Resazurin microplate test method for rapid determination of colistin resistance in carbapenem-resistant Acinetobacter baumanii isolates.

BACKGROUND: Colistin-resistant Acinetobacter baumannii isolates are extremely important pathogens for hospital-acquired infections. OBJECTIVE: To investigate the effectiveness of the resazurin microplate assay (REMA) for the rapid determination of colistin resistance. METHODS: Susceptibility for colistin was investigated in vitro by the broth microdilution method (BMD) and the resazurin microplate assay (REMA) on 106 carbapenem-resistant Acinetobacter baumannii isolates. RESULTS: The results of both test methods were compared, and the categorical agreement between them was found to be 100%. No minor, major, or very major discrepancy was observed between the 2 methods. CONCLUSIONS: The most important advantages of REMA are that the results are obtained within 6 hours compared to the reference method, that it is easy to evaluate because it is colorimetric, and that the susceptibility result can be reported to the clinician on the same day as bacterial identification.

Detoxification of aflatoxin M1 in different milk types using probiotics.

The aim of this study, research the potential use of probiotics in reducing the toxic effect of Aflatoxin M1 in cow milk, goat milk, sheep milk, and Phosphate-buffered saline (PBS). Milk and Phosphate-buffered saline were contaminated with Aflatoxin M1 at a concentration of 100 ppt. Then, various study groups were formed by adding Lactobacillus acidophilus DSMZ 20079, Lactobacillus rhamnosus GG, and Bifidobacterium bifidum DSMZ 20456 probiotic bacteria at a density of 108 CFU/ml. Then, working groups were stored for 1 day and Aflatoxin M1 levels were analyzed by an Enzyme-Linked Immunosorbent Assay kit. The binding level of Aflatoxin M1 by probiotic bacteria varies between 2.32-12.52% in Phosphate-buffered saline, 9.08-40.14% in cow milk, 15.01-38.01% in goat milk, and 32.49-42.90% in sheep milk. The highest binding level of Aflatoxin M1 was detected in sheep milk and the lowest in Phosphate-buffered saline. The binding ability of Aflatoxin M1 is ranked from highest to lowest in sheep milk, cow milk, and goat milk. The data obtained from this study is important because it is the first study to show that if sheep and goat milk is enriched with probiotics, it can reduce AFM1 exposure.

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.

The Effect of Inoculum Size on Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis.

Phenotypic drug susceptibility testing (DST) requires a standardized amount of inoculum to produce reproducible susceptibility results. The most critical step in the application of DST in Mycobacterium tuberculosis isolates is the preparation of the bacterial inoculum. In this study, the effect of bacterial inoculum prepared in various McFarland turbidities on primary antituberculosis drug susceptibility of M. tuberculosis strains was investigated. Five standard ATCC strains (ATCC 27294 [H37Rv], ATCC 35822 [izoniazid-resistant], ATCC 35838 [rifampicin-resistant], ATCC 35820 [streptomycin-resistant], ATCC 35837 [ethambutol-resistant]) were tested. Inoculums of McFarland standard of 0.5, 1, 2, 3, and 1:100 dilutions of 1 McFarland standard of each strain were used. The effect of inoculum size on DST results was determined by the proportion method in Lowenstein-Jensen (LJ) medium and nitrate reductase assay (NRA) in the LJ medium. In both test methods, the increase in inoculum size did not affect the DST results of the strains. On the contrary, DST results were obtained more rapidly as a result of the use of dense inoculum. DST results obtained in all McFarland turbidities were found to be 100% compatible with the recommended amount of inoculum, 1:100 dilution of 1 McFarland standard (inoculum size of gold standard method). In conclusion, the use of a high amount of inoculum did not change the drug susceptibility profile of tuberculosis bacilli. Minimizing manipulations during the inoculum preparation phase of susceptibility testing, this outcome will decrease the need for equipment and make the test application easier, particularly in developing countries. IMPORTANCE During DST application, it can be challenging to evenly homogenize TB cell clumps with lipid-rich cell walls. These experiments must be carried out under Biosafety Level-3 (BSL-3) laboratory conditions with personal protective equipment and taking safety precautions because the procedures applied at this stage cause the formation of bacillus-laden aerosols and carry a serious risk of transmission. Considering this situation, this stage is important given that it is not possible to establish a BSL-3 laboratory in poor and developing countries. Reducing the manipulations to be applied during the preparation of bacterial turbidity will minimize the risk of aerosol formation. Perhaps there will be no need to do these steps for susceptibility tests in these countries or even in developed countries.

Efflux pump effects on Mycobacterium tuberculosis drug resistance.

Resistance and tolerance to antituberculosis drugs have become serious problems in disease treatment. This multi-phase study investigated the contributions of efflux pumps to Mycobacterium tuberculosis drug resistance. In the first phase, the minimum inhibitory concentration (MIC) levels of antibiotics were determined. In the second phase, MIC levels were determined in the presence of the efflux pump inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP), verapamil, reserpine and thioridazine. In the third phase, MIC levels were reduced in 6 M. tuberculosis isolates in the presence of efflux pump inhibitors to determine the expression of putative efflux pump genes by reverse transcriptase-polymerase chain reaction (RT-PCR). MIC levels of fluoroquinolones decreased in 6 (6.52%) isolates, MIC of rifampicin in 4 (4.34%), and MIC of streptomycin in 3 (3.26%) in the presence of efflux pump inhibitors reserpine, CCCP and verapamil. The efflux pump inhibitors CCCP, verapamil, and reserpine changed MICs 2- to 16-fold. Overexpression of all 15 efflux pump genes was observed in 6 isolates with a reduction in MIC values in the presence of efflux pump inhibitors. The overexpression of efflux-related genes in resistant isolates suggests that efflux pumps are associated with resistance development.

Cytotoxicity of Acrylic Resins, Particulate Filler Composite Resin and Thermoplastic Material in Artificial Saliva with and without Melatonin.

There is limited information on the effect of melatonin on the cytotoxicity of dental materials. The study evaluated the cytotoxic effects of heat- and auto-polymerized acrylic resin, particulate filler composite resin and a thermoplastic material on L-929 fibroblast cell viability at different incubation periods in artificial saliva without and with melatonin. Disk-shaped specimens were prepared according to each manufacturer's instructions and divided into two groups to be stored either in artificial saliva (AS) and AS with melatonin (ASM). The measurements were performed using an MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide) assay, in which the L-929 mouse fibroblasts cell culture was used. For the MTT test, extracts were examined at 1, 24, 72 h and 1 and 2 weeks. Data were analyzed using 3-way ANOVA and Tukey's tests. No significant difference was found between groups AS and ASM (F = 0.796; p = 0.373). Incubation period significantly affected all materials tested (p < 0.001). Storing resin-based materials in artificial saliva with melatonin solution for 24 h may reduce cytotoxic effects on the fibroblast cells for which the highest effect was observed. Soaking resin prosthesis or orthodontic appliances in artificial saliva with melatonin at least 24 h before intraoral use or rinsing medium containing melatonin may be recommended for decreasing the cytotoxicity of dental resin materials.

Multicenter evaluation of AYC.2.2 agar for the isolation of mycobacteria from clinical samples.

PURPOSE: The aim of this multicenter study is to evaluate AYC.2.2 agar for the isolation of mycobacteria from clinical samples. METHODS: Totally 5559 media were tested in 7 centers. AYC.2.2 agar media for the study were prepared by C1 and sent to other centers under appropriate conditions. Other media except AYC.2.2 agar were purchased commercially. The media were subjected to routine laboratory operations in the center where they were sent. After the samples received for routine processing (in all centers, samples were processed with the same method (NALC-NaOH)), they were cultivated on routine media and AYC.2.2 agar afterward. RESULTS: C1: Average growth time was determined as 12.74±3.74 days with MGIT 960 system; 24.42±4.75 days with LJ and 24.37±4.96 days with AYC.2.2 agar. C2: Average growth time was determined as 18.25±9.32 days with TK-Medium, 28.73±7.44 days with LJ, and 31.72±6.35 days with AYC.2.2 agar. C3: Average growth time was determined as 20.48±7.24 days with Ogawa medium, 20.74±7.12 days with LJ, and 20.26±7.43 days with AYC.2.2 agar. C4: Average growth time was determined as 15.27±6.37 days with MGIT 960 system, 22.14±9.1 days with LJ, and 22±8.45 days with AYC.2.2 agar. C5: Average growth time was determined as 13±4.24 days with MGIT 960 system, 32.16±6.23 days with LJ, and 33±5.73 days with AYC.2.2 agar. C6: Average growth time was determined as 9±3.11 days with MGIT 960 system, 18.68±5.32 days with LJ, and 18.34±4.63 days AYC.2.2 agar. C7: Average growth time was determined as 14.74±7.65 with MGIT 960 system, 26.01±8.21 days with LJ, and 26.24±7.88 days with AYC.2.2 agar. CONCLUSIONS: In conclusion, similar results were obtained with LJ and Ogawa media and AYC.2.2 agar. Furthermore, more studies should be conducted for isolation of M. tuberculosis and performing antibiotic susceptibility tests using AYC.2.2 agar before it can be used as a routine media in the laboratories.

Rapid thermal inactivation of aerosolized SARS-CoV-2.

Airborne transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the leading mechanisms of spread, especially in confined environments. The study aims to assess the thermal inactivation of SARS-CoV-2 at high temperatures in the time scale of seconds. An electric heater with a coiled resistance wire is located perpendicularly to the airflow direction inside an air tunnel. The airflow rate through the tunnel was 0.6 m3/h (10 L/ min). SARS-CoV-2 were suspended in Dulbecco's modified Eagle's medium (DMEM) with 10 % fetal bovine serum (FBS), aerosolized by a nebulizer at a rate of 0.2 L/min and introduced to the airflow inside the heater with the use of a compressor and an aspirator. In the control experiment, with the heater off, SARS-CoV-2 passed through the system. In the virus inactivation test experiments, the heater's outlet air temperature was set to 150 ± 5 °C and 220 ± 5 °C, and the air traveling through the tunnel was exposed to heat for 1.44 s. An inline gelatine filter harvested SARS-CoV-2 that passed through the system. The viral titer obtained from the gelatine filter in the control experiment was about 5.5 log10 TCID50. The virus's loss in viability in test experiments at 150 °C and 220 °C were 99.900 % and 99.999 %, respectively. The results indicate that high-temperature thermal inactivation substantially reduces the concentration of SARS-CoV-2 in the air within seconds.

A novel agar base medium for drug susceptibility testing of Mycobacterium tuberculosis isolates.

Aim: In this study, it was aimed to evaluate AYC.2.2 agar for susceptibility testing of Mycobacterium tuberculosis clinical isolates against first-line drugs. Materials & methods: In the present study, 208 M. tuberculosis clinical isolates were tested on AYC.2.2 agar, which was previously validated for the first-line drugs isoniazid, rifampicin, streptomycin and ethambutol. Results: Specificity, sensitivity, positive predictive value, negative predictive value and agreement for isoniazid-rifampicin-ethambutol-streptomycin were 100-100-97.2-99.3%, 94.8-94.8-79.3-94.3%, 100-100-82.1-98.03%, 97.03-98.03-96.7-98.08%, 98.07-98.5-94.7-98.07%, respectively. Conclusion: Results had shown that the newly developed AYC.2.2 agar promises as an alternative medium that can be used to perform susceptibility testing of M. tuberculosis isolates. However, further multicenter studies are needed to be used in routine mycobacteriology laboratories.

Validation of a novel medium for drug susceptibility testing of Mycobacterium Tuberculosis against First- and Second-Line drugs: AYC.2.2 Agar and AYC.2.1 Broth.

Background: The aim of this study was the validation of AYC.2.2 agar and AYC.2.1 broth for the breakpoint values of first- and second-line drugs for Mycobacterium tuberculosis. Method: A total of 12 isolates including 5 reference strains and 7 well-defined clinical isolates were tested for their antituberculosis susceptibilities. Inhibitory effects of first- and second-line antituberculous drugs including isoniazid, rifampicin, streptomycin, ethambutol, amikacin, capreomycin, kanamycin, para-aminosalicylic acid, ethionamide, rifabutin, ofloxacin, levofloxacin, and moxifloxacin were tested. Results: According to the minimal inhibitory concentration values obtained in 7H10 agar, 7H9-S broth, AYC.2.2 agar, and AYC.2.1 broth, category agreement is 100%, and very major discrepancy (MAD), MAD, and minor discrepancy ratios were determined as 0 for all drugs. Conclusion: It was concluded that breakpoint values by CLSI recommendation for 7H10 agar can be also used for AYC.2.2 agar and AYC.2.1 broth. In addition, further multicenter studies are needed to use the new medium in routine mycobacteriology laboratories.

Tuberculosis and beta-lactam antibiotics.

Tuberculosis (TB) is one of the oldest health problems in the world and it remains unresolved. Multidrug-resistant-TB and extensively resistant-TB are a serious problem for control programs. The evaluation of available antibiotics has gained importance in recent years for the treatment of resistant TB. Beta-lactam antibiotics inhibit cell wall biosynthesis in the bacteria; the presence of beta-lactamase enzyme in TB bacilli raises the question of whether this group of antibiotics can be used in treatment. As a result, it has been reported that the combination of beta-lactam antibiotics with beta-lactamase is effective against Mycobacterium tuberculosis both in vitro and in vivo. The aim of this article is to review and discuss up-to-date knowledge and future perspective on beta-lactam antibiotics and TB.

Colorimetric methods for rapid determination of pyrazinamide resistance.

Background: Because pyrazinamide (PZA) is only effective for Mycobacterium tuberculosis at an acidic pH, susceptibility tests are more difficult to perform than those for other anti-tuberculosis (TB) drugs. The purpose of our work was to investigate the effectiveness of colorimetric methods to detect PZA susceptibility and to detect pncA gene mutations in resistant isolates by sequence analysis. Methods: In this study, 30 clinical isolates and 2 reference isolates were used, 15 of which were resistant to PZA. The PZA susceptibility of all the isolates was determined by the BACTEC MGIT 960 reference method. As colorimetric methods, Resazurin Microtiter Assay (REMA), Nitrate Reductase Assay (NRA), Malachite Green Decolorization Assay (MGDA), and Crystal Violet Decolorization Assay (CVDA) methods were included in the study. In addition, mutations in the pncA gene were investigated using sequence analysis in PZA-resistant isolates. Results: As a result of the comparison of the colorimetric methods with the reference method, agreement was determined as 93.3% in REMA and NRA, 90% in MGDA, and 93.3% in CVDA. In 13 of 15 resistant isolates, the pncA gene mutation was detected by sequence analysis. Conclusions: As a result of the work, the results from the colorimetric methods were found to be at a high level of concordance with the reference method. They are also inexpensive and easily applicable methods.

A new colorimetric method for rapid detection of ethambutol and streptomycin resistance in Mycobacterium tuberculosis: crystal violet decolorization assay (CVDA).

Streptomycin (STR) and ethambutol (EMB) are important drugs used for the treatment of tuberculosis. There is a need for fast, reliable and inexpensive methods for detecting resistance to these drugs. The aim of this study was to evaluate the performance of the crystal violet decolorization assay (CVDA) for the detection of STR and EMB resistance that is important drugs in tuberculosis treatment. In this study, drug susceptibility testing was performed on 140 Mycobacterium tuberculosis isolates provided from nine centers. Three tubes were used for each isolate. One of the tubes had a concentration of 2 mg/L STR and the other 5 mg/L EMB. The third was drug-free control tube. Sensitivity, specificity, positive predictive value (PPD), negative predictive value (NPD) and agreement for STR were found to be 81.8%, 94.6%, 87.8%, 91.5% and 90.57%, respectively. For EMB, sensitivity, specificity, PPD, NPD, and agreement were found to be 76%, 98.23%, 90.47%, 94.87% and 94.2%, respectively. The results were obtained in 11.3 ± 2.7 days (8-21 days). CVDA is rapid, reliable, inexpensive, and easy to perform for rapid detection of STR and EMB resistance, and it could be adapted for drug susceptibility testing.

Crystal violet decolorization assay for rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates: A multicenter study.

Background: Effective control of tuberculosis is achieved by early diagnosis and drug susceptibility testing for initiation of appropriate treatment. The performance of crystal violet decolorization assay (CVDA) for susceptibility testing of Mycobacterium tuberculosis to isoniazid (INH) and rifampicin (RIF) was compared in a multicenter study. Methods: Seventy-two M. tuberculosis isolates were tested in two phases by CVDA. Results: In Phase I, the specificity, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and agreement for INH were 100%, respectively. Specificity, sensitivity, PPV, NPV, and agreement for RIF were 98.2%, 100%, 94.1%, 100%, and 98.6%, respectively. In Phase II, specificity, sensitivity, PPV, NPV, and agreement were 98%, 100%, 95.4%, 100%, and 98.6% for INH, respectively. Specificity, sensitivity, PPV, NPV, and agreement for RIF were 96.3%, 88.2%, 88.2%, 96.3%, and 94.4%, respectively. Results in the study were obtained on average 10.9 ± 3.1 days in Phase I and 9.8 ± 2.2 days in Phase II. Conclusion: CVDA can be performed for drug susceptibility testing in developed and developing countries. In addition, further studies with larger sample size are needed for evaluation of this method.

An evaluation of false-positive rifampicin resistance on the Xpert MTB/RIF.

BACKGROUND: Mycobacterium tuberculosis (MTB) is one of the most significant causes of mortality and morbidity. Early diagnose is important especially in multiple drug resistant tuberculosis to avoid transmission. Traditional techniques requires at least one to three weeks for diagnosis of tuberculosis. Diagnostic delays with multiple drug resistant tuberculosis are associated with worse clinical outcomes and increased transmission The Xpert MTB/RIF assay is one of the new diagnostic device for the diagnosis of tuberculosis and rapid detection of rifampicin resistance. OBJECTIVE: We assessed the performance of Xpert MTB/RIF assay for detecting rifampicin resistance using phenotypic drug susceptibility tests as automated BD MGIT 960. METHODS: Total of 2136 specimens were included in the study. Xpert MTB/RIF testing was performed on samples, using version 4 cartridges, according to the manufacturer's recommendations. The MTBC culture and first-line phenotypic DST were performed in automated BD MGIT 960 (Becton & Dickinson, USA) according to the recommendations of the manufacturer. Agar proportion was used in the case of inconsistency for rifampicin resistance. FINDINGS: Thirty-four samples (19 respiratory and 15 nonrespiratory samples) were determined as positive for M. tuberculosis complex by Xpert MTB/RIF (Cepheid GeneXpert® System, USA). Xpert MTB/RIF assay detected 4/34 (11.7%) specimens as rifampicin resistant. One of the rifampicin resistant isolates was determined susceptible in MGIT 960 automated system. This isolate was also tested with agar proportion method and found susceptible to rifampicin. MAIN CONCLUSION: The Xpert MTB/RIF assay can be used as first-line assay for the detection of M. tuberculosis. However, microbiologists must be aware of the limitations of the assay.

An evaluation of false-positive rifampicin resistance on the Xpert MTB/RIF

BACKGROUND Mycobacterium tuberculosis (MTB) is one of the most significant causes of mortality and morbidity. Early diagnose is important especially in multiple drug resistant tuberculosis to avoid transmission. Traditional techniques requires at least one to three weeks for diagnosis of tuberculosis. Diagnostic delays with multiple drug resistant tuberculosis are associated with worse clinical outcomes and increased transmission The Xpert MTB/RIF assay is one of the new diagnostic device for the diagnosis of tuberculosis and rapid detection of rifampicin resistance. OBJECTIVE We assessed the performance of Xpert MTB/RIF assay for detecting rifampicin resistance using phenotypic drug susceptibility tests as automated BD MGIT 960. METHODS Total of 2136 specimens were included in the study. Xpert MTB/RIF testing was performed on samples, using version 4 cartridges, according to the manufacturer's recommendations. The MTBC culture and first-line phenotypic DST were performed in automated BD MGIT 960 (Becton & Dickinson, USA) according to the recommendations of the manufacturer. Agar proportion was used in the case of inconsistency for rifampicin resistance. FINDINGS Thirty-four samples (19 respiratory and 15 nonrespiratory samples) were determined as positive for M. tuberculosis complex by Xpert MTB/RIF (Cepheid GeneXpert® System, USA). Xpert MTB/RIF assay detected 4/34 (11.7%) specimens as rifampicin resistant. One of the rifampicin resistant isolates was determined susceptible in MGIT 960 automated system. This isolate was also tested with agar proportion method and found susceptible to rifampicin. MAIN CONCLUSION The Xpert MTB/RIF assay can be used as first-line assay for the detection of M. tuberculosis. However, microbiologists must be aware of the limitations of the assay.

Multicenter evaluation of crystal violet decolorization assay (CVDA) for rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis.

The aim of this multicenter study was to evaluate the performance of the crystal violet decolorization assay (CVDA) for detection of multidrug resistant tuberculosis (MDR-TB). This study was performed in 11 centers in two phases. A total of 156 isolates were tested for INH and RIF resistance. In the phase I, 106 clinical isolates were tested in the Center 1-7. In the phase 2, 156 clinical isolates were tested in the center 1-6, center 8-11. Eighty six of 156 tested isolates were the same in phase I. Agreements were 96.2-96.8% for INH and 98.1-98.7% for RIF in the phase I-II, respectively. Mean time to obtain the results in the phase I was 14.3 ± 5.4 days. In the phase II, mean time to obtain the results was 11.6 ± 3.5 days. Test results were obtained within 14days for 62.3% (66/106) of isolates in the phase I and 81.4% (127/156) of isolates in the phase II. In conclusion, CVDA is rapid, reliable, inexpensive, and easy to perform for rapid detection of MDR-TB isolates. In addition, it could be adapted for drug susceptibility testing with all drugs both in developed and developing countries.

Evaluation of crystal violet decolorization assay for minimal inhibitory concentration detection of primary antituberculosis drugs against Mycobacterium tuberculosis isolates

In this study we evaluated the crystal violet decolorization assay (CVDA) for detection of minimum inhibitory concentration (MIC) of antituberculosis drugs. 53 isolates were tested in this study and 13 of them were multidrug resistant (MDR) isolates. The antibiotics concentrations were 2-0.06 mg/L for isoniazid (INH) and rifampicin (RIF) and were 16-0.25 mg/L for streptomycin (STM) and ethambutol (EMB). Crystal violet (CV-25 mg/L) was added into the microwells on the seventh day of incubation and incubation was continued until decolorization. Decolorization of CV was the predictor of bacterial growth. Overall agreements for four drugs were detected as 98.1%, and the average time was detected as 9.5 ± 0.89 day after inoculation. One isolate for INH and two isolates for STM were determined resistant in the reference method, but susceptible by the CVDA. One isolate was susceptible to EMB by the reference method, but resistant by the CVDA. All results were concordant for RIF. This study shows that CVDA is a rapid, reliable and suitable for determination of MIC values of Mycobacterium tuberculosis. And it can be used easily especially in countries with limited-sources.

Evaluation of crystal violet decolorization assay for minimal inhibitory concentration detection of primary antituberculosis drugs against Mycobacterium tuberculosis isolates.

In this study we evaluated the crystal violet decolorization assay (CVDA) for detection of minimum inhibitory concentration (MIC) of antituberculosis drugs. 53 isolates were tested in this study and 13 of them were multidrug resistant (MDR) isolates. The antibiotics concentrations were 2-0.06 mg/L for isoniazid (INH) and rifampicin (RIF) and were 16-0.25 mg/L for streptomycin (STM) and ethambutol (EMB). Crystal violet (CV-25 mg/L) was added into the microwells on the seventh day of incubation and incubation was continued until decolorization. Decolorization of CV was the predictor of bacterial growth. Overall agreements for four drugs were detected as 98.1%, and the average time was detected as 9.5 ± 0.89 day after inoculation. One isolate for INH and two isolates for STM were determined resistant in the reference method, but susceptible by the CVDA. One isolate was susceptible to EMB by the reference method, but resistant by the CVDA. All results were concordant for RIF. This study shows that CVDA is a rapid, reliable and suitable for determination of MIC values of Mycobacterium tuberculosis. And it can be used easily especially in countries with limited-sources.

[Multicenter evaluation of the indirect nitrate reductase assay for the rapid detection of multidrug-resistant tuberculosis]. / Çok ilaca dirençli tüberkülozun hizli tespiti için dolayli nitrat redüktaz testinin çok merkezli degerlendirilmesi.

Multidrug-resistant tuberculosis (MDR-TB) is defined as resistance to at least isoniazid (INH) and rifampicin (RIF), and it complicates the implementation of tuberculosis control programmes. The rapid detection of MDR-TB is crucial to reduce the transmission of disease. The nitrate reductase assay (NRA) is one of the colorimetric susceptibility test methods for rapid detection of MDR-TB and based on the ability of reduction of nitrate to nitrite by Mycobacterium tuberculosis. The aim of this study was to evaluate the performance of the NRA for the rapid detection of MDR-TB. A total of 237 M.tuberculosis complex (MTC) isolates that were identified by the same method (BD MGIT(TM) TBc Identification Test, USA) from nine different medical centers in Turkey were included in the study. The susceptibility results of the isolates against INH and RIF obtained by reference test (Bactec MGIT(TM) 960, BD, USA) were then compared with NRA. In order to ensure consistency between centers, Löwenstein-Jensen (LJ) medium with antibiotics and without antibiotics (growth control) and Griess reagent solution were prepared in a single center (Ondokuz Mayis University School of Medicine, Medical Microbiology Department) and sent to all participant centers with the standardized test procedure. After the inoculation of bacteria into the test tubes, the tubes were incubated at 37°C, and after seven days of incubation, 500 µl Griess reagent was added to the LJ medium without antibiotics. If a color change was observed, an equal volume of Griess reagent was added to test LJ media with antibiotics. When a color change was observed in LJ media with antibiotics, it was considered that the isolate was resistant to tested antibiotics. Among 237 MTC isolates, 16 were resistant only to INH and nine were resistant only to RIF; 93 isolates (39.2%) were resistant (MDR) and 119 isolates (50.2%) were susceptible to both of the drugs determined with the reference susceptibility test. In the study, five INH-resistant isolates determined with reference method were found susceptible with NRT and eight INH-susceptible isolates determined with reference method were found resistant with NRT. In contrast, one RIF-resistant isolate determined with reference method was found susceptible with NRT and three RIF-susceptible determined isolates were found resistant with NRT. Accordingly, the concordance rate between the reference method and NRA were estimated as 94.5% for INH and 98.3% for RIF. The sensitivity, specificity, positive and negative predictive values of NRA were detected as 95.4%, 93.7%, 92.8% and 96% for INH, and 99%, 97.8%, 97.1% and 99.2% for RIF, respectively. The results of the 111 isolates were obtained on the seventh day, while the rest of the results were obtained between 10-14 days. In conclusion, the data of this multicenter study showed that NRA is a reliable, relatively inexpensive and practical method to perform for the rapid detection of MDR-TB.