To characterize the genetic determinants of resistance to antituberculosis drugs, we performed a genome-wide association study (GWAS) of 6,465 Mycobacterium tuberculosis clinical isolates from more than 30 countries. A GWAS approach within a mixed-regression framework was followed by a phylogenetics-based test for independent mutations. In addition to mutations in established and recently described resistance-associated genes, novel mutations were discovered for resistance to cycloserine, ethionamide and para-aminosalicylic acid. The capacity to detect mutations associated with resistance to ethionamide, pyrazinamide, capreomycin, cycloserine and para-aminosalicylic acid was enhanced by inclusion of insertions and deletions. Odds ratios for mutations within candidate genes were found to reflect levels of resistance. New epistatic relationships between candidate drug-resistance-associated genes were identified. Findings also suggest the involvement of efflux pumps (drrA and Rv2688c) in the emergence of resistance. This study will inform the design of new diagnostic tests and expedite the investigation of resistance and compensatory epistatic mechanisms.

Approximately 10 % of the Mycobacterium tuberculosis genome is made up of two families of genes that are poorly characterized due to their high GC content and highly repetitive nature. The PE and PPE families are typified by their highly conserved N-terminal domains that incorporate proline-glutamate (PE) and proline-proline-glutamate (PPE) signature motifs. They are hypothesised to be important virulence factors involved with host-pathogen interactions, but their high genetic variability and complexity of analysis means they are typically disregarded in genome studies. To elucidate the structure of these genes, 518 genomes from a diverse international collection of clinical isolates were de novo assembled. A further 21 reference M. tuberculosis complex genomes and long read sequence data were used to validate the approach. SNP analysis revealed that variation in the majority of the 168 pe/ppe genes studied was consistent with lineage. Several recombination hotspots were identified, notably pe_pgrs3 and pe_pgrs17. Evidence of positive selection was revealed in 65 pe/ppe genes, including epitopes potentially binding to major histocompatibility complex molecules. This, the first comprehensive study of the pe and ppe genes, provides important insight into M. tuberculosis diversity and has significant implications for vaccine development.

In recent years various novel DNA typing methods have been developed which are faster and easier to perform than the current internationally standardized IS6110restriction fragment length polymorphism typing method. However, there has been no overview of the utility of these novel typing methods, and it is largely unknown how they compare to previously published methods. In this study, the discriminative power and reproducibility of nine recently described PCR-based typing methods forMycobacterium tuberculosiswere investigated using the strain collection of the interlaboratory study of Kremer et al. (J. Clin. Microbiol. 37:2607-2618, 1999). This strain collection contains 90M. tuberculosiscomplex and 10 non-M. tuberculosiscomplex mycobacterial strains, as well as 31 duplicated DNA samples to assess reproducibility. The highest reproducibility was found with variable numbers of tandem repeat typing using mycobacterial interspersed repetitive units (MIRU VNTR) and fast ligation-mediated PCR (FLiP), followed by second-generation spoligotyping, ligation-mediated PCR (LM-PCR), VNTR typing using five repeat loci identified at the Queens University of Belfast (QUB VNTR), and the Amadio speciation PCR. Poor reproducibility was associated with fluorescent amplified fragment length polymorphism typing, which was performed in three different laboratories. The methods were ordered from highest discrimination to lowest by the Hunter-Gaston discriminative index as follows: QUB VNTR typing, MIRU VNTR typing, FLiP, LM-PCR, and spoligotyping. We conclude that both VNTR typing methods and FLiP typing are rapid, highly reliable, and discriminative epidemiological typing methods forM. tuberculosisand that VNTR typing is the epidemiological typing method of choice for the near future.

The reference standard methods for drug susceptibility testing (DST) of M. tuberculosis are very slow to give results, and due to the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis, there is an urgent demand for new, rapid and accurate DST methods, particularly in low-income countries. The nitrate reductase assay (NRA) has been proposed as a rapid method for the detection of resistance to rifampicin and isoniazid, but its accuracy has not been systematically evaluated. We performed a systematic review and meta-analysis to evaluate the accuracy of the NRA for the detection of rifampicin- and isoniazid-resistant tuberculosis. We searched Medline PubMed (NCBI), Global Health-CAB, EJS-E (EbscoHost), ISI Web, Web of Science and IFCC and contacted authors if additional information was required. Fifteen studies met our inclusion criteria for rifampicin resistance detection and 13 for isoniazid. Of these, the majority of the studies used culture isolates on solid medium, four used culture isolates on liquid medium and three used sputum samples. We applied the summary receiver operating characteristic (SROC) curve to perform meta-analysis and to summarize diagnostic accuracy. For rifampicin, the majority of the studies that applied NRA to isolates had a sensitivity and specificity >94% and for isoniazid, >92%. The three studies that applied NRA directly on sputum samples had a sensitivity and specificity that ranged between 88% and 100%. The SROC curve had an area of >0.99 for both drugs. There is evidence that NRA is highly sensitive and specific for the rapid detection of rifampicin and isoniazid resistance in culture isolates. More evidence is required for the NRA applied directly on sputum samples, but preliminary results appear promising and show a good sensitivity and specificity. Additional studies are required in countries with a high prevalence of MDR-TB and also cost-effectiveness analysis in order to obtain a complete picture on the utility of this method for rapid drug resistance detection in tuberculosis.