Indirect Drug Susceptibility Testing Research Articles

Effectiveness of GenoType MTBDRsl in excluding TB drug resistance in a clinical trial.

OBJECTIVES: To assess the performance of the GenoType MTBDRsl v1, a line-probe assay (LPA), to exclude baseline resistance to fluoroquinolones (FQs) and second-line injectables (SLIs) in the Standard Treatment Regimen of Anti-tuberculosis Drugs for Patients With MDR-TB 1 (STREAM 1) trial.METHODS: Direct sputum MTBDRsl results in the site laboratories were compared to indirect phenotypic drug susceptibility testing (pDST) results in the central laboratory, with DNA sequencing as a reference standard.RESULTS: Of 413 multidrug-resistant TB (MDR-TB) patients tested using MTBDRsl and pDST, 389 (94.2%) were FQ-susceptible and 7 (1.7%) FQ-resistant, while 17 (4.1%) had an inconclusive MTBDRsl result. For SLI, 372 (90.1%) were susceptible, 5 (1.2%) resistant and 36 (8.7%) inconclusive. There were 9 (2.3%) FQ discordant pDST/MTBDRsl results, of which 3 revealed a mutation and 5 (1.3%) SLI discordant pDST/MTBDRsl results, none of which were mutants on sequencing. Among the 17 FQ- and SLI MTBDRsl-inconclusive samples, sequencing showed 1 FQ- and zero SLI-resistant results, similar to frequencies among the conclusive MTBDRsl. The majority of inconclusive MTBDRsl results were associated with low bacillary load samples (acid-fast bacilli smear-negative or scantily positive) compared to conclusive results (P < 0.001).CONCLUSION: MTBDRsl can facilitate the rapid exclusion of FQ and SLI resistances for enrolment in clinical trials.

A successful introduction to a non-expert setting of the thin-layer agar Colour Test as an indirect phenotypic drug susceptibility test for Mycobacterium tuberculosis

ObjectivesWe evaluated the performance of the MDR/XDR-TB Colour Test (CT) as an in-house thin-layer agar-based indirect drug susceptibility test (DST) for Mycobacterium tuberculosis (MTB) in a non-expert setting in Estonia. MethodsAfter 2 days of hands-on training for laboratory technicians, 6 panels of 150 MTB isolates were cultured onto CT plates prepared in-house in 2 laboratories. Triplicate readings of 900 CT plates resulted in 18 DST patterns for each initial isolate. Time intervals to the results and for media preparation were estimated, and intra- and interobserver agreement, test sensitivities and specificities were calculated. BACTEC MGIT 960 DST was used as a reference. ResultsThe median time to produce DST results for isoniazid, rifampicin and levofloxacin was 13 days. CT sensitivity was 94.7% for levofloxacin, 95.8% for isoniazid and 97.3% for rifampicin. Test specificities were >97% for all 3 drugs. Interobserver agreement was 100% in Lab A and in Lab B >97% for levofloxacin and 99% for isoniazid and rifampicin. ConclusionsThe implementation of the CT into a new laboratory was straightforward with only minimal guidance required. This study proves that the CT is highly reproducible and easily interpreted by previously inexperienced personnel.

Non-commercial phenotypic assays for the detection of Mycobacterium tuberculosis drug resistance: a systematic review.

Several rapid non-commercial culture-based methods and assays for drug susceptibility testing (DST) of Mycobacterium tuberculosis have emerged over the last decades. The aim of the current review was to summarise evidence on the performance of microscopic observation of drug susceptibility (MODS), thin-layer agar (TLA) and colorimetric redox-indicator (CRI) assays for detection of resistance to first- and second-line anti-tuberculosis (TB) drugs. Forty-three publications satisfying selection criteria were selected for data extraction. MODS and CRI assays demonstrated pooled sensitivity and specificity of > 93% for the detection of resistance to rifampicin and isoniazid and confirmed their utility for an accurate detection of multidrug-resistant TB (MDR-TB) in various settings. Sensitivity and specificity values for indirect DST for ethambutol (EMB) using CRI assays were 94.0% and 82.0%, respectively, suggesting that CRIs could be used to rule out resistance to EMB. Performance for other drugs varied more substantially across the reports. There was no sufficient evidence on the performance of the TLA assay for making any conclusion on its utility for DST. Our data suggests that non-commercial assays could be used for a rapid and accurate DST in settings where the use of commercial World Health Organization-endorsed assays could be limited due to a variety of reasons including limited resources, laboratory facilities or trained personnel. While inexpensive and easy-to-perform MODS and TLA assays can be used in low-income settings, using CRI assays for determination of minimal inhibitory concentrations may be implemented in middle- and high-income countries with high MDR-TB burden to guide clinical management of TB patients.

Primary drug resistance in extra-pulmonary tuberculosis: a hospital-based prospective study from Pakistan.

SETTING: Tertiary level specialised tuberculosis (TB) hospital.OBJECTIVE: To determine the prevalence of antimicrobial resistance in new extra-pulmonary tuberculosis (EPTB) cases.DESIGN: Prospective cross-sectional study. Presumptive EPTB patients with enlarged lymph nodes or pleural effusion having no history of TB treatment were enrolled. Specimens were tested for smear, Xpert® MTB/RIF and culture. Indirect drug susceptibility testing (DST) was performed using MGIT 960 and line-probe assays (LPA).RESULTS: Among 671 cases, 255 were bacteriologically confirmed and 185 DSTs were performed. Multidrug resistance (MDR-TB) was reported in 2.2% (95%CI 0.6-5.4), any resistance to rifampicin (RMP) in 2.7% (95%CI 0.9-6.2), isoniazid (INH) in 7.6% (95%CI 4.1-12.4), ethambutol in 1.1% (95%CI 0.1-3.9), pyrazinamide in 2.2% (95%CI 0.9-5.5) and fluoroquinolones (FQ) in 6.0% (95%CI 3.0-10.4). The sensitivity and specificity of LPA-DST was 100% and >98.8% respectively for RMP, INH and FQ. Among 82 cases with RMP of the results available for all three methods used, five were reported to be resistant on Xpert but all five were susceptible on MGIT 960 and four on MTBDRplus.CONCLUSION: Prevalence of RMP resistance in new EPTB cases is 2.7% (95%CI 0.9-6.2). Caution is warranted for RMP resistance detected using Xpert in EPTB samples with a very low bacterial load.

Direct drug susceptibility testing of Mycobacterium tuberculosis using the proportional method: A multicenter study.

Conventional indirect drug susceptibility testing (DST) of Mycobacterium tuberculosis with solid media is inexpensive and reliable, but time-consuming. This study aimed to evaluate direct DST for testing sputum samples without culture to significantly reduce the time required to detect multidrug-resistant tuberculosis (MDR-TB). Direct and indirect DST of isoniazid (INH), rifampicin (RIF) and ethambutol (EMB) were performed on 334 sputum smear-positive specimens. There was full agreement between the results obtained from direct testing and after isolation of the bacteria by culture. Thus, the sensitivity and specificity were observed to be 100% for all three tested drugs when compared with indirect DST. In comparison with indirect DST, none of the samples with the direct method took >25days to report the DST (between 15-25days with a mean detection time of 20 days). Direct DST on solid media was shown to give reliable results at a much earlier stage than conventional phenotypic DST. The direct method was found to be more rapid, more accurate and simpler. In addition, it reduced the handling of pathogenic bacteria and thus reduced the bio hazards related to conventional DST.

Direct tuberculosis drug susceptibility testing: time-saving and cost-effective in detecting MDR-TB.

Recent advances have made molecular diagnosis of tuberculosis (TB) and drug susceptibility testing (DST) possible, but the high costs involved present a huge challenge. The refinement and improvement of affordable methods therefore remain a priority. Conventional indirect DST is inexpensive and reliable, but time-consuming. A direct DST method for the direct testing of sputum samples without culture has been developed to reduce the time required for DST, but there have been conflicting results. Direct and indirect DST against isoniazid and rifampicin were performed on 208 sputum smear-positive specimens, 186 from newly diagnosed patients and 22 from previously treated patients; respectively 169 and 180 of the direct and indirect DST results were reportable. In comparison with indirect DST, direct DST resulted in a saving of on average 10.5 days. The time to direct DST results was inversely correlated with the number of acid-fast bacilli in the sputum samples. Direct DST is highly sensitive, reliable, cost-effective and time-saving in comparison with indirect DST.

Molecular diagnostics for tuberculosis

The phenotypic methods of smear microscopy, culture and indirect drug susceptibility testing (DST) remain the 'gold standard' diagnostics for tuberculosis (TB) in 2015. However, this review demonstrates that genotypic methods are in the ascendancy. Current-generation nucleic acid amplification tests (NAATs) are important supplementary tests for the rapid direct detection of (multidrug-resistant) TB in specific clinical settings. Genotypic detection is already the preferred method of detecting rifampicin and pyrazinamide resistance. Next-generation NAATs able to detect about 10 colony forming units/mL of sputum could replace culture as the initial test for detecting TB. Whole genome sequencing could also plausibly replace phenotypic DST but much work is required in method standardisation, database development and elucidation of all resistance gene determinants. The challenge then will be to rollout these increasingly complex and expensive diagnostics in the low-income countries where TB is prevalent.

A field evaluation of the Hardy TB MODS Kit™ for the rapid phenotypic diagnosis of tuberculosis and multi-drug resistant tuberculosis.

BackgroundEven though the WHO-endorsed, non-commercial MODS assay offers rapid, reliable TB liquid culture and phenotypic drug susceptibility testing (DST) at lower cost than any other diagnostic, uptake has been patchy. In part this reflects misperceptions about in-house assay quality assurance, but user convenience of one-stop procurement is also important. A commercial MODS kit was developed by Hardy Diagnostics (Santa Maria, CA, USA) with PATH (Seattle, WA, USA) to facilitate procurement, simplify procedures through readymade media, and enhance safety with a sealing silicone plate lid. Here we report the results from a large-scale field evaluation of the MODS kit in a government service laboratory.Methods & Findings2446 sputum samples were cultured in parallel in Lowenstein-Jensen (LJ), conventional MODS and in the MODS kit. MODS kit DST was compared with conventional MODS (direct) DST and proportion method (indirect) DST. 778 samples (31.8%) were Mycobacterium tuberculosis culture-positive. Compared to conventional MODS the sensitivity, specificity, positive, and negative predictive values (95% confidence intervals) of the MODS Kit were 99.3% (98.3–99.8%), 98.3% (97.5–98.8%), 95.8% (94.0–97.1%), and 99.7% (99.3–99.9%). Median (interquartile ranges) time to culture-positivity (and rifampicin and isoniazid DST) was 10 (9–13) days for conventional MODS and 8.5 (7–11) for MODS Kit (p<0.01). Direct rifampicin and isoniazid DST in MODS kit was almost universally concordant with conventional MODS (97.9% agreement, 665/679 evaluable samples) and reference indirect DST (97.9% agreement, 687/702 evaluable samples).ConclusionsMODS kit delivers performance indistinguishable from conventional MODS and offers a convenient, affordable alternative with enhanced safety from the sealing silicone lid. The availability in the marketplace of this platform, which conforms to European standards (CE-marked), readily repurposed for second-line DST in the near future, provides a fresh opportunity for improving equity of access to TB diagnosis and first and second-line DST in settings where the need is greatest.

Evaluation of indirect drug susceptibility testing using the MODS assay for the detection of XDR-TB in China

The First Affiliated Hospital of Nanchang University and the Chest Hospital of Jiangxi Province, Jiangxi, China. To assess the performance and feasibility of the microscopic observation drug susceptibility (MODS) assay for the simultaneous detection of multidrug-resistant (MDR) and extensively drug-resistant tuberculosis (XDR-TB) in a high-burden, resource-limited setting. A total of 208 archived clinical isolates of Mycobacterium tuberculosis were used to compare MODS with the conventional proportion method for the rapid detection of resistance to rifampicin (RMP) and isoniazid (INH), as well as to the second-line drugs ofloxacin (OFX) and kanamycin (KM). Sensitivity was respectively 94.5%, 91%, 96.2% and 91.5% for RMP, INH, OFX and KM, and specificity was respectively 97.5%, 96%, 100% and 98.7%. Results for MODS were obtained in a median time of 7 days (range 5-17). The MODS assay offers a simple, rapid, economical and feasible method for the detection of M. tuberculosis resistance to first- and second-line drugs in resource-limited settings.

Performance of Cepheid® Xpert MTB/RIF® and TB-Biochip® MDR in two regions of Russia with a high prevalence of drug-resistant tuberculosis

The purpose of this study was to assess the performance of Cepheid® Xpert MTB/RIF® ("Xpert") and TB-Biochip® MDR ("TB-Biochip"). Sputum specimens from adults with presumptive tuberculosis (TB) were homogenized and split for: (1) direct Xpert and microscopy, and (2) concentration for Xpert, microscopy, culture [Lowenstein-Jensen (LJ) solid media and Mycobacteria Growth Indicator Tube® (MGIT)], indirect drug susceptibility testing (DST) using the absolute concentration method and MGIT, and TB-Biochip. In total, 109 of 238 (45.8 %) specimens were culture-positive for Mycobacterium tuberculosis complex (MTBC), and, of these, 67 isolates were rifampicin resistant (RIF-R) by phenotypic DST and 64/67 (95.5 %) were isoniazid resistant (INH-R). Compared to culture of the same specimen, a single direct Xpert was more sensitive for detecting MTBC [95.3 %, 95 % confidence interval (CI), 90.0-98.3 %] than direct (59.6 %, 95 % CI, 50.2-68.5 %) or concentrated smear (85.3 %, 95 % CI, 77.7-91.1 %) or LJ culture (80.8 %, 95 % CI, 72.4-87.5 %); the specificity was 86.0 % (95 % CI, 78.9-91.3 %). Compared with MGIT DST, Xpert correctly identified 98.2 % (95 % CI, 91.5-99.9 %) of RIF-R and 95.5 % (95 % CI, 85.8-99.2 %) of RIF-susceptible (RIF-S) specimens. In a subset of 104 specimens, the sensitivity of TB-Biochip for MTBC detection compared to culture was 97.3 % (95 % CI, 91.0-99.5 %); the specificity was 78.1 % (95 % CI, 61.5-89.9 %). TB-Biochip correctly identified 100 % (95 % CI, 94.2-100 %) of RIF-R, 94.7 % (95 % CI, 76.7-99.7 %) of RIF-S, 98.2 % (95 % CI, 91.4-99.9 %) of INH-R, and 78.6 % (95 % CI, 52.1-94.2 %) of INH-S specimens compared to MGIT DST. Xpert and Biochip were similar in accuracy for detecting MTBC and RIF resistance compared to conventional culture methods.

Direct Drug Susceptibility Testing of Mycobacterium tuberculosis for Rapid Detection of Multidrug Resistance Using the Bactec MGIT 960 System: a Multicenter Study

Conventional indirect drug susceptibility testing of Mycobacterium tuberculosis with liquid medium is well established and offers time-saving and reliable results. This multicenter study was carried out to evaluate if drug susceptibility testing (DST) can be successfully carried out directly from processed smear-positive specimens (direct DST) and if this approach could offer substantial time savings. Sputum specimens were digested, decontaminated, and concentrated by the laboratory routine procedure and were inoculated in Bactec MGIT 960 as well as Lowenstein-Jensen (LJ) medium for primary isolation. All the processed specimens which were acid-fast bacterium (AFB) smear positive were used for setting up direct DST for isoniazid (INH) and rifampin (RIF). After the antimicrobial mixture of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA) was added, the tubes were entered in the MGIT 960 instrument using the 21-day protocol (Bactec 960 pyrazinamide [PZA] protocol). Results obtained by direct DST were compared with those obtained by indirect DST to establish accuracy and time savings by this approach. Of a total of 360 AFB smear-positive sputum specimens set up for direct DST at four sites in three different countries, 307 (85%) specimens yielded reportable results. Average reporting time for direct DST was 11 days (range, 10 to 12 days). The average time savings by direct DST compared to indirect DST, which included time to isolate a culture and perform DST, was 8 days (range, 6 to 9 days). When results of direct DST were compared with those of indirect DST, there was 95.1% concordance with INH and 96.1% with rifampin. These findings indicate that direct DST with the Bactec MGIT 960 system offers further time savings and is a quick method to reliably detect multidrug resistance (MDR) cases.

Fast track method for the identification of multi–drug resistant tuberculosis on direct clinical specimen using combined drug media

Objective To combine isoniazid (INH) and rifampicin (RIF) in a single media to detect and evaluate multi-drug resistant Mycobacterium tuberculosis (MDR-TB) strains using clinical specimens by direct and indirect drug susceptibility testing (DST). Methods Drug susceptibility testing for INH and RIF using individual and combined drug media was performed by minimum inhibitory concentration (MIC) method on direct clinical specimens. Results The combined drug media showed complete concordance with individual drug media in the detection of MDR-TB by direct DST method and 89% efficiency with indirect DST method. Susceptibility results were available by 3 weeks after the receipt of clinical specimen using direct DST on combined drug media. Conclusions Combined drug media can be used as a fast track method in large scale studies warranting detection of MDR-TB.

An Integrated Approach to Rapid Diagnosis of Tuberculosis and Multidrug Resistance Using Liquid Culture and Molecular Methods in Russia

ObjectiveTo analyse the feasibility, cost and performance of rapid tuberculosis (TB) molecular and culture systems, in a high multidrug-resistant TB (MDR TB) middle-income region (Samara, Russia) and provide evidence for WHO policy change.MethodsPerformance and cost evaluation was conducted to compare the BACTEC™ MGIT™ 960 system for culture and drug susceptibility testing (DST) and molecular systems for TB diagnosis, resistance to isoniazid and rifampin, and MDR TB identification compared to conventional Lowenstein-Jensen culture assays.Findings698 consecutive patients (2487 sputum samples) with risk factors for drug-resistant tuberculosis were recruited. Overall M. tuberculosis complex culture positivity rates were 31.6% (787/2487) in MGIT and 27.1% (675/2487) in LJ (90.5% and 83.2% for smear-positive specimens). In total, 809 cultures of M. tuberculosis complex were isolated by any method. Median time to detection was 14 days for MGIT and 36 days for LJ (10 and 33 days for smear positive specimens) and indirect DST in MGIT took 9 days compared to 21 days on LJ. There was good concordance between DST on LJ and MGIT (96.8% for rifampin and 95.6% for isoniazid). Both molecular hybridization assay results correlated well with MGIT DST results, although molecular assays generally yielded higher rates of resistance (by approximately 3% for both isoniazid and rifampin).ConclusionWith effective planning and logistics, the MGIT 960 and molecular based methodologies can be successfully introduced into a reference laboratory setting in a middle incidence country. High rates of MDR TB in the Russian Federation make the introduction of such assays particularly useful.

Inter- and Intra-Assay Reproducibility of Microplate Alamar Blue Assay Results for Isoniazid, Rifampicin, Ethambutol, Streptomycin, Ciprofloxacin, and Capreomycin Drug Susceptibility Testing of Mycobacterium tuberculosis

The intersample and intrasample variability of the results obtained with the microplate Alamar blue assay for the indirect drug susceptibility testing of Mycobacterium tuberculosis was investigated. Between 1.2 and 8.5% of paired MICs differed by more than one twofold dilution, resulting in discordant susceptible-resistant designations at frequencies between 0.6% (rifampin) and 18.9% (ethambutol).

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis.

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and preventing new infections. Furthermore, the emergence of multidrug resistant tuberculosis (MDRTB) means that detection of drug resistance is necessary for stopping the spread of drug-resistant strains. The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of TB and MDRTB. The MODS assay is based on three principles: 1) mycobacterium tuberculosis (MTB) grows faster in liquid media than on solid media 2) microscopic MTB growth can be detected earlier in liquid media than waiting for the macroscopic appearance of colonies on solid media, and that growth is characteristic of MTB, allowing it to be distinguished from atypical mycobacteria or fungal or bacterial contamination 3) the drugs isoniazid and rifampicin can be incorporated into the MODS assay to allow for simultaneous direct detection of MDRTB, obviating the need for subculture to perform an indirect drug susceptibility test. Competing current diagnostics are hampered by low sensitivity with sputum smear, long delays until diagnosis with solid media culture, prohibitively high cost with existing liquid media culture methods, and the need to do subculture for indirect drug susceptibility testing to detect MDRTB. In contrast, the non-proprietary MODS method has a high sensitivity for TB and MDRTB, is a relatively rapid culture method, provides simultaneous drug susceptibility testing for MDRTB, and is accessible to resource-limited settings at just under $3 for testing for TB and MDRTB.

Rapid drug-susceptibility testing of Mycobacterium tuberculosis.

Three methods for rapidly determining the susceptibility of Mycobacterium Tuberculosis isolates to isoniazid, rifampin, ethambutol, streptomycin, and para-aminosalicylic acid were evaluated in a large-scale, blind study. Two of the methods measured evolution of CO2 from radio-labeled substrate (14CO2), and one method measured incorporation of 3H-uracil into ribonucleic acid. Rapid indirect drug-susceptibility test results for nearly 300 isolates were compared with those obtained using a standard modified proportion technique. The 3H-uracil uptake method proved to be unacceptable. Over-all, the results obtained using the 14CO2 methods and the standard method were similar. In general, there was greater agreement between the 14CO2 and proportion techniques with drug-susceptible strains than with drug-resistant strains. Among drug-resistant strains, both 14CO2 methods were more reliable for determining resistance to rifampin than to other drugs. This study demonstrates that large-scale, blind evaluations of new laboratory procedures are valuable. Our results indicate that methods relying o the enzymatic release of 14CO2 should be further refined and evaluated.