[Using of the Sensititre MycoTB Plate Method for the Detection of Anti-tuberculous Drug Susceptibility of Rifampin Resistant Mycobacterium tuberculosis Complex Isolates]. / Rifampisine Dirençli Mycobacterium tuberculosis Kompleks Izolatlarinin Antitüberküloz Ilaç Duyarliligi Için Sensititre MycoTB Plak Yönteminin Kullanilmasi.

Members of the Mycobacterium tuberculosis complex (MTBC) are the causative agents of tuberculosis (TB). Rifampin resistance in the clinical isolates of MTBC is an important indicator for multidrug resistant-TB (MDR-TB) cases. In this study, it was aimed to evaluate whether the Sensititre MycoTB plaque method is suitable for the routine use in determining drug susceptibility of rifampin resistant MTBC strains. Xpert MTBC/ RIF positive rifampin resistant 100 MTBC isolates were included in the study. Xpert MTBC/RIF (Cepheid, USA) test were performed after the samples were processed by homogenization and decontamination and acid-fast staining. Rifampin resistant clinical samples were cultured in automated MGIT/BACTEC 960 (Becton Dickinson, USA) system and acid fast bacteria (AFB) were investigated. The anti-TB drug susceptibility tests of all culture positive AFB and cord factor identified as MTBC by using a cart test (MPB64, Capilla TB-Neo, Tauns Laboratories, Inc., Numazu, Japan) were performed with the Löwenstein-Jensen proportion method (LJPM) and Sensititre MycoTB (Trek Diagnostic Systems, Cleveland, OH, USA) methods. For the comparison of the methods used, the tests were performed simultaneously. The standard LJPM was performed according to the previously described procedures by World Health Organization and the Sensititre MycoTB plate method was performed as defined by the manufacturer. The final concentrations of isoniazide, rifampin, rifabutin, ethambutol, ofloxacin, moxifloxacin amikacin, kanamycin, cycloserine, ethionamide and p-aminosalicylic acid in Löwenstein-Jensen media for LJPM were 0.2 µg/ml, 40.0 µg/ml, 20.0 µg/ml, 2.0 µg/ml, 2.0 µg/ml, 1.0 µg/ml, 4.0 µg/ml, 30.0 µg/ml, 30.0 µg/ml, 40.0 µg/ml, 40.0 µg/ml and 1.0 µg/ ml, respectively. The results were obtained in 14 days for all of the drugs in the Sensititre MycoTB plate method and in 28-42 days in the LJPM. In this study, the sensitivity and specificity percentages of the Sensititre MycoTB method and the categorical agreement between the two methods were calculated. The sensitivity and specificity percentages of the Sensititre MycoTB plate method were between 84.4-100% and 95.6- 100%, respectively. The categorical agreements between the two methods were 92-100% for the drugs tested in the study. Ethambutol was found to have the lowest sensitivity (84.4%) and specificity (95.6%). The sensitivities of isoniazide, ofloxacin, streptomycin, kanamycin, ethionamide and p-aminosalicylic acid were 98.8%, 90.0%, 94.3%, 87.5%, 91.7% and 95.6%, respectively, while rifampicin, rifabutin, moxifloxacin, amikacin, cycloserine were calculated as 100%. The specificities of isoniazid, rifampicin, rifabutin, ofloxacin, moxifloxacin, amikacin, kanamycin, and cycloserine were found to be 100%, streptomycin, ethionamide and p-aminosalicylic specificity were 96.9%, 97.4% and 98.9% respectively. The categorical agreement was 96-100% in all tested drugs except ethambutol (92%). As a result, although the cost is high, owing to the short incubation period, easy to perform, the possibility for evaluating both first and second line anti-TB drugs simultaneously, determination of minimum inhibitory concentration values of the drugs, long shelf life, high sensitivity, specificity and the categorical agreement values, the Sensititre MycoTB method was determined as an effective method that can be used especially in laboratories where the workload and the MDR-TB cases are high.

Optimization of isothermal amplification method for Mycobacteriumtuberculosisdetection and visualization method for fieldwork

Background/aim: Tuberculosis is still one of the most contagious diseases around the world. Key factors of tuberculosis control are rapid diagnostic, efficient treatment, and prevention of contamination by surveillance and monitoring. However, culture is the gold standard method for laboratory diagnosis of tuberculosis; the results are several weeks to obtain. In order to prevent contamination of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. The aim of this study is to optimize the loop-mediated isothermal amplification (LAMP) method, which provides a much faster and more sensitive result than the polymerase chain reaction (PCR) method and allows the replication of target nucleic acid sequences under isothermal conditions without the need for laboratory infrastructure. Materials and methods: Sputum samples were homogenized with 5% trypsin solution in CaCl2 to obtain DNA.DNA was purified using QIAGEN QIAamp DNA mini kit. LAMP primers were design using Primer explorer V5 program according to IS6110 gene of Mycobacterium tuberculosis. NEB Bst 3.0 DNA polymerase kit was used for LAMP reactions. Besides, LAMP reactions were compared with TaqMan based RT-PCR method using NEB's Taq polymerase kit. Finally, for visualization of LAMP products, lateral flow dipsticks that produced by Milenia Biotec, colorimetric 2X LAMP master mix that produced by NEB and 2% w/v agarose gel electrophoresis methods were used. Results: Optimum amplification temperature for LAMP was found to be 71.4 °C. The detection limit of the method was 102 CFU/mL and sensitivity was determined 100% compared to five different Mycobacterium species. Conclusion: The current study indicated that the LAMP-LFD and colorimetric LAMP protocol optimized with sputum samples can be reliable used as a rapid, sensitive and specific assay in the diagnosis of tuberculosis in the field.

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.

[Frequently Isolated Slow Growing Nontuberculous Mycobacteria from Pulmonary Samples and Evaluation of Drug Susceptibility Testing Results in a Referral Hospital in Turkey]. / Türkiye'de Bir Referans Hastanede Akciger Örneklerinden Siklikla Izole Edilen Yavas Üreyen Tüberküloz Disi Mikobakteriler ve Ilaç Duyarlilik Sonuçlarinin Degerlendirilmesi.

Most of the nontuberculous mycobacteria (NTM) are opportunistic pathogenic microorganisms and free-living in nature. NTM can cause a wide range of infections. However, pulmonary NTM disease is the most frequent clinical picture. The aim of this study was to identify and evaluate drug susceptibility of slow growing NTM isolated from pulmonary samples of patients prediagnosed as tuberculosis between 2014 and 2018 in Atatürk Chest Diseases and Chest Surgery Training and Research Hospital Microbiology Laboratory by a commercial microtube dilution plaque method. A total of 435 NTM strains obtained from suspected TB patients were included in the study. After the samples were processed by homogenization and decontamination and acid-fast staining, culture in two solid media (Löwenstein-Jensen, Ogawa) and in MGIT-BACTEC960 automated system were performed. Acid-fast bacilli isolated from culture media were identified by using cart test (MPB64, Capilla TB-Neo) and polymerase chain reaction (PCR) based reverse hybridization "line probe assay (LPA)" method (GenoType MycobacteriumCM/AS, Hain Lifescience, GmbH, Germany). After DNA isolation from the culture, PCR was performed by using the primers specific for mycobacterial 23S rRNA spacer region. PCR products were then hybridized with the probes specific for Mycobacterium species on nitrocellulose strips according to the recommendations of the manufacturer and the results were evaluated. In this study, Mycobacterium avium (n= 77, 17.7%), Mycobacterium intracellulare (n= 70, 16.1%), Mycobacterium szulgai (n= 19, 4.4%), Mycobacterium kansasii (n= 10, 2.3%) ve Mycobacterium smiae (n= 9, 2.1%) were isolated as slowly growing mycobacteria from the pulmonary patients. Susceptibility testing was performed in cation-adjusted Mueller-Hinton broth (CAMH), supplemented with "oleic acid, albumin dextrose catalase" according to CLSI/ M24-A2 guideline recommendations. For the antibiotic susceptibility test, ready-to-use plaque drugs for slow-growing mycobacteria (SLOMYCO-Sensititre, TREK Diagnostic Systems Ltd, UK), were used. M.intracellulare, M.avium, M.kansasii and M.smiae isolates were found to be sensitive to clarithromycin %100, %99, %100 and %100, respectively. For M.intracellulare and M.avium isolates, moxifloxacin and linezolid sensitivity values were found to be 91%, 64% and 80%, 74% respectively. M.kansasii isolates were more sensitive than M.simiae isolates to the most of the drugs. M.kansasii isolates, were susceptible to rifabutin, rifampin, moxifloxacin, amikacin, linezolid, trimethoprim-sulfamethoxazole (TMP-SMX), ciprofloxacin and etambutol, with the frequencies of 100%, 90%, 100%, 100%, 80%, 70% and 50%, respectively. The study showed that the species identification and drug susceptibility testing of frequently isolated slow-growing NTM's from pulmonary specimens could guide for the treatment.

[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.

[Tuberculosis Laboratory Surveillance Network (TuLSA) study group. The first step for national tuberculosis laboratory surveillance: Ankara, 2011]. / Ulusal Tüberküloz Laboratuvar Sürveyansina ilk adim: Ankara, 2011.

The most effective method for monitoring country-level drug resistance frequency and to implement the necessary control measures is the establishment of a laboratory-based surveillance system. The aim of this study was to summarize the follow up trend of the drug-resistant tuberculosis (TB) cases, determine the load of resistance and evaluate the capacities of laboratories depending on laboratory quality assurance system for the installation work of National Tuberculosis Laboratory Surveillance Network (TuLSA) which has started in Ankara in 2011. TuLSA studies was carried out under the coordination of National Tuberculosis Reference Laboratory (NRL) with the participation of TB laboratories and dispensaries. Specimens of TB patients, reported from health institutions, were followed in TB laboratories, and the epidemiological information was collected from the dispensaries. One isolate per patient with the drug susceptibility test (DST) results were sent to NRL from TB laboratories and in NRL the isolates were rechecked with the genotypical (MTBDRplus, Hain Lifescience, Germany) and phenotypical (MGIT 960, BD, USA) DST methods. Molecular epidemiological analysis were also performed by spoligotyping and MIRU/VNTR. Second-line DST was applied to the isolates resistant to rifampin. A total of 1276 patients were reported between January 1st to December 31th 2011, and 335 cases were defined as "pulmonary TB from Ankara province". The mean age of those patients was 43.4 ± 20 years, and 67.5% were male. Three hundred seventeen (94.6%) patients were identified as new cases. The average sample number obtained from pulmonary TB cases was 3.26 ± 2.88, and 229 (68.3%) of them was culture positive. DST was applied to all culture positive isolates; 90.4% (207/229) of cases were susceptible to the five drugs tested (ethambutol, isoniazid, pyrazinamide, rifampicin, streptomycin). Eight (3.5%) of the isolates were multidrug-resistant (MDR-TB), while no extensively drug-resistant strains were detected. MDR-TB is likely to occur in 63.3 times more among previously treated cases, and 73.3 times more in legal aliens. The achievement of therapy among pulmonary TB cases was 91.9%. Spoligotyping performed for 221 M.tuberculosis complex isolates, showed that all strains were clustered in nine groups. SIT 41 (105/221; 47.5%) was the most frequent spoligotype detected, and clustering rate based on MIRU-VNTR results were found as 16.3%. All of the clustered strains were sensitive while all of MDR-TB isolates showed specific MIRU-VNTR profiles. In conclusion, TuLSA studies started in Ankara in 2011 and the system is still expanding in the country. Our data obtained with TuLSA have been published as a regional surveillance data in the WHO Global Tuberculosis Report 2011, and as a national surveillance data in Global Tuberculosis Report 2012.

Distribution of nontuberculous Mycobacteria strains.

AIM: Mycobacteria other than tuberculosis (MOTT) cause increasingly serious infections especially in immunosuppressive patients by direct transmission from the environment or after colonization. However, identification of these species is difficult because of the cost and difficulties in defining to species level. Identification and distribution of these species can help clinician in the choice of treatment. MATERIALS AND METHODS: A total of 90 MOTT strains obtained from four different centers were included in the study. These strains were identified by sequence analysis of 16S rRNA and Hsp65 genetic regions. RESULTS: Accordingly, within the 90 MOTT strains, 17 different species were identified. In order of frequency, these species were M. gordonea (n = 21), M. abscessus (n = 13), M. lentiflavum (n = 9), M. fortuitum (n = 8), M. intracellulare (n = 6), M. kumamotonense (n = 6), M. neoaurum (n = 5), M. chimaera (n = 5), M. alvei (n = 5), M. peregrinum (n = 3), M. canariasense (n = 3), M. flavescens (n = 1), M. mucogenicum (n = 1), M. chelona (n = 1), M. elephantis (n = 1), M. terrae (n = 1) and M. xenopi (n = 1). Most frequently identified MOTT species according to the geographical origin were as follows: M. abscessus was the most common species either in Istanbul or Malatya regions (n = 6, n = 6, consequently). While M. kumamotonense was the most frequent species isolated from Ankara region (n = 6), M. gordonea was the most common for Samsun region (n = 14). CONCLUSION: Our study revealed that frequency of MOTT varies depending on the number of clinical samples and that frequency of these species were affected by the newly identified species as a result of the use of novel molecular methods. In conclusion, when establishing diagnosis and treatment methods, it is important to know that infections caused by unidentified MOTT species may vary according to the regions in Turkey. The results of the study showed that there were differences in the frequency of MOTT species in the different geographical regions of Turkey.

[Comparison and evaluation of Lowenstein-Jensen medium and 2% Ogawa medium for the diagnosis of tuberculosis]. / Tüberkülozun Tanisinda %2 Ogawa Besiyerinin Löwenstein-Jensen Besiyeri ile Karsilastirilmasi ve Degerlendirilmesi.

Accurate diagnosis of tuberculosis is based on the detection of the bacilli in the clinical specimen. The growth of mycobacteria in laboratory media is regarded as the gold standard for diagnosis and use of two different cultivation media, one of them being a liquid one, is recommended. In this study, the diagnostic values of egg-based Lowenstein-Jensen (LJ) medium used extensively all over the world and Ogawa (2%) medium were compared. A total of 7912 pulmonary and extrapulmonary clinical samples which belonged to 3311 patients with suspected tuberculosis, were enrolled in the National Tuberculosis Reference Laboratory in Refik Saydam National Public Health Agency during 2.5 years (January 2006- June 2008). The samples were processed by modified Petroff method (4% NaOH) for homogenization-decontamination-concentration on the same day and inoculated on two Ogawa and two LJ media. The cultures were incubated at 35-37°C for eight weeks and were controlled on the third day of incubation in terms of contamination. If no contamination were detected, the cultures were incubated for a total of eight weeks with regular weekly controls. The colonies detected in culture media were identified by biochemical tests, including paranitrobenzoic acid (PNB), niacin accumulation and nitrate reduction tests. Those identified as Mycobacterium tuberculosis complex were reported as positive. The rates of contamination was 3.1% for Ogawa medium and 4% for LJ medium. Culture results were found positive for 248 patients (7.5%). While 210 of these were positive by both of the media, 20 (8.1%) patients were detected positive only by LJ and 18 (7.3%) only by Ogawa medium. The sensitivity of LJ medium was 92.7% (230/248) and of Ogawa medium was 91.9% (228/248). When LJ medium was taken as the reference method, the sensitivity, specificity, positive and negative predictive values of Ogawa medium were 91.3%, 99.4%, 92.1% and 99.4%, respectively. It was concluded that, when low price and low contamination rates were taken into consideration, Ogawa medium, used together with a liquid medium + LJ medium, would increase the yield of mycobacteria from single samples (cerebrospinal fluid, biopsy, pus, etc.) sent to the laboratories.

[Evaluation of the distribution of non-tuberculous mycobacteria strains isolated in National Tuberculosis Reference Laboratory in 2009-2010, Turkey]. / Ulusal Tüberküloz Referans Laboratuvarinda 2009-2010 Yillarinda Tespit Edilen Tüberküloz Disi Mikobakterilerin Dagilimlarinin Irdelenmesi.

Non-tuberculous mycobacteria (NTM) are commonly encountered environmental bacteria, and most of them are associated with lung diseases. Diagnosis of infections caused by NTM is based on clinical, radiological and microbiological findings. The aim of this study was to investigate the distribution of non-tuberculous mycobacterial species isolated from clinical specimens as etiologic agents. The NTM strains isolated from clinical specimens in National Tuberculosis Reference Laboratory (NTRL), together with the strains that were sent to NTRL for the advanced identification of non-tuberculous mycobacterial species that have clinical or microbiological significance, were analysed retrospectively. The strains belonged to January 2009 - December 2010 period. If the same NTM type was isolated more than once in the clinical specimens of a patient, then it was defined microbiologically as a causative agent. Identification of mycobacteria species was performed by using a commercial line-probe assay (GenoType Mycobacterium CM/AS; Hain Lifescience, Germany). In our study, pulmonary and non-pulmonary samples obtained from 206 patients yielded mycobacterial growth in their cultures, and of them 24 (11.7%) were identified as NTM. On the other hand, 51 of the 101 samples sent to NTRL for identification were confirmed as NTM. Of the patients who were found to be infected with NTM (n= 75), 59 (78.7%) were male and the mean age was 50.9 ± 18.8 years. The most frequently identified NTM species was M.fortuitum (33.3%, n= 25), followed by M.abscessus (18.7%, n= 14), M.gordonae (10.7%, n= 8) and M.avium (%8; n= 6). The other types of NTM species identified in our laboratory were M.chelonae (n= 3), M.intracellulare (n= 3), M.kansasii (n= 3), M.peregrinum (n= 2), M.scrofulaceum (n= 2), M.szulgai (n= 2), M.celatum (n= 1), M.haemophilum (n= 1), M.smegmatis (n= 1) and M.xenopi (n= 1). Rapidly growing NTM species (M.fortuitum and M.abscessus) were the most frequent (52%) species isolated in our laboratory as the cause of non-tuberculous mycobacterial infection. Interestingly, the majority of M.fortuitum isolates (n= 21) which was the most common species identified in our laboratory, were those received from the peripheral laboratories. The most common species identified in our laboratory were rapidly growing NTM, however the countrywide distribution of the NTM species was found different than previously reported. In conclusion, further investigation of the non-tuberculous mycobacteria profile in adjunct with epidemiological data seems to be essential in our country.

Investigation of species distribution of nontuberculosis mycobacteria isolated from sputum samples in patients with suspected pulmonary tuberculosis.

Aims: Rapid and accurate identification of mycobacteria is important for the species-specific treatment of the disease. The aim of this study was the identification at the species level of 34 nontuberculous mycobacteria strains isolated from respiratory tract samples and 14 reference strains as by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Materials and Methods: Isolates derived from clinical specimens were subcultured in the Lowenstein-Jensen medium. Deoxyribonucleic acid isolation was carried out using the boiling method. PCR amplification was performed using primers specific to the hsp65 gene region. The PCR products were digested BstEII and HaEIII enzymes. All samples were studied comparatively by two different centers. Results: In our study, the most common species were found to be Mycobacterium intracellulare in 23.52% (8/34). The performance of the PCR-RFLP method in detecting mycobacteria was found to be 82.35%. Conclusions: The PCR-RFLP method is a rapid, cheap, and practical method for the identification of mycobacteria.

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.

[Quality assessment of microscopic examination in tuberculosis diagnostic laboratories: a preliminary study]. / Tüberküloz tani laboratuvarlarinda mikroskopi kalite degerlendirme pilot uygulamasi

Recently, the diagnosis of pulmonary tuberculosis (TB) has based on smear microscopy in the Direct Observed Treatment Strategy (DOTS) programme which provides the basis of treatment worldwide. Microscopic detection of AFB (Acid-Fast Bacilli) is one of the main components in the National TB Control Programmes (NTCP). Precision level in microscopy procedures and evaluations are the most important steps for accurate diagnosis of the disease and to initiate proper treatment. Therefore, the external quality assessment (EQA) is the most important implement to provide the reliability and validity of tests. In countries where NTCP are performed, this task is fulfilled by the National Reference Laboratories (NRL) according to the guidelines of the World Health Organization (WHO). For this purpose a pilot study was initiated by the central NRL of Turkey for EQA of AFB smear microscopy as part of the NTCP on January 1, 2005. A total of 5 laboratories of which 2 were district TB laboratories (A, B), 2 were tuberculosis control dispensaries (C, D), 1 was a national reference laboratory (E), participated in this study. Blind re-checking method (re-examination of randomly selected slides) was used for the evaluation, and the slides were sent to the central NRL with 3 months interval, four times a year, selected according to LQAS (Lot Quality Assurance Sampling) guides. In the re-evaluation of the slides, false positivity (FP), false negativity (FN) and quantification errors (QE) were noted. Laboratory A, sent totally 525 slides between January 1, 2005 and April 1, 2008. In the result of re-checking, 514 (97.9%) slides were found concordant, and 11 (2.1%) were discordant (10 FP, 1 FN). Laboratory B, participated in the study between October 1, 2005 and July 1, 2006 and of the 67 re-examined slides, 60 (89.5%) were concordant and 7 (10.5%) were discordant (2 FP, 0 FN, 5 QE). Laboratory C, sent 235 slides between January 1, 2005 and April 1, 2006; of them 218 (92.8%) were detected as compatible and 17 (7.2%) slides were incompatible (4 FP, 9 FN, 4 QE). Laboratory D, participated in QC for only once between January 1, 2008 and April 1, 2008; and all the 50 slides were found compatible, with no FP, FN and QE. Laboratory E, was included in the study between January 1, 2005 and January 1, 2008 and of the 696 re-checked slides, 690 (99.1%) were reported as compatible and 6 (0.9%) were incompatible (3 FN, 3 QE). Following EQA, on-site evaluation of the laboratories with major errors, was performed and necessary adjustments and training were done. In conclusion, external quality control measures for AFB microscopy is crucial and essential for the tuberculosis laboratory performances for accurate and reliable results.

[Identification and isolation of non-tuberculous mycobacteria from environmental samples]. / Çevre örneklerinden tüberküloz disi mikobakterilerin izolasyonu ve tanimlanmasi

Non-tuberculous mycobacteria (NTM) found frequently in tap water and environment cause important opportunistic infections in immunocompromised patients. The aim of this study was to isolate and identify non-tuberculous mycobacteria in soil, raw milk and water distribution system samples in Mersin (a province located at Mediterranean region of Turkey). A total of 101 water, 124 soil and 40 milk samples collected from the central part and suburban parts of Mersin during November 2003-May 2004 period were included in the study. Water samples were collected from 29 different water distribution systems; soil samples from different parks and gardens and milk samples from raw milks sold at different districts. After the samples were processed by homogenization and decontamination, acid-fast staining and culture into Löwenstein-Jensen medium were performed. Acid-fast bacilli isolated from culture medium were identified by using conventional methods, polymerase chain reaction (PCR)-RFLP (Restriction Fragment Length Polymorphism) and INNO-LIPA Mycobacteria methods. NTM were identified from 4.9% (5/101) of water samples and 0.8% (1/124) of soil samples by culture and PCR. No NTM were detected in the raw milk samples. Three of the NTM strains isolated from water samples were defined as Mycobacterium chelonae type III and two as Mycobacterium kansasii type II. One NTM strain isolated from soil was defined as Mycobacterium fortuitum. It was of note that two of the five NTM positive water samples were tap water samples collected from hospitals. It was concluded that NTM colonization/contamination of water and environment in the hospitals was a potential risk factor in terms of nosocomial infections. Thus surveillance cultures of the water systems and the medical devices in the hospital are necessary to fix the source of NTM, to identify and type the strains and to establish effective control measures such as sterilization, disinfection, maintenance and modernization of water systems.

[Resistance rates to major anti-tuberculosis drugs in Mycobacterium tuberculosis strains isolated from seven different regions of Turkey in 2003-2006 period]. / Ulkemizin yedi farkli bölgesinden 2003-2006 yillari arasinda izole edilen mycobacterium tuberculosis suslarinin majör antitüberkoloz ilaçlara direnç oranlari.

Multi-drug resistance in Mycobacterium tuberculosis (MDR-TB) is a global problem and has increased especially in areas where tuberculosis control programmes are inefficient. The aim of this study was to detect the resistance rates against isoniazide (INH), rifampisin (RMP), ethambutol (EMB) and streptomycin (SM) in M. tuberculosis strains collected from 7 different regions including 62 cities and sent to Refik Saydam Hygiene Center National Tuberculosis Reference and Research Laboratory. Of the patients included, 7.61% were children, 92.39% were adults; 76.16% were male and 23.84% were female. These strains were isolated from sputum (n = 885, 81.11%), gastric lavage (n = 49, 4.49%), pleural fluid (n = 43, 3.94%), urine (n = 30, 2.74%), bronchoalveolar lavage (n = 22, 2.01%), and other clinical samples (n = 62, 8.46%) such as cerebrospinal fluid, lymph node, abscess material, lung tissue. The susceptibilities of the 1091 M. tuberculosis strains against the major anti-tuberculosis drugs were determined by the proportion method in Lowenstein-Jensen medium. Three hundred ninety two of the isolates were from Central Anatolia, 146 from Black Sea, 419 from Aegean, 28 from Mediterranean, 20 from Marmara, 64 from Eastern Anatolia and 21 from South Eastern Anatolia regions of Turkey. The distribution of the strains according to years were as follows: 88 in 2003, 114 in 2004, 341 in 2005 and 548 in 2006. Resistance to at least one of the drugs tested was found in 264 (14.20%) strains. Overall drug resistance rates to INH, RMP, EMB and SM were 12.3%, 10.1%, 6% and 15.8%, respectively. MDR-TB rate was 10% for this 4 years study period. MDR-TB rate was detected as 13.2%, 9.7%, 10.1% and 9.6% in children, adult, male and female patients, respectively. MDR-TB rate did not exhibit a statistically significant difference in terms of sex, age and study years (p > 0.05). However, this rate showed statistically significant difference in terms of geographical regions (p < 0.05). This study emphasized that regular surveillance of M. tuberculosis resistance to the major anti-tuberculosis drugs will provide valuable data for the effective national control and treatment of tuberculosis.

[Evaluation of Quantiferon-TB Gold and tuberculin skin test in patients with tuberculosis, close contact of patients, health care workers and tuberculosis laboratory personnel]. / Tüberkülozlu hastalar, yakin temaslilari, saglik çalisanlari ve tüberküloz laboratuvari personelinde Quantiferon-TB Gold ve tüberkülin cilt testinin degerlendirilmesi.

Tuberculin skin test (TST) has been used effectively for a long time, despite inherent sensitivity and specificity limitations. Patients with a positive TST without active tuberculosis are identified as having latent tuberculosis infection. Identifying patients with latent tuberculosis infection with this test is an important part of control of the disease. A whole-blood inferferon gamma (IFN-γ) assay, the Quantiferon TB Gold test (QTG; Cellestis, Australia) which is a promising in vitro diagnostic test for the identification of latent tuberculosis infection (LTBI), has potential advantages over the TST. This test includes Myobacterium tuberculosis specific ESAT- 6 and CFP-10 antigens. The aim of this study was to compare the results obtained by QTG and TST in active tuberculosis (TB) patients, close contacts of patients, health care workers and tuberculosis laboratory personel. Twenty-six patients with active pulmonary TB, 6 close contacts of those patients, 11 health care workers with contact to TB patients and 8 TB reference laboratory personnel were included in the study. Prior to administration of the TST, blood samples were drawn from each participant for QTG test. All subjects were asked for BCG vaccination history and examined for a BCG scar. All individuals had a BCG scar. The QTG assay was performed in whole blood samples according to manufacturer's instructions. The agreement between TST and QTG was measured with kappa statistical analysis. In active TB patients (true-infected cases) TST (PPD) positivity was found 34.6% (9/26) while QTG positivity was 65.3% (17/26). Although the positivity rate was higher in QTG test, this difference was not found statistically significant (p > 0.001). TST and QTG positivity rates for health care workers, close house contact of TB patients and TB laboratory staff were as follows, respectively; 36% (4/11) and 27% (3/11); 16.6% (1/6) and 83% (5/6); 37.5% (3/8) and 75% (6/8). The mean PPD diameter was 11 mm in QTG negative group and 14 mm in QTG positive group with a statistically significant difference (p < 0.001). However, there was no statistical significance between QTG positive and negative groups by means of age (p ≥ 0.05) and gender (p < 0.001). In conclusion, QTG assay was superior to TST in its ability to detect LTBI and active TB infection, not to be affected with BCG vaccination, to discriminate responses due to non-tuberculous mycobacteria, and to avoid variability and subjectivity associated with application and reading the TST. Besides, QTG assay needs only one visit to the test unit. However, its being expensive than TST and requirement for special equipments and skilled laboratory personnel, are among the disadvantages of QTG assay.

[Susceptibilities of Mycobacterium tuberculosis strains collected from regional tuberculosis laboratories to major antituberculous drugs]. / Bölge tüberküloz laboratuvarlarindan gonderilen Mycobacterium tuberculosis suslarinin majör antitüberküloz ilaçlara duyarliliklari.

The aim of this study was to investigate the susceptibility rates of Mycobacterium tuberculosis strains sent to Refik Saydam Hygiene Center, Tuberculosis Reference and Research Laboratory, Ankara, from seven different regional tuberculosis laboratories between the 1999-2002 period against major antituberculous drugs. The sensitivities of a total 505 M. tuberculosis strains to isoniazid (INAH), rifampicin (RIF), streptomycin (SM) and ethambutol (EMB) were determined by using proportion method in Lowenstein-Jensen medium. Of the strains, 385 (76.2%) were found sensitive to all of the tested drugs, while 120 strains were resistant to at least one of the antituberculous drugs. The resistant strains showed 14 different resistance patterns. The resistance rates were detected as 13.3% for INAH and RIF (67 strains of each), 9.1% for SM (46 strains), and 3.4% (17 strains) for EMB. Multidrug resistant (INAH+RIF) M. tuberculosis was 7.9% (40 strains). The highest resistance rate to INAH, RIF and EMB (21.2%, 21.2% and 10.6%, respectively) was detected in the isolates which were sent from Bursa province (located in northwestern Turkey); the highest SM (18.8%) and multidrug resistance (INAH+RIF) rates (18.8% and 10.6%, respectively) were detected in the strains sent from Elazig and Van provinces (both located in eastern Turkey). Since the inappropriate use of the first and second line antituberculous drugs leads to the development and spread of the resistant strains, "Directly Observed Therapy Shortcourse (DOTS)" is a very important practice. Therefore regional tuberculosis laboratories should be worth considering as the chains of a well-organized national laboratory network, in order to detect the antituberculous drug resistance patterns of the M. tuberculosis strains over the country.

[The rates of resistance to second-line drugs in multidrug resistant Mycobacterium tuberculosis strains]. / Cok ilaca dirençli Mycobacterium tuberculosis suslarinin minör antitüberküloz ilaçlara duyarliliginin saptanmasi.

The treatment of multidrug resistant tuberculosis (MDR-TB) requires the use of minor antituberculosis drugs, thus susceptibilities against these minor drugs should be performed. The aim of this study was to establish and standardize the susceptibility testing methods for minor antituberculosis drugs in this laboratory and to obtain data about the resistance rates. Drug susceptibility tests were performed by proportion method in Lowenstein-Jensen media for 50 MDR Mycobacterium tuberculosis isolates that have been collected from different regions of our country. Resistance rates to ethionamide, cycloserin, kanamycin, p-aminosalicylic acid, ofloxacin, thiacetasone, and capreomycin were found as 22%, 8%, 6%, 6%, 2%, 2%, and 0%, respectively. Multidrug resistance was observed in 8% of the isolates. As a result, the resistance rates of multidrug resistant M. tuberculosis strains against the tested minor antituberculosis drugs seem to be low in this study.

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.

Molecular analysis of isoniazid, rifampin and streptomycin resistance in Mycobacterium tuberculosis isolates from patients with tuberculosis in Düzce, Turkey.

The aim of this study was to use DNA sequencing analysis to analyze the mutations in the most commonly targeted genes (katG, inhA, rpoB, rpsL) in isoniazid (INH)-, rifampin (RIF)- and streptomycin (SM)-resistant Mycobacterium tuberculosis strains obtained from subjects in Duzce, Turkey. Four isolates were found to be INH-resistant, 3 were RIF-resistant and 5 were SM-resistant, out of a total of 52 M. tuberculosis strains. In 3 of the 4 INH-resistant strains, a mutation in the katG gene in codon 315 appeared as AGC-->ACC (Ser-->Thr), and the other INH-resistant strain showed a mutation in the katG gene in codon 314 as ACC-->CCC (Thr-->Pro). There were no mutations in the inhA gene in INH-resistant isolates. Two of the 3 RIF-resistant strains were found to have mutations in the rpoB gene in codon 516 appearing as GAC-->GTC (Asp-->Val), and the other RIF-resistant strain has a mutation in the rpoB gene in codon 531 as TCG-->TTG (Ser-->Leu). These 3 RIF-resistant strains are also INH-resistant. All 5 SM-resistant strains have mutations in the rpsL gene in codon 43 appearing as AAG-->AGG (Lys-->Arg). Thus, we found common gene mutations that bring about the resistance of M. tuberculosis to antituberculosis drugs in all of our isolates from Duzce. To the best of our knowledge, the ACC-->CCC (Thr-->Pro) mutation in the katG gene in codon 314 has not been previously defined.

The resistance to major antituberculous drugs of Mycobacterium tuberculosis strains isolated from the respiratory system specimens of tuberculosis patients in Duzce, Turkey.

Through generally curable, tuberculosis (TB) is becoming increasingly resistant to commonly used antibiotics. Drug-resistant and multidrug-resistant (MDR)-TB is a consequence of monotherapy, insufficient drug therapy and national TB control programs. The present study was designed to reveal the resistance to major antimicrobial drugs (isoniazid [INH], streptomycin [SM], ethambutol [EMB], and rifampicin [RIF]) of Mycobacterium tuberculosis isolated from the respiratory specimens of TB patients in Duzce, Turkey. A total of 62 TB patients (46 male, 16 female; age: 17 - 75 mean: 42 +/- 15.9) were included in the study; 52 (83.8%) were new cases and susceptible to all anti-TB drugs, while 10 (16.2%) were previously treated cases. Antimicrobial susceptibility tests were performed by the proportion method in Löwenstein-Jensen medium. Fifty-two of the 62 (83.8%) isolated M. tuberculosis strains were found to be susceptible to all drugs, and 7 (11.3%), 5 (8%), and 3 (4.8%) were resistant to SM, INH, and RIF, respectively; 3 (4.8%) were MDR. There were no EMB-resistant strains. The results of this study show the presence of drug-resistant and MDR strains of TB at Duzce in the northwest part of Turkey.