MODS assay for the diagnosis of TB.
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Bacteriological Techniques↗
Biomedical subjects
Anandi Martin
Publications and source records attributed to Anandi Martin.
Colorimetric redox-indicator methods for the rapid detection of multidrug resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis.
OBJECTIVES: With the spread of multidrug-resistant tuberculosis (MDR-TB) there is increasing demand for new accurate and cost-effective tools for rapid drug susceptibility testing (DST), particularly for developing countries. The reference standard method used today for DST is very slow and cumbersome. Colorimetric assays using redox indicators have been proposed to be used in low-resource countries as rapid alternative culture methods for the detection of resistance especially to rifampicin and isoniazid. These methods appear as promising new tools but their accuracy has not been systematically evaluated. METHODS: We did a meta-analysis to evaluate the accuracy of the colorimetric assays for the detection of rifampicin and isoniazid-resistant tuberculosis among clinical isolates. We searched Medline, PubMed (NCBI), Global health-CAB, EJS-E (EbscoHost), ISI Web, Web of Science and IFCC databases and contacted authors if additional information was needed. RESULTS: Eighteen studies met our inclusion criteria for rifampicin resistance detection and 16 for isoniazid. We used a summary receiver operating characteristic (SROC) curve to perform meta-analysis and summarize diagnostic accuracy. For both drugs, all studies had a sensitivity and specificity that ranged between 89% and 100%. CONCLUSIONS: There is evidence that colorimetric methods are highly sensitive and specific for the rapid detection of MDR-TB. These new tools could offer affordable technologies for TB laboratories especially in places where resources are limited and where the prevalence of MDR-TB is important and make TB control efforts more effective. Additional studies are needed in high MDR prevalence countries and cost-effectiveness analysis to have more evidence on the utility of these methods. Future developments to detect resistance directly from smear-positive sputum specimens should be taken into consideration to speed up the process.
Antitubercular Agents↗
A new rapid and simple colorimetric method to detect pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide.
OBJECTIVES: The purpose of this study was to develop and assess a rapid method for pyrazinamide resistance detection in Mycobacterium tuberculosis using nicotinamide in a colorimetric resazurin assay. METHODS: We have tested M. tuberculosis isolates using nicotinamide in a 96-well format with the redox indicator resazurin (REMA) and compared results using the BACTEC 460-TB system with two concentrations of pyrazinamide (100 and 300 mg/L), as well as the Wayne method for detecting pyrazinamidase activity. Mutations in the pncA gene were detected by DNA sequencing of the pyrazinamide-resistant strains. RESULTS: Out of 95 clinical isolates of M. tuberculosis tested, 25 were determined to be resistant by the Wayne, BACTEC (300 mg/L), and the REMA nicotinamide methods. Using a nicotinamide MIC>250 mg/L as the cut-off for defining resistance, only one strain was falsely labelled as resistant. The REMA nicotinamide assay demonstrated a sensitivity of 100% and a specificity of 98%. The BACTEC (100 mg/L) falsely classified 8 strains as resistant. DNA sequencing detected mutations in 18/22 of the pncA genes from pyrazinamide-resistant strains. CONCLUSIONS: The REMA plate using nicotinamide to detect resistance to pyrazinamide is a simple and rapid method that could be useful in limited-resource countries.
Amidohydrolases↗
Evaluation of phage assay for rapid phenotypic detection of rifampicin resistance in Mycobacterium tuberculosis.
BACKGROUND: Conventional methods for susceptibility testing require several months before results can be reported. However, rapid methods to determine drug susceptibility have been developed recently. Phage assay have been reported as a rapid useful tools for antimicrobial susceptibility testing. The aim of this study was to apply the Phage assay for rapid detection of resistance on Mycobacterium tuberculosis strains in Cuba. METHODS: Phage D29 assay was performed on 102 M. tuberculosis strains to detect rifampicin resistance. The results were compared with the proportion method (gold standard) to evaluate the sensitivity and specificity of Phage assay. RESULTS: Phage assay results were available in 2 days whereas Proportion Methods results were obtain in 42 days. A total of 44 strains were detected as rifampicin resistant by both methods. However, one strains deemed resistant by Proportion Methods was susceptible by Phage assay. The sensitivity and specificity of Phage assay were 97.8 % and 100% respectively. CONCLUSION: Phage assay provides rapid and reliable results for susceptibility testing; it's easy to perform, requires no specialized equipment and is applicable to drug susceptibility testing in low income countries where tuberculosis is a major public health problem.
Antibiotics, Antitubercular↗
Application of the resazurin microtitre assay for detection of multidrug resistance in Mycobacterium tuberculosis in Algiers.
This study assessed the performance of a rapid, low-cost, colorimetric method, the resazurin microtitre assay (REMA) plate method, for the detection of resistance to isoniazid and rifampicin in 136 clinical isolates of Mycobacterium tuberculosis from two hospitals in Algiers. MICs were determined and the results were compared with those obtained with the conventional proportion method on Löwenstein-Jensen medium. Excellent results were obtained for the REMA plate method, with a sensitivity of 100 % for both isoniazid and rifampicin and a specificity of 98.3 and 99.2 %, respectively. The REMA plate method appears to be a reliable method for the rapid determination of multidrug-resistant tuberculosis and is a good alternative for use in resource-limited countries such as Algeria.
Algeria↗
Nitrate reductase assay for detection of drug resistance in Mycobacterium tuberculosis: simple and inexpensive method for low-resource laboratories.
The nitrate reductase assay (NRA) was used as an alternative method for detection of resistance to the first-line antituberculous drugs isoniazid, rifampicin, ethambutol and streptomycin. A total of 320 strains of Mycobacterium tuberculosis were studied and the results compared with the proportion method (PM) on Löwenstein-Jensen medium. The mean time to obtain results was 10 days and the overall agreement between the NRA and PM was 98.8 %. The NRA was easy to perform and represents a useful tool for rapid and accurate determination of drug-resistant M. tuberculosis strains in low-resource countries.
Antitubercular Agents↗
Comparative evaluation of the nitrate reduction assay, the MTT test, and the resazurin microtitre assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis.
OBJECTIVES: To evaluate the performance of three rapid low-cost methods for the detection of resistance to first-line drugs in Mycobacterium tuberculosis. METHODS: One hundred M. tuberculosis clinical isolates were tested by the nitrate reductase assay (NRA), the MTT test and the resazurin microtitre assay (REMA), and the results compared with those obtained with the gold standard proportion method (PM) on Lowenstein Jensen medium. RESULTS: The results using the three methods showed a good sensitivity and specificity between 94% and 100% for the detection of rifampicin and isoniazid resistance. Specificity for ethambutol and streptomycin using MTT and resazurin was low (58-89%). In contrast, NRA showed a good agreement for all first-line drugs tested. CONCLUSIONS: This study shows a high level of agreement of these three low-cost methods compared with the PM for rapid detection of rifampicin and isoniazid resistance. However, more standardization is needed for ethambutol and streptomycin using the MTT test and resazurin microtitre assay. The nitrate reductase assay might represent an inexpensive procedure for rapid detection of resistance to first-line drugs in low-resource countries.
Antitubercular Agents↗
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 Löwenstein-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.
Antitubercular Agents↗
In-house phage amplification assay is a sound alternative for detecting rifampin-resistant Mycobacterium tuberculosis in low-resource settings.
An in-house mycobacteriophage amplification assay for detecting rifampin-resistant Mycobacterium tuberculosis showed 100% sensitivity, 97.7% specificity, and 95.2% predictive value for resistance in a test of 129 isolates from a hot spot area of multidrug-resistant M. tuberculosis. The applicability of the test was demonstrated in the routine work flow of a low-resource reference laboratory.
Antibiotics, Antitubercular↗
Rapid detection of ofloxacin resistance in Mycobacterium tuberculosis by two low-cost colorimetric methods: resazurin and nitrate reductase assays.
We have evaluated the performance of two rapid, low-cost methods for the detection of ofloxacin (OFX) resistance with 95 Mycobacterium tuberculosis isolates from countries with high multidrug-resistant tuberculosis endemicity. Results obtained by nitrate reductase and resazurin assays showed 100% agreement with those of the proportion method on 7H11 agar using 2 mug of OFX/ml. We confirmed the resistance of all isolates found to be resistant to OFX by the Mycobacterium Growth Indicator Tube system, and complete agreement among all methods was observed. Nitrate reductase and resazurin assays are rapid, simple, low-cost methods and might become inexpensive alternative procedures for rapid detection of OFX resistance in low-resource countries.
Anti-Bacterial Agents↗
Profiling Mycobacterium ulcerans with hsp65.
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Bacterial Proteins↗
[Phenotypic and genotypic methods for detecting multidrug resistance in Mycobacterium tuberculosis].
BACKGROUND: Expeditious charactization of drug susceptibility in Mycobacterium tuberculosis is difficult and, calls for the design and evaluation of faster, cheaper and more effective new techniques. OBJECTIVE: The aim of the current study was to compare one genotypic and two phenotypic methods for rapid susceptibility detection of M. tuberculosis. MATERIAL AND METHODS: Twenty-one M. tuberculosis strains were evaluated by phenotypic methodologies of oxidation and reduction of Alamar blue and MTT in the presence of streptomycin, isoniazid, rifampin and ethambutol. In all tests, the proportion method was applied as the comparison standard. By means of receiver operative characteristic (ROC) analysis, the performance, predictive values and threshold values for all drugs were determined. In addition, the performance of PCR and reverse line blot hybridization in establishing predictive values for sensitivity and resistance were compared in contingency tables. RESULTS: The susceptibility patterns established by colorimetric techniques were obtained after seven days of incubation. The performances of these tests were excellent for all drugs-the areas under curves were >0.9, 100% of sensitivity (S) and specificity (E) >80%. The genotypic method of RFLP oligotyping detected multidrug resistance with S of 100% and E of 93%. Conclusion. The results indicated that Alamar blue, MTT and RFLP methodologies are rapid and useful tools for characterizing multidrug resistance in M. tuberculosis, particularly for those patients with high risk of developing multidrug resistant tuberculosis.
Antitubercular Agents↗
Multicenter evaluation of mycobacteria identification by PCR restriction enzyme analysis in laboratories from Latin America and the Caribbean.
The identification of mycobacterial species in clinical isolates is essential for making patient care decisions. Polymerase chain reaction (PCR) restriction enzyme analysis (PRA) is a simple and rapid identification method, based on amplification of 441 bp of the hsp65 gene and restriction with BstEII and HaeIII. As a contribution to the validation of PRA, a multicenter study was performed in eight laboratories located in Argentina, Brazil, Colombia, Chile, and Guadeloupe. Each laboratory received 18 coded isolates from the collection of the Institute of Tropical Medicine (Antwerp, Belgium), representing duplicates of nine laboratory strains: Mycobacterium terrae CIPT 140320001, Mycobacterium scrofulaceum CIPT 140220031, Mycobacterium flavescens ATCC 14474, Mycobacterium triviale ATCC 23292, Mycobacterium nonchromogenicum ATCC 19530, Mycobacterium chitae ATCC 19627, Mycobacterium abscessus ATCC 19977, Mycobacterium kansasii ATCC 12478, and Mycobacterium peregrinum ATCC 14467. A detailed protocol including amplification, enzymatic digestion, and gel preparation was provided to each laboratory. Two laboratories identified correctly all 18 (100%) isolates, one identified correctly 17 (94.5%), two identified 14 (77.7%), one identified 11 (61%), and two identified 8 (44.4%) isolates. Errors detected in laboratories with more than 77% accuracy were associated with electrophoresis running conditions and an unspecific amplicon produced by a single strain. Lower accuracy was mainly related to inappropriate use of DNA markers and insufficient training in interpretation of patterns. In conclusion, the PRA method was readily implemented in some Latin American and Caribbean laboratories of mycobacteria, but improvements in critical points, as gel running conditions and training in interpretiation of patterns, are needed in order to improve accuracy. In others, improvement in critical points is still necessary.
Bacterial Proteins↗
Rapid alternative methods for detection of rifampicin resistance in Mycobacterium tuberculosis.
OBJECTIVE: To study the performance of three rapid low cost methods for the detection of rifampicin resistance. METHODS: A panel of 20 coded Mycobacterium tuberculosis strains was tested blindly by the low cost methods: nitrate reductase, MTT and resazurin assays, and compared with the results obtained with the gold standard methods: the proportion method on Löwenstein-Jensen medium and the BACTEC TB 460 system. We have also tested two commercial tests: MGIT and INNO LiPA Rif.TB kit. RESULTS: Complete agreement was observed among all methods. CONCLUSION: These three simple methods might become inexpensive alternative procedures for rapid detection of rifampicin resistance in low-resource countries.
Antibiotics, Antitubercular↗
One-tube cell lysis and DNA extraction procedure for PCR-based detection of Mycobacterium ulcerans in aquatic insects, molluscs and fish.
The purpose of this study was to develop a simple procedure for cell lysis and DNA extraction for direct detection of Mycobacterium ulcerans in aquatic insects, gills and intestinal contents of fish, molluscs and human tissue samples using a nested PCR method specific for the insertion sequence IS2404. The simultaneous action of sodium N-lauroyl sarcosine, guanidinium isothiocyanate, chloroform and Tris-saturated phenol on mycobacteria, followed by a DNA purification method using mini-columns fitted with silica-cellulose membranes was successfully employed to extract DNA from cultured bacteria, environmental and human tissue samples. All specimens were collected from Buruli ulcer endemic regions. M. ulcerans DNA was detected in 11 of 57 aquatic insects, one of six molluscs and three of 15 fish, supporting the hypothesis that the fauna of major Buruli ulcer endemic foci in swampy terrain of tropical and subtropical regions can be a source of M. ulcerans infection.
Animals↗
A microplate indicator-based method for determining the susceptibility of multidrug-resistant Mycobacterium tuberculosis to antimicrobial agents.
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Antibiotics, Antitubercular↗
Resazurin microtiter assay plate testing of Mycobacterium tuberculosis susceptibilities to second-line drugs: rapid, simple, and inexpensive method.
The emergence of multidrug-resistant tuberculosis calls for new, rapid drug susceptibility tests. We have tested 150 Mycobacterium tuberculosis isolates against the second-line drugs ethionamide, kanamycin, capreomycin, ofloxacin, and para-aminosalicylic acid by the colorimetric resazurin microtiter assay and the proportion method. By visual reading, MICs were obtained after 8 days. A very good correlation between results by the colorimetric resazurin microtiter assay and the proportion method was obtained. The colorimetric resazurin microtiter assay is inexpensive, rapid, and simple to perform, and implementation of the assay is feasible for low-resource countries.
Antitubercular Agents↗
Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis.
A method for detecting multidrug-resistant Mycobacterium tuberculosis by using a reduction of resazurin is described. Eighty clinical isolates were evaluated against isoniazid and rifampin; results at 7 days were compared with those of the proportion method. Specificity and sensitivity were excellent. The method is simple, inexpensive, and rapid and might be used with other antituberculosis drugs.
Antitubercular Agents↗