Fast detection of Mycobacterium tuberculosis in culture-positive sputum samples by nitrate reductase activity

ABSTRACT Microscopy and bacterial culture are the main tools in the diagnosis of tuberculosis. Since the slow growth of Mycobacterium tuberculosis impairs rapid diagnosis strategies, especially in countries where the latter are the only available resources, the ongoing development of new and inexpensive tools based on mycobacterial metabolism optimizing growth detection with preliminary identification is greatly welcome. When compared to the other species from the M. tuberculosis complex, M. tuberculosis is a strong nitrate reducer. Current assay compares the nitrate reductase activity of M. tuberculosis from pulmonary specimens cultivated in nitrate-supplemented media. Fifty-five sputum samples were decontaminated and inoculated in conventional (Middlebrook 7H9, Ogawa Kudoh-OK) and in nitrate-supplemented media (Middlebrook 7H9-N, Ogawa Kudoh-N). An aliquot from the media directly reacted with Griess reagent (7H9-N and OK-N) every five days, or transferred to a nitrate substrate solution (7H9, OK). Nitrate to nitrite reduction was considered positive, revealed by the pink color, indicating bacterial growth. As reference method, the Mycobacteria Growth Indicator Tube (MGIT) was used for sensitivity calculations and statistical analysis. 7H9-N and OK-N assays proved to perform better in detecting M. tuberculosis than conventional assays (7H9 and OK). Indeed, broth nitrate-supplemented medium (7H9-N) was comparable to MGIT to detect M. tuberculosis, except in growth detection time. Results show that 7H9-N may be used as an alternative tool particularly in low-income countries since it is a simple and cheap technique, and does not restrict diagnosis to single-source products.

Resposta de híbridos de milho ao uso de piraclostrobina na ausência de doenças / Corn hybrids responses to pyraclostrobin use in disease absences

Objetivando-se avaliar o efeito da aplicação de piraclostrobina em diferentes épocas e combinações de aplicação em dois híbridos simples de milho cultivados na safra de verão, realizou-se um experimento no município de Jataí, Goiás. Adotou-se o delineamento de blocos ao acaso no esquema fatorial 2 x 9 (híbridos x aplicações de piraclostrobina), com 4 repetições. As aplicações foram realizadas em diferentes combinações de fungicidas: presença ou ausência de piraclostrobina + tiofanato metílico + fipronil (100 g i.a.100 kg semente-1) no tratamento de sementes (V0) combinado com a aplicação de piraclostrobina (150 g i.a.ha-1) antes ou após a adubação de cobertura (V4 ou V6) combinado ou não com a aplicação de piraclostrobina + ciproconazole (137,25 g i.a.ha-1) em pré-pendoamento (VT) e uma testemunha (sem uso de piraclostrobina). Foram avaliados: atividade da enzima redutase do nitrato, índice de clorofila, altura de plantas, altura de inserção de espiga, diâmetro de colmo, porcentagem de folhas senescentes, severidade de Puccinia polysora Underw, massa de mil grãos, densidade e produtividade de grãos. Submeteram-se os dados ao teste F para verificação de significância; e para comparação de médias utilizou-se o teste de Tukey, ambos a 5% de probabilidade. Para a maioria dos caracteres avaliados observou-se diferença entre híbridos. Não foi observado efeito das aplicações de piraclostrobina e interação entre os fatores em questão. Conclui-se que não há efeito benéfico ou deletério da aplicação de piraclostrobina em diferentes épocas e combinações de aplicação sobre os dois híbridos simples de milho cultivados na safra de verão.(AU)

An experiment was conducted in Jataí, Goiás, Brazil, aiming to evaluate the effects of the application of pyraclostrobin at different times and with different application combinations in two simple hybrids of corn grown in the summer season. A randomized block design was adopted in a factorial 2 x 9 (hybrid x pyraclostrobinapplications) design, with 4 replications. The applications were performed in different combinations: presence or absence of pyraclostrobin + thiophanate methyl + fipronil (100 g a.i.100 kg seed-1) in seed treatment (V0) combined with pyraclostrobin (150 g a.i.ha-1) application before or after topdressing (V4 or V6) combined or not with pyraclostrobin + cyproconazole (137.25 g a.i.ha-1) application in pre-bolting (VT) and a control (without use of pyraclostrobin). The following evaluations were performed: activity of the nitrate reductase enzyme, chlorophyll index, plant height, corn cob insertion height, stem diameter, percentage of senescent leaves, severity of Puccinia polysora Underw, thousand grain weight, grains density, and grains yield. An F test was performed to analyze the significance, and for a means comparison, a Tukey test was used, both at 5% probability. Differences among hybrids were observed for most of the evaluated traits. There were no effects of the applications of pyraclostrobin and no interaction between the factors in question. It can, therefore, be concluded that there are not beneficial or deleterious effects of the application of pyraclostrobin at different times and with different application combinations in two simple hybrids of corn grown in the summer season.(AU)

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)

Effect of cadaverine on Brassica juncea (L.) under multiple stress.

The cadaverine (Cad), an organic diamine was examined for its response on growth in salinity and metal stressed B. juncea cv RH-30 vis-à-vis compared the response of ammonium nitrate. The Cad (1 mM) application ameliorated the effect caused by salinity and metal stress on seed germination and plant growth. The plant growth recovery (dry biomass accumulation) was dependent on stress and diamine type. The higher growth recovery potential of Cad under both stresses was due to elevation in photosynthetic pigments, nitrate reductase activity and organic nitrogen as well as soluble protein, It is inferred that growth in stressed seedlings was mediated by Cad through lowering endogenous Cd/Pb and Na+/K+ level in leaf and shoot tissues.

Biosynthesis of silver nanoparticles from Trichoderma species.

A total of 75 isolates belonging to five different species of Trichoderma viz., T. asperellum, T. harzianum, T. longibrachiatum, T. pseudokoningii and T. virens were screened for the production of silver nanoparticles. Although all the isolates produced nanoparticles, T. virens VN-11 could produce maximum nanoparticles as evident from the UV-Vis study. The highest Plasmon band was observed at 420 nm at every 24 h that attained maximum intensity at 120 h (0.543). The high resolution transmission electron microscopy (HRTEM) further provided the morphology of the nanoparticles. These nanoparticles were found single or aggregated with round and uniform in shape and 8-60 nm in size. The nitrate reductase activity of VN-11 was found to be 150 nmol/h/mL which confirmed the production of silver nanoparticles through reduction of Ag+ to Ag0.

Allelopathic effects of extracts from fibrous roots of Coptis chinensis on two leguminous species / 中国中药杂志

An experiment was carried out to study the allelopathic effects of Coptis chinensis fibrous root extracts (CRE) on the germination and seedling growth of Vicia faba and Pisum sativum in order to alleviate the allelopathic effects and increase land productivity. The seeds of both garden pea (P. sativum) and broad been (V. faba) were germinated in CRE solution of various concentrations, the germination rate, seedling growth and related physiological indexes were measured. The result indicated that there were no significant effects of CRE in low concentrations on seed germination, including both the rate and index, and seed vitality and membrane permeability. With the increment of CRE concentrations, however, the high seed membrane permeability and germination inhibition were observed. For example, the germination rates were reduced by 23.4% (P. sativum) and 9.5% (V. faba), respectively, in CRE solution with 800 mg . L-1. Simultaneously, soluble sugars and the free amino acids in the seeds were lower than those in the control (without CRE) after soaking seeds in CRE solutions. In addition, the seedling growth and nitrate reductase activity were stimulated by CRE at low concentrations in contrast to high concentrations which behaved otherwise and inhibited the nutrient utilization in endosperm. Therefore, the large amount of allelochemicals released from the roots and remains of C. chinensis in soils could inhibit the seed germination and seedling growth of legumes, which may lead to decrease even fail crop yields after growing this medical plant.

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.

Manual de procedimientos para la evaluación externa de la calidad de la prueba rápida de susceptibilidad isoniacida y rifampicina, método GRIESS / Manual procedures for external quality assessment of rapid susceptibility testing of isoniazid and rifampicin, Griess method

El manual, es para dar a conocer los procedimientos técnicos de evaluación externa de la calidad de la prueba de Griess en los laboratorios de referencia de salud pública donde se ha validado e implementado esta prueba

Utilidad del ensayo de nitrato reductasa en la detección de Mycobacterium tuberculosis multirresistente en caso de recursos limitados / Utility of nitrate reductase assay for detection of multidrug-resistant Mycobacterium tuberculosis in a low resource setting

Introducción. El rendimiento de cualquier prueba rápida de sensibilidad a medicamentos para Mycobacterium tuberculosis puede cambiar cuando se pasa de la etapa de investigación a su implementación en la práctica de salud pública. Objetivo. Describir el rendimiento del ensayo de nitrato reductasa para detectar tuberculosis multirresistente en la rutina de trabajo de un laboratorio de referencia con recursos limitados. Materiales y métodos. Se comparó prospectivamente la utilidad del ensayo de nitrato reductasa con el método de las proporciones para la detección de resistencia a isoniacida y rifampicina en aislamientos de pacientes con riesgo de multirresistencia. Resultados. Comparando con el método de las proporciones, la sensibilidad del ensayo de nitrato reductasa fue de 86,8 % y 84,9 % para rifampicina e isoniacida, respectivamente; la especificidad fue 100 % para rifampicina e isoniacida. La sensibilidad del ensayo de nitrato reductasa fue significativamente mayor cuando se emplearon aislamientos en los cuales el tiempo entre la realización del cultivo y la inoculación del ensayo de nitrato reductasa no superaba 70 días: 94,3 % Vs. 78,1 % para resistencia a rifampicina usando aislamientos frescos o envejecidos, respectivamente; prueba exacta de Fisher, p=0,05; 94,4 % Vs. 74,3 % para resistencia a isoniacida usando aislamientos frescos o envejecidos, respectivamente, y prueba de ji al cuadrado, p=0,03. La proporción de resultados no interpretables en el ensayo de nitrato reductasa fue mayor en los aislamientos multirresistentes que en los otros Conclusiones. La experiencia con el ensayo de nitrato reductasa demuestra que produce resultados resistentes confiables. El uso de cultivos envejecidos conduciría a una mayor proporción de resultados falsos sensibles, mientras que su capacidad para detectar multirresistencia disminuye a consecuencia de una mayor proporción de resultados no interpretables.

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 multidrugresistant 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 nonmultidrug-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.

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.

Susceptibilidad de M. tuberculosis a drogas antituberculosas, determinada por dos técnicas, en el estado Sucre, Venezuela / usceptibility of M. tuberculosis to antituberculosis drugs as determined by two methods, in Sucre state, Venezuela

El objetivo de este trabajo fue evaluar la resistencia a isoniacida (INH), rifampicina (RIF), estreptomicina (STR) y etambutol (EMB) de 59 cepas de Mycobacterium tuberculosis, aisladas en el período agosto 2005-diciembre 2006, en el estado Sucre, Venezuela, empleando el método de proporciones de Canetti y de nitrato reductasa. Se encontró 6,3% de resistencia primaria y 14,3% de adquirida. Una cepa fue considerada MDR, al presentar resistencia a RIF e INH. Se comparó la prueba de nitrato reductasa con el método de las proporciones, encontrándose 100% de concordancia entre los resultados de los dos métodos para INH, RIF y EMB, y 95,65% para STR. Además, la prueba nitrato reductasa produjo resultados en 10 a 14 días, comparado con 42 días para el método de proporciones, por lo que la primera se postula como una alternativa muy valiosa para acortar el tiempo de respuesta en la valoración de la susceptibilidad de M. tuberculosis. La secuencia del gen rpoB en la cepa resistente a RIF demostró la presencia de una mutación no descrita anteriormente en la región hipervariable de 81 pares de bases, donde se ha reportado el mayor número de mutaciones de cepas resistentes a RIF. Esta mutación produjo un cambio en el codón 456 de TCG > CAG. Al comparar nuestros resultados con los hallados en el último estudio de prevalencia de resistencia realizado en el estado, se demuestra una disminución en la circulación de cepas resistentes en la zona de estudio.

The objective of this study was to evaluate the resistance to isoniazid (INH), rifampicin (RIF), streptomycin (STR) and ethambutol (EMB), with the Canetti’s proportions method (PM) and the nitrate reductase assay (NRA) of 59 clinical strains of Mycobacterium tuberculosis, isolated in the period of august 2005 to december 2006, in Sucre state, Venezuela. Primary and acquired drug resistance was 6.3% and 14.3%, respectively. Only one strain was found to be multidrug resistant (MDR). The overall agreement between the NRA and PM was 100% for INH, RIF and EMB, and 96% for STR. The time to obtain results was 10 to 14 days for the NRA, compared to 42 days for the PM. The NRA was easy to perform and therefore represents a useful tool for rapid and accurate determination of drug-resistant M. tuberculosis. The sequence of the rpoB gene of the RIF resistant strain demonstrated a never described mutation (change in the codon 456; TCG > CAG) in the hypervariable region of 81 base pairs where most of the mutations of the RIF resistant strains have been reported. Comparison of our results with those of the last resistance prevalence study carried out in the years 1998-1999, shows a decrease in the studied area.

Nodulação e atividade da nitrato redutase em função da aplicação de molibdênio em soja / Nodulation and nitrate reductase activity affected by molybdenum application in soybean

Entre os nutrientes essenciais para o processo de fixação biológica do nitrogênio pela soja, o molibdênio destaca-se por ser cofator da enzima nitrato redutase, afetando a sua atividade e, consequentemente, o processo de nodulação. O trabalho objetivou avaliar os efeitos da aplicação de molibdênio na nodulação de soja e na atividade da enzima nitrato redutase. O experimento foi conduzido em casa de vegetação, semeando-se a soja em vasos de 12 L e conduzindo-se duas plantas por vaso. Os tratamentos constaram de duas vias de aplicação (via semente e via foliar) e duas doses de molibdênio (12 e 24 g ha-1 via semente e 0, 30 e 60 g ha-1 via foliar) na forma de molibdato de amônio, mais a testemunha. Avaliou-se o número e massa seca de nódulos, teor de nitrogênio nas folhas e atividade da enzima nitrato redutase, em dois horários de coleta das folhas de soja (às 10 e 22 horas). Concluiu-se que a nodulação em soja é afetada pela dose e via de aplicação de molibdênio, sendo que maior número e massa de nódulos são obtidos com a aplicação de 24 g ha-1 do nutriente via semente. A atividade da enzima nitrato redutase é afetada pela adubação com molibdênio, independente da via de aplicação e do horário de coleta das folhas para avaliação, sendo crescente com a elevação das doses de Mo.

Among the nutrients that are essential for the biological nitrogen fixation by soybean plants, molybdenum stands out for being a cofactor of the nitrate reductase, affecting enzymatic activity and, consequently, the nodulation process. The research had as objective to evaluate the effects of molybdenum application on soybean nodulation and nitrate reductase activity. The experiment was conduced in greenhouse, sowing soybean in 12 L pots, with two plants per plot. The treatments consisted of two application via (with the seeds and leaf dressing) and two Nodulação e atividade molybdenum doses (12 and 24 g ha-1 with the seeds; 30 and 60 g ha-1 leaf dressing) in ammonium molybdate form, plus the control. The number and dry mass of nodules and nitrogen content in soybean leaves were evaluated. Samples of leaves for the evaluation of nitrate reductase activity were taken at 10 a.m. and 10 p.m. It was concluded that soybean nodulation is affected by Mo dose and application via, resulting in higher number and weight of nodules when it is applied with the seeds. The enzymatic activity of the nitrate reductase is influenced by Mo fertilization and it is higher for leaf dressing with the double of the recommended dose

Effects of NH4(+)-N/NO3(-)-N ratio in applied supplementary fertilizer on nitrogen metabolism, photosynthesis and growth of Isatis indigotica / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of NH4(+)-N/NO3(-)-N ratios in the applied supplementary fertilizer on the growth, nitrogen metabolis related enzymes activity and photosynthetic characteristics of Isatis indigotica.</p><p><b>METHOD</b>The sand culture experiment was conducted, and seedling of I. indigotica was fertilized with the mixed nutrition that containing the Hoagland's macro elements and the Aron's micro elements, the additional 63 mmol N was supplementary with the NH4(+)-N/NO3(-)-N ratio of 100:0, 75:25, 50:50, 25:75 and 0:100.</p><p><b>RESULT</b>The biomass of I. indigotica increased at first when the supplementary N of NH4(+)-N/NO3(-)-N ratio changed from 100:0 to 50:50 and decreased afterwards. The maximum value was at 50:50 and the minimum at 100: 0. With increasing the ratio of NO3(-)-N, the activity of nitrate reductase and glutamine synthetase increased and then decreased and the relationship between the activity and the ratio could be described with an approximate parabola curve. The net photosynthetic rate of I. indigotica was the highest at the NH4(+)-N/NO3(-)-N ratio of 75:25 and the lowest at 100:0.</p><p><b>CONCLUSION</b>Increasing the NO3(-)-N ratio properly was beneficial to promote the growth and improve the activity of nitrate reductase and glutamine synthetase and net photosynthetic rate of I. indigotica.</p>

Establishment and evaluation of nitrate reductase combined with mycobacteriophage assay / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a rapid, inexpensive, and simple drug susceptibility test (DST) for Mycobacterium tuberculosis (M. tb) and evaluate its feasibility.</p><p><b>METHOD</b>We used nitrate reductase combined with mycobacteriophage assay (PhaB-NRA) to test 49 clinical M. tb isolates of, and the results were compared with those of PhaB-NRA and traditional absolute concentration method.</p><p><b>RESULTS</b>The sensitivity, specificity, and accuracy of PhaB-NRA for rifampicin were 89.1%, 91.67%, and 89.8%; on the contrary, those of isonicotinyl hydrazide were 86.21%, 90.0%, and 87.8%, respectively. The coincidence between PhaB-NRA and traditional assay were 0.746 for rifampicin and 0.750 for isonicotinyl hydrazide.</p><p><b>CONCLUSIONS</b>PhaB-NRA is an inexpensive, rapid, and simple DST method. It is a promising rapid screening technique for DST of M. tb.</p>

Drug susceptibility testing of pulmonary tuberculosis by nitrate reductase assay

Treatment of multidrug-resistance tuberculosis [MDR-TB] remains a challenge and its success depends on how quickly a case of TB is identified as drug resistant and whether an effective drug therapy is available. As a drug susceptibility test [DST], nitrate reductase assay [NRA] might help in management of MDR-TB patients. This study was conducted to evaluate NRA as a drug susceptibility test [DST] for the four primary anti-tuberculosis drugs [Isoniazide [INH], Rifampicin [RIF], Streptomycin [SM] and Ethambutol [EMB]]. A total of 30 isolates of M. tuberculosis were subcultured for 28 day to be fresh isolates and subjected to NRA. The results were recorded after 10 days of incubation and compared to that of the proportion method [PM] as a gold standard. Susceptibility testing for INH and RIF revealed relatively higher efficiencies when compared to that of the PM. For INH and RIF, the efficiencies were 93.3% and 90%, respectively. The sensitivity of the NRA as a DST for INH was 89.4%, while that for RIF was 93.3%. The specificities of NRA for INH and RIF were 100% and 86.6%, respectively. The predictive value of sensitivity for INH was 84.6% and that of RIF was 92.8%. The predictive value of resistance for INH was a high as 100%, while that of RIF was 87.5%. Susceptibility testing for SM and EMB showed relatively lower efficiency compared to PM. The efficiencies of NRA for SM and EMB were 86.6% and 76.6%, respectively. The sensitivity of the NRA as a DST for SM was 78.9%, while that for EMB was 40%. The specificity of the NRA for SM was 100% and that of EMB was 95%. The predictive values of sensitivity for SM and EMB were 73.3% and 76%, respectively. The predictive value of resistance for SM was 100% and that of EMB was 80%. NRA is a simple, rapid and inexpensive technique of high performance when used as susceptibility testing for both INH and RIF, but was not good for SM and EMB

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...

Accumulation, tolerance and impact of aluminium, copper and zinc on growth and nitrate reductase activity of Ceratophyllum demersum (Hornwort).

Ceratophyllum demersum (homwort) was subjected to toxic concentrations of Al (3 and 9 mg l(-1)), Zn (3 and 9 mg l(-1)) and Cu (2.5 and 7 mg l(-1)) in culture solutions for 15 days. The higher dose of Al enhanced the chlorophyll content significantly (p<0.05) in the first 6 days of treatment while other treatments caused marked reductions. Nitrate reductase activity (NRA) was significantly reduced (p<0.05) byAl, Cu and Zn toxicity and ceased completely in plants treated with Cu by the 6th day of treatment. Dry biomass and relative growth rate were reduced significantly (p<0.05) by metal treatment. Tolerance index of the plant was low for Cu (21.62 and 13.43% at low and high doses, respectively) and moderate for Zn (63.74 and 54.85%) and Al (72.83 and 68.79%). Accumulation ofAl, Zn and Cu was threefold at higher doses compared with the lower doses but the bioconcentration factors (BCF) were very low indicating that this plant is not a hyper accumulator of these metals.

Leaf biochemistry of Lycopersicon esculentum Mill. at different stages of plant development as affected by mercury treatment.

The effect of mercury (Hg) on the biochemical parameters of Lycopersicon esculentum Mill leaf was studied. Application of mercuric chloride in varying concentrations (0.5, 1.0, 1.5 and 2.0 mM HgCl2 kg(-1) sand) caused significant reduction that went up to 89% and 72% chlorophyll a and chlorophyll b contents respectively (at flowering stage), 69% in carotenoid content, 64% in total soluble protein content and 91% in nitrate reductase activity (all at post-flowering stage). The amounts of nitrate and proline increased maximally (151% and 143% respectively) at the flowering stage, whereas total soluble sugar enhanced by 57% at the post-flowering stage. Changes observed in most of the parameters, were concentration dependent. Such studies seem to be able to discover suitable bioindicators of heavy metal pollution.