Nitrate Reductase Assay for Rapid Determination of Methicillin-Resistant Staphylococcus aureus Clinical Isolates.

OBJECTIVE: To evaluate the performance of nitrate reductase assay (NRA), a rapid, colorimetric method for the determination of methicillin resistance in Staphylococcus aureus isolates obtained from the culture collection of the Akdeniz University Hospital Central Laboratory, Antalya, Türkiye. MATERIALS AND METHODS: Identification for all 290 S aureus isolates at the species level was performed via matrix-assisted laser desorption/ionization-time of flight. Isolates were tested with NRA for methicillin resistance. The cefoxitin broth microdilution (BMD) method recommended by the Clinical and Laboratory Standards Institute was used as the reference method in the study. S aureus ATCC 29213 and S aureus ATCC 43300 strains were used for quality control. RESULTS: According to Food and Drug Administration criteria, the category agreement between NRA and BMD was found to be 100%. The essential agreement between both methods was determined to be 96.20%. There is no minor, major, or extremely major discrepancy between both methods. CONCLUSION: The results show that NRA is a rapid, practical, and reliable colorimetric method for detecting MRSA.

Genotype analysis of ofloxacin-resistant multidrug-resistant Mycobacterium tuberculosis isolates in a multicentered study from India.

Background & objectives: Drug resistance surveillance offers useful information on trends of drug resistance and the efficacy of control measures. Studies and reports of drug-resistant mutations and phenotypic assays thus become important. This study was conducted to investigate the molecular characteristics of ofloxacin (OFX)-resistant, multidrug-resistant tuberculosis (MDR-TB) isolates from different geographical regions of India and their association with strains of different genotypes. Further, the nitrate reductase assay (NRA) was tested against Mycobacteria Growth Indicator Tube(MGIT) for the determination of OFX resistance as an alternative and cost-effective method. Methods: A total of 116 Mycobacterium tuberculosis isolates were used to assess the mutations in the gyrA, gyrB genes and resistance levels to OFX. Mutational analysis in gyrA and gyrB genes and genotype analysis of M. tuberculosis isolates was done by gene-specific polymerase chain reaction (PCR) followed by DNA sequencing and spoligotyping, respectively. Results: Three (6.25%), 12 (44.44%) and 12 (29.27%) MDR-TB isolates from western, northern and southern India, respectively, were found to be OFX-resistant MDR-TB isolates. OFX resistance was observed to be significantly higher in MDR-TB cases for all study regions. Beijing genotypes from northern India were observed to be associated with OFX-resistant MDR-TB cases (P <0.05). Among 35 (30.15%) phenotypically OFX-resistant isolates, 22 (62.86%) had mutations in the gyrA gene and two (5.71%) isolates had mutations in the gyrB gene. Interpretation & conclusions: These results caution against the PCR-based prediction of OFX resistance patterns and highlight the need for searching other genetic loci for the detection of mutations conferring resistance to OFX in M. tuberculosis. Our study also showed the usefulness of NRA as an alternative method to detect OFX resistance.

[Experience of using TB test kit for the rapid drug susceptibility testing of M. tuberculosis.]

The results of the comparative testing of the susceptibility of M. tuberculosis clinical strains to isoniazid, streptomycin, rifampicin and ethambutol using the TB test kit, developed in SCRAMB, (Obolensk) and the absolute concentrations method; the TB test kit and the BACTEC MGIT 960 automated system are presented in the study. A total of 629 and 220 strains, respectively, were tested. A high degree of agreement of the results was shown: 89.1-98.6% for isoniazid, 96.2-98.0% for rifampicin, 91.5-98.2% for streptomycin and 89.1-95.9% for ethambutol. The smallest number of discrepancies in the results was obtained when comparing the TB test kit and BACTEC MGIT 960. The discrepant results analysis was performed by the proportion method, PCR sequencing, or re-testing on new lots of the TB test kit and Lowenstein-Jensen medium with anti-tuberculosis drugs, after which the sensitivity, the specificity and the efficiency of the TB test kit have exceeded 95 % for all anti-tuberculosis drugs. The turnaround time with the TB test kit (median 9.25-9.9 days, ranged from 8 to13 days) was significantly shorter than that with the absolute concentration method (median 21-23 days, ranged from 20 to 28 days) and is commensurate with the turnaround time with BACTEC MGIT 960 (average 7.2 days, ranged from 5 to 12 days). The TB test kit is easy to use, does not require expensive equipment and special staff training.

Evaluation of nitrate reductase assay in 7H11 agar for diagnosis of multi-drug-resistant tuberculosis in eastern Nepal.

BACKGROUND: Emergence of multi-drug-resistant tuberculosis is a serious challenge for successful global tuberculosis control. Early diagnosis of drug-resistant tuberculosis by direct nitrate reductase assay (NRA) aids in appropriate treatment and reduction in disease transmission, particularly in countries with high tuberculosis burden. The aim of this study was to evaluate the performance of NRA for direct detection of resistance to rifampicin and isoniazid in Mycobacterium tuberculosis in laboratories with limited resources. METHODS: Fifty-eight new smear-positive sputum samples were processed as per the guidelines of revised national tuberculosis control program, India. The performance of NRA on middlebrook 7H11 agar was evaluated for detection of rifampicin and isoniazid resistance directly on smear-positive sputum specimens, and the results were compared with conventional proportion method. Sensitivity and specificity of the test were compared with the gold standard proportion method. Mc Nemar chi-square test was used to find out the significant difference between two methods. RESULTS: Direct NRA for detection of rifampicin resistance was 85.7% sensitive and 100% specific, whereas sensitivity and specificity of isoniazid resistance were 87.5% and 100%, respectively. Agreement between NRA and proportion method was 98% for both the drugs. The mean days of drug susceptibility testing results were 19.3 days for NRA and 72 days for conventional proportion method. The results of NRA were available in 21 days for 83% of the samples. CONCLUSIONS: Direct NRA on middlebrook 7H11 medium is a highly sensitive, reliable, and significantly faster method to perform drug susceptibility testing. It has the potential to be implemented for rapid detection of multi-drug-resistant tuberculosis against insufficient resources.

A comparative study of phenotypic and genotypic first- and second-line drug resistance testing of Mycobacterium tuberculosis.

This study aimed to evaluate the frequency of resistance to first- and second-line drugs using phenotypic and genotypic methods and its correlation with resistance-linked mutations in Mycobacterium tuberculosis (M. tb) isolated in Iran. Three different methods, including the indirect proportion method(PM), direct and indirect nitrate reductase assay(NRA), and direct sequencing were used to assess drug resistance. In this study, sensitivity, specificity, agreement, costs, and turnaround time of these methods were compared in 395 smear positive isolates. Compared to the PM, the NRA and the direct sequencing methods demonstrated higher specificity, sensitivity, and agreement for detection of all anti-tuberculosis drugs. The NRA had a short turnaround time and was more cost-effective than the other methods. Mutations in codon 531 in rpoB, 315 in katG, 18 in rpsL, and 306 in embB were associated with high-level resistance to the first-line drugs, and mutations in codon 94 in gyrA, and A1401G in rrs were correlated with resistance to the second-line drugs. We found that the NRA is a highly sensitive, specific, inexpensive, and rapid test with strong potential to be a useful and interesting alternative tool, particularly in low-income countries. In addition, these molecular data will be helpful for developing new molecular methods for detecting first- and second-line drug-resistant M. tb.

Rapid drug-susceptibility testing of Mycobacterium tuberculosis clinical isolates to first-line antitubercular drugs by nitrate reductase assay: A comparison with proportion method.

OBJECTIVE/BACKGROUND: Early initiation of therapy in patients with tuberculosis is imperative for its control. Conventional methods of susceptibility testing such as the proportion method (PM) require visual detection and counting of colonies that takes up to 6weeks. Rapid and simple phenotypic methods that have been endorsed by the World Health Organization can serve as alternatives. METHODS: In this study, we evaluated the colorimetric nitrate reductase assay, which utilizes the detection of nitrate reduction as an indicator of growth much earlier compared with PM (within 7-14days). The susceptibility of 75 clinical isolates of Mycobacterium tuberculosis to four first-line antitubercular drugs was tested by nitrate reductase assay and compared with the standard PM. In this assay, inoculation was done on both drug-free and drug-containing Löwenstein-Jensen medium containing sodium nitrate. After incubation for 7-14days, reduction to nitrite was taken as an indicator of growth, which was detected by color change on addition of Griess reagent. RESULTS: Agreement between nitrate reductase assay and PM was 100% for rifampicin, 97.30% for isoniazid, 93.30% for streptomycin, and 98.60% for ethambutol. Cost/isolate with this assay was found to be approximately two times lesser than that of PM. All results were obtained in 7-14days by nitrate reductase assay, which was significantly rapid compared with 42days taken for obtaining results by PM. CONCLUSION: Nitrate reductase assay can be used as a rapid and inexpensive method for drug-susceptibility testing of M. tuberculosis for first-line antitubercular drugs without compromising accuracy of standard methods.

A Low Cost/Low Power Open Source Sensor System for Automated Tuberculosis Drug Susceptibility Testing.

In this research an open source, low power sensor node was developed to check the growth of mycobacteria in a culture bottle with a nitrate reductase assay method for a drug susceptibility test. The sensor system reports the temperature and color sensor output frequency change of the culture bottle when the device is triggered. After the culture process is finished, a nitrite ion detecting solution based on a commercial nitrite ion detection kit is injected into the culture bottle by a syringe pump to check bacterial growth by the formation of a pigment by the reaction between the solution and the color sensor. Sensor status and NRA results are broadcasted via a Bluetooth low energy beacon. An Android application was developed to collect the broadcasted data, classify the status of cultured samples from multiple devices, and visualize the data for the end users, circumventing the need to examine each culture bottle manually during a long culture period. The authors expect that usage of the developed sensor will decrease the cost and required labor for handling large amounts of patient samples in local health centers in developing countries. All 3D-printerable hardware parts, a circuit diagram, and software are available online.

Evaluation of four colourimetric susceptibility tests for the rapid detection of multidrug-resistant Mycobacterium tuberculosisisolates

The purpose of this study is to evaluate four rapid colourimetric methods, including the resazurin microtitre assay (REMA), malachite green decolourisation assay (MGDA), microplate nitrate reductase assay (MNRA) and crystal violet decolourisation assay (CVDA), for the rapid detection of multidrug-resistant (MDR) tuberculosis. Fifty Mycobacterium tuberculosisisolates were used in this study. Eighteen isolates were MDR, two isolates were only resistant to isoniazid (INH) and the remaining isolates were susceptible to both INH and rifampicin (RIF). INH and RIF were tested in 0.25 µg/mL and 0.5 µg/mL, respectively. The agar proportion method was used as a reference method. MNRA and REMA were performed with some modifications. MGDA and CVDA were performed as defined in the literature. The agreements of the MNRA for INH and RIF were 96% and 94%, respectively, while the agreement of the other assays for INH and RIF were 98%. In this study, while the specificities of the REMA, MGDA and CVDA were 100%, the specificity of the MNRA was lower than the others (93.3% for INH and 90.9% for RIF). In addition, while the sensitivity of the MNRA was 100%, the sensitivities of the others were lower than that of the MNRA (from 94.1-95%). The results were reported on the seventh-10th day of the incubation. All methods are reliable, easy to perform, inexpensive and easy to evaluate and do not require special equipment.

New Diagnostics for Childhood Tuberculosis.

The challenge of diagnosing childhood tuberculosis (TB) results from its paucibacillary nature and the difficulties of sputum collection in children. Mycobacterial culture, the diagnostic gold standard, provides microbiological confirmation in only 30% to 40% of childhood pulmonary TB cases and takes up to 6 weeks to result. Conventional drug susceptibility testing requires an additional 2 to 4 weeks after culture confirmation. In response to the low sensitivity and long wait time of the traditional diagnostic approach, many new assays have been developed. These new tools have shortened time to result; however, none of them offer greater sensitivity than culture.

Utilidad del método de la nitrato reductasa para la detección de resistencia a drogas antituberculosas de segunda línea / Usefulness of the nitrate reductase method to detect resistance to second-line anti-tuberculosis drugs

Introducción: el creciente hallazgo de cepas de Mycobacterium tuberculosis multidrogorresistentes extremadamente resistentes ratifica la importancia de ofrecer, de forma rápida, los resultados de susceptibilidad de M. tuberculosis a drogas de primera y segunda línea como única alternativa para evitar la transmisión. Objetivo: comparar el método de la nitrato reductasa y el de las proporciones para la detección de susceptibilidad a drogas antituberculosas de segunda línea en aislamientos clínicos de M. tuberculosis, recuperados de pacientes cubanos con tuberculosis multidrogorresistente. Métodos: se investigó, mediante el método de las proporciones en Löwenstein-Jensen y el de la nitrato reductasa, la susceptibilidad a la ofloxacina, la kanamicina y a la capreomicina en 34 aislamientos de M. tuberculosis multidrogorresistentes. Resultados: en tres aislamientos se evidenció un comportamiento extremadamente resistente por ambos métodos. Mediante el método de la nitrato reductasa los resultados estuvieron disponibles entre 7 y 14 días. La sensibilidad fue de 100 por ciento, 90,0 por ciento y 77,8 por ciento para la ofloxacina, la kanamicina y la capreomicina, respectivamente, mientras que la especificidad fue superior al 95,0 por ciento y el valor de kappa fue superior a 0,85 para las tres drogas. Conclusión: de acuerdo con los resultados alcanzados, consideramos que el método de la nitrato reductasa constituye una valiosa alternativa para la detección oportuna de tuberculosis extremadamente resistente en países con limitados recursos económicos(AU)

Introduction: the increase of multidrug resistant and extensively drug resistant tuberculosis underlines the urgent need to obtain early results of Mycobacterium tuberculosis susceptibility both to first and second line antituberculosis drugs in order to avoid dissemination of resistant isolates. Objective: the aim of this research was to compare the performance of the nitrate reductase assay and the proportion method for to detect the susceptibility to second line antituberculosis drugs in multidrug resistant clinical isolates of M. tuberculosis. Methods: the susceptibility to ofloxacin, kamamycin and capreomycin of 34 M. tuberculosis multidrug resistant isolates was investigated using the proportion method in Löwenstein-Jensen and the nitrate reductase assay. Results: three isolates were identified as extensively drug resistant by both methods. The results of the nitrate reductase assay were obtained between 7-14 days achieving 100 percent, 90.0 percent and 77.8 percent of sensitivity for ofloxacin, kamamycin and capreomycin, respectively while specificity was higher than 95.0 percent and kappa value was higher to 0,85 for all drugs. Conclusion: the nitrate reductase assay represents a useful tool for the rapid identification of extensively drug resistant tuberculosis in low resources setting(AU)

Detection of drug resistant Mycobacterium tuberculosis among patients with and without HIV infection in a rural setting / Detección de Mycobacterium tubersulosis resistente a los antibióticos entre pacientes con y sin infección por VIH en un área rural

OBJECTIVE: To analyse the sensitivity of Mycobacterium tuberculosis by nitrate reductase assay (NRA) and the Hain molecular line probe assay (LPA) in sputa of tuberculosis (TB)/HIV co-infected patients in Guyana. DESIGN: Sputum samples were collected from known TB patients at Georgetown Chest Clinic and were analysed at the Reference Laboratory, Guyana, over the period April 2010 to April 2011. RESULTS: Both methods recorded greater sensitivity for rifampin (RIF) than of isoniazid (INH). Both methods detected four RIF resistant, two INH resistant and two multi-drug resistant (MDR) strains and they had greater negative agreement indices than positive agreement indices. CONCLUSION: It was established that the sensitivity of Mycobacterium tuberculosis by the NRA and Hain LPA in TB/HIV co-infected patients has acceptable correlation and that HIV infection does not affect drug susceptibility testing.

OBJETIVO: Analizar la sensibilidad de Mycobacterium tuberculosis por medio del ensayo de nitrato reductasa (NRA) y el ensayo de sonda lineal (LPA) molecular de Hain en esputos de pacientes co-infectados TB/VIH en Guyana. DISEÑO: Muestras de esputo de pacientes de la Clínica del Tórax en Georgetown diagnosticados con tuberculosis, fueron analizadas en el Laboratorio de Referencias, en Guyana, en el período de abril de 2010 a abril de 2011. RESULTADOS: Ambos métodos registraron una mayor sensibilidad a la rifampicina (RIF) que a la isoniacida (INH). Ambos métodos detectaron cuatro cepas resistentes a RIF, dos resistentes a INH, y dos resistentes a mútiples medicamentos (RMM). Asimismo, presentaban mayores índices de concordancia negativa que de concordancia positiva. CONCLUSIÓN: Se estableció que la sensibilidad de Mycobacterium tuberculosis por medio del ensayo de NRA y el LPA de Hain en pacientes co-infectados TB/VIH, guarda una correlación aceptable, y que la infección por VIH no afecta la prueba de susceptibilidad a los medicamentos.

Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis

We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB.

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.

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 percent and 94 percent, 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.

Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis.

We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB.

A rapid detection of multidrug-resistant Mycobacterium tuberculosis by a nitrate reductase assay on blood agar

The susceptibility of 49 Mycobacterium tuberculosis clinical isolates to isoniazid (INH) and rifampisin (RIF) (28 multi-drug resistant-tuberculosis samples) was determined by a nitrate reductase assay (NRA) on blood agar. Agreement between the NRA and other testing methods was found to be 93.8 percent for both INH and RIF. The sensitivity, specificity, positive predictive value and negative predictive value for INH were 92.8 percent, 94.2 percent, 86.6 percent and 97 percent, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for RIF were 90.4 percent, 96.4 percent, 95 percent and 93.1 percent. In conclusion, we show here that blood agar can be used effectively for the NRA test.

Comparative evaluation of the Nitrate Reductase Assay and the Resazurin Microtitre Assay for drug susceptibility testing of Mycobacterium tuberculosis against first line anti-tuberculosis drugs

Tuberculosis remains as a serious infection disease of worldwide distribution, with high morbidity and mortality, mainly in low socio-economic condition countries. The state of emergency of tuberculosis caused by the resistant and multidrug-resistant (MDR) strains, became the main threat to the tuberculosis treatment and control programs. A fast detection method for the resistant strains will allow the implementation of an adequate treatment and contribute for controlling the dissemination of these resistant strains. This study evaluated the performance of the nitrate reductase assay in solid (NRA-LJ) and liquid (NRA-7H9) media, to determine the susceptibility to first line anti-tuberculosis drugs: isoniazid (INH), rifampicin (RMP), ethambutol (EMB) and streptomycin (SMR). Both methods NRA-LJ and NRA-7H9 were evaluated among 18 strains with a known susceptibility profile. The resazurin microtiter assay (REMA) was performed as a reference method. One hundred percent of accordance was observed between NRA-7H9 and REMA for the four tested drugs. When the NRA-LJ method was compared to REMA, the sensitivity and the specificity to INH, RMP, EMB and SMR were 100 percent, 100 percent, 85.7 percent, 76.9 percent and 80 percent, 100 percent, 75 percent and 80 percent, respectively. From the 57 clinical isolates of M. tuberculosis evaluated by NRA-7H9 and REMA, 56 (98.2 percent) were sensitive to all antibiotics tested (INH, RMP, EMB and SMR) by the NRA-7H9 method, while three of these strains were resistant to INH by REMA. One strain showed resistance to INH and RMP for both methods, and MIC of 1.0 µg/ml to INH for both methods, while MIC of 1.0 and 2.0 µg/ml to RMP for REMA and NRA-7H9, respectively. The three assays showed a high level of agreement for rapid detection of rifampicin and isoniazid resistance. Regarding rapidness, the detection of color change in the NRA method is within instants as compared to the overnight incubation required...

A tuberculose permanece como uma séria doença infecciosa, com distribuição mundial, alta morbidade e mortalidade, ocorrendo principalmente em paises com baixa condição econômica. O estado de emergência da tuberculose causada por cepas resistentes e multirresistentes tornou-se uma importante ameaça para o tratamento e programas de controle da tuberculose. Uma rápida detecção de cepas resistentes permitirá a implantação de um tratamento adequado e contribuirá para controlar a disseminação destas cepas. Este estudo avaliou a performace do ensaio nitrato redutase em meio sólido (NRA-LJ) e meio líquido (NRA-7H9), para determinar a sensibilidade frente aos fármacos antituberculosos de primeira linha: isoniazida (INH), rifampicina (RMP), etambutol (EMB) and estreptomicina (SMR). Ambos os métodos, NRA-LJ e NRA-7H9, foram avaliados com 18 cepas com conhecido perfil de sensibilidade. O ensaio de microplaca com resazurina (REMA) foi utilizado como método de referência. A concordância observada entre NRA-7H9 and REMA foi de 100 por cento para os quatro fármacos testados. Quando o método NRA-LJ foi comparado com o REMA, a sensibilidade e especificidade para INH RMP e SMR foram de 100 por cento, 100 por cento, 85,7 por cento, 76,9 por cento e 80 por cento, 100 por cento, 75 por cento and 80 por cento, respectivamente. Dos 57 isolados clinicos de M. tuberculosis avaliados por NRA-7H9 e REMA, 56 (98.2 por cento) foram sensíveis a todos antibióticos testados (INH, RMP, EMB e SMR) pelo método NRA-7H9, enquanto três destas cepas foram resistentes para INH pelo REMA. Uma cepa mostrou resistência para INH e RMP por ambos os métodos, e CMI de 1,0 µg/ml para INH para ambos os métodos, enquanto CMI de 1,0 e 2,0 µg/ml para RMP pelo REMA e NRA-7H9, respectivamente. Os três ensaios mostraram um alto nível ded concordância para uma rápida detecção de resistência a rifampicina e isoniazida. Com relação à rapidez na obtenção dos resultados, a detecção na mudança de...

Comparative evaluation of the Nitrate Reductase Assay and the Resazurin Microtitre Assay for drug susceptibility testing of Mycobacterium tuberculosis against first line anti-tuberculosis drugs.

Tuberculosis remains as a serious infection disease of worldwide distribution, with high morbidity and mortality, mainly in low socio-economic condition countries. The state of emergency of tuberculosis caused by the resistant and multidrug-resistant (MDR) strains, became the main threat to the tuberculosis treatment and control programs. A fast detection method for the resistant strains will allow the implementation of an adequate treatment and contribute for controlling the dissemination of these resistant strains. This study evaluated the performance of the nitrate reductase assay in solid (NRA-LJ) and liquid (NRA-7H9) media, to determine the susceptibility to first line anti-tuberculosis drugs: isoniazid (INH), rifampicin (RMP), ethambutol (EMB) and streptomycin (SMR). Both methods NRA-LJ and NRA-7H9 were evaluated among 18 strains with a known susceptibility profile. The resazurin microtiter assay (REMA) was performed as a reference method. One hundred percent of accordance was observed between NRA-7H9 and REMA for the four tested drugs. When the NRA-LJ method was compared to REMA, the sensitivity and the specificity to INH, RMP, EMB and SMR were 100%, 100 %, 85.7%, 76.9% and 80%, 100%, 75% and 80%, respectively. From the 57 clinical isolates of M. tuberculosis evaluated by NRA-7H9 and REMA, 56 (98.2%) were sensitive to all antibiotics tested (INH, RMP, EMB and SMR) by the NRA-7H9 method, while three of these strains were resistant to INH by REMA. One strain showed resistance to INH and RMP for both methods, and MIC of 1.0 µg/ml to INH for both methods, while MIC of 1.0 and 2.0 µg/ml to RMP for REMA and NRA-7H9, respectively. The three assays showed a high level of agreement for rapid detection of rifampicin and isoniazid resistance. Regarding rapidness, the detection of color change in the NRA method is within instants as compared to the overnight incubation required for the REMA test. NRA might represent an inexpensive and alternative assay for rapid detection of resistance in low-income countries.

Nitrate reductase assay for detection of drug resistance in Mycobacterium tuberculosis: simple and inexpensive method for low-resource laboratories / s. t

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 percent. 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(AU)