scholarly journals Characterization of katG, inhA, rpoB and pncA in Mycobacterium tuberculosis isolates from MDR-TB risk patients in Thailand

2020 ◽  
Vol 14 (03) ◽  
pp. 268-276
Author(s):  
Krairerk Suthum ◽  
Worada Samosornsuk ◽  
Seksun Samosornsuk
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Sequencing ◽  
Susceptibility Testing ◽  
Gene Mutations ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Common Mutation ◽  
Mdr Tb ◽  
Risk Patients

Introduction: Multidrug-resistant tuberculosis (MDR-TB) is commonly found in Thailand especially in the public health region 5, the Western region of Thailand. This study’s aim was to characterize katG, inhA, rpoB and pncA genes in Mycobacterium tuberculosis. Methodology: One hundred strains of Mycobacterium tuberculosis (MTB) were isolated from sputum samples of MDR-TB risk patients in the laboratory of the Office of Disease Prevention and Control 5th Ratchaburi province, Thailand from January to December 2015. Drug susceptibility testing (DST) was performed using a BACTEC MGIT 960 system. Furthermore, the genes katG, inhA, rpoB and pncA were characterized by DNA sequencing. Results: Of a total of 100 MTB samples which underwent drug susceptibility testing, 42% showed isoniazid (INH) and rifampicin (RIF) resistance, and a further 25% showed INH mono-resistance (25%). The most common gene mutations found using DNA sequencing were katG_Ser315Thr (70%), rpoB_Ser531leu (81%) and pncA_Ile31Thr (84%). The common mutation of pncA_Ile31Thr substitution was detected in 26 of 91 (29%) pyrazinamide (PZA) susceptible isolates. Conclusion: Using DNA sequencing to screen for gene mutations conferring drug resistance may be feasible and use less time than using DST to detect resistance patterns.

scholarly journals Phenotypic and molecular characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis isolates in Ningbo, China

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yang Che ◽  
Dingyi Bo ◽  
Xiang Lin ◽  
Tong Chen ◽  
Tianfeng He ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Sequencing ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Mdr Tb ◽  
Resistant Group ◽  
Susceptible Group

Abstract Background Detection of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis (TB) patients is critical, especially in dealing with multidrug-resistant Mycobacterium tuberculosis (MDR-TB) case. Up to date, PZA drug susceptibility testing (DST) has not been regularly performed in China. The prevalence and molecular characteristics of PZA resistance in M.tuberculosis isolates, especially MDR-TB have not been studied in Ningbo, China. This study aimed to analyze the phenotypic and molecular characterization of PZA resistance among MDR-TB isolates in Ningbo. Methods A total of 110 MDR-TB isolates were collected from the TB patients who were recorded at local TB dispensaries in Ningbo. All clinical isolates were examined by drug susceptibility testing and genotyping. DNA sequencing was used to detect mutations in the pncA gene associated with PZA resistance. Results The prevalence of PZA resistance among MDR-TB strains in Ningbo was 59.1%. With regard to the history and the outcome of treatments among MDR-TB cases, the percentages of re-treated MDR-TB patients in the PZA-resistant group and of successful patients in PZA-susceptible group were significantly higher than the ones in the PZA-susceptible group and in the PZA-resistant group, respectively (P = 0.027, P = 0.020). The results showed that the resistance of streptomycin (67.7% vs 46.7%, P = 0.027), ethambutol (56.9% vs 33.3%, P = 0.015), ofloxacin (43.1% vs 11.1%, P = 0.000), levofloxacin (43.1% vs 11.1%, P = 0.000), pre-XDR (pre-Xtensively Drug Resistance) (38.5% vs 15.6%, P = 0.009), were more frequently adverted among PZA-resistant isolates compared with PZA-susceptible isolates. In addition, 110 MDR-TB was composed of 87 (PZA resistant, 78.5%) Beijing strains and 23 (PZA resistant, 21.5%) non-Beijing strains. Fifty-four out of 65 (83.1%) PZA-resistant MDR strains harbored a mutation located in the pncA gene and the majority (90.7%) were point mutations. Compared with the phenotypic characterization, DNA sequencing of pncA has sensitivity and specificity of 83.1 and 95.6%. Conclusion The mutations within pncA gene was the primary mechanism of PZA resistance among MDR-TB and DNA sequencing of pncA gene could provide a rapid detection evidence in PZA drug resistance of MDR-TB in Ningbo.

scholarly journals Use of Genotype MTBDRplus Assay for Diagnosis of Multidrug-Resistant Tuberculosis in Nepal

2017 ◽  
Vol 2017 ◽  
pp. 1-5 ◽  
Author(s):  
Elina Maharjan ◽  
Narayan Dutt Pant ◽  
Sanjeev Neupane ◽  
Jyoti Amatya ◽  
Bhawana Shrestha
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
Conventional Drug ◽  
Mdr Tb ◽  
Genotype Mtbdrplus

The main aims of this study were to study the patterns of mutations in rpoB, katG, and inhA genes in Mycobacterium tuberculosis strains isolated from patients from Nepal and to evaluate the performance of genotype MTBDRplus assay, taking conventional drug susceptibility testing as gold standard for diagnosis of MDR-TB. A total of 69 Mycobacterium tuberculosis strains isolated from 73 smear positive sputum samples from patients suspected of suffering from multidrug-resistant tuberculosis were used in our study. The drug susceptibility pattern of Mycobacterium tuberculosis isolated from these sputum specimens was determined by using genotype MTBDRplus assay taking conventional drug susceptibility testing as reference. The sensitivity and specificity of the genotype MTBDRplus assay for the detection of MDR-TB were found to be 88.7% and 100%, respectively. 88.7% of the rifampicin resistant isolates had mutations in rpoB gene. Similarly, 79.7% and 9.4% of isoniazid resistant isolates had mutations in katG and inhA genes, respectively. Genotype MTBDRplus assay was found to be very rapid and highly sensitive and specific method for diagnosis of MDR-TB and will be very helpful for early diagnosis of MDR-TB in high tuberculosis burden countries.

scholarly journals Molecular characterisation of multidrug-resistantMycobacterium tuberculosisisolates from a high-burden tuberculosis state in Brazil

2019 ◽  
Vol 147 ◽  
Author(s):  
R. S. Salvato ◽  
S. Schiefelbein ◽  
R. B. Barcellos ◽  
B. M. Praetzel ◽  
I. S. Anusca ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Molecular Characterisation ◽  
Drug Resistant ◽  
Isoniazid Resistance ◽  
High Burden ◽  
Brics Countries ◽  
Mdr Tb

AbstractTuberculosis (TB) is the leading cause of death among infectious diseases worldwide. Among the estimated cases of drug-resistant TB, approximately 60% occur in the BRICS countries (Brazil, Russia, India, China and South Africa). Among Brazilian states, primary and acquired multidrug-resistant TB (MDR-TB) rates were the highest in Rio Grande do Sul (RS). This study aimed to perform molecular characterisation of MDR-TB in the State of RS, a high-burden Brazilian state. We performed molecular characterisation of MDR-TB cases in RS, defined by drug susceptibility testing, using 131Mycobacterium tuberculosis (M.tb)DNA samples from the Central Laboratory. We carried out MIRU-VNTR 24loci, spoligotyping, sequencing of thekatG,inhA andrpoB genes and RDRiosublineage identification. The most frequent families found were LAM (65.6%) and Haarlem (22.1%). RDRiodeletion was observed in 42 (32%) of theM.tbisolates. Among MDR-TB cases, eight (6.1%) did not present mutations in the studied genes. In 116 (88.5%)M.tbisolates, we found mutations associated with rifampicin (RIF) resistance inrpoB gene, and in 112 isolates (85.5%), we observed mutations related to isoniazid resistance inkatG andinhA genes. An insertion of 12 nucleotides (CCAGAACAACCC) at the 516 codon in therpoB gene, possibly responsible for a decreased interaction of RIF and RNA polymerase, was found in 19/131 of the isolates, belonging mostly to LAM and Haarlem families. These results enable a better understanding of the dynamics of transmission and evolution of MDR-TB in the region.

scholarly journals Non-pncAGene-Mutated but Pyrazinamide-Resistant Mycobacterium tuberculosis: Why Is That?

2017 ◽  
Vol 55 (6) ◽  
pp. 1920-1927 ◽  
Author(s):  
Jim Werngren ◽  
Erik Alm ◽  
Mikael Mansjö
Keyword(s):  
Susceptibility Testing ◽  
Gene Mutations ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Molecular Techniques ◽  
Content Type ◽  
Resistant Strains ◽  
Clear Majority

ABSTRACTPyrazinamide (PZA) is a key component for the effective treatment of drug-susceptible and PZA-susceptible multidrug-resistant (MDRPZA-S) tuberculosis (TB).pncAgene mutations are usually detected in a clear majority (>90%) of PZA-resistant strains but obviously not in all. Rapid and reliable PZA drug susceptibility testing (DST) is critical whenever PZA is to be used in a treatment regimen, not least for the treatment of MDRPZA-STB. In this study, we selected 26 PZA-resistant isolates reported to carry a wild-typepncAgene. To confirm resistance, susceptibility testing was repeated using 100 mg/liter and 200 mg/liter PZA for all the 26 isolates and Sanger sequencing was repeated on the 18 isolates that remained PZA resistant. Apart from the eight isolates initially misclassified as PZA resistant, the retests identified three factors responsible for the phenotype-genotype discrepancy:panDorrpsAmutations identified by whole-genome sequencing (WGS) (n= 7), heteroresistance (n= 8), and mixed populations withMycobacterium avium(n= 3). Additionally, we performed WGS on 400 PZA-susceptible isolates and 15 consecutive MDRPZA-Rclinical isolates. Of the 400 PZA-susceptible isolates, only 1 harbored a nonsynonymouspncAmutation (Thr87Met), whereas a nonsynonymousrpsAmutation was found in 17 isolates. None of these isolates carried a nonsynonymouspanDmutation, while all 15 of the MDRPZA-Risolates harbored a nonsynonymouspncAmutation. Our findings indicate that it is necessary to consider the occurrence ofpanDmutations in PZA-resistant isolates, as well as heteroresistance, for the development and evaluation of new molecular techniques to ensure high-quality DST performance. The identification of nonsynonymousrpsAmutations in both PZA-susceptible and PZA-resistant isolates also implies that further studies are needed in order to determine the role ofrpsAin PZA resistance.

scholarly journals rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates from rural areas of Zhejiang, China

2021 ◽  
Vol 49 (3) ◽  
pp. 030006052199759
Author(s):  
Mei-Chun Zeng ◽  
Qing-Jun Jia ◽  
Lei-Ming Tang
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rural Areas ◽  
Gene Mutations ◽  
Gene Sequencing ◽  
Rpob Gene ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Drug Resistant ◽  
Extensively Drug Resistant

Objective The aim was to analyze genetic mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis isolates (RIFR-MTB) from Zhejiang, China. Methods We prospectively analyzed RIFR-associated mutations in 13 rural areas of Zhejiang. Isolates were subjected to species identification, phenotype drug susceptibility testing (DST), DNA extraction, and rpoB gene sequencing. Results A total of 103 RIFR isolates were identified by DST (22 RIFR only, 14 poly-drug resistant, 49 multidrug resistant, 13 pre-extensively drug resistant [pre-XDR], and 5 extensively drug resistant [XDR]) from 2152 culture-positive sputum specimens. Gene sequencing of rpoB showed that the most frequent mutation was S450L (37.86%, 39/103); mutations P280L, E521K, and D595Y were outside the rifampicin resistance-determining region (RRDR) but may be associated with RIFR. Mutations associated with poly-drug resistant, pre-XDR, and XDR TB were mainly located at codon 445 or 450 in the RRDR. Conclusions The frequency of rpoB RRDR mutation in Zhejiang is high. Further studies are needed to clarify the relationships between RIFR and the TTC insertion at codon 433 in the RRDR and the P280L and D595Y mutations outside the RRDR.

scholarly journals Drug susceptibility patterns of Mycobacterium tuberculosis from adults with multidrug-resistant tuberculosis and implications for a household contact preventive therapy trial

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Anne-Marie Demers ◽  
◽  
Soyeon Kim ◽  
Sara McCallum ◽  
Kathleen Eisenach ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Sputum Sample ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Preventive Therapy ◽  
Drug Susceptibility Testing ◽  
Study Entry ◽  
Resistant Tuberculosis ◽  
Mdr Tb ◽  
Therapy Trial

Abstract Background Drug susceptibility testing (DST) patterns of Mycobacterium tuberculosis (MTB) from patients with rifampicin-resistant tuberculosis (RR-TB) or multidrug-resistant TB (MDR-TB; or resistant to rifampicin and isoniazid (INH)), are important to guide preventive therapy for their household contacts (HHCs). Methods As part of a feasibility study done in preparation for an MDR-TB preventive therapy trial in HHCs, smear, Xpert MTB/RIF, Hain MTBDRplus, culture and DST results of index MDR-TB patients were obtained from routine TB programs. A sputum sample was collected at study entry and evaluated by the same tests. Not all tests were performed on all specimens due to variations in test availability. Results Three hundred eight adults with reported RR/MDR-TB were enrolled from 16 participating sites in 8 countries. Their median age was 36 years, and 36% were HIV-infected. Routine testing on all 308 were confirmed as having RR-TB, but only 75% were documented as having MDR-TB. The majority of those not classified as having MDR-TB were because only rifampicin resistance was tested. At study entry (median 59 days after MDR-TB treatment initiation), 280 participants (91%) were able to produce sputum for the study, of whom 147 (53%) still had detectable MTB. All but 2 of these 147 had rifampicin DST done, with resistance detected in 89%. Almost half (47%) of the 147 specimens had INH DST done, with 83% resistance. Therefore, 20% of the 280 study specimens had MDR-TB confirmed. Overall, DST for second-line drugs were available in only 35% of the 308 routine specimens and 15% of 280 study specimens. Conclusions RR-TB was detected in all routine specimens but only 75% had documented MDR-TB, illustrating the need for expanded DST beyond Xpert MTB/RIF to target preventive therapy for HHC.

scholarly journals Transmission patterns of rifampicin resistant Mycobacterium tuberculosis complex strains in Cameroon: a genomic epidemiological study

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Matthias Merker ◽  
Nkongho F. Egbe ◽  
Yannick R. Ngangue ◽  
Comfort Vuchas ◽  
Thomas A. Kohl ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Prolonged Exposure ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
High Dose ◽  
Factors Affecting ◽  
Recent Transmission ◽  
Mdr Tb

Abstract Background Determining factors affecting the transmission of rifampicin (RR) and multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains under standardized tuberculosis (TB) treatment is key to control TB and prevent the evolution of drug resistance. Methods We combined bacterial whole genome sequencing (WGS) and epidemiological investigations for 37% (n = 195) of all RR/MDR-TB patients in Cameroon (2012–2015) to identify factors associated with recent transmission. Results Patients infected with a strain resistant to high-dose isoniazid, and ethambutol had 7.4 (95% CI 2.6–21.4), and 2.4 (95% CI 1.2–4.8) times increased odds of being in a WGS-cluster, a surrogate for recent transmission. Furthermore, age between 30 and 50 was positively correlated with recent transmission (adjusted OR 3.8, 95% CI 1.3–11.4). We found high drug-resistance proportions against three drugs used in the short standardized MDR-TB regimen in Cameroon, i.e. high-dose isoniazid (77.4%), ethambutol (56.9%), and pyrazinamide (43.1%). Virtually all strains were susceptible to fluoroquinolones, kanamycin, and clofazimine, and treatment outcomes were mostly favourable (87.5%). Conclusion Pre-existing resistance to high-dose isoniazid, and ethambutol is associated with recent transmission of RR/MDR strains in our study. A possible contributing factor for this observation is the absence of universal drug susceptibility testing in Cameroon, likely resulting in prolonged exposure of new RR/MDR-TB patients to sub-optimal or failing first-line drug regimens.

Effectiveness of GenoType MTBDRsl in excluding TB drug resistance in a clinical trial

2021 ◽  
Vol 25 (10) ◽  
pp. 839-845
Author(s):  
M. Ejo ◽  
A. Van Deun ◽  
A. Nunn ◽  
S. Meredith ◽  
S. Ahmed ◽  
...  
Keyword(s):  
Standard Treatment ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Central Laboratory ◽  
Mdr Tb ◽  
Smear Negative ◽  
Probe Assay ◽  
Standard Treatment Regimen

OBJECTIVES: To assess the performance of the GenoType MTBDRsl v1, a line-probe assay (LPA), to exclude baseline resistance to fluoroquinolones (FQs) and second-line injectables (SLIs) in the Standard Treatment Regimen of Anti-tuberculosis Drugs for Patients With MDR-TB 1 (STREAM 1) trial.METHODS: Direct sputum MTBDRsl results in the site laboratories were compared to indirect phenotypic drug susceptibility testing (pDST) results in the central laboratory, with DNA sequencing as a reference standard.RESULTS: Of 413 multidrug-resistant TB (MDR-TB) patients tested using MTBDRsl and pDST, 389 (94.2%) were FQ-susceptible and 7 (1.7%) FQ-resistant, while 17 (4.1%) had an inconclusive MTBDRsl result. For SLI, 372 (90.1%) were susceptible, 5 (1.2%) resistant and 36 (8.7%) inconclusive. There were 9 (2.3%) FQ discordant pDST/MTBDRsl results, of which 3 revealed a mutation and 5 (1.3%) SLI discordant pDST/MTBDRsl results, none of which were mutants on sequencing. Among the 17 FQ- and SLI MTBDRsl-inconclusive samples, sequencing showed 1 FQ- and zero SLI-resistant results, similar to frequencies among the conclusive MTBDRsl. The majority of inconclusive MTBDRsl results were associated with low bacillary load samples (acid-fast bacilli smear-negative or scantily positive) compared to conclusive results (P < 0.001).CONCLUSION: MTBDRsl can facilitate the rapid exclusion of FQ and SLI resistances for enrolment in clinical trials.

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