatitis A are rarely recognized as such. Detection of these infections is challenging because of the infection's long incubation period and patients' recall bias. Nevertheless, the complex food market might lead to reemergence of hepatitis A virus outside of disease-endemic areas. To assess the role of food as a source of infection, we combined routine surveillance with real-time strain sequencing in the Netherlands during 2008-2010. Virus RNA from serum of 248 (59%) of 421 reported case-patients could be sequenced. Without typing, foodborne transmission was suspected for only 4% of reported case-patients. With typing, foodborne transmission increased to being the most probable source of infection for 16%. We recommend routine implementation of an enhanced surveillance system that includes prompt forwarding and typing of hepatitis A virus RNA isolated from serum, standard use of questionnaires, data sharing, and centralized interpretation of data.