Drug-resistant Mycobacterium tuberculosis among Nepalese patients at a tuberculosis referral center.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB), characterized by isoniazid and rifampicin resistance, is caused by chromosomal mutations that restrict treatment options and complicate tuberculosis management. This study sought to investigate the prevalence of pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) tuberculosis, as well as mutation pattern, in Nepalese patients with MDR/rifampicin-resistant (RR)-TB strains. METHODS: A cross-sectional study was conducted on MDR/RR-TB patients at the German Nepal Tuberculosis Project from June 2017 to June 2018. The MTBDRsl line probe assay identified pre-XDR-TB and XDR-TB. Pre-XDR-TB included MDR/RR-TB with resistance to any fluoroquinolone (FLQ), while XDR-TB included MDR/RR-TB with resistance to any FLQ and at least one additional group A drug. Mutation status was determined by comparing bands on reaction zones [gyrA and gyrB for FLQ resistance, rrs for SILD resistance, and eis for low-level kanamycin resistance, according to the GenoType MTBDRsl VER 2.0, Hain Lifescience GmbH, Nehren, Germany definition of pre-XDR and XDR] to the evaluation sheet. SPSS version 17.0 was used for data analysis. RESULTS: Out of a total of 171 patients with MDR/RR-TB, 160 had (93.57%) had MTBC, of whom 57 (35.63%) had pre-XDR-TB and 10 (6.25%) had XDR-TB. Among the pre-XDR-TB strains, 56 (98.25%) were FLQ resistant, while 1 (1.75%) was SLID resistant. The most frequent mutations were found at codons MUT3C (57.14%, 32/56) and MUT1 (23.21%, 13/56) of the gyrA gene. One patient had SLID resistant genotype at the MUT1 codon of the rrs gene (100%, 1/1). XDR-TB mutation bands were mostly detected on MUT1 (30%, 3/10) of the gyrA and rrs, MUT3C (30%, 3/10) of the gyrA, and MUT1 (30%, 3/10) of the rrs. CONCLUSIONS: Pre-XDR-TB had a significantly higher likelihood than XDR-TB, with different specific mutation bands present in gyrA and rrs genes.

Whole-genome sequencing of clinical isolates from tuberculosis patients in India: real-world data indicates a high proportion of pre-XDR cases.

Treatment decisions for tuberculosis (TB) in the absence of full drug-susceptibility data can result in amplifying resistance and may compromise treatment outcomes. Genomics of Mycobacterium tuberculosis (M.tb) from clinical samples enables detection of drug resistance to multiple drugs. We performed whole-genome sequencing (WGS) for 600 clinical samples from patients with tuberculosis to identify the drug-resistance profile and mutation spectrum. We documented the reasons reported by clinicians for referral. WGS identified a high proportion (51%) of pre-extensively drug-resistant (pre-XDR) cases followed by multidrug-resistant tuberculosis (MDR-TB) (15.5%). This correlates with the primary reason for referral, as non-response to the first-line treatment (67%) and treatment failure or rifampicin resistance (14%). Multivariate analysis indicated that all young age groups (P < 0.05), male gender (P < 0.05), and Beijing strain (P < 0.01) were significant independent predictors of MDR-TB or MDR-TB+ [pre-extensively drug-resistant tuberculosis (XDR-TB) and XDR-TB]. Ser315Thr (72.5%) in the inhA gene and Ser450Leu in the rpoB gene (65.5%) were the most prevalent mutations, as were resistance-conferring mutations to pyrazinamide (41%) and streptomycin (61.33%). Mutations outside the rifampicin resistance-determining region (RRDR), Ile491Phe and Val170Phe, were seen in 1.3% of cases; disputed mutations in rpoB (Asp435Tyr, His445Asn, His445Leu, and Leu430Pro) were seen in 6% of cases, and mutations to newer drugs such as bedaquiline and linezolid in 1.0% and 7.5% of cases, respectively. This study on clinical samples highlights that there is a high proportion of pre-XDR cases and emerging resistance to newer drugs; ongoing transmission of these strains can cause serious threat to public health; and whole-genome sequencing can effectively identify and support precision medicine for TB. IMPORTANCE: The current study is based on real-world data on the TB drug-resistance profile by whole-genome sequencing of 600 clinical samples from patients with TB in India. This study indicates the clinicians' reasons for sending samples for WGS, which is for difficult-to-treat cases and/or relapse and treatment failure. The study reports a significant proportion of cases with pre-XDR-TB strains that warrant policy makers' attention. It reflects the current iterative nature of the diagnostic tests under programmatic conditions that leads to delays in appropriate diagnosis and empirical treatment. India had an estimated burden of 2.95 million TB cases in 2020 and 135,000 multidrug-resistant cases. However, WGS profiles of M.tb from India remains disproportionately poorly represented. This study adds a significant body of data on the mutation profiles seen in M.tb isolated from patients with TB in India, mutations outside the RRDR, disputed mutations, and resistance-conferring mutations to newer drugs such as bedaquiline and linezolid.

Whole genome sequencing for the prediction of resistant tuberculosis strains from northern India.

PURPOSE: Tuberculosis is an important public health problem among infectious diseases. The problem becomes more concerning with the emergence of MDR-TB and pre-XDR-TB. Whole genome sequencing (WGS) detection of resistance has recently gained popularity as it has advantages over other commercial techniques. METHODS: We performed in-house WGS followed by detailed analysis by an in-house pipeline to identify the resistance markers. This was accompanied by Phenotypic DST, and Sanger sequencing on all the 12 XDR, 06 pre-XDR, and 06 susceptible M. tb isolates. These results were collated with online M. tb WGS pipelines (TB profiler, PhyResSE, Mykrobe predictor) for comparative analysis. RESULTS: Following our in-house analysis, we observed 64 non-synonymous SNPs, fifteen synonymous SNPs, and five INDELs in 25 drug resistance-associated genes/intergenic regions (IGRs) in M. tb isolates. Sensitivity for detecting XDR is 33%, 58%, 83%, and 83%, respectively, using Mykrobe predictor, PhyResSE, TB-profiler, and in-house pipeline for WGS analysis, respectively. TB-profiler detected a rare mutation H70R in the gyrA gene in one pre-XDR isolate. Lineage 2.2.1 East-Asian (Beijing sublineage type) predominated (60%) in WGS data analysis of the XDR isolates. CONCLUSIONS: Our findings suggest that in-house analysis of WGS data and TB-profiler sensitivity was better for the detection of second-line resistance as compared to other automated tested tools. Frequent upgradation of newer mutations associated with resistance needs to be updated, as it potentiates tailored treatment for patients.

Defensins: A novel weapon against Mycobacterium tuberculosis?

Tuberculosis (TB) is a serious airborne communicable disease caused by organisms of the Mycobacterium tuberculosis (Mtb) complex. Although the standard treatment antimicrobials, including isoniazid, rifampicin, pyrazinamide, and ethambutol, have made great progress in the treatment of TB, problems including the rising incidence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), the severe toxicity and side effects of antimicrobials, and the low immunity of TB patients have become the bottlenecks of the current TB treatments. Therefore, both safe and effective new strategies to prevent and treat TB have become a top priority. As a subfamily of cationic antimicrobial peptides, defensins are rich in cysteine and play a vital role in resisting the invasion of microorganisms and regulating the immune response. Inspired by studies on the roles of defensins in host defence, we describe their research history and then review their structural features and antimicrobial mechanisms, specifically for fighting Mtb in detail. Finally, we discuss the clinical relevance, therapeutic potential, and potential challenges of defensins in anti-TB therapy. We further debate the possible solutions of the current application of defensins to provide new insights for eliminating Mtb.

[Study on the resistance of rifampicin-resistant Mycobacterium tuberculosis to anti-tuberculosis drugs in group A].

Objective: To summarize the resistance of rifampicin-resistant Mycobacterium tuberculosis to anti-tuberculosis drugs in group A. Methods: In the retrospective study, a total of 1 226 clinical isolates from suspected multidrug-resistant pulmonary tuberculosis patients in Beijing TB control system from 2016 to 2021 were identified as Mycobacterium tuberculosis (MTB) strains by MPB64 antigen detection test. Rifampicin-resistant tuberculosis (RR-TB) strains were screened by the phenotypic drug susceptibility using the proportion method. The drug susceptibilities of Levofloxacin(LFX), Moxifloxacin(MFX), Bedaquiline(BDQ) and Linezolid(LZD)were detected by the phenotypic drug susceptibility with microplate method. The drug resistance rate, drug resistance level and minimum inhibitory concentration (MIC) distribution of four anti-tuberculosis drugs in group A were analyzed. We calculated the demographic distribution of RR-TB, multidrug-resistant tuberculosis(MDR-TB), pre-extensively drug resistant tuberculosis (pre-XDR-TB), extensively drug resistant tuberculosis (XDR-TB) patients and the cross resistance of LFX and MFX, then summarized the drug-resistance spectrum of BDQ-resistant and LZD-resistant strains and the treatment outcome of RR-TB patients. Measurement data were expressed as rate or composition ratio,χ2 test was used between and within groups, and P<0.05 was considered statistically significant. Results: Among the 1 226 suspected multidrug-resistant pulmonary tuberculosis patients, the detection rates of RR/MDR/pre-XDR/XDR-TB patients were 20.8%(255/1 226), 15.2%(186/1 226), 5.7%(70/1 226), 0.5%(6/1 226), respectively. There were statistically significant differences in the distribution of patients with the four types of drug resistance in terms of age and treatment history (χ2=14.95, P=0.020;χ2=15.91, P=0.001). The drug resistance rates of LFX, MFX, BDQ and LZD in RR-TB patients were 27.5% (70/255), 27.5% (70/255), 0.4% (1/255) and 2.4% (6/255), respectively. The MICs of LFX, MFX and LZD-susceptible MTB were mainly at 0.25 mg/L, and the MIC of BDQ-susceptible MTB was mainly concentrated at 0.03 mg/L. 25.1% (64/255) of the RR MTB were resistant to both LFX and MFX, and 6 strains were resistant to LFX or MFX, showing incomplete two-way cross resistance. One BDQ-resistant strain and six LZD-resistant strains were detected. The treatment success rate of RR-TB patients was 74.4% (151/203), and there were statistically significant differences in treatment outcomes between resistant and sensitive patients on the LFX-containing treatment regimen (Fisher's exact test, P=0.012). Conclusions: The prevalence of fluoroquinolones (LFX and MFX) resistance in rifampicin-resistant MTB is very serious. LFX and MFX show incomplete bidirectional cross-resistance. BDQ and LZD have the most promising future in the treatment of MDR-TB. Improve drug-resistance testing will help to further improve the success rate of treatment.

Metabolomic analysis of Mycobacterium tuberculosis reveals metabolic profiles for identification of drug-resistant tuberculosis.

The detection of pre-extensively (pre-XDR) and extensively drug-resistant tuberculosis (XDR-TB) is challenging. Drug-susceptibility tests for some anti-TB drugs, especially ethambutol (ETH) and ethionamide (ETO), are problematic due to overlapping thresholds to differentiate between susceptible and resistant phenotypes. We aimed to identify possible metabolomic markers to detect Mycobacterium tuberculosis (Mtb) strains causing pre-XDR and XDR-TB. The metabolic patterns of ETH- and ETO-resistant Mtb isolates were also investigated. Metabolomics of 150 Mtb isolates (54 pre-XDR, 63 XDR-TB and 33 pan-susceptible; pan-S) were investigated. Metabolomics of ETH and ETO phenotypically resistant subgroups were analyzed using UHPLC-ESI-QTOF-MS/MS. Orthogonal partial least-squares discriminant analysis revealed distinct separation in all pairwise comparisons among groups. Two metabolites (meso-hydroxyheme and itaconic anhydride) were able to differentiate the pre-XDR and XDR-TB groups from the pan-S group with 100% sensitivity and 100% specificity. In comparisons of the ETH and ETO phenotypically resistant subsets, sets of increased (ETH = 15, ETO = 7) and decreased (ETH = 1, ETO = 6) metabolites specific for the resistance phenotype of each drug were found. We demonstrated the potential for metabolomics of Mtb to differentiate among types of DR-TB as well as between isolates that were phenotypically resistant to ETO and ETH. Thus, metabolomics might be further applied for DR-TB diagnosis and patient management.

In vitro activity of tetracycline analogs against multidrug-resistant and extensive drug resistance clinical isolates of Mycobacterium tuberculosis.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) has become a big threaten to global health. The current strategy for treatment of MDR-TB and extensive drug resistant tuberculosis (XDR-TB) is with low efficacy and high side effect. While new drug is fundamental for cure MDR-TB, repurposing the Food and Drug Administration (FDA)-approved drugs represents an alternative soluation with less cost. METHODS: The activity of 8 tetracycline-class antibiotics against mycobacterium tuberculosis (M.tb) were determined by Minimum Inhibitory Concentration (MIC) in vitro. A transposon M.smeg libraries was generated by using the Harm phage and then used to isolate the conditional growth mutants in doxycycline containing plate. Eleven mutants were isolated and genomic DNAs were extracted using the cetyltrimethyl ammonium bromide (CTAB) method and analyzed by whole genome sequencing. RESULTS: We found that three of eight drugs efficiently inhibited mycobacteria growth under the peak plasma concentration in the human body. Further tests showed these three tetracycline analogs (demeclocycline, doxycycline and methacycline) had antimicrobial activity against seven clinical isolates, including MDR and XDR strains. Among them, Doxycycline had the lowest MICs in all mycobacteria strains tested in this study. By using a transposon library, we identify the insertion of transposon in two genes, porin and MshA, associatewith the resistant to doxycycline. CONCLUSIONS: Our findings show that tetracycline analogs such as doxycycline, has bactericidal activity against not only drug sensitive M.tb, but also clinical MDR and XDR strains, provided proof of concept to repurpose doxycycline to fight MDR-TB and XDR-TB. Further investigations are warranted to clarify the underlying mechanism and optimize the strategy in combination with other anti-TB drugs.

Molecular Characterization of Resistance to Second-Line Anti-Mycobacterial Drugs among Clinical Isolates of Multidrug-Resistant Mycobacterium tuberculosis.

BACKGROUND: The emergence of multidrug resistance and extensively drug-resistant tuberculosis is a serious public health crisis. Using rapid and inexpensive molecular methods such as HRM assay in the detection of second-line drugs resistance in M. tuberculosis would be helpful in the treatment and control of XDR tuberculosis cases. METHODS: MDR-TB isolates were collected from Iranian tuberculosis laboratories. Drug susceptibility test performed via the indirect proportion method utilizing LJ Medium. Susceptibility to ciprofloxacin, ofloxacin, amikacin, kanamycin, and capreomycin, as second-line anti-tuberculosis agents were assessed. Single point mutations in gyrA, rrs and eis genes were detected via HRM assay and DNA sequencing. RESULTS: A DST test was performed for 56 MDR isolates and at least 27 (48.2%) isolates were resistant to CIP or OFL. Also, 14 (25%), 12 (21.4%), and 15 (26.7%) isolates were resistant to capreomycin, amikacin, and kanamycin, respectively. D94G, A90V, and G88C mutations were the most frequent mutations in gyrA gene. Also, A1401G mutation was detected more than the other mutations in rrs gene. CONCLUSIONS: The frequency of CIP/OFL and AMK/CAP/KAN-resistant TB is considerable among Iranian tuberculosis cases. HRM assay is a rapid and inexpensive test and can detect important mutation-based drug resistance in MDR-TB and XDR-TB isolates.

Availability and costs of medicines for the treatment of tuberculosis in Europe.

OBJECTIVES: To evaluate the access to comprehensive diagnostics and novel antituberculosis medicines in European countries. METHODS: We investigated the access to genotypic and phenotypic Mycobacterium tuberculosis drug susceptibility testing and the availability of antituberculosis drugs and calculated the cost of drugs and treatment regimens at major tuberculosis treatment centres in countries of the WHO European region where rates of drug-resistant tuberculosis are the highest among all WHO regions. Results were stratified by middle-income and high-income countries. RESULTS: Overall, 43 treatment centres from 43 countries participated in the study. For WHO group A drugs, the frequency of countries with the availability of phenotypic drug susceptibility testing was as follows: (a) 75% (30/40) for levofloxacin, (b) 82% (33/40) for moxifloxacin, (c) 48% (19/40) for bedaquiline, and (d) 72% (29/40) for linezolid. Overall, of the 43 countries, 36 (84%) and 24 (56%) countries had access to bedaquiline and delamanid, respectively, whereas only 6 (14%) countries had access to rifapentine. The treatment of patients with extensively drug-resistant tuberculosis with a regimen including a carbapenem was available only in 17 (40%) of the 43 countries. The median cost of regimens for drug-susceptible tuberculosis, multidrug-resistant/rifampicin-resistant tuberculosis (shorter regimen, including bedaquiline for 6 months), and extensively drug-resistant tuberculosis (including bedaquiline, delamanid, and a carbapenem) were €44 (minimum-maximum, €15-152), €764 (minimum-maximum, €542-15152), and €8709 (minimum-maximum, €7965-11759) in middle-income countries (n = 12) and €280 (minimum-maximum, €78-1084), €29765 (minimum-maximum, €11116-40584), and €217591 (minimum-maximum, €82827-320146) in high-income countries (n = 29), respectively. DISCUSSION: In countries of the WHO European region, there is a widespread lack of drug susceptibility testing capacity to new and repurposed antituberculosis drugs, lack of access to essential medications in several countries, and a high cost for the treatment of drug-resistant tuberculosis.

Antibiotic resistance acquisition versus primary transmission in the presentation of extensively drug-resistant tuberculosis.

Mycobacterium tuberculosis is the leading cause of mortality worldwide due to a single bacterial pathogen. Of concern is the negative impact that the COVID-19 pandemic has had on the control of tuberculosis (TB) including drug-resistant forms of the disease. Antimicrobial resistance increases the likelihood of worsened outcomes in TB patients including treatment failure and death. Multidrug-resistant (MDR) strains, resistant to first-line drugs isoniazid and rifampin, and extensively drug-resistant (XDR) strains with further resistance to second-line drugs (SLD), threaten control programs designed to lower TB incidence and end the disease as a public health challenge by 2030, in accordance with UN Sustainable Development Goals. Tackling TB requires an understanding of the pathways through which drug resistance emerges. Here, the roles of acquired resistance mutation, and primary transmission, are examined with regard to XDR-TB. It is apparent that XDR-TB can emerge from MDR-TB through a small number of additional resistance mutations that occur in patients undergoing drug treatment. Rapid detection of resistance, to first-line drugs and SLD, at the initiation of and during treatment, and prompt adjustment of regimens are required to ensure treatment success in these patients. Primary transmission is predicted to make an increasing contribution to the XDR-TB caseload in the future. Much work is required to improve the implementation of the World Health Organization-recommended infection control practices and block onward transmission of XDR-TB patients to contacts including health-care workers. Finally, limiting background resistance to fluoroquinolones in pre-XDR strains of M. tuberculosis will necessitate better antimicrobial stewardship in the broader use of this drug class.

A five-year review of prevalence and treatment outcomes of pre-extensively drug-resistant plus additional drug-resistant tuberculosis in the Henan Provincial Tuberculosis Clinical Medicine Research Centre.

OBJECTIVES: This study investigated the prevalence and significant clinical outcomes of pre-extensively drug-resistant plus additional drug-resistant tuberculosis (pre-XDR-plus) in Henan Provincial Chest Hospital between 2017 and 2021. METHODS: We analysed and summarized the drug sensitivity test (DST) results of clinical Mycobacterium tuberculosis (MTB) strains in TB patients seeking care in the Tuberculosis Clinical Medical Research Centre of Henan Province between 2017 and 2021. Medical records of pre-extensively drug-resistant plus additional drug-resistant TB patients were statistically analysed, including demographic characteristics, regimens, and outcomes. RESULTS: Of the 3689 Mycobacterium tuberculosis strains, 639 (17.32%), 353 (9.56%), and 109 (2.95%), multidrug-resistant tuberculosis (MDR-TB), pre-extensively drug-resistant tuberculosis (pre-XDR), and pre-XDR-plus, respectively. The proportion of MDR decreased from 19.1% in 2017 to 17.5% in 2021 (χ2 = 0.686, P = 0.407), the proportion of pre-XDR from 11.4% in 2017 to 9.0% in 2021 (χ2 = 2.39, P = 0.122), and pre-XDR-plus from 4.7% in 2017 to 1.8% in 2020, with the declining trend was significant (χ2 = 9.348, P = 0.002). The most commonly used anti-TB drugs were pyrazinamide (PZA, 37/46, 80.43%) and cycloserine (CS, 32/46, 69.57%), followed by linezolid (LZD, 25/46, 54.35%), protionamide (TH, 25/46, 54.35%), and para-aminosalicylic acid (PAS, 23/46, 50.00%). Patients receiving the LZD regimen were 5 times more likely to have a favourable outcome than those not receiving LZD (OR = 6.421, 95% CI 2.101-19.625, P = 0.001). Patients receiving a regimen containing CS were 4 times more likely to have a favourable outcome compared to those not taking CS (OR = 5.444, 95% CI 1.650-17.926, P = 0.005). CONCLUSIONS: Our data suggest that the population of pre-XDR-plus had significantly decreased over the past five years in the Henan Provincial Chest Hospital. The COVID-19 and flood disaster affect TB patients' selection of medical services. In addition, the pre-XDR-plus patients whose regimens contain LZD or CS were more likely to have favourable outcomes.

Mutation detection and minimum inhibitory concentration determination against linezolid and clofazimine in confirmed XDR-TB clinical isolates.

BACKGROUND: The emergence of multidrug-resistant tuberculosis (MDR-TB) has complicated the situation due to the decline in potency of second-line anti-tubercular drugs. This limits the treatment option for extensively drug-resistant tuberculosis (XDR-TB). The aim of this study was to determine and compare the minimum inhibitory concentration (MIC) by agar dilution and resazurin microtiter assay (REMA) along with the detection of mutations against linezolid and clofazimine in confirmed XDR-TB clinical isolates. RESULTS: A total of 169 isolates were found positive for Mycobacterium tuberculosis complex (MTBC). The MIC was determined by agar dilution and REMA methods. The isolates which showed non-susceptibility were further subjected to mutation detection by targeting rplC gene (linezolid) and Rv0678 gene (clofazimine). The MIC for linezolid ranged from 0.125 µg/ml to > 2 µg/ml and for clofazimine from 0.25 µg/ml to > 4 µg/ml. The MIC50 and MIC90 for linezolid were 0.5 µg/ml and 1 µg/ml respectively while for clofazimine both were 1 µg/ml. The essential and categorical agreement for linezolid was 97.63% and 95.26% and for clofazimine, both were 100%. The sequencing result of the rplC gene revealed a point mutation at position 460 bp, where thymine (T) was substituted for cytosine (C) while seven mutations were noted between 46 to 220 bp in Rv0678 gene. CONCLUSION: REMA method has been found to be more suitable in comparison to the agar dilution method due to lesser turnaround time. Mutations in rplC and Rv0678 genes were reasons for drug resistance against linezolid and clofazimine respectively.

The second national anti-tuberculosis drug resistance survey in Tanzania, 2017-2018.

OBJECTIVE: To determine the levels and patterns of resistance to first- and second-line anti-tuberculosis (TB) drugs among new and previously treated sputum smear positive pulmonary TB (PTB) patients. METHODS: We conducted a nationally representative cross-sectional facility-based survey in June 2017-July 2018 involving 45 clusters selected based on probability proportional to size. The survey aimed to determine the prevalence of anti-TB drug resistance and associated risk factors among smear positive PTB patients in Tanzania. Sputum samples were examined using smear microscopy, Xpert MTB/RIF, culture and drug susceptibility testing (DST). Logistic regression was used to account for missing data and sampling design effects on the estimates and their standard errors. RESULTS: We enrolled 1557 TB patients, including 1408 (90.4%) newly diagnosed and 149 (9.6%) previously treated patients. The prevalence of multidrug-resistant TB (MDR-TB) was 0.85% [95% confidence interval (CI): 0.4-1.3] among new cases and 4.6% (95% CI: 1.1-8.2) among previously treated cases. The prevalence of Mycobacterium tuberculosis strains resistant to any of the four first-line anti-TB drugs (isoniazid, rifampicin, streptomycin and ethambutol) was 1.7% among new TB patients and 6.5% among those previously treated. Drug resistance to all first-line drugs was similar (0.1%) in new and previously treated patients. None of the isolates displayed poly-resistance or extensively drug-resistant TB (XDR-TB). The only risk factor for MDR-TB was history of previous TB treatment (odds ratio = 5.7, 95% CI: 1.9-17.2). CONCLUSION: The burden of MDR-TB in the country was relatively low with no evidence of XDR-TB. Given the overall small number of MDR-TB cases in this survey, it will be beneficial focusing efforts on intensified case detection including universal DST.

Transcontinental spread and evolution of Mycobacterium tuberculosis W148 European/Russian clade toward extensively drug resistant tuberculosis.

Transmission-driven multi-/extensively drug resistant (M/XDR) tuberculosis (TB) is the largest single contributor to human mortality due to antimicrobial resistance. A few major clades of the Mycobacterium tuberculosis complex belonging to lineage 2, responsible for high prevalence of MDR-TB in Eurasia, show outstanding transnational distributions. Here, we determined factors underlying the emergence and epidemic spread of the W148 clade by genome sequencing and Bayesian demogenetic analyses of 720 isolates from 23 countries. We dated a common ancestor around 1963 and identified two successive epidemic expansions in the late 1980s and late 1990s, coinciding with major socio-economic changes in the post-Soviet Era. These population expansions favored accumulation of resistance mutations to up to 11 anti-TB drugs, with MDR evolving toward additional resistances to fluoroquinolones and second-line injectable drugs within 20 years on average. Timescaled haplotypic density analysis revealed that widespread acquisition of compensatory mutations was associated with transmission success of XDR strains. Virtually all W148 strains harbored a hypervirulence-associated ppe38 gene locus, and incipient recurrent emergence of prpR mutation-mediated drug tolerance was detected. The outstanding genetic arsenal of this geographically widespread M/XDR strain clade represents a "perfect storm" that jeopardizes the successful introduction of new anti-M/XDR-TB antibiotic regimens.

Effectiveness and safety of bedaquiline-containing regimens for treatment on patients with refractory RR/MDR/XDR-tuberculosis: a retrospective cohort study in East China.

OBJECTIVE: Refractory rifampicin-resistant/multidrug resistant/extensively-drug resistant tuberculosis (RR/MDR/XDR-TB) were defined as patients infected with Mycobacterium tuberculosis (MTB) resistant to rifampicin(RR-TB), or at least resistant to rifampicin and isoniazid (MDR-TB) or added resistant to fluoroquinolones (FQs) and one of second line injectable agents (XDR-TB), a patient for whom an effective regimen (fewer than 4 effective agents due to adverse events (AEs) or multiple drug resistances) cannot be developed. To compare the effectiveness and safety of bedaquiline (BDQ)-containing and BDQ-free regimens for treatment of patients with refractory RR/MDR/XDR-TB. METHODS: Patients with refractory RR/MDR/XDR-TB receiving BDQ-containing regimens (BDQ group, n = 102) and BDQ-free regimens (non-BDQ group, n = 100) satisfied with included criteria were strictly included in this retrospective historical control study across East China. Culture conversion, treatment outcome, cavity closing rate, and AEs were compared between two groups. RESULTS: The baseline characteristics involved all possible aspects of patients were well balanced between two groups (p > 0.05). Culture conversion rates in the BDQ group at month 3 (89.2% vs. 66.0%), month 6 (90.2% vs 72.0%), month 9 (91.2% vs. 66.0%), and month 12 (94.1% vs 65.0%) were all significantly higher than those in non-BDQ group (p < 0.001). Similar results were observed in the cavity closing rate at month 9 (19.6% vs 8.0%, p = 0.0) and month 12 (39.2% vs 15.0%, p < 0.001). Patients receiving BDQ-containing regimens had more treatment success than those receiving BDQ-free regimens (p < 0.001; cure rate, 69.6% vs. 45.0%; complete the treatment, 22.5% vs. 18.0%; treatment success, 92.2% vs. 63.0%); the use of BDQ and combined with Linezolid or Clofazimine or Cycloserine were identified as independent predictors of treatment success and no culture reversion (P < 0.05). AEs were similarly reported in 26.5% of patients in the BDQ group and 19.0% in the non-BDQ group (p = 0.2). CONCLUSIONS: BDQ-containing regimens resulted in better treatment outcomes and similar safety relative to BDQ-free regimens for patients with refractory pulmonary RR/MDR/XDR-TB.

A data compendium associating the genomes of 12,289 Mycobacterium tuberculosis isolates with quantitative resistance phenotypes to 13 antibiotics.

The Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) presents here a data compendium of 12,289 Mycobacterium tuberculosis global clinical isolates, all of which have undergone whole-genome sequencing and have had their minimum inhibitory concentrations to 13 antitubercular drugs measured in a single assay. It is the largest matched phenotypic and genotypic dataset for M. tuberculosis to date. Here, we provide a summary detailing the breadth of data collected, along with a description of how the isolates were selected, collected, and uniformly processed in CRyPTIC partner laboratories across 23 countries. The compendium contains 6,814 isolates resistant to at least 1 drug, including 2,129 samples that fully satisfy the clinical definitions of rifampicin resistant (RR), multidrug resistant (MDR), pre-extensively drug resistant (pre-XDR), or extensively drug resistant (XDR). The data are enriched for rare resistance-associated variants, and the current limits of genotypic prediction of resistance status (sensitive/resistant) are presented by using a genetic mutation catalogue, along with the presence of suspected resistance-conferring mutations for isolates resistant to the newly introduced drugs bedaquiline, clofazimine, delamanid, and linezolid. Finally, a case study of rifampicin monoresistance demonstrates how this compendium could be used to advance our genetic understanding of rare resistance phenotypes. The data compendium is fully open source and it is hoped that it will facilitate and inspire future research for years to come.

High fluoroquinolone resistance proportions among multidrug-resistant tuberculosis driven by dominant L2 Mycobacterium tuberculosis clones in the Mumbai Metropolitan Region.

BACKGROUND: Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains are a serious health problem in India, also contributing to one-fourth of the global MDR tuberculosis (TB) burden. About 36% of the MDR MTBC strains are reported fluoroquinolone (FQ) resistant leading to high pre-extensively drug-resistant (pre-XDR) and XDR-TB (further resistance against bedaquiline and/or linezolid) rates. Still, factors driving the MDR/pre-XDR epidemic in India are not well defined. METHODS: In a retrospective study, we analyzed 1852 consecutive MTBC strains obtained from patients from a tertiary care hospital laboratory in Mumbai by whole genome sequencing (WGS). Univariate and multivariate statistics was used to investigate factors associated with pre-XDR. Core genome multi locus sequence typing, time scaled haplotypic density (THD) method and homoplasy analysis were used to analyze epidemiological success, and positive selection in different strain groups, respectively. RESULTS: In total, 1016 MTBC strains were MDR, out of which 703 (69.2%) were pre-XDR and 45 (4.4%) were XDR. Cluster rates were high among MDR (57.8%) and pre-XDR/XDR (79%) strains with three dominant L2 (Beijing) strain clusters (Cl 1-3) representing half of the pre-XDR and 40% of the XDR-TB cases. L2 strains were associated with pre-XDR/XDR-TB (P < 0.001) and, particularly Cl 1-3 strains, had high first-line and FQ resistance rates (81.6-90.6%). Epidemic success analysis using THD showed that L2 strains outperformed L1, L3, and L4 strains in short- and long-term time scales. More importantly, L2 MDR and MDR + strains had higher THD success indices than their not-MDR counterparts. Overall, compensatory mutation rates were highest in L2 strains and positive selection was detected in genes of L2 strains associated with drug tolerance (prpB and ppsA) and virulence (Rv2828c). Compensatory mutations in L2 strains were associated with a threefold increase of THD indices, suggesting improved transmissibility. CONCLUSIONS: Our data indicate a drastic increase of FQ resistance, as well as emerging bedaquiline resistance which endangers the success of newly endorsed MDR-TB treatment regimens. Rapid changes in treatment and control strategies are required to contain transmission of highly successful pre-XDR L2 strains in the Mumbai Metropolitan region but presumably also India-wide.

Evaluation of the MolecuTech® REBA MTB-XMDR kit for detection of pre-extensively drug-resistant TB.

BACKGROUND: Rapid diagnosis of drug-resistant TB is critical for early initiation of effective therapy. YD Diagnostics in South Korea recently developed the MolecuTech® REBA MTB-XMDR test to rapidly detect multidrug-resistant TB (MDR-TB), pre-extensively drug-resistant TB (pre-XDR-TB) and resistance to second-line injectable drugs (SLIDs) simultaneously using a fully automated test platform. This study aimed to evaluate the MolecuTech® test for the detection of MDR- and pre-XDR-TB, as well as SLID resistance.METHODS: A total of 151 clinical Mycobacterium tuberculosis isolates from South Korea were tested using the MolecuTech test, and the results were analysed by comparing these with phenotypic drug susceptibility testing (pDST) and sequencing.RESULTS: Compared to pDST, the MolecuTech test showed a sensitivity and specificity of respectively 97.7% and 100.0% for rifampicin (RIF), 82.4% and 100.0% for isoniazid (INH), 97.5% and 97.2% for fluoroquinolones (FQs), and 94.0% and 98.8% for SLIDs. Concordances with the sequencing results of each resistance determinant were 99.3% for RIF, 96.7% for INH, 98.7% for FQs and 99.3% for SLIDs.CONCLUSION: The MolecuTech test is an efficient and reliable rapid molecular diagnostic tool for the simultaneous screening of MDR- and pre-XDR-TB.

High clustering rate and genotypic drug-susceptibility screening for the newly recommended anti-tuberculosis drugs among global extensively drug-resistant Mycobacterium tuberculosis isolates.

Multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) make TB difficult to control. Global susceptibility data for six newly recommended anti-TB drugs against M/XDR-TB are still limited. Using publicly available whole-genome sequences, we determined the proportion of 513 phenotypically XDR-TB isolates that carried mutations associated with resistance against these drugs (bedaquiline, clofazimine, linezolid, delamanid, pretomanid and cycloserine). Mutations of Rv0678 and Rv1979c were detected in 69/513 isolates (13.5%) for bedaquiline resistance and 79/513 isolates (15.4%) for clofazimine resistance with additional mmpL5 mutations. Mutations conferring resistance to delamanid were detected in fbiB and ddn genes for 11/513 isolates (2.1%). For pretomanid, a mutation was detected in the ddn gene for 3/513 isolates (0.6%). Nineteen mutations of pykA, cycA, ald, and alr genes, conferring resistance to cycloserine, were found in 153/513 isolates (29.8%). No known mutations associated with linezolid resistance were detected. Cluster analysis showed that 408/513 isolates fell within 99 clusters and that 354 of these isolates were possible primary drug-resistant TB (292 XDR-TB, 57 pre-XDR-TB and 5 MDR-TB). Clonal transmission of primary XDR isolates might contribute significantly to the high prevalence of DR-TB globally.

Laboratory-based study of drug resistance and genotypic profile of multidrug-resistant tuberculosis isolates in Salvador, Bahia, Brazil.

BACKGROUND: Surveillance of multidrug resistant/extensively drug-resistant tuberculosis (MDR/XDR-TB) is essential to guide disease dissemination control measures. Brazil contributes to a significant fraction of tuberculosis (TB) cases worldwide, but only few reports addressed MDR/XDR-TB in the country. METHODS: This cross-sectional, laboratory-based study describes the phenotypic resistance profiles of isolates obtained between January 2008 and December 2011 in Bahia, Brazil, and sociodemographic, epidemiological, and clinical characteristics (obtained from mandatory national registries) of the corresponding 204 MDR/XDR-TB patients. We analyzed the mycobacterial spoligotyping and variable number of tandem repeats of mycobacterial interspersed repetitive units in 12-loci profiles obtained from Salvador. RESULTS: MDR/XDR-TB patients were predominantly male, had a median age of 43 years, belonged to black ethnicity, and failed treatment before MDR-TB diagnosis. Nearly one-third of the isolates had phenotypic resistance (evaluated by mycobacteria growth indicator tube assay) to second-line anti-TB drugs (64/204, 31%), of which 22% cases (14/64) were diagnosed as XDR-TB. Death was a frequent outcome among these individuals and was associated with resistance to second-line anti-TB drugs. Most isolates successfully genotyped belonged to the Latin-American Mediterranean (LAM) Family, with an unprecedented high proportion of LAM10-Cameroon subfamily bacilli. More than half of these isolates were assigned to a unique cluster by the genotyping methods performed. Large clusters of identical genotypes were also observed among LAM SIT42 and SIT376 strains. CONCLUSIONS: We highlight the need for strengthening local and national efforts to perform early detection of TB drug resistance and to prevent treatment discontinuation to limit the emergence of drug-resistant strains.