[In vitro Activity of Rifabutin and Clofazimine to Macrolide-Resistant Mycobacterium abscessus Complex Clinical Isolates]. / Rifabutin ve Klofaziminin Makrolidlere Dirençli Mycobacterium abscessus Kompleks Klinik Izolatlarina In vitro Etkinligi.

Mycobacterium abscessus complex (MABSC) is one of the most resistant bacteria against antimicrobial agents. The number of agents that can be used by oral route, such as macrolides, is limited in antimicrobial therapy. In recent years, rifabutin and clofazimine have gained importance as they can be administered by oral route and have shown synergistic effects with macrolides and aminoglycosides. The aim of this study was to determine the in vitro activity of rifabutin and clofazimine against clinical isolates of MABSC resistant to macrolides. A total of 48 MABSC isolates obtained from respiratory tract and other clinical samples in the Tuberculosis Laboratories of the Faculty of Medicine of Manisa Celal Bayar and Ege Universities were included in the study. Subspecies differentiation and aminoglycoside and macrolide resistance of the isolates were determined by GenoType NTM-DR test. Rifabutin and clofazimine susceptibilities were determined by standard broth microdilution method. Of the MABSC isolates 42 were identified as M.abscessus subsp. abscessus, three as M.abscessus subsp. bolletii and three as M.abscessus subsp. massiliense. None of the isolates exhibited rrs and rrl mutations indicating acquired macrolide resistance and aminoglycoside resistance. However, the erm(41) T28 genotype which is associated with inducible macrolide resistance was detected in 41 (85%) of the strains. All M.abscessus subsp. massiliense isolates were found to be genotypically susceptible to macrolides. The minimum inhibitory concentration (MIC) range values for rifabutin were 0.0625 to 32 µg/mL, while for clofazimine, the range was 0.0625 to 1 µg/mL. Rifabutin MIC values were significantly higher (mean 5.98 µg/mL vs 0.5 µg/mL, p= 0.026) in the isolates with macrolide resistance. There was no correlation between macrolide resistance and clofazimine MIC values (mean 0.25 µg/mL vs. 0.214 µg/mL, p= 0.758). The MIC50 and MIC90 values for rifabutin were 1 and 8 µg/mL, respectively, while for clofazimine they were 0.25 and 0.5 µg/mL. Macrolide resistance was found to be higher in isolates with rifabutin MIC values above the MIC50 value (p= 0.045). In conclusion, the determination of higher rifabutin MIC values in isolates resistant to macrolides suggested that susceptibility testing should be performed before adding rifabutin to the treatment regimen. The low MIC values of clofazimine in all strains indicated that it may be used as a first choice in the combination therapy. However, further studies using a larger number of clinical isolates and applying genotypic and phenotypic susceptibility tests are needed to determine threshold MIC values to assist clinicians in making treatment decisions.

Investigation of miRNA and cytokine expressions in latent tuberculosis infection and active tuberculosis.

BACKGROUND: In tuberculsosis (TB), miRNA has been used as a biomarker to distinguish between healthy individuals and TB patients. The aim of this study was to investigate (i) the association of the miRNA and cytokine expression levels, the course of tuberculosis infection, clinical forms and response to treatment, and (ii) the effects of genotypic features of bacteria on the course of tuberculosis and the relationship between miRNA and cytokine expressions and bacterial genotypes. METHODS: A total of 200 cases (100: culture positive active tuberculosis, 50: quantiferon positive latent tuberculosis infection and 50: quantiferon negative healthy controls) were included in the study. For the tuberculosis group at the time of admission and after treatment, for the latent tuberculosis infection and healthy control groups at the time of admission, miRNA and cytokine expressions were determined. Genotyping of M.tuberculosis isolates was performed by spoligotyping method. RESULTS: While, in the comparison of miRNA expressions between the pretreatment patient group and the healthy control group, there was a statistically significant decrease in the expression of miR-454-3p, miR-15a-5p, miR-590-5p, miR-381, and miR-449a in the Pulmonary TB group, there was no significant change in miRNA expression in extrapulmonary TB patients. When the cytokine expressions of the patient group and the healthy control group were compared before treatment, the expressions of all cytokines in the patient group decreased. However, the only cytokine that showed a significantly lower expression was IL12A in PTB patients. DISCUSSION: There is no significant relationship between the clinical course of the disease, cytokine and miRNA expression, and the genotype of the bacteria.

[Friend or Foe? Evaluation of BCG Vaccine and Latent Tuberculosis Infection Effect in Patients Diagnosed with COVID-19 Infection]. / Dost mu Düsman mi? COVID-19 Enfeksiyonu Tanili Hastalarda BCG Asisi ve Latent Tüberküloz Enfeksiyonu Etkisinin Degerlendirilmesi.

While severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to spread rapidly worldwide, some issues such as the uncertainty of the disease progress, whether intensive care will be needed, and risk classification are still important for clinicians. It is notable that in countries where latent tuberculosis infection (LTBI) is common and participating in the national Bacillus Calmette-Guerin (BCG) vaccination program, the case-fatality rates are relatively low throughout the world. In this study, it was aimed to evaluate the effects of the BCG vaccine and LTBI status on the course of the disease in patients diagnosed with coronavirus-19 (COVID-19) infection and to compare the LTBI rate with people with and without COVID-19 infection. The patients diagnosed with COVID-19 infection who were hospitalized during a period of seven months between May 1st to December 1st, 2020 were investigated by the QuantiFERON-TB Gold Plus (QFT-Plus) test in the blood samples for the presence of LTBI. For the comparison of the patients diagnosed with COVID-19 and people without COVID-19 infections in terms of LTBI rate retrospectively; all consecutive patients who were sent blood samples to the mycobacteriology laboratory for the QFT-Plus test between January 2016 and December 2019 were included in the study. Demographic, clinical, radiological, laboratory, and follow-up data of the patients were obtained from the electronic patient file. A total of 170 patients (n= 9 8 male [57.6%], n= 72 female [42.3%], mean age= 53.5 ± 15.8 years) were enrolled. Twenty-five patients' (25/170 [14.7%]) QFT-plus tests were positive. When the cases with positive QFT-Plus test (n= 25) and the cases with negative QFT-Plus test (n = 145) were compared in terms of disease severity respectively; it was determined that mild/moderate patients were 18/25 (72%) and 108/145 (74.5%), severe patients were 7/25 (28%) and 37/145 (25.5%) (p= 0.988). When these two groups were compared in terms of the clinical course respectively; the need for intensive care was 6/25 (24%) and 34/145 (23.4%) (p= 1.00), oxygen therapy requirement was 13/25 (52%) and 49/145 (33.8%) (p= 0.128), and death was 5/25 (20%) and 18/145 (12.4%) (p= 0.341). QFT-Plus positivity was 25/170 (14.7%) in patients diagnosed with COVID-19, while in control group it was 198/496 (39.9%) (OR= 0.259, 95% CI [0.164-0.411], p<0.001). When the values were evaluated quantitatively, in the COVID-19 patient group, QFT-Plus T1/T2 (IU/ml) interferon (IFN)-É£ was 0.87 ± 1.52/0.62 ± 1.53, while in the control group it was 1.52 ± 3.69/1.50 ± 3.33 (p= 0.032, p= 0.04). There was no significant difference in the parameters investigated between 82 (48.2%) patients with BCG vaccine and those 88 (51.8%) without BCG vaccine. Although it was not statistically significant in our study, increased oxygen therapy requirement and higher mortality rates in the QFT-Plus positive group were remarkable. The detection of statistically significantly lower LTBI rates and T1-T2/IFN-É£ values in the COVID-19 group supported that SARS-CoV-2 infection may suppress lymphocyte functions in patients and IFN-É£ response. We believe that the results of our study are remarkably valuable, but more clinical studies are needed to elucidate the relationship between BCG vaccine, LTBI, and COVID-19 infection.

Osteomyelitis Caused by Bacillus Calmette-Guérin Vaccination in a Healthy Toddler.

Bacillus Calmette-Guérin (BCG) osteomyelitis in immunocompetent children is a rare complication of BCG immunization which presents with nonspecific findings and often leads to delayed diagnosis. We report a 1-year and 10-month-old male infant with complaining of knee pain and limping for 5 months. He received surgical debridement due to suspicion of malignancy but BCG osteomyelitis of the distal femur was diagnosed with the culture of the specimens which revealed to have Mycobacterium bovis-BCG strain. He was successfully treated with antituberculous therapy lasting for 1 year.

[Comparison of two methods for the detection of antituberculous drug susceptibility in Mycobacterium tuberculosis complex isolates: sensititre MycoTB MIC plate and the indirect agar proportion methods]. / Mycobacterium tuberculosis kompleks izolatlarinin antitüberküloz ilaç duyarliligini saptamada iki yöntemin karsilastirilmasi: sensititre MycoTB MIC plak ve indirekt agar proporsiyon yöntemleri.

Tuberculosis is still one of the most important public health problems worldwide. Due to the increase of multidrug-resistant (MDR) tuberculosis and extensively drug-resistant (XDR) tuberculosis cases, fast, practical and standardized methods need to be developed for the determination of drug resistance in Mycobacterium tuberculosis complex. The aim of this study was to compare the Sensititre MycoTB plate method with the gold standard agar proportion method (APM); in order to determine if the Sensititre MycoTB plate method is effective enough to be used instead of the APM. A total of 100 M.tuberculosis complex isolates, 36 with different resistance patterns and 64 sensitive to all first-line agents stored in culture collection of Ege University Faculty of Medicine Microbiology Department Laboratory, were used in the study. The susceptibility of rifampin, isoniazid, ethambutol, streptomycin, ofloxacin, moxifloxacin, amikacin, rifabutin, para-aminosalicylic acid, ethionamide, cycloserine and kanamycin for all the isolates were determined by APM and Sensititre MycoTB plate methods. APM was performed as defined by CLSI, Sensititre MycoTB plate method was performed as instructed by the manufacturer. Results were assessed in 95% confidence interval and sensitivity, specificity and categorical agreement values were determined. Results were obtained in 14 days using the Sensititre MycoTB plate method. Sensitivity and the specificity of the Sensititre MycoTB was between 86-100% and 95-100% respectively, while the categorical agreement between the two methods were 95-100% for the drugs tested. The sensitivities of the drugs were 100% except ethambutol and ethionamide. The specificity values for both of the drugs and categorical agreement were over 95%. All the drugs were in the 95% confidence interval (p< 0.001) and the greatest difference between the lower and upper levels of sensitivity and specificity confidence limits was found in ethambutol and ethionamide. MDR was detected in 16 isolates with the APM and in 20 isolates with the Sensititre MycoTB plate method. None of the isolates had XDR [in addition to MDR resistance against any fluoroquinolones and at least one of the second-line parenteral drugs (amikacin, kanamycin or capreomycin)]. As a result, owing to the short incubation period (14 days), high sensitivity, specificity and categorical agreement values, and the possibility for evaluating both first- and second-line agents and the quantitative values of drugs; the Sensititre MycoTB method was determined as an effective method that can be used especially in laboratories where the rate of MDR-TB isolates are high.

[Culture proven extra pulmonary tuberculosis: drug susceptibility and genetic profile analysis]. / Kültür ile kanitlanmis akciger disi tüberküloz: ilaç duyarliligi ve genetik profil analizi.

INTRODUCTION: Tuberculosis is seen generally in the lungs. Besides, all organs in the body can be affected by tuberculosis. Diagnosis of extra pulmonary tuberculosis (EPTB) is more difficult than pulmonary tuberculosis (PTB). Although, the isolation of Mycobacterium tuberculosis is gold standard of diagnosis of EPTB, the rate of bacteriologic isolation is low especially in EPTB. If M. tuberculosis is detected, it gives some information about the epidemiological features of the disease and drug susceptibility. MATERIALS AND METHODS: In this study, extra pulmonary samples isolated M. tuberculosis in mycobacteriology laboratory were evaluated between 2009-2016. The identification of the genotype of isolated bacteria and drug sensitivity tests were conducted. Spoligotyping was accomplished using a standard technique as described previously. RESULT: During the study period, M. tuberculosis were cultured in 171 extra pulmonary samples of 165 patients (75 male, 90 female, mean age: 53.35 ± 19.92). Initial direct microscopically examination was revealed M. tuberculosis in 44 patients. There were more than one extra pulmonary organ involvement in six patients. The most common EPTB forms were lymph node TB in 60 patients, pleural tuberculosis in 32 patients and bone tuberculosis in 25 patients. Immunosuppression was detected in 44 (%26.6) patients. Among these, seven patients were infected with HIV. In 21 of 175 samples, drug resistance was detected. Rifampicin resistance in 7 samples, high level isoniazid resistance in 11 samples and rifampicin plus isoniazid resistance (multiply drug resistance) in 6 samples were demonstrated. One hundred thirty-five clinical isolates were cultured from tuberculosis patient's different samples, of which the genetic profile was determined by using Spoligotyping. The major Spoligotypes were T (n= 62; 45.9%), LAM7-TUR (ST41) (n= 11; 8.1%) and H1 (n= 9; 6%) genotypes. CONCLUSIONS: The most common EPTB form was lymph node tuberculosis in culture proven patients. In these patients group, multiply drug resistance rate was low (3.6%). Spoligotypes T (45.9%) was detected as most common genetic profile.

In vitro effects of ciprofloxacin, levofloxacin and moxifloxacin on Mycobacterium tuberculosis isolates.

INTRODUCTION: Increased tuberculosis prevalence, and isolation of multidrug resistant (MDR) Mycobacterium tuberculosis strains frequently as causative organisms from tuberculosis infections are resulted in increasing need of new anti-tuberculosis drugs. Nowadays, fluoroquinolones known to have fewer side effects than the other drugs used in treatment of tuberculosis are sometimes assessed even as first-line anti-tuberculosis drugs due to their in vitro and in vivo strong activity. It was aimed in this study to investigate phenotypically the fluoroquinolone susceptibility in MDR and non-MDR M. tuberculosis isolates. MATERIALS AND METHODS: A total of 126 MDR and non-MDR M. tuberculosis isolates from mycobacteriology laboratory of two hospitals in the Aegean Region of Turkey were included in the study. Ciprofloxacin (CIP), levofloxacin (LEV) and moxifloxacin (MXF) susceptibilities were assessed by agar proportion method according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. RESULT: Twelve (15.2%), 5 (6.3%) and 4 (5.1%) of the MDR M. tuberculosis strains were resistant to CIP, LEV, MXF, respectively [resistance breakpoints (µg/mL); CIP (> 2), LEV (> 1), MXF (> 0.5)] while non-MDR strains were susceptible to CIP, LEV, MXF. CONCLUSIONS: Consequently, although high fluoroquinolone susceptibilities were evaluated as a pleasing data in this study, to preserve their efficiency for many years steadily, quinolone usage and resistance increment in MDR M. tuberculosis isolates should be monitored elaborately.

Evaluation of the performance of MALDI-TOF MS and DNA sequence analysis in the identification of mycobacteria species

Background/aim: Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is an alternative way of identifying mycobacteria via the analysis of biomolecules. It is being increasingly used in routine microbiology practice since it permits early, rapid, and cost-effective identification of pathogens of clinical importance. In this study, we aimed to evaluate the efficacy of phenotypic identification of mycobacteria by the MALDI-TOF MS MBT Mycobacteria Library (ML) 4.0 (Bruker, Daltonics) compared to standard sequence analysis. Materials and methods: A total of 155 Mycobacterium clinical and external quality control isolates, comprising nontuberculous mycobacteria (NTM) (n = 95) and the Mycobacterium tuberculosis complex (MTC) (n = 60), were included in the study. Results: Identification by MBT ML4.0 was correctly performed in 100% of MTC and in 91% of NTM isolates. All of the MTC isolates were correctly differentiated from NTM isolates. Conclusion: Based on our results, MBT ML4.0 may be used reliably to identify both NTM and MTC.

[Molecular epidemiology of human Mycobacterium bovis infection in Aegean Region, Turkey]. / Ege Bölgesi'nde insan Mycobacterium bovis enfeksiyonlarinin moleküler epidemiyolojisi.

Mycobacterium bovis, which has a broader host spectrum as opposed to Mycobacterium tuberculosis that generally causes disease in humans, mainly leads to chronic progressive pulmonary disease in a large number of domestic and wild mammals, particularly cattle. The term "zoonotic tuberculosis (TB)" is used to describe M.bovis infection in humans. "Zoonotic TB" can not be differentiated clinically, radiologically or pathologically from TB caused by M.tuberculosis. The aim of this study was to evaluate the role of M.bovis in epidemiology of human TB in Aegean Region, Turkey and to determine M.bovis genotypes responsible for human TB. Thirteen M.bovis isolates identified by spoligotyping from 482 M.tuberculosis complex isolates obtained from clinical specimens sent for routine mycobacteriological examination to the Mycobacteriology Laboratory in Medical Microbiology Department of Ege University Faculty of Medicine Hospital between 2009 and 2014 were included in the study. Drug susceptibility tests of the isolates were performed by the BACTEC MGIT 960 automated system. It was determined that 9 (63.6%) of the 13 spoligotyped M.bovis isolates in this study were ST685 (SB0288), 1 (7.7%) was ST 1118 (SB0989) and 1 (7.7%) was ST820 (SB0856), for two isolates there were no registered data in SpolDB4 and Mbovis.org databases. All the isolates were susceptible to first-line antituberculosis drugs. It was determined that 13 M.bovis isolates examined in the study accounted for 2.7% of the 482 M.tuberculosis complex isolates spoligotyped in the same period. In this study, it was determined that 8 (%61.5) of 13 patients was male, 5 (38.5%) of them was female, 9 (69.2%) of the 13 patients had pulmonary TB and 4 (30.8%) had extra pulmonary TB. Seven of nine patients with pulmonary TB and two of the four patients with extrapulmonary TB were living in the rural area, and two patients with pulmonary TB had occupational exposure. Although ST683 (SB0140) is widely seen in the world among human isolates, it was not detectedin this study and other studies conducted in Turkey. In contrast, ST685 (SB0288) and ST1118 (SB0989), which have been reported very few in the world, found to be predominant in this study. This result suggested that they may be unique spoligotypes emerging in Anatolia. In conclusion, collaborative molecular epidemiological studies are needed in conjunction with researchers working in medicine and veterinary fields to determine precisely the importance of zoonotic TB in human TB in our country, to determine the route of transmission to humans and risk factors for zoonotic TB infections, to identify the dominant types between humans and animals and to understand the phylogeographic relationships of the strains in our country.

[Evaluation of QuantiFERON®-TB Gold in Tube Test and Tuberculin Skin Test in the diagnosis of Mycobacterium tuberculosis infection]. / Mycobacterium tuberculosis enfeksiyonu tanisinda QuantiFERON®-TB Gold in Tube Testi ve Tüberkülin Deri Testinin degerlendirilmesi.

The aims of this study were to evaluate the sensitivity of QuantiFERON®-TB Gold in Tube (QFT) test and its agreement with the tuberculin skin test (TST), to investigate possible factors associated with indeterminate QFT test results and to explore the relationship between latent tuberculosis infection (LTBE) prevalence and the rate of tuberculosis (TB) cases in our region. 1455 cases with QFT test performed in Ege University Faculty of Medicine Hospital between 2013 and 2015 were included in the study and simultaneously TST results of 268 of 1455 cases were reached. TST results were evaluated according to both ≥ 10 mm and ≥ 15 mm cut-off values. The QFT results of the cases were compared according to their gender, age groups and clinical characteristics with chi-square test. Stratified analyses were also conducted according to age groups. Multivariate logistic regression was used to analyse factors associated with QFT positivity and indeterminate QFT results. Cohen's kappa was used to test the agreement between QFT and TDT, overall and stratified according to age groups. Among 1455 cases, 396 (27.2%) were QFT positive and 120 (8.2%) had an indeterminate QFT result. When the indeterminate results were excluded, QFT positivity was found as 29.7%. The highest indeterminate results were determined among 0-4 year-old and ≥ 65 year-old groups as 17.6% and 12.1%, respectively and lowest among the 55-64 age group as 4%. The comparison of the cases without any cellular immunity defect and the patients with hematologic malignancies or immune deficiency and patients under immunosuppressive treatment had two and 2.44 times more indeterminate QFT results, respectively. Among 268 cases with TST results reached, QFT positivity was 30.6%; 38.1% for TST ≥ 10 mm and 25.7% for TST ≥ 15. After the exclusion of indeterminate results, the agreement between QFT and TST ≥ 10 mm was 71.3% for positive cases and 75.5% for negative cases. The highest agreement between QFT and TST ≥ 10 mm was in the age group 35-64 and lowest in the age group ≥ 65. Among 43 culture-positive cases, 32 had QFT positive, six negative and five indeterminate results. When indeterminate results were excluded, the sensitivity of thetest was 84.2% (32/38) among culture-positive active TB cases. TST results were available for 17 of the culture-positive cases, among them QFT sensitivity was 76.5% (13/17), TST sensitivity 70.6% (12/17) and the sensitivity of both tests was 88.2% (15/17). The ratio of QFT positivity has increased as the age increased. Interestingly, QFT positivity was higher among females than males in the 15-34 age group and higher among males in the 35-64 age group. The rates of QFT positivity were lower among immunocompromised patients. When QFT and TST positivities were compared with the rate of TB cases among age groups, QFT positivity was observed as parallel to the rate of TB cases. In conclusion, although the sensitivity of QFT was higher than TST, it was found that it could not be considered as a gold standard in LTBE diagnosis. As active TB cases originate from the LTBE pool, QFT test results might be considered a better indicator of active TB development risk.

Genotype distribution of Mycobacterium tuberculosis in theAegean Region and associated demographic factors

Background/aim: The Aegean Region is the second-ranking region in Turkey according to the Human Development Index and population density and it hosts 1/8 of Turkey's population. Izmir is the largest city of the region, receiving internal migration both from inside and outside the region. The tuberculosis incidence in Izmir is lower than overall in Turkey: 17.7/100,000 in 2011. Our aims were to determine genotypes of Mycobacterium tuberculosis isolates; to explore possible associations between genotypes with case-demographic data, clinical presentation, and antimicrobial susceptibility patterns; and to determine variations in genotype distribution of strains isolated in Ege University Hospital, Izmir. Materials and methods: Forty-nine M. tuberculosis isolates from 49 patients in 1996-2000 and 421 M. tuberculosis isolates from 421 patients in 2009-2014 were spoligotyped. Drug susceptibility testing and demographic data of the 421 isolates were investigated. Chi-square, Student's t, and Mann-Whitney U tests were used for analyses. Results: Among the 470 M. tuberculosis strains, 132 different spoligopatterns were identified and 46 different clusters for 384 strains were determined. The most predominant spoligotypes were ST53 (n = 116; 24.7%) and ST41 (n = 38; 8.1%), followed by ST50 (5.7%), ST284 (4.7%), and ST4 (4.3%), respectively. ST53 was the most predominant type in both sexes. Multidrug resistance (MDR) was determined in 12 isolates, of which six were ST1.Conclusion: As a consequence of worldwide migration and increasing status of HIV-infected hosts, the increasing prevalence of Beijing strains with higher MDR rates may threaten disease control programs. With its increasing trend, ST284 could replace ST41 in the following years in this region.

Mycobacterium celeriflavum sp. nov., a rapidly growing scotochromogenic bacterium isolated from clinical specimens.

Six strains of a rapidly growing scotochromogenic mycobacterium were isolated from pulmonary specimens of independent patients. Biochemical and cultural tests were not suitable for their identification. The mycolic acid pattern analysed by HPLC was different from that of any other mycobacterium. Genotypic characterization, targeting seven housekeeping genes, revealed the presence of microheterogeneity in all of them. Different species were more closely related to the test strains in various regions: the type strain of Mycobacterium moriokaense showed 99.0 % 16S rRNA gene sequence similarity, and 91.5-96.5 % similarity for the remaining six regions. The whole genome sequences of the proposed type strain and that of M. moriokaense presented an average nucleotide identity (ANI) of 82.9 %. Phylogenetic analysis produced poorly robust trees in most genes with the exception of rpoB and sodA where Mycobacterium flavescens and Mycobacterium novocastrense were the closest species. This phylogenetic relatedness was confirmed by the tree inferred from five concatenated genes, which was very robust. The polyphasic characterization of the test strains, supported by the ANI value, demonstrates that they belong to a previously unreported species, for which the name Mycobacterium celeriflavum sp. nov. is proposed. The type strain is AFPC-000207(T) ( = DSM 46765(T) = JCM 18439(T)).

[Distribution of nontuberculous mycobacteria isolated from clinical specimens and identified with DNA sequence analysis]. / Klinik örneklerden soyutlanan ve DNA dizi analizi ile tanimlanan tüberküloz disi mikobakterilerin dagilimi.

The aims of the study were to perform the identification of nontuberculous mycobacteria (NTM) isolated from different clinical specimens in the Mycobacteriology Laboratory of Celal Bayar University, Manisa (located at Aegean region of Turkey), by DNA sequence analysis, and to discuss the epidemiological aspects of the data obtained. Out of 5122 clinical specimens sent to the laboratory with the initial diagnosis of tuberculosis in the period April 2007 to July 2011, M.tuberculosis complex and NTM were identified in 225 (4.39%) and 126 (2.46%) samples, respectively. DNA sequence analysis by targeting hsp65 and 16S rDNA gene regions was performed on 101 of the NTM strains in Mycobacteriology Laboratory of Ege University, Izmir. DNA sequence analysis data was evaluated using RIDOM and GenBLAST data bases. NTM strains were identified as 40 M.porcinum (39.60%), 36 M.lentiflavum (35.65%), six M.abscessus (5.64%), five M.peregrinum (4.95%), four M.gordonae (3.96%), three M.fortuitum (2.97%), two M.chelonae (1.98%), and one for each M.alvei (0.99%), M.scrofulaceum (0.99%), M.kansasii (0.99%) species. Two strains which were both 95-98% compatible with other mycobacteria in the data bases could not be identified with certainty. Seventy-two (94.73%) strains of M.lentiflavum and M.porcinum, which were the most frequent (75.24%) species in the study, were isolated from bronchoalveolar lavage (BAL) specimens. The remaining 99 strains examined could not be proven as the cause of the disease due to absence of patients' clinical data, whereas two M.abscessus strains isolated from the sputum were considered as the cause of the disease according to the ATS/IDSA criteria. The isolation rate of NTM in 2010 was found significantly higher (5.33%) than previous years. Review of the 2010 data showed that all strains of M.porcinum and M.lentiflavum, which were the most frequently identified strains were isolated from BAL specimens. This situation is in line with the start of using of an automatic bronchoscope washing machine in our hospital in the same year. In conclusion, NTM were isolated in 2.46% of the clinical specimens of the patients with the initial diagnosis of tuberculosis and these strains belonged to 10 different NTM species. The two NTM species most frequently isolated in our study were M.lentiflavum and M.porcinum which are known for their potential to cause human infections and antibiotic resistance. As these strains were mostly isolated in BAL specimens, it is concluded that automatic bronchoscope washing machines and water delivery system in the hospitals should be examined in terms of contamination by NTM. The isolated NTM strains could not be distinguished as the cause of the disease or a contaminant, which is the limiting factor in this study. However, knowing that the environmental mycobacteria can cause hospital infections, the data obtained in this study can contribute to epidemiology of NTM infections in Turkey.

[Rifampicin-resistant Mycobacterium bovis BCG strain isolated from an infant with NEMO mutation]. / NEMO mutasyonlu bir bebekten izole edilen rifampisine dirençli Mycobacterium bovis BCG susu.

It is well known that disseminated Mycobacterium bovis BCG infection is developed after BCG vaccination in infants with congenital cellular immune deficiencies such as mutations in genes along the interleukin (IL)-12/interferon (IFN)-γ pathway and mutations in nuclear factor-kB essential modulator (NEMO). In this report, a rifampicin-resistant M.bovis BCG strain isolated from an infant with NEMO defect was presented. An 8-month-old male infant with NEMO defect admitted to the pediatric outpatient clinic of our hospital with fever, generalized lymphadenopathy and hepatosplenomegaly. Microscopic examination of the smears prepared from lymph node and liver biopsy specimens revealed abundant amount (3+) of acid-fast bacilli (AFB). Rifampicin-susceptible Mycobacterium tuberculosis complex (MTC) was detected by real-time PCR (GeneXpert MTB/RIF; Cepheid, USA) in the samples. The growth of mycobacteria was determined on the 20th day of culture performed in MGIT960 system (Becton Dickinson, USA). The isolate was identified as M.bovis BCG by GenoType MTBC kit (Hain Lifescience, Germany) and defined as M.bovis BCG [SIT 482 (BOV_1)] by spoligotyping. In the primary anti-tuberculosis drug susceptibility test performed by MGIT960 system, the isolate was found susceptible to rifampicin (RIF), isoniazid (INH), streptomycin (STM) and ethambutol (EMB). Then anti-tuberculosis treatment was started to the patient. However, the patient at the age of 2 years, re-admitted to the hospital with the complaint of hepatosplenomegaly. Smear of spontaneously draining abscess material obtained from subcutaneous nodules revealed intensive AFB positivity (3+) once again. In the present instance RIF-resistant MTC was detected with GeneXpert system in the specimen. The growth of mycobacteria was determined on the 13th day of culture and isolate was identified as M.bovis BCG. The present isolate was found susceptible to INH, STM and EMB but resistant to RIF. A mutation in the rpoB gene (codon 531, S531L) associated with RIF resistance was detected by using the partial sequencing of the rpoB gene. Patient died due to disseminated bovis BCG infection and multiple organ failure. To our knowledge, there are only six RIF-resistant M.bovis BCG strains isolated from patients in the literature. However, this is the first RIF-resistant M.bovis BCG strain isolated from a NEMO-deficient patient.

[Investigation of extensive drug resistance in multidrug resistance tuberculosis isolates]. / Çok Ilaca Dirençli Tüberküloz Izolatlarinda Yaygin Ilaç Direncinin Arastirilmasi

Increasing number of drug resistant tuberculosis (TB) cases, observed in recent years, is an important public health problem. Extensively drug resistant TB (XDR-TB) is the development of resistance against any fluoroquinolones and at least one of the injectable second line anti-TB drugs in addition to resistance against isoniazide and rifampicin which are the first line anti-TB drugs [definition of multidrug resistant TB (MDR-TB)]. Anti-TB therapy failed with first-line anti-TB drugs due to MDR-TB cases is being planned according to second-line anti-TB drug susceptibility test results if available and if not, standart treatment protocols are used. Although it is recommended that individual anti-TB therapy should be designed according to the isolate's susceptibility test results, standart therapeutic protocols are always needed since second-line anti-TB drug susceptibility testing generally could not be performed in developing countries like Turkey. For this reason, nationwide and regional surveillance studies to determine the resistance patterns are always needed to make decisions about the standard therapy algorithms. In this study, it was aimed to investigate the presence of extensive drug resistance among 81 MDR-TB isolates obtained from various health care facilities from Istanbul, Izmir and Manisa and to determine the XDR-TB incidence in Marmara and Aegean regions. Furthermore, we aimed to provide epidemiological data to clinicians to support their choice of second-line anti-TB drugs for MDR-TB infections. Susceptibility testing of isolates for the first and the second-line anti-TB drugs were performed by using modified Middlebrook 7H9 broth in fluorometric BACTEC MGIT 960 system (Becton Dickinson, USA). Eighty-one MDR-TB isolates included in this study were isolated from 43 (53.1%) patients residing in Istanbul, 26 (32.1%) in Izmir and 12 (14.8%) in Manisa provinces. We could not find any isolate consistent with XDR-TB definition in this study. Second-line drug resistance rates of MDR-TB isolates to amikacin and kanamycin were 1.2%, ofloxacin and levofloxacin were 2.5%, capreomycin was 14.8%, ethionamide was 37% whereas linezolid resistance was not detected. Statistically significant correlation was detected between resistance rates of these antibiotic pairs; levofloxacin-ofloxacin (p< 0.01), amikacin-kanamycin (p= 0.01) and streptomycin-ethionamide (p= 0.04). In our study, extensive drug resistance was not encountered in any MDR-TB isolates while high resistance rates was observed against ethionamide and capreomycin. It can be concluded that parenteral aminoglycosides amikasin and kanamycin, fluoroquinolones and linezolid seemed to be reliable anti-TB agents in MDR-TB treatment, however, further larger scale studies are needed.

Evaluation of antimicrobial susceptibilities of rapidly growing mycobacteria by Sensititre RAPMYCO panel.

This study used Sensititre RAPMYCO to test the activities of amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, linezolid, sulfamehoxazole, tigecycline and tobramycin against 25 clinical isolates of rapidly growing mycobacteria (RGM), including the common disease producing species Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium fortuitum and Mycobacterium peregrinum. Analysis of the four different RGM species showed that isolates of M. fortuitum and M. peregrinum were more susceptible than M. abscessus and M. chelonae. Different antimicrobials showed a variable sensitivity in all strains. Therefore, each species and strain must be individually evaluated, and it is always advisable to perform in vitro sensitivity tests before the treatment of infections due to RGM.

Evaluation of the GeneXpert MTB/RIF assay for rapid diagnosis of tuberculosis and detection of rifampin resistance in pulmonary and extrapulmonary specimens.

Mycobacterium tuberculosis remains one of the most significant causes of death from an infectious agent. The rapid diagnosis of tuberculosis and detection of rifampin (RIF) resistance are essential for early disease management. The GeneXpert MTB/RIF assay is a novel integrated diagnostic device for the diagnosis of tuberculosis and rapid detection of RIF resistance in clinical specimens. We determined the performance of the MTB/RIF assay for rapid diagnosis of tuberculosis and detection of rifampin resistance in smear-positive and smear-negative pulmonary and extrapulmonary specimens obtained from possible tuberculosis patients. Two hundred fifty-three pulmonary and 176 extrapulmonary specimens obtained from 429 patients were included in the study. One hundred ten (89 culture positive and 21 culture negative for M. tuberculosis) of the 429 patients were considered to have tuberculosis. In pulmonary specimens, sensitivities were 100% (27/27) and 68.6% (24/35) for smear-positive and smear-negative specimens, respectively. It had a lower sensitivity with extrapulmonary specimens: 100% for smear-positive specimens (4/4) and 47.7% for smear-negative specimens (21/44). The test accurately detected the absence of tuberculosis in all 319 patients without tuberculosis studied. The MTB/RIF assay also detected 1 RIF-resistant specimen and 88 RIF-susceptible specimens, and the results were confirmed by drug susceptibility testing. We concluded that the MTB/RIF test is a simple method, and routine staff with minimal training can use the system. The test appeared to be as sensitive as culture with smear-positive specimens but less sensitive with smear-negative pulmonary and extrapulmonary specimens that include low numbers of bacilli.

Evaluation of the performance of QuantiFERON®-TB Gold plus test in active tuberculosis patients.

The aim was to evaluate the sensitivity and the possible factors affecting the sensitivity of the QuantiFERON®-TB Gold Plus (QFT-Plus) assay in culture-positive active TB (Tuberculosis) patients, to investigate the possible causes of negative and indeterminate results in active TB patients, and to compare the QFT-Plus results of active TB patients and latent tuberculosis infection (LTBI) cases. The QFT-Plus assay was performed in 46 active TB patients and 64 LTBI. The sensitivity of the test was found as 79.5% in all culture-positive patients, 72.7% in the immunocompromised patients, and 86.4% in the non-immunocompromised patients. Compared to active TB, individuals with LTBI had a lower T-cell response and lower IFN-É£ concentrations. It was determined that the immunocompromisation reduced the sensitivity of the test and the secreted IFN-É£ concentrations and increased the indeterminate results in patients with active TB. There was no difference in secreted IFN-É£ concentrations between M. tuberculosis clones, but higher IFN-É£ concentrations in patients infected with M. tuberculosis strains compared to patients infected with zoonotic strains. Compared with active TB, response to "only to TB2" was significantly higher in LTBI. In conclusion, it was concluded that TB2 tube increased sensitivity in LTBI but may not contribute to sensitivity in active TB.

A new nomenclature for the livestock-associated Mycobacterium tuberculosis complex based on phylogenomics.

Background:  The bacteria that compose the Mycobacterium tuberculosis complex (MTBC) cause tuberculosis (TB) in humans and in different animals, including livestock. Much progress has been made in understanding the population structure of the human-adapted members of the MTBC by combining phylogenetics with genomics. Accompanying the discovery of new genetic diversity, a body of operational nomenclature has evolved to assist comparative and molecular epidemiological studies of human TB. By contrast, for the livestock-associated MTBC members, Mycobacterium bovis, M. caprae and M. orygis, there has been a lack of comprehensive nomenclature to accommodate new genetic diversity uncovered by emerging phylogenomic studies. We propose to fill this gap by putting forward a new nomenclature covering the main phylogenetic groups within M. bovis, M. caprae and M. orygis. Methods:  We gathered a total of 8,736 whole-genome sequences (WGS) from public sources and 39 newly sequenced strains, and selected a subset of 829 WGS, representative of the worldwide diversity of M. bovis, M. caprae and M. orygis. We used phylogenetics and genetic diversity patterns inferred from WGS to define groups. Results:  We propose to divide M. bovis, M. caprae and M. orygis in three main phylogenetic lineages, which we named La1, La2 and La3, respectively. Within La1, we identified several monophyletic groups, which we propose to classify into eight sublineages (La1.1-La1.8). These sublineages differed in geographic distribution, with some being geographically restricted and others globally widespread, suggesting different expansion abilities. To ease molecular characterization of these MTBC groups by the community, we provide phylogenetically informed, single nucleotide polymorphisms that can be used as barcodes for genotyping. These markers were implemented in KvarQ and TB-Profiler, which are platform-independent, open-source tools. Conclusions:  Our results contribute to an improved classification of the genetic diversity within the livestock-associated MTBC, which will benefit future molecular epidemiological and evolutionary studies.