Prospective multicentre evaluation of the direct nitrate reductase assay for the rapid detection of extensively drug-resistant tuberculosis.

OBJECTIVES: To perform a multicentre study evaluating the performance of the direct nitrate reductase assay (NRA) for the detection of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis in sputum samples. METHODS: The study was conducted in six laboratories performing tuberculosis diagnosis that were located in six different countries. The NRA was performed directly on sputum samples in parallel with the reference method used at each site. Detection of resistance was performed for rifampicin, isoniazid, ofloxacin and kanamycin. RESULTS: Excellent agreement was obtained for all drugs tested at the majority of sites. The accuracy was 93.7%-100% for rifampicin, 88.2%-100% for isoniazid, 94.6%-100% for ofloxacin and 100% for kanamycin. The majority of NRA results were available at day 21 for sites 1, 2 and 5. Site 3 had a turnaround time of 13.9 days, at site 4 it was 18.4 days and at site 6 it was 16.2 days. The contamination rate ranged between 2.5% and 12%. CONCLUSIONS: Rapid detection of drug resistance by the direct NRA on sputum smear-positive samples was accurate and easy to implement in clinical diagnostic laboratories, making it a good alternative for rapid screening for MDR and XDR tuberculosis.

[In Vitro Effect of Ankaferd Blood Stopper®, a Plant Extract Against Mycobacterium tuberculosis Isolates]. / Bir Bitki Ekstresi Olan Ankaferd Blood Stopper®'in Mycobacterium tuberculosis Izolatlarina Karsi In Vitro Etkinligi.

Treatment of drug-resistant Mycobacterium tuberculosis infections requires combination of anti-tuberculosis drugs which have several toxic side effects. Thus there is a need for safer and effective new drugs. Ankaferd Blood Stopper® (ABS), which is a mixture of plant extracts prepared from Alpinia officinarum, Glycyrrhiza glabra, Thymus vulgaris, Urtica dioica and Vitis vinifera, has homeostatic and antibacterial effects. Standard solutions of ABS are already being used topically for post-traumatic and post-operative bleeding control in our country. This study was aimed to evaluate the in vitro activity of ABS against M.tuberculosis isolates. A total of 57 clinical isolates [17 multidrug resistant (MDR), 11 resistant to only isoniazid (INH), one resistant to INH and streptomycin (STR), two resistant only to STR, two resistant only to ETM, and 24 susceptible to all drugs] and three standard strains [H37Rv (susceptible to all drugs), ATCC 35822 (INH-resistant), ATCC 35820 (STR-resistant)] were included in the study. Agar dilution method was used to detect the MIC values of ABS. In the study, ABS MIC value was determined as 10.94 µg/ml for M.tuberculosis H37Rv strain which was susceptible to all anti-tuberculosis drugs, whereas it was determined as 21.88 µg/ml for INH-resistant ATCC 35822 and STR-resistant ATCC 35820 strains. The MIC values for 24 susceptible clinical isolates were as follows; 10.94 µg/ml (n= 17), 21.88 µg/ml (n= 6) and < 1.37 µg/ml (n= 1). When evaluating 17 MDR clinical isolates, MIC values were determined as 5.47 µg/ml (n= 1), 10.94 µg/ml (n= 5) and 21.88 µg/ml (n= 11). MIC values were ranging between < 1.37-21.88 µg/ml among 11 INH-resistant isolates. These isolates were susceptible to other first line anti-tuberculosis drugs. MIC value of one isolate resistant to both of INH and STR was determined as 21.88 µg/ml. MIC value of the two sole STR-resistant isolates was 21.88 µg/ml. MIC values of the two sole ETM-resistant isolates were determined as 21.88 µg/ml and 10.94 µg/ml. MIC50 and MIC90 values for the tested bacteria were 10.94 µg/ml and 21.88 µg/ml, respectively. It was concluded that 16 fold diluted concentration of the topically used ABS solution was found to be active against tuberculosis bacilli in vitro. Thus ABS might be used as a supportive agent together with anti-tuberculous drugs during debridement of multiple drug-resistant M.tuberculosis caused osteomyelitis and lymphadenitis lesions.

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.

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% for both INH and RIF. The sensitivity, specificity, positive predictive value and negative predictive value for INH were 92.8%, 94.2%, 86.6% and 97%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for RIF were 90.4%, 96.4%, 95% and 93.1%. In conclusion, we show here that blood agar can be used effectively for the NRA test.

[Evaluation of blood agar medium for the growth of mycobacteria]. / Mikobakterilerin Üretilmesinde Kanli Agar Besiyerinin Degerlendirilmesi.

This study was aimed to evaluate the performance of blood agar for the growth of mycobacteria from clinical specimens sent to Mycobacteriology Laboratory of Samsun Chest Diseases Hospital. One hundred fifty six clinical specimens including 123 sputum, 28 bronchoalveolar lavage (BAL) and 5 pleural fluid specimens were inoculated in Löwenstein-Jensen (LJ), BACTEC MGIT 960 system (Becton Dickinson, USA) and blood agar following decontamination process. The specimens were also simultaneously examined for the presence of acid-fast bacilli (AFB). Thirty five mycobacteria strains (33 Mycobacterium tuberculosis and 2 atypical mycobacteria) grew in blood agar, 38 (36 M.tuberculosis and 2 atypical mycobacteria) in LJ media and 46 (44 M.tuberculosis and 2 atypical mycobacteria) in BACTEC MGIT 960 system. Among 29 AFB negative specimens, 20 revealed growth in both blood agar and LJ medium and 27 in MGIT system. AFB positive 20 samples yielded growth in 15 samples in blood agar, 18 in LJ medium and 19 in MGIT system. Among the total of 156 samples, contamination was observed in 15 (9.6%) samples in blood agar, 16 (10.2%) in LJ medium and 18 (11.5%) in MGIT system. Growth time was 5-35 days (mean 18 ± 7.4), 11-35 days (mean 19 ± 5.9) and 5-15 days (mean 10 ± 2.4) for blood agar, LJ medium and BACTEC MGIT 960 system, respectively. The three samples which revealed contamination in BACTEC MGIT 960 system, grew successfully in both blood agar and LJ medium without contamination. In one sample, growth was observed only in LJ medium but neither in blood agar nor BACTEC MGIT 960 system. However, in another sample, growth was observed only in blood agar while no growth was detected in LJ or BACTEC MGIT 960 system. Six samples yielded mycobacteria only in BACTEC MGIT 960 system. These results indicated that simultaneous use of one liquid and one solid medium to grow mycobacteria from the clinical samples seemed to be complementary. Blood agar was a promising choice since it was found to be as effective as LJ medium for the growth of mycobacteria, however, this issue needs to be further evaluated in a multicentre study with a larger specimen collection.

Resistance for anti-tuberculosis drugs in central Black Sea region of Turkey.

One of the primary aims in tuberculosis (TB) management is to detect new cases as early as possible, and instigate the most appropriate therapy, for which it is important to know the characteristics of TB drug resistance in society. The aim of our study was to determine the resistance status of tuberculosis in the Samsun region of Turkey. To achieve that, the medical records of 1,029 pulmonary tuberculosis patients admitted to Samsun Chest Diseases and Chest Surgery Hospital between 2004 and 2006 were analyzed for drug resistance characteristics. In order to define the problem, isolates were tested on Lowenstein-Jensen medium. For drug susceptibility testing, isoniazid (I), streptomycin (S), ethambutol (E), rifampicin (R) and the radiometric Bactec 460 TB system were used. Eighty-six percent (86%) of the cases (623/721) were new patients, and 13.5% (98/721) were previously treated cases. One hundred and thirty-four (134) of the 721 patients (18.6%) had resistance to one or more drugs. Resistance to any drug was determined in 16.9% (105/623) cases of new patients. I resistance was 13.2%, any R resistance was 2.9%, and multi-drug resistance (MDR) was 1.9%. In previously treated cases, resistance to any drug was 29.6%, any I resistance was 26.5%, any R resistance was 15.3%, and MDR was 13.3%. It was concluded that resistance to anti-tuberculosis drugs is an important problem in Samsun.

Comparative study for determination of Mycobacterium tuberculosis susceptibility to first- and second-line antituberculosis drugs by the Etest using 7H11, blood, and chocolate agar.

We investigated the performance of blood and chocolate agar as alternatives to Middlebrook 7H11 agar for testing the susceptibility of Mycobacterium tuberculosis to first-and second-line drugs by the Etest method. A total of 39 strains of M. tuberculosis including 22 multidrug-resistant M. tuberculosis strains and 17 susceptible strains were tested. In conclusion, our results showed that chocolate agar gave insufficient growth, needing up to 21 days of incubation, while results on blood agar were comparable to those on Middlebrook 7H11 agar and can be further explored as an alternative for Etest-based susceptibility testing of M. tuberculosis.

[Susceptibility testing of Mycobacterium tuberculosis isolates to primary antituberculous drugs on chocolate agar: a preliminary study]. / Mycobacterium tuberculosis izolatlarinin primer antitüberküloz ilaçlara duyarliliklarinin çikolatali agarda saptanmasi: Bir ön çalisma.

The aim of this study was to evaluate the use of chocolate agar as an alternative medium instead of Middlebrook 7H10 agar, for the susceptibility testing of Mycobacterium tuberculosis strains against isoniazid (INH), rifampicin (RIF), streptomycin (STR) and ethambutol (ETM). The susceptibility results obtained by chocolate agar were compared with the results of BACTEC 460 TB (Becton Dickinson, Sparks, MD, USA) system which was accepted as the reference method. A total of 25 M. tuberculosis clinical isolates were included to the study and susceptibility testing was performed on malachite green added-chocolate agar with some modifications of proportion method recommended by NCCLS. In our study when comparing the results obtained by chocolate agar with the results of BACTEC 460 TB system, the concordance rates for INH, STR, RIF and ETM were found as 88%, 88%, 84% and 72%, respectively. The specificity, sensitivity, positive and negative predictive values of susceptibility testing on chocolate agar have been detected as 82.3%, 100%, 72.7% and 100% for INH; 78.5%, 100%, 78.5% and 100% for RIF; 83.3%, 84.2%, 94.1% and 62.5% for STR; 25%, 94.1%, 72.7% and 66.6% for ETM, respectively. The results of the susceptibility testing performed on chocolate agar were obtained on the 21st day of incubation for all isolates. In conclusion, the data from our study suggested that chocolate agar can be used as an alternative medium for the susceptibility testing of M. tuberculosis, however, further studies with more isolates are needed for the standardisation of the method.

[Evaluation of phenol ammonium sulfate sedimentation method for diagnosis of pulmonary tuberculosis]. / Fenol amonyum sülfat sedimentasyon yönteminin pulmoner tüberküloz tanisi için degerlendirilmesi.

In the study, the results of culture have been accepted as a gold standard and the specificity and sensitivity of phenol ammonium sulfate sedimentation method has been evaluated. When it is evaluated according to the results of culture, it has been found that the specificity and sensitivity of phenol ammonium sulfate sedimentation method is 90%, the specificity of the process which is made through the N-acetyl-L-cysteine NaOH method is 90% and the sensitivity of it is 85%. In conclusion, the phenol ammonium sulfate method seems to be as a secure method that can only be used in the laboratories in which microscopic studies are made.

[Histopathologically diagnosed pulmonary complicated hydatid cyst cases]. / Histopatolojik olarak tani konulan komplike akciger kist hidatik olgulari.

OBJECTIVE: Hydatid cyst disease is caused by the metacestod form of Echinococcosis granulosus from cestods. Pulmonary hydatid cyst is the second most frequent form of the disease after the liver involvement and may open into the bronchial or pleural space by perforation and may cause complications. The aim of the study was to evaluate the clinical features and the frequency of the complicated pulmonary hydatid cyst disease. METHODS: Fifteen hydatid cyst patients were evaluated according to socio-demographical, clinical and radiological findings between 2009 and 2011 retrospectively. Hydatid cyst diseases were diagnosed histopathologically after chest surgery. Diagnostic difficulties and clinical features were analysed in four complicated pulmonary hydatid cyst cases. RESULTS: Pneumothorax, pleural effusion, lung abscess, and hemoptysis were observed in four complicated cases. The complicated cases were diagnosed after surgery. Eleven of lung cysts were intact, radiological and histopathological features were typical for images of hydatid cyst disease and reported as compatible with the clinical diagnosis. A synchronized liver and pulmonary hydatid cyst was evaluated as a morbidity factor. CONCLUSION: Hydatid cyst should be considered in the differential diagnosis of uncertain chest pathologies, especially in rural areas where the disease is endemic.

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.

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.

Two new colorimetric methods for early detection of vancomycin and oxacillin resistance in Staphylococcus aureus.

We developed two colorimetric methods for the detection of vancomycin- and oxacillin-resistant Staphylococcus aureus in

Susceptibilities of Mycobacterium tuberculosis to isoniazid and rifampin on blood agar.

In this study, blood agar was used instead of 7H10 agar for the susceptibility testing of 34 clinical isolates of Mycobacterium tuberculosis to isoniazid (INH) and rifampin (RIF) in accordance with the NCCLS. The BACTEC 460 TB system (Becton Dickinson, Sparks, Md.) was used as a "gold standard." Results for both media were in agreement for RIF and INH at 100 and 94.1%, respectively. For INH, the specificity, sensitivity, positive predictive value, and negative predictive value were found to be 71.4, 100, 93.1, and 100%, respectively, while these values were 100% for RIF. In addition, the results of the susceptibility test performed with blood agar were obtained on day 14 of incubation. In conclusion, results were obtained much earlier with blood agar (2 weeks) than with 7H10 agar (3 weeks), and the results of this study suggest that blood agar may be used as an alternative medium for the susceptibility testing of M. tuberculosis to INH and RIF.