GenoType MTBDRplus Assay Research Articles

Rapid Screening of MDR-TB in Cases of Extra Pulmonary Tuberculosis Using Geno Type MTBDRplus.

BackgroundDrug resistance in tuberculosis is a major public health challenge in developing countries. The limited data available on drug resistance in extra pulmonary tuberculosis stimulated us to design our study on anti-tuberculosis drug resistance pattern in cases of extra pulmonary tuberculosis in a tertiary referral hospital of North India. We performed Geno Type MTBDRplus assay in comparison with conventional drug susceptibility testing by proportion method to study the mutation patterns in rpoB, katG and inhA genes.MethodsA total of 510 extra pulmonary samples were included in this study. After the smear microscopy, all the specimens were subjected for culture on Lowenstein Jensen (LJ) media. Phenotypic drug susceptibility testing (DST) was performed on LJ media for all the MTB isolates and compared with the results of Geno Type MTBDRplus assay which was performed with the DNA isolated from the culture by conventional method.ResultsOf 510 specimens cultured, the total culture positivity obtained was 11.8% (60) encompassing 54 (10.6%) Mycobacterium tuberculosis and 6 (1.2%) non-tubercular mycobacteria (NTM). DST results by Geno Type MTBDRplus assay and solid culture methods were compared in 51 MTB isolates excluding the two Rif indeterminate and one invalid test. Geno Type MTBDRplus accurately identified 13 of 14 rifampicin-resistant strains, 14 of 15 isoniazid-resistant strains and 13 of 14 as multi drug resistant tuberculosis (MDR-TB) in comparison with conventional method. Sensitivity and specificity were 92.86% and 97.30% respectively for detection of RIF resistance, 93.33% and 94.44% respectively for detection of INH resistance, 92.86% and 97.30% respectively for detection of MDR-TB, while the overall concordance of Geno Type MTBDRplus assay with conventional DST was 94.11%. The turn-around time for performing Geno Type MTBDRplus assay test was 48 hours.ConclusionThe problem of MDR in extra pulmonary tuberculosis (EPTB) cannot be overlooked and due attention on patients should be given. Routine use of Geno Type MTBDRplus assay for the diagnosis of MDR-EPTB can substantially reduce the time between diagnosis and drug therapy. Culture along with Geno Type MTBDRplus assay could be a solution for rapid and accurate diagnosis of MDR-TB in low bacillary non sputum specimens.

Evaluation of the GenoType MTBDRplus assay for detection of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis isolates in central Ethiopia

Objective/Background: Multidrug-resistant tuberculosis (MDR-TB) is growing globally and becoming a major challenge for national TB control programs. Therefore, rapid identification of MDR strains of Mycobacterium tuberculosis and monitoring their transmission could contribute significantly to the control of TB. The GenoType MTBDRplus assay has been recommended by the World Health Organization to identify rifampicin (RIF)- and isoniazid (INH)-resistant M. tuberculosis isolates. This study was carried out to evaluate the performance of the GenoType MTBDRplus assay for the detection of RIF- and INH-resistant M. tuberculosis isolates in central Ethiopia. Methods: A total of 279 M. tuberculosis strains isolated from active TB cases in central Ethiopia were evaluated for their drug sensitivity by the conventional drug-susceptibility test (DST) and compared with data derived from the GenoType MTBDRplus assay. The DST served as the gold standard for evaluating the GenoType MTBDRplus assay. Results: The sensitivity and specificity of the GenoType MTBDRplus assay for the detection of RIF-resistant M. tuberculosis isolates were 80.0% and 99.6%, respectively. Its sensitivity and specificity for the detection of INH-resistant M. tuberculosis isolates were 82.7% and 99.6%, respectively, whereas they were 75.0% and 100%, respectively, for the detection of MDR M. tuberculosis strains. The concordances of the GenoType MTBDRplus assay and the conventional DST for the detection of RIF and INH susceptibility were 80% (8/10) and 86.2% (25/29), respectively. Furthermore, the concordance of the two tests for the detection of MDR M. tuberculosis strains was 75%. Specific mutations were detected in 55.6% (5/9) of the RIF-resistant isolates, with the highest mutation rate (33.3%) for the rpoB gene (Codon S531L). For INH-resistant isolates, the highest mutation rate (88.8%) related to a katG mutation (Codon S315T1). Conclusion: The findings of this study revealed that the GenoType MTBDRplus assay has high sensitivity and specificity for the detection of RIF and INH resistance. These preliminary data support the notion that the assay should be considered as an alternative to the DST for the characterization of MDR in M. tuberculosis isolates and the control of TB.

GenoType MTBDRplus Assay for Rapid Detection of Multidrug Resistance in Mycobacterium tuberculosis: A Meta-Analysis.

BackgroundThere is an urgent demand for rapid and accurate drug-susceptibility testing for the detection of multidrug-resistant tuberculosis. The GenoType MTBDRplus assay is a promising molecular kit designed for rapid identification of resistance to first-line anti-tuberculosis drugs, isoniazid and rifampicin. The aim of this meta-analysis was to evaluate the diagnostic accuracy of GenoType MTBDRplus in detecting drug resistance to isoniazid and rifampicin in comparison with the conventional drug susceptibility tests.MethodsWe searched PubMed, EMBASE, and Cochrane Library databases to identify studies according to predetermined criteria. A total of 40 studies were included in the meta-analysis. QUADAS-2 was used to assess the quality of included studies with RevMan 5.2. STATA 13.0 software was used to analyze the tests for sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and area under the summary receiver operating characteristic curves. Heterogeneity in accuracy measures was tested with Spearman correlation coefficient and Chi-square.ResultsPatient selection bias was observed in most studies. The pooled sensitivity (95% confidence intervals were 0.91 (0.88–0.94) for isoniazid, 0.96 (0.95–0.97) for rifampicin, and 0.91(0.86–0.94) for multidrug-resistance. The pooled specificity (95% CI) was 0.99 (0.98–0.99) for isoniazid, 0.98 (0.97–0.99) for rifampicin and 0.99 (0.99–1.00) for multidrug-resistance, respectively. The area under the summary receiver operating characteristic curves ranged from 0.99 to 1.00.ConclusionThis meta-analysis determined that GenoType MTBDRplus had good accuracy for rapid detection of drug resistance to isoniazid and/or rifampicin of M. tuberculosis. MTBDRplus method might be a good alternative to conventional drug susceptibility tests in clinical practice.

Primary Multidrug Resistant Tuberculosis and Utility of Line Probe Assay for Its Detection in Smear-Positive Sputum Samples in a Tertiary Care Hospital in South India.

In a high tuberculosis burdened country like India, rapid, cost-effective, and reliable diagnostic tools for tuberculosis are an urgent need of the hour to prevent inappropriate treatment strategies and further spread of resistance. This study aimed to estimate the proportion of new smear-positive tuberculosis cases with primary resistance to rifampicin and/or isoniazid as well as identify the common mutations associated with it. Sputum of 200 newly diagnosed smear-positive cases of 1+ score and above was directly subjected to Line Probe Assay using the GenoType MTBDRplus assay kit. All samples were inoculated onto solid media and 61 samples were inoculated in automated liquid culture also. The Line Probe Assay gave hundred percent interpretable results with 2.5% of the study population showing resistant pattern. Only 1% of the cases were primary multidrug resistant tuberculosis and 1.5% showed isoniazid monoresistance. S531L and C15T were the most common genetic mutations seen for rifampicin and isoniazid resistance, respectively. 40% had absent rpoB wild type 8 band indicating probable silent mutation after clinical correlation. The average turnaround time for Line Probe Assay was far less (3.8 days) as compared to solid and liquid cultures (35.6 days and 13.5 days, resp.).

Diagnostic usefulness of the GenoType MTBDRplus assay for detecting drug-resistant tuberculosis using AFB smear-negative specimens with positive TB-PCR result

Background The aim of this study was to evaluate the diagnostic accuracy of the GenoType MTBDRplus assay in detecting drug-resistant tuberculosis (DR-TB) by using acid-fast bacilli (AFB) smear-negative specimens with positive TB-PCR results. Methods The MTBDRplus assay was performed with 2 different categories of 117 samples, including AFB smear-positive specimens (n = 53) and AFB smear-negative specimens (n = 64), which exhibited positive TB-PCR results, at a single institution. The results were retrospectively compared with the results of the phenotypic drug susceptibility test (DST), for reference. Results A total of 105 tests were finally analyzed. Of these, 54 tests were conducted using AFB smear-negative specimens with positive TB-PCR results. The MTBDRplus assay for these 54 samples demonstrated a sensitivity of 100%, specificity of 98%, positive predictive value (PPV) of 75%, and negative predictive value (NPV) of 100% in detecting rifampicin resistance. With these same species, the sensitivity, specificity, PPV, and NPV values for the MTBDRplus assay were 83.3%, 97.9%, 83.3%, and 97.9%, respectively, for the detection of isoniazid resistance. The overall correlation between the MTBDRplus assay and phenotypic DST demonstrated excellent agreement for detection of rifampicin resistance (κ = 0.847) and for detection of INH resistance (κ = 0.812), respectively. Conclusions The MTBDRplus assay can be used effectively even on AFB smear-negative specimens from TB patients, when the TB-PCR is positive. This result might help clinicians to manage patients with suspected DR-TB in difficult situations.

The Second Zambian National Tuberculosis Drug Resistance survey - a comparison of conventional and molecular methods.

The prevalence of MDR-TB in Zambia was estimated to be 1.8% in 2001. A second drug resistance survey was conducted in 2008 to determine trends; the use of the Genotype MTBDRplus assay was applied to compare results to the gold standard. A two-stage cluster sampling, with health facilities as primary sampling units. Processed sputum specimens were inoculated on solid media for culture; heat-inactivated bacterial suspensions from sputum samples were tested on a commercial line probe assay for the identification of rifampicin and isoniazid resistance. A total of 917 patients with TB were enrolled and 883 (96.3%) analysed. A total of 574 (65%) had LJ results and 824 (93.3%) had results from MTBDRplus assay. The median age was 32, and 63.3% were males. MDR-TB according to LJ-based DST was 1.1% (CI 0.1-2.4) whereas according to MDTBDRplus assay was 1.6% (CI 0.6-2.6). Isoniazid monoresistance in new cases was 2.4% (CI 0.613-4.26) based on LJ results and 5.0% (CI 3.2-6.7) based on the MTBDRplus; in retreatment cases, it was 4.4% (CI 0.3-8.6) and 2.40% (CI <0.1-5.1) on LJ and MTBDRplus, respectively. Rifampicin monoresistance in new cases was 0.1% (CI <0.1-0.4) based on LJ and 0.6% (CI 0.01-1.1) based on the MTBDRplus; in retreatment cases, it was 0% (CI 0-3.8) and 1.8% (CI <0.1-4.0) on LJ and MTBDRplus, respectively. There were no XDR-TB cases found and no association between MDR-TB and HIV. There was no increase in MDR-TB prevalence in Zambia from 2001 to 2008; results from the two methods were similar. Molecular methods were quicker and simpler to use.

Phenotypic and genotypic analysis of anti-tuberculosis drug resistance in Mycobacterium tuberculosis isolates in Myanmar.

BackgroundTuberculosis (TB) is one of the most serious health problems in Myanmar. Because TB drug resistance is associated with genetic mutation(s) relevant to responses to each drug, genotypic methods for detecting these mutations have been proposed to overcome the limitations of classic phenotypic drug susceptibility testing (DST). We explored the current estimates of drug-resistant TB and evaluated the usefulness of genotypic DST in Myanmar.MethodsWe determined the drug susceptibility of Mycobacterium tuberculosis isolated from sputum smear-positive patients with newly diagnosed pulmonary TB at two main TB centers in Myanmar during 2013 by using conventional phenotypic DST and the GenoType MTBDRplus assay (Hain Lifescience, Germany). Discrepant results were confirmed by sequencing the genes relevant to each type of resistance (rpoB for rifampicin; katG and inhA for isoniazid).ResultsOf 191 isolates, phenotypic DST showed that 27.7% (n=53) were resistant to at least one first-line drug and 20.9% (n=40) were resistant to two or more, including 18.3% (n=35) multidrug-resistant TB (MDR-TB) strains. Monoresistant strains accounted for 6.8% (n=13) of the samples. Genotypic assay of 189 isolates showed 17.5% (n=33) MDR-TB and 5.3% (n=10) isoniazid-monoresistant strains. Genotypic susceptibility results were 99.5% (n=188) concordant and agreed almost perfectly with phenotypic DST (kappa=0.99; 95% confidence interval 0.96-1.01).ConclusionsThe results highlight the burden of TB drug resistance and prove the usefulness of the genotypic DST in Myanmar.

Performance of Genotype MTBDRplus in the Detection of Resistance to Rifampicin and Isoniazid Among Clinical Mycobacteria Isolates in Ilorin, Nigeria.

Performance of Genotype MTBDRplus assay against Lowenstein Jensen (LJ) proportion method of Drug Susceptibility Testing (DST) in detection of resistance among clinical mycobacteria isolates to rifampicin (RMP) and isoniazid (INH) was evaluated in Ilorin, Nigeria. This retrospective study characterized one hundred mycobacteria isolates from pulmonary TB patients, stored on LJ medium and subcultured unto fresh LJ slants before being genotyped using MTBDRplus assay. DST was performed on the isolates against RMP, INH, Ethambutol and Streptomycin. Genotype MTBDRplus identified 97% and 3% of the 100 isolates as Mycobacterium tuberculosis Complex (MTBC) and Non-Tuberculous Mycobacteria (NTM) respectively. Fourteen of the isolates, (14%) were resistant to RMP while 86% were sensitive by the genotypic method. Three of these 14 RMP-resistant isolates were NTMs while 11 were MTBC. Twelve (12%) of the 100 isolates were resistant to INH. Three INH-resistant isolates were NTMs, and 9 were MTBC. Phenotypically and genotypically, the 3 NTMs were resistant to RMP and INH and ten of the 97 MTBC strains were RMP-resistant. One RMP-phenotypically-sensitive strain was genotypically resistant to RMP. Six of the MTBC isolates were resistant to both RMP and INH by both methods. Most mutations occurred in the S-531L and S315T1 codons of rpoB and KatG genes of RMP and INH, respectively. The high specificity and positive predictive values recorded by MTBDRplus in our study make it suitable for use in the programmatic management of drug-resistant TB in resource-limited settings.

Xpert MTB/RIF and GenoType MTBDRplus assays for the rapid diagnosis of bone and joint tuberculosis

Bone and joint tuberculosis (BJTB) constitutes about 10-20% of the extrapulmonary tuberculosis (EPTB) cases in China. The GenoType MTBDRplus assay (MTBDR) has been endorsed by the World Health Organization (WHO) for the diagnosis of pulmonary TB (PTB), while the Xpert MTB/RIF assay (Xpert) has also been endorsed by the WHO for the diagnosis of both PTB and EPTB. The diagnostic utility of these two techniques for BJTB was investigated prospectively. Sixty pus specimens were obtained from orthopedic patients. Smear, culture, Xpert, and MTBDR assays were performed for each specimen, and MGIT 960-based drug susceptibility testing (DST) was conducted for all of the isolates recovered. The diagnostic efficiency of Xpert and MTBDR was evaluated on the basis of bacteriological examination and the composite reference standard (CRS). Fifty of the 60 patients were considered to have BJTB according to the CRS. The sensitivities of smear, culture, Xpert, and MTBDR were 26% (13/50), 48% (24/50), 82% (41/50), and 72% (36/50) respectively, while the specificities of all of the tests were 100% (10/10). Xpert was 100% concordant with MGIT 960-based DST for the detection of rifampicin resistance. MTBDR had a sensitivity of 83.3% and a specificity of 100% for the detection of rifampicin resistance and a sensitivity of 85.7% and specificity of 100% for the detection of isoniazid resistance. With their high sensitivities, short turnaround times, and ability to diagnose TB and detect drug resistance simultaneously, both Xpert and MTBDR are feasible as diagnostic tools for BJTB in clinical practice.

Mal de Pott en un indígena colombiano

Approximately 2 million people die each year from tuberculosis. One third of the world´s population is estimated to be infected with the tuberculosis bacillus, although only 5-10% will develop the disease in their lifetime. The disease progression risk depends on endogenous and exogenous factors. Indigenous communities are a high-risk group for infection and development of tuberculosis. In addition to factors such as geographical isolation, social and cultural neglect and malnutrition, susceptibility to genetic polymorphisms has been identified in them. Spinal tuberculosis is the most destructive form of the disease, which represents approximately half of all cases of skeletal tuberculosis. The case of an HIV negative, indigenous Colombian man is presented. His diagnosis was done based on clinical and image findings, and it was confirmed with the rapid molecular assay Genotype MTBDRplus® and IS6110 PCR.The culture in solid media was negative after 16 weeks. We briefly discuss the pathogenesis, diagnosis and treatment. Finally, we comment on some aspects of the situation of tuberculosis among indigenous Colombian communities.

Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay

Purpose: There is scarcity of prevalence data of multi-drug–resistant tuberculosis (MDR-TB) data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. Materials and Methods: All MDR suspects in five districts of New Delhi were referred to the laboratory from 1st October 2011 to 31st December 2012 as per criterion defined by Programmatic Management of Drug Resistant Tuberculosis (PMDT). GenoType MTBDRplus assay was performed on 2182 samples or cultures and mutations in the rpoB gene for rifampicin (RIF) and katG and inhA genes for isoniazid (INH) were analyzed. Results: A total of 366 (16.8%) MDR-TB cases were diagnosed. MDR rate was found to be 32%, 16.6% and 10.2% during criterion A, B and C respectively. The most common mutation detected for RIF was S531L (59.0%) and for INH was S315T1 (88.3%). Mutations S531L and S315T1 occurred significantly higher in MDR strains as compared to RIF mono-resistant and INH mono-resistant strains, respectively. Average laboratory turn-around time (TAT) for dispatch of result to districts for test conducted on samples was 4.4 days. Conclusion: GenoType MTBDRplus is a useful assay for rapid detection of MDR-TB. The common mutations for RIF and INH were similar to those seen in other regions. However, mutations determining MDR strains and mono-resistant strains differed significantly for both RIF and INH.

Defining multidrug-resistant tuberculosis: correlating GenoType MTBDRplus assay results with minimum inhibitory concentrations

This study correlates MICs of rifampicin (RIF) and isoniazid (INH) with GenoType MTBDRplus assay results for drug-resistant Mycobacterium tuberculosis (MTB) clinical isolates. MICs of RIF and INH were established for 84 and 90 isolates, respectively, testing 7 concentrations of each drug. Genotypic resistance to each drug was determined by GenoType MTBDRplus assay with 50 representative mutations confirmed by pyrosequencing, with mutations in the rpoB gene associated with RIF resistance and mutations in the katG and/or inhA genes associated with INH resistance. Based upon the correlation of MICs with specific genetic profiles, relative resistance levels were established for each isolate. Results indicate that MTB phenotypic resistance, currently based upon the testing of isolate susceptibility to a single drug concentration, may be more accurately profiled via quantitative MICs, and therefore, the correlation of molecular diagnostic results with specific MICs may allow for more optimal treatment of infections.

Utility of GenoType MTBDRPlus Assay for Direct Detection and Drug Susceptibility Testing of Mycobacterium tuberculosis from Probable Tuberculous Meningitis Patients

Aims: To evaluate GenoType ® MTBDRplus line probe assay as a diagnostic tool for detection of Mycobacterium tuberculosis and drug susceptibility testing from cerebrospinal fluid of probable tuberculous meningitis patients. Study Design: A prospective, double blind study. Place and Duration of study: Dept. of Microbiology and Neurology, Institute of Human Behavior Original Research Article Gupta et al.; BJMMR, 8(9): 772-780, 2015; Article no.BJMMR.2015.505 773 and Allied sciences, Delhi, India between February 2014 to October 2014. Methodology: Cerebrospinal fluid collected from 107 probable meningitis patients with diagnostic score >10 were subjected to smear microscopy, automated liquid culture (BACTEC MGIT 960) and Polymerase chain reaction (IS6110). All the samples were also subjected to GenoType ® MTBDRplus line probe assay for detecting M. tuberculosis and drug susceptibility. Drug susceptibility testing of all the M. tuberculosis isolates was done by BACTEC MGIT 960 and GenoType ® MTBDRplus line probe assay. Results: The sensitivity, specificity of the assay for M. tuberculosis detection was 49.5%, 100% against clinical diagnosis as reference standard and 68.9%, 100% against definitive diagnosis as reference standard. A diagnostic accuracy of 56.8% (kappa 0.22), 75% (kappa 0.46), were seen in patients with probable and confirmed diagnosis respectively. The drug susceptibility results for Isoniazid and Rifampicin could be delineated in only 39.2% of patients. Conclusion: This assay proved to have better sensitivity, diagnostic accuracy than smear microscopy and automated liquid culture for early detection of M. tuberculosis from probable tuberculous meningitis patients and has comparable sensitivity to culture (39.2%) for detection of drug susceptibility (though on different isolates). Rapid turnaround time and user friendliness makes it an acceptable assay for simultaneous early detection of M. tuberculosis and its drug susceptibility for better patient management.

Diagnosis of Pulmonary Tuberculosis Using Genotype MTBDRplus Assay in Three Local Government Primary Health Centres of Osun State, Nigeria- a Pilot Study

Study background: Tuberculosis including multi-drug resistant form is not adequately diagnosed in spite of the fact that it constitutes a major public health challenge. This study was carried out to obtain data on laboratory diagnosis of pulmonary tuberculosis at the three local government areas in Osun state, Nigeria. Methods: Six month pilot study was carried out at Iwo, Irewole and Ede North LGAs, all located in Osun state, Nigeria. Socio-demographic and clinical information of the subjects were obtained using a pre-tested questionnaire. Sputum samples were collected from symptomatic pulmonary tuberculosis patients, stained with Zeihl-Neelsen (ZN) reagents and cultured on egg-based Lowenstein-Jensen medium. The medium was incubated at 37°C for eight weeks. Acid Fast Bacilli was confirmed by repeat Z-N staining. Drug resistance testing of the isolates was done using Genotype MTBDRplus. Serum was screened for HIV test using recombinant ELISA. Those that were screened positive were retested using Capillus HIV 1 and 2. Results: Of the 75 subjects studied, seven (9.3%) were over 60 years while the majority, 45 (76.0%) were aged 21-60 years. Thirty-four (45.3%) had their sputum positive for acid fast bacilli (AFB) while 24 (32.0%) were culture positive. Among the 24 isolates, 6 were identified to be non tuberculos mycobacteria (NTM) by molecular methods. The HIV prevalence rate was observed to be 18.6% among all the study participants. Six out of 34 AFB positive persons (17.6%) were also positive for HIV specific antibody, while eight (19.5%) of the AFB negative persons were positive for HIV. There is no significant difference in the incidence of TB between the HIV infected and uninfected groups (p>0.05).Among the 18 mycobacterial isolates (excluding NTM), one strain (5.5%) was found to be resistant to rifampicin and isoniazid (Multi-Drug Resistant-MDR) while three were resistant to isoniazid alone.. Conclusion: This pilot study reveals the existence of MDR-TB and warrants well designed studies to ascertain the magnitude of the problem.

Evaluation of genetic mutations associated with mycobacterium tuberculosis resistance to isoniazid, rifampicin (MTBDR plus), fluoroquinolone and injectable second-line drugs (MTBDRsl)

Aims and objectivesTuberculosis (TB) is one of the major public health problems in Armenia. In 2009, TB incidence in Armenia was reported to be as high as 45.5 per 100,000 population, and TB mortality was 3.9 per 100,000 population. Only 35% of estimated new sputum smear-positive pulmonary TB patients are notified annually. From 1729 TB cases notified in 2010, only 329 patients were sputum smear-positive. The treatment success rate of new sputum smear-positive pulmonary TB patients was 76%, which is below the WHO target of 85%. Poor treatment outcome is partially explained by the high prevalence of drug resistant forms of TB. According to the 2007 drug resistance survey, MDR-TB among never-treated patients was 9.4% and 43.2% among previously treated cases with 4% of XDR cases. This represents an enormous public health challenge for Armenia; early identification and effective treatment of patients with MDR-TB is crucial to prevent further spread of the disease. MethodsA total of 583 specimens are sent to the NRL Armenia being either acid-fast bacterium positive or negative, but culture positive. Rifampin and isoniazid resistance was performed on 77 out of the 583 specimens of DR TB clinical isolates to analyze resistance for FQL, AM/CP and EMB.The DNA preparation method used was as described and recommended by the manufacturer (MTBDR plus version 1.0 and MTBDR sl version 2.0 Hain Lifescience, Nehren).For 99 (88.4%) and 160 (76.2%) strains, the mutations causing rifampicin and isoniazid resistance were located in the codon of rpoB S531L and katG S315T 1, respectively. Regarding isoniazid, 14 (6.6%) strains have a mutation in the inhA regulatory region (C15T) and 31 (14.7%) in both katG and InhA.Mutations detected in the FQ, EMB and rrs-resistant respectively are: 13 (16.9%) strains with the majority of mutations (10 [13%]) in the codon of 94 of the gyrA gen; 20 (26%) strains, with the majority of mutations (7 [9.1%]) in the codon of 306 of the EMB gen; and 13 (16.8%) strains with the majority of mutations (9 [11.6%]) in the codon of 1401 of the rrs gen.Comparing with phenotypically DST sensitivity and specificity for RMP was 97.6% (69 strains) and 100%, respectively. False resistance was detected for 2 strains containing missed bands of rpoB (W6, 7, 8) and 1 strain W7. Sensitivity and specificity for INH was 98.33%, 87.5% (69 strains). From 2 strains, 1 was false resistant (missing of WT 1, 2 bands) and 1 false sensitive results for INH. The sensitivity and specificity for AM/CM was 80%, 100%, and for the FQL 85.7%, 96.72%, respectively (69 strains). ConclusionRapid and accurate detection of resistance to first- and second-line anti-TB drugs is the key to successful therapy and interruption of the transmission chain of MDRTB strains. In summary, this data represent an important addition to the rare epidemiological data concerning resistance patterns of MTB in Armenia and showed that the application of the GenoType MTBDRplus and GenoType MTBDRsl assays might be useful additional tools to allow for a rapid and safe diagnosis of MDR and XDR TB. In mutations associated with katG, high dosages of isoniazid in future will be considered for treatment.

Assessment of the genotype MTBDRplus assay for rifampin and isoniazid resistance detection on sputum samples in Cote d'Ivoire.

We conducted an evaluation study on the GenoType MTBDRplus assay's ability to detect mutations conferring resistance to rifampin and isoniazid directly from sputum taken from 120 smear positive pulmonary patients from tuberculosis (TB) centers in Cote d'Ivoire. The sputum was decontaminated by N-acetyl-l-cysteine (NALC) and comparatively analyzed with the MTBDRplus assay version 2.0 and the mycobacterial growth indicator tube (MGIT) 960 automated drug susceptibility testing (MGIT-DST). The Gene-Xpert Mycobacterium tuberculosis (MTB)/rifampicin (RIF) assay was performed for 21 sputa with absence of hybridization for at least one rpoB wild-type probes. Four and seven, respectively, discordant and concordant results were also analyzed. The mutations in the rpoB gene were 21 (17.5%), 20 (16.7%), 7 (5.8%), and 10 (8.3%), respectively, for D516V, H526Y, H526D, and S531L. S315T mutation in katG gene associated or not with mutation in promoter of inhA was detected in 76 (63.3%) of the sputum. Compared to MGIT-DST, the sensitivity and specificity of the MTBDRplus for rifampin resistance detection were 100% (75-100%) and 73.2% (61.3-84%), respectively. For isoniazid resistance detection, the sensitivity and specificity were, respectively, 95% (90-‌99) and 95.1% (88.5-100%). Interpretation of 16 sputa without hybridization of rpoB wild-type probe 8 compared to those obtained with MGIT-DST and GeneXpert MTB/RIF was discordant and concordant, respectively, for 11 and 5.

A 24-well plate assay for simultaneous testing of first and second line drugs against Mycobacterium tuberculosis in a high endemic setting

BackgroundEarly detection of drug resistance is one of the priorities of tuberculosis (TB) control programs as drug resistance is increasing. New molecular assays are only accessible for a minority of the second line drugs and their availability in high endemic settings is also hampered by high cost and logistic challenges. Therefore, we evaluated a previously developed method for drug susceptibility testing (DST) including both first- and second line anti-TB drugs for use in high endemic areas.ResultsBaseline mycobacterial isolates from 78 consecutive pulmonary TB patients from Addis Ababa, Ethiopia who were culture positive for Mycobacterium tuberculosis at the end of a two-month directly observed treatment short course (DOTS) were included. The isolates were simultaneously tested for isoniazid, rifampicin, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide and para-aminosalicylic acid susceptibility using the indirect proportion method adopted for 24-well agar plates containing Middlebrook 7H10 medium. Applying the 24-well plate assay, 43 (55.1%) isolates were resistant to one or more of the first line drugs tested (isoniazid, rifampicin and ethambutol). MDR-TB was identified in 20.5% of this selected group and there was a perfect correlation for rifampicin resistance with the results from the genotype MTBDRplus assay. All isolates were susceptible to aminoglycosides and fluoroquinolones in agreement with the genotype MTBDRsl assay. The only tested second line drug associated to resistance was ethionamide (14.1% resistant). The method was reproducible with stable results for internal controls (one multi-drug resistant (MDR) and one pan-susceptible strain (H37Rv) and DST results could be reported at two weeks.ConclusionsThe 24-well plate method for simultaneous DST for first- and second line drugs was found to be reproducible and correlated well to molecular drug susceptibility tests. It is likely to be useful in high-endemic areas for surveillance as well as for the detection of second line drug resistance in targeted groups such as in those who fail empirical MDR treatment.

Significant clinical impact of a rapid molecular diagnostic test (Genotype MTBDRplus assay) to detect multidrug-resistant tuberculosis.

There are limited data on the clinical impact of rapid diagnostic tests to detect multidrug-resistant tuberculosis (MDR-TB). We sought to determine whether the use of a molecular diagnostic test to detect MDR-TB improves clinical outcomes. A quasi-experimental study was conducted to analyze the impact of the Genotype MTBDRplus assay on clinical outcomes among patients with culture-confirmed pulmonary MDR-TB. Patients received treatment at the National Center for Tuberculosis and Lung Diseases in Tbilisi, Georgia. Time to MDR-TB treatment initiation, culture conversion, and infection control measures were compared to a time period prior to the implementation of the molecular test. Of 152 MDR-TB patients, 72 (47%) were from prior to and 80 (53%) following implementation of the MTBDRplus assay ("post-implementation group"). Patients in the post-implementation group initiated a second-line treatment regimen more rapidly than those in the pre-implementation group (18.2 vs 83.9 days, P < .01). Among patients admitted to a "drug-susceptible" tuberculosis ward, those from the post-implementation group spent significantly fewer days on the drug-susceptible ward compared to patients in the pre-implementation group (10.0 vs 58.3 days, P < .01). Among patients with 24 weeks follow-up (n = 119), those in the post-implementation group had a higher rate of culture conversion at 24 weeks (86% vs 63%, P < .01) and a more rapid rate of time to culture conversion (adjusted hazard ratio [aHR] 4.15, 95% confidence interval [CI], 2.5-6.9). The implementation of a rapid molecular diagnostic test led to significant clinical improvements including reduced time to initiation of MDR-TB treatment, culture conversion, and improved infection control practices.

Molecular approaches for detection of the multi-drug resistant tuberculosis (MDR-TB) in Bangladesh.

The principal obstacles in the treatment of tuberculosis (TB) are delayed and inaccurate diagnosis which often leads to the onset of the drug resistant TB cases. To avail the appropriate treatment of the patients and to hinder the transmission of drug-resistant TB, accurate and rapid detection of resistant isolates is critical. Present study was designed to demonstrate the efficacy of molecular techniques inclusive of line probe assay (LPA) and GeneXpert MTB/RIF methods for the detection of multi-drug resistant (MDR) TB. Sputum samples from 300 different categories of treated and new TB cases were tested for the detection of possible mutation in the resistance specific genes (rpoB, inhA and katG) through Genotype MTBDRplus assay or LPA and GeneXpert MTB/RIF tests. Culture based conventional drug susceptibility test (DST) was also carried out to measure the efficacy of the molecular methods employed. Among 300 samples, 191 (63.7%) and 193 (64.3%) cases were found to be resistant against rifampicin in LPA and GeneXpert methods, respectively; while 189 (63%) cases of rifampicin resistance were detected by conventional DST methods. On the other hand, 196 (65.3%) and 191 (63.7%) isolates showed isoniazid resistance as detected by LPA and conventional drug susceptibility test (DST), respectively. Among the drug resistant isolates (collectively 198 in LPA and 193 in conventional DST), 189 (95.6%) and 187 (96.9%) were considered to be MDR as examined by LPA and conventional DST, respectively. Category-II and -IV patients encountered higher frequency of drug resistance compared to those from category-I and new cases. Considering the higher sensitivity, specificity and accuracy along with the required time to results significantly shorter, our study supports the adoption of LPA and GeneXpert assay as efficient tools in detecting drug resistant TB in Bangladesh.

Usefulness of Genotype MTBDRplus assay in acid-fast bacilli positive smear specimens in Almeria, Spain

IntroductionThe urgent need for operational research evaluating test performance in a real-world setting has been highlighted. The purpose of this study was therefore to evaluate the performance of MTBDRplus assay. MaterialsAccording to the reference method, of the 155 clinical specimens with valid results, 147 were susceptible to rifampicin (RIF) and isoniazid (INH), with 4 being multi-drug resistant (MDR) and 4 with isolated resistance to isoniazid (INH). ResultsThe results of the MTBDRplus assay were 100% concordant for the MDR and mono-resistant to INH specimens. However, the MTBDRplus assay showed a resistance pattern to RIF in one specimen which was classified as susceptible by the reference method. The majority of the specimens (118/75.6%) were also tested using the MTBDRplus method after culture on Lowenstein-Jensen media, showing 100% agreement with the results of the test directly from the specimens. An MTBDRplus test result was available within an average of 8 days. ConclusionsOverall, MTBDR results showed excellent results when compared with the reference method and achieved a significant time-reduction.