BackgroundMycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate knowledge of the rate of change in the genome over relevant timescales is required.MethodsWe attempted to estimate a molecular clock by sequencing 199 isolates from epidemiologically linked tuberculosis cases, collected in the Netherlands spanning almost 16 years.ResultsMultiple analyses support an average mutation rate of ~0.3 SNPs per genome per year. However, all analyses revealed a very high degree of variation around this mean, making the confirmation of links proposed by epidemiology, and inference of novel links, difficult. Despite this, in some cases, the phylogenetic context of other strains provided evidence supporting the confident exclusion of previously inferred epidemiological links.ConclusionsThis in-depth analysis of the molecular clock revealed that it is slow and variable over short time scales, which limits its usefulness in transmission studies. However, the superior resolution of whole genome sequencing can provide the phylogenetic context to allow the confident exclusion of possible transmission events previously inferred via traditional DNA fingerprinting techniques and epidemiological cluster investigation. Despite the slow generation of variation even at the whole genome level we conclude that the investigation of tuberculosis transmission will benefit greatly from routine whole genome sequencing.

Background: Tuberculosis (TB) is a major health problem and HIV is the major cause of the increase in TB. Sub-Saharan Africa is endemic for both TB and HIV infection. Determination of the prevalence of M. tuberculosis strains and their drug susceptibility is important for TB control.

The European Centre for Disease Prevention and Control (ECDC) initiated a project on the molecular surveillance of multi-and extensively drug-resistant tuberculosis (MDR-/XDR-TB) transmission in the European Union (EU) in the period from 2009 to 2011. In total, 2,092 variable number of tandem repeat (VNTR) patterns of MDR-/XDR-TB Mycobacterium tuberculosis isolates were collected, originating from 24 different countries in the period 2003 to 2011. Of the collected VNTR patterns, 45% (n=941) could be assigned to one of the 79 European multiple-country molecular fingerprint clusters and 50% of those (n=470) belonged to one extremely large cluster caused by Beijing strains of one genotype. We conclude that international transmission of MDR-/XDR-TB plays an important role in the EU, especially in the eastern part, and is significantly related to the spread of one strain or clone of the Beijing genotype. Implementation of international cluster investigation in EU countries should reveal underlying factors of transmission, and show how TB control can be improved regarding case finding, contact tracing, infection control and treatment in order to prevent further spread of MDR-/XDR-TB in the EU.

f Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra-and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter-and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future.

Tap and shower water at two locations in the Netherlands was examined for the presence of rapidly growing nontuberculous mycobacteria. Cultures yielded Mycobacterium peregrinum, M. salmoniphilum, M. llatzerense, M. septicum, and three potentially novel species, a distribution different from that in clinical samples.