Multidrug-resistant Tuberculosis Isolates Research Articles

Predicting differential rifamycin resistance in clinical Mycobacterium tuberculosis isolates by specific rpoB mutations.

Rifampin (RMP) resistant Mycobacterium tuberculosis is usually assumed to be resistant to all rifamycins. Increasing evidence indicates, however, that some rpoB mutations, detectable by rapid molecular diagnostics, confer resistance to RMP but not to rifabutin (RBT), suggesting that RBT may be effective for the treatment of M. tuberculosis with these mutations. To determine if specific rpoB mutations reliably predict differential phenotypic resistance to RMP and RBT. We selected 60 clinical M. tuberculosis isolates from a repository of multinational multidrug-resistant tuberculosis isolates and stratified them into two groups: 1) those with rpoB mutations suspected to confer differential resistance to RMP and RBT, and 2) those expected to be cross-resistant to RMP and RBT. These assumptions were tested by comparing the phenotypic susceptibilities of RMP/RBT with those predicted by mutations in the rpoB gene. Of 20 suspected RMP-resistant/RBT-susceptible isolates, 15 were RMP-resistant but RBT-susceptible, 3 were RMP- and RBT-susceptible, and 2 were cross-resistant to both RMP and RBT. In comparison, 40 of 40 suspected cross-resistant isolates were both RMP- and RBT-resistant. Our data support the association between specific rpoB mutations and differential resistance of M. tuberculosis to RMP and RBT. Clinical studies are required to investigate the efficacy of RBT in the treatment of M. tuberculosis harboring these mutations.

Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates

The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n = 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 82% (n = 88), 83% (n = 88), and 89% (n = 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.

Optimization of Annealing Temperature for Amplification of 507 bp fragment of rpoB Gene of Clinical Multidrug-Resistant Mycobacterium tuberculosis Isolate 86

The gene of rpoB is the primary gene that is well known as a surrogate marker in MDR-TB (Multidrug-Resistant Tuberculosis) detection. The mutation of rpoB was studied around the world in its core region called RRDR (Rifampicin Resistance Determining Region). To learn the mutation in this fragment, PCR (Polymerase Chain Reaction) is the most common method used. This study was purposed to optimize the annealing temperature in amplifying the 507 bp fragment of rpob gene containing RRDR of isolate 86.DNA of MDR-TB isolate 86 was isolated by using Boom method for further amplified by PCR. The oligonucleotide primers used in this study were FrTB and RrTB. Eight different annealing temperature were used to optimize the amplification of rpoB gene : 53, 55, 57, 58, 59, 60, 61, and 63oC. Detection of PCR products was done with 1,5% agarose gel electrophoresis.Agarose gel electrophoresis of PCR products showed that 507 bp of rpoB gene could be produced at all annealing temperatures. A faint amplified product was observed at temperature 53 and 55oC. However, at temperature 58 and 60oC more intense bands were observed. In conclusion, the best annealing temperature was at 60oC in producing the 507 bp fragment.

In Vitro Evaluation of Linezolid and Meropenem Against Clinical Isolates of Multi Drug Resistant Tuberculosis By Mycobacterial Growth Indicator Tube (MGIT) 960.

To evaluate the in vitro effectiveness of multiple breakpoint concentrations of newer antituberculosis agents (Linezolid and Meropenem) against Multi Drug-Resistant Tuberculosis (MDR-TB) isolates. A descriptive cross-sectional study. Microbiology Department, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from September 2011 to August 2013. A total of 100 MDR-TB isolates recovered during the study period were subjected to susceptibility testing against multiple breakpoint concentrations of Linezolid (LZD) and Meropenem (MER). The breakpoint concentration used for LZD were 0.5, 1.0 and 2.0 µg/ml, while for MER were 4.0, 8.0 and 16 µg/ml. Mycobacterial Growth Indicator Tube (MGIT) 960 system was used to carry out drug susceptibility testing as per recommended protocol. At break point concentration of 0.5 µg/ml, 80 out of 100 (80%) MDR-TB isolates were susceptible to LZD while at breakpoint concentration of 1.0 µg/ml and 2.0 µg/ml, 96/100, (96%) of MDR-TB isolates were susceptible. For MER, at breakpoint concentrations of 4.0 µg/ml no MDR-TB isolate was susceptible, while at 8.0 µg/ml 3/100, (3%) and at 16.0 µg/ml 11/100, (11%) of MDR-TB isolates were susceptible. LZD was found to have excellent in vitro efficacy as 96% of MDR-TB isolates were susceptible at breakpoint concentration of 1.0 µg/ml or more. In case of MER it was found that in vitro susceptibility improved as the break point concentrations were increased.

Extensively and pre-extensively drug resistant tuberculosis in clinical isolates of multi-drug resistant tuberculosis using classical second line drugs (levofloxacin and amikacin).

To find out the frequency of Extensively Drug Resistant (XDR) and pre-XDR tuberculosis in clinical isolates of Multi-Drug Resistant (MDR) Tuberculosis (TB) by determining the susceptibilities against Levofloxacin and Amikacin (classical second line antituberculosis drugs). Adescriptive cross-sectional study. Microbiology Department, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from September 2011 to August 2013. Amikacin (AK) and Levofloxacin (LEVO) were obtained in chemically pure form from Sigma (Taufkirchen, Germany). The breakpoint concentration used for AK was 1.0 µg/ml and for LEVO 2.0 µg/ml. Mycobacterial Growth Indicator Tube (MGIT) 960 system was used to carry out drug susceptibility testing as per recommended protocol. A total of 3 MDR-TB isolates (3%) turned out to be XDR-TB based upon simultaneous resistance to injectable second line antituberculosis drug AK and one of the fluoro-quinolones (LEVO). A total of 24 MDR-TB isolates (24%) were found to be pre-XDR based upon resistance to LEVO alone. Treatment status record of patients with XDR and pre-XDRTB isolates revealed that majority of patients had received fluoroquinolones (FQs) during the course of treatment. XDR-TB has started to emerge in MDR-TB isolates in our set up. The worrying sign is the high frequency of pre-XDR tuberculosis. Urgent steps need to be taken to stem the tide of pre-XDR-TB in our population. It is thus recommended to develop facilities to carry out drug susceptibility testing to monitor the status of pre-XDR and XDR-TB in our population.

The impact of isoniazid resistant Mycobacterium tuberculosis on the epidemic of multi-drug resistant tuberculosis

Objective To investigate the impact of isoniazid (INH)-resistant Mycobacterium tuberculosis (Mtb) on the prevalence and dissemination of multi-drug resistant tuberculosis (MDR-TB). Methods A total of 251 patients diagnosed with tuberculosis in designated hospitals of Guanyun, Jiangsu and Deqing, Zhejiang from 2010 to 2011 were included in the study. The drug susceptibility tests (DST) were performed on all the Mtb isolates available from the sputum cultures. Mycobacteral interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) was conducted for genotyping for all available Mtb isolates. Chi-square test, Fisher exact test, ANOVA and non-conditional Logistic regression modelling were applied for data analysis. Results Among 251 patients with Mtb isolates and DST results available, 72(28.7%) were resistant to INH, including 13 were INH mono-drug resistant. Of the remaining 59 INH-resistant Mtb, 34(13.5%) were resistant to rifampin TB and 25 were resistant to streptomycin and/or ethambutol. The clustering analysis based on MIRU-VNTR genotyping revealed 29 clustered genotypes (including 105 isolates) and 146 unique genotypes (including 119 isolates). Twenty-five clusters contained drug resistant Mtb and 16 clusters of them comprised by 37 INH-resistant isolates and 20 MDR-TB isolates, which accounted for 51.4% of the INH-resistant isolates and 58.8% of the MDR-TB isolates. Single factor analysis showed that sex, age, previous tuberculosis treatment history and sputum smear results were all related to INH-resistant tuberculosis and MDR-TB (all P<0.05). Multiple factors analysis showed that previous tuberculosis treatment history was risk factor of MDR-TB (OR=8.40, 95%CI: 3.342-21.105), while the risk factors of INH-resistant tuberculosis were previous tuberculosis treatment history (OR=3.52, 95%CI: 1.570-7.910), pulmonary caviry (OR=2.27, 95%CI: 1.075-4.799) and sputum smear results (OR=0.50, 95%CI: 0.275-0.892). Conclusions That INH-resistant strain may evolve to the MDR-TB after recent transmission is a possible trend. Patients with previous treatment history and advanced age are at high risk of INH-resistant tuberculosis and MDR-TB. Key words: Mycobacterium tuberculosis; Isoniazid; Tuberculosis, multidrug-resistant

Analysis of embCAB mutations associated with ethambutol resistance in multidrug-resistant mycobacterium tuberculosis isolates from China.

Ethambutol (EMB) plays a pivotal role in the chemotherapy of drug-resistant tuberculosis (TB), including multidrug-resistant tuberculosis (MDR-TB). Resistance to EMB is considered to be caused by mutations in the embCAB operon (embC, embA, and embB). In this study, we analyzed the embCAB mutations among 139 MDR-TB isolates from China and found a possible association between embCAB operon mutation and EMB resistance. Our data indicate that 56.8% of MDR-TB isolates are resistant to EMB, and 82.2% of EMB-resistant isolates belong to the Beijing family. Overall, 110 (79.1%) MDR-TB isolates had at least one mutation in the embCAB operon. The majority of mutations were present in the embB gene and the embA upstream region, which also displayed significant correlations with EMB resistance. The most common mutations occurred at codon 306 in embB (embB306), followed by embB406, embA(-16), and embB497. Mutations at embB306 were associated with EMB resistance. DNA sequencing of embB306-497 was the best strategy for detecting EMB resistance, with 89.9% sensitivity, 58.3% specificity, and 76.3% accuracy. Additionally, embB306 had limited value as a candidate predictor for EMB resistance among MDR-TB infections in China.

Diagnosis of Multi-drug Resistant Tuberculosis Mutations Using Hain Line Probe Assay and GeneXpert: A Study Done in Zimbabwe

Background and Aims: Tuberculosis (TB) is a global public health problem and one of the leading causes of death. Worldwide, 31% of all estimated new TB cases are from Africa. Zimbabwe is one of the 22 high TB burden countries. Multi-drug resistant TB (MDR-TB) poses challenges in TB control, hence the need for rapid laboratory diagnosis of MDR-TB for optimal treatment and reducing spread. The study aim was to investigate genetic mutations associated with MDR-TB isolates from various Harare clinics using the GeneXpert MTB/RIF® by Cepheid and Original Research Article Racheal et al.; BJMMR, 5(8): 1044-1052, 2015; Article no.BJMMR.2015.115 1045 Genotype MTBDRplus, to improve the diagnosis and management of MDR-TB. Methods: Samples from adults aged 16 years and older, recruited from several polyclinics in the southern suburbs of Harare were used for our study. All laboratory tests prior to this study had been carried out at Biomedical research and training institute’s level three bio-safety TB laboratory from January 2008-August 2012. Ethical approval was sought from BRTI Institutional review board. A total of 69 (37 MDR-TB and 32 non MDR-TB) archived isolates processed on Genotype MTBDRplus (Hains) and corresponding 39 sputum were processed on the GeneXpert. Mutations on rpoB, katG and inhA genes were observed. The gold standard was culture. Diagnostic accuracy of both methods and their level of agreement were calculated. Results: Of the 37/69 isolates screened by culture for MDR-TB, 88.4% were confirmed by MTBDR plus line probe assay (Hains). Within the 39 isolates tested using the Xpert MTB/RIF (GeneXpert) assay 12 were true MDR-TB. Over 8 single nucleotide polymorphisms were observed on the three genes conferring Rifampicin and Isoniazide drug resistance. The Hains and GeneXpert had an almost perfect agreement with a kappa value of 0.82. Conclusion: Genetic markers can be used in the diagnosis of MDR-TB, to complement phenotypic methods such as culture. Using the commercial methods, Hains and GeneXpert, 88.494.2% of drug resistance maybe detected. Furthermore, we recommend sequencing so as to identify novel mutations and to design a kit that is custom made for the population.

Second-line drug resistance patterns among patients with multidrug-resistant tuberculosis of Gujarat, western India

Background: Lack of laboratory diagnostic capacity is a crucial barrier preventing an effective response to early and appropriate case detection and treatment of multidrug-resistant tuberculosis (MDR-TB). To date, limited representative drug resistance data are available from India on second-line drug (SLD) resistance. Objectives: To study SLD resistance patterns in MDR-TB patients of Gujarat, western India. Materials and Methods: Antituberculosis drug susceptibility testing of 50 MDR-TB isolates particularly for SLDs, namely p-aminosalicylic acid, ciprofloxacin, amikacin, D-cycloserine, kanamycin, and ethionamide, was conducted on solid Lowenstein–Jensen medium using the 1% proportion sensitivity method. Results: Of 50 MDR-TB isolates tested, 24 (24/50, 48%) were found to be resistant to one or more SLDs. Of 50 MDR-TB isolates, 18 (18/50, 36%) showed resistance to ethionamide, 13 (13/50, 26%) to D-cycloserine, 11 (11/50, 22%) to ciprofloxacin, 7 (7/50, 14%) to kanamycin, 6 (6/50, 12%) to p-aminosalicylic acid, and 5 (5/50, 10%) to Amikacin. Conclusion: High levels of SLD resistance were found to be present in this study. There is an urgent need for more control and rational use of the widely available fluoroquinolones and other SLDs outside of the Revised National Tuberculosis Control Programme, by both the public and the private health sectors in India.

Detection of novel and reported mutations in the rpoB, katG and inhA genes in multidrug-resistant tuberculosis isolates: A hospital-based study.

The objective of this study was to detect mutations associated with isoniazid (INH) and rifampicin (RIF) resistance in Mycobacterium tuberculosis isolates from newly diagnosed and previously treated tuberculosis patients using a PCR-based DNA sequencing technique. Phenotypic drug susceptibility testing was performed using a BACTEC™ MicroMGIT Culture System in 354 M. tuberculosis isolates. Among the 354 isolates, 18 were multidrug-resistant tuberculosis (MDR-TB). PCR-based DNA sequencing was performed targeting the rpoB gene for RIF and the whole of the katG gene and the promoter and coding region of the inhA gene for INH. Results were analysed using MultAlin analysis to identify the presence of polymorphisms or mutations by comparing with already available GenBank sequences. Only 37.5% of RIF-resistant isolates showed the presence of the most commonly reported mutation (Ser531Leu). The most commonly reported mutation (Ser531Leu) was detected in six MDR-TB isolates. The frequency of mutations associated with INH resistance was 31.5% (17/54) and 29.6% (16/54) for katG and inhA, respectively. Comparing the relative distribution of mutations in the two target loci revealed that 12 isolates (22.2%) had a mutation in both katG and inhA. Apart from previously reported mutations in the katG gene, there were three novel deletion and six novel substitution mutations. As reported in previous studies, Ser531Leu was the most common mutation detected in RIF-resistant isolates. The genetic mechanism of INH resistance in M. tuberculosis is highly complex involving several genes, and much remains to be explored to achieve a better understanding of this complex mechanism.

In vitro synergistic activity of clofazimine and other antituberculous drugs against multidrug-resistant Mycobacterium tuberculosis isolates

Clofazimine (CLO) is a promising candidate drug for use in the management of multidrug-resistant tuberculosis (MDR-TB) patients. In this study, the minimum inhibitory concentration (MIC) method was used to investigate drug susceptibility to CLO as well as potential synergies between CLO and other antituberculous drugs, including ethambutol (EMB), levofloxacin (LEV), moxifloxacin (MOX), amikacin (AMK) and capreomycin (CAP), among MDR-TB isolates from China. A total of 195 MDR-TB isolates were collected from the national drug resistance survey conducted in China. Of the 195 MDR-TB isolates, 54 (27.7%) were classified as CLO-resistant, whilst 141 (72.3%) were CLO-susceptible with MICs of ≤1μg/mL. In addition, the prevalence of CLO-resistant isolates among the extensively drug-resistant (XDR)-TB group was 61.5% (8/13), which was significantly higher than that of the MDR-TB group (23.0%) (P=0.006). When fractional inhibitory concentration indexes (FICIs) were calculated for 24 isolates, synergy was found in 11 isolates (45.8%) against the CLO/EMB combination, 6 (25.0%) against the CLO/LEV combination, 8 (33.3%) against the CLO/MOX combination, 4 (16.7%) against the CLO/AMK combination and 5 (20.8%) against the CLO/CAP combination. In addition, <15% of MDR-TB isolates showed antagonistic effects against these five combinations. In conclusion, these findings demonstrate that the combination of CLO and EMB shows better synergism than the other combinations containing CLO. The CLO/MOX combination is more likely to show synergy against MDR-TB isolates than the CLO/LEV combination. Taken together, we suggest that CLO, in combination with EMB or MOX, may be a promising drug regimen for the treatment of MDR-TB.

Mutation pattern in rifampicin resistance determining region of rpoB gene in multidrug-resistant Mycobacterium tuberculosis isolates from Pakistan

The current study was undertaken to characterize the RRDR rpoB gene mutations among the rifampicin-resistant Mycobacterium tuberculosis (MTB) isolates from Pakistan. Rifampicin mutation patterns were analyzed by using PCR followed by rpoB gene sequencing. Among the 1080 referred TB cases, 63 (6%) were resistant against at least one first-line TB drug. Out of these 63 resistant isolates, 24 isolates (38%) were found to be resistant to isoniazid and rifampicin. Sequence analysis of multidrug-resistant tuberculosis (MDR-TB) isolates detected a single mutation in the RRDR region of the rpoB gene at codon 531, 516, 512, 528 and 533; however, 5 MDR-TB isolates lack any mutation in the RRDR region. A double mutation was observed in 1 MDR-TB isolate at codon 512 and 516 which are reported for the first time from Pakistan. Moreover, in 1 isolate a novel silent mutation was observed at codon 528. Further studies about these mutations may be helpful in the development of diagnostic tools for the detection of MTB in a high TB endemic area like Pakistan.

In vitro bactericidal assay of linezolid on multidrug-resistant tuberculosis and nontuberculous mycobacteria

Objective To investigate the mycobactericidal efficacy of linezolid on multidrug-resistant tuberculosis (MDR-TB)and nontuberculous mycobacteria (NTM) in vitro. Methods Seven hundred and sixty-two Mycobacterium tuberculosis and 20 NTM isolates from Hangzhou were identified by using hsp65 gene sequencing from March to June, 2013. Seventy MDR-TB isolates, which were screened by using proportion method, and twenty NTM isolates were tested the MIC to linezolid，and the differences of MICs to linezolid between MDR-TB and NTM werw analyzed in vitro. Results All 70 MDR-TB isolates were inhibited at concentrations of ≤1 mg/L by linezolid, MIC50 was 0.5 mg/L and MIC90 1 mg/L. Of all 8 isolates inhibited at concentration of 1 mg/L，there were 6 isolates resistant to isoniazid, rifampin, streptomycin and ethambutol, suggesting this resistance pattern may reduce sensitivity to linezolid. Among the NTM isolates, 18 Mycobacterium intracellulare isolates, whose MICs to linezolid at 8-16 mg/L, were non-resistant isolates, while 2 Mycobacterium abscessus isolates displaying MICs＞32 mg/L, were resistant to linezolid. Conclusions Linezolid showed great activity to MDR-TB and there were no linezolid-resistant MDR-TB isolates identified in this study. However, MICs of MDR-TB against linezolid increased with their resistant numbers to first-line agents. This study also showed that different NTM species have varied sensitivity to linezolid. （Chin J Lab Med,2014,37:535-538） Key words: Mycobacterium tuberculosis; Nontuberculous mycobacteria; Drug resistance, multiple, bacterial

Trends in childhood drug-resistant tuberculosis in South Africa: a window into the wider epidemic?

Childhood tuberculosis (TB) is a marker of TB transmission within a community. We present the fourth consecutive survey of children with culture-confirmed TB at a hospital in Cape Town, South Africa, from 2009 to 2011. In comparison to the previous survey, the rate of multidrug-resistant TB (MDR-TB) has stabilised and the human immunodeficiency virus infection rate has declined. We also report on the first systematic surveillance of resistance to second-line drugs. Two concerns following from this are the high rate (22%) of ofloxacin resistance in MDR-TB isolates, and the discordance between isolates with an inhA promoter region mutation, usually implying ethionamide (ETH) resistance, and phenotypic ETH results in these isolates showing ETH susceptibility.

Multi-drug resistant Mycobacterium tuberculosis complex genetic diversity and clues on recent transmission in Punjab, Pakistan

Multi-Drug Resistant Tuberculosis (MDR-TB), i.e. bacilli resistant to rifampicin (RIF) and isoniazid (INH), is a major Public Health concern in Pakistan according to WHO estimates (3.5% and 32% of new and retreated cases, respectively). Previous Pakistanis reports identified a correlation between being MDR and belonging to Beijing or EAI lineages in one study, and belonging to “H4”-Ural Euro-American sublineage in another study. In addition, MDR-TB transmission was suspected in Karachi.We tested MDR characteristics on a Punjab sample of 278 clinical isolates (without selection for Multi-Drug Resistance) including new and retreated cases collected from 2008 to 2012. All samples were characterized by a new, microbead-based method named “TB-SPRINT” (molecular diagnostic including spoligotype identification, and genetic resistance determinants to first-line anti-TB drugs RIF and INH). Isolates from 2011 to 2012 (n=100) were further analyzed using 24-loci MIRU-VNTR.We detected 8.7% MDR isolates (CI95%=[5.0; 12.5]), mainly among CAS lineage that predominates in this central-East region of Pakistan. Out of 20 MDR-TB cases, 12 different TB-SPRINT profiles were identified, limiting the suspicion of MDR-TB transmission. 24 MIRU-VNTR confirmed the unrelatedness of isolates with different TB-SPRINT profiles and discriminated 3 isolates with identical TB-SPRINT profiles.In conclusion, our study did not confirm any of the correlations between Multi-Drug Resistance and lineage or sublineage in Punjab, Pakistan. MDR-TB isolates were diverse indicating that transmission is not pervasive. TB-SPRINT proved useful as a first step for detecting MDR-TB likely transmission events, before more extensive genotyping such as 15 or 24 MIRU-VNTR and thorough epidemiological investigation.

Survey of tuberculosis drug resistance among Tibetan refugees in India.

Tuberculosis (TB) is a major health problem among Tibetans living in exile in India. Although drug-resistant TB is considered common in clinical practice, precise data are lacking. To determine the proportion of drug-resistant cases among new and previously treated Tibetan TB patients. In a drug resistance survey in five Tibetan settlements in India, culture and drug susceptibility testing (DST) for first-line drugs were performed among all consecutive new and previously treated TB cases from April 2010 to September 2011. DST against kanamycin (KM), ethionamide, para-aminosalicylic acid and ofloxacin (OFX) was performed on multidrug-resistant TB (MDR-TB) isolates. Of 307 patients enrolled in the study, 264 (193 new and 71 previously treated) were culture-positive and had DST available. All patients tested for the human immunodeficiency virus (n = 250) were negative. Among new TB cases, 14.5% had MDR-TB and 5.7% were isoniazid (INH) monoresistant. Among previously treated cases, 31.4% had MDR-TB and 12.7% were INH-monoresistant. Of the MDR-TB isolates, 28.6% of new and 26.1% of previously treated cases were OFX-resistant, while 7.1% of new cases and 8.7% of previously treated cases were KM-resistant. Three patients had extensively drug-resistant TB. MDR-TB is common in new and previously treated Tibetans in India, who also show additional complex resistance patterns. Of particular concern is the high percentage of MDR-TB strains resistant to OFX, KM or both.

Molecular Characterization of Multidrug-Resistant Mycobacterium tuberculosis Isolates from China

To investigate the molecular characterization of multidrug-resistant tuberculosis (MDR-TB) isolates from China and the association of specific mutations conferring drug resistance with strains of different genotypes, we performed spoligotyping and sequenced nine loci (katG, inhA, the oxyR-ahpC intergenic region, rpoB, tlyA, eis, rrs, gyrA, and gyrB) for 128 MDR-TB isolates. Our results showed that 108 isolates (84.4%) were Beijing family strains, 64 (59.3%) of which were identified as modern Beijing strains. Compared with the phenotypic data, the sensitivity and specificity of DNA sequencing were 89.1% and 100.0%, respectively, for isoniazid (INH) resistance, 93.8% and 100.0% for rifampin (RIF) resistance, 60.0% and 99.4% for capreomycin (CAP) resistance, 84.6% and 99.4% for kanamycin (KAN) resistance, and 90.0% and 100.0% for ofloxacin (OFX) resistance. The most prevalent mutations among the MDR-TB isolates were katG315, inhA15, rpoB531, -526, and -516, rrs1401, eis-10, and gyrA94, -90, and -91. Furthermore, there was no association between specific resistance-conferring mutations and the strain genotype. These findings will be helpful for the establishment of rapid molecular diagnostic methods to be implemented in China.

Molecular Detection of Fluoroquinolone Resistance in Multidrug-ResistantMycobacterium tuberculosisIsolates

Background: Fluoroquinolones (FQs) are important drugs for treating multidrug-resistant tuberculosis (MDR-TB). However, due to widespread use of FQs, the resistance rates to FQs have been increasing among Mycobacterium tuberculosis. Rapid and reliable FQ drug susceptibility testing (DST) is crucial for successful treatment of MDR-TB. In this study, the feasibility of molecular detection of FQ resistance was evaluated. Methods: A total of 95 MDR-TB isolates were collected from Jan through Oct 2009 at the Korean Institute of Tuberculosis. DST for ofloxacin (OFL), levofloxacin, and moxifloxacin was performed using the Lowenstein-Jensen media absolute concentration method. Minimum inhibitory concentrations (MIC) of these were determined using the broth microdilution method. DNA was extracted from cultured isolates using bead beating method. The quinolone resistance-determining region (QRDR) of gyrA and gyrB were amplified and those sequences were analyzed. Results: Of 95 isolates, 79 were resistant to at least one of FQs. Of these, 71 (89.9%) harbored mutation in the QRDR of gyrA or gyrB. None of FQ susceptible strains possessed any mutation in gyrA or gyrB. Mutations in codon 94 of gyrA were most common; only two isolates had mutation in only the gyrB gene. OFL MICs for isolates with gyrA mutation ranged from 1 to 32 μg/mL, but FQ susceptible isolates showed MICs ranging from ≤0.06 to 0.5 μg/mL. Conclusion: Mutation analysis of QRDR of gyrA and gyrB showed 89.9% sensitivity and 100% specificity for detecting FQ resistance in MDR-TB. Therefore, molecular DST can be useful for rapid detection of FQ resistance in MDR-TB. (Ann Clin Microbiol 2014; 17:80-85)

High clustering rates of multidrug-resistant Mycobacterium tuberculosisgenotypes in Panama

BackgroundTuberculosis continues to be one of the leading causes of death worldwide and in the American region. Although multidrug-resistant tuberculosis (MDR-TB) remains a threat to TB control in Panama, few studies have focused in typing MDR-TB strains. The aim of our study was to characterize MDR Mycobacterium tuberculosis clinical isolates using PCR-based genetic markers.MethodsFrom 2002 to 2004, a total of 231 Mycobacterium tuberculosis isolates from TB cases country-wide were screened for antibiotic resistance, and MDR-TB isolates were further genotyped by double repetitive element PCR (DRE-PCR), (GTG)5-PCR and spoligotyping.ResultsA total of 37 isolates (0.85%) were resistant to both isoniazid (INH) and rifampicin (RIF). Among these 37 isolates, only two (5.4%) were resistant to all five drugs tested. Dual genotyping using DRE-PCR and (GTG)5-PCR of MDR Mycobacterium tuberculosis isolates revealed eight clusters comprising 82.9% of the MDR-TB strain collection, and six isolates (17.1%) showed unique fingerprints. The spoligotyping of MDR-TB clinical isolates identified 68% as members of the 42 (LAM9) family genotype.ConclusionOur findings suggest that MDR Mycobacterium tuberculosis is highly clustered in Panama’s metropolitan area corresponding to Panama City and Colon City, and our study reveals the genotype distribution across the country.

KatG Gene as a Surrogate Molecular Marker Leading to Cause Drug Resistance in Mycobacterium Tuberculosis Isolates

Multi Drug Resistant Tuberculosis (MDR-TB) is an emerging problem of great importance to public health worldwide. Resistance to Rifampicin (RIF) and Isoniazid (INH) are considered as surrogate markers for MDR-TB. Resistance in <i>Mycobacterium tuberculosis</i> to Rifampicin is reported to be conferred by mutation in an<i> </i>81bp rifampicin resistance determining region (RRDR) of the β subunit of RNA polymerase encoded by <i>rpoB</i> gene corresponding to codons 507-533. Resistance to Isoniazid is conferred by mutation in Catalase-peroxidase gene (<i>katG)</i>, alkyl hydroperoxidase gene (<i>aphC</i>) and enoyl acyl reductase gene (<i>inhA).</i><i> </i>The aim of this study was to identify the mutation conferring resistance to INH and by automated DNA sequencing. Seventy-four isolates of <i>M. </i><i>tuberculosis</i> were tested phenotypically with four anti tuberculosis drugs namely Rifmapicin (RIF), Isoniazid (INH), Ethambutol (EMB) and Streptomycin(SM) by absolute concentration method. Three isolates from pulmonary tuberculosis (PTB) patients were resistant to RIF with minimum inhibitory concentration (MIC) greater than or equal to 128µg/ml and to INH with MIC greater than are equal to 5 µg/ml. The genome of these three MDR-TB isolates were amplified by Polymerase Chain Reaction (PCR) and DNA sequencing was performed. All three isolates showed a point mutation at codon 315 (Ser ® Thr) in the sequenced <i>katG</i> region for INH resistance, but did not show any mutation in the 81bp hotspot sequenced <i>rpoB </i>region for resistance and promoter region of <i>inhA</i> and<i> aphC</i> for INH resistance. Two of three isolates were from patients who had not taken treatment previously which is of great concern with respect to public health.