Resistance Of Mycobacterium Tuberculosis Isolates Research Articles

Efflux pump gene single nucleotide variants associated with resistance in Mycobacterium tuberculosis isolates with discrepant drug genotypes

IntroductionSingle nucleotide variants (SNVs) in Mycobacterium tuberculosis (MTB) genomes can predict multi-drug resistance (MDR) but not all phenotype – genotype correlations can be explained. We investigated SNVs in efflux pumps (EP) in the context of MTB drug resistance. MethodsWe analysed 2221 MTB genomes from 1432 susceptible and 200 MDR, 172 pre-XDR (extensively drug resistant) and 417 XDR isolates. Analysis of 47 EP genes was conducted using MTB-VCF, an in-house bioinformatics pipeline. SNVs were categorized according to their SIFT/Polyphen scores. Resistance genotypes were also called using the TB-Profiler tool. ResultsGenome comparisons between susceptible and DR isolates identified 418 unique SNVs in EP of which; 53.5% were in MDR, 68.9% in pre-XDR and 61.3% in XDR isolates. Twenty EP had unique SNVs with a high SIFT/PolyPhen score, comprising 38 unique SNVs. Sixteen SNVs across 12 EP genes were significantly associated with drug resistance and enriched in preXDR and XDR strains. These were 12 previously reported SNVs (in Rv0191, Rv0507, Rv0676, Rv1217, Rv1218, Rv1273, Rv1458, Rv1819 and Rv2688) and four novel SNVs (in Rv1877 and Rv2333). We investigated the 16 SNVs in 52 MDR isolates with phenotype-genotype discrepancy to rifampicin (RIF), isoniazid (INH) and flouroquinolones. SNVs associated with RIF and INH (Rv1217_1218, Rv1819, Rv0450, Rv1458, Rv3827, Rv0507, Rv0676, Rv1273 and Rv2333), and with fluoroquinolone (Rv2688) resistance were present in these discrepant strains. ConclusionsConsidering SNVs in efflux pumps as part of MTB genome-based resistance interpretation may add value especially in evaluation of XDR resistance in strains with phenotype-genotype discrepancies.

Drug resistance characteristics of Mycobacterium tuberculosis isolates obtained between 2018 and 2020 in Sichuan, China.

We investigated the drug resistance of Mycobacterium tuberculosis isolates from patients with tuberculosis (TB) and HIV, and those diagnosed with only TB in Sichuan, China. TB isolates were obtained from January 2018 to December 2020 and subjected to drug susceptibility testing (DST) to 11 anti-TB drugs and to GeneXpert MTB/RIF testing. The overall proportion of drug-resistant TB (DR-TB) isolates was 32.1% (n = 10 946). HIV testing was not universally available for outpatient TB cases, only 29.5% (3227/10 946) cases had HIV testing results. The observed proportion of multidrug-resistant TB (MDR-TB) isolates was almost double than that of the national level, with approximately 1.5% and 0.1% of the isolates being extensively drug resistant and universally drug resistant, respectively. The proportions of resistant isolates were generally higher in 2018 and 2019 than in 2020. Furthermore, the sensitivities of GeneXpert during 2018-2020 demonstrated a downward trend (80.9, 95% confidence intervals (CI) 76.8-85.0; 80.2, 95% CI 76.4-84.1 and 75.4, 95% CI 70.7-80.2, respectively). Approximately 69.0% (7557/10 946) of the TB cases with DST results were subjected to GeneXpert detection. Overall, the DR-TB status and the use of GeneXpert in Sichuan have improved, but DR-TB challenges remain. HIV testing for all TB cases is recommended.

Next-generation sequencing to characterise pyrazinamide resistance in Mycobacterium tuberculosis isolates from two Balkan countries

Next-generation sequencing (NGS) can provide a comprehensive analysis of the genetic alterations most commonly linked with pyrazinamide (PZA) resistance. However, there are no studies reporting the molecular background of PZA resistance in Mycobacterium tuberculosis (TB) isolates from the Balkan Peninsula. We aimed to examine the feasibility of full-length analysis of a gene linked with PZA resistance (pncA) using Ion Torrent technology compared with phenotypic BACTEC MGIT 960 drug susceptibility testing (DST) in clinical TB isolates from two countries of the Balkan Peninsula. Between 1996 and 2017, we retrospectively selected 61 TB isolates. To identify gene variants related to drug resistance in genomic DNA extracted from TB isolates, AmpliSeq libraries were generated automatically using an AmpliSeq™ Kit for Chef DL8 and Ion AmpliSeq TB Research Panel. Of the 61 TB isolates, 56 were phenotypically resistant to any antibiotic. Among them, 38/56 isolates (67.9%) were phenotypically resistant to pyrazinamide, and pncA mutations were detected in 34/38 cases (89.5%). A mutation in the pncA promoter region was the most prevalent genetic alteration, detected in eight TB isolates. Comparison of NGS data with conventional BACTEC MGIT 960 DST revealed very strong agreement (91.8%) between the two methods in identifying PZA resistance, with high sensitivity (89.5%) and specificity (95.7%) for NGS. Detection of PZA resistance using NGS appears to be a valuable tool for surveillance of TB drug resistance in the Balkan Peninsula, with great potential to provide useful information at least 1 weak earlier than is possible with phenotypic DST.

Anti-Tuberculosis Drugs against the Resistance Level of Mycobacterium tuberculosis isolates

Prolonged MDR-TB therapy can have side effects, namely a correlation between cure rates and changes in bacterial profiles related to resistance to anti-tuberculosis drugs (ATD) which can affect the incidence rate of MTB and MDR-TB in a region. The research objective was to analyze the effectiveness of the type of ATD against the resistance level of Mycobacterium tuberculosis (MTB) isolates with the incidence of tuberculosis (TB) and MDR-TB. The research method used was a retrospective cohort based on tracing medical record data at the Surakarta City Center General Hospital 2016 until 2017 with total sampling. The independent variable of this study was the type of ATD, while the dependent variable was the resistance level of MTB isolates. The characteristics of the most patient respondent suspect TB were male with the level of resistance of MTB isolates to ATD relatively varied. The results of the analysis of different tests showed a p value of 0.000 so that the p value was 0.05, so there was an effect of the type of ATD (Strepttomycin, Isoniazid, Rifampicin and Ethambutol) on the resistance of TB isolates from patients with suspected TB. This is useful to determine the success of TB therapy in terms of mortality and the effectiveness of therapy in TB patients.

Correlating genetic mutations with isoniazid phenotypic levels of resistance in Mycobacterium tuberculosis isolates from patients with drug-resistant tuberculosis in a high burden setting.

We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in Mycobacterium tuberculosis isolates from drug-resistant TB patients from São Paulo state, Brazil. Isolates resistant to the critical concentration of isoniazid in MGIT (0.1µg/mL) were screened for mutations in katG 315 codon, inhA promoter region and rpoB RRDR by MTBDRplus assay and subjected to determination of isoniazid resistance levels by MGIT 960. Discordances were resolved by Sanger sequencing. Among the 203 isolates studied, 109 (54%) were isoniazid-monoresistant, 47 (23%) MDR, 29 (14%) polydrug-resistant, 12 (6%) pre-XDR and 6 (3%) XDR. MTBDRplus detected isoniazid mutations in 75% (153/203) of the isolates. Sequencing of the entire katG and inhA genes revealed mutations in 18/50 wild-type isolates by MTBDRplus (10 with novel mutations), resulting in a total of 32/203 (16%) isolates with no mutations detected. 81/83 (98%) isolates with katG 315 mutations alone had intermediate resistance. Of the 66 isolates with inhA C-15T mutation alone, 51 (77%) showed low-level, 14 (21%) intermediate and 1 (2%) high-level resistance. 5/6 (83%) isolates with mutations in both katG and inhA had high-level resistance. Inferred mutations corresponded to 22% (16/73) of all mutations found in rpoB. Mutations detected in katG regions other than codon 315 in this study might be potential new isoniazid resistance markers and could explain phenotypic resistance in some isolates without katG and inhA classic mutations. In our setting, 16% of isoniazid-resistant isolates, some with high-level resistance, presented no mutations either in katG or inhA.

Molecular Characteristics and Drug Resistance of Mycobacterium tuberculosis Isolate Circulating in Shaanxi Province, Northwestern China.

Objective: Shaanxi is the most highly populated province with high burdens of tuberculosis in northwestern China. The aim of this study was to investigate the molecular characteristics and drug resistance of Mycobacterium tuberculosis isolates from Shaanxi province of China in 2018. Methods: Phenotypic drug susceptibility testing and spoligotyping methods were performed on 518 M. tuberculosis isolates; drug-resistant isolates were sequenced in 11 drug loci, including katG, inhA, oxyR-ahpC, rpoB, embB, rpsL, rrs1 (nucleotides 388-1084), gyrA, gyrB, rrs2 (nucleotides 1158-1674), and eis. Results: The prevalences of isoniazid, rifampicin, ethambutol, streptomycin, ofloxacin, and kanamycin resistance were 22.0%, 19.3%, 7.9%, 23.8%, 10.4%, and 3.3%, respectively. The Beijing family (82.8%) was the predominant genotype, followed by the T (9.3%), H (0.6%), CAS (0.4%), LAM (0.4%), and U (0.4%) families. The percentage of Beijing genotype in a central area (88.1%) was higher than in the south (77.3%) and the north area (80.1%) (p < 0.05), while the sex, age, and treatment history between Beijing and non-Beijing family were not statistically different. Mutation analysis found that the most prevalent mutations were katG315, rpoB531, embB306, rpsL43, gyrA94, and rrs1401; the Beijing family exhibited a high rate of isoniazid-resistant isolates carrying katG315 mutations (p < 0.05). Furthermore, compared with the phenotypic data, the sensitivities of isoniazid, rifampicin, ethambutol, streptomycin, ofloxacin, and kanamycin resistance by sequencing base on 11 loci were 85.1%, 94.0%, 53.7%, 74.8%, 77.8%, and 64.7%, respectively. Conclusions: Shaanxi has a serious epidemic of drug-resistant tuberculosis, Beijing family is the predominant genotype, and the distribution showed geographic diversity. The prevalence of Beijing genotypes has a tendency to promote the transmission of high-level isoniazid-resistant M. tuberculosis. Besides, the hot spot regions localized in the embB, rrs2, and eis gene appear not to serve as excellent biomarkers for predicting ethambutol and kanamycin resistance in Shaanxi.

Performance of the GenoType MTBDRsl V 2.0 for detecting second-line drugs resistance of Mycobacterium tuberculosis isolates in Tunisia

Rapid detection of the second-line drug (SLD) resistant tuberculosis (TB) strains is challenging to prescribe an immediate adequate treatment and limit the transmission of SLD resistant strains.The study aimed to evaluate the performance of GenoType MTBDRsl V2.0 compared to phenotypic drug susceptibility testing (pDST:MGIT960) to detect resistance to SLD of Mycobacterium tuberculosis (MTB) isolates in Tunisia, between May 2015 and December 2019.As a matter of fact, 103 rifampicin-resistant and multidrug-resistant MTB strains were included. Discrepancies between pDST and MTBDRsl were solved by whole genome sequencing.Compared to pDST, MTBDRsl V2.0 showed a sensitivity of 92.8% (68.5%–98.7%) in detecting resistance to fluoroquinolones. As for second-line injectable drugs, it presented a sensitivity of 80.0% (49.0%–94.3%). MTBDRsl had sensitivities of 100.0% (67.5%–100.0%), 75.0% (40.9%–92.8%) and 100.0% (60.9%–100.0%) respectively for kanamycin, capreomycin and amikacin. The specificity was 100.0% for all the drugs evaluated.As for diagnosing XDR-TB, it had a sensitivity of 57.1% (25.0%–84.1%) and a specificity of 100.0% (96.1%–100.0%).MTBDRsl V2.0 showed a high performance in detecting SLD resistance with a short turnaround time compared with pDST, which made it possible to start an early treatment and to maintain a low prevalence of SLD resistance and XDR-TB in Tunisia.

Linezolid resistance in Mycobacterium tuberculosis isolates at a tertiary care centre in Mumbai, India.

Background & objectives:Linezolid (LZD) is increasingly being used in tuberculosis (TB) treatment. However, LZD resistance has already been reported, which is highly alarming, given its critical therapeutic role. This study was aimed to phenotypically and genotypically assess LZD resistance in Mycobacterium tuberculosis (MTB) isolates at a laboratory in a tertiary care centre in Mumbai, India.Methods:A sample of 32 consecutive LZD-resistant MTB isolates identified by liquid culture susceptibility testing was subjected to whole-genome sequencing (WGS) on the Illumina NextSeq platform. Sequences were analyzed using BioNumerics software to predict resistance for 12 antibiotics within 15 min.Results:Sixty eight of the 2179 isolates tested for LZD resistance by MGIT-based susceptibility testing (June 2015 to June 2016) were LZD-resistant. Thirty two consecutive LZD-resistant isolates were analyzed by WGS to screen for known mutations conferring LZD resistance. WGS of 32 phenotypically LZD-resistant isolates showed that C154R in the rplC gene and G2814T in the rrl gene were the major resistance determinants.Interpretation & conclusions:LZD resistance poses an important risk to the success of treatment regimens, especially those designed for resistant isolates; such regimens are extensively used in India. As LZD-containing regimens increase in prominence, it is important to support clinical decision-making with an improved understanding of the common mutations conferring LZD resistance and their frequency in different settings.

Genome sequencing of Mycobacterium tuberculosis clinical isolates revealed isoniazid resistance mechanisms undetected by conventional molecular methods

A combination of targeted molecular methods and phenotypic drug-susceptibility testing is the most widely used approach to detect drug resistance in Mycobacterium tuberculosis isolates. We report the delay in the introduction of an efficient anti-tuberculous drug regimen because of a M. tuberculosis strain displaying a high level of resistance to isoniazid, in the absence of the common mutations associated with isoniazid-resistance, including katG mutations and inhA promoter mutations. Whole-genome sequencing (WGS) identified a large loss-of-function insertion (>1000 pb) at the end of katG in the isolate together with a −57C>T ahpC mutation, a resistance mechanism that would have remained undetected by a conventional molecular targeted approach. A retrospective search using publicly available WGS data of more than 1200 isoniazid-resistant isolates and a similar sized control dataset of isoniazid-susceptible isolates revealed that most (22/31) isoniazid-resistant, KatG loss-of-function mutants had an associated rare ahpC promoter mutation. In contrast, only 7 of 1411 isoniazid-susceptible strains carried a rare ahpC promoter mutation, including shared mutations with the 31 isoniazid-resistant KatG loss-of-function mutants. These results indicate that rare ahpC promoter mutations could be used as a proxy for investigating simultaneous KatG loss-of-function or missense mutations. In addition, WGS in routine diagnosis would improve drug susceptibility testing in M. tuberculosis clinical isolates and is an efficient tool for detecting resistance mechanisms undetected by conventional molecular methods.

Frequency of genomic mutations mediating resistance of Mycobacterium tuberculosis isolates to rifampicin in Northern Morocco.

ABSTRACTDrug resistant tuberculosis (DR-TB) is challenging particularly in developing countries. As such, a previous investigation gave the first insight into the mutational status of the Rifampicin Resistance Determining Region (RRDR) of rpoB gene among a restricted number of MTB patients’ residents in the Northern Morocco. The purpose of this study was to investigate rpoB mutation types and frequencies associated with resistance to Rifampicin in a larger panel of MTB patients and to evaluate the usefulness of these mutations to improve the diagnosis of resistance to Rifampicin. A panel of 301 consecutive sputum samples belonging to patients suscpected of having TB from Northern Morocco was collected at the Pasteur Institute of Tangier between 2014-2017. Samples were subjected to conventionel microbiological tests. Evaluation of rpoB muational status was assessed by PCR amplification and sequencing of the RRDR of the rpoB gene. DST results showed that 26.4% of strains were MDR. Sequencing results reported single point mutations in 36 of 65 RIFR isolates of which two had two mutations. Aminoacid substitutions in the codon Ser531Leu occurred at the highest frequency (34.46%). Overall, 10 aminoacid substitutions have been registered, and the H526S substitution was reported for the first time. The present study highlighted that resistance to RIF is a reliable marker of MDR-TB, the common mutations successfully detected in the rpoB 531, rpoB526 and rpoB516 codons provide a foundation for the implementation of molecular approaches such as Hain and GeneXpert as a routine tests to detect DR-TB. However, considerable work is still necessary to identify extensive mutations associated with DR-TB.

Prevalence of isoniazid resistance-conferring mutations associated with multidrug-resistant tuberculosis in Free State Province, South Africa.

Multidrug- and extensively drug-resistant tuberculosis (MDR-TB and XDR- TB) threaten local and global control of the disease. The molecular line-probe assay (LPA) provides rapid diagnosis and early management of MDR-TB. The LPA detects mutations of katG and inhA genes associated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The katG and inhA genes are associated with high- and low-level INH resistance, respectively, as well as cross-resistance to ethionamide in the case of inhA gene mutations. Patients with MDR-TB due to an inhA mutation could benefit from the use of high-dose INH - instead of ethionamide - in their MDR-TB regimen. To determine the frequencies of katG and inhA mutations that conferred INH resistance among MDR-TB isolates during 2014 - 2016 in Free State (FS) Province of South Africa. We retrospectively reviewed MDR-TB isolates assayed with GenoType MTBDRplus (Hain Lifescience, Germany) (LPA) at the central TB laboratory of Universitas Academic Hospital, Bloemfontein, FS, and calculated the frequencies of katG and inhA mutations. Among 918 MDR-TB isolates, the prevalence of katG, inhA and katG plus inhA mutations was 63.9%, 13.4% and 22.7%, respectively. Approximately 60% (n=536; 58.4%) of the isolates were obtained from male patients. The patients' ages ranged from 1 to 89 (median 37) years. The Xhariep district had the highest incidence of INH resistance-conferring mutations in the province. katG-associated mutations are the predominant INH resistance-conferring mechanism among MDR-TB isolates in the FS. Patients infected with isolates that harbour the katG mutation are unlikely to benefit from high-dose INH therapy in the bedaquiline (BDQ)-containing modified short MDR-TB regimen. They may, however, benefit from the inclusion of ethionamide in the regimen. Dual katG and inhA gene mutations make these patients unlikely to respond to either high-dose INH or ethionamide and should now be considered for either the BDQ-containing long MDR-TB regimen or an individualised treatment regimen, depending on fluoroquinolone susceptibility. Clinicians should familiarise themselves with interpreting various INH resistance-conferring mutation results and their implications for management of MDR-TB treatment.

Rapid detection of drug-resistant Mycobacterium tuberculosis directly from clinical specimens using allele-specific polymerase chain reaction assay.

Background & objectives:Rapid detection of drug resistance in Mycobacterium tuberculosis (MTB) is essential for the efficient control of tuberculosis. Hence, in this study a nested-allele-specific (NAS) PCR, nested multiple allele-specific PCR (NMAS-PCR) and multiple allele-specific (MAS) PCR assays were evaluated that enabled detection of the most common mutations responsible for isoniazid (INH) and rifampicin (RIF) resistance in MTB isolates directly from clinical specimens.Methods:Six pairs of primers, mutated and wild type, were used for the six targets such as codon 516, 526 and 531 of rpoB, codon 315 of katG and C15-T substitution in the promoter region of mabA-inhA using allele-specific (AS) PCR assays (NAS-PCR, NMAS-PCR and MAS-PCR). The performance of AS PCR method was compared with phenotypic drug susceptibility testing (DST).Results:The usefulness of AS PCR assays was evaluated with 391 clinical specimens (251 Acid fast bacilli smear positive and MTB culture positive; 93 smear negative and MTB culture positive; 47 smear positive and MTB culture negative) and 344 MTB culture positive isolates. With culture-based phenotypic DST as a reference standard, the sensitivity and specificity of the NAS-PCR, NMAS-PCR and MAS-PCR assay for drug resistance-related genetic mutation detection were 98.6 and 97.8 per cent for INH, 97.5 and 97.9 per cent for RIF and 98.9 and 100 per cent for multidrug resistance (MDR).Interpretation & conclusions:The performance of AS PCR assays showed that those could be less expensive and technically executable methods for rapid detection of MDR-TB directly from clinical specimens.

A First Insight into the katG and rpoB Gene Mutations of Multidrug-Resistant Mycobacterium tuberculosis Strains from Ecuador.

The aim of this study was to characterize the most frequent mutations associated with rifampicin (RIF) and isoniazid (INH) resistance of Mycobacterium tuberculosis isolates from Ecuador. Sequence analysis of 40 strains, resistant for the tuberculosis drugs INH, RIF, or for both showed that of the 31 strains with resistance to INH, 20 strains (64.5%) carried a mutation in the katG gene (codon 315). Eight INH-resistant strains carried a mutation in the katG gene at codon 463. This katG463 mutation, considered a phylogenetic marker, was exclusively found in INH-resistant strains and not in 121 INH-susceptible strains. Of the 35 strains resistant to RIF, 33 (93.9%) had mutations in the hot spot region of the rpoB gene, predominantly in codons 531, 516, and 526. Our results show that sequence-based detection for drug resistance of the katG will identify, respectively, 64.5% or, considering katG463 as a marker, 90.3% of the INH-resistant strains. Sequencing of the hot spot region of the rpoB gene will detect 94.3% of the RIF drug-resistant isolates in Ecuador. This is appropriate for fast screening for drug resistance with the GeneXpert MTB/RIF assay or by direct sequencing of a part of the genes katG and rpoB of PCR products obtained from DNA isolation from primary cultures.

Trend of multidrug and fluoroquinolone resistance in Mycobacterium tuberculosis isolates from 2010 to 2014 in Korea: a multicenter study.

Background/AimsThis study was conducted to evaluate the recent prevalence and trend of anti-tuberculosis (TB) drug resistance with a focus on multidrug-resistance (MDR) and fluoroquinolone resistance in South Korea.MethodsWe retrospectively reviewed the drug susceptibility testing results of culture-confirmed Mycobacterium tuberculosis isolates collected from 2010 to 2014 at seven tertiary hospitals in South Korea.ResultsA total of 5,599 cases were included: 4,927 (88.0%) were new cases and 672 (12.0%) were previously treated cases. The MDR rate has significantly decreased from 6.0% in 2010 to 3.0% in 2014 among new cases, and from 28.6% in 2010 to 18.4% in 2014 among previously treated cases (p < 0.001 and p = 0.027, respectively). The resistance rate to any fluoroquinolone was 0.8% (43/5,221) in non-MDR-TB patients, as compared to 26.2% (99/378) in MDR-TB patients (p < 0.001). There was no significant change in the trend of fluoroquinolone resistance among both nonMDR-TB and MDR-TB patients. Among the 43 non-MDR-TB patients with fluoroquinolone resistance, 38 (88.4%) had fluoroquinolone mono-resistant isolates.ConclusionsThe prevalence of MDR-TB has significantly decreased from 2010 to 2014. The prevalence of fluoroquinolone resistance among non-MDR-TB patients was low, but the existence of fluoroquinolone mono-resistant TB may be a warning on the widespread use of fluoroquinolone in the community.

Prevalence of mutations in genes associated with isoniazid resistance in Mycobacterium tuberculosis isolates from re-treated smear-positive pulmonary tuberculosis patients: A meta-analysis.

The prevalence of isoniazid (INH) monoresistance is high in India. In this study, molecular epidemiological characteristics associated with INH resistance mutations in Mycobacterium tuberculosis in codon katG315 and in the promoter region of the inhA gene were investigated. Sputum specimens of smear-positive tuberculosis (TB) patients were subjected to GenoType MTBDRplus assay to identify katG and inhA mutations in M. tuberculosis. In addition to the current study, 17 publications assessed 14100 genotypically resistant M. tuberculosis isolates for mutations in katG inclusive of codon 315. In total, 1821 (11.8%) of 15438 INH-resistant strains had detectable mutations: 71.0% in katG315 and 29.0% in the inhA promoter region. The prevalence of IHN monoresistance was 89.1% in the economically-active age group, 0.4% in the paediatric age group and 10.5% in those aged >60years; the rate in males and females was 12.0% and 10.8%, respectively. Meta-analysis derived a pooled katGS315T resistant TB prevalence of 67.3% (95% CI 59.3-75.4%) with Q=732.19, I2=98.35% and P=0.000 for treated TB cases. INH resistance was spread widely and transmission of INH-resistant isolates, especially with katG315T mutation, was confirmed. Therefore, it is important to diagnose katG315T mutants among INH-resistant strains as it may be a risk factor for subsequent development of multidrug-resistant TB (MDR-TB). Prompt detection of patients with INH-resistant strains would expedite modification of treatment regimens, and appropriate infection control measures could be taken in time to diminish the risk of further development and transmission of MDR-TB.

Genetic sequencing for surveillance of drug resistance in tuberculosis in highly endemic countries: a multi-country population-based surveillance study

SummaryBackgroundIn many countries, regular monitoring of the emergence of resistance to anti-tuberculosis drugs is hampered by the limitations of phenotypic testing for drug susceptibility. We therefore evaluated the use of genetic sequencing for surveillance of drug resistance in tuberculosis.MethodsPopulation-level surveys were done in hospitals and clinics in seven countries (Azerbaijan, Bangladesh, Belarus, Pakistan, Philippines, South Africa, and Ukraine) to evaluate the use of genetic sequencing to estimate the resistance of Mycobacterium tuberculosis isolates to rifampicin, isoniazid, ofloxacin, moxifloxacin, pyrazinamide, kanamycin, amikacin, and capreomycin. For each drug, we assessed the accuracy of genetic sequencing by a comparison of the adjusted prevalence of resistance, measured by genetic sequencing, with the true prevalence of resistance, determined by phenotypic testing.FindingsIsolates were taken from 7094 patients with tuberculosis who were enrolled in the study between November, 2009, and May, 2014. In all tuberculosis cases, the overall pooled sensitivity values for predicting resistance by genetic sequencing were 91% (95% CI 87–94) for rpoB (rifampicin resistance), 86% (74–93) for katG, inhA, and fabG promoter combined (isoniazid resistance), 54% (39–68) for pncA (pyrazinamide resistance), 85% (77–91) for gyrA and gyrB combined (ofloxacin resistance), and 88% (81–92) for gyrA and gyrB combined (moxifloxacin resistance). For nearly all drugs and in most settings, there was a large overlap in the estimated prevalence of drug resistance by genetic sequencing and the estimated prevalence by phenotypic testing.InterpretationGenetic sequencing can be a valuable tool for surveillance of drug resistance, providing new opportunities to monitor drug resistance in tuberculosis in resource-poor countries. Before its widespread adoption for surveillance purposes, there is a need to standardise DNA extraction methods, recording and reporting nomenclature, and data interpretation.FundingBill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development.

The occurrence and frequency of genomic mutations that mediate Isoniazid and Rifampicin resistance in Mycobacterium tuberculosis isolates from untreated pulmonary Tuberculosis cases in urban Blantyre, Malawi.

The emergence and spread of drug-resistant Tuberculosis (TB) is a major public health threat. TB resistance originates in the course of treatment due to genomic mutations in Mycobacterium tuberculosis (MTB). An increase in new cases with drug-resistant TB could be an indicator of high levels of circulating resistant strains. This study was conducted to determine the occurrence and frequency of genomic mutations that mediate Isoniazid (INH) and Rifampicin (RIF) resistance among isolates from untreated TB cases in urban Blantyre, Malawi. A cross-sectional retrospective study was conducted on a panel of 141(n=141) MTB clinical isolates recovered between June 2010 and January 2012 from >2+ Ziehl-Neelsen smear positive new pulmonary-TB patients with no history of treatment. Frozen isolates were revived using the BACTEC MGIT detection system. DNA was extracted using GenoLyse DNA extraction kit and detection of genomic mutations was carried out using the GenoType MTBDRplus Ver 2.0 assay. Out of the 141 isolates studied, 3 (2.1%) were found carrying mutations in the katG gene that confer resistance to Isoniazid (INH). No mutations were detected in the inhA promoter region gene that confer weak INH resistance or in the rpoB gene that confer Rifampicin resistance. All katG mutant genes had a S315T1 single point mutation, a genomic alteration that mediates high INH resistance. The katG mutant gene conferring resistance to INH was the only genomic mutation observed among the isolates studied and the frequency of occurrence was low. Our findings suggest low levels of circulating drug-resistant MTB strains in urban Blantyre, Malawi.

Initial second-line drug resistance of Mycobacterium tuberculosis isolates from Sudanese retreatment-patients

SettingMultidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) are a major public health threat. ObjectiveThis study aimed to determine resistance patterns to second line anti-TB drugs (SLDs), and to determine the frequency of extensively drug resistant Mycobacterium tuberculosis (XDR-TB). DesignDuring the period from July 2009 to July 2010; sputum specimens were collected from TB retreatment patients; isolates were tested for sensitivity to first line anti-TB drugs by the 1% proportion method; MDR strains were tested for second line anti-TB drugs sensitivity by 1% proportion method and by version 1. Hain GenoType MTBDRsl Assay. ResultsOne hundred and forty three mycobacterial isolates were successfully recovered from a total of 239 specimens (143/239; 59.8%). Fifty six strains were rifampicin resistant (RR); of these 54 were multi-drug resistant (MDR); two were RIF/INH-resistant mycobacterium other than tuberculosis (MOTT). Five of MDR (5/50; 10%) showed resistance to at least one second line drug and one isolate (1/50; 2%) was XDR. The XDR strain was concordantly detected by the two methods. ConclusionInitial resistance to second line anti-TB drugs among MDR-TB patients is at 10% levels and XDR-TB is prevalent at low levels (2%). Nevertheless; without great efforts from national tuberculosis control program (NTP) this figure can fuel the TB epidemics in Sudan.

Characterization of gyrA and gyrB mutations associated with fluoroquinolone resistance in Mycobacterium tuberculosis isolates from Morocco

Fluoroquinolones (FQs) are the cornerstone of treatment for drug-resistant tuberculosis (TB). They are the most effective second-line antimycobacterial drugs and are recommended for the treatment of multidrug-resistant TB (MDR-TB). However, it is widely accepted that FQ resistance is high among MDR-TB isolates. Thus, characterisation of mutations conferring resistance to FQs will be of a great interest for effective and efficient management of TB resistance in Morocco. A laboratory collection of 30 Mycobacterium tuberculosis isolates previously characterised as phenotypically and genotypically MDR as well as 20 randomly selected pan-susceptible isolates were included in this retrospective study. The mutation profiles associated with resistance to FQs were assessed by PCR and DNA sequencing. Target sequences for two genes (gyrA and gyrB) were examined. All strains had their fingerprint previously established by spoligotyping. Molecular analyses showed that 30% of the MDR-TB isolates harboured FQ resistance mutations in gyrA, with the most prevalent being an alanine to threonine at position 90 (Ala90Thr) (56%; 5/9). None of the isolates harboured mutations in gyrB. All gyrA resistance mutant strains belonged to the LAM lineage, mostly LAM9, raising the possible emergence of a specific clone (gyrA mutant/LAM9). The results of this preliminary study highlight the high prevalence of FQ resistance among MDR-TB isolates in Morocco and consequently the need for rapid detection of FQ resistance once MDR-TB is confirmed to adjust treatment in a timely manner and to interrupt the propagation of more severe forms of M. tuberculosis drug resistance.

Population-based resistance of Mycobacterium tuberculosis isolates to pyrazinamide and fluoroquinolones: results from a multicountry surveillance project

SummaryBackgroundPyrazinamide and fluoroquinolones are essential antituberculosis drugs in new rifampicin-sparing regimens. However, little information about the extent of resistance to these drugs at the population level is available.MethodsIn a molecular epidemiology analysis, we used population-based surveys from Azerbaijan, Bangladesh, Belarus, Pakistan, and South Africa to investigate resistance to pyrazinamide and fluoroquinolones among patients with tuberculosis. Resistance to pyrazinamide was assessed by gene sequencing with the detection of resistance-conferring mutations in the pncA gene, and susceptibility testing to fluoroquinolones was conducted using the MGIT system.FindingsPyrazinamide resistance was assessed in 4972 patients. Levels of resistance varied substantially in the surveyed settings (3·0–42·1%). In all settings, pyrazinamide resistance was significantly associated with rifampicin resistance. Among 5015 patients who underwent susceptibility testing to fluoroquinolones, proportions of resistance ranged from 1·0–16·6% for ofloxacin, to 0·5–12·4% for levofloxacin, and 0·9–14·6% for moxifloxacin when tested at 0·5 μg/mL. High levels of ofloxacin resistance were detected in Pakistan. Resistance to moxifloxacin and gatifloxacin when tested at 2 μg/mL was low in all countries.InterpretationAlthough pyrazinamide resistance was significantly associated with rifampicin resistance, this drug may still be effective in 19–63% of patients with rifampicin-resistant tuberculosis. Even though the high level of resistance to ofloxacin found in Pakistan is worrisome because it might be the expression of extensive and unregulated use of fluoroquinolones in some parts of Asia, the negligible levels of resistance to fourth-generation fluoroquinolones documented in all survey sites is an encouraging finding. Rational use of this class of antibiotics should therefore be ensured to preserve its effectiveness.FundingBill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development.