Multidrug-resistant Tuberculosis Isolates Research Articles

Frequency of first and second-line drug resistance-associated mutations among resistant Mycobacterium tuberculosis clinical isolates from São Paulo, Brazil.

BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB) has increased considerably worldwide.OBJECTIVES Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution.METHODS Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB).FINDINGS Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression.MAIN CONCLUSIONS The present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.

Antituberculosis drug resistance in isolates of Mycobacterium tuberculosis complex in southeast Spain.

The aim of this study was to determine resistance to antituberculosis (anti-TB) drugs in Mycobacterium tuberculosis complex isolates from patients diagnosed with Tuberculosis (TB) in southeast Spain and to study related epidemiological factors. This retrospective study analysed 5-year data (2012-2016) obtained in southeast Spain for a total equivalent population of 1 735 608 inhabitants. Clinical samples were examined from 557 patients with suspected pulmonary TB (n=470; 84.4%) or extrapulmonary TB (n=87; 15.6%), taking into account patient age, sex, human immunodeficiency virus (HIV) infection, country of birth and prior anti-TB treatment. TB was found more frequently in men than in women (66.6% vs. 33.4%), and the age group with the most cases (43.7%) was 36-55 years. Among the first-line anti-TB drugs, 7.0% of patients harboured isolates resistant to isoniazid (INH) and 1.6% to rifampicin (RIF); moreover, 1.4% of isolates were multidrug-resistant TB (MDR-TB) and 0.7% were extensively drug-resistant TB. There was a statistically significant relationship (P=0.028) between MDR-TB isolates and non-Spanish-born patients, but not between the latter and INH resistance. Resistance to INH and RIF was observed at levels similar to those published nationwide, with rates of MDR-TB being somewhat lower. Rates of HIV/TB co-infection have decreased considerably between 2012 and 2016.

Synthesis and biological evaluation of 2,4,5-trisubstituted thiazoles as antituberculosis agents effective against drug-resistant tuberculosis

The dormant and resistant form of Mycobacterium tuberculosis presents a challenge in developing new anti-tubercular drugs. Herein, we report the synthesis and evaluation of trisubstituted thiazoles as antituberculosis agents. The SAR study has identified a requirement of hydrophobic substituent at C2, ester functionality at C4, and various groups with hydrogen bond acceptor character at C5 of thiazole scaffold. This has led to the identification of 13h and 13p as lead compounds. These compounds inhibited the dormant Mycobacterium tuberculosis H37Ra strain and M. tuberculosis H37Rv selectively. Importantly, 13h and 13p were non-toxic to CHO cells. The 13p showed activity against multidrug-resistant tuberculosis isolates.

RpoB, katG and inhA mutations in multi-drug resistant strains of Mycobacterium tuberculosis clinical isolates from southeast Mexico

IntroductionPrevious knowledge of molecular mechanisms related with multi-drug resistances in tuberculosis is important if molecular diagnostic procedures want to be used in specific geographical regions. For that reason, the aim of this study was to investigate the mutations at rpoB, katG and inhA in multi-drug resistant tuberculosis isolates from Southeast Mexico. MethodsIsolates of tuberculosis with a confirmed resistance against rifampicin and isoniazid were collected and sequencing analysis was performed of the rpoB rifampicin resistance-determining region, the katG and the encoding region of inhA. ResultOf 74 isolates with multidrug resistance, 34 (46%) presented six mutations in katG; the most abundant was katG315 in 29 (39%) isolates. At inhA, nine (11%) isolates presented three mutations; the most frequent was inhA21, located in five (6%) strains. Eleven polymorphisms were observed at rpoB in 61 (82%) isolates, prevailing rpoB531 and rpoB 526 in 48 (64%) and ten (12%) isolates, respectively. Eleven double combinations were observed in 39 (52%) isolates, the most common of which was rpoB531+katG315, found in 22 (29%) strains. ConclusionThis study provides valuable information on the diversity of polymorphisms in genes related to multidrug-resistant tuberculosis, as well as the presence of new mutations not previously described; this information should be considered in the implementation of molecular diagnostic tests.

Characterisation of drug resistance-associated mutations among clinical multidrug-resistant Mycobacterium tuberculosis isolates from Hebei Province, China.

Multidrug-resistant tuberculosis (MDR-TB) is a major public-health problem in China. However, there is little information on the molecular characterisation of clinical MDR-TB isolates in Hebei Province. In this study, 123 MDR-TB isolates were identified in sputum cultures using traditional drug susceptibility testing. The isolates were analysed for mutations in seven genes associated with resistance to antituberculous four drugs: katG and inhA promoter for isoniazid (INH); rpoB for rifampicin (RIF); gyrA and gyrB for ofloxacin (OFLX); and rrs and eis promoter for kanamycin (KAN). All strains were genotyped by spoligotyping and 15-loci MIRU-VNTR analysis. A total of 39 distinct mutations were found at the seven loci in 114/123 (92.7%) MDR-TB isolates. Frequencies of INH, RIF, OFLX and KAN resistance-associated mutations were 82.1% (101/123), 83.7% (103/123), 92.1% (35/38) and 76.2% (16/21), respectively. The most prevalent mutations involved in resistance were: Ser315Thr in katG (70/123; 56.9%) and C(-15)T in inhA (15/123; 12.2%) for INH; Ser531Leu in rpoB (72/123; 58.5%) for RIF; Asp94Gly in gyrA (10/38; 26.3%) for OFLX; and A1401G in rrs (12/21; 57.1%) for KAN. Four novel gyrB mutants (Leu442Leu, Ser447Phe, Asn499Thr and Ala504Val) were identified. Mutations in katG, rpoB (or both) and the inhA promoter showed a sensitivity of 75.6% and specificity of 97.0% for detection of MDR-TB. DNA sequencing of the seven loci was 57.1% sensitive and 91.0% specific for prediction of XDR-TB isolates. These results may be of value in rapid molecular detection of MDR- and XDR-TB isolates in clinical samples in Hebei Province.

Cross-Border Movement of Highly Drug-Resistant Mycobacterium tuberculosis from Papua New Guinea to Australia through Torres Strait Protected Zone, 2010-2015.

In this retrospective study, we used whole-genome sequencing (WGS) to delineate transmission dynamics, characterize drug-resistance markers, and identify risk factors of transmission among Papua New Guinea residents of the Torres Strait Protected Zone (TSPZ) who had tuberculosis diagnoses during 2010–2015. Of 117 isolates collected, we could acquire WGS data for 100; 79 were Beijing sublineage 2.2.1.1, which was associated with active transmission (odds ratio 6.190, 95% CI 2.221–18.077). Strains were distributed widely throughout the TSPZ. Clustering occurred more often within than between villages (p = 0.0013). Including 4 multidrug-resistant tuberculosis isolates from Australia citizens epidemiologically linked to the TSPZ into the transmission network analysis revealed 2 probable cross-border transmission events. All multidrug-resistant isolates (33/104) belonged to Beijing sublineage 2.2.1.1 and had high-level isoniazid and ethionamide co-resistance; 2 isolates were extensively drug resistant. Including WGS in regional surveillance could improve tuberculosis transmission tracking and control strategies within the TSPZ.

Second line drug sensitivity test of multidrug resistant tuberculosis (MDR-TB) isolates using proportional method

Drug resistant Tuberculosis (DR-TB) continue to present challenges to the TB control programmes with Multidrug resistant TB (MDR-TB) becoming a major public health threat predisposing individuals to develop Extensively drug resistant TB (XDR-TB), and Totally dug resistant TB (XXDR-TB). The World Health Organization reemphasizes the need for drug Susceptibility testing (DST) for appropriate treatment of individual cases in order to minimize rapid transmission of tuberculosis especially . DR-TB. The study aimed at performing second line drug sensitivity test of MDR-TB isolates to some of the major second line anti-TB drugs. Samples for the study comprised of 22 purposely selected MDR-TB isolates obtained from sputum samples of new AFB smear positive (MDR-TB) patients that were brought to the North West Zonal Tuberculosis Reference Laboratory located at Aminu Kano Teaching Hospital, Kano. The isolates were re-confirmed as MDR-TB using Line prove assay (LPA), and subjected to second line drug sensitivity testing using Proportional methods to some of the second line anti-TB drugs (Ofloxacin, Capreomycin, Ethionamide, & Kanamycin). The results of the study revealed that all the 22 isolates were re-confirmed as MDR-TB using the LPA. The results further shows that all the 22 isolates were sensitive to Ofloxacin (100%) and Capreomycin (100%), followed by Kanamycin (90.9%) and Aminkacin (86.36%) and none of them was identified as XDR-TB. Most of the isolates 16 (72.72%) were however resistant to Ethionamide with only 6 (27.27%) of them being . sensitive. The study revealed absence of XDR-TB among the studied isolates as all of them were sensitive to Ofloxacin and Capreomycin, however majority of them were resistant to Ethionamide and very few to Amikasin and Kanamycin. The study identifies the need for conducting drug susceptibility tests for isolates from patients identified as new AFB smear positive for appropriate treatment so as to reduce transmission of DR-TB.KEYWORDS: Second Line Drug Sensitivity Test, Proportional Method, MDR-TB

Drug resistance profiles and trends in drug-resistant tuberculosis at a major hospital in Guizhou Province of China.

ObjectivesTuberculosis (TB) is the leading cause of death from infectious diseases in the world, with an estimated 1.6 million deaths from TB in 2017. The objectives of this study were to determine drug resistance profiles of Mycobacterium tuberculosis clinical isolates and to analyze the trends in drug-resistant and multidrug-resistant tuberculosis (MDR-TB) from 2008 to 2015 at a major hospital in Guizhou, a high-TB burden and resource-limited province of China.Patients and methodsA total of 462 clinical isolates were collected from patients with pulmonary TB during the period from January 2013 to December 2015 and used for determining drug resistance profiles against four first-line and six second-line anti-TB drugs, and the results were compared with those of two previous studies.ResultsExactly 4.4% of new and 44.1% of previously treated TB cases were MDR/rifampicin-resistant TB (RR-TB), which were higher than the 2017 global average numbers of 3.5% and 18%, respectively. There were many drug-resistant patterns among MDR-TB isolates and most of them were resistant to three or four anti-TB drugs. The trends in drug-resistant and MDR-TB declined at the hospital from 2008 to 2015.ConclusionResults of this study show that the prevalence of MDR/RR-TB at a major hospital in Guizhou is higher than the global average and confirm the existence of heterogeneity in drug resistance patterns of MDR-TB isolates. Additionally, three practical measures have played an important role in the overall decline of MDR-TB at the hospital from 2008 to 2015.

Genetic Characterization of Second-Line Drug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis from the Northern Region of India

Rapid detection of drug resistance in Mycobacterium tuberculosis is important for the successful treatment of tuberculosis. Fluoroquinolone and aminoglycoside resistance detection by molecular methods becomes more complex due to cross resistance among them. Thus, we aimed to determine cross-resistance and mutations in resistance genes for these drugs. A total of 336 multidrug-resistant tuberculosis (MDR-TB) cases received in Mycobacteriology laboratory were screened for phenotypic drug sensitivity testing for second-line drugs, i.e., ofloxacin, amikacin, kanamycin, and capreomycin. Molecular characterization of resistance was done by DNA sequencing of gyrA gene for fuoroquinolones (FQ), and multiplex allele-specific polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (RFLP) of rrs gene for aminoglycosides. Of 336 MDR-TB isolates, 12 were extensively drug-resistant tuberculosis and 219 were sensitive to all the drugs tested. Ofoxacin, amikacin, kanamycin, and capreomycin resistance was detected in 101 (30.1%), 23 (6.8%), 27 (8.1%), and 19 (5.6%) cases, respectively. Eight different mutations were detected in gyrA gene in ofloxacin-resistant isolates and A1401G nucleotide change in rrs gene were seen in 55.6% (15/27), 65.2% (15/23), and 68.4% (13/29) for kanamycin-, amikacin-, and capreomycin-resistant isolates, respectively. Information on second-line drug resistance-associated mutations could potentially be used for development of newer rapid diagnostic tests.

The impact of combined gene mutations in inhA and ahpC genes on high levels of isoniazid resistance amongst katG non-315 in multidrug-resistant tuberculosis isolates from China

Whole-genome sequencing was used to analyze the profiles of isoniazid (INH) resistance-related mutations among 188 multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB) and mono-INH-resistant isolates collected in a recent Chinese national survey. Mutations were detected in 18 structural genes and two promoter regions in 96.8% of 188 resistant isolates. There were high mutation frequencies in katG, the inhA promoter, and ahpC-oxyR regulator regions in INH-resistant isolates with frequencies of 86.2%, 19.6%, and 18.6%, respectively. Moreover, a high diversity of mutations was identified as 102 mutants contained various types of single or combined gene mutations in the INH-resistant group of isolates. The cumulative frequencies of katG 315 or inhA-P/inhA mutations was 68.1% (128/188) for the INH-resistant isolates. Of these isolates, 46 isolates (24.5% of 188) exhibited a high level of resistance. A high level of resistance was also observed in 21 isolates (11.2% of 188) with single ahpC-oxyR mutations or a combination of ahpC-oxyR and katG non-315 mutations. The remaining 17 mutations occurred sporadically and emerged in isolates with combined katG mutations. Such development of INH resistance is likely due to an accumulation of mutations under the pressure of drug selection. Thus, these findings provided insights on the levels of INH resistance and its correlation with the combinatorial mutation effect resulting from less frequent genes (inhA and/or ahpC). Such knowledge of other genes (apart from katG) in high-level resistance will aid in developing better strategies for the diagnosis and management of TB.

Compensatory evolution drives multidrug-resistant tuberculosis in Central Asia.

Bacterial factors favoring the unprecedented multidrug-resistant tuberculosis (MDR-TB) epidemic in the former Soviet Union remain unclear. We utilized whole genome sequencing and Bayesian statistics to analyze the evolutionary history, temporal emergence of resistance and transmission networks of MDR Mycobacterium tuberculosis complex isolates from Karakalpakstan, Uzbekistan (2001-2006). One clade (termed Central Asian outbreak, CAO) dating back to 1974 (95% HPD 1969-1982) subsequently acquired resistance mediating mutations to eight anti-TB drugs. Introduction of standardized WHO-endorsed directly observed treatment, short-course in Karakalpakstan in 1998 likely selected for CAO-strains, comprising 75% of sampled MDR-TB isolates in 2005/2006. CAO-isolates were also identified in a published cohort from Russia (2008-2010). Similarly, the presence of mutations supposed to compensate bacterial fitness deficits was associated with transmission success and higher drug resistance rates. The genetic make-up of these MDR-strains threatens the success of both empirical and standardized MDR-TB therapies, including the newly WHO-endorsed short MDR-TB regimen in Uzbekistan.

RpoB, katG and inhA mutations in multi-drug resistant strains of Mycobacterium tuberculosis clinical isolates from southeast Mexico

IntroductionPrevious knowledge of molecular mechanisms related with multi-drug resistances in tuberculosis is important if molecular diagnostic procedures want to be used in specific geographical regions. For that reason, the aim of this study was to investigate the mutations at rpoB, katG and inhA in multi-drug resistant tuberculosis isolates from Southeast Mexico. MethodsIsolates of tuberculosis with a confirmed resistance against rifampicin and isoniazid were collected and sequencing analysis was performed of the rpoB rifampicin resistance-determining region, the katG and the encoding region of inhA. ResultOf 74 isolates with multidrug resistance, 34 (46%) presented six mutations in katG; the most abundant was katG315 in 29 (39%) isolates. At inhA, nine (11%) isolates presented three mutations; the most frequent was inhA21, located in five (6%) strains. Eleven polymorphisms were observed at rpoB in 61 (82%) isolates, prevailing rpoB531 and rpoB 526 in 48 (64%) and ten (12%) isolates, respectively. Eleven double combinations were observed in 39 (52%) isolates, the most common of which was rpoB531+katG315, found in 22 (29%) strains. ConclusionThis study provides valuable information on the diversity of polymorphisms in genes related to multidrug-resistant tuberculosis, as well as the presence of new mutations not previously described; this information should be considered in the implementation of molecular diagnostic tests.

Characterization of mutations conferring streptomycin resistance to multidrug-resistant Mycobacterium tuberculosis isolates from Myanmar

Numerous studies report that mutations of rpsL (encoding the S12 protein), rrs (encoding 16S rRNA) and gidB (encoding rRNA methyltransferase) are responsible for conferring resistance to streptomycin (STR), which is usually used in both multidrug-resistant tuberculosis (MDR-TB) treatments and re-treatments in Myanmar. The aim of this study was to explore the variation and frequency of mutations in rpsL, rrs and gidB in 141 STR-resistant MDR-TB isolates from Myanmar. Most isolates belonged to the Beijing genotype (105, 74.5%). Moreover, mutations in rpsL were identified in 69.5% (98/141) of the STR-resistant isolates, where the most prevalent (92.0%, 90/98) and significantly associated mutation with the Beijing genotype (P < 0.001) was Lys43Arg. Fifteen different mutations in gidB were found in 16.3% (23/141) of the isolates, and most of them were novel mutations. Moreover, based on our results, we suggest A276C nucleotide substitution in gidB as a phylogenetic marker for the Beijing family in Myanmar. Sequence analysis of rpsL, rrs and gidB with a sensitivity of 83.7% satisfactorily predicted STR resistance in Myanmar isolates. However, in 16.3% (23/141) of the isolates, none of the examined genes showed mutation. Hence, further studies are strongly recommended to elucidate other possible resistance mechanisms. The present findings may be useful in developing molecular STR susceptibility assays, which in turn could contribute to develop TB treatments and control strategies in Myanmar.

Multi- and Extensively Drug Resistant Mycobacterium tuberculosis in South Africa: a Molecular Analysis of Historical Isolates.

Modern advances in genomics provide an opportunity to reinterpret historical bacterial culture collections. In this study, genotypic antibiotic resistance profiles of Mycobacterium tuberculosis isolates from a historical 20-year-old multidrug-resistant tuberculosis (MDR-TB) culture collection in South Africa are described. DNA samples extracted from the phenotypically MDR-TB isolates (n = 240) were assayed by Hain line probe assay (LPA) for the confirmation of MDR-TB and by Illumina Miseq whole-genome sequencing (WGS) for the characterization of mutations in eight genes (rpoB, katG, inhA, rpsL, pncA, embB, gyrA, and rrs) that are known to code for resistance to commonly used anti-TB agents. LPA identified 71.3% of the TB isolates as MDR-TB, 18.3% as rifampin (RIF) monoresistant, 2% as isoniazid (INH) monoresistant, and 8.3% as susceptible to both RIF and INH (RIF+INH). In a subset of 42 randomly selected isolates designated as RIF+INH resistant by Löwenstein-Jensen (LJ) culture in 1993, LPA and WGS results confirmed MDR-TB. In all five INH-monoresistant isolates by LPA and in all but one (the wild type) of the 34 successfully sequenced RIF-monoresistant isolates, WGS revealed matching mutations. Only 26% of isolates designated as susceptible by LPA, however, were found to be wild type by WGS. Novel mutations were found in the rpoB (Thr480Ala, Gln253Arg, Val249Met, Val251Tyr, Val251Phe), katG (Trp477STOP, Gln88STOP, Trp198STOP, Trp412STOP), embB (Thr11Xaa, Gln59Pro), and pncA (Thr100Ile, Thr159Ala, Ala134Arg, Val163Ala, Thr153Ile, DelGpos7, Phe106Ser) genes. Three MDR-TB isolates showed mutations in both the gyrA and rrs genes, suggesting that extensively drug-resistant tuberculosis existed in South Africa well before its formal recognition in 2006.

Second Line Drugs Resistant Mycobacterium Tuberculosis in Multi-Drug Resistant Tuberculosis Patients

With an increase in Multi-drug resistant tuberculosis (MDR-TB), there is a need of second line drug susceptibility test that helps in early diagnosis and minimize the risk of other powerful drug resistant Mycobacterium tuberculosis. The aim of this study was to determine second line drugs (ofloxacin, kanamycin, capreomycin) resistance pattern in MDR-TB isolates and to determine the prevalence of pre-Extensively drug resistant tuberculosis (pre-XDR-TB) and XDR-TB in MDR-TB patients. The study was conducted from February to September 2015 at National Tuberculosis Centre, Thimi, Bhaktapur. MDR-TB (resistant to isoniazid and rifampicin) patients’ sputum samples were processed by Modified Petroff’s method. Out of 92 samples, 57 were found culture positive. Following the species identification of culture positive MDR-TB isolates, second line drug susceptibility test was performed by conventional proportion method. Of 57 MDR-TB isolates, 22 (38.59%) showed resistance to ofloxacin (Ofx), 9 (15.79%) to capreomycin (Cm) and 9 (15.79%) to kanamycin (Km). One XDR-TB (1.8%) resistant to all drugs was detected. Of the remaining, 21(36.8%) were resistant to ofloxacin only and 8(15.4%) were resistant to two drugs i.e.29 (50.9%) were pre-XDR-TB. The prevalence of pre-XDR-TB and XDR-TB was found to be 50.88% and 1.75% respectively. The resistance pattern of second line anti-tuberculosis drugs showed higher ofloxacin resistance in MDR-TB patients. In a nutshell, MDR-TB cases need urgent and timely susceptibility report for second line anti-tuberculosis drugs to help the clinicians start proper drug combinations to treat MDR-TB patients. Journal of Institute of Science and Technology Volume 22, Issue 2, January 2018, page: 168-174

Genetic diversity of drug and multidrug-resistant Mycobacterium tuberculosis circulating in Veracruz, Mexico.

BackgroundMexico is one of the most important contributors of drug and multidrug-resistant tuberculosis in Latin America; however, knowledge of the genetic diversity of drug-resistant tuberculosis isolates is limited.MethodsIn this study, the genetic structure of 112 Mycobacterium tuberculosis strains from the southeastern Mexico was determined by spoligotyping and 24-loci MIRU-VNTRs.FindingsThe results show eight major lineages, the most of which was T1 (24%), followed by LAM (16%) and H (15%). A total of 29 (25%) isolates were identified as orphan. The most abundant SITs were SIT53/T1 and SIT42/LAM9 with 10 isolates each and SIT50/H3 with eight isolates. Fifty-two spoligotype patterns, twenty-seven clusters and ten clonal complexes were observed, demonstrating an important genetic diversity of drug and multidrug-resistant tuberculosis isolates in circulation and transmission level of these aggravated forms of tuberculosis. Being defined as orphan or as part of an orphan cluster, was a risk factor for multidrug resistant-tuberculosis (OR 2.5, IC 1.05–5.86 and OR 3.3, IC 1–11.03, respectively). Multiple correspondence analyses showed association of some clusters and SITs with specific geographical locations.ConclusionsOur study provides one of the most detailed description of the genetic structure of drug and multidrug-resistant tuberculosis strains in southeast Mexico, establishing for the first time a baseline of the genotypes observed in resistant isolates circulating, however further studies are required to better elucidate the genetic structure of tuberculosis in region and the factors that could be participating in their dispersion.

Molecular Screening Versus Phenotypic Susceptibility Testing of Multidrug-Resistant Mycobacterium tuberculosis Isolates for Streptomycin and Ethambutol.

Proper management of multidrug-resistant tuberculosis (MDR-TB) requires accurate drug susceptibility testing (DST) of Mycobacterium tuberculosis isolates to other (ethambutol [EMB], pyrazinamide, and streptomycin [SM]) first-line drugs. This study compared the performance of Mycobacterium Growth Indicator Tube (MGIT) 960 system for DST of MDR-TB isolates with polymerase chain reaction (PCR) sequencing of embB, rpsL, and rrs genes for detecting resistance to EMB and SM. MDR-TB strains (n = 60) and 25 pansusceptible M. tuberculosis isolates collected during 2011-2016 were tested. Phenotypic DST was performed by MGIT 960 system by using SIRE drug kit. EMB and SM resistance-conferring mutations in embB and rpsL+rrs genes, respectively, were detected by PCR sequencing. No mutations were detected in pansusceptible isolates. Among 60 MDR-TB strains, 35 of 40 SM-resistant and none of 20 SM-susceptible isolates contained rpsL and/or rrs mutations (κ = 0.82, very good agreement). However, all 18 EMB-resistant MDR-TB strains and 33 of 42 EMB-susceptible MDR-TB strains contained an embB mutation (κ = 0.14, poor agreement). Thus, 40 of 60 (67%) and 35 of 60 (58%) isolates were resistant to SM (p = 0.451), while 18 of 60 (30%) and 51 of 60 (85%) isolates were resistant to EMB (p = 0.000) by MGIT 960 system and PCR sequencing, respectively. MGIT 960 system showed acceptable performance for DST for SM; however, it performed poorly for EMB as many MDR-TB strains with embB mutations, which confer low-level resistance to EMB, were detected as EMB susceptible. Molecular screening for resistance-conferring mutations in embB gene is thus superior to MGIT 960 system when accurate EMB susceptibility results are needed for proper management of MDR-TB patients.

Prevalence and molecular characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis isolates from Southern China

BackgroundPyrazinamide (PZA) plays a unique role in the treatment for multidrug-resistant tuberculosis (MDR-TB) in both first- and second-line regimens. The aim of this study was to investigate the prevalence and molecular characterization of PZA resistance among MDR-TB isolates collected in Chongqing municipality.MethodsA total of 133 MDR-TB isolates were collected from the smear-positive tuberculosis patients who were registered at local TB dispensaries of Chongqing. PZA susceptibility testing was determined with a Bactec MGIT 960 system. In addition, the genes conferring for PZA resistance were screened by DNA sequencing.ResultsOf these 133 MDR-TB isolates, 83 (62.4%) were determined as PZA-resistant by MGIT 960. In addition, streptomycin- (83.1% vs. 56.0%, P < 0.01), ofloxacin- (51.8% vs. 18.0%, P < 0.01), kanamycin- (22.9% vs. 2.0%, P < 0.01), amikacin- (18.1% vs. 2.0%, P = 0.01), capromycin-resistance (12.0% vs. 2.0%, P = 0.05), were more frequently observed among PZA-resistant isolates compared with PZA-susceptible isolates. Sequence analysis revealed that 73 out of 83 (88.0%) MDR strains harbored a mutation located in the pncA gene, including 55 (75.3%, 55/73) of single nucleotide substitutions and 18 (24.7%, 18/73) of frameshift mutation, while no genetic mutation associated with PZA resistance was found in the rpsA gene. The pncA expression of strains harboring substitution from A to G at position −11 in the promoter region of pncA was significantly lower than that of H37Rv (P < 0.01).ConclusionsIn conclusion, our data have demonstrated that the analysis of the pncA gene rather than rpsA gene provides rapid and accurate information regarding PZA susceptibility for MDR-TB isolates in Chongqing. In addition, loss of pncA expression caused by promoter mutation confers PZA resistance in MDR-TB isolates.

Correlation between the BACTEC MGIT 960 culture system with Genotype MTBDRplus and TB-SPRINT in multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil.

BACKGROUNDThe accurate detection of multidrug-resistant tuberculosis (MDR-TB) is critical for the application of appropriate patient treatment and prevention of transmission of drug-resistant Mycobacterium tuberculosis isolates. The goal of this study was to evaluate the correlation between phenotypic and molecular techniques for drug-resistant tuberculosis diagnostics. Molecular techniques used were the line probe assay genotype MTBDRplus and the recently described tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT) bead-based assay. Conventional drug susceptibility testing (DST) was done on a BACTECTM MGIT 960 TB.METHODWe studied 80 M. tuberculosis complex (MTC) clinical isolates from Minas Gerais state, of which conventional DST had classified 60 isolates as MDR and 20 as drug susceptible.FINDINGSAmong the 60 MDR-TB isolates with MGIT as a reference, sensitivity, specificity, accuracy, and kappa for rifampicin (RIF) resistance using TB-SPRINT and MTBDRplus, were 96.7% versus 93.3%, 100.0% versus 100.0%, 97.5% versus 95.0% and 0.94 versus 0.88, respectively. Similarly, the sensitivity, specificity, accuracy, and kappa for isoniazid (INH) resistance were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. Finally, the sensitivity, specificity, accuracy, and kappa for MDR-TB were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively.MAIN CONCLUSIONSBoth methods exhibited a good correlation with the conventional DST. We suggest estimating the cost-effectiveness of MTBDRplus and TB-SPRINT in Brazil.

Genotyping and molecular detection of multidrug-resistant Mycobacterium tuberculosis among tuberculosis lymphadenitis cases in Addis Ababa, Ethiopia.

Multidrug-resistant tuberculosis (MDR-TB) has emerged as a major public health problem. Drug-resistance surveillance data show that 3.9% of new and 21% of previously treated TB cases were estimated to have had rifampicin/ multidrug-resistant tuberculosis (MDR/RR-TB) in 2015. This implies that the MDR-TB is increasing alarmingly. Hence, a better understanding of drug resistance mechanisms and genotypes associated with multidrug resistance in M. tuberculosis is crucial for improving diagnostic and therapeutic methods to treat individuals with MDR-TB. The aim of this study was to analyze molecular drug resistance mutations of MDR-TB isolates from the cases of TB-lymphadenitis in relation to its genetic lineages.A cross-sectional study was conducted on culture positive cases from July to October, 2014 in Addis Ababa, Ethiopia. Sixty isolates were included to analyze drug resistance mutated gene responsible for MDR-TB in relation to its molecular genotyping. Mycobacterial culture, GenoTypeMTBDR plus and Spoligotyping were used to undertake the study.Of 60 TBLN isolates, 8.3% were identified MDR-TB cases and one isolate was isoniazid mono-resistant. Eleven isolates in T3-ETH genetic sub lineage were sensitive to both RMP and INH, while only 2 isolates were MDR-TB. Most of the RMP- resistant isolates showed mutation in codon S531L and all isolates mutated in the katG gene conferring INH resistant strains had mutations in codon of S315T1.Screening for the rpoB and katG gene mutation of tuberculosis lymphadenitis is useful in Ethiopia for an early detection and treatment of MDR-TB. Besides, there is a drug resistance variation among different lineages of Tuberculosis lymphadenitis which has important consequences for the development of efficient control strategies.