Discriminatory ability of hypervariable variable number tandem repeat loci in population-based analysis of Mycobacterium tuberculosis strains, London, UK

To address conflicting results about the stability of variable number tandem repeat (VNTR) loci and their value in prospective molecular epidemiology of Mycobacterium tuberculosis, we conducted a large prospective population-based analysis of all M. tuberculosis strains in a metropolitan setting. Optimal and reproducible conditions for reliable PCR and fragment analysis, comprising enzymes, denaturing conditions, and capillary temperature, were identified for a panel of hypervariable loci, including 3232, 2163a, 1982, and 4052. A total of 2,261 individual M. tuberculosis isolates and 265 sets of serial isolates were analyzed by using a standardized 15-loci VNTR panel, then an optimized hypervariable loci panel. The discriminative ability of loci varied substantially; locus VNTR 3232 varied the most, with 19 allelic variants and Hunter-Gaston index value of 0.909 . Hypervariable loci should be included in standardized panels because they can provide consistent comparable results at multiple settings, provided the proposed conditions are adhered to

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

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

Survival of drug resistant tuberculosis patients in Lithuania: retrospective national cohort study

The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement FP7-223681.

An Integrated Approach to Rapid Diagnosis of Tuberculosis and Multidrug Resistance Using Liquid Culture and Molecular Methods in Russia

Objective: To analyse the feasibility, cost and performance of rapid tuberculosis (TB) molecular and culture systems, in a high multidrug-resistant TB (MDR TB) middle-income region (Samara, Russia) and provide evidence for WHO policy change. Methods: Performance and cost evaluation was conducted to compare the BACTEC™ MGIT™ 960 system for culture and drug susceptibility testing (DST) and molecular systems for TB diagnosis, resistance to isoniazid and rifampin, and MDR TB identification compared to conventional Lowenstein-Jensen culture assays. Findings: 698 consecutive patients (2487 sputum samples) with risk factors for drug-resistant tuberculosis were recruited. Overall M. tuberculosis complex culture positivity rates were 31.6% (787/2487) in MGIT and 27.1% (675/2487) in LJ (90.5% and 83.2% for smear-positive specimens). In total, 809 cultures of M. tuberculosis complex were isolated by any method. Median time to detection was 14 days for MGIT and 36 days for LJ (10 and 33 days for smear positive specimens) and indirect DST in MGIT took 9 days compared to 21 days on LJ. There was good concordance between DST on LJ and MGIT (96.8% for rifampin and 95.6% for isoniazid). Both molecular hybridization assay results correlated well with MGIT DST results, although molecular assays generally yielded higher rates of resistance (by approximately 3% for both isoniazid and rifampin). Conclusion: With effective planning and logistics, the MGIT 960 and molecular based methodologies can be successfully introduced into a reference laboratory setting in a middle incidence country. High rates of MDR TB in the Russian Federation make the introduction of such assays particularly useful. © 2009 Balabanova et al

Performance of the Genotype® MTBDRPlus assay in the diagnosis of tuberculosis and drug resistance in Samara, Russian Federation

<p>Abstract</p> <p>Background</p> <p>Russia is a high tuberculosis (TB) burden country with a high prevalence of multidrug resistant tuberculosis (MDRTB). Molecular assays for detection of MDRTB on clinical specimens are not widely available in Russia.</p> <p>Results</p> <p>We performed an evaluation of the GenoType^®MTBDRplus assay (HAIN Lifescience GmbH, Germany) on a total of 168 sputum specimens from individual patients at a public health laboratory in Central Russia, as a model of a middle income site in a region with high levels of drug resistance. Phenotypic drug resistance tests (DST) were performed on cultures derived from the same sputum specimens using the BACTEC 960 liquid media system.</p> <p>Interpretable GenoType^®MTBDRplus results were obtained for 154(91.7%) specimens with readability rates significantly higher in sputum specimens graded 2+ and 3+ compared to 1+ (RR = 1.17 95%CI 1.04–1.32). The sensitivity and specificity of the assay for the detection of rifampicin (RIF) and isoniazid (INH) resistance and MDR was 96.2%, 97.4%, 97.1% and 90.7%, 83.3%, 88.9% respectively. Mutations in codon 531 of the <it>rpoB </it>gene and codon 315 of the <it>katG </it>gene dominated in RIF and INH resistant strains respectively. Disagreements between phenotypical and molecular tests results (12 samples) could be explained by the presence of rare mutations in strains circulating in Russia and simultaneous presence of resistant and sensitive bacilli in sputum specimens (heteroresistance).</p> <p>Conclusion</p> <p>High sensitivity, short turnaround times and the potential for screening large numbers of specimens rapidly, make the GenoType^®MTBDRplus assay suitable as a first-line screening assay for drug resistant TB.</p

Tuberculosis in UK cities: workload and effectiveness of tuberculosis control programmes

<p>Abstract</p> <p>Background</p> <p>Tuberculosis (TB) has increased within the UK and, in response, targets for TB control have been set and interventions recommended. The question was whether these had been implemented and, if so, had they been effective in reducing TB cases.</p> <p>Methods</p> <p>Epidemiological data were obtained from enhanced surveillance and clinics. Primary care trusts or TB clinics with an average of > 100 TB cases per year were identified and provided reflections on the reasons for any change in their local incidence, which was compared to an audit against the national TB plan.</p> <p>Results</p> <p>Access to data for planning varied (0-22 months). Sputum smear status was usually well recorded within the clinics. All cities had TB networks, a key worker for each case, free treatment and arrangements to treat HIV co-infection. Achievement of targets in the national plan correlated well with change in workload figures for the commissioning organizations (Spearman's rank correlation R = 0.8, P < 0.01) but not with clinic numbers. Four cities had not achieved the target of one nurse per 40 notifications (Birmingham, Bradford, Manchester and Sheffield). Compared to other cities, their loss to follow-up during treatment was usually > 6% (χ²= 4.2, P < 0.05), there was less TB detected by screening and less outreach. Manchester was most poorly resourced and showed the highest rate of increase of TB. Direct referral from radiology, sputum from primary care and outreach workers were cited as important in TB control.</p> <p>Conclusion</p> <p>TB control programmes depend on adequate numbers of specialist TB nurses for early detection and case-holding.</p> <p>Please see related article: <url>http://www.biomedcentral.com/1741-7015/9/127</url></p

Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance: a retrospective cohort study

Background Diagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drug-susceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistance-determining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all first-line and second-line drugs for tuberculosis. Methods Between Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identified from the drug-resistance scientific literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance. Findings We characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7–93·7) and 98·4% specificity (98·1–98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identified among mutations under selection pressure in non-candidate genes. Interpretation A broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workflows, phasing out phenotypic drug-susceptibility testing while reporting drug resistance early

Genetic variation of Mycobacterium tuberculosis circulating in Kharkiv Oblast, Ukraine

<p>Abstract</p> <p>Background</p> <p>A persistent increase of tuberculosis cases has recently been noted in the Ukraine. The reported incidence of drug-resistant isolates of <it>M. tuberculosis </it>is growing steadily; however, data on the genetic variation of isolates of <it>M. tuberculosis </it>circulating in northern Ukraine and on the spectrum and frequency of occurrence of mutations determining resistance to the principal anti-tuberculosis drugs isoniazid and rifampicin have not yet been reported.</p> <p>Methods</p> <p>Isolates of <it>M. tuberculosis </it>from 98 tuberculosis patients living in Kharkiv Oblast (Ukraine) were analyzed using VNTR- and RFLP-IS6110-typing methods. Mutations associated with resistance to rifampicin and isoniazid were detected by RFLP-PCR methods, and also confirmed by sequencing.</p> <p>Results</p> <p>We identified 75 different genetic profiles. Thirty four (34%) isolates belonged to the Beijing genotype and 23 (23%) isolates belonged to the LAM family. A cluster of isolates belonging to the LAM family had significant genetic heterogeneity, indicating that this family had an ancient distribution and circulation in this geographical region. Moreover, we found a significant percentage of the isolates (36%) belonged to as yet unidentified families of <it>M. tuberculosis </it>or had individual non-clustering genotypes. Mutations conferring rifampicin and isoniazid resistance were detected in 49% and 54% isolates, respectively. Mutations in codon 531 of the <it>rpoB </it>gene and codon 315 of the <it>katG </it>gene were predominant among drug-resistant isolates. An association was found for belonging to the LAM strain family and having multiple drug resistance (R = 0.27, p = 0.0059) and also for the presence of a mutation in codon 531 of the <it>rpoB </it>gene and belonging to the Beijing strain family (R = 0.2, p = 0.04).</p> <p>Conclusions</p> <p>Transmission of drug-resistant isolates seems to contribute to the spread of resistant TB in this oblast. The Beijing genotype and LAM genotype should be seen as a major cause of drug resistant TB in this region.</p