Molecular Drug Susceptibility Testing Research Articles

Evaluation of Five User-Friendly Whole Genome Sequencing Software for Mycobacterium tuberculosis in Clinical Application.

Whole genome sequencing (WGS) is an increasingly useful tool for tuberculosis (TB) diagnosis and disease management. In this study, we evaluated the utility of user-friendly WGS tools in reporting resistance profiles and identifying lineages of clinical TB isolates from South Korea. Forty clinical samples from TB patients showing discrepancies between their rapid molecular and conventional drug susceptibility tests were used in this study. Among these clinical isolates, 37 strains were successfully evaluated via WGS software, using the GenTB, TB Profiler, PhyResSE, CASTB, and Mykrobe. More accurate and faster susceptibility results could be obtained with isoniazid than with rifampin. Using the phenotypic test as the gold standard, the isoniazid concordance rate between phenotypic drug susceptibility test (DST) and WGS (GenTB: 45.9%, TB profiler: 40.5%, PhyResSE: 40.5%, CASTB: 48.6%, and Mykrobe: 43.2%) was much higher than between phenotypic DST and rapid molecular genotypic DST (18.9%) among the 37 strains. In contrast, the rifampin concordance rate between phenotypic DST and WGS and that between phenotypic DST and rapid molecular genotypic DST was similar (81.1-89.2%). We also found novel mutations associated with INH in katG and ahpC gene region, not covered by the line probe assay. In addition, lineage analysis identified 81.1% of these samples as L2 East Asian lineage strains, and 18.9% as L4 Euro-American lineage strains. WGS may play a pivotal role in TB diagnosis and the detection of drug resistance, genetic diversity, and transmission dynamics in the near future because of its accuracy, speed, and extensibility.

Whole-Genome Sequencing to Identify Missed Rifampicin and Isoniazid Resistance Among Tuberculosis Isolates-Chennai, India, 2013-2016.

India has a high burden of drug-resistant tuberculosis (DR TB) and many cases go undetected by current drug susceptibility tests (DSTs). This study was conducted to identify rifampicin (RIF) and isoniazid (INH) resistance associated genetic mutations undetected by current clinical diagnostics amongst persons with DR TB in Chennai, India. Retrospectively stored 166 DR TB isolates during 2013–2016 were retrieved and cultured in Löwenstein-Jensen medium. Whole genome sequencing (WGS) and MGIT DST for RIF and INH were performed. Discordant genotypic and phenotypic sensitivity results were repeated for confirmation and the discrepant results considered final. Further, drug resistance-conferring mutations identified through WGS were analyzed for their presence as targets in current WHO-recommended molecular diagnostics. WGS detected additional mutations for rifampicin and isoniazid resistance than WHO-endorsed line probe assays. For RIF, WGS was able to identify an additional 10% (15/146) of rpoB mutant isolates associated with borderline rifampicin resistance compared to MGIT DST. WGS could detect additional DR TB cases than commercially available and WHO-endorsed molecular DST tests. WGS results reiterate the importance of the recent WHO revised critical concentrations of current MGIT DST to detect low-level resistance to rifampicin. WGS may help inform effective treatment selection for persons at risk of, or diagnosed with, DR TB.

Predictors and Trends of MDR/RR-TB in Shenzhen China: A Retrospective 2012-2020 Period Analysis.

PurposeWe analyzed the trends and predictors of multidrug-resistant (MDR) or rifampicin-resistant (RR) tuberculosis (TB) in culture-positive cases in Shenzhen during 2012–2020, after the implementation of improved strategies (scale-up molecular drug susceptibility testing [mDST], expansion of DST eligibility, and generous reimbursement of MDR-TB outpatient care costs).Materials and MethodsWe retrospectively extracted and analyzed data from the TB Information System on drug-resistant pulmonary tuberculosis diagnosed in Shenzhen during the 2012–2020 period. We analyzed trends in RR- and MDR-TB rates in new cases during 2012–2018 and 2018–2020 periods, and among previously-treated cases during 2012–2017 and 2017–2020 periods, using Cochran-Armitage tests. We generated multivariate logistic regression models to analyze demographic predictors of MDR/RR-TB rates.ResultsWe found 21,367 positive mycobacterial cultures in Shenzhen during the 2012–2020 period, and 19,951 (93.4%) were identified as Mycobacterium tuberculosis and had DST results (92.0% of those were mDST-based). Of these patients with DST results, 1630 (8.2%) were RR-TB, and 1142 (5.7%) were MDR-TB. Of the RR-TB, 70% were MDR-TB. The MDR/RR-TB rate in new TB cases increased significantly during the 2012–2018 period (Ptrend < 0.05), but it decreased in the 2018–2020 period (Ptrend > 0.05, with a significant trend for MDR-TB). Among previously treated cases, the temporal MDR/RR-TB rate trends did not differ significantly (Ptrend > 0.05). Our multivariate analysis showed that age younger than 30 years, housework service/unemployment, local residency, and previous TB treatment were all predictors of MDR/RR-TB. The percentage of patients with MDR-TB on treatment increased from 49.4% in 2012 to 70.5% in 2020. The treatment success rate of patients with MDR-TB during the 2012–2018 period was 71%.ConclusionDuring the study period in Shenzhen, the cases of MDR/RR-TB were detected, and the treatment enrollment increased and the MDR-TB rates decreased gradually after 2017. Decreasing trends may reflect the efficacy of improved strategies; however, their long-term impact on the MDR-TB burden remains to be investigated. The predictors of MDR-TB identified in our study should be considered when developing targeted MDR-TB control strategies.

Genetic variants and their association with phenotypic resistance to bedaquiline in Mycobacterium tuberculosis: a systematic review and individual isolate data analysis

SummaryBackgroundBedaquiline is a crucial drug for control of rifampicin-resistant tuberculosis. Molecular drug resistance assays could facilitate effective use of bedaquiline and surveillance of drug resistance emergence. To facilitate molecular assay development, we aimed to identify genomic markers of bedaquiline resistance.MethodsIn this systematic review and individual isolate analysis, we searched Europe PubMed Central and Scopus for studies published from the inception of each database until Oct 19, 2020, that assessed genotypic and phenotypic bedaquiline resistance in clinical or non-clinical Mycobacterium tuberculosis isolates. All studies reporting on the assessment of variants in the four genes of interest (Rv0678, atpE, pepQ, and Rv1979c) and phenotypic bedaquiline data in both clinical and non-clinical samples were included. We collated individual isolate data from eligible studies to assess the association between genomic variants with phenotypic bedaquiline resistance, using a standardised method endorsed by WHO. Risk of bias of the extracted data was independently assessed by two authors using the Quality Assessment of Diagnostic Accuracy Studies tool for clinical studies and Systematic Review Center for Laboratory Animal Experimentation tool for animal studies. The primary outcome was to identify mutations associated with resistance in four genes of interest (Rv0678, atpE, pepQ, and Rv1979c); for each genomic variant, the odds ratio (OR), 95% CI, and p value were calculated to identify resistance markers associated with bedaquiline resistance. This study is registered with PROSPERO, CRD42020221498.FindingsOf 1367 studies identified, 41 published between 2007 and 2020 were eligible for inclusion. We extracted data on 1708 isolates: 1569 (91·9%) clinical isolates and 139 (8·1%) non-clinical isolates. We identified 237 unique variants in Rv0678, 14 in atpE, 28 in pepQ, and 11 in Rv1979c. Most clinical isolates with a single variant reported in Rv0678 (229 [79%] of 287 variants), atpE (14 [88%] of 16 variants), pepQ (32 [100%] of 32 variants), or Rv1979c (115 [98%] of 119 variants) were phenotypically susceptible to bedaquiline. Except for the atpE 187G→C (OR ∞, [95% CI 13·28–∞]; p<0·0001) and Rv0678 138_139insG (OR 6·91 [95% CI 1·16–47·38]; p=0·016) variants, phenotypic–genotypic associations were not significant (p≥0·05) for any single variant in Rv0678, atpE, pepQ, and Rv1979c.InterpretationAbsence of clear genotypic–phenotypic associations for bedaquiline complicates the development of molecular drug susceptibility tests. A concerted global effort is urgently needed to assess the genotypic and phenotypic drug susceptibility of M tuberculosis isolates, especially in patients who have received unsuccessful bedaquiline-containing regimens. Treatment regimens should be designed to prevent emergence of bedaquiline resistance and phenotypic drug susceptibility tests should be used to guide and monitor treatment.FundingResearch Foundation Flanders, South African Medical Research Council, Department of Science and Innovation - National Research Foundation, National Institute of Health Institute of Allergy and Infectious Diseases, and Doris Duke Charitable Foundation.

基于PARMS技术的结核分枝杆菌利福平耐药基因 rpoB 分子标记的开发

The purpose of this study is to provide a simple and reliable genetic typing approach for molecular drug susceptibility test of Mycobacterium tuberculosis, through the developing of fluorescence molecular marker of rifampicin resistance gene rpoB. Eleven fluorescent molecular markers of the rpoB gene were established by using the sequence difference between the amino acid positions 531, 526, 516, 511 and 513 of rpoB gene of rifampicin-resistant strains and the alleles of rifampicin-sensitive strains, combined with the PARMS technique (Penta-primer amplification refractory mutation system). We used 104 clinical isolates of Mycobacterium tuberculosis to validate this marker and it was verified by sequencing as 100% correct. These samples were also tested with proportional drug sensitivity test. The coincidence rate was 94.23%. The molecular markers had high reliability for genotyping of rpoB gene. It can also detect low-concentration drug-resistant samples (511/533 unit point mutations) whose phenotypic susceptibility cannot be detected. The eleven sets of fluorescent molecular markers could cover 92%-96% of rpoB gene mutation types of rifampicin-resistant strains, and provide new idea for rapid detection of rifampin-resistant Mycobacterium tuberculosis.

Diagnostic accuracy of the FluoroType MTB and MTBDR VER 2.0 assays for the centralized high-throughput detection of Mycobacterium tuberculosis complex DNA and isoniazid and rifampicin resistance

ObjectivesTo evaluate the accuracy of two new molecular diagnostic tests for the detection of drug-resistant tuberculosis, the FluoroType MTB and MTBDR VER 2.0 assays, in combination with manual and automated DNA extraction methods. MethodsSputa from 360 Xpert Ultra Mycobacterium tuberculosis complex (MTBC)-positive patients and 250 Xpert Ultra MTBC-negative patients were tested. GenoType MTBDRplus served as reference for MTBC and drug resistance detection. Sanger sequencing was used to resolve discrepancies. ResultsFluoroType MTB VER 2.0 showed similar MTBC sensitivity compared with FluoroType MTBDR VER 2.0 (manual DNA extraction: 91.6% (294/321) versus 89.8% (291/324); p 0.4); automated DNA extraction: 92.1% (305/331) versus 87.7% (291/332); p 0.05)). FluoroType MTBDR VER2.0 showed comparable diagnostic accuracy to FluoroType MTBDR VER1.0 as previously reported for the detection of MTBC and rifampicin and isoniazid resistance. ConclusionsThe FluoroType MTB and MTBDR VER 2.0 assays together with an automated DNA extraction and PCR set-up platform may improve laboratory operational efficiency for the diagnosis of MTBC and resistance to rifampicin and isoniazid and show promise for the implementation in a centralized molecular drug susceptibility testing model.

Tuberculosis Drug Susceptibility, Treatment, and Outcomes for Belarusian HIV-Positive Patients with Tuberculosis: Results from a National and International Laboratory.

Background To cure drug-resistant (DR) tuberculosis (TB), the antituberculous treatment should be guided by Mycobacterium tuberculosis drug-susceptibility testing (DST). In this study, we compared conventional DST performed in Minsk, Belarus, a TB DR high-burden country, with extensive geno- and phenotypic analyses performed at the WHO TB Supranational Reference Laboratory in Copenhagen, Denmark, for TB/HIV coinfected patients. Subsequently, DST results were related to treatment regimen and outcome. Methods Thirty TB/HIV coinfected patients from Minsk were included and descriptive statistics applied. Results Based on results from Minsk, 10 (33%) TB/HIV patients had drug-sensitive TB. Two (7%) had isoniazid monoresistant TB, 8 (27%) had multidrug-resistant (MDR) TB, 5 (17%) preextensive drug-resistant (preXDR) TB, and 5 (17%) had extensive drug-resistant (XDR) TB. For the first-line drugs rifampicin and isoniazid, there was DST agreement between Minsk and Copenhagen for 90% patients. For the second-line anti-TB drugs, discrepancies were more pronounced. For 14 (47%) patients, there were disagreements for at least one drug, and 4 (13%) patients were classified as having MDR-TB in Minsk but were classified as having preXDR-TB based on DST results in Copenhagen. Initially, all patients received standard anti-TB treatment with rifampicin, isoniazid, pyrazinamide, and ethambutol. However, this was only suitable for 40% of the patients based on DST. On average, DR-TB patients were changed to 4 (IQR 3-5) active drugs after 1.5 months (IQR 1-2). After treatment adjustment, the treatment duration was 8 months (IQR 2-11). Four (22%) patients with DR-TB received treatment for >18 months. In total, sixteen (53%) patients died during 24 months of follow-up. Conclusions We found high concordance for rifampicin and isoniazid DST between the Minsk and Copenhagen laboratories, whereas discrepancies for second-line drugs were more pronounced. For patients with DR-TB, treatment was often insufficient and relevant adjustments delayed. This example from Minsk, Belarus, underlines two crucial points in the management of DR-TB: the urgent need for implementation of rapid molecular DSTs and availability of second-line drugs in all DR-TB high-burden settings. Carefully designed individualized treatment regimens in accordance with DST patterns will likely improve patients' outcome and reduce transmission with drug-resistant Mycobacterium tuberculosis strains.

Drug-resistant TB prevalence study in 5 health institutions in Haiti.

Tuberculosis (TB) is the leading infectious cause of death in the world. Multi-drug resistant TB (MDR-TB) is a major public health problem as treatment is long, costly, and associated to poor outcomes. Here, we report epidemiological data on the prevalence of drug-resistant TB in Haiti. This cross-sectional prevalence study was conducted in five health centers across Haiti. Adult, microbiologically confirmed pulmonary TB patients were included. Molecular genotyping (rpoB gene sequencing and spoligotyping) and phenotypic drug susceptibility testing were used to characterize rifampin-resistant MTB isolates detected by Xpert MTB/RIF. Between April 2016 and February 2018, 2,777 patients were diagnosed with pulmonary TB by Xpert MTB/RIF screening and positive MTB cultures. A total of 74 (2.7%) patients were infected by a drug-resistant (DR-TB) M. tuberculosis strain. Overall HIV prevalence was 14.1%. Patients with HIV infection were at a significantly higher risk for infection with DR-TB strains compared to pan-susceptible strains (28.4% vs. 13.7%, adjusted odds ratio 2.6, 95% confidence interval 1.5-4.4, P = 0.001). Among the detected DR-TB strains, T1 (29.3%), LAM9 (13.3%), and H3 (10.7%) were the most frequent clades. In comparison with previous spoligotypes studies with data collected in 2000-2002 and in 2008-2009 on both sensitive and resistant strains of TB in Haiti, we observed a significant increase in the prevalence of the drug-resistant MTB Spoligo-International-Types (SIT) 137 (X2 clade: 8.1% vs. 0.3% in 2000-02 and 0.9% in 2008-09, p<0.001), 5 (T1 clade: 6.8% vs 1.9 in 2000-02 and 1.7% in 2008-09, P = 0.034) and 455 (T1 clade: 5.4% vs 1.6% and 1.1%, P = 0.029). Newly detected spoligotypes (SIT 6, 7, 373, 909 and 1624) were also recorded. This study describes the genotypic and phenotypic characteristics of DR-TB strains circulating in Haiti from April 2016 to February 2018. Newly detected MTB clades harboring multi-drug resistance patterns among the Haitian population as well as the higher risk of MDR-TB infection in HIV-positive people highlights the epidemiological relevance of these surveillance data. The importance of detecting RIF-resistant patients, as proxy for MDR-TB in peripheral sites via molecular techniques, is particularly important to provide adequate patient case management, prevent the transmission of resistant strains in the community and to contribute to the surveillance of resistant strains.

Refining MDR-TB treatment regimens for ultra short therapy (TB-TRUST): study protocol for a randomized controlled trial

BackgroundMultidrug-resistant tuberculosis (MDR-TB) are unsatisfied to treat, pressing more effective and innovative treatment regimens. New efficient regimens for MDR-TB have obtained high treatment success rates. However, those regimens without drug susceptibility testing (DST) are also likely to contribute to the emergence of resistance. Precision treatments guided by DST might optimize the patients’ treatment outcome individually and minimize resistance amplification.MethodsTB-TRUST is a phase III, multicenter, open-label, randomized controlled clinical trial of non-inferiority comparing the treatment success rate between the World Health Organization (WHO) shorter regimen and the refined ultra-short regimen for fluoroquinolones and second-line injectable drugs susceptible rifampicin-resistant TB. The control arm uses the WHO injectable-containing shorter regimen for 36–44 weeks depending on time of sputum smear conversion. The investigational arm uses a refined ultra-short regimen guided by molecular DST to pyrazinamide via whole-genome sequencing (WGS) to optimize the treatment of pyrazinamide-susceptible patients with levofloxacin, linezolid, cycloserine and pyrazinamide for 24–32 weeks and pyrazinamide-resistant with levofloxacin, linezolid, cycloserine and clofazimine for 36–44 weeks. The primary outcome is the treatment success rate without relapse at 84 weeks after treatment initiation. Secondary outcomes include the time of sputum culture conversion and occurrence of adverse events. Assuming α = 0.025 level of significance (one-sided test), a power of 80%, a < 10% difference in treatment success rate between control arm and investigational (80% vs. 82%), and a 5% lost follow-up rate, the number of participants per arm to show non-inferiority was calculated as 177(354 in total).DiscussionRapid molecular testing distinguishes patients who are eligible for shorter regimen with fluoroquinolone and the WGS-guided results shorten the treatment to 6 months for pyrazinamide susceptible patients. It’s foreseeable that not only novel developed medicines, but also traditional powerful medicines with the susceptibility confirmed by DST are the key factors to ensure the effect of anti-MDR-TB drugs. As a DST-guided precision treatment, TB-TRUST are expected to optimize therapy outcome in more patients who cannot afford the expensive new medicines and minimize and even avoid resistance amplification with the rational use of anti-TB drugs.Trail registrationClinicalTrial.gov, NCT03867136. Registered on March 7, 2019.

Accuracy of molecular drug susceptibility testing amongst tuberculosis patients in Karakalpakstan, Uzbekistan.

In this retrospective study, we evaluated the diagnostic accuracy of molecular tests (MT) for the detection of DR-TB, compared to the gold standard liquid-based drug susceptibility testing (DST) in Karakalpakstan. A total of 6670 specimens received in the Republican TB No 1 Hospital Laboratory of Karakalpakstan between January and July 2017 from new and retreatment patients were analysed. Samples were tested using Xpert MTB/RIF and line probe assays (LPA) for the detection of mutations associated with resistance. The sensitivity and specificity of MTs were calculated relative to results based on DST. The accuracy of MT for detection of rifampicin resistance was high, with sensitivity and specificity over 98%. However, we observed reduced sensitivity of LPA for detection of resistance; 86% for isoniazid (95% CI 82-90%), 86% for fluoroquinolones (95% CI 68-96%), 70% for capreomycin (95% CI 46-88%) and 23% for kanamycin (95% CI 13-35%). We show that MTs are a useful tool for rapid and safe diagnosis of DR-TB; however, clinicians should be aware of their limitations. Although detection of rifampicin resistance was highly accurate, our data suggest that resistance mutations circulating in the Republic of Karakalpakstan for other drugs were not detected by the methods used here. This merits further investigation.

Epidemiological Characteristics of Rifampicin-Resistant Tuberculosis in Students - China, 2015-2019.

What is already known about this topic? The number of students with tuberculosis (TB) has been increasing since 2015. However, the prevalence of rifampicin-resistant tuberculosis (RR-TB) among student population is unclear. What is added by the report? The number of students with RR-TB has significantly increased from 2015 to 2019, especially in the western region of China. The majority of patients were college students. Students with RR-TB were mainly new patients. What are the implications for public health practice? The following measures are recommended: strengthening TB surveillance in schools, promoting the application of convenient and rapid molecular drug susceptibility testing tools, and actively carrying out drug resistance screening and follow ups for cohabiting children of adult RR-TB patients.

Towards Unified Data Exchange Formats for Reporting Molecular Drug Susceptibility Testing.

With the rapid development of new advanced molecular detection methods, identification of new genetic mutations conferring pathogen resistance to an ever-growing variety of antimicrobial substances will generate massive genomic datasets for public health and clinical laboratories. Keeping up with specialized standard coding for these immense datasets will be extremely challenging. This challenge prompted our effort to create a common molecular resistance Logical Observation Identifiers Names and Codes (LOINC) panel that can be used to report any identified antimicrobial resistance pattern. To develop and utilize a common molecular resistance LOINC panel for molecular drug susceptibility testing (DST) data exchange in the U.S. National Tuberculosis Surveillance System using California Department of Public Health (CDPH) and New York State Department of Health as pilot sites. We developed an interface and mapped incoming molecular DST data to the common molecular resistance LOINC panel using Health Level Seven (HL7) v2.5.1 Electronic Laboratory Reporting (ELR) message specifications through the Orion Health™ Rhapsody Integration Engine v6.3.1. Both pilot sites were able to process and upload/import the standardized HL7 v2.5.1 ELR messages into their respective systems; albeit CDPH identified areas for system improvements and has focused efforts to streamline the message importation process. Specifically, CDPH is enhancing their system to better capture parent-child elements and ensure that the data collected can be accessed seamlessly by the U.S. Centers for Disease Control and Prevention. The common molecular resistance LOINC panel is designed to be generalizable across other resistance genes and ideally also applicable to other disease domains. The study demonstrates that it is possible to exchange molecular DST data across the continuum of disparate healthcare information systems in integrated public health environments using the common molecular resistance LOINC panel.

The frequency of point mutations associated with resistance to isoniazid and rifampin among clinical isolates of multidrug-resistant Mycobacterium tuberculosis in the west of Iran

Global increasing broad-spectrum resistance to many antibiotics established Tuberculosis (TB) infections as worldwide health issue. The rising prevalence of multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) isolates around the world is a therapeutic challenge. Monitoring of resistance to anti-TB drugs is essential to implement the strategies to control tuberculosis.This study was aimed to identify the point mutations associated with resistance to isoniazid (INH) and rifampin (RIF) antibiotics by Polymerase chain reaction (PCR) and reverse hybridization methods in clinical isolated samples of MDR Mycobacterium tuberculosis in the west of Iran.Phenotypic and biochemical methods were used for the identification of TB strains. Then proportion and GeneXpert methods were used to determine resistance isolates. The DNA was extracted, and finally, PCR and reverse hybridization methods were performed to identify the mutations.Among 54 isolates of MTB, 22 isolates were INH and RIF susceptible, and 32 isolates were diagnosed as MDR isolates. Of 32 MDR isolates, 22 cases (%70) had a mutation in katG codon of 315, 1 case inhA (%3) and 9 (%28) isolated had no detectable mutation. About rifampin, the mutations associated with rpoB gene in codons 531, 516 and 626 comprised (n = 21, %66), (n = 7, %22) and (n = 1, %3) respectively and in 3 (%9) isolates no mutations were found. The sensitivity, specificity and accuracy for INH (%71, %100, %83) and rifampin (%91, %100, %94) obtained, also PPV and NPV were determined for INH (%100, %70), RIF (%100, %88) respectively.Our results showed that MDR isolates with mutations in the katG 315 and rpoB 531 regions are epidemic strains in our study area. Also, our results suggest that TB Resistance line probe assay (AID) method has high sensitivity for detection of point mutations among isoniazid- and rifampin- resistant TB isolates. However, gold-standard phenotypic methods must always confirm the molecular drug susceptibility test results.

MDR M. tuberculosis outbreak clone in Eswatini missed by Xpert has elevated bedaquiline resistance dated to the pre-treatment era

BackgroundMultidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are threatening the control of MDR tuberculosis (MDR-TB) in Eswatini.MethodsWe investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009–2010; (2) MDR strains from the Nhlangano region, 2014–2017).ResultsMDR strains in collection 1 had a high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n = 28; gCL10, n = 40). All gCL10 isolates, which likely emerged around 1993 (95% highest posterior density 1987–1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2.ConclusionThe high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.

Multidrug-resistant tuberculosis in the Kharkiv Region, Ukraine.

OBJECTIVE: To document the level of drug resistance in MDR-TB patients and to characterize management capacities for their medical care and MDR-TB treatment outcomes in the Kharkiv region of Ukraine. This area has one of the highest frequencies of MDR-TB worldwide.METHODS: A retrospective observational cohort study was performed on registry data from the regional anti-TB dispensary in Kharkiv. All microbiologically confirmed MDR-TB patients registered in 2014 were included. Diagnostic, treatment and post-treatment follow-up data were analysed.RESULTS: Of 169 patients with MDR-TB, 55.0% had pre-extensively drug-resistant (pre-XDR) or XDR resistant patterns. Rapid molecular diagnosis by GeneXpert and liquid M. tuberculosis cultures were only available for 66.9% and 56.8% of patients, respectively. Phenotypic drug-susceptibility testing (DST) for high priority TB drugs (bedaquiline, linezolid, clofazimine) were not available. DST for later generation fluroquinolones was available only in 53.2% of patients. 50.9% of patients had less than 4 drugs in the treatment regimen proven to be effective by DST. More than 23.1% of patients with MDR-TB failed their treatment and only 45.0% achieved a cure.CONCLUSION: The high prevalence of MDR-TB and poor MDR-TB treatment outcomes in the Kharkiv region, is associated with substantial shortages in rapid molecular and phenotypic DST, a lack of high priority MDR-TB drugs, poor treatment monitoring and follow-up capacities.

Improved treatment outcome of multidrug-resistant tuberculosis with the use of a rapid molecular test to detect drug resistance in China

ObjectivesNumerous studies investigate the advantages of rapid molecular drug susceptibility testing (DST) in comparison to phenotypic DST, but the clinical impact on treating multi/extensively drug resistant TB(M/XDR-TB) is less studied. Therefore, we examined how molecular DST testing may improve MDR-TB treatment management and outcome in Chinese settings. MethodsWe performed a comparative study of patient cohorts before and after the implementation of molecular DST diagnosis with Genotype MTBDRsl/MTBDRplus assay in two Chinese hospitals. We collected clinical information including time to sputum culture conversion and final treatment outcome. ResultsIn total, 242 MDR-TB patients were studied including 114 before (pre-implementation group) and 128 after the implementation (post-implementation group) of molecular DST. Time to MDR-TB diagnosis was significantly reduced for patients in the post-implementation group, as compared to the pre-implementation group (median,16 vs 62 days; P < 0.001). Patients with early available molecular DST results had a more rapid culture conversion (aHR1.94 95% CI: 1.37-2.73; median,12 vs 24 months, respectively; P < 0.001) and higher rate of treatment success (68% vs 47%, P < 0.01). ConclusionsThe use of molecular DST in routine care for MDR-TB diagnosis as compared to phenotypic DST was associated with a decreased time to culture conversion and improved treatment outcome, highlighting its important clinical value.

Risk factors for drug-resistant tuberculosis at a referral centre in Toronto, Ontario, Canada: 2010-2016.

Drug-resistant tuberculosis (TB) poses a major public health concern worldwide. However, no studies have addressed risk factors for drug resistance in Ontario, which has its own unique profile of immigrants. We evaluated demographic and clinical risk factors for drug-resistant TB among patients treated at West Park Healthcare Centre, located in Toronto, Ontario (Canada). All patients who were diagnosed with TB and treated at West Park Healthcare Centre between January 2010 and December 2016 were included in this retrospective cohort study. Characteristics of patients with isoniazid mono-resistant (INH-R) TB and multidrug resistant (MDR) TB were compared to patients with drug-susceptible TB with bivariate and multivariable logistic regression. Risk factors for INH-R TB included younger age (younger than 35 years), prior TB treatment, non-diabetic and birth in a non-South-East Asian country, but only the latter two factors were significant in multivariable analysis. On the other hand, we found younger generation (younger than 65 years), birth in European region, recent arrival to Canada (fewer than 120 months), prior treatment and human immunodeficiency virus (HIV) infection were associated with MDR-TB, among which younger age (younger than 35 years), more recent immigration (fewer than 24 months), prior treatment and HIV infection were significant in multivariable analysis. These findings may be of use to TB clinicians in the province by informing the initial empiric antibiotic regimen prescribed while awaiting phenotypic drug susceptibility testing and assisting in decisions regarding whether to request rapid molecular drug susceptibility testing.

Phylogenomic assessment of drug-resistant Mycobacterium tuberculosis strains from Beira, Mozambique

BackgroundMozambique is a high-burden tuberculosis (TB) country where TB/HIV co-infection and drug resistant TB (DR-TB) incidence is increasing. Whole genome sequencing (WGS) comprehensively describes the molecular epidemiology of TB, allows prediction of DR-TB phenotypes, lineages strains identification and better understanding of transmission chains. ObjectiveTo describe genetic diversity of DR-TB Mycobacterium tuberculosis isolated in Beira, Mozambique. MethodsDescriptive cross-sectional study with 35 M. tuberculosis isolates, resistant to at least one first-line drug on molecular drug-susceptibility tests (DST). Variant identification, DR prediction and phylogenetic analysis provided by WGS, drug-susceptibility pattern compared to line-probe assay (LPA): Genotype MTBDRTMplus and MTBDRTMsl. FindingsLineage 4 (L4) was the most prevalent: 25 (71.4%) isolates; 5 (14.3%) L1 and 5 (14.3%) L2. WGS showed 33/35 (94.3%) isolates resistant to at least one drug, two pan-susceptible isolates that were previously diagnosed as DR-TB with genotype MTBDRplus. Concordance between WGS and LPA: 88.6% for isoniazid (INH), 85.7% to rifampicin (RPM), 91.4% for quinolones and 100% to second line injectable drugs. There were three possible TB transmission chains, 10 strains showing recent transmission. ConclusionWGS provided reliable information about the most frequent lineages related to DR-TB in Beira, Mozambique: L4.3 (LAM), L2 (Beijing) and L1 (EAI) and possible recent transmission chain.

Isoniazid-resistant Mycobacterium tuberculosis: prevalence, resistance spectrum and genetic determinants of resistance

The lack of simple, rapid diagnostic tests for isoniazid-resistant rifampicin-susceptible tuberculosis infection (Hr-TB) can result in low treatment efficacy and further amplification of drug resistance. Based on the clinical data, this study sought to estimate the prevalence of Hr-TB in the general population and characterize the phenotypic susceptibility and genetic determinants of isoniazid resistance in M. tuberculosis strains. Molecular-genetic and culture-based drug susceptibility tests were performed on M. tuberculosis isolates and M. tuberculosis DNA obtained from the patients with pulmonary TB undergoing treatment at the Central Tuberculosis Research Institute between 2011 and 2018. The tests revealed that Hr-TB accounted for 12% of all TB cases in the studied sample. Hr-TB strains were either resistant to isoniazid only (45%) or had multiple resistance to 2–6 anti-TB agents. Resistance to isoniazid was caused by mutations in the katG gene. Based on the literature analysis and our own observations, we emphasize the importance of developing simple molecular drug susceptibility tests capable of detecting simultaneous resistance to rifampicin and isoniazid and the necessity of their translation into clinical practice.

Impact of Molecular Drug Susceptibility Testing on the Time to Multidrug-resistant Tuberculosis Treatment Initiation.

BackgroundThe purpose of this study was to evaluate the current status and trends in the coverage of molecular drug susceptibility testing (mDST), and the impact of mDST on the time to multidrug-resistant tuberculosis (MDR-TB) treatment initiation in Korea.MethodsWe included confirmed rifampin-resistant (RR)/MDR-TB patients who submitted application forms for novel drug uses to the National TB Expert Review Committee from September 1, 2016 to November 30, 2019. We retrospectively reviewed their medical records.ResultsOf the 621 MDR/RR-TB patients, mDST was performed in 442 (71.2%); Xpert MTB/RIF (Xpert) alone in 109 (17.6%), MTBDRplus line probe assay (LPA) alone in 199 (32.0%), and both Xpert and LPA in 134 (21.6%) patients. The coverage rate of mDST has gradually increased to 70% in 2015, 50.7% in 2016, 67.9% in 2017, 75.2% in 2018, and 79.4% in 2019 (P for trend < 0.001). Median time to MDR-TB treatment initiation was 35 days (interquartile range25–75 0–72), which has gradually decreased during the study period (P < 0.001). Independent predictors of shorter time to MDR-TB treatment initiation were retreatment case (adjusted hazard ratio [aHR], 1.30; 95% confidence interval [CI], 1.10–1.54), Xpert testing (aHR, 2.42; 95% CI, 2.03–2.88), and LPA testing (aHR, 1.83; 95% CI, 1.55–2.16). Transfer to another healthcare facility was inversely related to shorter time to treatment initiation (aHR, 0.74; 95% CI, 0.63–0.88).ConclusionmDST coverage is gradually increasing and contributes to reducing the time to MDR-TB treatment initiation. Further efforts are needed to achieve universal access to mDST and to properly integrate mDST into routine clinical practice.