Maxine Caws

Mutations Prevalent among Rifampin- and Isoniazid-Resistant Mycobacterium tuberculosis Isolates from a Hospital in Vietnam

ABSTRACT Vietnam is ranked 13th among the WHO list of 22 high-burden countries, based upon estimated total number of tuberculosis cases. Despite having a model national tuberculosis program, consistently achieving and exceeding WHO targets for detection and cure, drug-resistant and multidrug-resistant tuberculosis cases continue to rise. Rapid multidrug-resistant tests applicable in this setting, coupled with effective treatment regimens, would be a useful tool in reversing this trend, allowing early identification of patients with multidrug-resistant tuberculosis and avoiding resistance-amplifying regimens. Sequencing of consecutive isolates identified by the National Tuberculosis Program showed 89% of isoniazid-resistant isolates could be detected by targeting just 2 codons, katG 315 and −15C→T in the inhA promoter, while rifampin resistance will be more complex to detect, with many different mutation and insertion events in rpoB. The most prevalent rifampin resistance-conferring mutations, as in other countries, were in rpoB codons 531 (43%), 526 (31%), and 516 (15%). However, a hybridization-based resistance test with probes targeting the 5 most common mutations would only detect 78% of rifampin-resistant isolates. Overall, these data suggest that rifampin resistance may be used as a surrogate marker for multidrug-resistant tuberculosis and that a sensitivity of between 70 to 80% may be possible for rapid molecular detection of multidrug-resistant tuberculosis in this setting.

GeneXpert MTB/Rif to Diagnose Tuberculous Meningitis: Perhaps the First Test but not the Last

Tuberculous meningitis (TBM) is the most severe form of tuberculous with substantial mortality. In May 2015, 54 researchers from 10 countries met in Da Lat, Vietnam, to discuss advances in TBM. Among the attendees were researchers involved in pivotal studies on the use of Xpert MTB/Rif for TBM diagnosis. Attendees discussed the 2014 World Health Organization strong recommendation favoring the use of Xpert in preference to conventional microscopy and culture as the initial diagnostic test for cerebrospinal fluid (CSF) if the sample volume is low or if additional specimens cannot be obtained to make a quick diagnosis. Attendees were concerned that the limitations of Xpert testing for TBM are not emphasized. Clear guidance is needed for the investigational pathway for TBM, including recommendations on the diagnostic package of investigations, which does not stop with Xpert testing. Second, emphasis on the large CSF volumes (ideally 8-10 mL) needed for Xpert testing is required. Guidelines should also emphasize that TBM is a medical emergency and early treatment reduces mortality.

Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis

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.A GWAS of multi- and extensively drug-resistant tuberculosis using 6,465 Mycobacterium tuberculosis clinical isolates from more than 30 countries identifies novel mutations associated with resistance. The capacity to detect resistance in particular to ethionamide, pyrazinamide, capreomycin, cycloserine and paraaminosalicylic acid was enhanced by inclusion of insertions and deletions.

Standardized Methods for Enhanced Quality and Comparability of Tuberculous Meningitis Studies

Summary This viewpoint defines a tuberculous meningitis core dataset, including demographic and clinical information, key patient management and monitoring data, and standardized reporting of patient outcomes. Wide adoption of standardized methods will provide a robust evidence base to improve patient outcomes.

MARCO variants are associated with phagocytosis, pulmonary tuberculosis susceptibility and Beijing lineage.

Macrophage receptor with collagenous structure (MARCO) has an important role in the phagocytosis of Mycobacterium tuberculosis (M. tuberculosis). We hypothesized that MARCO polymorphisms are associated with phagocytosis, tuberculosis (TB) disease susceptibility and presentation, and infecting lineage. We used a human cellular model to examine how MARCO genotype mediates the immune response; a case–control study to investigate tuberculosis host genetic susceptibility; and a host–pathogen genetic analysis to study host–pathogen interactions. Two MARCO heterozygous (AG) genotypes (single-nucleotide polymorphisms rs2278589 and rs6751745) were associated with impaired phagocytosis of M. tuberculosis trehalose 6,6-dimycolate-cord factor and β-glucan-coated beads in macrophages. The heterozygous genotypes of rs2278589 and rs6751745 were also associated with increased risk of pulmonary TB (PTB; rs2278589, P=0.001, odds ratio (OR)=1.6; rs6751745, P=0.009, OR=1.4), and with severe chest X-ray abnormalities (P=0.007, OR=1.6). These two genotypes were also associated with the Beijing lineage (rs2278589, P=0.001, OR=1.7; rs6751745, P=0.01, OR=1.5). Together, these results suggest that MARCO polymorphisms may regulate phagocytosis of M. tuberculosis and susceptibility and severity of PTB. They also suggest MARCO genotype and Beijing strains may interact to increase the risk of PTB.

Frequent transmission of the Mycobacterium tuberculosis Beijing lineage and positive selection for the EsxW Beijing variant in Vietnam

To examine the transmission dynamics of Mycobacterium tuberculosis (Mtb) isolated from tuberculosis patients in Ho Chi Minh City, Vietnam, we sequenced the whole genomes of 1,635 isolates and compared these with 3,144 isolates from elsewhere. The data identify an underlying burden of disease caused by the endemic Mtb lineage 1 associated with the activation of long-term latent infection, and a threefold higher burden associated with the more recently introduced Beijing lineage and lineage 4 Mtb strains. We find that Beijing lineage Mtb is frequently transferred between Vietnam and other countries, and detect higher levels of transmission of Beijing lineage strains within this host population than the endemic lineage 1 Mtb. Screening for parallel evolution of Beijing lineage-associated SNPs in other Mtb lineages as a signal of positive selection, we identify an alteration in the ESX-5 type VII-secreted protein EsxW, which could potentially contribute to the enhanced transmission of Beijing lineage Mtb in Vietnamese and other host populations.Genomic analysis of Mycobacterium tuberculosis (Mtb) isolated from tuberculosis patients identifies the transmission dynamics of Mtb in Vietnam including frequent transmission of Beijing lineage and positive selection for EsxW Beijing variant.

The Application of GeneXpert MTB/RIF for Smear-Negative TB Diagnosis as a Fee-Paying Service at a South Asian General Hospital

The GeneXpert MTB/RIF assay (Xpert) is a novel automated diagnostic tool for tuberculosis but its optimal placement in the healthcare system has not been determined. The objective of this study was to determine the possibility of additional case detection for pulmonary tuberculosis (PTB) by offering Xpert to smear-negative patients in a low-HIV burden setting with no Mycobacterium tuberculosis (M.tb.) culture facilities. Patients routinely presenting with symptoms suggestive of PTB with negative smears were offered single Xpert test on a fee-paying basis. Data were retrospectively reviewed to determine case detection in patients tested from February to December 2013. Symptoms associated with a positive test were analysed to determine if refinement of clinical criteria would reduce unnecessary testing. 258 smear-negative patients were included and M.tb. was detected in 55 (21.32%, n = 55/258). Using standard clinical assessment for selection, testing 5 patients detected one case of smear-negative PTB. These results demonstrate that fee-paying Xpert service in low-income setting can increase TB case confirmation substantially and further systematic studies of health economic implications should be conducted to determine optimal implementation models to increase access to Xpert in low- and middle-income countries.

Genomic analysis of Mycobacterium tuberculosis reveals complex etiology of tuberculosis in Vietnam including frequent introduction and transmission of Beijing lineage and positive selection for EsxW Beijing variant

Tuberculosis (TB) is a leading cause of death from infectious disease and the global burden is now higher than at any point in history. Despite coordinated efforts to control TB transmission, the factors contributing to its successful spread remain poorly understood. Vietnam is identified as one of 30 high burden countries for TB and MDR-TB with an incidence of 137 TB cases per 100,000 individuals in 2015. Recent phylogenomic analyses of the causative agent Mycobacterium tuberculosis (Mtb) in other high-prevalence regions have provided insights into the complex processes underlying TB transmission. Here we examine the transmission dynamics of Mtb isolated from TB patients in Ho Chi Minh City (HCMC), Vietnam via whole genome analysis of 1,635 isolates and comparison with 3,085 isolates from other locations. The genomic data reveal an underlying burden of disease caused by endemic Mtb Lineage 1 associated with activation of long-term latent infection, on top of which is overlaid a three-fold higher burden associated with introduction of exotic Lineage 2 and 4 Mtb strains. We identify frequent transfer of Beijing lineage Mtb into the country, which are associated with higher levels of transmission in this host population than endemic Lineage 1 Mtb. We identify a mutation in the secreted protein EsxW in Beijing strains that also appears to be under positive selection in other Mtb lineages, which could potentially contribute to the enhanced transmission of the Beijing lineage in Vietnamese and other host populations.

Tuberculosis in South Asia: a tide in the affairs of men

BackgroundTuberculosis (TB) remains the most common cause of infectious disease deaths worldwide. What is perhaps less appreciated is that the caseload of tuberculosis patients in South Asia is staggering.South Asia has almost 40% of the global TB burden with 4,028,165 cases in 2015. This region also has a disproportionate share of TB deaths (681,975 deaths, 38% of the global burden). Worldwide just 12.5% of TB cases are in HIV positive individuals, but much research and investment has focused on HIV-associated TB. Only 3.5% of patients with tuberculosis in South Asia have HIV co-infection. Not surprisingly with such a huge burden of disease, this region has an estimated 184,336 multi drug resistant (MDR) cases among notified TB cases which accounts for a third of global MDR burden. Crucially, at least 70% of the estimated MDR cases remain untreated in this region and MDR treatment success ranged from only 46% for India to 88% for Sri Lanka in the 2012 cohort that received treatment. This region represents many of the drivers of the modern TB epidemic: rapid urbanization and high density populations with dramatically rising incidence of diabetes, a burgeoning and largely unregulated private sector with escalating drug resistance and high air pollution both outdoor and household.ConclusionFrom bacterial biochemistry to policy implementation, we suggest ways in which South Asia can seize the opportunity lead global TB elimination by demonstrating feasibility in some of the world’s most densely populated cities and remotest reaches of the Himalayas. Clearly political will is essential, but we cannot defeat TB without understanding how to eliminate it in South Asia.

Global expansion of Mycobacterium tuberculosis lineage 4 shaped by colonial migration and local adaptation

Repeated emergence, not international dissemination, is behind the rise of multidrug-resistant lineage 4 tuberculosis. On the basis of population genomic and phylogeographic analyses of 1669 Mycobacterium tuberculosis lineage 4 (L4) genomes, we find that dispersal of L4 has been completely dominated by historical migrations out of Europe. We demonstrate an intimate temporal relationship between European colonial expansion into Africa and the Americas and the spread of L4 tuberculosis (TB). Markedly, in the age of antibiotics, mutations conferring antimicrobial resistance overwhelmingly emerged locally (at the level of nations), with minimal cross-border transmission of resistance. The latter finding was found to reflect the relatively recent emergence of these mutations, as a similar degree of local restriction was observed for susceptible variants emerging on comparable time scales. The restricted international transmission of drug-resistant TB suggests that containment efforts at the level of individual countries could be successful.