GenoType MTBDRsl Assay Research Articles

Determination of MICs of levofloxacin for Mycobacterium tuberculosis with gyrA mutations.

The purpose of the present study was to correlate gyrA mutations found in Mycobacterium tuberculosis isolates using the GenoType(®) MTBDRsl assay with minimum inhibitory concentrations of the fluoroquinolone levofloxacin (LVX). Of 123 archived clinical M. tuberculosis isolates evaluated, 93 isolates had an Ala90Val, Ser91Pro, Asp94Ala, Asn/Tyr, Gly or His mutation and 30 were wild-type. Phenotypically, gyrA mutations Ala90Val, Ser91Pro or Asp94Ala showed a low level of resistance to LVX, while Asp94Asn/Tyr, Asp94Gly or Asp94His mutations had high-level resistance.

Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second-line injectables

Objective/backgroundThe in vitro drug-susceptibility testing of Mycobacterium tuberculosis reports isolates as resistant or susceptible on the basis of single critical concentrations. It is evident that drug resistance in M. tuberculosis is quite heterogeneous, and involves low level, moderate level, and high level of drug-resistant phenotypes. Thus, the aim of our study was to correlate rrs (X52917) and eis (AF144099) promoter mutations, found in M. tuberculosis isolates, with corresponding minimum inhibitory concentrations of amikacin, kanamycin, and capreomycin. MethodsNinety M. tuberculosis clinical isolates were analyzed in this study. The minimum inhibitory concentrations were determined by MGIT 960 for 59 isolates with resistance-associated mutations in the rrs and eis promoter gene regions, and 31 isolates with wild-type sequences, as determined by the GenoType MTBDRsl (version 1) assay. ResultsThe rrs A1401G mutation was identified in 48 isolates resistant to the second-line injectables. The eis promoter mutations C-14T (n=3), G-10C (n=3), G-10A (n=3), and C-12T (n=2) were found within 11 isolates with various resistance profiles to the second-line injectables. Thirty-one isolates had wild-type sequences for the rrs and eis promoter gene regions of interest, one of which was amikacin, kanamycin, and capreomycin resistant. The isolates with the rrs A1401G mutation had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of >40mg/L, >20mg/L, and 5–15mg/L, respectively. The isolates with eis promoter mutations had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of 0.25–1.0mg/L, 0.625–10mg/L, and 0.625–2.5mg/L, respectively. ConclusionThis study provides a preliminary basis for the prediction of phenotypic-resistance levels to the second-line injectables based upon the presence of genetic mutations associated with amikacin, kanamycin, and capreomycin resistance. The results suggest that isolates with eis promoter mutations have consistently lower resistance levels to amikacin, kanamycin, and capreomycin than isolates with the rrs A1401G mutation.

Frequency and geographic distribution of gyrA and gyrB mutations associated with fluoroquinolone resistance in clinical Mycobacterium tuberculosis isolates: a systematic review.

BackgroundThe detection of mutations in the gyrA and gyrB genes in the Mycobacterium tuberculosis genome that have been demonstrated to confer phenotypic resistance to fluoroquinolones is the most promising technology for rapid diagnosis of fluoroquinolone resistance.MethodsIn order to characterize the diversity and frequency of gyrA and gyrB mutations and to describe the global distribution of these mutations, we conducted a systematic review, from May 1996 to April 2013, of all published studies evaluating Mycobacterium tuberculosis mutations associated with resistance to fluoroquinolones. The overall goal of the study was to determine the potential utility and reliability of these mutations as diagnostic markers to detect phenotypic fluoroquinolone resistance in Mycobacterium tuberculosis and to describe their geographic distribution.ResultsForty-six studies, covering four continents and 18 countries, provided mutation data for 3,846 unique clinical isolates with phenotypic resistance profiles to fluoroquinolones. The gyrA mutations occurring most frequently in fluoroquinolone-resistant isolates, ranged from 21–32% for D94G and 13–20% for A90V, by drug. Eighty seven percent of all strains that were phenotypically resistant to moxifloxacin and 83% of ofloxacin resistant isolates contained mutations in gyrA. Additionally we found that 83% and 80% of moxifloxacin and ofloxacin resistant strains respectively, were observed to have mutations in the gyrA codons interrogated by the existing MTBDRsl line probe assay. In China and Russia, 83% and 84% of fluoroquinolone resistant strains respectively, were observed to have gyrA mutations in the gene regions covered by the MTBDRsl assay.ConclusionsMolecular diagnostics, specifically the Genotype MTBDRsl assay, focusing on codons 88–94 should have moderate to high sensitivity in most countries. While we did observe geographic differences in the frequencies of single gyrA mutations across countries, molecular diagnostics based on detection of all gyrA mutations demonstrated to confer resistance should have broad and global utility.

Correlating Minimum Inhibitory Concentrations of ofloxacin and moxifloxacin with gyrA mutations using the genotype MTBDRsl assay

To correlate gyrA mutations found on the Genotype MTBDRsl assay in Mycobacterium tuberculosis (MTB) isolates with Minimum Inhibitory Concentrations (MICs) to the fluoroquinolones compounds ofloxacin (OFX) and moxifloxacin (MXF). MICs for OFX and MXF were ascertained for 93 archived clinical MTB isolates that showed gyrA mutations at Ala90Val, Ser91Pro, Asp94Ala, Asn/Tyr, Gly and His. Thirty fluoroquinolones susceptible isolates as determined by presence of all wild-type gyrA bands on the Genotype MTBDRsl assay were also included. gyrA mutations at Ala90Val (n=25), Ser91Pro (n=6), Asp94Ala (n=4), Asp94Asn/Tyr (n=13), Asp94Gly (n=42) and Asp94His (n=3) were observed. Isolates with mutations at Ala90Val or Ser91Pro had MIC90 of 4.0μg/ml and 1.0μg/ml for OFX and MXF, respectively, and isolates with mutations at Asp 94Ala, Asn/Tyr, Gly and His had MIC90 of 8.0μg/ml, and 2.5μg/ml for OFX and MXF, respectively. MTB MICs were found to be consistently lower for MXF than for OFX among isolates with the same gyrA mutation (e.g. Ala90Val). The majority of MTB isolates containing mutations at Asp94Ala, Asn/Tyr, Gly and His in gyrA were associated with a moderate level of resistance to MXF (MIC=2.5μg/ml), although 3 isolates with the mutations Asp94Asn/Tyr/Gly were associated with a high level of resistance to both fluoroquinolones (MXF MICs=5.0-8.0μg/ml, OFX MICs=≥10.0μg/ml).

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.

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.

Evaluation ofMycobacterium tuberculosisdrug susceptibility in clinical specimens from Nigeria using genotype MTBDRplus and MTBDRsl assays

The incidence of tuberculosis (TB) and especially multidrug-resistant TB (MDR) continues to increase alarmingly worldwide, and reliable and fast diagnosis of MDR is essential for the adequate treatment of patients. In contrast to the standard culture methods, nucleid acid amplification tests (NAATs) provide information about presence of Mycobacterium tuberculosis complex (MTBC) DNA and a potential resistance pattern within hours. We analyzed specimens of 110 patients from Nigeria comparing culture-based drug susceptibility testing (DST) to NAAT assays detecting isoniazid (INH), rifampicin (RMP) (GenoType MTBDRplus), and ethambutol (EMB) (GenoType MTBDRsl) resistance. Compared to DST, the GenoType MTBDRplus and MTBDRsl showed a specificity of 100% (86.3-100) and a sensitivity of 86% (42.1-99.6%) for detection of INH and a specificity of 100% (86.3-100) and a sensitivity of 83% (35.9-99.6%) for detection of RMP, and a sensitivity 100% (47.8-100%) for EMB resistance. However, in two strains, the NAAT assays provided false susceptible results as the mutations causing resistance were in genomic regions not covered by the probes of the GenoType MTBDRplus assay. We show that, in combination to DST, application of the GenoType MTBDRplus and GenoType MTBDRsl assays might be a useful additional tool to allow a rapid and safe diagnosis of MDR and extensively drug-resistant (XDR) MTBC.

Evaluation of Genotype MTBDRsl Assay to Detect Drug Resistance Associated with Fluoroquinolones, Aminoglycosides and Ethambutol on Clinical Sediments

BackgroundThe emergence of resistant tuberculosis (TB) is a major setback to the global control of the disease as the treatment of such resistance is complex and expensive. Use of direct detection of mutations by molecular methods could facilitate rapid diagnosis of resistance to offset diagnostic delays. We evaluated the performance of the Genotype MTBDRsl (Hain Life Sciences) for the detection of second line resistant TB directly from stored smear positive sputum sediments.Methodology/Principal FindingsThe assay showed a diverse range of sensitivity and specificity, 91.26% [95% CI, 84–96] and 95.5% [95% CI, 87–99] for FQ (PPV ∼97% & NPV ∼ 87.67%), 56.19% [95%CI, 46–66] and 81% [95%CI, 66–91] for EMB (PPV ∼ 88.06% & NPV ∼ 43.21%) and 100% for SLD. Diagnostic accuracy for FQ, SLD and EMB was 94%, 100% and 63.51%, respectively. 1.17% (2/170) were heteroresistance strains, where the heteroresistance was linked to rrs gene. A varying rate of validity was observed 100% (170/170) for FQ, 94.11% (160/170) for EMB, 88.23% (150/170) for SLD.Conclusions/SignificanceGenotype MTBDRsl is simple, rapid, economical assay that can be used to detect commonly known resistance associated with Fluoroquinolone, second line injectable drugs and ethambutol. The assay detects the targeted resistance in less time as compared to phenotypic DST. But due to low NPV to FQ (88%) and EMB (43.21%), the assay results must be interpreted in coordination with the phenotypic DST.

Diagnostic Accuracy of the GenoType MTBDR sl Assay for Rapid Diagnosis of Extensively Drug-Resistant Tuberculosis in HIV-Coinfected Patients

The Russian Federation is a high-tuberculosis (TB)-burden country with high rates of Mycobacterium tuberculosis multidrug resistance (MDR) and extensive drug resistance (XDR), especially in HIV-coinfected patients. Rapid and reliable diagnosis for detection of resistance to second-line drugs is vital for adequate patient management. We evaluated the performance of the GenoType MTBDRsl (Hain Lifescience GmbH, Nehren, Germany) assay on smear-positive sputum specimens obtained from 90 HIV-infected MDR TB patients from Russia. Test interpretability was over 98%. Specificity was over 86% for all drugs, while sensitivity varied, being the highest (71.4%) for capreomycin and lowest (9.4%) for kanamycin, probably due to the presence of mutations in the eis gene. The sensitivity of detection of XDR TB was 13.6%, increasing to 42.9% if kanamycin (not commonly used in Western Europe) was excluded. The assay is a highly specific screening tool for XDR detection in direct specimens from HIV-coinfected TB patients but cannot be used to rule out XDR TB.

Detection of Resistance to Second-Line Antituberculosis Drugs by Use of the Genotype MTBDR sl Assay: a Multicenter Evaluation and Feasibility Study

The rate of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has been steadily increasing in countries of the former USSR. The availability of rapid and reliable methods for the detection of drug resistance to second-line drugs is vital for adequate patient management. We evaluated the performance of the Genotype MTBDRsl assay compared to that of phenotypic drug susceptibility testing (Becton Dickinson Bactec MGIT 960 system) with a test panel of 200 Mycobacterium tuberculosis isolates at four sites in Eastern Europe. The interpretability of the Genotype MTBDRsl assay was over 95%. The sensitivity for the detection of resistance to fluoroquinolones, ethambutol, amikacin, and capreomycin varied between 77.3% and 92.3%; however, it was much lower for kanamycin (42.7%). The sensitivity for the detection of XDR TB was 22.6%. The test specificity was over 82% for all drugs. The assay presents a good screening tool for the rapid detection of resistance to individual second-line drugs and can be recommended for use in countries with a high burden of MDR/XDR TB. The sensitivity for the detection of kanamycin resistance needs improvement.

Detection of drug-resistant mycobacterium tuberculosis strains isolated in Serbia by the GenoType MTBDRsl assay

The new GenoType MTBDRsl assay (Hain Lifescience) detects mutations most frequently associated with resistance to fluoroquinolones (FLQ), aminoglycosides/cyclic peptides (AG/CP), and ethambutol (EMB) and thus, enables rapid identification of extensively drug-resistant (XDR) Mycobacterium tuberculosis strains. A set of 19 multidrug-resistant (MDR) strains isolated in Serbia in 2011 was tested by the MTBDRsl assay and by conventional drug susceptibility testing (DST). The sensitivity and specificity of the MTBDRsl assay were as follows: 100% for both for FLQ; 100% for both for AG/ CP; and 58.3% and 85.7%, respectively, for EMB. The sensitivity for detection of XDR strains was 100%. Mutations in the gyrA, rrs, and embB genes established in local M. tuberculosis strains resistant to the respective drugs have been reported as the most prevalent in other studies as well. We recommend the MTBDRsl assay as a screening test for the preliminary detection of XDR-TB cases in Serbia, but not as a replacement of the conventional second-line DST.

GenoType MTBDR sl for Molecular Detection of Second-Line-Drug and Ethambutol Resistance in Mycobacterium tuberculosis Strains and Clinical Samples

The purpose of this study was to evaluate the GenoType MTBDRsl assay (Hain Lifescience GmbH, Nehren, Germany) for its ability to detect resistance to fluoroquinolones (FLQ), injectable second-line antibiotics [kanamycin (KM) and capreomycin (CM)], and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains and directly in clinical samples. A total of 34 clinical strains were characterized with the Bactec 460 TB system. Fifty-four clinical samples from 16 patients (5 were smear negative and 49 were smear positive) were also tested directly. The corresponding isolates of the clinical specimens were also analyzed with the Bactec 460TB. When there was a discrepancy between assays, pyrosequencing was performed. The overall rates of concordance of the MTBDRsl and the Bactec 460TB for the detection of FLQ, KM/CM, and EMB susceptibility in clinical strains were 72.4% (21/29), 88.8% (24/27), and 67.6% (23/34), whereas for clinical samples, rates were 86.5% (45/52), 92.3% (48/52), and 56% (28/50), respectively. In conclusion, the GenoType MTBDRsl assay may be a useful tool for making early decisions regarding KM/CM susceptibility and to a lesser extent regarding FLQ and EMB susceptibility. The test is able to detect mutations in both clinical strains and samples with a short turnaround time. However, for correct management of patients with extensively drug-resistant tuberculosis, results must be confirmed by a phenotypical method.

Feasibility of the GenoType MTBDR sl Assay for Fluoroquinolone, Amikacin-Capreomycin, and Ethambutol Resistance Testing of Mycobacterium tuberculosis Strains and Clinical Specimens

The new GenoType Mycobacterium tuberculosis drug resistance second line (MTBDRsl) assay (Hain Lifescience, Nehren, Germany) was tested on 106 clinical isolates and directly on 64 sputum specimens for the ability to detect resistance to fluoroquinolones, injectable drugs (amikacin or capreomycin), and ethambutol in Mycobacterium tuberculosis strains. A total of 63 strains harboring fluoroquinolone, amikacin/capreomycin, or ethambutol resistance and 43 fully susceptible strains were comparatively analyzed with the new MTBDRsl assay, by DNA sequencing, and by conventional drug susceptibility testing in liquid and solid media. No discrepancies were obtained in comparison with the DNA sequencing results. Fluoroquinolone resistance was detected in 29 (90.6%) of 32, amikacin/capreomycin resistance was detected in 39/39 (84.8%/86.7%) of 46/45, and ethambutol resistance was detected in 36 (69.2%) of 52 resistant strains. A total of 64 sputum specimens (42 smear positive, 12 scanty, and 10 smear negative) were tested with the new MTBDRsl assay, and the results were compared with those of conventional drug susceptibility testing. Fluoroquinolone resistance was detected in 8 (88.9%) of 9, amikacin/capreomycin resistance was detected in 6/7 (75.0%/87.5%) of 8, and ethambutol resistance was detected in 10 (38.5%) of 26 resistant strains. No mutation was detected in susceptible strains. The new GenoType MTBDRsl assay represents a reliable tool for the detection of fluoroquinolone and amikacin/capreomycin resistance and to a lesser extent also ethambutol resistance. In combination with a molecular test for detection of rifampin and isoniazid resistance, the potential for the detection of extensively resistant tuberculosis within 1 to 2 days can be postulated.