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

Analysis of gene mutations associated with isoniazid, rifampicin and ethambutol resistance among Mycobacterium tuberculosis isolates from Ethiopia

<p>Abstract</p> <p>Background</p> <p>The emergence of drug resistance is one of the most important threats to tuberculosis control programs. This study was aimed to analyze the frequency of gene mutations associated with resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among <it>Mycobacterium tuberculosis </it>isolates from Northwest Ethiopia, and to assess the performance of the GenoType^®MTBDRplus and GenoType^®MTBDRsl assays as compared to the BacT/ALERT 3D system.</p> <p>Methods</p> <p>Two hundred sixty <it>Mycobacterium tuberculosis </it>isolates from smear positive tuberculosis patients diagnosed between March 2009 and July 2009 were included in this study. Drug susceptibility tests were performed in the Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University Hospital of Leipzig, Germany.</p> <p>Results</p> <p>Of 260 isolates, mutations conferring resistance to INH, RMP, or EMB were detected in 35, 15, and 8 isolates, respectively, while multidrug resistance (MDR) was present in 13 of the isolates. Of 35 INH resistant strains, 33 had mutations in the <it>katG </it>gene at Ser315Thr 1 and two strains had mutation in the <it>inhA </it>gene at C15T. Among 15 RMP resistant isolates, 11 had <it>rpoB </it>gene mutation at Ser531Leu, one at His526Asp, and three strains had mutations only at the wild type probes. Of 8 EMB resistant strains, two had mutations in the <it>embB </it>gene at Met306Ile, one at Met306Val, and five strains had mutations only at the wild type probes. The GenoType^®MTBDRplus assay had a sensitivity of 92% and specificity of 99% for INH resistance, and 100% sensitivity and specificity to detect RMP resistance and MDR. The GenoType^®MTBDRsl assay had a sensitivity of 42% and specificity of 100% for EMB resistance.</p> <p>Conclusion</p> <p>The dominance of single gene mutations associated with the resistance to INH and RMP was observed in the codon 315 of the <it>katG </it>gene and codon 531 of the <it>rpoB </it>gene, respectively. The GenoType^®MTBDRplus assay is a sensitive and specific tool for diagnosis of resistance to INH, RMP and MDR. However, the GenoType^®MTBDRsl assay shows limitations in detecting resistance to EMB.</p

Drug Resistant Mycobacterium tuberculosis of the Beijing Genotype Does Not Spread in Sweden

BACKGROUND: Drug resistant (DR) and multi-drug resistant (MDR) tuberculosis (TB) is increasing worldwide. In some parts of the world 10% or more of new TB cases are MDR. The Beijing genotype is a distinct genetic lineage of Mycobacterium tuberculosis, which is distributed worldwide, and has caused large outbreaks of MDR-TB. It has been proposed that certain lineages of M. tuberculosis, such as the Beijing lineage, may have specific adaptive advantages. We have investigated the presence and transmission of DR Beijing strains in the Swedish population. METHODOLOGY/PRINCIPAL FINDINGS: All DR M. tuberculosis complex isolates between 1994 and 2008 were studied. Isolates that were of Beijing genotype were investigated for specific resistance mutations and phylogenetic markers. Seventy (13%) of 536 DR strains were of Beijing genotype. The majority of the patients with Beijing strains were foreign born, and their country of origin reflects the countries where the Beijing genotype is most prevalent. Multidrug-resistance was significantly more common in Beijing strains than in non-Beijing strains. There was a correlation between the Beijing genotype and specific resistance mutations in the katG gene, the mabA-inhA-promotor and the rpoB gene. By a combined use of RD deletions, spoligotyping, IS1547, mutT gene polymorphism and Rv3135 gene analysis the Beijing strains could be divided into 11 genomic sublineages. Of the patients with Beijing strains 28 (41%) were found in altogether 10 clusters (2-5 per cluster), as defined by RFLP IS6110, while 52% of the patients with non-Beijing strains were in clusters. By 24 loci MIRU-VNTR 31 (45%) of the patients with Beijing strains were found in altogether 7 clusters (2-11 per cluster). Contact tracing established possible epidemiological linkage between only two patients with Beijing strains. CONCLUSIONS/SIGNIFICANCE: Although extensive outbreaks with non-Beijing TB strains have occurred in Sweden, Beijing strains have not taken hold, in spite of the proximity to high prevalence countries such as Russia and the Baltic countries. The Beijing sublineages so far introduced in Sweden may not be adapted to spread in the Scandinavian population

Pre-Existing Isoniazid Resistance, but Not the Genotype of Mycobacterium Tuberculosis Drives Rifampicin Resistance Codon Preference in Vitro

Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF)-resistant mutants of Mycobacterium tuberculosis. We characterised two series of spontaneous RIF-resistant in vitro mutants from isoniazid (INH)-sensitive and -resistant laboratory strains and clinical isolates, representing various M. tuberculosis genotypes. The first series were selected from multiple parallel 1 ml cultures and the second from single 10 ml cultures. RIF-resistant mutants were screened by Multiplex Ligation-dependent Probe Amplification (MLPA) or by sequencing the rpoB gene. For all strains the mutation rate for RIF resistance was determined with a fluctuation assay. The most striking observation was a shift towards rpoB-S531L (TCG→TTG) mutations in a panel of laboratory-generated INH-resistant mutants selected from the 10-ml cultures (p<0.001). All tested strains showed similar mutation rates (1.33×10−8 to 2.49×10−7) except one of the laboratory-generated INH mutants with a mutation rate measured at 5.71×10−7, more than 10 times higher than that of the INH susceptible parental strain (5.46–7.44×10−8). No significant, systematic difference in the spectrum of rpoB-mutations between strains of different genotypes was observed. The dramatic shift towards rpoB-S531L in our INH-resistant laboratory mutants suggests that the relative fitness of resistant mutants can dramatically impact the distribution of (subsequent) mutations that accumulate in a M. tuberculosis population, at least in vitro. We conclude that, against specific genetic backgrounds, certain resistance mutations are particularly likely to spread. Molecular screening for these (combinations of) mutations in clinical isolates could rapidly identify these particular pathogenic strains. We therefore recommend that isolates are screened for the distribution of resistance mutations, especially in regions that are highly endemic for (multi)drug resistant tuberculosis

Extremely high prevalence of multidrug resistant tuberculosis in Murmansk, Russia: a population-based study

Drug resistance and molecular epidemiology of tuberculosis (TB) in the Murmansk region was investigated in a 2-year, population-based surveillance of the civilian population. During 2003 and 2004, isolates from all culture-positive cases were collected (n = 1,226). Prevalence of multi-drug resistance (MDR) was extremely high, as 114 out of 439 new cases (26.0%), and 574 out of 787 previously treated cases (72.9%) were resistant to at least isoniazid (INH) and rifampin (RIF). Spoligotyping of the primary MDR-TB isolates revealed that most isolates grouped to the Beijing SIT1 genotype (n = 91, 79.8%). Isolates of this genotype were further analyzed by IS6110 RFLP. Sequencing of gene targets associated with INH and RIF resistance further showed that the MDR-TB strains are highly homogeneous as 78% of the MDR, SIT1 strains had the same resistance-conferring mutations. The genetic homogeneity of the MDR-TB strains indicates that they are actively transmitted in Murmansk</p

Tuberculosis in a southern Brazilian prison

The occurrence of tuberculosis (TB) in prisons has been described as an alarming public health problem in many countries, especially in developing nations. The objective of this study was to conduct a survey among prisoners with TB respiratory symptoms in order to estimate the incidence of the disease, to analyze the drug susceptibility profile and genotype the isolates of Mycobacterium tuberculosis in the city of Charqueadas, southern of Brazil. The TB incidence was 55/1,900 inhabitants in the prison; this corresponds to an incidence of 3,789/100,000 inhabitants, with a prevalence of 72/1,900 (4,960/100,000 inhabitants). Drug susceptibility test was performed and, among the analyzed isolates, 85% were susceptible to all drugs tested and 15% were resistant to at least one drug, of which 89% were resistant only to isoniazid (INH) or in combination with another drug. The genotype classification of spoligotyping analysis showed that 40% of the isolates belong to LAM family, 22% to T family, 17.5% to Haarlem family, 12.5% to U family and 3% to X family. The shared international spoligotypes most frequently found were 729 (27%), 50 (9.5%), 42 (8%), 53 (8%) and 863 (8%). In conclusion, it was observed that TB in this specific population had been caused, mostly, by strains that have been transmitted in the last few years, as demonstrated by the large level of genotype clustering. In addition, it was found specific large clusters, which were not often found in the general population from the same period and in the same region