Molecular detection of multidrug resistance pattern and associated gene mutations in M. tuberculosis isolates from newly diagnosed pulmonary tuberculosis patients in Addis Ababa, Ethiopia.
ABSTRACT: INTRODUCTION:Multi-drug resistance is a major challenge in the control of tuberculosis. Despite newer modalities for diagnosis and treatment, people are still suffering from this disease. Understanding the common gene mutations conferring rifampicin and isoniazid resistance is crucial for the implementation of effective molecular tools at local and national levels. Hence, this study aimed to evaluate the molecular detection of rifampicin and isoniazid-resistant gene mutations in M.tuberculosis isolates in Addis Ababa, Ethiopia. METHOD:Health Center-based cross-sectional study was conducted between January and September 2017 in Addis Ababa, Ethiopia. The collected sputum samples were processed for mycobacterial isolation and Region of difference 9 based polymerase chain reaction for species identification. To characterize the rifampicin and isoniazid-resistant M. tuberculosis isolates, a molecular genetic assay (GenoType MTBDRplus) was used; the assay is based on DNA-STRIP technology. RESULT:Culture positivity was confirmed in 82.6% (190/230) of smear-positive newly diagnosed pulmonary tuberculosis cases enrolled in the study. From 190 isolates 93.2% were sensitive for both rifampicin and isoniazid, and 6.8% of the isolates were resistant to at least one of the tested anti-TB drugs. Gene mutations were observed in all studied multidrug resistance-associated gene loci (rpoB, katG, and inhA). Two isolates exhibited heteroresistance, a mutated, as well as wild type sequences, were detected in the respective strains. MDR-TB case was observed in 1.1% (2/190) of the cases. All the MDR-TB cases were positive for HIV and found to have a history of prior hospital admission. CONCLUSION:In our finding a relatively high prevalence of any drug resistance was observed and the overall prevalence of multidrug-resistant tuberculosis was 1.1%.The majority of drug-resistant isolates demonstrated common mutations. Heteroresistant strains were detected, signaling the existence of an M.tuberculosis population with variable responses to anti-tuberculosis drugs or of mixed infections.
Project description:<h4>Background</h4>Multidrug drug-resistant tuberculosis (MDR-TB) is a major health problem and seriously threatens TB control and prevention efforts globally. Ethiopia is among the 30th highest TB burden countries for MDR-TB with 14% prevalence among previously treated cases. The focus of this study was on determining drug resistance patterns of Mycobacterium tuberculosis among MDR-TB suspected cases and associated risk factors.<h4>Methods</h4>A cross-sectional study was conducted in Addis Ababa from June 2015 to December 2016. Sputum samples and socio-demographic data were collected from 358 MDR-TB suspected cases. Samples were analyzed using Ziehl-Neelsen technique, GeneXpert MTB/RIF assay, and culture using Lowenstein-Jensen and Mycobacterial growth indicator tube. Data were analyzed using SPSS version 23.<h4>Results</h4>A total of 226 the study participants were culture positive for Mycobacterium tuberculosis, among them, 133 (58.8%) participants were males. Moreover, 162 (71.7%) had been previously treated for tuberculosis, while 128 (56.6%) were TB/HIV co-infected. A majority [122 (54%)] of the isolates were resistant to any first-line anti-TB drugs. Among the resistant isolates, 110 (48.7%) were determined to be resistant to isoniazid, 94 (41.6%) to streptomycin, 89 (39.4%) to rifampicin, 72 (31.9%) to ethambutol, and 70 (30.9%) to pyrazinamide. The prevalence of MDR-TB was 89 (39.4%), of which 52/89 (58.4%) isolates were resistance to all five first-line drugs. Risk factors such as TB/HIV co-infection (AOR = 5.59, p = 0.00), cigarette smoking (AOR = 3.52, p = 0.045), alcohol drinking (AOR = 5.14, p = 0.001) hospital admission (AOR = 3.49, p = 0.005) and visiting (AOR = 3.34, p = 0.044) were significantly associated with MDR-TB.<h4>Conclusions</h4>The prevalence of MDR-TB in the study population was of a significantly high level among previously treated patients and age group of 25-34. TB/HIV coinfection, smoking of cigarette, alcohol drinking, hospital admission and health facility visiting were identified as risk factors for developing MDR-TB. Therefore, effective strategies should be designed considering the identified risk factors for control of MDR-TB.
Project description:BACKGROUND:Pakistan is among top five high burden countries for tuberculosis and drug resistant TB. Among rifampicin sensitive new pulmonary TB (PTB), prevalence of isoniazid resistance is 8.3% (95%CI: 7.0-10.7) and resistance to fluoroquinolone is higher (11·1%, 95%CI: 7·8-14·3) than isoniazid resistance. METHOD:Five year retrospective data (2015-2019) of drug susceptibility testing (DST) for Mycobacterium tuberculosis isolates, performed using recommended phenotypic (pDST) and/or genotypic (gDST) methods was analyzed stratified by rifampicin results for isoniazid resistance profiles and associated levofloxacin and pyrazinamide resistance. FINDINGS:DST data was analyzed from 11045 TB patients. Isolates were tested using pDST (87%), gDST (92%) and both methods (79.5%). For both rifampicin and isoniazid, a significant difference (P < .001) was noted between resistance detected by pDST and gDST. Among isolates, tested by both methods (8787), 49% were resistant to rifampicin and 51.7% to isoniazid with discordance in resistant results of 15.8% for each, with 13.2% (570) of rifampicin resistance reported sensitive by pDST and 14.2% (660) of isoniazid resistance missed by gDST. Estimated isoniazid resistance among rifampicin sensitive new PTB, extrapulmonary TB and previously treated PTB was 9.8% (95%CI: 8.7-11.1), 6.8% (95%CI: 5.4-8.5) and 14.6% (95%CI: 11.8-17.9) respectively. Significant differences were reported between the genotypic profile of isoniazid resistance associated with rifampicin-resistant and sensitive isolates including detectable mutations (87% vs 71.6%), frequency of inhA (7.6% and 30.2%) and katG mutations (76.1% vs 41.2%) respectively. Among rifampicin resistant and sensitive isolates, a significantly higher level of resistance to levofloxacin and pyrazinamide was seen associated with isoniazid resistance. CONCLUSION:There are risks and many challenges in implementing WHO recommended treatment for isoniazid resistant tuberculosis. The laboratory based surveillance can complement random surveys in country specific planning for TB diagnostics and appropriate treatment regimens.
Project description:BACKGROUND:Accurate diagnosis of tuberculosis, especially by using rapid molecular assays, can reduce transmission of drug resistant tuberculosis in communities. However, the frequency of resistance conferring mutations varies with geographic location of Mycobacterium tuberculosis, and this affects the efficiency of rapid molecular assays in detecting resistance. This has created need for characterizing drug resistant isolates from different settings to investigate frequencies of resistance conferring mutations. Here, we describe the prevalence and patterns of rifampicin- and isoniazid- resistance conferring mutations in isolates from Uganda, which could be useful in the management of MDR-TB patients in Uganda and other countries in sub-Saharan Africa. RESULTS:Ninety seven M. tuberculosis isolates were characterized, of which 38 were MDR, seven rifampicin-resistant, 12 isoniazid-mono-resistant, and 40 susceptible to rifampicin and isoniazid. Sequence analysis of the rpoB rifampicin-resistance determining region (rpoB/RRDR) revealed mutations in six codons: 588, 531, 526, 516, 513, and 511, of which Ser531Leu was the most frequent (40%, 18/45). Overall, the three mutations (Ser531Leu, His526Tyr, Asp516Tyr) frequently associated with rifampicin-resistance occurred in 76% of the rifampicin resistant isolates while 18% (8/45) of the rifampicin-resistant isolates lacked mutations in rpoB/RRDR. Furthermore, sequence analysis of katG and inhA gene promoter revealed mainly the Ser315Thr (76%, 38/50) and C(-15)T (8%, 4/50) mutations, respectively. These two mutations combined, which are frequently associated with isoniazid-resistance, occurred in 88% of the isoniazid resistant isolates. However, 20% (10/50) of the isoniazid-resistant isolates lacked mutations both in katG and inhA gene promoter. The sensitivity of sequence analysis of rpoB/RRDR for rifampicin-resistance via detection of high confidence mutations (Ser531Leu, His526Tyr, Asp516Tyr) was 81%, while it was 77% for analysis of katG and inhA gene promoter to detect isoniazid-resistance via detection of high confidence mutations (Ser315Thr, C(-15)T, T(-8)C). Furthermore, considering the circulating TB genotypes in Uganda, the isoniazid-resistance conferring mutations were more frequent in M. tuberculosis lineage 4/sub-lineage Uganda, perhaps explaining why this genotype is weakly associated with MDR-TB. CONCLUSION:Sequence analysis of rpoB/RRDR, katG and inhA gene promoter is useful in detecting rifampicin/isoniazid resistant M. tuberculosis isolates in Uganda however, about ?20% of the resistant isolates lack known resistance-conferring mutations hence rapid molecular assays may not detect them as resistant.
Project description:With increasing incidence of multidrug-resistant tuberculosis (MDR-TB), accurate drug susceptibility testing (DST) of Mycobacterium tuberculosis to first-line drugs has become crucial for proper patient management. We evaluated concordance of DST results for 70 M. tuberculosis isolates across two phenotypic and two molecular methods: BACTEC 460TB, MGIT 960 system, GenoType MTBDRplus and DNA sequencing of gene segments most commonly implicated in conferring resistance to anti-TB drugs. Most (84%) M. tuberculosis isolates were multidrug-resistant. Twenty-four isolates yielded discrepant DST results. For rifampicin, isoniazid and streptomycin, 96%, 97% and 93% of isolates, respectively, were susceptible or resistant by all four methods, whereas for ethambutol, this agreement was observed for only 76% of isolates (P<0.05 for rifampicin or isoniazid or streptomycin versus ethambutol). Occurrence of rare mutations in three isolates that confer low-level resistance caused lower agreement for rifampicin among the four methods (kappa coefficient (?) range, 0.84 to 0.95). For isoniazid, there was perfect agreement among phenotypic methods and molecular methods (?, 1.00) but lower agreement between phenotypic and molecular methods. Three isolates were detected as polydrug-resistant by MGIT 960 system but as multidrug-resistant by DNA sequence-based method. The agreement was higher for streptomycin among the two phenotypic methods (?, 0.97) while targeted sequencing yielded lower agreement (? range, 0.86 to 0.89). The discrepancy for ethambutol resulted largely due to lower concordance of MGIT 960 results (? range, 0.53 to 0.64). The MGIT 960 system is an accurate method for DST of M. tuberculosis against isoniazid and streptomycin while the results of rifampicin susceptibility should be complemented with DNA sequencing-based method when the suspicion for resistance is high. The possibility of false susceptibility to ethambutol with MGIT 960 system suggests that molecular or other phenotypic methods may be more useful when accurate ethambutol susceptibility results are warranted.
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.
Project description:<h4>Background</h4>Antimicrobial resistance is a global concern of increasing significance. Multidrug resistant tuberculosis (MDR-TB) is spreading worldwide. It is important to monitor trends of antimycobacterial resistance. This is particularly true for high TB burden countries such as Ethiopia where disproportionally less drug sensitivity data are reported from.<h4>Methods</h4>The prevalence of drug resistance was assessed with the line probe assay GenoType MTBDR<i>plus</i> in a set of 161?<i>M. tuberculosis</i> strains that were selected from four common lineages and sub-lineages previously identified in Ethiopia. Most of the tested <i>M. tuberculosis</i> isolates had been genotyped by established Spoligotyping and MIRU-VNTR typing methods.<h4>Results</h4>The proportion of MDR-TB among the isolates was 3.1%. Mono-resistance was 1.2% to rifampicin and 4.3% to isoniazid, and resistance to either of the two first line drugs was 8.7%. Strains of Lineage 4 had the highest resistance rate (13.6%) followed by Lineage 3 (4.9%). None of the isolates representing Lineages 1 and Lineage 7 were drug resistant. Multidrug resistance among pulmonary TB and TB lymphadenitis clinical isolates was 2.8 and 3.7%, respectively. Drug resistance of strains carrying the most prevalent spoligotype in Ethiopia - SIT149 - was further explored. Stratification by MIRU-VNTR identified one genotype with a high rate of drug resistance against Rifampicin and Isoniazid and circulation of a potential MDR-TB clone is proposed.<h4>Conclusion</h4>Although the strain selection was not fully randomized, the overall <i>M. tuberculosis</i> drug resistance rate in this strain set was 8.7% while the rate of MDR was 3.1%. In parallel, we identified a sub-lineage that showed a high rate of resistance to both rifampicin and isoniazid. These resistant strains may belong to a clone of <i>M. tuberculosis</i> that is circulating in the highlands of Ethiopia.
Project description:Twenty isolates ofMycobacterium tuberculosis resistant to rifampicin(RIF), isoniazid(INH) and streptomycin(STR) were analysed by Polymerase Chain Reaction (PCR) amplification of rpoB, katG and rrs genes to evaluate comparative diagnostic significance of genetic assays. Mutations were identified by single strand conformation polymorphism (SSCP) and cleavase fragment length polymorphism (CFLP) and were confirmed by DNA sequencing. SSCP of 4 RIF resistant and 14 INH resistant isolates showed an extra peak at the level of 75-bp and 85-bp respectively, while 2 STR resistant isolates showed 2 peaks with 9 bases difference. CFLP showed a different pattern among RIF, INH and STR sensitive and resistant isolates Thus SSCP and CFLP can be used as alternative diagnostic methods for identification of mutations in RIF, INH and STR resistant strains of M.tuberculosis.
Project description:BACKGROUND: The aim of this study was to investigate the molecular characteristics of isoniazid resistant Mycobacterium tuberculosis (MTB), as well as its contribution to the dissemination of multi-drug resistant TB (MDR-TB) in rural areas of eastern China. METHODS: A population-based epidemiological study was conducted in two rural counties of eastern China from 2004 to 2005. In total, 131 isoniazid resistant MTB isolates were molecularly characterized by DNA sequencing and genotyped by IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. RESULTS: The katG315Thr mutation was observed in 74 of 131 isoniazid resistant isolates and more likely to be MDR-TB (48.6%) and have mutations in rpoB gene (47.3%). Spoligotyping identified 80.2% of isoniazid resistant MTB isolates as belonging to the Beijing family. Cluster analysis by genotyping based on IS6110 RFLP, showed that 48.1% isoniazid resistant isolates were grouped into 26 clusters and katG315Thr mutants had a significantly higher clustering proportion compared to those with katG wild type (73%.vs.18%; OR, 12.70; 95%CI, 6.357-14.80). Thirty-one of the 53 MDR-TB isolates were observed in 19 clusters. Of these clusters, isoniazid resistance in MDR-TB isolates was all due to the katG315Thr mutation; 18 clusters also contained mono-isoniazid resistant and other isoniazid resistant isolates. CONCLUSIONS: These results highlighted that isoniazid resistant MTB especially with katG315Thr is likely to be clustered in a community, develop extra resistance to rifampicin and become MDR-TB in Chinese rural settings.
Project description:Drug-resistant tuberculosis (TB) is a major public health problem. Clinical Mycobacterium tuberculosis (MTB) isolate with Extensively drug-resistant tuberculosis (MTB-XDR) profile was subjected to whole-genome sequencing using a next-generation sequencing platform (NGS) Roche 454 GS FLX+ followed by bioinformatics sequence analysis. Quality of read was checked by FastQC, paired-end reads were trimmed using Trimmomatic. De novo genome assembly was conducted using Velvet v.1.2.10. The assembled genome of XDR-TB-1599 strain was functionally annotated using the PATRIC platform. Analysis of de novo assembled genome was performed using ResFinder, CARD, CASTB and TB-Profiler tools. MIRU_VNTR genotyping on 12 loci and spoligotyping have been performed for XDR-TB-1599 isolate. M. tuberculosis XDR-TB-1599 strain yielded an average read depth of 21-fold with overall 4 199 325?bp. The assembled genome contains 5528 protein-coding genes, including key drug resistance and virulence-associated genes and GC content of 65.4%. We identified that all proteins encoded by this strain contain conserved domains associated with the first-line anti-tuberculosis drugs such as rifampicin, isoniazid, streptomycin and ethionamide. TB-Profiler had higher average concordance results with phenotypic DST (drug susceptibility testing) in comparison with ResFinder, CARD, CASTB profiling to first-line (75% vs 50%) and second-line (25% vs 0%) of anti-TB drugs, correspondingly. To our knowledge, this is the first report of a highly annotated and characterized whole-genome sequence and de novo assembled XDR-TB M.tuberculosis strain isolated from a sputum of new TB case-patient from Kazakhstan performed on Roche 454 GS FLX+ platform. This report highlights an important role of whole-genome sequencing technology and analysis as an advanced approach for drug-resistance investigations of circulated TB isolates.
Project description:<h4>Background</h4>Increasing incidence of multidrug-resistant Mycobacterium tuberculosis infections is hampering global tuberculosis control efforts. Kuwait is a low-tuberculosis-incidence country, and ~?1% of M. tuberculosis strains are resistant to rifampicin and isoniazid (MDR-TB). This study detected mutations in seven genes predicting resistance to rifampicin, isoniazid, pyrazinamide, ethambutol and streptomycin in MDR-TB strains. Sequence data were combined with spoligotypes for detecting local transmission of MDR-TB in Kuwait.<h4>Methods</h4>Ninety-three MDR-TB strains isolated from 12 Kuwaiti and 81 expatriate patients and 50 pansusceptible strains were used. Phenotypic drug susceptibility was determined by MGIT 460 TB/960 system. Mutations conferring resistance to rifampicin, isoniazid, pyrazinamide, ethambutol and streptomycin were detected by genotype MTBDRplus assay and/or PCR sequencing of three rpoB regions, katG codon 315 (katG315)?+?inhA regulatory region, pncA, three embB regions and rpsL?+?rrs-500-900 regions. Spoligotyping kit was used, spoligotypes were identified by SITVIT2, and phylogenetic tree was constructed by using MIRU-VNTRplus software. Phylogenetic tree was also constructed from concatenated sequences by MEGA7 software. Additional PCR sequencing of gidB and rpsA was performed for cluster isolates.<h4>Results</h4>Pansusceptible isolates contained wild-type sequences. Mutations in rpoB and katG and/or inhA were detected in 93/93 and 92/93 MDR-TB strains, respectively. Mutations were also detected for pyrazinamide resistance, ethambutol resistance and streptomycin resistance in MDR-TB isolates in pncA, embB and rpsL?+?rrs, respectively. Spoligotyping identified 35 patterns with 18 isolates exhibiting unique patterns while 75 isolates grouped in 17 patterns. Beijing genotype was most common (32/93), and 11 isolates showed nine orphan patterns. Phylogenetic analysis of concatenated sequences showed unique patterns for 51 isolates while 42 isolates grouped in 16 clusters. Interestingly, 22 isolates in eight clusters by both methods were isolated from TB patients typically within a span of 2 years. Five of eight clusters were confirmed by additional gidB and rpsA sequence data.<h4>Conclusions</h4>Our study provides the first insight into molecular epidemiology of MDR-TB in Kuwait and identified several potential clusters of local transmission of MDR-TB involving 2-6 subjects which had escaped detection by routine surveillance studies. Prospective detection of resistance-conferring mutations can identify possible cases of local transmission of MDR-TB in low MDR-TB settings.