A Rapid and Sensitive Diagnostic Screening Assay for Detection of Mycobacteria Including Mycobacterium tuberculosis Directly from Sputum without Extraction.

We report a novel approach utilising a real-time PCR screening assay targeting a 53 bp tandemly repeated element present at various loci within the Mycobacterium tuberculosis (Mtb) genome. Positive samples were identified within a discriminatory melting curve range of 90-94°C, with results obtained in under one hour directly from decontaminated sputum samples without extraction. A panel of 89 smear-positive sputa were used for analytical validation of the assay with 100% concordance, with sensitivity matching that of culture. Cross reactivity was detected within a narrow range of mycobacteria other than tuberculosis (MOTT) (five sputa, three in silico), with the highest sensitivity within M. avium complex (MAC). A year-long head to head evaluation of the test with the GeneXpert platform was carried out with 104 consecutive samples at the Royal Free Hospital, UK. Receiver operating characteristics (ROC) analysis of the data revealed that the two tests are approximately equivalent in sensitivity, with the area under the curve being 0.85 and 0.80 for the GeneXpert and our assay, respectively, indicating that the test would be a cost effective screen prior to GeneXpert testing.

IS6110-based global phylogeny of Mycobacterium tuberculosis.

IS6110, a Mycobacterium tuberculosis complex species-specific insertion element, is targeted primarily for DNA fingerprinting of M. tuberculosis strains. The number and chromosomal positions of copies of this element have been found to be highly variable between unrelated strains and have been exploited for molecular epidemiological purpose but the utility of IS6110 as an informative marker of strain phylogeny has yet to be demonstrated. In the current study, a recently proposed IS6110-targetting PCR based typing methodology, fluorescent amplified fragment length polymorphism (fAFLP) was applied to a global panel of 166 of the clinically more predominant 'modern' strains characterised by spoligotype and, where available, Variable Number Tandem Repeat (VNTR) to identify potentially evolutionarily informative common fragments that could define strains as belonging to established genetic lineages. These common fragments are hereby proposed to be ancestral insertion sites present in common ancestors of these strains rather than preferential insertion sites or 'hot spots'. Results indicate that the exact same spoligotype and VNTR-defined lineages are reflected in the fragment patterns but with greater resolution and are able to clearly define the very distinct Haarlem, LAM, X and also the currently ill-defined T and S and lineages and spoligotypes designated as U, or unknown, without ambiguity. The biogeographical patterns generated reflect the migration of mankind across the globe and indicate that only four successful clones (or individual bacteria) gave rise to virtually all of the tuberculosis in Europe and the Americas. Potential lies in the application of the data to determine IS6110 evolutionary events that have occurred during the evolution of these lineages.

Comparison of four-colour IS6110-fAFLP with the classic IS6110-RFLP on the ability to detect recent transmission in the city of Barcelona, Spain.

The purpose of the study was to compare the IS6110-RFLP (RFLP) results obtained in a previous epidemiological study in the city of Barcelona, Spain [Borrell S, Espanol M, Orcau A, Tudo G, March F, Cayla JA, et al. Factors associated with differences between conventional contact tracing and molecular epidemiology in study of tuberculosis transmission and analysis in the city of Barcelona, Spain. J Clin Microbiol 2009 Jan;47(1):198-204.] with the results obtained with IS6110-fAFLP, [Thorne N, Evans JT, Smith EG, Hawkey PM, Gharbia S, Arnold C. An IS6110-targeting fluorescent amplified fragment length polymorphism alternative to IS6110-restriction fragment length polymorphism analysis for Mycobacterium tuberculosis DNA fingerprinting. Clin Microbiol Infect 2007 Oct;13(10):964-70.] on the ability to detect recent transmission. fAFLP was applied to DNA samples of RFLP clustered strains, with and without known epidemiological links, with the additional inclusion of four nucleotide-specific fluorophores to further increase the discrimination of the fragments obtained. Four-colour fAFLP was performed on 123 RFLP clustered strains with no epidemiological link (NELC) and on 28 epidemiologically linked RFLP clustered (ELC) strains grouped into 48 and 13 clusters respectively. Clustering results obtained by the two methods were highly congruent in ELC strains with fAFLP allocating 92.3% of the ELCs. For the NELCs, RFLP results were confirmed in 39/48 (81.2%) of fAFLP-clusters with 0-1 different fragments and 9/48 (18.8%) differed in 2-4 fragments, which are considered genetically related but not recently transmitted. In conclusion, overestimation of recent tuberculosis transmission can occur because of the inaccurate analysis of RFLP results. Four-colour fAFLP allows us to differentiate between recent transmission strains and epidemiologically unrelated but genetically related strains.

Mathematical modelling of Mycobacterium tuberculosis VNTR loci estimates a very slow mutation rate for the repeats.

Minisatellites are highly variable tandem repeats used for over 20 years in humans for DNA fingerprinting. In prokaryotes fingerprinting techniques exploiting VNTR (variable number of tandem repeats) polymorphisms have become widely used recently in bacterial typing. However although many investigations into the mechanisms underlying minisatellite variation in humans have been performed, relatively little is known about the processes that mediate bacterial minisatellite polymorphism. An understanding of this is important since it will influence how the results from VNTR experiments are interpreted. The minisatellites of Mycobacterium tuberculosis are well characterized since they are some of the few polymorphic loci in what is otherwise a very homogeneous organism. Using VNTR results from a well-defined and characterized set of M. tuberculosis strains we show that the repeats at a locus are likely to evolve by stepwise contraction or expansion in the number of repeats. A stochastic continuous-time population mathematical model was developed to simulate the evolution of the repeats. This allowed estimation of the tendency of the repeats to increase or decrease and the rate at which they change. The majority of loci tend to lose rather than gain repeats. All of the loci mutate extremely slowly, with an average rate of 2.3 x 10(-8), which is 350 times slower than that of a set of VNTR repeats with similar diversity observed experimentally in Escherichia coli. This suggests that the VNTR profile of a strain of M. tuberculosis will be indicative of its clonal lineage and will be unlikely to vary in epidemiologically-related strains.

Evolutionary clues from comparative analysis of Mycobacterium tuberculosis variable-number tandem repeat sequences within genetic families.

Mycobacterium tuberculosis is one of the most successful bacterial pathogens in the history of mankind, and the study of the evolutionary and genetic behaviour of this organism is ongoing. The potential of variable-number tandem repeat (VNTR) regions to provide insight into the evolutionary past of this organism has yet to be evaluated. The aim of this study was to investigate diversity occurring within VNTR loci within established evolutionary lineages. Mycobacterial interspersed repetitive unit (MIRU) 4 and 26 nucleotide sequencing was undertaken revealing significant differences in VNTR discriminatory power and distribution of allelic types within defined sub-lineages, within a moderately discriminant and a highly discriminant locus, respectively. These findings indicate potential allele bias at these loci within the defined groups dependent on the genotype of their progenitor strain, and the patterns of distribution suggest that a step-wise contraction/expansion process is responsible for VNTR repeat evolution. The results also identified a possible incidence of genetic drift within major genetic group 2 (MGG2), which has a point mutation at locus MIRU26 in a clonal subgroup.

Evolution of short sequence repeats in Mycobacterium tuberculosis.

Whole genome comparison has revealed the presence of short sequence repeats (also called mycobacterial interspersed repeat units and variable number tandem repeat units) used for genotyping schemes. In this study, we have used deletion analysis, single nucleotide polymorphism data and spoligotype taken from published data from others to investigate the evolution of selected repeats that form the common denominators of the majority of established schemes. Analysis of the number of repeats per locus from over 400 isolates revealed that the general trend globally appears to be loss of repeats in modern strains compared with ancestral strains.