Experiences from 4 Years of Organization of an External Quality Assessment for Mycobacterium tuberculosis Whole-Genome Sequencing in the European Union/European Economic Area.

Here, we report the development and key features of the first external quality assessment (EQA) scheme for Mycobacterium tuberculosis whole-genome sequencing (WGS). The results of four rounds (2017 to 2020) of implementation within the European tuberculosis reference laboratories network (ERLTB-Net-2) are presented and discussed. EQA panels comprising 10 genomic DNAs were distributed to ERLTB-Net 2 laboratories volunteering to participate in this exercise. Since 2018, five FASTQ files were added to better assess the dry WGS processes, and in 2020, three of the five files were replaced by synthetic files (providing additional flexibility for the mutations included in the panels). Ten National tuberculosis reference laboratories participated in all four EQA rounds, and seven participated in at least one. High-confidence resistance mutations were correctly identified by all laboratories, but challenges remained with respect to the identification of mixed loci and interpretation of rare mutations. M. tuberculosis genotyping and clustering analysis was >90% accurate for pure samples with the main challenges being related to the analysis of mixed genotypes and DNA FASTQ files. The development and implementation of this WGS EQA scheme has contributed to the continuous improvement in performance of participating laboratories in M. tuberculosis WGS and data analysis. This scheme can serve as a model of comprehensive quality assessment for M. tuberculosis WGS that can be replicated in different settings worldwide. IMPORTANCE The wider availability of whole-genome sequencing (WGS) coupled to new developments in bioinformatic tools and databases to interpret Mycobacterium tuberculosis complex WGS data has accelerated the adoption of this method for the routine prediction of antimycobacterial drug resistance and genotyping, thus necessitating the establishment of a comprehensive external quality control system. Here, we report 4 years of development and results from such a panel.

Evaluation of a lateral-flow nanoparticle fluorescence assay for TB infection diagnosis.

BACKGROUND: Programmatic management of TB infection is a critical component of the WHO End TB Strategy. Interferon-gamma release assays (IGRAs) overcome some limitations of the tuberculin skin test, but implementation of IGRA testing in low-resource settings is challenging.METHODS: In this feasibility study, we evaluated performance of a novel digital lateral-flow assay, the QIAreach® QuantiFERON® TB (QIAreach-QFT) test, against the QuantiFERON®-TB Gold Plus (QFT-Plus) assay. A population with a mix of risk factors for TB infection (111 donors) were sampled over multiple days. A total of 207 individual blood samples were tested according to the manufacturer´s instructions.RESULTS: The overall percentage agreement was 95.6% (two-sided 95% CI 91.8-98), with a positive percentage agreement (i.e., sensitivity) of 100% (95% CI 94.7-100) and a negative percentage agreement (i.e., specificity) of 95.6% (95% CI 90.6-98.4). All QFT-Plus positive specimens with TB1-Nil and TB2-Nil values less than 1 IU/ml tested positive on QIAreach-QFT.CONCLUSIONS: QIAreach QFT is a deployable, accurate testing solution for decentralised testing. It has the potential to overcome key hurdles for TB infection screening in high-burden settings thus helping to achieve the WHO End TB programme goals.

Role and value of whole genome sequencing in studying tuberculosis transmission.

BACKGROUND: Tuberculosis (TB) remains a serious public health threat worldwide. Theoretically ultimate resolution of whole genome sequencing (WGS) for Mycobacterium tuberculosis complex (MTBC) strain classification makes this technology very attractive for epidemiological investigations. OBJECTIVES: To summarize the evidence available in peer-reviewed publications on the role and place of WGS in detection of TB transmission. SOURCES: A total of 69 peer-reviewed publications identified in Pubmed database. CONTENT: Evidence from >30 publications suggests that a cut-off value of fewer than six single nucleotide polymorphisms between strains efficiently excludes cases that are not the result of recent transmission and could be used for the identification of drug-sensitive isolates involved in direct human-to-human TB transmission. Sensitivity of WGS to identify epidemiologically linked isolates is high, reaching 100% in eight studies with specificity (17%-95%) highly dependent on the settings. Drug resistance and specific phylogenetic lineages may be associated with accelerated mutation rates affecting genetic distances. WGS can be potentially used to distinguish between true relapses and re-infections but in high-incidence low-diversity settings this would require consideration of epidemiological links and minority alleles. Data from four studies looking into within-host diversity highlight a need for developing criteria for acceptance or rejection of WGS relatedness results depending on the proportion of minority alleles. IMPLICATIONS: WGS will potentially allow for more targeted public health actions preventing unnecessary investigations of false clusters. Consensus on standardization of raw data quality control processing criteria, analytical pipelines and reporting language is yet to be reached.

Diagnostic performance and impact of routinely implemented Xpert® MTB/RIF assay in a setting of high incidence of drug-resistant TB in Odessa Oblast, Ukraine.

OBJECTIVES AND METHODS: The Xpert® MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) has been in routine use in Odessa Oblast, a region with the highest tuberculosis (TB) incidence in Ukraine, since 2013. We assessed the performance of the assay in routine settings and evaluated its effect on treatment outcomes. RESULTS: The sensitivity of Xpert for TB detection was 93.7% (1165/1243) and 69.5% (448/645) for smear-positive and smear-negative sputum specimens, respectively, and its sensitivity for rifampicin resistance was 93.4% (1212/1298). Median time to TB detection using the Xpert assay was 0 days. Treatment initiation within 1 week increased the proportion of successful outcomes (60.1% versus 25.9%, RR = 1.86, 95%CI = 1.46-2.42), but the introduction of Xpert MTB/RIF has not led to a significant improvement in treatment outcomes (57.2% versus 46.2%; RR = 0.93, 95%CI = 0.77-1.12). CONCLUSION: Performance characteristics of the Xpert assay demonstrated during its routine implementation in an area of high TB and drug-resistant TB incidence in Ukraine were in line with those demonstrated in similar settings elsewhere. Rollout of rapid molecular testing may lead to better treatment results provided that it is implemented in conjunction with other programmatic improvements.

Discordance in latent tuberculosis (TB) test results in patients with end-stage renal disease.

OBJECTIVES: This natural experiment was designed to assess the impact of exposure to an active case of tuberculosis (TB) on a group of immunosuppressed individuals, with end-stage renal disease over an extended follow-up. STUDY DESIGN: Close contacts of people with sputum smear-positive Mycobacterium tuberculosis are at high risk of infection, particularly immunosuppressed individuals. An infectious TB healthcare worker worked in a renal dialysis unit for a month before diagnosis, with 104 renal dialysis patients, was exposed for ≥8 h. METHODS: Patients were informed and invited for screening 8-10 weeks postexposure. They either underwent standard two-step assessment with tuberculin skin test (TST) and QuantiFERON®-TB Gold (Cellestis GmbH; QFN) interferon-gamma release assay (IGRA) or after consent, enrolled in a study where these two tests were performed simultaneously with T-SPOT®-TB (Oxford Immunotec Ltd; TSPOT). Patients within the study were followed up for 2 years from exposure, with QFN and TSPOT repeated at months 3 and 6 from the first testing. RESULTS: Of 104 exposed individuals, 75 enrolled in the study. There was a high degree of discordance among QFN, TSPOT and TST. This was seen at both the first time point and also over time in subjects who were retested. No patients had active TB at the baseline testing. None received treatment for latent TB infection. Over the following 2 years, no one developed TB disease. CONCLUSION: This study suggests that there is a low risk of progression to active TB in low-incidence countries even in high-risk groups. This plus the degree of the test result discordance emphasises the complexities of managing TB in such settings as it is unclear which of these tests, if any, provides the best diagnostic accuracy.

Time-and-motion tool for the assessment of working time in tuberculosis laboratories: a multicentre study.

SETTING: Implementation of novel diagnostic assays in tuberculosis (TB) laboratory diagnosis requires effective management of time and resources. OBJECTIVE: To further develop and assess at multiple centres a time-and-motion (T&M) tool as an objective means for recording the actual time spent on running laboratory assays. DESIGN: Multicentre prospective study conducted in six European Union (EU) reference TB laboratories. RESULTS: A total of 1060 specimens were tested using four laboratory assays. The number of specimens per batch varied from one to 60; a total of 64 recordings were performed. Theoretical hands-on times per specimen (TTPS) in h:min:s for Xpert® MTB/RIF, mycobacterial interspersed repetitive unit-variable number of tandem repeats genotyping, Ziehl-Neelsen staining and manual fluorescence microscopy were respectively 00:33:02 ± 00:12:32, 00:13:34 ± 00:03:11, 00:09:54 ± 00:00:53 and 00:06:23 ± 00:01:36. Variations between laboratories were predominantly linked to the time spent on reporting and administrative procedures. Processing specimens in batches could help save time in highly automated assays (e.g., line-probe) (TTPS 00:14:00 vs. 00:09:45 for batches comprising 7 and 31 specimens, respectively). CONCLUSIONS: The T&M tool can be considered a universal and objective methodology contributing to workload assessment in TB diagnostic laboratories. Comparison of workload between laboratories could help laboratory managers justify their resource and personnel needs for the implementation of novel, time-saving, cost-effective technologies, as well as identify areas for improvement.

Diagnostic accuracy of the Xpert® MTB/RIF cycle threshold level to predict smear positivity: a meta-analysis.

SETTING: Xpert® MTB/RIF is the most widely used molecular assay for rapid diagnosis of tuberculosis (TB). The number of polymerase chain reaction cycles after which detectable product is generated (cycle threshold value, CT) correlates with the bacillary burden.OBJECTIVE To investigate the association between Xpert CT values and smear status through a systematic review and individual-level data meta-analysis. DESIGN: Studies on the association between CT values and smear status were included in a descriptive systematic review. Authors of studies including smear, culture and Xpert results were asked for individual-level data, and receiver operating characteristic curves were calculated. RESULTS: Of 918 citations, 10 were included in the descriptive systematic review. Fifteen data sets from studies potentially relevant for individual-level data meta-analysis provided individual-level data (7511 samples from 4447 patients); 1212 patients had positive Xpert results for at least one respiratory sample (1859 samples overall). ROC analysis revealed an area under the curve (AUC) of 0.85 (95%CI 0.82-0.87). Cut-off CT values of 27.7 and 31.8 yielded sensitivities of 85% (95%CI 83-87) and 95% (95%CI 94-96) and specificities of 67% (95%CI 66-77) and 35% (95%CI 30-41) for smear-positive samples. CONCLUSION: Xpert CT values and smear status were strongly associated. However, diagnostic accuracy at set cut-off CT values of 27.7 or 31.8 would not replace smear microscopy. How CT values compare with smear microscopy in predicting infectiousness remains to be seen.

Whole genome sequencing of Mycobacterium tuberculosis for detection of drug resistance: a systematic review.

OBJECTIVES: We conducted a systematic review to determine the diagnostic accuracy of whole genome sequencing (WGS) of Mycobacterium tuberculosis for the detection of resistance to first- and second-line anti-tuberculosis (TB) drugs. METHODS: The study was conducted according to the criteria of the Preferred Reporting Items for Systematic Reviews group. A total of 20 publications were included. The sensitivity, specificity, positive-predictive value and negative-predictive value of WGS using phenotypic drug susceptibility testing methods as a reference standard were determined. RESULTS: Anti-TB agents tested included all first-line drugs, a variety of reserve drugs, as well as new drugs. Polymorphisms in a total of 53 genes were tested for associations with drug resistance. Pooled sensitivity and specificity values for detection of resistance to selected first-line drugs were 0.98 (95% CI 0.93-0.98) and 0.98 (95% CI 0.98-1.00) for rifampicin and 0.97 (95% CI 0.94-0.99) and 0.93 (95% CI 0.91-0.96) for isoniazid, respectively. Due to high heterogeneity in study designs, lack of data, knowledge of resistance mechanisms and clarity on exclusion of phylogenetic markers, there was a significant variation in analytical performance of WGS for the remaining first-line, reserved drugs and new drugs. CONCLUSIONS: Whole genome sequencing could be considered a promising alternative to existing phenotypic and molecular drug susceptibility testing methods for rifampicin and isoniazid pending standardization of analytical pipelines. To ensure clinical relevance of WGS for detection of M. tuberculosis complex drug resistance, future studies should include information on clinical outcomes.

What are the most efficacious treatment regimens for isoniazid-resistant tuberculosis? A systematic review and network meta-analysis.

INTRODUCTION: Consensus on the best treatment regimens for patients with isoniazid-resistant TB is limited; global treatment guidelines differ. We undertook a systematic review and meta-analysis using mixed-treatment comparisons methodology to provide an up-to-date summary of randomised controlled trials (RCTs) and relative regimen efficacy. METHODS: Ovid MEDLINE, the Web of Science and EMBASE were mined using search terms for TB, drug therapy and RCTs. Extracted data were inputted into fixed-effects and random-effects models. ORs for all possible network comparisons and hierarchical rankings for different regimens were obtained. RESULTS: 12 604 records were retrieved and 118 remained postextraction, representing 59 studies-27 standalone and 32 with multiple papers. In comparison to a baseline category that included the WHO-recommended regimen for countries with high levels of isoniazid resistance (rifampicin-containing regimens using fewer than three effective drugs at 4 months, in which rifampicin was protected by another effective drug at 6 months, and rifampicin was taken for 6 months), extending the duration of rifampicin and increasing the number of effective drugs at 4 months lowered the odds of unfavourable outcomes (treatment failure or the lack of microbiological cure; relapse post-treatment; death due to TB) in a fixed-effects model (OR 0.31 (95% credible interval 0.12-0.81)). In a random-effects model all estimates crossed the null. CONCLUSIONS: Our systematic review and network meta-analysis highlight a regimen category that may be more efficacious than the WHO population level recommendation, and identify knowledge gaps where data are sparse. SYSTEMATIC REVIEW REGISTRATION NUMBER: PROSPERO CRD42014015025.

The Colour Test for drug susceptibility testing of Mycobacterium tuberculosis strains.

SETTING: Tartu, Estonia. OBJECTIVE: To assess the performance and feasibility of the introduction of the thin-layer agar MDR/XDR-TB Colour Test (Colour Test) as a non-commercial method of drug susceptibility testing (DST). DESIGN: The Colour Test combines the thin-layer agar technique with a simple colour-coded quadrant format, selective medium to reduce contamination and colorimetric indication of bacterial growth to simplify interpretation. DST patterns for isoniazid (INH), rifampicin (RMP) and ciprofloxacin (CFX) were determined using the Colour Test for 201 archived Mycobacterium tuberculosis isolates. Susceptibilities were compared to blinded DST results obtained routinely using the BACTEC™ Mycobacteria Growth Indicator Tube™ (MGIT) 960 to assess performance characteristics. RESULTS: In all, 98% of the isolates produced interpretable results. The average time to positivity was 13 days, and all results were interpretable. The Colour Test detected drug resistance with 98% sensitivity for INH, RMP and CFX and 99% for multidrug-resistant tuberculosis. Specificities were respectively 100% (95%CI 82-100), 88% (95%CI 69-97) and 91% (95%CI 83-96) and 90% (95%CI 74-98). Agreement between the Colour Test and BACTEC MGIT 960 were respectively 98%, 96%, 94% and 97%. CONCLUSION: The Colour Test could be an economical, accurate and simple technique for testing tuberculosis strains for drug resistance. As it requires little specialist equipment, it may be particularly useful in resource-constrained settings with growing drug resistance rates.

Diagnosis of tuberculosis and drug resistance: what can new tools bring us?

This is an exciting time for tuberculosis (TB) diagnostics. The technology for rapid diagnosis of TB and rifampicin (RMP) resistance in pulmonary sputum smear-positive specimens is well advanced, and assays have high specificity with good sensitivity. Nevertheless, the current sensitivity of TB detection means that these assays still cannot replace the standard diagnostic methods for TB or conventional drug susceptibility testing (DST). In extra-pulmonary specimens, the performance of molecular tools varies and should be considered separately for each specimen type. Evidence for the use of these assays for TB and drug resistance detection in individuals co-infected with TB and the human immunodeficiency virus (HIV) is limited. As the positive predictive value for RMP resistance reaches ≥ 90% only when the prevalence of RMP resistance in new TB patients is >15%, which is rare globally, many cases with such resistance will be false-resistant, emphasising the need for a secondary confirmative test. Similarly, increased (or incorrect) diagnosis of TB may compromise programme effectiveness by increasing the numbers of individuals requiring anti-tuberculosis treatment, unless it is carefully planned. For the future, 1) assays with greater sensitivity for TB detection are needed; 2) rapid diagnostics for paediatric TB are important, and there is a need for carefully designed studies, including those involving HIV-positive children; 3) more clinical data need to be obtained from longitudinal studies, especially related to the influence of rapid diagnostics on disease outcome; and 4) point-of-care tests using untreated sputum, blood or urine and little or no equipment would be of immeasurable benefit. Although great progress has been made, we are not there yet.

Performance of the GenoType® MTBDRPlus assay in routine settings: a multicenter study.

Former Soviet Union countries including the Baltic States (Latvia, Lithuania, and Estonia) are hot spots for an emerging epidemic of drug resistant tuberculosis (TB). As a part of the development of a co-ordinated network of centers for diagnostic trials across Eastern Europe we conducted a retrospective multicenter analysis of the performance of the GenoType® MTBDRPlus assay for TB identification and susceptibility to isoniazid (INH) and rifampicin (RIF) in routine settings. A total of 1,045 primary samples, 1045 TB cultures derived from these specimens and 306 separate M. tuberculosis isolates tested in 2007-2010 at four participating sites (Tartu, Estonia; Riga, Latvia; Vilnius, Lithuania; and Samara, Russian Federation) were included in the analysis. The pooled sensitivity and specificity values for RIF and INH were 95.3% and 95.5%, 89.9 and 87.1%, respectively; there were no statistically significant variations in performance across sites. The proportion of multidrug resistant (MDR) strains in the collections ranged from 21.8% (in Estonia) to 55.9% (in Russia). In a routine non-trial context, the assay reliably detected both rifampicin and isoniazid resistance. The absence of statistically significant differences between sites suggested that the comparable performance obtained using these assays has helped demonstrate the formation of a successful diagnostic trial network.

Evaluation of two molecular assays for rapid detection of mycobacterium tuberculosis resistance to fluoroquinolones in high-tuberculosis and -multidrug-resistance Settings.

The Russian Federation is a high-tuberculosis (TB)-burden country with high rates of drug resistance, including multidrug and extensive drug resistance to TB (M/XDRTB). Rapid diagnosis of resistance to fluoroquinolones (FQs) using molecular assays is essential for the implementation of appropriate drug regimens and prevention of the transmission of XDR strains. A total of 51 individual MDRTB strains were tested by pyrosequencing of the quinolone resistance determining region of the gyrA gene and the GenoType MTBDRsl assay (Hain Lifescience, GmbH, Nehren, Germany), and the results were evaluated against those obtained by phenotypic drug susceptibility testing (DST). Mutations were detected in 25 (78.1%) FQ-resistant strains, with the majority of mutations (n = 19 [73.0%]) found in codon 94 of the gyrA gene; the novel mutation 1457 Câ†’Τ was found in the gyrB gene. Three mixed allelic variants were detected, which is a well-known phenomenon in areas with high TB and drug-resistant TB rates. The sensitivity and specificity of pyrosequencing (86.2 and 100%, respectively) and MTBDRsl (86.2 and 100%, respectively) were high; however, the results for 5.9% of the analyzed strains were unreadable when MTBDRsl was used. The MTBDRsl and pyrosequencing assays offer a rapid and accurate means for diagnosing resistance to FQs in high-TB-burden areas.

Typing Mycobacterium tuberculosis using variable number tandem repeat analysis.

DNA-based typing has contributed to the understanding of M. tuberculosis epidemiology and evolution. IS6110 RFLP was the first method described and has been used in many epidemiologic investigations. Technological difficulties have hampered the widespread establishment of this method, and it has been found to be of little use in evolutionary studies. PCR-based methods such as spoligotyping and variable number tandem repeat (VNTR) analysis largely overcome these difficulties. Spoligotyping alone is of limited value in epidemiologic investigations due to low discrimination but can be useful in evolutionary studies. Panels of VNTR loci selected from the 59 polymorphic VNTRs described to date have been shown to be useful in both epidemiologic and evolutionary studies. A VNTR type is identified by, first, amplifying a series of PCR fragments each encompassing a different VNTR locus and, second, determining the PCR fragment sizes from which the number of repeats present is calculated. The repeat number present at a series of loci is used as numerical code to describe a type. This chapter describes a high-throughput automated method for VNTR analysis at 15 loci using a capillary fragment analyzer and a manual method using agarose gel analysis.

The added value of a European Union tuberculosis reference laboratory network--analysis of the national reference laboratory activities.

National reference laboratories (NRL) and other laboratories are the cornerstones of well-functioning tuberculosis programmes and surveillance activities. However, the scope and activity of NRL services for mycobacterial identification and drug susceptibility testing (DST) has not been examined in detail across the European Union (EU), nor has the added value of cooperation and networking at the European level been explored with regard to strengthening laboratory services. Therefore, the European Centre for Disease Prevention and Control (ECDC) has commissioned a survey to explore these issues and to identify areas of work that could bring added value by supporting networking activities of tuberculosis (TB) reference laboratories in the EU. Structured questionnaires were sent to TB reference laboratory experts in the EU and European Economic Area (EEA) countries, and in three additional countries selected on the basis of their networking activities with EU projects and other initiatives (Switzerland, Croatia and Israel). The compiled results describe the activities and structure of 32 NRLs (29 countries replied, a response rate of 91%). The analysis of the survey led to the following recommendations for strengthening TB laboratory services: (1) implementing of the published European standards for TB laboratory services with respect to infrastructure, national reference functions, biosafety, human resources, quality assurance, operational research (including evaluation of new medical diagnostics), accuracy and speed, appropriately trained staff; (2) ensuring that laboratories only perform activities for which they have demonstrated proficiency; (3) implement validated and standardised second-line drug susceptibility testing (DST), including drugs used to define extensively drug-resistant tuberculosis (XDR TB); (4) aiming to identify Mycobacterium tuberculosis complex (MTBC) and rifampicin (RIF) resistance in over 90% of cultures and cases from smear-positive sputum directly within one to two working days. To realise some of the above recommendations and to strengthen links of TB surveillance and microbiology activities in the EU, a list of suggested generic areas of activities for an EU network of reference laboratories is presented. Such a network would build on and link to existing networks and initiatives at the European and global level.

Molecular epidemiology and prevalence of mutations conferring rifampicin and isoniazid resistance in Mycobacterium tuberculosis strains from the southern Ukraine.

Understanding the molecular epidemiology of tuberculosis (TB) and mutations in genes associated with drug resistance may contribute to the development of appropriate interventions to improve tuberculosis control. A structured questionnaire was used to collect basic epidemiological data from 589 patients with radiologically confirmed TB in the Odessa and Nikolaev regions of the Ukraine in 2003-2004. A non-commercial reverse hybridisation assay and DNA sequencing were used to detect mutations associated with rifampicin and isoniazid resistance. Genotyping was performed using multilocus variable number tandem repeat (VNTR) typing and spoligotyping. Mutations conferring rifampicin and isoniazid resistance were detected in 32.9% and 44.0%, respectively, of 225 Mycobacterium tuberculosis isolates from individual consecutive patients. Mutations in codon 531 and codon 315 of the rpoB and katG genes, respectively, were predominant among drug-resistant isolates. Multidrug (MDR) resistance rates were significantly higher among former prison inmates compared with non-prisoners (54.8% vs. 27.3%; RR 2.01; 95% CI 1.35-2.97) and the prevalence of mutations was higher in Beijing strains sharing the VNTR signature 223325173533424 than in other Beijing strains (71.4% vs. 45.7%; RR 1.74; 95% CI 1.17-2.57), suggesting that this group may be responsible for rapid transmission of MDR TB in the southern Ukraine.

The Directly Observed Therapy Short-Course (DOTS) strategy in Samara Oblast, Russian Federation.

BACKGROUND: The World Health Organisation (WHO) defines Russia as one of the 22 highest-burden countries for tuberculosis (TB). The WHO Directly Observed Treatment Short Course (DOTS) strategy employing a standardised treatment for 6 months produces the highest cure rates for drug sensitive TB. The Russian TB service traditionally employed individualised treatment. The purpose of this study was to implement a DOTS programme in the civilian and prison sectors of Samara Region of Russia, describe the clinical features and outcomes of recruited patients, determine the proportion of individuals in the cohorts who were infected with drug resistant TB, the degree to which resistance was attributed to the Beijing TB strain family and establish risk factors for drug resistance. METHODS: Prospective study. RESULTS: 2,099 patients were recruited overall. Treatment outcomes were analysed for patients recruited up to the third quarter of 2003 (n = 920). 75.3% of patients were successfully treated. Unsuccessful outcomes occurred in 7.3% of cases; 3.6% of patients died during treatment, with a significantly higher proportion of smear-positive cases dying compared to smear-negative cases. 14.0% were lost and transferred out. A high proportion of new cases (948 sequential culture-proven TB cases) had tuberculosis that was resistant to first-line drugs; (24.9% isoniazid resistant; 20.3% rifampicin resistant; 17.3% multidrug resistant tuberculosis). Molecular epidemiological analysis demonstrated that half of all isolated strains (50.7%; 375/740) belonged to the Beijing family. Drug resistance including MDR TB was strongly associated with infection with the Beijing strain (for MDR TB, 35.2% in Beijing strains versus 9.5% in non-Beijing strains, OR-5.2. Risk factors for multidrug resistant tuberculosis were: being a prisoner (OR 4.4), having a relapse of tuberculosis (OR 3.5), being infected with a Beijing family TB strain (OR 6.5) and having an unsuccessful outcome from treatment (OR 5.0). CONCLUSION: The implementation of DOTS in Samara, Russia, was feasible and successful. Drug resistant tuberculosis rates in new cases were high and challenge successful outcomes from a conventional DOTS programme alone.