Delamanid-containing regimens and multidrug-resistant tuberculosis: A systematic review and meta-analysis.

INTRODUCTION: Multidrug-resistant tuberculosis (MDR-TB) is a life-threatening condition needing long poly-chemotherapy regimens. As no systematic reviews/meta-analysis is available to comprehensively evaluate the role of delamanid (DLM), we evaluated its effectiveness and safety. METHODS: We reviewed the relevant scientific literature published up to January 20, 2022. The pooled success treatment rate with 95% confidence intervals (CI) was assessed using a random-effect model. We assessed studies for quality and bias, and considered P<0.05 to be statistically significant. RESULTS: After reviewing 626 records, we identified 25 studies that met the inclusion criteria, 22 observational and 3 experimental, with 1276 and 411 patients, respectively. In observational studies the overall pooled treatment success rate of DLM-containing regimens was 80.9% (95% CI 72.6-87.2) with no evidence of publication bias (Begg's test; P >0.05). The overall pooled treatment success rate in DLM and bedaquiline-containing regimens was 75.2% (95% CI 68.1-81.1) with no evidence of publication bias (Begg's test; P >0.05). In experimental studies the pooled treatment success rate of DLM-containing regimens was 72.5 (95% CI 44.2-89.8, P <0.001, I2: 95.1%) with no evidence of publication bias (Begg's test; P >0.05). CONCLUSIONS: In MDR-TB patients receiving DLM, culture conversion and treatment success rates were high despite extensive resistance with limited adverse events.

The 7H11 Agar Medium Supplemented with Calf Bovine Serum for Susceptibility Testing of Mycobacterium tuberculosis Isolates Against Pyrazinamide.

Despite its importance, pyrazinamide (PZA) is a blind spot in drug susceptibility testing in tuberculosis laboratories. The aim of this study was to set up a reliable agar-based proportion method for detection of PZA-resistant phenotypes using Middlebrook 7H11 agar supplemented with calf bovine serum (CBS) compared with albumin/dextrose/catalase (ADC) enrichment and pncA/rpsA sequencing results. The 7H11 agar medium supplemented with 10% ADC or 10% CBS (pH 6.2) and 100 µg/mL PZA was used to detect PZA resistance among 64 Mycobacterium tuberculosis isolates. Sanger sequencing and whole-genome sequencing were performed to track mutations in the pncA, rpsA, and their upstream regions. A total of 43 rifampicin/multidrug-resistant, 20 drug-susceptible, and 1 isoniazid mono-resistant M. tuberculosis isolates were investigated. The 7H11+ADC and 7H11+CBS could detect 22 and 23 PZA-resistant strains, respectively. With the same specificity, the sensitivity and accuracy of 7H11+CBS was found to be a little greater than 7H11+ADC in PZA resistance detection compared with sequencing results. Twenty-four mutant strains were found to have different mutations in pncA-upstream, pncA and rpsA genes, in which Gly97Asp was the most dominant mutation. The results obtained from 7H11+CBS were comparable to the results of 7H11+ADC. Therefore, the 7H11 agar proportion method would be a less-expensive test using CBS and produces reliable results.

Assessment of the GenoType MTBDRsl VER 2.0 compared to the phenotypic drug susceptibility testing and whole genome sequencing for the rapid detection of resistance to fluoroquinolone and second-line injectable drugs among rifampicin-resistant Mycobacterium tuberculosis isolates.

Molecular techniques have considerable advantages for rapid detection, a reduction of infectiousness, prevention of further resistance development and surveillance of drug-resistant TB. MTBDRsl VER 2.0 was used to detect resistance to second-line anti-tuberculosis drugs on 35 rifampicin-resistant M. tuberculosis (RR-MTB) isolates compared to the minimum inhibitory concentrations (MICs) and whole genome sequencing (WGS). The MTBDRsl VER 2.0 (Hain Life Science, Nehren, Germany) and WGS (San Diego, CA, USA) were performed for tracing mutations in resistant-related genes involved in resistance to fluoroquinolone (FLQ) and second-line injectable drugs. The broth microdilution method using 7H9 Middlebrook media supplemented with OADC was used to determine the MICs. The MTBDRsl VER 2.0 correctly detected 5/6 (83.3%) of FLQ-resistant strains. The MUT1 A1401G (seven strains) and MUT2 G1484T (one strain) mutations in rrs gene were detected in eight AMK/KAN/CAP-resistant strains. Four low-level KAN-resistant strains with the G-10A/C-12T (three strains) and eis C-14T (one strain) mutations in eis gene was diagnosed using MTBDRsl VER 2.0. Five errors were found in detecting resistance to kanamycin and capreomycin compared to the phenotypic drug susceptibility testing and WGS. Failling wild-type bands without improved mutant bands did not indicate a reliable resistance. WGS could efficiently resolve the discrepancies of the results. MTBDRsl showed better performance in detecting XDR strains than pre-XDR.

Drug Susceptibility Testing of Mycobacterium Simiae: An Emerging Pathogen in Iran.

INTRODUCTION: Mycobacterium simiae is an emerging pathogen in Iran and little is known about drug susceptibility patterns of this pathogen. MATERIALS AND METHODS: Twenty-five clinical isolates of M. simiae from 80 patients with confirmed NTM pulmonary disease were included in this study. For drug susceptibility testing (DST), proportional and broth microdilution methods were used according to the clinical and laboratory standards institute (CLSI) guideline. RESULTS: All clinical isolates of M. simiae were resistant to isoniazid, rifampicin, ethambutol, streptomycin, amikacin, kanamycin, ciprofloxacin, and clarithromycin. They also were highly resistant to ofloxacin (80%). Susceptibility to ofloxacin was only noted in the 5 isolates. CONCLUSION: Clinical isolates of M. simiae were multidrug-resistant, and had different drug susceptibility patterns than previously published studies. DST results can assist in selecting more appropriate treatment regimens. Newer drugs with proven clinical efficacy correlating with in vitro susceptibility should be substituted with first- and second-line anti-TB drug testing.

Spoligotype and Drug Susceptibility Profiles of Mycobacterium tuberculosis Complex Isolates in Golestan Province, North Iran.

INTRODUCTION: Despite the moderate incidence of tuberculosis (TB) in many parts of Iran, Golestan province had a permanently higher TB incidence rate than the national average. Moreover, Golestan province receives immigrants, mainly from TB-endemic areas of Iran and neighbor countries. Here, we aimed to characterize the circulating Mycobacterium tuberculosis complex (MTBC) isolates in terms of the spoligotype and drug resistance patterns, across Golestan province. MATERIALS AND METHODS: A set of 166 MTBC isolates was collected during July 2014 to July 2015 and subjected to drug susceptibility testing for first- and second-line anti-TB drugs and spoligotyping. RESULTS: Of 166 MTBC isolates, 139 (83.7%) isolates were assigned to 28 spoligotype international types (SITs). The most frequent SITs were SIT127/Ural-2 (n=25, 15.1%), followed by SIT1/Beijing (n=21, 12.7%) and SIT3427/Ural-2 (n=18, 10.8%). The set of 18 isolates (10.8%) showed resistance to at least one drug, which mainly belonged to SIT1/Beijing (n=7, 38.9%), orphan patterns (n=4, 22.2%) and SIT357/CAS1-Delhi (n=3, 16.7%). In addition, four isolates (2.4%) were resistant to pyrazinamide. The analysis of mutation corresponded to resistance to rifampin and isoniazid showed that two isolates had Ser531Leu substitution in rpoB, four isolates had Ser315Thr substitution in katG and one isolate had [C(-15)T] in inhA locus. CONCLUSION: High diversity in spoligotypes of the MTBC isolates and lack of dominant genotype might be due to residence of immigrants in this region and consequent reactivation of latent infection. In addition, due to the presence of extensively drug-resistant (XDR) isolates in Golestan province, it is important to conduct future studies to determine transmission pattern of drug-resistant isolates in this region.

Molecular identification of mutations conferring resistance to rifampin, isoniazid and pyrazinamide among Mycobacterium tuberculosis isolates from Iran.

Here, we aimed to determine the susceptibility of 70 Mycobacterium tuberculosis isolates obtained from different regions of the country to 8 anti-tuberculosis (anti-TB) drugs and possible underlying mechanisms causing resistance to rifampin, isoniazid, and pyrazinamide. The susceptibility of 70 isolates of M. tuberculosis to anti-TB drugs was tested using proportion method. Strains showing resistance to the first line anti-TB drugs were subjected to PCR amplification and sequencing of the rpoB, katG, ahpC, pncA genes, inhA promoter and oxyR-ahpC intergenic regions to detect resistance conferring mutations. Overall, 77.1% and 77.1% of isolates were resistant to at least one of the tested first- and second-line drugs, respectively. Within the rpoB gene the highest rate of mutation was found in codons 531(450) (56.3%), and 533(452) (12.5%). Also, codons 315 (42.4%) of katG, positions -48, -72 and -77 of oxyR-ahpC (total= 3, 9.1%) and -15 of inhA promoter region (33.3%) were the most altered positions in isoniazid resistant isolates. Only a single mutation was detected for pncA among resistant isolates. High prevalence of resistance to essential anti-TB drugs among M. tuberculosis strains isolated from retreated tuberculosis cases is alarming issue necessitating immediate action to prevent the spread of drug resistant isolates in the country.

Low diagnostic accuracy of Xpert MTB/RIF assay for extrapulmonary tuberculosis: A multicenter surveillance.

Diagnostic accuracy of Xpert MTB/RIF assay for pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB) has not been investigated in Iran. This study was aimed to assess the diagnostic accuracy of Xpert MTB/RIF assay for both PTB and EPTB. A total of 2111 clinical samples (1218 pulmonary and 838 extra-pulmonary) were collected from 16 medical centers during the study period and were analyzed for detection of PTB and EPTB by both Xpert MTB/RIF assay and standard conventional methods (culture and direct smear microscopy). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of Xpert MTB/RIF assay for PTB were found to be 95.5%, 96.7%, 83.8%, and 99.1% respectively. For EPTB, the sensitivity, specificity, PPV and NPV of Xpert MTB/RIF assay counted for 76.5%, 95.9%, 62%, and 97.9% respectively. Xpert MTB/RIF assay found to be highly sensitive, specific and comparable to standard conventional methods for the diagnosis of PTB. However, the sensitivity and specificity of Xpert MTB/RIF for EPTB specimens were highly variable; thus, Xpert MTB/RIF cannot be recommended to replace standard conventional tests for diagnosis of EPTB.

Direct drug susceptibility testing of Mycobacterium tuberculosis using the proportional method: A multicenter study.

INTRODUCTION: Conventional indirect drug susceptibility testing (DST) of Mycobacterium tuberculosis with solid media is inexpensive and reliable, but time-consuming. This study aimed to evaluate direct DST for testing sputum samples without culture to significantly reduce the time required to detect multidrug-resistant tuberculosis (MDR-TB). METHODS: Direct and indirect DST of isoniazid (INH), rifampicin (RIF) and ethambutol (EMB) were performed on 334 sputum smear-positive specimens. RESULTS: There was full agreement between the results obtained from direct testing and after isolation of the bacteria by culture. Thus, the sensitivity and specificity were observed to be 100% for all three tested drugs when compared with indirect DST. In comparison with indirect DST, none of the samples with the direct method took >25days to report the DST (between 15-25days with a mean detection time of 20 days). CONCLUSIONS: Direct DST on solid media was shown to give reliable results at a much earlier stage than conventional phenotypic DST. The direct method was found to be more rapid, more accurate and simpler. In addition, it reduced the handling of pathogenic bacteria and thus reduced the bio hazards related to conventional DST.

Expression analysis of 10 efflux pump genes in multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis clinical isolates.

OBJECTIVES: Active extrusion of antituberculosis drugs via efflux pumps (EPs) has been suggested as contributing to drug resistance in Mycobacterium tuberculosis. This study was conducted to determine the role of 10 drug efflux transporters in the development of drug resistance in a series of clinical M. tuberculosis isolates. METHODS: A total of 31 clinical M. tuberculosis isolates without drug exposure [21 multi/extensively drug-resistant (M/XDR-TB) and 10 drug-susceptible isolates] were studied. The expression profile of 10 EP genes, including efpA, mmr, stp, drrA, drrB, mmpL7, Rv1250, Rv1634, Rv2994 and Rv1258c, was investigated against the H37Rv standard strain by quantitative reverse transcription PCR (RT-qPCR). RESULTS: Among the 21M/XDR-TB isolates, 10 showed significantly increased levels of gene expression (>4-fold) for at least one of the studied EPs. Moreover, of the isolates with overexpressed genes, three and seven lacked genetic alterations in the surveyed regions of the rpoB+katG+inhA and katG+inhA genes, respectively. Whilst no elevation was observed in the expression of mmr, Rv1250, Rv1634 and Rv1258c genes in any of the isolates, drrA, stp and drrB were found to be the most commonly overexpressed, being overexpressed in seven, five and three isolates, respectively. Decreased minimum inhibitory concentrations (MICs) of rifampicin, but not isoniazid, were observed in the presence of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). CONCLUSION: Overexpression of EP genes can contribute to the emergence of a MDR phenotype in M. tuberculosis. Inhibition of EPs may provide a promising strategy for improving tuberculosis treatment outcomes in patients infected with M/XDR-TB isolates.

Molecular detection of rifampin, isoniazid, and ofloxacin resistance in Iranian isolates of Mycobacterium tuberculosis by high-resolution melting analysis.

BACKGROUND: The emergence of drug resistance among Mycobacterium tuberculosis (MTB) strains is a serious health concern worldwide. The development of rapid molecular diagnostic methods in recent years has a significant impact on the early detection of resistance to major anti-TB drugs in MTB isolates, which helps in employing appropriate treatment regimen and prevents the spread of drug-resistant strains. This study was designed to evaluate the efficacy of real-time PCR and high-resolution melting (HRM) curve analysis for the determination of resistance to rifampin (RIF), isoniazid (INH), and ofloxacin (OFX) in MTB isolates and to investigate their resistance-related mutations. METHODS: HRM analysis was performed to screen 52 (32 drug-resistant and 20 fully susceptible) MTB clinical isolates for mutations in rpoB, katG, mab-inhA, and gyrA genes. The HRM results were then confirmed by DNA sequencing. RESULTS: In total, 32 phenotypically resistant isolates, comprising 18 RIF-, 16 INH-, and five OFX- resistant strains, were investigated. HRM analysis successfully identified 15 out of 18 mutations in rpoB, 14 out of 16 mutations in katG and mab-inhA, and four out of five mutations in gyrA conferring resistance to RIF, INH, and OFX, respectively. The obtained sensitivity and specificity, respectively, for HRM in comparison with phenotypic susceptibility testing were found to be 83.3% and 100% for RIF, 87.5% and 100% for INH, and 80% and 100% for OFX. In five resistant strains (12.8%), no mutation was detected by using HRM and DNA sequencing. CONCLUSION: HRM assay is a rapid, accurate, and cost-effective method possessing high sensitivity and specificity for the determination of antibiotic resistance among MTB clinical isolates and screening of their associated mutations. This method can generate results in a shorter period of time than taken by the phenotypic susceptibility testing and also allows for timely treatment and prevention of the emergence of possible MDR strains.