Mutation and Transmission Profiles of Second-Line Drug Resistance in Clinical Isolates of Drug-Resistant Mycobacterium tuberculosis From Hebei Province, China.
ABSTRACT: The emergence of drug-resistant tuberculosis (TB) is involved in ineffective treatment of TB, especially multidrug resistant/extensively resistant TB (MDR/XDR-TB), leading to acquired resistance and transmission of drug-resistant strains. Second-line drugs (SLD), including both fluoroquinolones and injectable drugs, were commonly proved to be the effective drugs for treatment of drug-resistant TB. The purpose of this study was to investigate the prevalence of SLD-resistant strains and its specific mutations in drug-resistant Mycobacterium tuberculosis clinical isolates, and to acknowledge the transmission pattern of SLD resistance strains in Hebei. The genes gyrA, gyrB, rrs, eis promoter and tlyA of 257 drug-resistant clinical isolates were sequenced to identify mutations that could be responsible for resistance against fluoroquinolones and second-line injectable drugs. Each isolate was genotyped by Spoligotyping and 15-loci MIRU-VNTR. Our results indicated that 48.2% isolates were resistant to at least one of five SLD. Of them, 37.7% isolates were resistant to fluoroquinolones and 24.5% isolates were resistant to second-line injectable drugs. Mutations in genes gyrA, gyrB, rrs, eis promoter and tlyA were detected in 73 (75.3%), 7 (7.2%), 24 (38.1%), 5 (7.9%), and 3 (4.8%) isolates, respectively. The most prevalent mutations were the D94G (23.7%) in gyrA gene and the A1401G (33.3%) in rrs gene. A combination of gyrA, rrs and eis promoter can act as a valuable predicator for predicting XDR phenotype. These results highlight the development of rapid diagnosis are the effective manners for the control of SLD-TB or XDR-TB.
Project description:Nearly 5% of all Mycobacterium tuberculosis strains worldwide are resistant at least to rifampicin and isoniazid (multidrug-resistant tuberculosis, MDR-TB). Inclusion of a fluoroquinolone and an injectable agent (kanamycin, amikacin or capreomycin) in multidrug therapy is crucial for proper treatment of MDR-TB. The incidence of MDR-TB in Kuwait is ~1%. MDR-TB strains additionally resistant to fluoroquinolones and injectable agents are defined as extensively drug-resistant (XDR-TB) strains and have been detected in >55 countries. Infections with XDR-TB strains have very poor prognosis. This study detected the occurrence of gyrA mutations associated with fluoroquinolone resistance among MDR-TB strains in Kuwait.Direct DNA sequencing of quinolone resistance-determining region of gyrA gene was performed to detect fluoroquinolone resistance-associated mutations in 85 MDR-TB strains isolated from 55 TB patients and 25 pansusceptible M. tuberculosis strains. For isolates exhibiting gyrA mutations, 3'-end of rrs (16S rRNA) was sequenced for the detection of XDR-TB. Fingerprinting of fluoroquinolone resistant MDR-TB strains was performed by detecting mutations in three (81 bp hot-spot, N-terminal and cluster II) regions of rpoB, katG codon 315 and inhA-regulatory region, polymorphisms at gyrA codon 95 and katG codon 463 by DNA sequencing and by double-repetitive-element PCR for determining strain relatedness. None of the pansusceptible but six of 85 MDR-TB strains contained gyrA mutations. Only gyrA codon 94 was mutated in all six (D94A in one and D94G in five) strains. Three of six mutant strains were recovered from the same patient while three other strains represented individual patient isolates. Fingerprinting studies identified all individual patient isolates as epidemiologically distinct strains. All six strains with a gyrA mutation contained wild-type rrs sequence.Although fluoroquinolones are generally not used for chemotherapy of TB and drug susceptibility testing for second-line drugs is not carried out in Kuwait, four of 55 (7%) individual patient MDR-TB strains contained mutations in gyrA gene. The data advocate routine drug susceptibility testing for this important second-line drug for proper management of MDR-TB in Kuwait. Lack of mutations in 3'-end of rrs gene that confer resistance to injectable agents reduce the likelihood of occurrence of XDR-TB, at present, in Kuwait.
Project description:Resistance to anti-tuberculosis (TB) drugs has been a great challenge for global TB control. Limited tools have been developed to detect resistance to second-line drugs that are associated with extensively drug-resistant (XDR) TB. In this study we aimed to develop a simple and widely applicable assay for detecting mutations associated with second-line drug resistance in Mycobacterium tuberculosis.Three dually labelled probes targeting gyrA, rrs and the promoter of eis were designed to detect resistance to fluoroquinolones and second-line injectable agents (capreomycin, amikacin and kanamycin). A triplex reaction with all three probes and corresponding primers was first tested against 13 isolates with different mutations in the targeted regions. Then, the triplex assay was applied to 109 second-line drug-resistant isolates in a blind manner and the results were compared with the sequencing data.All mutations in the targeted regions of 13 representative isolates could be detected through significant Tm reductions of the corresponding probe compared with the wild-type control. The detection results with 109 isolates were 100% concordant with sequencing data. Twelve ofloxacin-resistant isolates were detected as heteroresistant, indicating the coexistence of mutant and wild-type strains or the existence of different gyrA mutations.We have developed a simple and widely applicable assay to detect second-line drug resistance of M. tuberculosis. This method, combined with assays for detecting first-line drug resistance, provides an efficient and reliable tool to diagnose multidrug-resistant TB and XDR-TB.
Project description:There is limited data on the use of Genotype MTBDRslVersion 1 (MTBDRsl V1) as an initial rapid screening test to rule out XDR-TB and most importantly its performance in various genotypes of Mycobacterium tuberculosis is scarcely studied. A total of 359 MDR-TB isolates were tested for gene mutations representing second line drug resistance, using the MTBDRsl_V.1 and the results were compared with phenotypic method (Bactec MGIT-960 system) for second-line drug (SLD) susceptibility testing. Genetic lineages of all these isolates were also determined using spoligotyping and SITVIT2 WEB database. The MTBDRsl V1 detected mutations in the gyrA, rrs, and emb genes in 108 (30%), 2 (0.5%) and 129 (35.9%) isolates, respectively. Remaining 120 (33.4%) had no second line drug (SLD) resistance. In 17 (4.7%) isolates mutations were detected in both gyrA and rrs genes. Its concordance with MGIT-960 culture drug susceptibility testing (DST) was 97% and 94.1%, 93.5%, 60.5% and 50% for the detection of XDR-TB, pre-XDR, Ethambutol, and Aminoglycosides/Cyclopeptides resistance. The Beijing lineage was predominant (46%) between both the pre-XDR/XDR-TB isolates. We conclude that MTBDRsl is useful for rapid detection of SLD resistance. Also in pre-XDR and XDR-TB isolates the frequency of relevant genetic mutations was significantly higher in the Beijing strains.
Project description:BACKGROUND:Pulmonary tuberculosis is a leading cause of morbidity and mortality in developing countries. Drug resistance, a huge problem in this contagious disease, is driven by point mutations in the Mycobacterium tuberculosis genome however, their frequencies vary geographically and this affects applicability of molecular diagnostics for rapid detection of resistance. Here, we report the frequency and patterns of mutations associated with resistance to second-line anti-TB drugs in multidrug-resistant (MDR) M. tuberculosis isolates from eSwatini, Somalia and Uganda that were resistant to a second-line anti-TB drug. METHODS:The quinolone resistance determining region (QRDR) of gyrA/gyrB genes and the drug resistance associated fragment of rrs gene from 80 isolates were sequenced and investigated for presence of drug resistance mutations. Of the 80 isolates, 40 were MDR, of which 28 (70%) were resistant to a second-line anti-TB injectable drug, 18 (45%) were levofloxacin resistant while 12 (30%) were extensively drug resistant (XDR). The remaining 40 isolates were susceptible to anti-TB drugs. MIRU-VNTR analysis was performed for M/XDR isolates. RESULTS:We successfully sub-cultured 38 of the 40?M/XDR isolates. The gyrA resistance mutations (Gly88Ala/Cys/Ala, Ala90Val, Ser91Pro, Asp94Gly/Asn) and gyrB resistance mutations (Asp500His, Asn538Asp) were detected in 72.2% (13/18) and 22.2% (4/18) of the MDR and levofloxacin resistant isolates, respectively. Overall, drug resistance mutations in gyrA/gyrB QRDRs occurred in 77.8% (14/18) of the MDR and levofloxacin resistant isolates. Furthermore, drug resistance mutations a1401g and g1484?t in rrs occurred in 64.3% (18/28) of the MDR isolates resistant to a second-line anti-TB injectable drug. Drug resistance mutations were not detected in drug susceptible isolates. CONCLUSIONS:The frequency of resistance mutations to second-line anti-TB drugs in MDR-TB isolates resistant to second line anti-TB drugs from eSwatini, Somalia and Uganda is high, implying that rapid molecular tests are useful in detecting second-line anti-TB drug resistance in those countries. Relatedly, the frequency of fluoroquinolone resistance mutations in gyrB/QRDR is high relative to global estimates, and they occurred independently of gyrA/QRDR mutations implying that their absence in panels of molecular tests for detecting fluoroquinolone resistance may yield false negative results in our setting.
Project description:Molecular diagnostic methods based on the detection of mutations conferring drug resistance are promising technologies for rapidly detecting multidrug-/extensively drug-resistant tuberculosis (M/XDR TB), but large studies of mutations as markers of resistance are rare. The Global Consortium for Drug-Resistant TB Diagnostics analyzed 417 Mycobacterium tuberculosis isolates from multinational sites with a high prevalence of drug resistance to determine the sensitivities and specificities of mutations associated with M/XDR TB to inform the development of rapid diagnostic methods. We collected M/XDR TB isolates from regions of high TB burden in India, Moldova, the Philippines, and South Africa. The isolates underwent standardized phenotypic drug susceptibility testing (DST) to isoniazid (INH), rifampin (RIF), moxifloxacin (MOX), ofloxacin (OFX), amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) using MGIT 960 and WHO-recommended critical concentrations. Eight genes (katG, inhA, rpoB, gyrA, gyrB, rrs, eis, and tlyA) were sequenced using Sanger sequencing. Three hundred seventy isolates were INHr, 356 were RIFr, 292 were MOXr/OFXr, 230 were AMKr, 219 were CAPr, and 286 were KANr. Four single nucleotide polymorphisms (SNPs) in katG/inhA had a combined sensitivity of 96% and specificities of 97 to 100% for the detection of INHr. Eleven SNPs in rpoB had a combined sensitivity of 98% for RIFr. Eight SNPs in gyrA codons 88 to 94 had sensitivities of 90% for MOXr/OFXr. The rrs 1401/1484 SNPs had 89 to 90% sensitivity for detecting AMKr/CAPr but 71% sensitivity for KANr. Adding eis promoter SNPs increased the sensitivity to 93% for detecting AMKr and to 91% for detecting KANr. Approximately 30 SNPs in six genes predicted clinically relevant XDR-TB phenotypes with 90 to 98% sensitivity and almost 100% specificity.
Project description:BACKGROUND: The steady rise in the spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) requires rapid and reliable methods to identify resistant strains. The current molecular methods to detect MTB resistance to second-line drugs either do not cover an extended spectrum of mutations to be identified or are not easily implemented in clinical laboratories. A rapid molecular technique for the detection of resistance to second-line drugs in M. tuberculosis has been developed using hybridisation analysis on microarrays. METHODS: The method allows the identification of mutations within the gyrA and gyrB genes responsible for fluoroquinolones resistance and mutations within the rrs gene and the eis promoter region associated with the resistance to injectable aminoglycosides and a cyclic peptide, capreomycin. The method was tested on 65 M. tuberculosis clinical isolates with different resistance spectra that were characterised by their resistance to ofloxacin, levofloxacin, moxifloxacin, kanamycin and capreomycin. Also, a total of 61 clinical specimens of various origin (e.g., sputum, bronchioalveolar lavage) were tested. RESULTS: The sensitivity and specificity of the method in the detection of resistance to fluoroquinolones were 98% and 100%, respectively, 97% and 94% for kanamycin, and 100% and 94% for capreomycin. The analytical sensitivity of the method was approximately 300 genome copies per assay. The diagnostic sensitivity of the assay ranging from 67% to 100%, depending on the smear grade, and the method is preferable for analysis of smear-positive specimens. CONCLUSIONS: The combined use of the developed microarray test and the previously described microarray-based test for the detection of rifampin and isoniazid resistance allows the simultaneous identification of the causative agents of MDR and XDR and the detection of their resistance profiles in a single day.
Project description:The country of Georgia has a high burden of multi- and extensively drug-resistant tuberculosis (XDR-TB). To evaluate whether mutations in gyrB and eis genes increased the sensitivity of detection of phenotypic resistance to ofloxacin and kanamycin or capreomycin compared to use of the first-generation MTBDRsl assay alone, which tests for mutations in gyrA and rrs genes, a retrospective study of stored Mycobacterium tuberculosis isolates was performed. All isolates underwent DNA sequencing of resistance-determining regions. Among 112 M. tuberculosis isolates with DNA extraction data, targeted sequencing was successfully performed for each gene as follows: for gyrA, 98% sensitivity; for gyrB, 96%; for rrs, 93%; for the eis gene and its promoter, 93%. The specificity and hence the positive predictive value of gyrA and gyrB mutations for detecting ofloxacin resistance were 100%. The addition of gyrB mutations increased the sensitivity of phenotypic ofloxacin resistance detection by 13% (75% to 88%). All rrs resistance-conferring mutations were A1401G, and this mutation had low sensitivity (40% and 18%) and high specificity (95% and 100%) in predicting phenotypic capreomycin and kanamycin resistance, respectively. The eis C-14T mutation increased the sensitivity of phenotypic kanamycin resistance detection by 9% (18% to 27%) and was found solely in kanamycin phenotypic resistance isolates. Our data showed that the inclusion of eis C-14T and gyrB mutations in addition to rrs and gyrA mutations improves the sensitivity of detection of phenotypic ofloxacin and kanamycin resistance, respectively.
Project description:Detecting resistance to fluoroquinolones (FQ) and second-line injectable drugs (amikacin [AMK], kanamycin [KAN], and capreomycin [CAP]) is crucial given the worldwide increase in the incidence of extensively drug-resistant tuberculosis (XDR-TB). A new version of the GenoType MTBDRsl test (v2.0) has been developed to improve the detection of resistance to FQ (involving gyrA and gyrB mutations) and to second-line injectable drugs (involving rrs and eis promoter mutations) in Mycobacterium tuberculosis A collection of 127 multidrug-resistant (MDR) M. tuberculosis complex strains was tested using the first (v1) and second (v2.0) versions of the MTBDRsl test, as well as DNA sequencing. The specificities in resistance detection of v1 and v2.0 were similar throughout, whereas the levels of sensitivity of v2.0 were superior for FQ (94.8% versus 89.6%) and KAN (90.5% versus 59.5%) but similar for AMK (91.3%) and CAP (83.0%). The sensitivity and specificity of v2.0 were superior to those of v1 for the detection of pre-XDR strains (83.3% versus 75.0% and 88.6% versus 67.1%, respectively), whereas the sensitivity of v2.0 was superior to that of v1 only for the detection of XDR strains (83.0% versus 49.1%). In conclusion, MTBDRsl v2.0 is superior to MTBDRsl v1 and efficiently detects the most common mutations involved in resistance to FQ and aminoglycosides/CAP. However, due to mutations not recognized by v2.0 or to the presence of resistance mechanisms not yet characterized (particularly mechanisms related to monoresistance to aminoglycosides or CAP), the results for wild-type strains obtained with MTBDRsl v2.0 should be confirmed by further DNA sequencing and phenotypic drug susceptibility testing.
Project description:Pakistan ranks 5th among the world's highest tuberculosis (TB) burden countries alongside the 6th among countries with the highest burden of drug-resistant TB, including multi-drug resistant (MDR)-TB. Methods for rapid and reliable drug susceptibility testing (DST) are prerequisite for the prompt institution of effective anti-TB treatment. The aim of this study was to evaluate the efficiency of Genotype MTBDRplus and MTBDRsl assays for the detection of MDR and (pre-) extensively drug-resistant (XDR-TB) isolates in Pakistan. The study included 47 pre-XDR and 6 XDR-TB isolates, recovered from 53 patients from Pakistan. Conventional DST was performed using the standard 1% proportion method on the Löwenstein-Jensen medium. For molecular determination of drug resistance, GenoType MTBDRplus and GenoType MTBDRsl assays (Hain Lifescience, Germany) were used. To evaluate discrepancies between conventional and molecular DST results, mutation profiling was performed by amplifying and sequencing seven genetic loci, i.e., katG, inhA, and mabA-inhA promoter, rpoB, gyrA, embB, rrs. The sensitivity of Genotype MTBDRplus was 71.7% for isoniazid (INH) and 79.2% for rifampicin (RIF). Sequence analysis revealed non-synonymous mutations in 93.3 and 27.3% of isolates phenotypically resistant to INH and RIF, respectively, albeit susceptible when tested by GenoType MTBDRplus. GenoType MTBDRsl had a sensitivity of 73.6, 64.7, 20, 25, and 100% for the detection of fluoroquinolones, ethambutol, kanamycin, amikacin, and capreomycin resistance, respectively. Upon sequencing, mutations were detected in 20, 77.8%, and all isolates phenotypically resistant to aminoglycosides, ethambutol, and fluoroquinolones, respectively, yet declared as susceptible with GenoType MTBDRsl. Low sensitivities seriously impede the large-scale application of the Genotype MTBDRplus and MTBDRsl assays. Unless further optimized, the currently available line-probe assays should rather be auxiliary to the conventional, phenotype-based methods in the detection of MDR- and XDR-TB in Pakistan.
Project description:The emergence and transmission of multidrug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (M.tb) strains is a threat to global tuberculosis (TB) control. The early detection of drug resistance is critical for patient management. The aim of this study was to determine the proportion of isolates with additional second-line resistance among rifampicin and isoniazid resistant and MDR-TB isolates. A total of 66 M.tb isolates received at the National Tuberculosis Reference Laboratory between March 2012 and October 2013 with resistance to isoniazid, rifampicin or both were analyzed in this study. The genotypes of the M.tb isolates were determined by spoligotyping and second-line drug susceptibility testing was done using the Hain Genotype MTBDRsl line probe assay version 2.0. The treatment outcomes were defined according to the Botswana national and World Health Organization (WHO) guidelines. Of the 57 isolates analyzed, 33 (58%) were MDR-TB, 4 (7%) were additionally resistant to flouroquinolones and 3 (5%) were resistant to both fluoroquinolones and second-line injectable drugs. The most common fluoroquinolone resistance-conferring mutation detected was gyrA A90V. All XDR-TB cases remained smear or culture positive throughout the treatment. Our study findings indicate the importance of monitoring drug resistant TB cases to ensure rapid detection of second-line drug resistance.