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The purpose of this simple study was to characterize a panel of clinical isolates of Mycobacterium tuberculosis obtained from the Western Cape region of South Africa where new clinical vaccine trials are beginning, in the low dose aerosol guinea pig infection model. Most of the strains tested grew well in the lungs and other organs of these animals, and in most cases gave rise to moderate to very severe lung damage. We further observed that the current BCG vaccine was highly protective against two randomly selected strains, giving rise to significantly prolonged survival., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Children with presumed tuberculosis who are in contact with a multidrug-resistant source case should be treated according to the drug susceptibility of the source case's isolate. However, it is important to obtain a microbiologic diagnosis as it is possible for the child to have a different susceptibility profile to the source case. We present 2 such cases.

We report treatment outcomes from a retrospective cohort of patients with isoniazid-monoresistant tuberculosis in rural South Africa. Sixteen percent of patients had poor outcomes, 61% of whom progressed to multidrug-resistant tuberculosis. These data reveal the need for early identification and aggressive follow-up of isoniazid monoresistance to increase treatment success.

Despite numerous intervention strategies, including the direct observed short-course treatment strategy and improved diagnostic methods, the incidence of multidrug-resistant and extensively drug-resistant tuberculosis (TB) continues to rise globally. Many treatment policies are based on the model that acquisition of drug resistance in already infected individuals drives the drug-resistant TB epidemic, hence the focus on drug-resistance testing of retreatment cases. However, molecular epidemiology and mathematical modeling suggest that the majority of multidrug-resistant TB cases are due to ongoing transmission of multidrug-resistant strains. This is most likely the result of diagnostic delay, thereby emphasizing the need for rapid diagnostics and comprehensive contact tracing, as well as active case finding. Current diagnosis of TB in low-income, high-burden regions relies on smear microscopy and clinical signs and symptoms. However, this smear-centered approach has many pitfalls, including low sensitivity in HIV patients and children, the inability of smear to reveal drug-resistance patterns, and the need for sampling on consecutive days. In order to address these limitations, efforts have been made to expand access to Mycobacterium tuberculosis culture and drug susceptibility testing. However, the slow growth rate of the causative agent, M. tuberculosis, contributes to significant diagnostic delay. Molecular-based diagnostic methods, targeting mutations that are known to confirm drug resistance, are capable of significantly reducing diagnostic delay. Two such methods, the line-probe assay and the real-time PCR-based Xpert® MTB/RIF assay, have been described. The latter test shows particular promise for smear-negative and extrapulmonary specimens. This may prove especially useful in settings where co-infection rates with HIV are high. However, since most research focuses on the performance of both of these assays, further investigations need to be done regarding the impact of the routine implementation of these assays on TB control programs and the cost effectiveness thereof.

Recent developments in the field of TB diagnostics, including the introduction of the Xpert MTB/RIF assay in field testing, raise the hope for faster and more accurate identification of active TB patients. However, there are still many issues that need to be addressed as no point-of-care tests are yet available. Furthermore, no tests are available which are universally applicable to all patients. Improvements in the microbiological and molecular-based approaches are promising and the diagnostic pipeline is encouraging. Host markers associated with active disease may hold promise, especially in situations where sputum diagnostics are problematic, including in children, HIV-infected individuals and in the case of extrapulmonary TB.

Members of the Mycobacterium tuberculosis complex contain the transposable element IS6110 which, due to its high numerical and positional polymorphism, has become a widely used marker in epidemiological studies. Here, we review the evidence that IS6110 is not simply a passive or 'junk' DNA sequence, but that, through its transposable activity, it is able to generate genotypic variation that translates into strain-specific phenotypic variation. We also speculate on the role that this variation has played in the evolution of M. tuberculosis and conclude that the presence of a moderate IS6110 copy number within the genome may provide the pathogen with a selective advantage that has aided its virulence.

Anti-tuberculosis drugs are a two-edged sword. While they destroy pathogenic M. tuberculosis they also select for drug resistant bacteria against which those drugs are then ineffective. Global surveillance has shown that drug resistant Tuberculosis is widespread and is now a threat to tuberculosis control programs in many countries. Application of molecular methods during the last decade has greatly changed our understanding of drug resistance in tuberculosis. Application of molecular epidemiological methods was also central to the description of outbreaks of drug resistance in Tuberculosis. This review describes recommendations for Tuberculosis treatment according to the WHO guidelines, the drug resistance problem in the world, mechanisms of resistance to first line and second line drugs and applications of molecular methods to detect resistance causing gene mutations. It is envisaged that molecular techniques may be important adjuncts to traditional culture based procedures to rapidly screen for drug resistance. Prospective analysis and intervention to prevent transmission may be particularly helpful in areas with ongoing transmission of drug resistant strains as recent mathematical modeling indicate that the burden of MDR-TB cannot be contained in the absence of specific efforts to limit transmission.

In order to fully understand the global tuberculosis (TB) epidemic it is important to investigate the population structure and dissemination of the causative agent that drives the epidemic. Mycobacterium tuberculosis strain family 11 (F11) genotype isolates (found in 21.4% of all infected patients) are at least as successful as the Beijing genotype family isolates (16.5%) in contributing to the TB problem in some Western Cape communities of South Africa. This study describes key molecular characteristics that define the F11 genotype. A data-mining approach coupled with additional molecular analysis showed that members of F11 can easily and uniquely be identified by PCR-based techniques such as spoligotyping and dot blot screening for a specific rrs491 polymorphism. Isolates of F11 not only are a major contributor to the TB epidemic in South Africa but also are present in four different continents and at least 25 other countries in the world. Careful study of dominant compared to rare strains should provide clues to their success and possibly provide new ideas for combating TB.

Glutathione S-transferase (GST) and arylamine N-acetyltransferase 2 (NAT2) metabolise many environmental and chemotherapeutic agents, which influence susceptibility to disease. Polymorphisms in these enzymes result in different host phenotypes and contribute to different disease profiles or responses to toxic or chemotherapeutic agents, depending on their frequency in different populations. GST and NAT2 polymorphisms were investigated in different population groups, including African populations, and a range of allelic frequencies have been observed. The GSTM1 null genotype frequency, reported in this paper in two South African ethnic groups, is the lowest reported (0.19-0.21). In contrast, these same groups have a high GSTT1 null frequency (0.41-0.54), which is considerably higher than in African-Americans, or other Africans. The GSTT1 null frequency is comparable to the Chinese, a population with a very high oesophageal cancer incidence, similar to that in the African group. The frequency of the GSTPi Val105 variant in the South African Xhosas was also high (0.53), differing significantly from the low frequency in other Africans. These variants could therefore be associated with high cancer susceptibility. In addition, the high proportion of NAT2 "fast" alleles may partially explain the high tuberculosis prevalence in South Africans, due to reduced isoniazid efficacy in the presence of rapid acetylation.

BackgroundGrowth-based drug susceptibility testing (DST) is the reference standard for diagnosing drug-resistant tuberculosis (TB), but standard time to result (TTR) is typically ≥ 3 weeks. Rapid tests can reduce that TTR to days or hours, but accuracy may be lowered. In addition to the TTR and test accuracy, the cost of a diagnostic test may affect whether it is adopted in clinical settings. We examine the cost-effectiveness of rapid diagnostics for extremely drug-resistant TB (XDR-TB) in three different high-prevalence settings.Methods1128 patients with confirmed TB were enrolled at clinics in Mumbai, India; Chisinau, Moldova; and Port Elizabeth, South Africa. Patient sputum samples underwent DST for first and second line TB drugs using 2 growth-based (MGIT, MODS) and 2 molecular (Pyrosequencing [PSQ], line-probe assays [LPA]) assays. TTR was the primary measure of effectiveness. Sensitivity and specificity were also evaluated. The cost to perform each test at each site was recorded and included test-specific materials, personnel, and equipment costs. Incremental cost-effectiveness ratios were calculated in terms of $/day saved. Sensitivity analyses examine the impact of batch size, equipment, and personnel costs.ResultsOur prior results indicated that the LPA and PSQ returned results in a little over 1 day. Mean cost per sample without equipment or overhead was $23, $28, $33, and $41 for the MODS, MGIT, PSQ, and LPA, respectively. For diagnosing XDR-TB, MODS was the most accurate, followed by PSQ, and LPA. MODS was quicker and less costly than MGIT. PSQ and LPA were considerably faster but cost more than MODS. Batch size and personnel costs were the main drivers of cost variation.ConclusionsMultiple factors must be weighed when selecting a test for diagnosis of XDR-TB. Rapid tests can greatly improve the time required to diagnose drug-resistant TB, potentially improving treatment success, and preventing the spread of XDR-TB. Faster time to result must be weighed against the potential for reduced accuracy, and increased costs.Trial registrationClinicalTrials.gov Identifier: NCT02170441 .

BackgroundRapid molecular diagnostics, with their ability to quickly identify genetic mutations associated with drug resistance in Mycobacterium tuberculosis clinical specimens, have great potential as tools to control multi- and extensively drug-resistant tuberculosis (M/XDR-TB). The Qiagen PyroMark Q96 ID system is a commercially available pyrosequencing (PSQ) platform that has been validated for rapid M/XDR-TB diagnosis. However, the details of the assay's diagnostic and technical performance have yet to be thoroughly investigated in diverse clinical environments.MethodsThis study evaluates the diagnostic performance of the PSQ assay for 1128 clinical specimens from patients from three areas of high TB burden. We report on the diagnostic performance of the PSQ assay between the three sites and identify variables associated with poor PSQ technical performance.ResultsIn India, the sensitivity of the PSQ assay ranged from 89 to 98 % for the detection of phenotypic resistance to isoniazid, rifampicin, fluoroquinolones, and the injectables. In Moldova, assay sensitivity ranged from 7 to 94 %, and in South Africa, assay sensitivity ranged from 71 to 92 %. Specificity was high (94-100 %) across all sites. The addition of eis promoter sequencing information greatly improved the sensitivity of kanamycin resistance detection in Moldova (7 % to 79 %). Nearly all (89.4 %) sequencing reactions conducted on smear-positive, culture-positive specimens and most (70.8 %) reactions conducted on smear-negative, culture-positive specimens yielded valid PSQ reads. An investigation into the variables influencing sequencing failures indicated smear negativity, culture negativity, site (Moldova), and sequencing of the rpoB, gyrA, and rrs genes were highly associated with poor PSQ technical performance (adj. OR > 2.0).ConclusionsThis study has important implications for the global implementation of PSQ as a molecular TB diagnostic, as it demonstrates how regional factors may impact PSQ diagnostic performance, while underscoring potential gene targets for optimization to improve overall PSQ assay technical performance.Trial registrationClinicalTrials.gov ( #NCT02170441 ). Registered 12 June 2014.

Accurate identification of drug-resistantMycobacterium tuberculosisis imperative for effective treatment and subsequent reduction in disease transmission. Line probe assays rapidly detect mutations associated with resistance and wild-type sequences associated with susceptibility. Examination of molecular-level performance is necessary for improved assay result interpretation and for continued diagnostic development. Using data collected from a large, multisite diagnostic study, probe hybridization results from line probe assays, MTBDRplusand MTBDRsl, were compared to those of sequencing, and the diagnostic performance of each individual mutation and wild-type probe was assessed. Line probe assay results classified as resistant due to the absence of wild-type probe hybridization were compared to those of sequencing to determine if novel mutations were inhibiting wild-type probe hybridization. The contribution of absent wild-type probe hybridization to the detection of drug resistance was assessed via comparison to a phenotypic reference standard. In our study, mutation probes demonstrated significantly higher specificities than wild-type probes and wild-type probes demonstrated marginally higher sensitivities than mutation probes, an ideal combination for detecting the presence of resistance conferring mutations while yielding the fewest number of false-positive results. The absence of wild-type probe hybridization without mutation probe hybridization was determined to be primarily the result of failure of mutation probe hybridization and not the result of novel or rare mutations. Compared to phenotypic culture-based drug susceptibility testing, the absence of wild-type probe hybridization without mutation probe hybridization significantly contributed to the detection of phenotypic rifampin and fluoroquinolone resistance with negligible increases in false-positive results.

Reliable molecular diagnostics, which detect specific mutations associated with drug resistance, are promising technologies for the rapid identification and monitoring of drug resistance in Mycobacterium tuberculosis isolates. Pyrosequencing (PSQ) has the ability to detect mutations associated with first- and second-line anti-tuberculosis (TB) drugs, with the additional advantage of being rapidly adaptable for the identification of new mutations. The aim of this project was to evaluate the performance of PSQ in predicting phenotypic drug resistance in multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) clinical isolates from India, South Africa, Moldova, and the Philippines. A total of 187 archived isolates were run through a PSQ assay in order to identify M. tuberculosis (via the IS6110 marker), and to detect mutations associated with M/XDR-TB within small stretches of nucleotides in selected loci. The molecular targets included katG, the inhA promoter and the ahpC-oxyR intergenic region for isoniazid (INH) resistance; the rpoB core region for rifampin (RIF) resistance; gyrA for fluoroquinolone (FQ) resistance; and rrs for amikacin (AMK), capreomycin (CAP), and kanamycin (KAN) resistance. PSQ data were compared to phenotypic mycobacterial growth indicator tube (MGIT) 960 drug susceptibility testing results for performance analysis. The PSQ assay illustrated good sensitivity for the detection of resistance to INH (94%), RIF (96%), FQ (93%), AMK (84%), CAP (88%), and KAN (68%). The specificities of the assay were 96% for INH, 100% for RIF, FQ, AMK, and KAN, and 97% for CAP. PSQ is a highly efficient diagnostic tool that reveals specific nucleotide changes associated with resistance to the first- and second-line anti-TB drug medications. This methodology has the potential to be linked to mutation-specific clinical interpretation algorithms for rapid treatment decisions.

BackgroundThe aim of this study was to compare the performance of several recently developed assays for the detection of multi- and extensively drug-resistant tuberculosis (M/XDR-TB) in a large, multinational field trial.MethodsSamples from 1,128 M/XDR-TB suspects were examined by Line Probe Assay (LPA), Pyrosequencing (PSQ), and Microscopic Observation of Drug Susceptibility (MODS) and compared to the BACTEC MGIT960 reference standard to detect M/XDR-TB directly from patient sputum samples collected at TB clinics in India, Moldova, and South Africa.ResultsSpecificity for all three assays was excellent: 97-100% for isoniazid (INH), rifampin (RIF), moxifloxacin (MOX) and ofloxacin (OFX) and 99-100% for amikacin (AMK), capreomycin (CAP) and kanamycin (KAN) resistance. Sensitivities were lower, but still very good: 94-100% for INH, RIF, MOX and OFX, and 84-90% for AMK and CAP, but only 48-62% for KAN. In terms of agreement, statistically significant differences were only found for detection of RIF (MODS outperformed PSQ) and KAN (MODS outperformed LPA and PSQ) resistance. Mean time-to-result was 1.1 days for LPA and PSQ, 14.3 days for MODS, and 24.7 days for MGIT.ConclusionsAll three rapid assays evaluated provide clinicians with timely detection of resistance to the drugs tested; with molecular results available one day following laboratory receipt of samples. In particular, the very high specificity seen for detection of drug resistance means that clinicians can use the results of these rapid tests to avoid the use of toxic drugs to which the infecting organism is resistant and develop treatment regiments that have a higher likelihood of yielding a successful outcome.

BackgroundDrug-resistant tuberculosis (DR-TB) remains a threat to global public health, owing to the complexity and delay of diagnosis and treatment. The Global Consortium for Drug-resistant Tuberculosis Diagnostics (GCDD) was formed to develop and evaluate assays designed to rapidly detect DR-TB, so that appropriate treatment might begin more quickly. This paper describes the methodology employed in a prospective cohort study for head-to-head assessment of three different rapid diagnostic tools.MethodsSubjects at risk of DR-TB were enrolled from three countries. Data were gathered from a combination of patient interviews, chart reviews, and laboratory testing from each site's reference laboratory. The primary outcome of interest was reduction in time from specimen arrival in the laboratory to results of rapid drug susceptibility tests, as compared with current standard mycobacterial growth indicator tube (MGIT) drug susceptibility tests.ResultsSuccessful implementation of the trial in diverse multinational populations is explained, in addition to challenges encountered and recommendations for future studies with similar aims or populations.ConclusionsThe GCDD study was a head-to-head study of multiple rapid diagnostic assays aimed at improving accuracy and precision of diagnostics and reducing overall time to detection of DR-TB. By conducting a large prospective study, which captured epidemiological, clinical, and biological data, we have produced a high-quality unique dataset, which will be beneficial for analyzing study aims as well as answering future DR-TB research questions. Reduction in detection time for XDR-TB would be a major public health success as it would allow for improved treatment and more successful patient outcomes. Executing successful trials is critical in assessment of these reductions in highly variable populations.Trial registrationClinicalTrials.gov NCT02170441.

Molecular diagnostic methods based on the detection of mutations conferring drug resistance are promising technologies for rapidly detecting multidrug-/extensively drug-resistant tuberculosis (M/XDR TB), but large studies of mutations as markers of resistance are rare. The Global Consortium for Drug-Resistant TB Diagnostics analyzed 417 Mycobacterium tuberculosis isolates from multinational sites with a high prevalence of drug resistance to determine the sensitivities and specificities of mutations associated with M/XDR TB to inform the development of rapid diagnostic methods. We collected M/XDR TB isolates from regions of high TB burden in India, Moldova, the Philippines, and South Africa. The isolates underwent standardized phenotypic drug susceptibility testing (DST) to isoniazid (INH), rifampin (RIF), moxifloxacin (MOX), ofloxacin (OFX), amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) using MGIT 960 and WHO-recommended critical concentrations. Eight genes (katG, inhA, rpoB, gyrA, gyrB, rrs, eis, and tlyA) were sequenced using Sanger sequencing. Three hundred seventy isolates were INHr, 356 were RIFr, 292 were MOXr/OFXr, 230 were AMKr, 219 were CAPr, and 286 were KANr. Four single nucleotide polymorphisms (SNPs) in katG/inhA had a combined sensitivity of 96% and specificities of 97 to 100% for the detection of INHr. Eleven SNPs in rpoB had a combined sensitivity of 98% for RIFr. Eight SNPs in gyrA codons 88 to 94 had sensitivities of 90% for MOXr/OFXr. The rrs 1401/1484 SNPs had 89 to 90% sensitivity for detecting AMKr/CAPr but 71% sensitivity for KANr. Adding eis promoter SNPs increased the sensitivity to 93% for detecting AMKr and to 91% for detecting KANr. Approximately 30 SNPs in six genes predicted clinically relevant XDR-TB phenotypes with 90 to 98% sensitivity and almost 100% specificity.

Background: South Africa shows one of the highest global burdens of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). Since 2002, MDR-TB in South Africa has been treated by a standardized combination therapy, which until 2010 included ofloxacin, kanamycin, ethionamide, ethambutol and pyrazinamide. Since 2010, ethambutol has been replaced by cycloserine or terizidone. The effect of standardized treatment on the acquisition of XDR-TB is not currently known. Methods: We genetically characterized a random sample of 4,667 patient isolates of drug-sensitive, MDR and XDR-TB cases collected from three South African provinces, namely, the Western Cape, Eastern Cape and KwaZulu-Natal. Drug resistance patterns of a subset of isolates were analyzed for the presence of commonly observed resistance mutations. Results: Our analyses revealed a strong association between distinct strain genotypes and the emergence of XDR-TB in three neighbouring provinces of South Africa. Strains predominant in XDR-TB increased in proportion by more than 20-fold from drug-sensitive to XDR-TB and accounted for up to 95% of the XDR-TB cases. A high degree of clustering for drug resistance mutation patterns was detected. For example, the largest cluster of XDR-TB associated strains in the Eastern Cape, affecting more than 40% of all MDR patients in this province, harboured identical mutations concurrently conferring resistance to isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, ethionamide, kanamycin, amikacin and capreomycin. Conclusions: XDR-TB associated genotypes in South Africa probably were programmatically selected as a result of the standard treatment regimen being ineffective in preventing their transmission. Our findings call for an immediate adaptation of standard treatment regimens for M/XDR-TB in South Africa. [ABSTRACT FROM AUTHOR]

Genetically related Mycobacterium tuberculosis strains with alterations at codon 516 in the rpoB gene were observed amongst a substantial number of patients with drug resistant tuberculosis in the Eastern Cape Province (ECP) of South Africa. Mutations at codon 516 are usually associated with lower level rifampicin (RIF) resistance, while susceptibility to rifabutin (RFB) remains intact. This study was conducted to assess the rationale for using RFB as a substitution for RIF in the treatment of MDR and XDR tuberculosis outbreaks. Minimum inhibitory concentrations (MICs) of 34 drug resistant clinical isolates of M tuberculosis were determined by MGIT 960 and correlated with rpoB mutations. RFB MICs ranged from 0.125 to 0.25 µg/ml in the 34 test isolates thereby confirming phenotypic susceptibility as per critical concentration (CC) of 0.5 µg/ml. The corresponding RIF MICs ranged between 5 and 15 µg/ml, which is well above the CC of 1.0 µg/ml. Molecular-based drug susceptibility testing provides important pharmacogenetic insight by demonstrating a direct correlation between defined rpoB mutation and the level of RFB susceptibility. We suggest that isolates with marginally reduced susceptibility as compared to the epidemiological cut-off for wild-type strains (0.064 µg/ml), but lower than the current CC (≤0.5 µg/ml), are categorised as intermediate. Two breakpoints (0.064 µg/ml and 0.5 µg/ml) are recommended to distinguish between susceptible, intermediate and RFB resistant strains. This concept may assist clinicians and policy makers to make objective therapeutic decisions, especially in situations where therapeutic options are limited. The use of RFB in the ECP may improve therapeutic success and consequently minimise the risk of ongoing transmission of drug resistant M. tuberculosis strains. [ABSTRACT FROM AUTHOR]

Drug-resistant tuberculosis is a threat to tuberculosis control programs and community health. This growing problem mirrors the increasing incidence of tuberculosis in general. Public health problems include the absence of early diagnosis and effective treatment. The real need is to identify tuberculosis patients far earlier, particularly those with drug-resistant strains, and to begin appropriate therapy, which is of the shortest possible duration with minimal risk of acquiring further drug resistance or permitting further transmission. This article will address the epidemic of drug resistance and discuss some of the inherent difficulties in the treatment of drug-resistant tuberculosis. We highlight some of the controversies and new findings in this area, as well as future perspectives requiring more active interventions, in addition to new technology and developments. [ABSTRACT FROM AUTHOR]

Management of Tuberculosis is complicated by the emergence of drug resistant strains of Mycobacterium tuberculosis and this poses a threat to the success of Tuberculosis control programmes. Drug susceptibility testing by culture is time-consuming and technically difficult. It is known that resistance to drugs is due to a number of genomic mutations in specific genes of M. tuberculosis . These mutations in combination with molecular techniques can be used as markers for drug resistance, since drug susceptible isolates lack the corresponding gene mutations. This review focuses on molecular mechanisms, methods and applications as a possible new diagnostic tool for the early molecular detection of drug resistance in M. tuberculosis . [ABSTRACT FROM AUTHOR]

A letter to the editor is presented which investigates the association of Rv2629 191A/C nucleotide change with rifampicin resistance in Mycobacterium tuberculosis.

A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds. [Copyright &y& Elsevier]