Declining trend of resistance to first-line anti-tubercular drugs in clinical isolates of Mycobacterium tuberculosis in a tertiary care north Indian hospital after implementation of revised national Tuberculosis control programme

Trends showing drug-resistance pattern are needed to understand direction of tuberculosis (TB) control programme. The drug-resistance pattern in state of Uttar Pradesh, India, is not documented. Here we are reporting the prevalence of multi-drug-resistant (MDR) and drug-resistant TB in previously treated cases of pulmonary tuberculosis following launch of revised national TB control programme (RNTCP) in whole of Uttar Pradesh. Isolates of Mycobacterium tuberculosis, from patients of pulmonary tuberculosis, who were treated with antitubercular drugs for more than 4 weeks, were tested for resistance to first-line drugs; streptomycin (S), Ethambutol (E), Rifampicin (R) and isoniazid (H) over a period of 4 years, 2009-2012. Total 2496 isolates of M. tuberculosis were tested, of which 1139 isolates (45.6%) were pan-sensitive and 370 (14.8%) were pan-resistant. Total 695 isolates (27.8%) were MDR. Maximum resistance was with Isoniazid (n = 1069, 42.8%) followed by streptomycin (n = 840, 33.7%), rifampicin (n = 742, 29.7%), and ethambutol (n = 613, 24.6%). A decline in number of MDR strains and individual drug resistance was seen. Total MDR strains in the year 2009, 2010, 2011 and 2012 were 35.6%, 30.8%, 26.7% and 22.8% respectively. The drug resistance pattern reported from time to time may vary substantially. The decline in drug resistance visible over last four years, after implementation of DOTS, appears promising.     

Evaluation of the AID TB Resistance Line Probe Assay for Rapid Detection of Genetic Alterations Associated with Drug Resistance in Mycobacterium tuberculosis Strains

The rapid accurate detection of drug resistance mutations in Mycobacterium tuberculosis is essential for optimizing the treatment of tuberculosis and limiting the emergence and spread of drug-resistant strains. The TB Resistance line probe assay from Autoimmun Diagnostika GmbH (AID) (Strassburg, Germany) was designed to detect the most prevalent mutations that confer resistance to isoniazid, rifampin, streptomycin, amikacin, capreomycin, fluoroquinolones, and ethambutol. This assay detected resistance mutations in clinical M. tuberculosis isolates from areas with low and high levels of endemicity (Switzerland, n = 104; South Africa, n = 52) and in selected Mycobacterium bovis BCG 1721 mutant strains (n = 5) with 100% accuracy. Subsequently, the line probe assay was shown to be capable of rapid genetic assessment of drug resistance in MGIT broth cultures, the results of which were in 100% agreement with those of DNA sequencing and phenotypic drug susceptibility testing. Finally, the line probe assay was assessed for direct screening of smear-positive clinical specimens. Screening of 98 clinical specimens demonstrated that the test gave interpretable results for >95% of them. Antibiotic resistance mutations detected in the clinical samples were confirmed by DNA sequencing. We conclude that the AID TB Resistance line probe assay is an accurate tool for the rapid detection of resistance mutations in cultured isolates and in smear-positive clinical specimens.     

Detecting mutation pattern of drug-resistant Mycobacterium tuberculosis isolates in Himachal Pradesh using GenoType® MTBDRplus assay

Context: Tuberculosis (TB) is a major public health problem in India and a principal cause of death in adults, especially among the economically productive age group. India accounts for one-fifth of the global burden of TB. It is estimated that about 40% of Indian population is infected with TB bacillus. The GenoType® MTBDRplus molecular method allows rapid diagnosis of the clinical samples and detection of the most common mutations in the genes associated with rifampicin (R) and isoniazid (H) resistance. Aims: To study the drug resistance and mutational patterns in multidrug-resistant (MDR) suspects clinical strains using GenoType® MTBDRplus assay. Subjects and Methods: A total of 770 sputum samples of the MDR-TB suspects were included in this study, which were received at Intermediate Reference Laboratory, Government TB Sanatorium, Dharampur, Solan, Himachal Pradesh from the Designated Microscopy Centres of Himachal Pradesh for the culture and susceptibility testing. All the 521 Mycobacterium tuberculosis complex (MTBC) strains were subjected to GenoType® MTBDRplus (HAIN Lifescience) assay to detect molecular resistance pattern to first line anti-tubercular drugs (isoniazid and rifampicin). Results: Of 770 samples, 556 (72.20%) were from male and 214 (27.80%) were from female. Among the 521 MTBC strains, 19.76% were found to be MDR and mono-resistance to isoniazid and rifampicin was detected in 8.63% and 6.14% strains respectively. About 74.81%, 76.35% and 5.40% strains harboured known mutation in rpoB, katG and inhA genes respectively. Conclusions: In rpoB gene, the most common mutation is associated with S531 L region. The GenoType® MTBDRplus assay is a rapid test for the detection of the most common mutations in MDR-TB strains. In our study, unknown rpoB gene mutations were found in 25.18% strains that may further be detected by gene sequencing.     

Extremely high prevalence of multidrug resistant tuberculosis in Murmansk, Russia: a population-based study

Drug resistance and molecular epidemiology of tuberculosis (TB) in the Murmansk region was investigated in a 2-year, population-based surveillance of the civilian population. During 2003 and 2004, isolates from all culture-positive cases were collected (n?=?1,226). Prevalence of multi-drug resistance (MDR) was extremely high, as 114 out of 439 new cases (26.0%), and 574 out of 787 previously treated cases (72.9%) were resistant to at least isoniazid (INH) and rifampin (RIF). Spoligotyping of the primary MDR-TB isolates revealed that most isolates grouped to the Beijing SIT1 genotype (n?=?91, 79.8%). Isolates of this genotype were further analyzed by IS6110 RFLP. Sequencing of gene targets associated with INH and RIF resistance further showed that the MDR-TB strains are highly homogeneous as 78% of the MDR, SIT1 strains had the same resistance-conferring mutations. The genetic homogeneity of the MDR-TB strains indicates that they are actively transmitted in Murmansk.     

Multicenter Evaluation of Anyplex Plus MTB/NTM MDR-TB Assay for Rapid Detection of Mycobacterium tuberculosis Complex and Multidrug-Resistant Isolates in Pulmonary and Extrapulmonary Specimens

The rapid diagnosis of tuberculosis (TB) and the detection of drug-resistant Mycobacterium tuberculosis strains are critical for successful public health interventions. Therefore, TB diagnosis requires the availability of diagnostic tools that allow the rapid detection of M. tuberculosis and drug resistance in clinical samples. Here, we performed a multicenter study to evaluate the performance of the Seegene Anyplex MTB/NTM MDR-TB assay, a new molecular method based on a multiplex real-time PCR system, for detection of Mycobacterium tuberculosis complex (MTBC), nontuberculous mycobacteria (NTM), and genetic determinants of drug resistance. In total, the results for 755 samples (534 pulmonary and 221 extrapulmonary samples) were compared with the results of smears and cultures. For pulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 86.4% and 75.0%, respectively, and the specificities were 99% and 99.4%. For extrapulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 83.3% and 50.0%, respectively, and the specificities of both were 100%. The negative and positive predictive values of the Anyplex assay for pulmonary specimens were 97% and 100%, respectively, and those for extrapulmonary specimens were 84.6% and 100%. The sensitivities of the Anyplex assay for detecting isoniazid resistance in MTBC strains from pulmonary and extrapulmonary specimens were 83.3% and 50%, respectively, while the specificities were 100% for both specimen types. These results demonstrate that the Anyplex MTB/NTM MDR-TB assay is an efficient and rapid method for the diagnosis of pulmonary and extrapulmonary TB and the detection of isoniazid resistance.     

Molecular epidemiology and prevalence of mutations conferring rifampicin and isoniazid resistance in Mycobacterium tuberculosis strains from the southern Ukraine

Understanding the molecular epidemiology of tuberculosis (TB) and mutations in genes associated with drug resistance may contribute to the development of appropriate interventions to improve tuberculosis control. A structured questionnaire was used to collect basic epidemiological data from 589 patients with radiologically confirmed TB in the Odessa and Nikolaev regions of the Ukraine in 2003-2004. A non-commercial reverse hybridisation assay and DNA sequencing were used to detect mutations associated with rifampicin and isoniazid resistance. Genotyping was performed using multilocus variable number tandem repeat (VNTR) typing and spoligotyping. Mutations conferring rifampicin and isoniazid resistance were detected in 32.9% and 44.0%, respectively, of 225 Mycobacterium tuberculosis isolates from individual consecutive patients. Mutations in codon 531 and codon 315 of the rpoB and katG genes, respectively, were predominant among drug-resistant isolates. Multidrug (MDR) resistance rates were significantly higher among former prison inmates compared with non-prisoners (54.8% vs. 27.3%; RR 2.01; 95% CI 1.35-2.97) and the prevalence of mutations was higher in Beijing strains sharing the VNTR signature 223325173533424 than in other Beijing strains (71.4% vs. 45.7%; RR 1.74; 95% CI 1.17-2.57), suggesting that this group may be responsible for rapid transmission of MDR TB in the southern Ukraine.     

Development of a Luminex-Based Multiplex Assay for Detection of Mutations Conferring Resistance to Echinocandins in Candida glabrata

Echinocandins are the recommended treatment for invasive candidiasis due to Candida glabrata. Resistance to echinocandins is known to be caused by nonsynonymous mutations in the hot spot-1 (HS1) regions of the FKS1 and FKS2 genes, which encode a subunit of the β-1,3-glucan synthase, the target of echinocandins. Here, we describe the development of a microsphere-based assay using Luminex MagPix technology to identify mutations in the FKS1 HS1 and FKS2 HS1 domains, which confer in vitro echinocandin resistance in C. glabrata isolates. The assay is rapid and can be performed with high throughput. The assay was validated using 102 isolates that had FKS1 HS1 and FKS2 HS1 domains previously characterized by DNA sequencing. The assay was 100% concordant with DNA sequencing results. The assay was then used for high-throughput screening of 1,032 C. glabrata surveillance isolates. Sixteen new isolates with mutations, including a mutation that was new to our collection (del659F), were identified. This assay provides a rapid and cost-effective way to screen C. glabrata isolates for echinocandin resistance.     

Comparison of two commercially available DNA line probe assays for detection of multidrug-resistant Mycobacterium tuberculosis

Two commercially available DNA line probe assays, Genotype MTBDR and INNO-LiPA Rif. TB, were evaluated for their abilities to detect resistance to isoniazid (INH) and rifampin (RIF) in 52 Mycobacterium tuberculosis isolates. The test results were compared to those obtained by phenotypic drug susceptibility testing and sequencing. Compared to the results of phenotypic drug susceptibility testing, the Genotype MTBDR test results were concordant for INH for 47 of the 52 (90.4%) isolates, and both the Genotype MTBDR and the INNO-LiPA Rif. TB test results were concordant for RIF for 51 of the 52 (98.1%) isolates. The Genotype MTBDR test results correlated with the sequencing results for 48 of the 52 (92.3%) isolates and the INNO-LiPA Rif. TB results for 50 of the 52 (96.2%) isolates. Both assays are useful for the rapid screening of M. tuberculosis isolates obtained from patients suspected of having multidrug-resistant tuberculosis, but the GenoType MTBDR assay has the advantage of being able to detect resistance to both INH and RIF simultaneously.     

Prevalence,associated factors,outcomes and transmission of extensively drug-resistant tuberculosis among multidrug-resistant tuberculosis patients in São Paulo,Brazil: a cross-sectional study

ObjectivesTo describe the prevalence, associated factors, treatment outcomes and transmission of extensively drug-resistant (XDR) tuberculosis (TB) in the state of São Paulo, Brazil, for 2011 to 2013.MethodsDrug susceptibility testing to first- and second-line drugs was performed by BACTEC MGIT 960 and molecular typing, by IS6110 restriction fragment length polymorphism. Clinical, epidemiologic and demographic data were obtained from surveillance information systems for TB. Patients were divided into three groups: multidrug resistant (MDR) TB (resistance to at least isoniazid and rifampicin), pre–XDR-TB (MDR-TB resistant to a fluoroquinolone or to at least one of the second-line injectable drugs) and XDR-TB (MDR-TB resistant to a fluoroquinolone and to at least one of the second-line injectables).ResultsAmong the 313 MDR-TB patients identified, the prevalence of XDR-TB and pre–XDR-TB was 10.2% (n = 32) and 19.2% (n = 60), respectively. Compared to MDR-TB patients, XDR-TB patients were more likely to be female (odds ratio (OR) = 2.74, 95% confidence interval (CI), 1.29–5.83), have a history of TB (OR = 5.16; 95% CI, 1.52–17.51) and present higher death rates (OR= 3.74; 95% CI 1.70–8.25). XDR-TB transmission was observed in households, between neighbours and between a patient and a healthcare worker in a hospital.ConclusionsThe prevalence of XDR-TB in the state of São Paulo is close to that estimated globally. Most of the XDR-TB patients were treated previously for TB and presented the lowest successful outcome rates. Because transmission of XDR-TB occurred, it is important that timely diagnosis of drug resistance is performed.     

Use of the genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis complex isolates

A commercially available DNA strip assay (Genotype MTBDR; Hain Lifescience, Nehren, Germany) was evaluated for its ability to detect mutations conferring resistance to rifampin (RMP) and isoniazid (INH) in clinical Mycobacterium tuberculosis complex isolates. A total of 103 multidrug-resistant (MDR; i.e., at least resistant to RMP and INH) and 40 fully susceptible strains isolated in Germany in 2001 in which resistance mutations have been previously defined by DNA sequencing and real-time PCR analysis were investigated. The Genotype MTBDR assay identified 102 of the 103 MDR strains with mutations in the rpoB gene (99%) and 91 strains (88.4%) with mutations in codon 315 of katG. All 40 susceptible strains showed a wild-type MTBDR hybridization pattern. The concordance between the MTBDR assay and the DNA sequencing results was 100%. Compared to conventional drug susceptibility testing, the sensitivity and specificity were 99 and 100% for RMP resistance and 88.4 and 100% for INH resistance, respectively. In conclusion, the MTBDR assay is a rapid and easy-to-perform test for the detection of the most common mutations found in MDR M. tuberculosis strains that can readily be included in a routine laboratory work flow.     

Evaluation of two tuberculosis PCR assays for routine use in a clinical setting of low population and low tuberculosis prevalence

Today, there are numerous different molecular diagnostic assays for the detection of tuberculosis (TB), allowing the optimization of rapid detection of TB according to the clinical need. In this study, two high‐throughput TB PCR assays with combined antimicrobial resistance detection, Anyplex? II MTB/MDR (Seegene) and RealTime MTB + RealTime MTB RIF/INH Resistance (Abbott Molecular), were evaluated for routine use in a clinical setting of low population and low TB prevalence in Finland. The RealTime MTB assay was 100% concordant (22/22 positive, n = 169) with the reference methods (culture and Xpert MTB/RIF PCR assay, Cepheid). However, with a limitation of four separate PCR cycles per kit, the routine use in a low TB‐prevalence setting would easily lead to wasting most of the RIF/INH Resistance reagents. The Anyplex? II MTB/MDR assay usability was more adaptive to suit the clinical setting but the assay sensitivity was considerably lower (86%, 19/22 positive, n = 76) being closer to the sensitivity of smear microscopy. The findings of this study suggest that the evaluated high‐throughput MTB/MDR assays are evidently suboptimal for routine use in a low population, low TB‐prevalence setting. In addition, neither of the two assays covers non‐tuberculous mycobacteria and could therefore not fully replace acid‐fast staining as the initial screening method.     

Performance of the Genotype® MTBDRPlus assay in the diagnosis of tuberculosis and drug resistance in Samara, Russian Federation

Background

Russia is a high tuberculosis (TB) burden country with a high prevalence of multidrug resistant tuberculosis (MDRTB). Molecular assays for detection of MDRTB on clinical specimens are not widely available in Russia.

Results

We performed an evaluation of the GenoType^® MTBDRplus assay (HAIN Lifescience GmbH, Germany) on a total of 168 sputum specimens from individual patients at a public health laboratory in Central Russia, as a model of a middle income site in a region with high levels of drug resistance. Phenotypic drug resistance tests (DST) were performed on cultures derived from the same sputum specimens using the BACTEC 960 liquid media system. Interpretable GenoType^® MTBDRplus results were obtained for 154(91.7%) specimens with readability rates significantly higher in sputum specimens graded 2+ and 3+ compared to 1+ (RR = 1.17 95%CI 1.04–1.32). The sensitivity and specificity of the assay for the detection of rifampicin (RIF) and isoniazid (INH) resistance and MDR was 96.2%, 97.4%, 97.1% and 90.7%, 83.3%, 88.9% respectively. Mutations in codon 531 of the rpoB gene and codon 315 of the katG gene dominated in RIF and INH resistant strains respectively. Disagreements between phenotypical and molecular tests results (12 samples) could be explained by the presence of rare mutations in strains circulating in Russia and simultaneous presence of resistant and sensitive bacilli in sputum specimens (heteroresistance).

Conclusion

High sensitivity, short turnaround times and the potential for screening large numbers of specimens rapidly, make the GenoType^® MTBDRplus assay suitable as a first-line screening assay for drug resistant TB.     

Concordance between Molecular and Phenotypic Testing of Mycobacterium tuberculosis Complex Isolates for Resistance to Rifampin and Isoniazid in the United States

Multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis complex (MTBC) are defined by resistance to at least rifampin (RMP) and isoniazid (INH). Rapid and accurate detection of multidrug resistance is essential for effective treatment and interruption of disease transmission of tuberculosis (TB). Overdiagnosis of MDR TB may result in treatment with second-line drugs that are more costly, less effective, and more poorly tolerated than first-line drugs. CDC offers rapid confirmation of MDR TB by the molecular detection of drug resistance (MDDR) for mutations associated with resistance to RMP and INH along with analysis for resistance to other first-line and second-line drugs. Simultaneously, CDC does growth-based phenotypic drug susceptibility testing (DST) by the indirect agar proportion method for a panel of first-line and second-line antituberculosis drugs. We reviewed discordance between molecular and phenotypic DST for INH and RMP for 285 isolates submitted as MTBC to CDC from September 2009 to February 2011. We compared CDC''s results with those from the submitting public health laboratories (PHL). Concordances between molecular and phenotypic testing at CDC were 97.4% for RMP and 92.5% for INH resistance. Concordances between CDC''s molecular testing and PHL DST results were 93.9% for RMP and 90.0% for INH. Overall concordance between CDC molecular and PHL DST results was 91.7% for RMP and INH collectively. Discordance was primarily attributable to the absence of known INH resistance mutations in isolates found to be INH resistant by DST and detection of mutations associated with low-level RMP resistance in isolates that were RMP susceptible by phenotypic DST. Both molecular and phenotypic test results should be considered for the diagnosis of MDR TB.     

Sputum microbiota as a potential diagnostic marker for multidrug-resistant tuberculosis

The prevalence of drug-resistant Mycobacterium tuberculosis (Mtb) strains makes disease control more complicated, which is the main cause of death in tuberculosis (TB) patients. Early detection and timely standard treatment are the key to current prevention and control of drug-resistant TB. In recent years, despite the continuous advancement in drug-resistant TB diagnostic technology, the needs for clinical rapid and accurate diagnosis are still not fully met. With the development of sequencing technology, the research of human microecology has been intensified. This study aims to use 16 rRNA sequencing technology to detect and analyze upper respiratory flora of TB patients with anti-TB drug sensitivity (DS, n = 55), monoresistance isoniazide (MR-INH, n = 33), monoresistance rifampin (MR-RFP, n = 12), multidrug resistance (MDR, n = 26) and polyresistance (PR, n = 39) in southern China. Potential microbial diagnostic markers for different types of TB drug resistance are searched by screening differential flora, which provides certain guiding significance for drug resistance diagnosis and clinical drug use of TB. The results showed that the pulmonary microenvironment of TB patients was more susceptible to infection by external pathogens, and the infection of different drug-resistant Mtb leads to changes in different flora. Importantly, seven novel microorganisms (Leptotrichia, Granulicatella, Campylobacter, Delfitia, Kingella, Chlamydophila, Bordetella) were identified by 16S rRNA sequencing as diagnostic markers for different drug resistance types of TB. Leptotrichia, Granulicatella, Campylobacter were potential diagnostic marker for TB patients with INH single-resistance. Delftia was a potential diagnostic marker for TB patients with RFP single drug-resistance. Kingella and Chlamydophila can be used as diagnostic markers for TB patients with PR. Bordetella can be used as a potential diagnostic marker for identification of TB patients with MDR.     

Primary drug resistance to anti-tuberculosis drugs in major towns of Amhara region, Ethiopia

Drug resistance is a major obstacle to effective TB control program performance. In this study, we assessed the prevalence of primary drug resistance in Mycobacterium tuberculosis (Mtb) isolates in Amhara Region, Ethiopia. A total of 112 Mtb isolates from cases with newly diagnosed pulmonary TB were subjected to drug susceptibility testing (DST) in a cross-sectional study. Isolates were tested for sensitivity to isoniazid, rifampicin, ethambutol, and streptomycin using the MGIT 960 protocol. A total of 93 Mtb isolates yielded valid DST results and 28 (30.1%) were resistant to one or more of first line anti-TB drugs. One isolate (1.0%) was multi-drug resistant (MDR), five (5.4%) were classified as poly-resistant and 22 showed single drug resistance to either streptomycin (n = 19) or isoniazid (n = 3). Isolates from HIV-positive patients were more likely to be resistant to at least one of the four anti-TB drugs compared with HIV-negative individuals (odds ratio 2.76, 95% confidence interval 1.06-7.22; p = 0.03). The study showed a high prevalence of primary drug resistance. Even though the prevalence of MDR was low, conditions that can contribute to the development of MDR are increasing. Therefore, regular monitoring of drug resistance and enhanced implementation of TB/HIV collaborative activities in the study region are imperative.     

The management of tuberculosis: epidemiology, resistance and monitoring

This PhD thesis is based on 5 studies conducted in the period 2006-2010 during my employment at the International Reference Laboratory of Mycobacteriology, Statens Serum Institut. The overall aim was to assess tuberculosis (TB) treatment in Denmark with specific focus on the risk of relapse of TB disease, and to analyse treatment outcome of patients with multidrug-resistant (MDR) or isoniazid-resistant TB. The project established the need for rapid methods to detect resistance and follow-up of treatment. A rapid method to detect drug resistance was optimised and evaluated for use directly in clinical specimens. The studies were based on data from the Mycobacterial registry in the period 1992-2007, which included the results from microscopy, culture, drug-susceptibility and restriction fragment length polymorphism (RFLP). Information on dates of death/emigration were taken from the CPR-registry and treatment from surveillance data and patient records. The rate of recurrent TB was found to be low in Denmark, during 13.5 years of follow-up. Relapse accounted for 1.3% of the recurrent cases and reinfection was rare, only in 0.5% cases. The relapse hazard increased up to four years after diagnosis. Cavitary disease was associated with relapse as opposed to reinfection and may need prolonged treatment and closer monitoring. The incidence of MDR-TB and isoniazid resistance was confirmed to be low. Successful short- and long-term treatment outcome of MDR-TB and isoniazid-resistant TB was high. High- and low-level isoniazid resistance did not affect treatment outcome. A multiplex PCR hybridization mutation analysis, that simultaneously detects the most frequent rpoB and katG gene mutations conferring rifampin and high-level isoniazid resistance, was optimized for direct use and evaluated in smear-positive specimens as opposed to slow conventional drug-susceptibility testing (DST). The second-generation rifampin and isoniazid resistance mutation assay additionally included detection of mutations within the inhA gene conferring low-level isoniazid resistance. This assay was found to be rapid (< 48 h) and easy to perform in isolates and clinical specimens. A high concordance between mutation and conventional DST results was found for rifampin, while results varied for isoniazid . The mutation analysis identified all MDR-TB cases and the majority of isoniazid-resistant cases in Denmark. Standard 6-month multiple anti-TB drug therapy is necessary to treat drug-susceptible TB. Drug-resistant TB often requires therapy adjustments and extended treatment. MDR-TB particularly poses therapeutic challenges. Rapid detection of resistance mutations directly in smear-positive patient specimens may improve MDR-TB patient treatment, although the impact on isoniazid-resistant TB treatment outcome remains to be determined. The mutation assay is a rapid supplement to the gold standard conventional DST in high-income countries such as Denmark, while in low-income countries it can be used for preliminary DST. The assay may be applied to smear-positive samples from patients suspected of treatment failure, recurrent TB, drug-resistant TB exposure or originating from countries with high levels of DR. The new extended mutation assay has proved to be a useful tool, which has now been included in the World Health Organization's policy to combat and prevent new cases of MDR and extensively drug-resistant TB.     

Evaluation of the GenoType MTBDR assay for detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis complex isolates

Detection of drug resistance plays a critical role in tuberculosis treatment. The aim of this study was to evaluate the performance of GenoType Mycobacteria Drug Resistance (MTBDR) assay (Hain Lifescience, Germany) and to compare it with radiometric BACTEC 460 TB system (Becton Dickinson, USA) for the detection of rifampicin (RIF) and isoniazid (INH) resistance in 84 Mycobacterium tuberculosis complex (MTBC) isolates. RIF resistance was identified in 6 of 7 (85.7%) isolates and INH resistance was identified in 8 of 14 (57.1%) isolates by the GenoType MTBDR assay. Compared with BACTEC system, the sensitivity, specificity, positive predictive value and negative predictive values were 85.7%, 98.7%, 85.7% and 98.7% for RIF resistance; and 57.1%, 100%, 100% and 92.1% for INH resistance, respectively. GenoType MTBDR assay is reliable when tested specimen is resistant to the tested drugs. Although test was more successful in the detection of RIF resistance, it exhibited low sensitivity for the detection of INH resistance.     

Magnitude of Gene Mutations Conferring Drug Resistance in Mycobacterium Tuberculosis Isolates from Lymph Node Aspirates in Ethiopia

Objective: Resistance to drugs is due to particular genomic mutations in the specific genes of Mycobacterium tuberculosis. Timely genetic characterization will allow identification of resistance mutations that will optimize an effective antibiotic treatment regimen. We determine the magnitude of gene mutations conferring resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among tuberculosis (TB) lymphadenitis patients.Methods: A cross sectional prospective study was conducted among 226 M.tuberculosis isolates from culture positive lymph node aspirates collected from TB lymphadenitis patients between April 2012 and May 2012. Detection of mutations conferring resistance to drugs was carried out using GenoType^® MTBDRplus and GenoType® MTBDRsl assay.Results: Out of the 226 strains, mutations conferring resistance to INH, RMP, multidrug resistance tuberculosis (MDR-TB) and EMB were 8, 3, 2 and 2 isolates, respectively. There was no isolated strain that showed mutation in the inhA promoter region gene. All INH resistant strains had mutations in the katG gene at codon 315 with amino acid change of S315T1. Among rifampicin resistant strains, two isolates displayed mutations at codon 531 in the rpoB gene with amino acid change of S531L and one isolate was by omission of wild type probes at Q513L. According to mutations associated with ethambutol resistance, all of the isolates had mutations in the embB gene with aminoacid change of M306I. All isolates resistant to INH, RMP and MDR using BacT/AlerT 3D system were correctly identified by GenoType® MTBDRplus assay.Conclusion: We observed mutations conferring resistance to INH at S315T1 of the katG gene, RMP at S531L and Q513L in the rpoB genes and EMB at M306I of the embB gene. In the absence of conventional drug susceptibility testing, the effort to develop easy, rapid and cost effective molecular assays for drug resistance TB monitoring is definitely desirable and the GenoType® MTBDRplus assay was found to be a useful method for diagnosis of resistance to INH, RMP and MDR from lymph node aspirates. Further molecular cluster analysis to determine transmission dynamics of mutated strain is required.     

The use of E-test for the drug susceptibility testing of Mycobacterium tuberculosis – A solution or an illusion?

Aim: To evaluate E-test as a tool for rapid determination of drug susceptibility against the conventional LJ method focusing on reliability, expense, ease of standardization and performance of the technique in low resource settings. Materials and Methods: A total of 74 clinical isolates (2004-2005) of Mycobacterium tuberculosis were tested using E-test for susceptibility to streptomycin (STM), isoniazid (INH), rifampicin (RIF) and ethambutol (EMB) by E-strip and LJ (LJPM) proportion methods. Results: The LJPM method, the gold standard, detected resistance against STM in 16.2%, INH in 40.5%, RIF in 18.9% and EMB in 27% cases. In comparison, the resistance values showed by E-test was 66.67% for STM, 57.14% for INH 71.43% for RIF and 80% for EMB. The susceptible correlation was 90.32% for STM, 73.91% for INH, 93.33% for RIF and 59.26% for EMB. E-test correctly identified only eight of the 12 (66.6%) MDR isolates and wrongly identified four isolates which were not MDR. The overall agreement between the two methods was only 48.6%. Resistant isolates showed false positive resistance observed while using E-strip towards all the drugs. Conclusion: E-strips are not quite feasible as a replacement for LJ-proportion method on a large scale due to high risk of cross contamination, laboratory infection, expense associated with it and high false positive resistance observed to all first line drugs. However, the good correlation observed for RIF between the two methods indicates that E-test could contribute to the role in rapid screening of MDR TB isolates as rifampicin mutations are invariably observed in MDR TB isolates.     

Phenotypic and genotypic characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis clinical isolates in Hangzhou,China

ObjectivesPyrazinamide (PZA) is a crucial first-line tuberculosis (TB) drug recommended for both drug-susceptible and multidrug-resistant Mycobacterium tuberculosis. This study aimed to evaluate the performance of the sequencing method of pncA, rpsA and panD mutations in detecting PZA resistance in multidrug-resistant (MDR) TB isolates.MethodsWe sequenced the pncA, rpsA and panD genes and performed PZA susceptibility tests across 291 MDR-TB isolates to evaluate the performance of the sequencing method of these genes in detecting PZA resistance.ResultsResults showed that 145 (90.0%) of 161 PZA phenotypic resistant isolates had mutations in pncA. Among the 16 isolates (10.0%) which did not have mutations in pncA, ten and three isolates had mutations in rpsA and panD, respectively. The sequencing method for detecting mutations in pncA alone had 90.1% (95% confidence interval (CI), 84.4–94.2) sensitivity and 92.3% (95% CI, 86.3–96.3) specificity. The combination of all three genes increased the sensitivity from 90.1% (95% CI, 84.4–94.2) to 98.1% (95% CI, 94.7–99.6) (p < 0.001) while the specificity remained unchanged. In 120 PZA-susceptible and 16 PZA-resistant isolates without pncA mutations, rpsA/panD mutations were correlated with PZA resistance.ConclusionsPZA resistance was largely associated with mutations in pncA. Mutations in rpsA and panD were also associated with PZA resistance in MDR isolates expressing wild-type pncA. The detection of mutations in pncA, rpsA and panD can be useful for the determination of PZA resistance.