Challenging the gold standard: the limitations of molecular assays for detection of Mycobacterium tuberculosis heteroresistance.

OBJECTIVES: Heteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. In Mycobacterium tuberculosis (M. tb), heteroresistance poses a challenge in diagnosis and has been linked with poor treatment outcomes. We compared the analytical sensitivity of molecular methods, such as GeneXpert and whole genome sequencing (WGS) in detecting heteroresistance when compared with the 'gold standard' phenotypic assay: the agar proportion method (APM). METHODS: Using two rounds of proficiency surveys with defined monoresistant BCG strains and mixtures of susceptible/resistant M. tb, we determined the limit of detection (LOD) of known resistance associated mutations. RESULTS: The LOD for rifampin-R (RIF-R) detection was 1% using APM, 60% using GeneXpert MTB/RIF, 10% using GeneXpert MTB/RIF Ultra and 10% using WGS. While WGS could detect mutations beyond those associated with RIF resistance, the LOD for these other mutations was also 10%. Additionally, we observed instances where laboratories did not report resistance in the majority population, yet the mutations were present in the raw sequence data. CONCLUSION: The gold standard APM detects minority resistant populations at a lower proportion than molecular tests. Mycobacterium bovis BCG strains with defined resistance and extracted DNA from M. tb provided concordant results and can serve in quality control of laboratories offering molecular testing for resistance. Further research is required to determine whether the higher LOD of molecular tests is associated with negative treatment outcomes.

Whole genome sequence of Mycobacterium kansasii isolates of the genotype 1 from Brazilian patients with pulmonary disease demonstrates considerable heterogeneity.

Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.

The epidemic of multidrug resistant tuberculosis in China in historical and phylogenetic perspectives.

OBJECTIVES: For the past decade, the epidemic of multidrug resistance tuberculosis (MDR-TB) stays high in China. We investigated the possible driving forces behind the epidemics from phylogenetic and historical perspectives. METHODS: 420 representative strains were selected from the first national drug resistance survey based on their genotypes, drug susceptibility patterns and geographic information. We reconstructed the phylogeny by whole genome sequencing and compared it to the global phylogeny including MDR outbreaks reported in other settings. We estimated the historical trajectory of population dynamics by Bayesian Skygrid plot for all strains and MDR-TB alone. Integrating geographic information and mutations in drug resistance related genes, we investigated the spatial scale of transmission, recent selection of drug resistant mutant, and mechanism for fitness restoration. RESULTS: Three new subgroups within Beijing clade are described for the first time, but none of the MDR-TB outbreak strains reported in other high MDR-TB burden settings is identified. The overall epidemics experienced two successive phases of expansion at different rates between 1660s and 1950s, followed by a sharp decline till today. Four fifths of the clustered MDR-TB strains suggest transmission of DR strains and nearly half suggest recent selection of (additional) mutations in rpoB. Among all identified transmission events, about one fifth occurred between far distant locations. Possible intergenic and intragenic compensatory mutations both presented in our dataset at comparable frequencies. CONCLUSIONS: MDR-TB epidemic in China is not yet driven by the spread of a few highly successful clonal expansions but by repeated emergence of smaller and currently less successful clusters. However, internal migration and undertreatment could escalate MDR-TB epidemic. To prevent generating of drug resistance and restoration of fitness as well as to stop transmission of MDR-TB at early stage, national TB control program needs to strengthen management of floating populations and promote universal drug susceptibility testing in China.

A cluster of multidrug-resistant Mycobacterium tuberculosis among patients arriving in Europe from the Horn of Africa: a molecular epidemiological study.

BACKGROUND: The risk of tuberculosis outbreaks among people fleeing hardship for refuge in Europe is heightened. We describe the cross-border European response to an outbreak of multidrug-resistant tuberculosis among patients from the Horn of Africa and Sudan. METHODS: On April 29 and May 30, 2016, the Swiss and German National Mycobacterial Reference Laboratories independently triggered an outbreak investigation after four patients were diagnosed with multidrug-resistant tuberculosis. In this molecular epidemiological study, we prospectively defined outbreak cases with 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) profiles; phenotypic resistance to isoniazid, rifampicin, ethambutol, pyrazinamide, and capreomycin; and corresponding drug resistance mutations. We whole-genome sequenced all Mycobacterium tuberculosis isolates and clustered them using a threshold of five single nucleotide polymorphisms (SNPs). We collated epidemiological data from host countries from the European Centre for Disease Prevention and Control. FINDINGS: Between Feb 12, 2016, and April 19, 2017, 29 patients were diagnosed with multidrug-resistant tuberculosis in seven European countries. All originated from the Horn of Africa or Sudan, with all isolates two SNPs or fewer apart. 22 (76%) patients reported their travel routes, with clear spatiotemporal overlap between routes. We identified a further 29 MIRU-VNTR-linked cases from the Horn of Africa that predated the outbreak, but all were more than five SNPs from the outbreak. However all 58 isolates shared a capreomycin resistance-associated tlyA mutation. INTERPRETATION: Our data suggest that source cases are linked to an M tuberculosis clone circulating in northern Somalia or Djibouti and that transmission probably occurred en route before arrival in Europe. We hypothesise that the shared mutation of tlyA is a drug resistance mutation and phylogenetic marker, the first of its kind in M tuberculosis sensu stricto. FUNDING: The Swiss Federal Office of Public Health, the University of Zurich, the Wellcome Trust, National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC), the Medical Research Council, BELTA-TBnet, the European Union, the German Center for Infection Research, and Leibniz Science Campus Evolutionary Medicine of the Lung (EvoLUNG).

Comparison of different treatments for isoniazid-resistant tuberculosis: an individual patient data meta-analysis.

BACKGROUND: Isoniazid-resistant, rifampicin-susceptible (INH-R) tuberculosis is the most common form of drug resistance, and is associated with failure, relapse, and acquired rifampicin resistance if treated with first-line anti-tuberculosis drugs. The aim of the study was to compare success, mortality, and acquired rifampicin resistance in patients with INH-R pulmonary tuberculosis given different durations of rifampicin, ethambutol, and pyrazinamide (REZ); a fluoroquinolone plus 6 months or more of REZ; and streptomycin plus a core regimen of REZ. METHODS: Studies with regimens and outcomes known for individual patients with INH-R tuberculosis were eligible, irrespective of the number of patients if randomised trials, or with at least 20 participants if a cohort study. Studies were identified from two relevant systematic reviews, an updated search of one of the systematic reviews (for papers published between April 1, 2015, and Feb 10, 2016), and personal communications. Individual patient data were obtained from authors of eligible studies. The individual patient data meta-analysis was performed with propensity score matched logistic regression to estimate adjusted odds ratios (aOR) and risk differences of treatment success (cure or treatment completion), death during treatment, and acquired rifampicin resistance. Outcomes were measured across different treatment regimens to assess the effects of: different durations of REZ (≤6 months vs >6 months); addition of a fluoroquinolone to REZ (fluoroquinolone plus 6 months or more of REZ vs 6 months or more of REZ); and addition of streptomycin to REZ (streptomycin plus 6 months of rifampicin and ethambutol and 1-3 months of pyrazinamide vs 6 months or more of REZ). The overall quality of the evidence was assessed using GRADE methodology. FINDINGS: Individual patient data were requested for 57 cohort studies and 17 randomised trials including 8089 patients with INH-R tuberculosis. We received 33 datasets with 6424 patients, of which 3923 patients in 23 studies received regimens related to the study objectives. Compared with a daily regimen of 6 months of (H)REZ (REZ with or without isoniazid), extending the duration to 8-9 months had similar outcomes; as such, 6 months or more of (H)REZ was used for subsequent comparisons. Addition of a fluoroquinolone to 6 months or more of (H)REZ was associated with significantly greater treatment success (aOR 2·8, 95% CI 1·1-7·3), but no significant effect on mortality (aOR 0·7, 0·4-1·1) or acquired rifampicin resistance (aOR 0·1, 0·0-1·2). Compared with 6 months or more of (H)REZ, the standardised retreatment regimen (2 months of streptomycin, 3 months of pyrazinamide, and 8 months of isoniazid, rifampicin, and ethambutol) was associated with significantly worse treatment success (aOR 0·4, 0·2-0·7). The quality of the evidence was very low for all outcomes and treatment regimens assessed, owing to the observational nature of most of the data, the diverse settings, and the imprecision of estimates. INTERPRETATION: In patients with INH-R tuberculosis, compared with treatment with at least 6 months of daily REZ, addition of a fluoroquinolone was associated with better treatment success, whereas addition of streptomycin was associated with less treatment success; however, the quality of the evidence was very low. These results support the conduct of randomised trials to identify the optimum regimen for this important and common form of drug-resistant tuberculosis. FUNDING: World Health Organization and Canadian Institutes of Health Research.

Whole genome sequence of Mycobacterium kansasii isolates of the genotype 1 from Brazilian patients with pulmonary disease demonstrates considerable heterogeneity

Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.