Multidrug-resistant Mycobacterium Tuberculosis Clinical Isolates Research Articles

Evaluation of the accuracy of the microplate alamar blue assay and the proportion method for the prompt detection of Mycobacterium tuberculosis and susceptibility of multidrug-resistant Mycobacterium tuberculosis clinical isolates

Aims and objectives: Tuberculosis (TB) is estimated to have caused the deaths of 1 billion people in the last 200 years. Methods: In the first step, the microplate alamar blue assay (MABA) was used for the detection of seventy-eight clinical isolates from Golestan Regional Tuberculosis Reference Laboratory (GRTRL) and the results were compared with the proportion method. In second step, the MABA was used for the susceptibility testing of 35 clinical isolates and the results were compared with the proportion method. Results: For the MABA, the sensitivity was 100 (90.97-100), specificity was 74.36 (57.87-86.96), positive predictive value (PPV) was 79.59 (65.66-89.76), negative predictive value (NPV) was 100 (88.06-100). The concentrations of isoniazid (INH) and rifampicin (RMP) ranged from 1.0-0.01 μg.mL and 2.0-0.03 μg.mL, respectively. For the proportion method, the INH concentration was between 1.0-0.03 μg.mL and the RMP concentration was between 2.0-0.06 μg.mL. For the MABA to RMP, the sensitivity was 100 (89.11-100), specificity was 100 (29.24-100), PPV was 100 (89.11-100), NPV was 100 (29.24-100), and for the MABA to INH, the sensitivity was 84.38 (67.21-94.72), specificity was 66.67 (9.43-99.16), PPV was 96.43 (81.65-99.91), NPV was 28.57 (3.67-70.96). Conclusion: We found the high accuracy between the MABA-RMP and the proportion method. In fact, the rapid and low-cost MABA assay is inexpensive and alternative assays for the detection of RMP resistant tuberculosis in low-income countries.

Phenotypic and Genotypic Characterization of Streptomycin-Resistant Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates in Southern China.

Streptomycin (STR) is the first antibiotic used in the treatment of tuberculosis (TB) and the earliest antituberculosis drug with acquired resistance developed by Mycobacterium tuberculosis. The high prevalence of such resistance in many parts of the world limits its use for treating multidrug-resistant (MDR) TB. The aims of this study are to characterize of mutations in rpsL, rrs, and gidB genes in MDR M. tuberculosis isolates originating from southern China and to investigate possible relationship between mutations and strain genotypes for precise diagnosis and treatment. Sequences of rpsL, rrs, and gidB genes and the resistance profiles were analyzed for 218 MDR M. tuberculosis isolates. Our study showed that 68.35% of MDR M. tuberculosis isolates were resistant to STR and 89.91% of STR-resistant (STRR) isolates were Beijing lineage strains. Mutations were observed in STRR MDR M. tuberculosis isolates at the following rates: 72.48% in rpsL, 36.91% in rrs, and 15.44% in gidB. Compared with the phenotypic data, the combination of mutations in rpsL, rrs, and gidB has sensitivity and specificity of 96.64% and 100.00%, respectively. The most common mutations in STRR isolates were rpsL128,263 and rrs514,1401, of which rpsL128 showed association with Beijing lineage (p < 0.001). It is noteworthy that a1401g mutation was present in rrs, while MDR M. tuberculosis isolates were resistant to both STR and amikacin. Twenty two novel mutations were found in STRR isolates. These findings could be helpful to develop rapid molecular diagnostic methods and understand STR resistance in China for developing TB precision medicine and disturbance of drug-resistant TB transmission.

Detection of Novel Gene Mutations Associated with Pyrazinamide Resistance in Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates in Southern China.

ObjectivePyrazinamide (PZA) is a cornerstone of modern tuberculosis regimens. This study aimed to investigate the performance of genotypic testing of pncA+ upstream region, rpsA, panD, Rv2783c, and clpC1 genes to add insights for more accurate molecular diagnosis of PZA-resistant (R) Mycobacterium tuberculosis.MethodsDrug susceptibility testing, sequencing analysis of PZA-related genes including the entire operon of pncA (Rv2044c-pncA-Rv2042c) and PZase assay were performed for 448 M. tuberculosis clinical isolates.ResultsOur data showed that among 448 M. tuberculosis clinical isolates, 113 were MDR, 195 pre-XDR and 70 XDR TB, while the remaining 70 strains had other combinations of drug-resistance. A total of 60.04% (269/448) M. tuberculosis clinical isolates were resistant to PZA, of which 78/113 were MDR, 119/195 pre-XDR and 29/70 XDR TB strains. PZAR isolates have predominance (83.3%) of Beijing genotype. Genotypic characterization of Rv2044c-pncA-Rv2042c revealed novel nonsynonymous mutations in Rv2044c with negative PZase activity which led to confer PZAR. Compared with phenotypic data, 84.38% (227/269) PZAR strains with mutations in pncA+ upstream region exhibited 83.64% sensitivity but the combined evaluation of the mutations in rpsA 2.60% (7/269), panD 1.48% (4/269), Rv2783c 1.11% (3/269) and Rv2044c 0.74% (2/269) increased the sensitivity to 89.59%. Fifty-seven novel mutations were identified in this study. Interestingly, a frameshift deletion (C−114del) in upstream of pncAwt nullified the effect of A−11G mutation and induced positive PZase activity, divergent from five PZase negative A−11G PZAR mutants. Twenty-six PZAR strains having wild-type-sequenced genes with positive or negative PZase suggest the existence of unknown resistance mechanisms.ConclusionOur study revealed that PZAR rate in MDR and pre-XDR TB was markedly higher in southern China. The concomitant evaluation of pncA+ UFR, rpsA, panD, Rv2783c, and Rv2044c provides more dependable genotypic results of PZA resistance. Fifty-seven novel mutations/indels in this study may play a vital role as diagnostic markers. The upstream region of pncA and PZase regulation are valuable to explore the unknown mechanism of PZA-resistance.

Phenotypic and genotypic characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis clinical isolates in Hangzhou, China

ObjectivesPyrazinamide (PZA) is a crucial first-line tuberculosis (TB) drug recommended for both drug-susceptible and multidrug-resistant Mycobacterium tuberculosis. This study aimed to evaluate the performance of the sequencing method of pncA, rpsA and panD mutations in detecting PZA resistance in multidrug-resistant (MDR) TB isolates. MethodsWe sequenced the pncA, rpsA and panD genes and performed PZA susceptibility tests across 291 MDR-TB isolates to evaluate the performance of the sequencing method of these genes in detecting PZA resistance. ResultsResults showed that 145 (90.0%) of 161 PZA phenotypic resistant isolates had mutations in pncA. Among the 16 isolates (10.0%) which did not have mutations in pncA, ten and three isolates had mutations in rpsA and panD, respectively. The sequencing method for detecting mutations in pncA alone had 90.1% (95% confidence interval (CI), 84.4–94.2) sensitivity and 92.3% (95% CI, 86.3–96.3) specificity. The combination of all three genes increased the sensitivity from 90.1% (95% CI, 84.4–94.2) to 98.1% (95% CI, 94.7–99.6) (p < 0.001) while the specificity remained unchanged. In 120 PZA-susceptible and 16 PZA-resistant isolates without pncA mutations, rpsA/panD mutations were correlated with PZA resistance. ConclusionsPZA resistance was largely associated with mutations in pncA. Mutations in rpsA and panD were also associated with PZA resistance in MDR isolates expressing wild-type pncA. The detection of mutations in pncA, rpsA and panD can be useful for the determination of PZA resistance.

Identification and Analysis of Mutations in the KatG Gene in Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates

A major role in the development of resistance of Mycobacterium tuberculosis to isoniazid (INH) is attributed to mutations in the katG gene coding for the catalase/peroxidase, an enzyme required for obtaining a pharmacologically active form of the drug. Analysis of mutations in the katG gene in M. tuberculosis strains may contribute to the development of reliable and rapid tests for detection of INH resistance. The aim of the study was to identify and characterize mutations in the katG gene in multidrug-resistant M. tuberculosis clinical isolates. The study included 46 strains of M. tuberculosis, recovered from MDR-TB patients in Poland in 2004. Mutations in the katG gene were detected by comparing DNA sequences with the corresponding sequence of a wild-type reference laboratory strain (M. tuberculosis H37Rv). The obtained results were interpreted in the context of MIC values of INH and catalase activity of the strains tested. A total of 43 (93%) strains contained mutations in the katG gene. The most frequently observed were mutations at codon 315, found in 34 (74%) strains. Mutations at other codons were rare: 4 strains contained mutations at codon 463, 2 at codon 131 and another 2 at codon 234. Mutations at codons 68, 91, 101, 126, 128 and 194 were found in single strains only. Two strains, for which no mutations at codon 315 of the katG gene were identified, had a unique translation termination mutation, which would invariably result in polypeptide truncation leading to the generation of dysfunctional catalase polypeptides. Both these strains presented the highest MIC values for INH (80 and 100 μg/mL) and showed a complete loss of catalase activity. For the remaining 41 strains with katG mutations, the MICs of INH were within the range 0.2-10 μg/mL. Thirty-six (88%) of those strains retained their catalase activity. Mutations at codon 315 within the katG gene, depending on their type might be useful for the prediction of INH resistance. Whereas the missense mutations do not affect the catalase activity or the level of INH resistance, the nonsense mutations result in high-level resistance to INH and a total loss of catalase activity.

A Two-Step Strategy for Molecular Typing of Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates from Poland

Tuberculosis (TB) continues to be one of the most challenging public health problems in the world. An important contributor to the global burden of the disease is the emergence and spread of drug-resistant and particularly multidrug-resistant Mycobacterium tuberculosis strains (MDR), defined as being resistant to at least isoniazid and rifampicin. In recent years, the introduction of different DNA-based molecular typing methods has substantially improved the knowledge of the epidemiology of TB. The purpose of this study was to employ a combination of two PCR-based genotyping methods, namely spoligotyping and IS6110-Mtb1/Mtb2 PCR to investigate the clonal relatedness of MDR M. tuberculosis clinical isolates recovered from pulmonary TB patients from Poland. Among the 50 isolates examined, 28 (56%) were clustered by spoligotyping, whereas IS6110-Mtb1/Mtb2 PCR resulted in 16 (32%) clustered isolates. The isolates that clustered in both typing methods were assumed to be clonally related. A two-step strategy consisting of spoligotyping as a first-line test, performed on the entire pool of isolates, and IS6110-Mtb1/Mtb2 PCR typing as a confirmatory subtyping method, performed only within spoligotype-defined clusters, is an efficient approach for determining clonal relatedness among M. tuberculosis clinical isolates.

Clinical Isolates of Mycobacterium tuberculosis in Four European Hospitals Are Uniformly Susceptible to Benzothiazinones

The new antitubercular drug candidate 2-[2-S-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one (BTZ043) targets the DprE1 (Rv3790) subunit of the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase. To monitor the potential development of benzothiazinone (BTZ) resistance, a total of 240 sensitive and multidrug-resistant Mycobacterium tuberculosis clinical isolates from four European hospitals were surveyed for the presence of mutations in the dprE1 gene and for BTZ susceptibility. All 240 strains were susceptible, thus establishing the baseline prior to the introduction of BTZ043 in clinical trials.

Activity against multidrug-resistant Mycobacterium tuberculosis in Mexican plants used to treat respiratory diseases.

The increase of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) demands the search for alternative antimycobacterial drugs. The aim of this study was to evaluate plants used in Mexican traditional medicine to treat respiratory diseases for activity against MDR-TB. A group of 22 plants was screened for activity against Mycobacterium tuberculosis H37Rv and Mycobacterium avium at concentrations from 50 to 200 microg/mL. The antimycobacterial effect was determined by a microcolorimetric assay with Alamar blue dye. None of the aqueous extracts had antimycobacterial activity. Hexane extracts from Artemisia ludoviciana, Chamaedora tepejilote, Lantana hispida, Juniperus communis and Malva parviflora, and methanol extracts from Artemisia ludoviciana and Juniperus communis inhibited the growth of Mycobacterium tuberculosis. Mycobacterium avium was inhibited by Juniperus communis hexane extract and by Malva parviflora methanol extract. The active extracts were tested against monoresistant variants of Mycobacterium tuberculosis H37Rv (isoniazid, rifampin, streptomycin and ethambutol resistant) and the hexane extract of Lantana hispida showed the best activity. Lantana hispida hexane extract was also active against a group of MDR-TB clinical isolates. In contrast, it did not inhibit the growth of non-tuberculous mycobacteria. The hexane extract of Lantana hispida was fractionated by column chromatography and one of its fractions (FVI) inhibited the growth of all the MDR-TB clinical isolates at concentrations up to 25 microg/mL. This study supports the fact that selecting plants by ethnobotanical criteria enhances the probability of finding species with activity against mycobacteria, and our results point to Lantana hispida as an important source of potential compounds against MDR-TB.