RIF-resistant Isolates Research Articles

Unveiling the complexity of rifampicin drug susceptibility testing in Mycobacterium tuberculosis: comparative analysis with next-generation sequencing.

Introduction. The discordance between phenotypic and molecular methods of rifampicin (RIF) drug susceptibility testing (DST) in Mycobacterium tuberculosis poses a significant challenge, potentially resulting in misdiagnosis and inappropriate treatment.Hypothesis/gap statement. A comparison of RIF phenotypic and molecular methods for DST, including whole genome sequencing (WGS), may provide a better understanding of resistance mechanisms.Aim. This study aims to compare RIF DST in M. tuberculosis using two phenotypic and molecular methods including the GeneXpert RIF Assay (GX) and WGS for better understanding.Methodology. The study evaluated two phenotypic liquid medium methods [Lowenstein-Jensen (LJ) and Mycobacterium Growth Indicator Tube (MGIT)], one targeted molecular method (GX), and one WGS method. Moreover, mutational frequency in ponA1 and ponA2 was also screened in the current and previous RIF resistance M. tuberculosis genomic isolates to find their compensatory role.Results. A total of 25 RIF-resistant isolates, including nine from treatment failures and relapse cases with both discordant and concordant DST results on LJ, MGIT and GX, were subjected to WGS. The phenotypic DST results indicated that 11 isolates (44%) were susceptible on LJ and MGIT but resistant on GX. These isolates exhibited multiple mutations in rpoB, including Thr444>Ala, Leu430>Pro, Leu430>Arg, Asp435>Gly, His445>Asn and Asn438>Lys. Conversely, four isolates that were susceptible on GX and MGIT but resistant on LJ were wild type for rpoB in WGS. However, these isolates possessed several novel mutations in the PonA1 gene, including a 10 nt insertion and two nonsynonymous mutations (Ala394>Ser, Pro631>Ser), as well as one nonsynonymous mutation (Pro780>Arg) in PonA2. The discordance rate of RIF DST is higher on MGIT than on LJ and GX when compared to WGS. These discordances in the Delhi/CAS lineages were primarily associated with failure and relapse cases.Conclusion. The WGS of RIF resistance is relatively expensive, but it may be considered for isolates with discordant DST results on MGIT, LJ and GX to ensure accurate diagnosis and appropriate treatment options.

Patterns and profiles of drug resistance-conferring mutations in Mycobacterium tuberculosis genotypes isolated from tuberculosis-suspected attendees of spiritual holy water sites in Northwest Ethiopia.

This study examined the patterns and frequency of genetic changes responsible for resistance to first-line (rifampicin and isoniazid), fluoroquinolones, and second-line injectable drugs in drug-resistant Mycobacterium tuberculosis (MTB) isolated from culture-positive pulmonary tuberculosis (PTB) symptomatic attendees of spiritual holy water sites (HWSs) in the Amhara region. From June 2019 to March 2020, a cross-sectional study was carried out. A total of 122 culture-positive MTB isolates from PTB-suspected attendees of HWSs in the Amhara region were evaluated for their drug resistance profiles, and characterized gene mutations conferring resistance to rifampicin (RIF), isoniazid (INH), fluoroquinolones (FLQs), and second-line injectable drugs (SLIDs) using GenoType®MTBDRplus VER2.0 and GenoType®MTBDRsl VER2.0. Drug-resistant MTB isolates were Spoligotyped following the manufacturer's protocol. Genetic changes (mutations) responsible for resistance to RIF, INH, and FLQs were identified in 15/122 (12.3%), 20/122 (16.4%), and 5/20 (25%) of MTB isolates, respectively. In RIF-resistant, rpoB/Ser531Lue (n = 12, 80%) was most frequent followed by His526Tyr (6.7%). Amongst INH-resistant isolates, katG/Ser315Thr1 (n = 19, 95%) was the most frequent. Of 15 MDR-TB, the majority (n = 12, 80%) isolates had mutations at both rpoB/Ser531Leu and katG/Ser315Thr1. All 20 INH and/or RIF-resistant isolates were tested with the MTBDRsl VER 2.0, yielding 5 FLQs-resistant isolates with gene mutations at rpoB/Ser531Lue, katG/Ser315Thr1, and gyrA/Asp94Ala genes. Of 20 Spoligotyped drug-resistant MTB isolates, the majority (n = 11, 55%) and 6 (30%) were SIT149/T3-ETH and SIT21/CAS1-Kili sublineages, respectively; and they were any INH-resistant (mono-hetero/multi-). Of 15 RIF-resistant (RR/MDR-TB) isolates, 7 were SIT149/T3-ETH, while 6 were SIT21/CAS1-Kili sublineages. FLQ resistance was detected in four SIT21/CAS1-Kili lineages. In the current study, the most common gene mutations responsible for resistance to INH, RIF, and FLQs were identified. SIT149/T3-ETH and SIT21/CAS1-Kili constitute the majority of drug-resistant TB (DR-TB) isolates. To further understand the complete spectrum of genetic changes/mutations and related genotypes, a sequencing technology is warranted.

Insights into the in-vitro Susceptibility and Drug-Drug Interaction Profiles Against Drug-Resistant and Susceptible Mycobacterium tuberculosis Clinical Isolates in Amhara, Ethiopia.

In Ethiopia, tuberculosis (TB) is a major public health problem. The aim of the study was to determine the in vitro susceptibility level of drugs and drug interaction profiles against drug-resistant and susceptible M. tuberculosis clinical isolates. A laboratory-based cross-sectional study was conducted between January 2023 and August 2023. GenoType MTBDRplus v.2.0 was facilitated in genetic mutation detection. Minimum inhibitory concentration (MIC) was determined using resazurin microtitre assay (REMA), while fractional inhibitory concentration index (FICI) using resazurin drug combination microtitre assay (REDCA) for in vitro quantitative susceptibility and drug interaction prediction. Among 32 clinical isolates, a total of 14 (43.8%) RIF, 20 (62.5%) INH, 2 (6.3%) EMB-related resistant and 14 (43.8%) MDR isolates were identified. Five of RIF-resistant isolates (55.6%) carrying rpoB common mutations at codon S450L were associated with high levels of RIF-resistance with MICs of ≥ 2μg/mL, whereas 100% of isolates harboring rpoB substitutions at codons D435V and H445Y were linked with moderate or low-level RIF-resistance in the MIC ranges from 0.5 to 1μg/mL. A proportion of 81.8% of isolates harboring katG S315T mutations were associated with high-level INH resistance (MIC ≥ 1μg/mL), while the 18.2% of isolates with S315T katG mutations and 100% of isolates with inhA C-15T mutations were linked to the low-level of INH resistance with MIC variability from 0.25 to 0.5μg/mL. Our results indicated that most FICIs of the dual drugs INH+RIF and INH+LEV combination for 9 (28.1%) and 4 (12.5%) INH-resistant isolates, respectively, were ≤0.5, whereas triple drugs INH+RIF+EMB, INH+RIF+LEV and INH+EMB+LEV combination for 6 (18.8%), 11 (34.4%) and 8 (25%) INH-resistant isolates were from 0.62 to 0.75, all showed synergistic effect. The study highlights that isolates with rpoB S450L and katG S315T substitutions were associated with high level of RIF and INH resistance. It is concluded that REDCA can quantitatively determine anti-mycobacterial synergy and that LEV being of potential use against INH-resistant isolates including MDR-TB when combined with RIF+INH and INH+EMB.

Drug resistance and the genotypic characteristics of rpoB and katG in rifampicin- and/or isoniazid-resistant Mycobacterium tuberculosis isolates in central Vietnam.

Tuberculosis (TB) and drug-resistant TB (DR-TB) are national health burdens in Vietnam. In this study, we investigated the prevalence of rifampicin (RIF) and/or isoniazid (isonicotinic acid hydrazide, INH) resistance in patients with suspected TB, and applied appropriate techniques to help rapidly target DR-TB. In total, 1,547 clinical specimens were collected and cultured using the BACTEC MGIT system (Becton Dickinson and Co.). A resazurin microtiter assay (REMA) was used to determine the proportions of RIF and/or INH resistance. A real-time polymerase chain reaction panel with TaqMan probes was employed to identify the mutations of rpoB and katG associated with DR-TB in clinical isolates. Genotyping of the identified mutations was also performed. A total of 468 Mycobacterium tuberculosis isolates were identified using the REMA. Of these isolates, 106 (22.6%) were found to be resistant to 1 or both antibiotics. Of the resistant isolates, 74 isolates (69.8%) were resistant to isoniazid (INH) only, while 1 isolate (0.94%) was resistant to RIF only. Notably, 31 isolates (29.24%) were resistant to both antibiotics. Of the 41 phenotypically INH-resistant isolates, 19 (46.3%) had the Ser315Thr mutation. There were 8 different rpoB mutations in 22 (68.8%) of the RIF-resistant isolates. The most frequently detected mutations were at codons 531 (37.5%), 526 (18.8%), and 516 (6.3%). To help prevent new cases of DR-TB in Vietnam, it is crucial to gain a comprehensive understanding of the genotypic DR-TB isolates.

Isoniazid resistance profile in rifampicin resistant Mycobacterium tuberculosis

Background: Multidrug-resistant tuberculosis (MDR-TB) is a global public health problem. Rifampicin (RIF) resistance has been used as a surrogate marker for MDR-TB but isoniazid (INH) resistance within RIF resistance cases is little known. This study aimed to determine the proportion of INH resistance among RIF-resistant MTB. Methods: In this cross-sectional study, from March 2021 to February 2022, 53 RIF-resistant MTB isolates in sputum samples detected by Xpert-MTB RIF assay were enrolled. All samples were tested for mutation in katG (codon 315) and inhA promoter (-5, -8, -15 and -16) genes to detect INH resistance by real-time PCR. Statistical analysis was done using IBM SPSS (version 26). Results: Out of 53 RIF-resistant samples, 15.1% were sensitive to INH, and the rest had concomitant resistance to INH. The proportion of newly diagnosed and previously treated cases was nearly equal, and most of the previously treated cases (92.9%) received treatment regularly. INH-resistant cases were mostly previously treated (55.5%), whereas sensitive cases were mostly newly diagnosed (62.5%). KatG was found to be the prominent mutation, with or without in combination with inhA mutation. Conclusion: A considerable number of RIF-resistant isolates did not show concomitant resistance to INH. Most of the INH-resistant isolates were associated with katG mutation. Evaluation of INH resistance before using high-dose INH will help to avoid dose-dependent toxicity in MDR-TB patients. Bangabandhu Sheikh Mujib Medical University Journal 2023;16(3): 160-166

Profile and Frequency of Mutations Conferring Drug-Resistant Tuberculosis in the Central, Southeastern and Eastern Ethiopia.

Advances in molecular tools that assess genes harboring drug resistance mutations have greatly improved the detection and treatment of drug-resistant tuberculosis (DR-TB). This study was conducted to determine the frequency and type of mutations that are responsible for resistance to rifampicin (RIF), isoniazid (INH), fluoroquinolones (FLQs) and second-line injectable drugs (SLIDs) in Mycobacterium tuberculosis (MTB) isolates obtained from culture-positive pulmonary tuberculosis (TB) patients in the central, southeastern and eastern Ethiopia. In total, 224 stored culture-positive MTB isolates from pulmonary TB patients referred to Adama and Harar regional TB laboratories between August 2018 and January 2019 were assessed for mutations conferring RIF, INH, FLQs and SLIDs resistance using GenoType®MTBDRplus (MTBDRplus) and GenoType®MTBDRsl (MTBDRsl). RIF, INH, FLQs and SLIDs resistance-conferring mutations were identified in 88/224 (39.3%), 85/224 (38.0%), 7/77 (9.1%), and 3/77% (3.9%) of MTB isolates, respectively. Mutation codons rpoB S531L (59.1%) for RIF, katG S315T (96.5%) for INH, gyrA A90V (42.1%) for FLQs and WT1 rrs (100%) for SLIDs were observed in the majority of the isolates tested. Over a 10th of rpoB mutations detected in the current study were unknown. In this study, the most common mutations conferring drug resistance to RIF, INH, FLQs were identified. However, a significant proportion of RIF-resistant isolates manifested unknown rpoB mutations. Similarly, although few in number, all SLID-resistant isolates had unknown rrs mutations. To further elucidate the entire spectrum of mutations, tool such as whole-genome sequencing is imperative. Furthermore, the expansion of molecular drug susceptibility testing services is critical for tailoring patient treatment and preventing disease transmission.

Evaluation of a Molecular Test for Detection of Mycobacterium tuberculosis Isolates Resistant to Rifampicin and Isoniazid.

Multidrug-resistant tuberculosis (MDR-TB) is increasing worldwide and is a major cause of death in many countries. It has become a major challenge for national tuberculosis control programs. Therefore, rapid identification of MDR strains of Mycobacterium tuberculosis and monitoring of their transmission could contribute significantly to the fight against tuberculosis. The GenoType MTBDRplus assay has been recommended by the World Health Organization to identify rifampicin (RIF)- and isoniazid (INH)-resistant M. Tuberculosis isolates. The objectives of this study were to evaluate the performance of the GenoType MTBDRplus test in the detection of rifampicin and isoniazid resistance of M. tuberculosis isolates in a Moroccan hospital and then to determine the frequency of mutations associated with resistance to these two major anti-tuberculosis drugs. This is a retrospective study conducted at the bacteriology department of the Mohammed V military hospital over a period of one year from 01/01/2018 to 12/31/2019. A total of 92 isolates of M. tuberculosis from pulmonary and extra-pulmonary specimens were evaluated for drug susceptibility by MGIT™ 960 AST system and compared to the GenoType MTBDRplus assay. The MGIT™ 960 AST system was used as the gold standard for the evaluation of the GenoType MTBDRplus assay. Sensitivity and specificity of the GenoType MTBDRplus assay for the detection of RIF-resistant M. tuberculosis isolates were 83.33% and 100%, respectively. Its sensitivity and specificity for the detection of INH-resistant M. tuberculosis were 88.23% and 100% respectively. The concordances of the GenoType MTBDRplus assay and the MGIT™ 960 AST system for the detection of sensitivity to RIF and INH were 99% (1/92) and 98% (2/92), respectively. Among the five RIF-resistant isolates, the MUT3 mutation in the rpoB gene (codon S531L mutation) was present in 80% of isolates, whereas mutations in the rpoB MUT1 gene were present in only one (20%) RIF-resistant isolate. INH resistance was detected in 15 isolates, of which nine isolates (60%) had specific mutations of the katG gene (codon S315T1) and conferred a high level of resistance to INH. The results of this study have shown that the GenoType MTBDRplus test has a high sensitivity and specificity for the detection of resistance to RIF and INH.

Discordant results between genotypic and phenotypic assays (Xpert MTB/RIF vs. BACTEC MGIT 960 system) for detection of RIF-resistant Mycobacterium tuberculosis isolates in a high burden region.

Clinical isolates with discordant phenotypic and genotypic results were submitted to DNA sequencing to identify which were genuinely resistant to rifampin and determine the frequency of silent and disputed mutations in our region. We present the retrospective analysis of all the culture-proven TB cases tested with the Xpert®MTB/RIF assay at the Tuberculosis Clinic and Laboratory of the Tijuana General Hospital, Mexico. Clinical isolates showing a discrepancy between phenotypic and molecular tests were analyzed by DNA sequencing. Thirteen isolates tested as rifampin susceptible on the MGIT system were rifampin-resistant according to Xpert®MTB/RIF assay. DNA sequencing showed that seven (53.8%) isolates had a silent (P514P) mutation; three isolates showed different missense (L511P, D516Y, and S531L) mutations. Three isolates showed no mutations. The existence of heteroresistance and silent or disputed mutations warrants that all rifampin-resistance cases diagnosed with the Xpert®MTB/RIF should be referred to specialized centers for DNA sequencing.

Profiling and identification of novel rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis clinical isolates from Pakistan

IntroductionRifampicin (RIF) is one of the most effective anti-tuberculosis first-line drugs prescribed along with isoniazid. However, the emergence of RIF resistance Mycobacterium tuberculosis (MTB) isolates is a major issue towards tuberculosis (TB) control program in high MDR TB-burdened countries including Pakistan. Molecular data behind phenotypic resistance is essential for better management of RIF resistance which has been linked with mutations in rpoB gene. Since molecular studies on RIF resistance is limited in Pakistan, the current study was aimed to investigate the molecular data of mutations in rpoB gene behind phenotypic RIF resistance isolates in Pakistan. MethodA total of 322 phenotypically RIF-resistant isolates were randomly selected from National TB Reference Laboratory, Pakistan for sequencing while 380 RIF resistance whole-genome sequencing (WGS) of Pakistani isolates (BioProject PRJEB25972), were also analyzed for rpoB mutations. ResultAmong the 702 RIF resistance samples, 675 (96.1%) isolates harbored mutations in rpoB in which 663 (94.4%) were detected within the Rifampicin Resistance Determining Region (RRDR) also known as a mutation hot spot region, including three novel. Among these mutations, 657 (97.3%) were substitutions including 603 (89.3%) single nucleotide polymorphism, 49 (7.25%) double and five (0.8%) triple. About 94.4% of Phenotypic RIF resistance strains, exhibited mutations in RRDR, which were also detectable by GeneXpert. ConclusionMutations in the RRDR region of rpoB is a major mechanism of RIF resistance in MTB circulating isolates in Pakistan. Molecular detection of drug resistance is a faster and better approach than phenotypic drug susceptibility testing to reduce the time for transmission of RIF resistance strains in population. Such insights will inform the deployment of anti-TB drug regimens and disease control tools and strategies in high burden settings, such as Pakistan.

Rifampicin resistance in Mycobacterium tuberculosis in Iran: a two-centre study

Multidrug-resistant tuberculosis remains a challenge. In this study, we investigated the incidence of rifampicin (RIF) resistance in Mycobacterium tuberculosis in a large number of pulmonary specimens.A two-center study in Tehran, the capital of Iran, was performed with 6624 pulmonary samples of patients with tuberculosis (TB) who were subjected to detection of RIF-resistant TB by GeneXpert MTB/RIF assay between May 2014 and July 2018. Conventional drug susceptibility testing was performed to confirm the results.Xpert MTB/RIF identified a total of 96 positives for M. tuberculosis, of which 5 (5.3%) samples were found to be RIF-resistant TB. All RIF-resistant and sensitive isolates detected by GeneXpert were phenotypically confirmed by drug susceptibility testing.These results indicated that the Xpert MTB/RIF test can be used as a rapid diagnostic method and can potentially decrease the morbidity associated with diagnostic delay and mistreatment.

Analysis of drug resistance and mutation profiles in Mycobacterium tuberculosis isolates in a surveillance site in Beijing, China

ObjectiveThis study analyzed drug resistance and mutations profiles in Mycobacterium tuberculosis isolates in a surveillance site in Huairou District, Beijing, China.MethodsThe proportion method was used to assess drug resistance profiles for four first-line and seven second-line anti-tuberculosis (TB) drugs. Molecular line probe assays were used for the rapid detection of resistance to rifampicin (RIF) and isoniazid (INH).ResultsAmong 235 strains of M. tuberculosis, 79 (33.6%) isolates were resistant to one or more drugs. The isolates included 18 monoresistant (7.7%), 19 polyresistant (8.1%), 28 RIF-resistant (11.9%), 24 multidrug-resistant (MDR) (10.2%), 7 pre-extensively drug-resistant (XDR, 3.0%), and 2 XDR strains (0.9%). A higher rate of MDR-TB was detected among previously treated patients than among patients with newly diagnosed TB (34.5% vs. 6.8%). The majority (62.5%) of RIF-resistant isolates exhibited a mutation at S531L in the DNA-dependent RNA polymerase gene. Meanwhile, 62.9% of INH-resistant isolates carried a mutation at S315T1 in the katG gene.ConclusionOur results confirmed the high rate of drug-resistant TB, especially MDR-TB, in Huairou District, Beijing, China. Therefore, detailed drug testing is crucial in the evaluation of MDR-TB treatment.

Interpretation of Discordant Rifampicin Susceptibility Test Results Obtained Using GeneXpert vs Phenotypic Drug Susceptibility Testing.

BackgroundThe 3-month difference in turnaround time between Xpert and conventional phenotypic drug susceptibility testing (pDST) causes patient treatment challenges when pDST rifampin (RIF) susceptibility results and earlier Xpert results disagree, resulting in unnecessary tuberculosis (TB) patient exposure to toxic second-line drugs. Here, the prevalence of discordant RIF susceptibility test results, specifically Xpert (resistant) vs pDST (susceptible) results, was determined.MethodsTuberculosis patients enrolled between January 2015 and June 2018 at Beijing Chest Hospital who consecutively tested positive for RIF resistance using Xpert then negative using pDST were studied. DNA sequences and minimal inhibitory concentration (MIC) results provided insights for understanding discordant results.ResultsOf 26 826 patients with suggestive TB symptoms undergoing Xpert MTB/RIF testing, 728 diagnosed as RIF-resistant were evaluated. Of these, 118 (16.2%) exhibiting Xpert RIF resistance and phenotypic RIF susceptibility yielded 104 successfully subcultured isolates; of these, 86 (82.7%) harbored rpoB gene RIF resistance–determining region mutations and 18 (17.3%) did not. The Leu511Pro (25.0%) and Leu533Pro (17.3%) mutations were most frequently associated with discordant RIF susceptibility test results. Of the 86 isolates with rpoB mutations, 42 (48.8%) with MICs ≤1.0 mg/L were assigned to the RIF-susceptible group, with Leu511Pro being the most common mutation observed. Isolates with a very low bacterial load were most frequently misdiagnosed as RIF-resistant by Xpert.ConclusionsApproximately one-sixth of RIF-resistant TB isolates identified via Xpert yielded discordant pDST results due to questionable interpretation of specific “disputed” mutations. Thus, a diagnostic flowchart should be used to correctly interpret Xpert RIF resistance results to best guide patient treatment.

Xpert Mycobacterium tuberculosis/Rifampicin-Detected Rifampicin Resistance is a Suboptimal Surrogate for Multidrug-resistant Tuberculosis in Eastern Democratic Republic of the Congo: Diagnostic and Clinical Implications.

Rifampicin (RIF) resistance is highly correlated with isoniazid (INH) resistance and used as proxy for multidrug-resistant tuberculosis (MDR-TB). Using MTBDRplus as a comparator, we evaluated the predictive value of Xpert MTB/RIF (Xpert)-detected RIF resistance for MDR-TB in eastern Democratic Republic of the Congo (DRC). We conducted a cross-sectional study involving data from new or retreatment pulmonary adult TB cases evaluated between July 2013 and December 2016. Separate, paired sputa for smear microscopy and MTBDRplus were collected. Xpert testing was performed subject to the availability of Xpert cartridges on sample remnants after microscopy. Among 353 patients, 193 (54.7%) were previously treated and 224 (63.5%) were MTBDRplus TB positive. Of the 224, 43 (19.2%) were RIF monoresistant, 11 (4.9%) were INH monoresistant, 53 (23.7%) had MDR-TB, and 117 (52.2%) were RIF and INH susceptible. Overall, among the 96 samples detected by MTBDRplus as RIF resistant, 53 (55.2%) had MDR-TB. Xpert testing was performed in 179 (50.7%) specimens; among these, 163 (91.1%) were TB positive and 73 (44.8%) RIF resistant. Only 45/73 (61.6%) Xpert-identified RIF-resistant isolates had concomitant MTBDRplus-detected INH resistance. Xpert had a sensitivity of 100.0% (95% CI, 92.1-100.0) for detecting RIF resistance but a positive-predictive value of only 61.6% (95% CI, 49.5-72.8) for MDR-TB. The most frequent mutations associated with RIF and INH resistance were S531L and S315T1, respectively. In this high-risk MDR-TB study population, Xpert had low positive-predictive value for the presence of MDR-TB. Comprehensive resistance testing for both INH and RIF should be performed in this setting.

Molecular Analysis of Rifampicin Resistance Conferring Mutations in Mycobacterium tuberculosis

Mycobacterium tuberculosis resistance to rifampicin is mainly mediated through mutations in the rpoB gene. The effects of rpoB mutations are relieved by secondary mutations in rpoA or rpoC genes. This study aims to identify mutations in rpoB, rpoA, and rpoC genes of Mycobacterium tuberculosis isolates and clarify their contribution to rifampicin resistance. Seventy isolates were identified by acid-fast bacilli smear, Genexpert assay, and growth on Lowenstein Jensen medium. Drug susceptibility, testing was performed by the proportional method. DNA extraction, PCR, and sequencing were accomplished for the entire rpoA, rpoB, and rpoC genes. Twenty-three isolates (32.85%) showed resistance to rifampicin by either proportion method or Genexpert assay. Sequence analysis of the rpoB gene revealed fourteen different mutation patterns. Inside the rifampicin resistance determining region (RRDR), codons: S531L, D516V were highly mutated with frequencies of (21.73%, 17.39%) respectively. Outside the RRDR, there were nine different types of mutations, and M479L was the most prevalent one. Out of 23 RIF resistant isolates, seven isolates (30.43%) carried mutations in the rpoA gene, and twelve isolates (52.17%) harbored a mutation in rpoC. Most of the mutations were identified for the first time in this study. The current study demonstrated that mutations in rpoB, rpoA, and rpoC contributed to RIF resistance in Mycobacterium tuberculosis and this new finding may be relevant to realize how compensatory mutations in the rpoA and rpoC genes restore the fitness cost caused by rifampin resistance-conferring mutations in rpoB.

Mutations in rpoB and katG genes and the inhA operon in multidrug-resistant Mycobacterium tuberculosis isolates from Zambia

ObjectivesIt is established that resistance to rifampicin (RIF) in 90% of RIF-resistant Mycobacterium tuberculosis isolates is attributable to point mutations in the rpoB gene, whilst 50–95% of M. tuberculosis resistance to isoniazid (INH) is caused by mutations in the katG gene. However, the patterns and frequencies of mutations vary by geographical region. In Zambia, the genetic mechanisms of resistance of M. tuberculosis to RIF and INH were unreported before this study. MethodsUsing gene sequencing, the rpoB, katG and inhA genes of 99 multidrug-resistant M. tuberculosis (MDR-TB) and 49 pan-susceptible M. tuberculosis isolates stored at a tuberculosis reference laboratory from 2013 to 2016 were analysed and were compared with published profiles from other African countries. ResultsOf the 99 MDR-TB isolates, 95 (96.0%) carried mutations in both rpoB and katG. No mutations were detected among the pan-susceptible isolates. The most common mutations among RIF- and INH-resistant isolates were in codon 531 of the rpoB gene (55.6%; 55/99) and codon 315 of the katG gene (94.9%; 94/99), respectively. Distinctly, katG mutations were predominantly high among Zambian isolates (96.0%) compared with other countries in the region. ConclusionResistance-associated mutations to RIF and INH circulating in Zambia are similar to those reported globally, therefore these data validate the applicability of molecular diagnostic tools in Zambia. However, katG mutations were predominantly high among M. tuberculosis isolates in this study compared with other regional countries and might distinguish cross-boundary transmission of MDR-TB from other African nations.

Genotypic characterization of ‘inferred’ rifampin mutations in GenoType MTBDRplus assay and its association with phenotypic susceptibility testing of Mycobacterium tuberculosis.

In GenoType MTBDRplus assay [line probe assay (LPA)], when Mycobacterium tuberculosis (M. tuberculosis) sample DNA fails to hybridize to at least 1 rpoB wild-type probe and any mutation probe, it is inferred as rifampin (RIF)-resistant. In this study, we sought to identify such ‘inferred’ mutations in M. tuberculosis isolates (n = 203) by rpoB gene sequencing and determined their association with phenotypic resistance. D516Y, H526N, L511P mutations were associated with both phenotypically sensitive (59%, n = 38/64) and resistant (23.7%, n = 33/139) antimicrobial susceptibility testing (AST) results, whereas S531W mutation was associated with only RIF-resistant isolates (33%, n = 46/139). These results demonstrated that, at standard drug concentrations, some ‘inferred’ mutations may be missed by RIF-AST (phenotypically sensitive). The use of LPA permits identification of these RIF-resistant isolates, and incorporation of additional mutation probes (e.g., S531W) could further increase LPA specificity. Further studies are needed to establish the significance of the type of ‘inferred’ mutation with clinical/treatment outcomes.

Detection of Mutations in 81-bpRifampin Resistance Determining Region (RRDR) of rpoB gene in Mycobacterium tuberculosis using GeneXpert MTB/RIF in Clinical Specimens from Quetta, Pakistan

GeneXpert MTB/RIF has revolutionized the tuberculosis diagnosis by simultaneous detection of Mycobacterium tuberculosis and resistance to RIF (rifampicin), a surrogate marker for multidrug-resistant TB in less than two hours. The RIF-resistance pattern in Balochistan, Pakistan, is not documented. This study was aimed to detect RIF-resistant TB and mutations in RNA polymerase beta (rpoB) gene of M. tuberculosis within 81-bp RRDR in Quetta, Pakistan using GeneXpert® MTB/RIF assay. In total, 2300 clinical specimens were collected from suspected TB patients at Fatima Jinnah General and Chest Hospital Quetta, Pakistan between January and August 2017. These specimens were analyzed by GeneXpert® MTB/RIF assay. The data was statistically analyzed using SPSS software. Out of 2300 clinical specimens, M. tuberculosis was positive in in 899 (39.1%) cases by GeneXpert® MTB/RIF assay [positive respiratory cases 42.9% (871/2032) and non-respiratory 10.4% (28/268) with statistically significant difference (χ2= 104.5, p<0.001)]. Among 899 MTB positive cases, 46 (5.1%) were RIF-resistance caused by various rpoB gene mutations within 81-bp RRDR. Most of the RIF-resistant isolates were observed to harbor mutationsin Probe E 78.3% (n=36) whereas mutations in Probe A, B, D were observed 2.2% (n=1), 4.3% (n=2), and 6.5% (n=3), respectively. However, none of cases had RIF-resistance associated with Probe C. Out of 46 RRD cases, 21 (45.7 %) were males and 25 (54.3 %) were females. Additionally, Xpert® test showed higher detection rate than fluorescent microscopy (39.1% vs 31.2%, P<0.05) and detected MTB in 186 (11.8%) smear-negative specimens. Among 42 confirmed TB patients had MDR contact and eight patients were co-infected with HIV. In conclusion, 5.1% of the TB patients showed rifampicin resistance. The most frequent rpoB genetic mutations were observed in codons 531/533 (Probe E, 78.3%) whereas the least within the sequence 511 (Probe A, 2.2%).

High incidence of drug-resistant Mycobacterium tuberculosis in Hainan Island, China.

To assess the proportion of drug-resistant tuberculosis (TB) cases and to identify independent risk factors associated with drug-resistant TB in Hainan. Descriptive analysis of demographic and clinical data of culture-positive TB patients to assess the trends in drug-resistant TB at the Provincial Clinical Center on Tuberculosis of Hainan between 2014 and 2017. 994 patients were recruited into the study. Overall, the proportion of patients resistant to at least one TB drug tested was 36.1% (359/994). The most frequent resistance was to isoniazid (INH, 29.8%), followed by rifampin (RIF, 29.3%), streptomycin (19.3%), ofloxacin (OFX, 17.4%), ethambutol (9.5%) and kanamycin (KAN, 3.2%). Of 291 RIF-resistant isolates, 228 (78.4%) were also resistant to INH, while the remaining 63 (21.6%) were susceptible to INH. Among those with multidrug-resistant tuberculosis (MDR-TB), 41.2% had additional resistance to OFX and 3.9% to KAN. 8.8% of MDR-TB patients were affected by extensively drug-resistant (XDR-TB). Females were more likely to infected with MDR-TB than males, and young people (<20years old) were more likely to have MDR-TB; patients exhibited decreasing MDR-TB risk with increasing age. Our data provide the first primary understanding of the drug-resistant TB epidemic in Hainan. The high incidence of drug resistance, especially RIF and FQ resistance, highlight the importance of interventions for preventing epidemics of drug-resistant TB. Younger age is an independent predictor of MDR-TB, reflecting the potential transmission in this population.

MYCOBACTERIUM TUBERCULOSIS;

Objectives: The primary objective of this study is to determine the frequency of rpoB gene mutations within 81-bp RRDR in isolates from Quetta, Pakistan using GeneXpert® MTB/RIF assay. Background: Multi drug resistance is one of the major obstacles in the control of tuberculosis throughout the globe. Study Design: A cross sectional experimental study was designed. Setting: A total of 500 clinical specimens obtained from suspected TB patients at Provincial TB Reference Laboratory, Fatima Jinnah General and Chest Hospital Quetta, Pakistan Period: From January to July 2017. Methodology: Were analyzed by GeneXpert® MTB/RIF assay. Statistical analysis of the data was performed using SPSS version 20. Results: Out of total 500 samples, MTB was detected in 211 (42%) cases by GeneXpert® MTB/RIF assay [positive pulmonary cases 48.8% (206/422) and extra-pulmonary 6.4% (5/78)]. Among 211 MTB positive cases, the assay detected 11 (5.2%) cases with RIF-resistance caused by various rpoB gene mutations within 81-bp RRDR. All the eleven RIF-resistant isolates were found to have mutations only in Probe E and none of cases had RIF-resistance associated with probes A, B, C, and D. Out of 11 RRD cases, 4 (2%) were males and 7 (3.3%) were females. New TB cases were 3 (1.4%) and previously treated cases were 8 (3.8%). Conclusion: In our settings 11/211 (5.2%) of the TB patients showed rifampicin resistance. Probe E mutations (also called codons 531 and 533) were the only rpoB gene mutations detected by GeneXpert® MTB/RIF assay. No mutations were detected in the codons 511, 513, 516, 518, 522 and 526 sequences.

Modulatory effects of verapamil in rifampicin activity against Mycobacterium tuberculosis.

To evaluate modulatory effect of verapamil (VP) in rifampicin (RIF) activity and its effect in efflux pumps (EPs) transcript levels in Mycobacterium tuberculosis. RIF and VP minimal inhibitory concentration, combinatory effect and detection of mutations were determined in 16 isolates. EPs transcript levels were determined in four isolates by real-time PCR after exposure to drugs. VP showed good combinatory effect among RIF-resistant isolates. This effect was also observed in the relative transcript levels of EPs, mainly after 72h of exposure, depending on the EP gene, genotype and the resistance profile of the isolate. Additional regulatory mechanisms in the EP activities, as well as, interactions with other drug-specific resistance mechanisms need further investigation in M. tuberculosis.