Nitrate Reductases Are Relocalized to the Nucleus by AtSIZ1 and Their Levels Are Negatively Regulated by COP1 and Ammonium.

Nitrate reductases (NRs) catalyze the first step in the reduction of nitrate to ammonium. NR activity is regulated by sumoylation through the E3 ligase activity of AtSIZ1. However, it is not clear how NRs interact with AtSIZ1 in the cell, or how nitrogen sources affect NR levels and their cellular localization. Here, we show that the subcellular localization of NRs is modulated by the E3 SUMO (Small ubiquitin-related modifier) ligase AtSIZ1 and that NR protein levels are regulated by nitrogen sources. Transient expression analysis of GFP fusion proteins in onion epidermal cells showed that the NRs NIA1 and NIA2 localize to the cytoplasmic membrane, and that AtSIZ1 localizes to the nucleoplasm, including nuclear bodies, when expressed separately, whereas NRs and AtSIZ1 localize to the nucleus when co-expressed. Nitrate did not affect the subcellular localization of the NRs, but it caused AtSIZ1 to move from the nucleus to the cytoplasm. NRs were not detected in ammonium-treated cells, whereas the localization of AtSIZ1 was not altered by ammonium treatment. NR protein levels increased in response to nitrate but decreased in response to ammonium. In addition, NR protein levels increased in response to a 26S proteasome inhibitor and in cop1-4 and DN-COP1-overexpressing transgenic plants. NR protein degradation occurred later in cop1-4 than in the wild-type, although the NR proteins did not interact with COP1. Therefore, AtSIZ1 controls nuclear localization of NR proteins, and ammonium negatively regulates their levels. The function and stability of NR proteins might be post-translationally modulated by ubiquitination.

Measuring nitrate reductase activity from human and rodent tongues.

Reduction of salivary nitrate to nitrite by oral microbes expressing nitrate-reductase has emerged as a crucial pathway in systemic NO homeostasis in humans and other mammals. Selective depletion of oral microbes prevents dietary nitrate-dependent lowering of blood pressure, inhibition of platelet aggregation and ischemic injury. To date, most studies interrogate enterosalivary nitrate reduction by following changes in saliva or plasma nitrite and NO-signaling (functional) end points. Little is known about whether, and if so how, nitrate-reductase enzymatic activity per se (i.e. independent of nitrate levels) is a variable and may account for any individual to individual variation. Here, we describe a minimally invasive protocol that allows for NR activity determination from human, rat and mouse tongue scrapes/swabs. We validate this method using selective application of antiseptic agents to the distal tongue surface which decreased NR activity by >80% and show that bacterial number is a significant variable in measured NR activities between males and females. Also, we show that NR activity is >80% lower in smokers (humans) and after bromine gas exposure (mice), suggesting that exposure to inhaled reactive substances inhibit NR activity identifying a potentially new mechanism by which environmental toxicants promote dysfunction in NO-bioavailability. The described method will facilitate studies testing whether NR specific activity is a variable in different pathophysiologic settings, and in turn how this activity modulates enterosalivary nitrate-reduction.

In situ assay of nitrate reductase activity using portable water bath.

In environmental research (i.e., plant ecophysiology, environmental microbiology, and environmental chemistry), some assays require incubation of samples at controlled temperature and darkness. Until now, due to a lack of equipment providing such possibility in situ, researchers had to move collected samples to the laboratory for incubation. Obviously, a delayed incubation and the ex situ conditions could seriously affect the assays' results. A good example of analysis where water bath use is needed is the nitrate reductase activity (NRA) in vivo assay where plant tissue samples are incubated in buffer solution at a predetermined temperature. We designed a transportable water bath with a temperature control which enables in situ measurements in many types of environmental studies. The presented device is small in size featuring a thermally insulated chamber and an electronically controlled thermostat system powered by a 12-V battery. Due to its modular design, it can be transported comfortably in difficult terrain. The incubation process can be carried out continuously in stable temperature and darkness. In order to examine the field usability of the presented device, we conducted measurements of plant nitrate reductase activity in difficult field conditions. The in situ assays were carried out at high altitudes in the Karkonosze mountains, SW Poland. The NRA was studied in two alpine species (Deschampsia caespitosa and Homogyne alpina). Our results showed low NR activity in H. alpina (mean 0.31 µM NO2 g-1 DW h-1) and higher NRA in D. caespitosa (mean 2.7 µM NO2 g-1 DW h-1). The obtained results were highly reproducible and had small variability (low standard error values).

Nitrate reductase assay for rapid detection of isoniazid, rifampin, ethambutol, and streptomycin resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis.

Colorimetric phenotypic tests recently gained interest because traditional primary drug susceptibility testing of Mycobacterium tuberculosis isolates takes a long time. We used meta-analysis techniques to review the reliability and accuracy of the nitrate reductase assay (NRA), which is one of the most popular colorimetric methods to detect resistance to first-line drugs. Medline, PubMed, ISI Web, Web of Science, and Google Scholar were used to search for studies enrolled in the meta-analysis. The analysis included 35 studies for isoniazid (INH), 38 for rifampin (RIF), and 22 for ethambutol (EMB) and streptomycin (STR). Summary receiver operating characteristic (SROC) curves were applied to summarize diagnostic accuracy. The meta-analyses were performed by the use of Meta-DiSc software (version 1.4) and were focused on sensitivity and specificity values for measurements of accuracy. The pooled sensitivities were 96% for INH, 97% for RIF, 90% for EMB, and 82% for STR. The pooled specificities for INH, RIF, EMB, and STR were 99%, 100%, 98%, and 96%, respectively. The times required to obtain results were between 5 and 28 days by the direct NRA and between 5 and 14 days by the indirect test. In conclusion, the present meta-analysis showed that the NRA is a reliable low-cost rapid colorimetric susceptibility test that can be used for the detection of multidrug-resistant (MDR) tuberculosis, including detection of EMB resistance. However, the test appears to have a relatively low sensitivity for STR and needs further improvement.

Biochemical and proteomic analyses reveal that Populus cathayana males and females have different metabolic activities under chilling stress.

Male and female poplars (Populus cathayana Rehd.) respond differently to environmental stresses. However, little is known about sex-dependent responses to chilling at the proteome level. To better understand these differences, a comparative proteomics investigation combined with a biochemical approach was used in the current study. Three-month-old poplar cuttings were treated at 25 or 4 °C for 14 days. Results revealed significant sexual differences in nitrogen metabolic enzymes and free amino acid components in response to chilling. The chilling-treated males showed higher activities of nitrate reductase and glutamine synthetase and higher contents of reduced glutathione, serine, arginine, leucine, glycine, proline and methionine than chilling-treated females. A total of 65 chilling-responsive spots were found, of which 48 showed significant sexual differences. These proteins are involved in photosynthesis, carbon and energy metabolism, metabolic processes of proteins, lipid metabolism, vitamin metabolism, stress defense, and gene expression regulation. The study shows that males have more effective metabolic processes and protective systems to chilling than females.

The manual mycobacteria growth indicator tube and the nitrate reductase assay for the rapid detection of rifampicin resistance of M. Tuberculosis in low resource settings.

BACKGROUND: Tuberculosis (TB) is a disease of poverty that contributes significantly to ill-health in developing countries. Drug resistant TB is a major challenge to disease control. Early diagnosis and rapid determination of drug sensitivity is of paramount importance in eradication of TB. Although automated liquid culture based methods are available for rapid detection of drug resistance, the high cost of these tests prevent them from being used routinely in low resource settings. This study compares two phenotypic methods, the manual Mycobacteria Growth Indicator Tube (MGIT) and the Nitrate Reductase Assay (NRA) in liquid medium, with the agar proportion method (APM), the gold standard for susceptibility testing of Mycobacterium tuberculosis. METHODOLOGY: Fourteen day old M. tuberculosis strains (n=373) grown on solid media were used for drug susceptibility testing by APM, NRA and the manual MGIT method. Rifampicin free and rifampicin incorporated (final concentration, 1 µg/ml) media were inoculated with the recommended concentrations of mycobacterial suspensions and incubated at 37°C in 5% CO2. In the APM, the proportion of colonies in the drug containing medium was determined. In the NRA, the colour change in the medium was compared with a standard colour series after day 6 and day 12 of incubation. Growth in the MGIT was detected using the manual MGIT reader from day 2 onwards. The 2 methods were compared with the gold standard, APM to determine sensitivity and specificity and agreement between the methods was calculated using kappa statistics. RESULTS: Thirty one (31) rifampicin resistant isolates were identified. When compared with the APM, the sensitivity of detection of rifampicin resistance was 85% for the NRA and 93% for the manual MGIT and the specificity was 99% and 100% respectively. Both assays, NRA (κ=0.86) and manual MGIT method (κ= 0.94) were in excellent agreement with the APM. The mean turnaround time for manual MGIT method and NRA were 08 days and 10 days respectively. CONCLUSION: The NRA in liquid medium and manual MGIT are useful alternatives to APM for drug susceptibility testing of M. tuberculosis in low resource settings.

Restoration of taxonomic and functional genes after bioaugmentation of petroleum contaminated soil.

Soil microbial ecosystems are responsive to environmental changes that underpin the biological functions of the soil. The present study was conducted to profile variations in the microbial ecological system of remediated soil (R) and petroleum contaminated soil (P) based on comparisons with soil that had not been contaminated (N), using a cloning library of taxonomic genes (16S rRNA gene for bacteria and 18S rRNA gene for eukaryotes) and functional genes (nifH, amoA and narG). The results showed that N and R had a similar distribution in both the taxonomic genes and functional genes for bacteria and eukaryotes, which were dominated by Proteobacteria and Arthropoda, respectively. Phylogenetic analysis based on the nifH gene showed that the sequences from the three soils were clustered into six taxonomic groups, Actinobacteridae, and Alpha-, Beta-, Gamma- and Delta-proteobacteria, as well as an unclassified group. Evaluation of the amoA gene revealed that all sequences derived from the three samples belonged to Betaproteobacteria. The R and N soil had similar Shannon-Wiener diversity index (H') values, both of which were significantly higher than that of the P soil. The most abundant bacterial phylotype identified in the N and R soils were the same and were related to an uncultured bacterial clone (GAN-SB17, FN423475). None of the narG genes were found in the P soil. Similar results in terms of distribution, composition and the related index were obtained for nifH and amoA. These parameters may comprise a biological ecology index that may be applied to aid the design, implementation and evaluation of soil bioremediation.

Vegetated ditches for treatment of surface water with highly fluctuating water regime.

Vegetated drainage ditches (VDD) as a type of constructed wetland primarily serve to remove and store excess water associated with irrigation and storm events. Current research suggests using a VDD as an additional practice in the mitigation of surface water pollution. The VDD for water treatment of the Glinscica River was constructed in 2006. The efficiency of the system was evaluated in 2008 and 2009 regarding the reduction of SS, COD, BOD5, NH4-N, NO3-N, NO2-N, TN, ON and TP. The microbiological association developed in the VDD was analyzed with a focus on the identification and quantification of the narG gene as a denitrification indicator. This paper discusses the efficiency of pollution removal and the distribution of the narG gene within the VDD. The results showed that the highly fluctuating water regime was the main reason for the even distribution and abundance of the narG gene throughout the system, regardless of oxygen saturation or the nutrient status of the wastewater. With the exception of SS, pollutant concentrations met the permitted outflow levels.

Chronic toxicity of a triazole fungicide tebuconazole on the growth and metabolic activities of heterocystous, nitrogen-fixing paddy field cyanobacterium, Westiellopsis prolifica Janet.

This study explored the chronic and harmful effects of different doses of the triazole fungicide, tebuconazole, on the growth, and metabolic and enzymatic variations in the filamentous paddy field cyanobacterium, Westiellopsis prolifica Janet. The growth of the cyanobacterium was determined by an estimation of the change in pigment contents. Chlorophyll-a, carotenoids and accessory pigments such as phycocyanin, allophycocyanin and phycoerythrin, were shown to decline over a 16-day period by a factor of 92%, 93%, 83%, 95% and 100%, respectively, with increasing doses of the fungicide. Metabolic and enzymatic activities were also adversely affected. Over the 16 days, a gradual rise in the total phenol content was recorded when Westiellopsis prolifica Janet was treated with 60 ppm of the fungicide, despite the reduction in carbohydrates, proteins and amino acids by 96%, 92% and 90%, respectively. Moreover, the enzymes nitrate reductase (NR), glutamine synthetase (GS) and succinate dehydrogenase (SDH) also registered reductions of 93%, 90% and 98%, respectively. This study indicates that tebuconazole, although an important fungicide used extensively in rice fields, exhibits an inhibitory effect on the growth and metabolic activities of Westiellopsis prolifica Janet and hence possibly on other varieties as well.

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

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.

Multicentre study of nitrate reductase assay for rapid detection of rifampicin-resistant M. tuberculosis.

SETTING: Four regional laboratories belonging to the Mycobacteria Reference Laboratory of São Paulo State, Brazil. OBJECTIVE: To evaluate the nitrate reductase assay (NRA) for rifampicin (RMP) susceptibility testing of Mycobacterium tuberculosis directly from clinical sputum samples of patients with pulmonary tuberculosis (TB). DESIGN: Performance of the NRA for detection of M.tuberculosis susceptibility to RMP was evaluated with 210 clinical sputum samples received by the participating laboratories during 2005 and 2006 and compared with the results of the direct proportion method. RESULTS: Susceptibility tests performed using the NRA and the direct proportion method showed 204 susceptible isolates and six isolates resistant to RMP by both methods. NRA sensitivity and specificity for RMP was 100%. The NRA results of susceptibility tests against RMP were available in 15 days for 87% of the samples. The results showed that NRA may yield a rapid answer in determining resistance for the majority of sputum samples with smear results reported as 3+ and 2+. CONCLUSION: The results demonstrate the feasibility of NRA for screening resistant strains in sputum samples from patients with pulmonary TB. NRA represents a rapid and low-cost alternative method that might be used in microbiological laboratories where resources are scarce.

The nitrate reductase assay for the rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis.

OBJECTIVES: The reference standard methods for drug susceptibility testing (DST) of M. tuberculosis are very slow to give results, and due to the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis, there is an urgent demand for new, rapid and accurate DST methods, particularly in low-income countries. The nitrate reductase assay (NRA) has been proposed as a rapid method for the detection of resistance to rifampicin and isoniazid, but its accuracy has not been systematically evaluated. METHODS: We performed a systematic review and meta-analysis to evaluate the accuracy of the NRA for the detection of rifampicin- and isoniazid-resistant tuberculosis. We searched Medline PubMed (NCBI), Global Health-CAB, EJS-E (EbscoHost), ISI Web, Web of Science and IFCC and contacted authors if additional information was required. Fifteen studies met our inclusion criteria for rifampicin resistance detection and 13 for isoniazid. Of these, the majority of the studies used culture isolates on solid medium, four used culture isolates on liquid medium and three used sputum samples. We applied the summary receiver operating characteristic (SROC) curve to perform meta-analysis and to summarize diagnostic accuracy. RESULTS: For rifampicin, the majority of the studies that applied NRA to isolates had a sensitivity and specificity >94% and for isoniazid, >92%. The three studies that applied NRA directly on sputum samples had a sensitivity and specificity that ranged between 88% and 100%. The SROC curve had an area of >0.99 for both drugs. CONCLUSIONS: There is evidence that NRA is highly sensitive and specific for the rapid detection of rifampicin and isoniazid resistance in culture isolates. More evidence is required for the NRA applied directly on sputum samples, but preliminary results appear promising and show a good sensitivity and specificity. Additional studies are required in countries with a high prevalence of MDR-TB and also cost-effectiveness analysis in order to obtain a complete picture on the utility of this method for rapid drug resistance detection in tuberculosis.

28-homobrassinolide protects chickpea (Cicer arietinum) from cadmium toxicity by stimulating antioxidants.

In the present experiment the seeds of Cicer arietinum (L.) cv. Uday were inoculated with specific Rhizobium grown in sandy loam soil and were allowed to grow for 15 days. At this stage, the seedlings were supplied with 0, 50, 100 or 150 microM of cadmium in the form of cadmium chloride and sprayed with 0.01 microM of 28-homobrassinolide (HBL) at 30-day stage. The data indicated that plant fresh and dry mass, number of nodules, their fresh and dry mass, leghemoglobin content, nitrogen and carbohydrate content in the nodules, leaf chlorophyll content, nitrate reductase and carbonic anhydrase activities decreased proportionately with the increasing concentrations of cadmium but the content of proline and the activities of catalase, peroxidase and superoxide dismutase increased. The ill effect, generated by cadmium, was overcome if the stressed plants were sprayed with HBL.

Detection of ofloxacin resistance by nitrate reductase assay in Mycobacterium tuberculosis isolates from extrapulmonary tuberculosis.

CONTEXT: Increased use of fluoroquinolones to treat community-acquired infections has led to the decreased susceptibility to Mycobacterium tuberculosis. There is a paucity of data on ofloxacin (OFX) resistance detection by nitrate reductase assay (NRA). Hence, the present study was carried out to find the efficacy of NRA for detection of OFX resistance in M. tuberculosis isolated from extrapulmonary tuberculosis (EPTB) cases. AIMS: (1) To compare sensitivity, specificity and median time required to obtain results by NRA with economic variant proportion method (PM) for detection of OFX resistance.(2) To determine the extent of OFX resistance in clinical isolates of M. tuberculosis. SETTINGS AND DESIGN: Seventy-three M. tuberculosis isolates from cases of EPTB were subjected to economic variant of PM for isoniazid, rifampicin and OFX. NRA was done for detection of OFX resistance. SUBJECTS AND METHODS: Seventy-three isolates from clinical samples of suspected EPTB received in the Department of Microbiology were included in the study. Drug susceptibility test was performed on Lowenstein-Jensen medium with and without drugs. STATISTICAL ANALYSIS USED: Of turnaround time was done by Mann-Whitney test on SPSS (version 19, released in 2010, IBM Corp, Armonk NY),P < 0.05. RESULTS: OFX resistance was seen in nine isolates. The sensitivity and specificity of OFX resistance by NRA was 100% and 96.87%, respectively. Median time required to obtain results by NRA was 10 days as compared to 28 days by PM. CONCLUSIONS: NRA is a specific and sensitive method for detection of OFX resistance in resource-restricted settings.

Applying Taguchi design and large-scale strategy for mycosynthesis of nano-silver from endophytic Trichoderma harzianum SYA.F4 and its application against phytopathogens.

Development of reliable and low-cost requirement for large-scale eco-friendly biogenic synthesis of metallic nanoparticles is an important step for industrial applications of bionanotechnology. In the present study, the mycosynthesis of spherical nano-Ag (12.7 ± 0.8 nm) from extracellular filtrate of local endophytic T. harzianum SYA.F4 strain which have interested mixed bioactive metabolites (alkaloids, flavonoids, tannins, phenols, nitrate reductase (320 nmol/hr/ml), carbohydrate (25 µg/µl) and total protein concentration (2.5 g/l) was reported. Industrial mycosynthesis of nano-Ag can be induced with different characters depending on the fungal cultivation and physical conditions. Taguchi design was applied to improve the physicochemical conditions for nano-Ag production, and the optimum conditions which increased its mass weight 3 times larger than a basal condition were as follows: AgNO3 (0.01 M), diluted reductant (10 v/v, pH 5) and incubated at 30 °C, 200 rpm for 24 hr. Kinetic conversion rates in submerged batch cultivation in 7 L stirred tank bioreactor on using semi-defined cultivation medium was as follows: the maximum biomass production (Xmax) and maximum nano-Ag mass weight (Pmax) calculated (60.5 g/l and 78.4 g/l respectively). The best nano-Ag concentration that formed large inhibition zones was 100 µg/ml which showed against A.alternate (43 mm) followed by Helminthosporium sp. (35 mm), Botrytis sp. (32 mm) and P. arenaria (28 mm).

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.

Evaluation of the direct nitrate reductase assay for rapid detection of extensively drug-resistant tuberculosis.

SETTING: Jiangxi, China. OBJECTIVE: To evaluate the performance of the direct nitrate reductase assay (D-NRA) for rapid, low-cost detection of multidrug-resistant (MDR-) and extensively drug-resistant tuberculosis (XDR-TB) in high-burden, resource-limited settings. METHODS: A total of 225 smear-positive sputum samples were collected from consecutive drug-resistant TB subjects. Samples were processed at the Province TB Reference Laboratory and tested for susceptibility to rifampicin (RMP), isoniazid (INH), ofloxacin (OFX), kanamycin (KM) and capreomycin (CPM) by D-NRA, using the indirect Löwenstein-Jensen proportion method (LJ-PM) as reference. RESULTS: Of the 225 smear-positive sputum samples, 214 isolates were identified as Mycobacterium tuberculosis and analysed for further comparison. The sensitivity of the D-NRA in the detection of resistance to RMP, INH, OFX, KM and CPM was respectively 95.1% (97/102), 93.1% (135/145), 97.4% (76/78), 88.9% (40/45) and 90.6% (29/32); specificity was respectively 100% (112/112), 97.1% (67/69), 100% (136/136), 98.8% (167/169) and 96.7% (176/182). The median time to culture positivity was significantly shorter for NRA than for the indirect LJ-PM (14 days vs. 70 days, P < 0.001). CONCLUSION: D-NRA showed high sensitivity and specificity in the rapid diagnosis of MDR- and XDR-TB in a high-burden, resource-limited setting.

Rapid, inexpensive MIC determination of Mycobacterium tuberculosis isolates by using microplate nitrate reductase assay.

A rapid colorimetric, microplate-based nitrate reductase assay (NRA) method for antibiotic susceptibility profile of clinical isolates of Mycobacterium tuberculosis was compared with Alamar Blue assay (ABA). The results obtained by both the methods were also compared with conventional agar proportion method. The overall agreement between the results obtained by NRA and ABA was 99% and 98%, respectively, when compared with agar proportion method. Reproducible results for all the isolates were obtained within 8 days by NRA as well as ABA. The specificity of NRA method for M. tuberculosis makes it more suitable method for MIC determination.

Evaluation of direct microplate nitrate reductase assay as a rapid method for the detection of multiple and extensively tuberculosis drug resistance.

INTRODUCTION: Reports of Mycobacterium tuberculosis resistant to multiple drugs are increasing globally and laboratories are becoming increasingly aware of the need for drug susceptibility testing. In recent years, due to the long time required by conventional drug susceptibility testing, new approaches have been proposed for faster detection of drug resistance, such as the nitrate reductase assay, considered fast and inexpensive, making it a good diagnostic tool for low resource countries. OBJECTIVE: The present study proposed a fast direct colorimetric drug susceptibility testing method in a microplate format using solid medium. MATERIALS AND METHODS: The diagnostic accuracy was evaluated by comparing the proportion method with the direct nitrate reductase assay in plates. Frozen sputum samples, known to be positive, were decontaminated and processed by Petroff method. The decontaminated suspension was used to perform direct nitrate reductase assay in 7H11 medium using 1 µ g/ml rifampicin (RIF), 0.2 µg/ml isoniazid (INH), 2 µg/ml ofloxacin (OFX), 6 µg/ml kanamycin (KAN), 2 µg/ml amikacin (AMK) and 10 µg/ml capreomycin (CAP). Eighty-four samples were tested and the results for 69% of them were available within 21 days. RESULTS: The sensitivity and specificity compared to the proportion method, was 98.5% and 100% for INH, 98.3% and 96.2% for RIF, 91.7% and 100% for KAN, 78.8% and 97.3% for OFX, 100% and 100% for AMK and CAP, respectively. CONCLUSION: The results lead to the conclusion that direct nitrate reductase assay, in this new format, is an accurate, quick and inexpensive method to determine the susceptibility profile of M. tuberculosis and may become an alternative for countries with limited resources.

Predictive value of direct nitrate reductase assay and its clinical performance in the detection of multi- and extensively drug-resistant tuberculosis.

Conventional culture and drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and time consuming. For this reason alternative rapid culture and DST techniques are urgently needed to shorten the time for drug-resistance detection. A total of 222 smear-positive sputum samples were evaluated by the direct nitrate reductase assay (D-NRA) on Lowenstein-Jensen medium, for the rapid and simultaneous detection of resistance to isoniazid, rifampicin, kanamycin and ofloxacin. p-Nitrobenzoic acid was also included for identification of the M. tuberculosis complex. Results were compared with the BACTEC MGIT 960 as gold standard. The general performance of the D-NRA was very good, reaching a global value of 97 %. D-NRA had a turn-around time of 16.9 days to obtain results while that of the indirect MGIT 960 system was 29 days. D-NRA is a low-cost technology, easy to set up in clinical laboratories and suitable to be used for DST of M. tuberculosis in all smear-positive samples.