Evaluation of the fully automated Bactec MGIT 960 system for the susceptibility testing of Mycobacterium tuberculosis to first-line drugs: a multicenter study

The accuracy of the Bactec MGIT 960 system for susceptibility testing of 177 clinical isolates of Mycobacterium tuberculosis to first line drugs (isoniazid, rifampicin, ethambutol and streptomycin) was compared with the agar reference method. The sensitivity, the ability to detect resistance, of the MGIT system was 100%, while the specificity, the ability to detect susceptibility, ranged from 98.6% to 100% for all drugs tested.     

Standardization of broth microdilution method for Mycobacterium tuberculosis

Indirect drug susceptibility tests of Mycobacterium tuberculosis was done to investigate the accuracy and feasibility of a broth microdilution method (BMM) for determining minimal inhibitory concentrations of conventional drugs against M. tuberculosis. Test drugs included isoniazid (H), rifampicin (R), ethambutol (E), streptomycin (S) and pyrazinamide (Z). Fifty isolates of M. tuberculosis from patients who had never received drug therapy, and H37Rv strain for control, were evaluated in the system. When comparing this method with the gold standard proportional method in Lowenstein-Jensen medium, sensitivity of 100% for all drugs and specifities of 91, 100, 96, 98 and 85% were observed respectively for H, R, E, S and Z. The BMM was read faster (14-20 days) than the proportional method (20-28 days). The microdilution method evaluated allows the testing of multiple drugs in multiple concentrations. It is easy to perform and does not require special equipment or expensive supplies. In contrast to radiometric method it does not use radioactive material.     

Drug susceptibility testing of Mycobacterium tuberculosis by the broth microdilution method with 7H9 broth

In this study, we have evaluated the broth microdilution method (BMM) for susceptibility testing of Mycobacterium tuberculosis. A total of 43 clinical isolates of M. tuberculosis and H37Rv as a control strain were studied. All isolates were tested by the proportion method and the BMM for isoniazid (INH), rifampicin (RIF), streptomycin (STR), and ethambutol (ETM). The proportion method was carried out according to the National Committee for Clinical Laboratory Standards (NCCLS) on L?wenstein-Jensen (LJ) medium. The BMM was carried out using 7H9 broth with 96 well-plates. All strains were tested at 3.2-0.05 micro g/ml, 16-0.25 micro g/ml, 32-0.5 micro g/ml, and 32-0.5 micro g/ml concentrations for INH, RIF, STR, and ETM, respectively. When the BMM was compared with the proportion method, sensitivity was 100, 100, 96.9, and 90.2%, while specificity was 100, 85.7, 90.9, and 100% for INH, RIF, STR, and ETM, respectively. The plates were examined 7, 10, 14, and 21 days after incubation. The majority of the result were obtained at 14th days after incubation, while the proportion method result were ended in 21-28 days. According to our results, it may be suggested that the BMM is suitable for early determining of multidrug-resistance-M. tuberculosis strains in developed or developing countries.     

Comparison of the mycobacteria growth indicator tube with radiometric and solid culture for isolation of mycobacteria from clinical specimens and susceptibility testing of Mycobacterium tuberculosis

We compared the mycobacteria growth indicator tube (MGIT) system with the BACTEC 460 TB and Loewenstein-Jensen (LJ) systems for the recovery of mycobacteria (acid-fast bacilli [AFB]) from 600 clinical specimens. A total of 50 AFB (32 Mycobacterium tuberculosis complex, 10 M. avium complex, 3 M. gordonae, 3 M. xenopi, 1 M. terrae and 1 M. fortuitum) were detected. MGIT recovered 50 isolates of AFB (100% sensitivity), and BACTEC 460 TB and LJ recovered 49 (98% sensitivity) and 19 (38% sensitivity) AFB isolates, respectively. The mean times to detect mycobacteria were 10, 10 and 25 days for MGIT, BACTEC 460, and LJ slants. All isolates of M. tuberculosis complex were tested for susceptibility to streptomycin, isoniazid, rifampin, and ethambutol with the MGIT and BACTEC 460 TB. Both systems yielded identical susceptibility data with different mean times to report (5.38 days for MGIT versus 7.33 days for BACTEC 460 TB, P<0.05). The results suggest that MGIT is equivalent to BACTEC 460 TB in its ability to support the growth of mycobacteria, but significantly more efficient than LJ. MGIT may also be used for susceptibility testing of primary antituberculosis drugs.     

Multicenter evaluation of the nitrate reductase assay for drug resistance detection of Mycobacterium tuberculosis

The performance of the nitrate reductase assay was evaluated in a multicenter laboratory study to detect resistance of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin, isoniazid, ethambutol and streptomycin using a set of coded isolates. Compared with the gold standard proportion method on L?wenstein-Jensen medium, the assay was highly accurate in detecting resistance to rifampicin, isoniazid and ethambutol with an accuracy of 98%, 96.6% and 97.9%, respectively. For streptomycin, discrepant results were obtained with an overall accuracy of 85.3%. The assay proved easy to be implemented in countries with limited laboratory facilities.     

Direct nitrate reductase assay versus microscopic observation drug susceptibility test for rapid detection of MDR-TB in Uganda

The most common method for detection of drug resistant (DR) TB in resource-limited settings (RLSs) is indirect susceptibility testing on Lowenstein-Jensen medium (LJ) which is very time consuming with results available only after 2-3 months. Effective therapy of DR TB is therefore markedly delayed and patients can transmit resistant strains. Rapid and accurate tests suitable for RLSs in the diagnosis of DR TB are thus highly needed. In this study we compared two direct techniques--Nitrate Reductase Assay (NRA) and Microscopic Observation Drug Susceptibility (MODS) for rapid detection of MDR-TB in a high burden RLS. The sensitivity, specificity, and proportion of interpretable results were studied. Smear positive sputum was collected from 245 consecutive re-treatment TB patients attending a TB clinic in Kampala, Uganda. Samples were processed at the national reference laboratory and tested for susceptibility to rifampicin and isoniazid with direct NRA, direct MODS and the indirect LJ proportion method as reference. A total of 229 specimens were confirmed as M. tuberculosis, of these interpretable results were obtained in 217 (95%) with either the NRA or MODS. Sensitivity, specificity and kappa agreement for MDR-TB diagnosis was 97%, 98% and 0.93 with the NRA; and 87%, 95% and 0.78 with the MODS, respectively. The median time to results was 10, 7 and 64 days with NRA, MODS and the reference technique, respectively. The cost of laboratory supplies per sample was low, around 5 USD, for the rapid tests. The direct NRA and MODS offered rapid detection of resistance almost eight weeks earlier than with the reference method. In the study settings, the direct NRA was highly sensitive and specific. We consider it to have a strong potential for timely detection of MDR-TB in RLS.     

Evaluation of nitrate reduction assay, resazurin microtiter assay and microscopic observation drug susceptibility assay for first line antitubercular drug susceptibility testing of clinical isolates of M. tuberculosis

Background

Drug resistant tuberculosis (TB) is a growing concern worldwide. Early detection of multidrug-resistant Mycobacterium tuberculosis is of primary importance for both patient management and infection control. Optimal method for identifying drug-resistant M. tuberculosis in a timely and affordable way in resource-limited settings is not yet available.

Aim

This study evaluated; nitrate reductase assay (NRA), resazurin microtiter assay (REMA) and microscopic observation drug susceptibility assay (MODS) against the conventional 1% proportion method (PM) for the detection of resistance to first line antitubercular drugs, in M. tuberculosis clinical isolates.

Methods

A total of one hundred and five clinical isolates of M. tuberculosis; 50 pan sensitive and 55 pan resistant were tested with NRA, REMA and MODS. The 1% proportion method on Lowenstein-Jensen medium was used as reference test.

Results

Of all three methods which were tested NRA was found to be most sensitive and specific. Sensitivity for rifampicin resistance detection was 100%, 94.55% and 92.73% by NRA, REMA and MODS respectively. NRA and REMA were found to be 100% specific, while the MODS was 98% specific for detection of rifampicin resistance. Test results with all these methods were obtained within 8-14 days.

Conclusion

Rapid non-conventional and inexpensive methods may serve as a replacement for 1% proportion method in resource limited settings.     

High Isoniazid Resistance Rates in Rifampicin Susceptible Mycobacterium tuberculosis Pulmonary Isolates from Pakistan

Background

Rapid new diagnostic methods (including Xpert MTB/RIF assay) use rifampicin resistance as a surrogate marker for multidrug resistant tuberculosis. Patients infected with rifampicin susceptible strains are prescribed first line anti-tuberculosis therapy. The roll out of such methods raises a concern that strains with resistance to other first line anti-tuberculosis drugs including isoniazid will be missed and inappropriate treatment given. To evaluate implications of using such methods review of resistance data from high burden settings such as ours is essential.

Objective

To determine resistance to first line anti-tuberculosis drugs amongst rifampicin susceptible pulmonary Mycobacterium tuberculosis (MTB) isolates from Pakistan.

Materials and Methods

Data of pulmonary Mycobacterium tuberculosis strains isolated in Aga Khan University Hospital (AKUH) laboratory (2009–2011) was retrospectively analyzed. Antimicrobial susceptibility profile of rifampicin susceptible isolates was evaluated for resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin.

Results

Pulmonary specimens submitted to AKUH from 2009 to 2011 yielded 7738 strains of Mycobacterium tuberculosis. These included 54% (n 4183) rifampicin susceptible and 46% (n: 3555) rifampicin resistant strains. Analysis of rifampicin susceptible strains showed resistance to at least one of the first line drugs in 27% (n:1133) of isolates. Overall isoniazid resistance was 15.5% (n: 649), with an isoniazid mono-resistance rate of 4% (n: 174). Combined resistance to isoniazid, pyrazinamide, and ethambutol was noted in 1% (n: 40), while resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin was observed in 1.7% (n: 70) of strains.

Conclusions

Our data suggests that techniques (including Xpert MTB/RIF assay) relying on rifampicin susceptibility as an indicator for initiating first line therapy will not detect patients infected with MTB strains resistant to other first line drugs (including isoniazid). The roll out of these techniques must therefore be accompanied by strict monitoring ensuring early resistance detection to increase chances of improved patient outcomes.     

Multi Drug and Other Forms of Drug Resistant Tuberculosis Are Uncommon among Treatment Na?ve Tuberculosis Patients in Tanzania

Background

Surveillance and effective management of drug resistance is important to sustaining tuberculosis (TB) control efforts. We aimed to determine resistance rates to first line anti tuberculosis drugs and to describe factors associated with the resistance to any of the first line anti tuberculosis drugs in Dar es Salaam Tanzania.

Materials

Newly diagnosed, TB patients with neither history of tuberculosis treatment nor isoniazid prophylaxis were included into the study. Sputum specimens were cultured on either mycobacteria growth indicator tube 960 (MGIT 960) or Lowenstein Jenstein (LJ) medium supplemented with either glycerol (GLJ) or pyruvate (PLJ). Drug susceptibility for isoniazid, rifampicin, streptomycin and ethambutol was determined by either Lowenstein–Jensen (LJ) medium or mycobacteria growth indicator tube 960 (MGIT 960).

Results

A total of 933 newly diagnosed TB patients, were included into the study. Multi drug resistance (MDR) tuberculosis was detected among 2 (0.2%) patients. Resistance to any of the four tested drugs was detected among 54 (5.8%) patients. Mono-resistance to isoniazid, rifampicin, streptomycin and ethambutol were 21(2.3%), 3 (0.3%), 13 (1.4%), 9 (1.0%) respectively.

Conclusion

Primary resistance to first line anti tuberculosis drugs is still low in this setting. Continued vigilance including periodic national surveillance of anti-tuberculosis resistance is recommended.     

Comparative evaluation under routine conditions of the nitrate reduction assay, the proportion assay and the MGIT 960 assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis

The performance of the nitrate reductase assay (NRA) was compared with the proportion method (PM) on Lowenstein-Jensen medium and the BACTEC MGIT960 assay under routine conditions using 160 clinical isolates of Mycobacterium tuberculosis with a high proportion of resistant strains. The mean time to obtain results was 8.8 days and the overall agreements between NRA and PM and NRA and M960 were 95% and 94%, respectively. NRA was easy to perform and represents a useful tool for the rapid screening of drug-resistant M. tuberculosis strains in low-resource countries.     

Evaluation of BACTEC MGIT 960 System for Testing Susceptibility of Mycobacterium tuberculosis to First-Line Drugs in China

Background

The purpose of this study was to evaluate the performance of the BACTEC MGIT 960 (M960) system compared with the proportion method (PM) on Löwenstein-Jensen (L-J) medium in a peripheral laboratory in China for the testing of Mycobacterium tuberculosis (MTB) susceptibility to streptomycin (SM), isoniazid (INH) rifampicin (RIF) and ethambutol (EMB) a combination known as SIRE.

Methods

The susceptibility of 205 clinical isolates of MTB to SM, INH, RIF and EMB was performed with the M960 system. The drugs were tested at the following concentrations: 1.0 µg/ml for SM, 0.1 µg/ml for INH, 1.0 µg/ml for RIF, and 5.0 µg/ml for EMB. The results were compared with those obtained by the L-J PM. The L-J PM at an arbiter site was used to resolve any discordant results.

Results

The overall consistency was 96.6% and concordance values were 95.6% for SM, 97.6% for INH, 98.0% for RIF and 95.1% for EMB. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the M960 system for PM (the standard method) was 95.6%, 97.3%, 96.2% and 96.9% respectively, and the sensitivity were 93.3% for SM, 96.9% for INH, 97.4% for RIF and 94.6% for EMB, the specificity were 96.9% for SM, 98.2% for INH, 98.4% for RIF and 95.5% for EMB, the PPV were 94.6% for SM, 97.9% for INH, 97.4% for RIF and 94.6% for EMB, the NPV were 96.2% for SM, 97.3% for INH, 98.4% for RIF and 95.5% for EMB. The turnaround time with the M960 system (median 8.0 days, ranged from 5 to 14 days) was significantly shorter than that with the PM (28 days or 42 days).

Conclusion

There was a substantial degree of agreement between the two methods. The M960 system was a reliable and rapid method for SIRE susceptibility testing of tuberculosis in China.     

Detection of mutations associated with multidrug-resistant Mycobacterium tuberculosis clinical isolates

Antimicrobial resistance was studied in 100 Mycobacterium tuberculosis strains selected randomly from sputum cultures of newly diagnosed tuberculosis patients. Resistance of the isolates to rifampicin, isoniazid, and ethambutol was tested by both drug susceptibility testing (DST) and allele-specific PCR (AS-PCR). A total of 19 (19%) isolates were found resistant to at least one of the antituberculosis drugs investigated by PCR compared with 14 (14%) resistant isolates detected by DST. Eleven mutations were detected by AS-PCR in the rpoB gene (codons 516, 526, and 531), associated with rifampicin resistance, a marker of multidrug-resistant tuberculosis (MDR-TB), 14 mutations in the katG gene codon 315 that confers resistance to isoniazid, and nine mutations in the embB gene codon 306 that confers resistance to ethambutol. Mutations in the six multidrug-resistant isolates were confirmed by DNA sequencing. Results were compared with phenotypic DST data. Nineteen different mutation types to at least one of the drugs were found; six isolates (6%) were classified as MDR-TB, defined as resistance to at least rifampicin and isoniazid. The rates of concordance of the PCR with the phenotypic susceptibility test were 71.4, 54.5, and 44.4 for isoniazid, rifampicin, and ethambutol, respectively. These results highlight the importance of molecular epidemiology studies of tuberculosis in understudied regions with a tuberculosis burden to uncover the true prevalence of the MDR-TB.     

Bacteriostatic and bactericidal activity of antituberculosis drugs against Mycobacterium tuberculosis, Mycobacterium avium-Mycobacterium intracellulare complex and Mycobacterium kansasii in different growth phases.

Bacteriostatic and bactericidal activities of rifampicin, isoniazid, streptomycin, enviomycin and ethambutol against Mycobacterium tuberculosis, Mycobacterium avium--M. intracellulare complex and Mycobacterium kansasii were studied in different growth phases. Bacteriostatic activities of the drugs were similar in different growth phases, except isoniazid. M. tuberculosis was much less susceptible to isoniazid in the lag phase than in the log and the stationary phases. In contrast, bactericidal activity was influenced by the growth phase. M. tuberculosis was killed by isoniazid, streptomycin and rifampicin. The bactericidal activity of isoniazid was strongest. The bactericidal activity of isoniazid and streptomycin was most marked in the log phase. M. avium complex and M. kansasii resisted the bactericidal activity, but some strains of M. avium complex were killed by streptomycin and enviomycin, and the activities of these two drugs were most marked in the lag phase.     

Utility of nitrate reductase assay for detection of multidrug-resistant Mycobacterium tuberculosis in a low resource setting

Introduction. The performance of a drug susceptibility test may change when moving from the research stage to implementation on a population level in actual public health practice. Objective. The performance of a rapid drug susceptibility test was described for detecting multidrug-resistant Mycobacterium tuberculosis when implemented in the routine workflow of a low-resource reference laboratory. Materials and methods. A prospective study was done comparing the performance of the nitrate reductase assay with the conventional proportion method for rifampicin and isoniazid on 364 isolates were obtained from multidrug-resistant tuberculosis risk patients referred from diffrent Colombian laboratories. Results. When compared with the proportion method, the nitrate reductase assay sensitivity was 86.8% and 84.9% for rifampicin and isoniazid, respectively, whereas nitrate reductase assay specificity was 100% for isoniazid and rifampicin. Nitrate reductase assay sensitivity was significantly higher when the age of isolate was less than 70 days. A sensitivity of 94.4% dropped to 78.1% for rifampicin resistance for fresh and old isolates, respectively (Fisher exact test, p=0.05). For isoniazid resistance using fresh and old isolates, 94.7% vs.74.3% sensitivities, were achieved (chi square test, p=0.03). The proportion of nitrate reductase assay ambiguous results was significantly higher in multidrug-resistant than in non-multidrug-resistant isolates (17.6% vs. 4.0%, chi square test, p<0.005). Conclusions. The nitrate reductase assay demonstrated provided reliable results for antibiotic resistance. However, using old cultures leds to a higher proportion of false sensitive results; furthermore, the nitrate reductase assay capability to detect multidrug-resistant tuberculosis decreased due to a higher proportion of non-interpretable results.     

Photochromogenic and scotochromogenic mycobacteria: their clinical significance

In the period 1973--1977, Mycobacterium tuberculosis was isolated by cultivation in 4408 cases from the clinical specimens of patients with positive X-ray findings. On the basis of atypical colony morphology or pigment formation, 263 other mycobacterial strains were identified: of these 23 were photochromogenic and belonged to Mycobacterium kansasii. The strains were cultured on several occasions from the specimens of 4 patients with broncho-pulmonary mycobacteriosis. The strains were resistant to isoniazid and streptomycin, sensitive to ethambutol and rifampicin. A total of 18 scotochromogenic isolates cultured from 14 patients with positive X-ray findings were identified as Mycobacterium aquae (M. gordonae) and its variants: strains showing slow Tween hydrolysis and 1 strain of rapid growth. In 5 cases M. tuberculosis was also obtained, indicating the presence of a mixed mycobacterial population. All scotochromogens were resistant to isoniazid and sensitive to ethambutol, with the exception of two strains sensitive to rifampicin.     

Low Resistance to First and Second Line Anti-Tuberculosis Drugs among Treatment Naive Pulmonary Tuberculosis Patients in Southwestern Uganda

BackgroundThere are limited data on region-specific drug susceptibility of tuberculosis (TB) in Uganda. We performed resistance testing on specimens collected from treatment-naive patients with pulmonary TB in Southwestern Uganda for first and second line anti-TB drugs. We sought to provide data to guide regional recommendations for empiric TB therapy.MethodsArchived isolates, obtained from patients at Mbarara Regional Referral Hospital from February 2009 to February 2013, were tested for resistance to isoniazid and rifampicin using the MTBDRplus and Xpert MTB/RIF assays. A subset of randomly selected isolates was tested for second line agents, including fluoroquinolones (FQs), aminoglycosides, cyclic peptides, and ethambutol using the MTBDRsl assay. We performed confirmatory testing for FQ resistance using repeated MTBDRsl, the Mycobacteria growth indicator tube (MGIT) assay, and sequencing of the gyrA and gyrB genes.ResultsWe tested isolates from 190 patients. The cohort had a median age of 33 years (IQR 26-43), 69% (131/190) were male, and the HIV prevalence was 42% (80/190). No isolates (0/190) were rifampicin-resistant and only 1/190 (0.5%) was isoniazid-resistant. Among 92 isolates tested for second-line drug resistance, 71 (77%) had interpretable results, of which none were resistant to aminoglycosides, cyclic peptides or ethambutol. Although 7 (10%) initially tested as resistant to FQs by the MTBDRsl assay, they were confirmed as susceptible by repeat MTBDRsl testing as well as by MGIT and gyrase gene sequencingConclusionWe found no MDR-TB and no resistance to ethambutol, FQs, or injectable anti-TB drugs in treatment naïve patients with pulmonary TB in Southwestern Uganda. Standard treatment guidelines for susceptible TB should be adequate for most patients with TB in this population. Where possible, molecular susceptibility testing methods should be routinely validated by culture methods.     

Antituberculotic drug resistance in a Turkish state hospital with national data

Antituberculosis drug resistance is a major factor threatening the success of tuberculosis control programmes. The aim of this study was to reveal the patterns of antituberculosis drug resistance in a secondary hospital in Turkey and to compare with national data. The results of BACTEC MGIT 960 system for susceptibility testing were retrospectively analysed on 76 clinical Mycobacterium tuberculosis complex isolates from different patients. The mean age of 48 men (63.2%) and 28 women was 37 and 39, respectively. Overall resistance rate to isoniazid was 14.5%, followed by streptomycin 9.2%, ethambutol 6.9% and rifampin 5.3%. Female sex and diabetes mellitus but not the presence of cavitary lesion or radiological involvement was a risk factor for the development of drug resistance. Anemia, leukocytosis or thrombocytosis was not associated with the drug resistance. In conclusions, further studies should be conducted regularly to monitor drug resistance in Turkey in order to manage effectively national tuberculosis control efforts.     

Proficiency testing of conventional drug susceptibility tests of Mycobacterium tuberculosis

Proficiency testing of indirect drug susceptibility tests of Mycobacterium tuberculosis was begun in 1985 by the Laboratory Centre for Disease Control (LCDC) with the participation of Provincial Public Health Laboratories in Canada. Comparable sets of 60 cultures of Mycobacterium tuberculosis representing 30 strains were distributed by LCDC to the participating laboratories to be tested for drug susceptibility against isoniazid, streptomycin, rifampin, and ethambutol using conventional methodologies. Intralaboratory agreement values determined by comparing results obtained on sets of duplicate cultures were high and were found to vary little from drug to drug and from laboratory to laboratory. Interlaboratory agreement was determined by comparing results reported by participating laboratories to those obtained by the Reference Laboratory. Agreement percentages were found to be lower for drug-resistant cultures than for drug-susceptible cultures. The reliability of drug susceptibility testing results was higher for isoniazid and rifampin, than for ethambutol and streptomycin. This study shows that the higher subsidiary drug concentrations do not compare well with main drug concentrations, especially in the case of streptomycin and ethambutol. The significance of the higher subsidiary concentrations in in vitro susceptibility testing is therefore in need of clarification. The proficiency testing results obtained in this study compare favorably with those reported in other developed countries despite the fact that a variety of testing procedures are used throughout the country.     

Modified inoculum for radiometric susceptibility testing ofMycobacterium tuberculosis

Two-hundred and fifteen isolates ofMycobacterium tuberculosis were evaluated with the BACTEC 460 radiometric method for susceptibility to isoniazid, rifampin, ethambutol, and streptomycin (SM); a revised protocol for inoculum preparation was used. Fresh clinical isolates were subcultured into 7H9 broth and then photometrically adjusted to the equivalent of a 0.5 McFarland standard, one-half the recommended inoculum density. This method produced an overall 98.3% correlation with a conventional agar method. The sensitivity of this procedure was good for all drugs tested except for the lowest concentration of SM (2 g/ml). Specificity was excellent for all drugs tested. After repeat testing, only four discrepancies were found, yielding a 99.8% correlation between the two systems. The time required for susceptibility tests averaged 4.6 days. This method for inoculum preparation effectively minimized the number of susceptibility tests exceeding the threshold value before the fourth day of incubation. This allowed for definite trends of the growth index values to become established before interpretation of results.     

Comparative evaluation of the microplate nitrate reductase assay and the rezasurin microtitre assay for the rapid detection of multidrug resistant Mycobacterium tuberculosis clinical isolates

The microplate nitrate reductase assay (MNRA) and the rezasurin microtitre assay (REMA) were used for the susceptibility testing of 73 clinical isolates and the results were compared with those that were obtained using the Bactec 460 TB and Bactec MGIT 960 systems. The REMA and the MNRA were performed in 96-well plates. For the REMA, the concentrations of isoniazid (INH) and rifampicin (RIF) ranged from 1.0-0.01 μg/mL and 2.0-0.03 μg/mL, respectively. For the MNRA, the INH concentration was between 1.0-0.03 μg/mL and the RIF concentration was between 2.0-0.06 μg/mL. For the MNRA, the sensitivity, specificity, positive predictive value, negative predictive value and INH/RIF agreement were 100/95.6, 97.6/100, 96.8/100, 100/98 and 98.6/98.6, respectively, and for the REMA, they were 100/91.3, 90.4/100, 88.5/100, 100/96.1 and 94.5/97.2, respectively. Our data suggest that these two rapid, low-cost methods may be inexpensive, alternative assays for the rapid detection of multidrug resistant tuberculosis in low-income countries.