Whole genome sequencing as the ultimate tool to diagnose tuberculosis

AbstractIn the past two decades, DNA techniques have been increasingly used in the laboratory diagnosis of tuberculosis (TB). The (sub) species of the Mycobacterium tuberculosis complex are usually identified using reverse line blot techniques. The resistance is predicted by the detection of mutations in genes associated with resistance. Nevertheless, all cases are still subjected to cumbersome phenotypic resistance testing. The production of a strain-characteristic DNA fingerprint, to investigate the epidemiology of TB, is done by the 24-locus variable number tandem repeat (VNTR) typing. However, most of the molecular techniques in the diagnosis of TB can eventually be replaced by whole genome sequencing (WGS). Many international TB reference laboratories are currently working on the introduction of WGS; however, standardization in the international context is lacking. The European Centre for Infectious Disease Prevention and Control in Stockholm, Sweden organizes a yearly round of quality control on VNTR typing and in 2015 for the first time also WGS. In this first proficiency study, only three out of eight international TB laboratories produced WGS results in line with those of the reference laboratory. The whole process of DNA isolation, purification, quantification, sequencing, and analysis/interpretation of data is still under development. In this presentation, many aspects will be covered that influence the quality and interpretation of WGS results. The turn-around-time, analysis, and utility of WGS will be discussed. Moreover, the experiences in the use of WGS in the molecular epidemiology of TB in The Netherlands are detailed. It can be concluded that many difficulties still have to be conquered. The state of the art is that bacteria still have to be cultured to have sufficient quality and quantity of DNA for succesful WGS. The quality of sequencing has improved significantly over the past 7years, and the detection of mutations has, therefore, become more reliable. The resistance mutations detected in WGS are in line with the ones visualized in reverse line blot techniques. The turnover in the genome of M. tuberculosis is very low, ∼0.3–0.5 mutations per genome per year. However, there is a wide variation in the occurrence of mutations per strain and genotype. Still, the resolution of WGS in epidemiological typing is higher than that in VNTR typing; previously suggested epidemiological links by VNTR typing are sometimes refuted on the basis of WGS. Although WGS offers the highest resolution in typing, in a country like The Netherlands, there are many strains with a limited genetic distance up to 100 mutations, without an apparent epidemiological link between the respective cases. These lookalikes are presumably even more prevalent in settings where predominant genotypes of M. tuberculosis are circulating. In summary, WGS seems to yield a more reliable prediction of resistance by the (lack of) detection of mutations in all 25 genes ever associated with resistance. This may within a short while prevent the need for many phenotypic resistance tests. Although more robust algorithms need to be developed, the recognition of the (sub) species in the M. tuberculosis complex seems possible. The first detailed studies on the population structure of M. tuberculosis strains in The Netherlands provide more resolution in typing but also an interesting observation that a part of the strains are genetically so conserved that they are separated by less than 100 mutations. This demands a more extended and accurate validation and understanding of the utility of WGS in the epidemiology of TB

Towards standardisation:comparison of five whole genome sequencing (WGS) analysis pipelines for detection of epidemiologically linked tuberculosis cases

BackgroundWhole genome sequencing (WGS) is a reliable tool for studying tuberculosis (TB) transmission. WGS data are usually processed by custom-built analysis pipelines with little standardisation between them.AimTo compare the impact of variability of several WGS analysis pipelines used internationally to detect epidemiologically linked TB cases.MethodsFrom the Netherlands, 535 Mycobacterium tuberculosis complex (MTBC) strains from 2016 were included. Epidemiological information obtained from municipal health services was available for all mycobacterial interspersed repeat unit-variable number of tandem repeat (MIRU-VNTR) clustered cases. WGS data was analysed using five different pipelines: one core genome multilocus sequence typing (cgMLST) approach and four single nucleotide polymorphism (SNP)-based pipelines developed in Oxford, United Kingdom; Borstel, Germany; Bilthoven, the Netherlands and Copenhagen, Denmark. WGS clusters were defined using a maximum pairwise distance of 12 SNPs/alleles.ResultsThe cgMLST approach and Oxford pipeline clustered all epidemiologically linked cases, however, in the other three SNP-based pipelines one epidemiological link was missed due to insufficient coverage. In general, the genetic distances varied between pipelines, reflecting different clustering rates: the cgMLST approach clustered 92 cases, followed by 84, 83, 83 and 82 cases in the SNP-based pipelines from Copenhagen, Oxford, Borstel and Bilthoven respectively.ConclusionConcordance in ruling out epidemiological links was high between pipelines, which is an important step in the international validation of WGS data analysis. To increase accuracy in identifying TB transmission clusters, standardisation of crucial WGS criteria and creation of a reference database of representative MTBC sequences would be advisable

Prevalence and characterization of heterogeneous VNTR clusters comprising drug susceptible and/or variable resistant Mycobacterium tuberculosis complex isolates in the Netherlands from 2004-2016.

Introduction: The variable number of tandem repeats (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, also heterogeneous clusters are 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).Methods: In the period 2004-2016, 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 (varying) 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 cut-off for WGS clustering.Results: In total, 4,661/9,072 (51%) isolates were clustered in 985 different VNTR clusters, of which 217 (22%) were heterogeneous. Patient characteristics associated with heterogeneous clustering were non-Dutch ethnicity (OR 1.46 [1.22-1.75]), asylum seeker (OR 1.51 [1.24-1.85]), extra pulmonary TB (OR 1.26 [1.09-1.46]), previous TB diagnosis (OR 1.38 [1.04-1.82]), and non-contact of a TB patient (OR 1.35 [1.08-1.69]). With WGS, 34% of heterogeneous and 78% of homogeneous isolates from 2016 remained clustered.Conclusion: Heterogeneous VNTR clusters are common, but seem to be explained by a substantial degree of false clustering by VNTR when 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.]

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

Towards standardisation: comparison of five whole genome sequencing (WGS) analysis pipelines for detection of epidemiologically linked tuberculosis cases.

BackgroundWhole genome sequencing (WGS) is a reliable tool for studying tuberculosis (TB) transmission. WGS data are usually processed by custom-built analysis pipelines with little standardisation between them.AimTo compare the impact of variability of several WGS analysis pipelines used internationally to detect epidemiologically linked TB cases.MethodsFrom the Netherlands, 535 Mycobacterium tuberculosis complex (MTBC) strains from 2016 were included. Epidemiological information obtained from municipal health services was available for all mycobacterial interspersed repeat unit-variable number of tandem repeat (MIRU-VNTR) clustered cases. WGS data was analysed using five different pipelines: one core genome multilocus sequence typing (cgMLST) approach and four single nucleotide polymorphism (SNP)-based pipelines developed in Oxford, United Kingdom; Borstel, Germany; Bilthoven, the Netherlands and Copenhagen, Denmark. WGS clusters were defined using a maximum pairwise distance of 12 SNPs/alleles.ResultsThe cgMLST approach and Oxford pipeline clustered all epidemiologically linked cases, however, in the other three SNP-based pipelines one epidemiological link was missed due to insufficient coverage. In general, the genetic distances varied between pipelines, reflecting different clustering rates: the cgMLST approach clustered 92 cases, followed by 84, 83, 83 and 82 cases in the SNP-based pipelines from Copenhagen, Oxford, Borstel and Bilthoven respectively.ConclusionConcordance in ruling out epidemiological links was high between pipelines, which is an important step in the international validation of WGS data analysis. To increase accuracy in identifying TB transmission clusters, standardisation of crucial WGS criteria and creation of a reference database of representative MTBC sequences would be advisable

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 tuberculosisisolates are routinely subjected to variable-number tandem-repeat (VNTR) typing to investigateM. tuberculosistransmission. 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 40M. tuberculosisisolates 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 theM. tuberculosiscases, 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 directM. tuberculosistransmission 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