[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.

Safety and effectiveness of a 2009 H1N1 vaccine in chronic kidney disease children.

BACKGROUND/AIMS: Influenza vaccination is the most effective method for preventing influenza infection and its complications. The risk groups are children with chronic kidney diseases (CKDs) such as children on peritoneal dialysis (PD) and hemodialysis (HD), predialysis CKD (pCKD) and renal transplant (RTx) patients and immunosuppressed children. The aim of the present study was to assess the safety and immunogenicity of a single administration of a monovalent inactivated pandemic (H1N1) 2009 vaccine in children with CKD. METHODS: Patients were given a single intramuscular injection of 0.5 ml of monovalent inactivated vaccine. RESULTS: Totally, there were 25 pediatric patients with a diagnosis of CKD (16 PD, 2 HD, 3 pCKD and 4 RTx). Seroconversion was observed in 15 of the 16 patients with PD. Seroconversion occurred in all patients undergoing pCKD and HD. However, 3 of the RTx patients were seronegative. CONCLUSION: This study demonstrated a high level of immunogenicity and safety of an (H1N1) 2009 influenza vaccine in children with CKD.

[Comparison of two different real-time PCR systems in postmortem diagnosis of tuberculosis in paraffin-embedded tissues]. / Postmortem tüberküloz tanisinda parafi ne gömülü dokularda iki farkli gerçek zamanli PCR sisteminin karsilastirilmasi

Tuberculosis (TB) is one of those infections with high morbidity and mortality in all around the world. Hundreds of people died from this disease without diagnosed or due to resistant strains in Turkey. Therefore, it is important to identify postmortem cases who have died from tuberculosis. Molecular methods have been widely used as well as conventional methods in the diagnosis of tuberculosis. The aim of this study was to compare the two different real-time polymerase chain reaction (Rt-PCR) system in the postmortem diagnosis of Mycobacterium tuberculosis infections in paraffin-embedded tissues. A total of 40 paraffin-embedded tissue samples [lung (n= 35), brain (n= 2), heart (n= 2), lymph node (n= 1)] in which histopathologic findings consistent with TB (necrotizing granulomatous inflammation, gelatinous caseous pneumonia, necrotic fibrous nodul) obtained from 37 autopsy cases (31 male, 6 female; age range: 25-85 yrs) were included in the study. Paraffin-embedded tissues were deparafinized with xylene and ethyl alcohol and then DNA isolation was done with QIAsymphony DSP Virus/Pathogen Midi kit in the QIAsymphony device. DNA amplification process was performed by Rt-PCR using the kit Artus® M. tuberculosis RG-PCR in the Rotor-Gene® Q device (Qiagen, Germany). Likewise, after deparafinization process, samples placed in the cartridge and isolation and Rt-PCR was performed by Xpert® MTB/RIF (Cepheid, USA) system, simultaneosly. Seventeen and 20 out of the 40 paraffin-embedded tissues yielded positive results with Qiagen and Xpert system, respectively. M.tuberculosis DNA was found positive in 13 (32.5%) and negative in 16 (40%) of the samples by both of the systems, exhibiting 72.5% (29/40) of concordance. On the other hand, seven (17.5%) samples that were positive with Xpert system yielded negative result with the Qiagen, while four (10%) samples that were positive with Qiagen yielded negative result with the Xpert system. Of the 20 positive cases detected with Xpert MTB/RIF system, 15 were found rifampicin-susceptible, and three were rifampicin-resistant. In two samples in which M. tuberculosis DNA was low positive, rifampicin resistance could not be detected. The identification of M.tuberculosis infections in postmortem cases will contribute epidemiological data in Turkey. In these cases, effective sampling and diagnosing of M.tuberculosis infections by acid-fast stain and culture methods are crucial. However, in cases without microbiological sampling the detection of M.tuberculosis DNA in paraffin-embedded tissues with PCR, although there are differences between PCR systems has diagnostic value. In conclusion, our data indicated that Xpert MTB/RIF system is more favourable to detect M.tuberculosis DNA in paraffin-embedded tissues, with the advantages of determination of rifampicin resistance, and detection of more positive results within a shorter time.

[Investigation of the presence of Mycobacterium tuberculosis in the lymph node aspirates of the suspected tularemia lymphadenitis cases]. / Tularemi lenfadeniti süphesi ile alinan lenf aspirati örneklerinde Mycobacterium tuberculosis s varliginin arastirilmasi

Recently reports of cervical tuberculous lymphadenitis and oropharyngeal tularemia which are the most common infectious causes of granulomatous lymphadenitis, have been significantly increased in Turkey. The differentiation of cervical tuberculous lymphadenitis and oropharyngeal tularemia is usually confusing on the basis of clinical and histopathological findings. Thus, in tularemia endemic areas, the patients are more commonly evaluated in terms of tularemia lymphadenitis leaving tuberculosis out. The aim of this study was to investigate the presence of Mycobacterium tuberculosis in cervical lymph node aspirates, obtained from tularemia suspected cases. A total of 105 oropharyngeal tularemia-suspected cases which were found negative for Francisella tularensis by bacteriological (culture), molecular (PCR) and serological (microagglutination) methods, were included in the study. The samples had been previously studied at National Tularemia Reference Laboratory, Turkish Public Health Institution, between 2009-2011. The study samples were evaluated in terms of M.tuberculosis by culture and real-time PCR (rtPCR) methods in the National Tuberculosis Reference Laboratory. Both Lowenstein-Jensen (LJ) medium and liquid-based MGIT (BD, USA) automated culture system were used for mycobacterial culture. Samples that yielded mycobacterial growth were identified as M.tuberculosis by immunochromotographic test (BD, USA). The lymph node aspirates of 65 patients who were F.tularensis PCR negative but antibody positive, were used as the control group. As a result, M.tuberculosis was found to be positive in 9 (8.6%) of 105 tularemia-negative lymph node aspirates, sent to our laboratory from different geographic regions for the investigation of tularemia. Six of the M.tuberculosis positive cases were male and the age range of the patients was 26-85 years. The presence of M.tuberculosis was detected only by culture in two samples, only by rtPCR in five samples and both by culture and rtPCR in two samples. M.tuberculosis was not identified in the control group specimens. Three of the samples which revealed tuberculosis, were from the tularemia endemic areas. In conclusion, the data of this preliminary study indicated that tuberculous lymphadenitis should be kept in mind in suspected tularemia cases and those patients should also be investigated simultaneously for the presence of tuberculous lymphadenitis.

[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.

Differential expression of maturation and activation markers on NK cells in patients with active and latent tuberculosis.

NK cells were recently suggested to be important for the initial control of M. tuberculosis infection. The phenotypes of the 3 main NK blood subsets, CD56bright , CD56dim , and CD56neg cells, were characterized by flow cytometry in a cohort of 81 prospectively enrolled subjects (21 untreated patients with active tuberculosis -aTB-, 35 latently TB infected -LTBI- subjects, and 25 non-infected controls), using 9 different mAbs added to whole blood. Compared to LTBI subjects, patients with aTB had lower proportions of total NK cells, lower proportions and numbers of CD56neg cells expressing early maturation markers (CD161, NKp30, NKp46), but higher density of NKp30 and NKp46 expression on both CD56neg and CD56dim subsets, associated with higher expression of granzymes A/B. They also had higher proportions of activated CD69pos cells within all 3 NK cell subsets and, the percentage of CD69pos CD56dim cells among CD69pos and/or NKG2Cpos NK cells was identified as a potential biomarker to discriminate aTB from LTBI. LTBI subjects were in contrast characterized by higher expression of late maturation markers (CD57, KIR molecules) on the CD56neg subset, by higher proportions of NKG2Cpos KIRpos CD56dim NK cells, and by higher in vitro IFN-γ production than patients with aTB. Thus, the in-depth phenotypic characterization of blood NK cell subsets provides new insights on possible functional modifications and the potential role of NK cells in the control of M. tuberculosis infection in humans.

Distinct Expression Patterns of Interleukin-22 Receptor 1 on Blood Hematopoietic Cells in SARS-CoV-2 Infection.

The new pandemic virus SARS-CoV-2 is characterized by uncontrolled hyper-inflammation in severe cases. As the IL-22/IL-22R1 axis was reported to be involved in inflammation during viral infections, we characterized the expression of IL-22 receptor1, IL-22 and IL-22 binding protein in COVID-19 patients. Blood samples were collected from 19 non-severe and 14 severe patients on the day they presented (D0), at D14, and six months later, and from 6 non-infected controls. The IL-22R1 expression was characterized by flow cytometry. Results were related to HLA-DR expression of myeloid cells, to plasma concentrations of different cytokines and chemokines and NK cells and T lymphocytes functions characterized by their IFN-γ, IL-22, IL-17A, granzyme B and perforin content. The numbers of IL-22R1+ classical, intermediate, and non-classical monocytes and the proportions of IL-22R1+ plasmacytoid DC (pDC), myeloid DC1 and DC2 (mDC1, mDC2) were higher in patients than controls at D0. The proportions of IL-22R1+ classical and intermediate monocytes, and pDC and mDC2 remained high for six months. High proportions of IL-22R1+ non-classical monocytes and mDC2 displayed HLA-DRhigh expression and were thus activated. Multivariate analysis for all IL-22R1+ myeloid cells discriminated the severity of the disease (AUC=0.9023). However, correlation analysis between IL-22R1+ cell subsets and plasma chemokine concentrations suggested pro-inflammatory effects of some subsets and protective effects of others. The numbers of IL-22R1+ classical monocytes and pDC were positively correlated with pro-inflammatory chemokines MCP-1 and IP-10 in severe infections, whereas IL-22R1+ intermediate monocytes were negatively correlated with IL-6, IFN-α and CRP in non-severe infections. Moreover, in the absence of in vitro stimulation, NK and CD4+ T cells produced IFN-γ and IL-22, and CD4+ and CD8+ T cells produced IL-17A. CD4+ T lymphocytes also expressed IL-22R1, the density of its expression defining two different functional subsets. In conclusion, we provide the first evidence that SARS-CoV-2 infection is characterized by an abnormal expression of IL22R1 on blood myeloid cells and CD4+ T lymphocytes. Our results suggest that the involvement of the IL-22R1/IL-22 axis could be protective at the beginning of SARS-CoV-2 infection but could shift to a detrimental response over time.

Chemerin plasma levels are increased in COVID-19 patients and are an independent risk factor of mortality.

Background: Chemerin is an extracellular protein with chemotactic activities and its expression is increased in various diseases such as metabolic syndrome and inflammatory conditions. Its role in lung pathology has not yet been extensively studied but both known pro- and anti-inflammatory properties have been observed. The aim of our study was to evaluate the involvement of the chemerin/ChemR23 system in the physiopathology of COVID-19 with a particular focus on its prognostic value. Methods: Blood samples from confirmed COVID-19 patients were collected at day 1, 5 and 14 from admission to Erasme Hospital (Brussels - Belgium). Chemerin concentrations and inflammatory biomarkers were analyzed in the plasma. Blood cells subtypes and their expression of ChemR23 were determined by flow cytometry. The expression of chemerin and ChemR23 was evaluated on lung tissue from autopsied COVID-19 patients by immunohistochemistry (IHC). Results: 21 healthy controls (HC) and 88 COVID-19 patients, including 40 in intensive care unit (ICU) were included. Plasma chemerin concentration were significantly higher in ICU patients than in HC at all time-points analyzed (p<0.0001). Moreover, they were higher in deceased patients compared to survivors (p<0.05). Logistic univariate regression and multivariate analysis demonstrated that chemerin level at day 14 of admission was an independent risk factor for death. Accordingly, chemerin levels correlated with inflammatory biomarkers such as C-reactive protein and tumor necrosis factor α. Finally, IHC analysis revealed a strong expression of ChemR23 on smooth muscle cells and chemerin on myofibroblasts in advanced acute respiratory distress syndrome (ARDS). Discussion: Increased plasma chemerin levels are a marker of severity and may predict death of COVID-19 patients. However, multicentric studies are needed, before chemerin can be considered as a biomarker of severity and death used in daily clinical practice. Further studies are also necessary to identify the precise mechanisms of the chemerin/ChemR23 system in ARDS secondary to viral pneumonia.

Evaluation of respiratory viral pathogens in acute asthma exacerbations during childhood.

OBJECTIVE: Common upper respiratory tract viruses are the most frequent and important causes of asthma exacerbations in both children and adults. Prospective epidemiologic studies report that up to 80% of childhood exacerbations are associated with viral upper respiratory tract infections. MATERIALS AND METHODS: The study group consisted of 104 children with asthma aged 3-17 years who received treatment for asthma exacerbations in our clinic between September 2009 and 2010. Nasopharyngeal and nasal swabs were obtained from all patients during an acute attack, and from the control group (31 subjects). These specimens were investigated for the presence of viral respiratory pathogens using a real-time multiplex PCR method. The patients were compared for the presence of respiratory pathogens and factors related to the severity of the asthma exacerbation. RESULTS: A pathogenic respiratory virus was detected in 53.8% of patients in the acute exacerbation group. The most commonly encountered viral agent was Rhinovirus (35.6%). Patients who had an acute exacerbation with or without a detectable viral pathogen were compared according to the severity of the exacerbation, the need for systemic steroids, and hospitalization rates. No statistically significant difference was found. CONCLUSION: Although viral upper respiratory tract infections are the most common cause of asthma exacerbations, the severity level of the exacerbation seems to be independent of whether a respiratory virus has been detected.

Serological response to influenza vaccine after hematopoetic stem cell transplantation.

Vaccination is the best strategy to prevent influenza infection that is a potential cause of morbidity and mortality in immunosuppressed patients. Here, we evaluated the factors that may affect serological response to influenza vaccine in patients who have undergone hematopoetic stem cell transplantation (HSCT). Sixty-one HSCT recipients were included in the study during the 2007-2008 influenza season. Serum samples prior to vaccination and 6-10 weeks after vaccination were collected. Samples were assayed for antibodies to influenza virus A/H1N1, A/H3N2, and B strains by hemagglutination-inhibition assay. The patients were followed in terms of clinical symptoms up to the next influenza season and for adverse effects within a month after vaccination. Overall, pre-vaccine seroprotection rate against all vaccine antigens (A/H1N1, A/H3N2, and B antigens) was 45.1%, post-vaccine seroprotection rate 91% and seroconversion rate was 28.3%. Seroconversion rates were found to be low against B in patients who were vaccinated in the late influenza season (p = 0.018; respectively). Five patients (10.9%) had no immune response against H1N1. Adverse events were reported in 19.6% (n = 9/46) of the patients. In conclusion, the patients should be vaccinated as early as possible in the influenza season, before they are exposed to the virus.

[Evaluation of PCR and ELISA-IgM results in the laboratory diagnosis of Crimean-Congo haemorrhagic fever cases in 2008 in Turkey]. / 2008 yili Kirim-Kongo kanamali atesi olgularinin laboratuvar tanisinda PCR ve ELISA-IgM sonuçlarinin irdelenmesi.

Crimean-Congo hemorrhagic fever (CCHF) is a fatal zoonotic viral haemorrhagic infection described in Africa, Asia, Eastern Europe, and the Middle East. CCHF virus (CCHFV) classified in Bunyaviridae family and Nairovirus genus, is transmitted to humans by tick (Hyalomma and Ixodid) bites and human to human transmission may occur by direct contact with blood or other infected tissues. The disease became endemic and a public health problem since 2002 outbreak in Turkey. The specific laboratory diagnosis and confirmation of the disease is performed in Refik Saydam National Public Health Agency, by using molecular and serological methods. For this purpose serum and/or plasma samples from suspected CCHF patients are submitted to the reference laboratory with an official "possible case report form". According to the algorithm in our laboratory, the first samples which were sent from possible acute cases were searched initially by an in-house real time-polymerase chain reaction (PCR) method and those which were found negative with PCR, were then studied by in-house ELISA method in terms of CCHF-IgM antibodies. In 2008, a total of 4634 samples obtained from 2855 CCHF suspected patients have been examined for the positivity of CCHFV, and 1315 (46%) cases were found to be positive by molecular and/or serologic methods. The aim of this study was to evaluate the results of 726 cases whose at least 2 samples were sent to laboratory, with at least 1 positivity in at least 1 clinical sample with either PCR or IgM ELISA, or both, and with complete informations in possible case report form, during 2008 in Turkey. The positive results were also analyzed according to the starting date of the complaints and the date samples received in order to evaluate the positivity rates of molecular and serological methods with regard to the time. The first serum samples in 94.1% (683/726) of cases were found to be positive with PCR and/or ELISA-IgM methods. PCR positivity was found as 78.1% (567/726), while CCHFV-IgM positivity was detected in 116 (72.9%) in the remaining 159 PCR negative samples. In the first sera, PCR and ELISA results were evaluated in relation to the start of complaints and the date samples received. After the onset of symptoms, PCR positivity was determined as 83.4% in the samples taken in the first 5 days, and reduces to 67.5% in the samples between 6-10 days. The detection rate of CCHFV-IgM increases up to 95% when PCR positivity rate decreases after the 5th day. As expected, positivity is determined to be high by PCR in the first days, and ELISA-IgM after the 5th day. In conclusion, recording clinical data such as the onset of disease and the date of sample received ensure the accurate evaluation of the disease and the laboratory results are reliably accomplished in a short time.

[Investigation of bactericidal effect and nitric oxide responses of Caco-2 epithelial cells and THP-1 macrophage cells against Streptococcus pyogenes and Escherichia coli]. / Caco-2 epitel hücresi ve THP-1 makrofaj hücrelerinin Streptococcus pyogenes ve Escherichia coli'ye karsi bakterisidal etki ve nitrik oksit yanitlarinin arastirilmasi.

Epithelial cells and macrophages contribute to innate immune responses by cell to cell interactions and releasing proinflammatory mediators. In this study, we aimed to illuminate the underlying mechanisms of contribution of macrophages and epithelial cells in the struggle against pathogens. Therefore, Streptococcus pyogenes and Escherichia coli activated Caco-2 cells and THP-1 macrophage-like cells were investigated for their bactericidal activities and nitric oxide (NO) production when they were alone, in contact with eachother and when their contact was blocked by continuing exposure of soluble mediators. Caco-2 epithelial cells and THP-1 macrophage cells were used as effector cells and S. pyogenes or E. coli as target cells and were incubated at an effector cell/target cell ratio of 1/1 in duplication in 24-well plates. After 5 hours incubation, supernatants were collected from each well for growth inhibition assay and were inoculated onto blood agar plates for S. pyogenes and eosin methylene blue agar plates for E. coli. Following overnight incubation at 37 degrees C, bactericidal effect rate was calculated by counting the colony forming units. The supernatants were also collected after 5 and 24 hours incubation for measurement of NO production by using Griess reagent. Bactericidal effect of Caco-2 cells alone against S. pyogenes and E. coli were found 21.9% and 36.2%, when seperated from THP-1 cells via an insert was 31.8% and 30.5%, and when cell-cell contact was established with THP-1 cells was 24.4% and 55.7%, respectively. Bactericidal effect of THP-1 cells alone against S. pyogenes and E. coli was 27.7% and 63.9%, when seperated from Caco-2 cells via an insert was %27.5 and 43.6%, and when cell-cell contact was established with Caco-2 cells was 24.4% and 55.7%, respectively. As a result, we found that Caco-2 epithelial cells and THP-1 macrophage cells had an antibacterial effect against S. pyogenes and E. coli (p < 0.05), and this effect was higher in macrophage cells than epithelial cells. NO levels in epithelial and/or macrophage cell culture supernatants collected after exposure to S. pyogenes and E. coli were significantly higher for S. pyogenes at 5 hours incubation and for E. coli at 24 hours incubation (p < 0.05). Morever, it can be concluded that macrophages played a more active role than epithelial cells in bactericidal effect and NO response. Besides, epithelial cells and macrophages activated each other more when they were in contact than when they were alone or when their contact was blocked.

[Influenza surveillance results in 2007-2008 winter season in nine provinces of Turkey]. / 2007-2008 kis sezonunda türkiye'nin dokuz ilinde influenza sürveyans sonuçlari.

Influenza virus infections constitute a serious public health problem owing to their epidemic and pandemic potential. Turkish Ministry of Health established the national influenza surveillance programme in two institutes to detect the virus types leading to the illness and the efficiency of the seasonal vaccine. Influenza surveillance is performed by Refik Saydam Hygiene Center, National Influenza Laboratory in nine provinces (which are located at central, northeast, south and east parts of Turkey) and by Istanbul University, Medical Faculty, Virology Laboratory in five provinces (which are located at west and northwest parts of Turkey). These two centers are the members of international information networks. The surveillance was aimed to contribute to the detection of influenza viruses with pandemic potential and also to determine the predominant strain circulating in Turkey. During November 2007-May 2008 period a total of 1157 clinical specimens collected from 90 health centers which were the representatives of nine provinces (Ankara, Samsun, Trabzon, Erzurum, Adana, Konya, Diyarbakir, Malatya and Van) were investigated for the presence of influenza virus and other respiratory viruses (Parainfluenza virus types 1-3, Respiratory Synctial Virus and Adenovirus). Samples were identified and subtyped by both molecular (real-time PCR) and cell culture techniques (MDCK and Hep-2). Influenza virus and at least one of the other respiratory viruses were detected in 321 (27.7%) and two different viruses in 16 of the specimens (total= 337). When all the specimens were considered, the most frequently identified virus was influenza A (n=188, 16.2%), H1N1 being 6.3% and H3N2 9.9%.The rate of identification for influenza B was 7.6% (n=88), for parainfluenza was 2.3% (n=27), for adenovirus was 2% (n=24) and for RSV was 0.9% (n=10). When only the positive specimens (n=337) were evaluated, influenza A was again the most frequently (55.7%) encountered virus, H1N1 being 38.8% and H3N2 61.2% of all. Influenza B was in the second rank with 26.1% frequency among the positive specimens. The results showed that influenza activity started around November and ended around May. When the distribution of influenza viruses were analysed according to months, Influenza A H1N1 predominated in January, influenza A H3N2 in December and February. influenza B viruses started to increase in February, and were also detected in May. The 2007-2008 influenza season in Turkey was characterized by moderate clinical activity, and a predominance of influenza A H3N2. These results indicate good match between the vaccine virus strains and the reported virus strains.

[Investigation of the bactericidal effect of urinary epithelial cells against different doses of Escherichia coli]. / Uriner epitel hücrelerinin farkli antijenik miktarlardaki Escherichia coli'ye karsi bakterisidal etkisinin arastirilmasi.

Epithelial cells take part in the stimulation of immune system during the conversion of nonspecific immune response to the adaptive one in the mucosal immune system. Epithelial cells also have critical roles in designating immune homeostasis towards immune regulation or tolerance. Its response type is determined according to the dose and structure of the antigen and the way it is presented. In this study, we aimed to evaluate the response of human urinary epithelial cells to different doses of Escherichia coli K12 by means of their bactericidal effect. Urine was collected from 16 healthy volunteers and urinary epithelial cells were prepared. The cells (effector) were stimulated with bacteria (target) in microplates for one hour with different effector/target cell ratios. At the end of the incubation period, the bactericidal effects were calculated and compared with microorganism controls. Mann-Whitney U test was used for statistical analysis. Urinary epithelial cells which were stimulated by E. coli K12 showed dose dependent bactericidal effect, calculated mean value for bactericidal effects were 60.7% at the 1/100.000 effector/target cell ratio and 11.3% 1/1.000.000 effector/target cell ratio, indicating that the bactericidal effect was dose dependent.

[Investigation of bactericidal effect and cytokine response of the oral epithelial cells against different antigenic doses of Streptococcus pyogenes]. / Insan agiz epitel hücresinin farkli antijenik miktarlardaki Streptococcus pyogenes'e karsi bakterisidal etkisinin ve sitokin yanitinin arastirilmasi.

Epithelial cells (EC) have an important role in the constitution of both innate and acquired immune responses. The aim of this study was to investigate the alterations of bactericidal effects and cytokine production patterns of human oral epithelial cells (OEC) against different doses of Streptococcus pyogenes. For this purpose, OEC have been stimulated with S. pyogenes with an effector/target (E/T) cell ratio of 1/1, 1/100, 1/1.000 and 1/10.000, and bactericidal effects and interleukin (IL)-6, IL-8 and IL-10 levels were detected in the first and sixth hours of incubation. The mean rates of bactericidal effect detected in the first and sixth hours were 38.7% and 54.5%, respectively. The bactericidal effects observed at 1/1 E/T cell ratio in the first hour, and at 1/1 and 1/100 E/T cell ratio in the the sixth hour were found significantly higher then the other cells ratios (p<0.05). Time- and dose-depended differences were detected in the cytokine responses of OEC for different S. pyogenes concentrations. IL-6 levels produced by stimulated OEC were found higher, and IL-8 levels were found lower then the levels which were produced by unstimulated OEC (p<0.05, p<0.001, respectively) in the first hour, while there were no change in IL-10 levels after stimulation with different bacterial concentrations (p>0.05). At the sixth hour there were no differences in the IL-6 levels produced by stimulated and unstimulated cells, while the levels of IL-8 produced by stimulated cells were found lower then the levels produced by unstimulated cells in the E/T cell ratio of 1/100, 1/1000 and 1/10.000 (p<0.05, p<0.01, p<0.01, respectively). Nevertheless IL-10 levels in the E/T cell ratio of 1/100 and 1/1.000 were statistically higher then the levels produced by unstimulated cells (p<0.05, p<0.05). As a result OEC stimulated with S. pyogenes showed dose dependent manner in bactericidal effect and cytokine production. It is suggested that epithelial cells stimulation with different doses of antigen contributed to the immune system activation or tolerance.

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.

[Bactericidal effects of human oral and urinary system epithelial cells against different Escherichia coli strains]. / Insan agiz ve uriner sistem epitel hücrelerinin farkli Escherichia coli suslarina karsi bakterisidal etkisinin arastirilmasi.

The functions of epithelial cells are more than a physical selective barrier for protection from mucosal pathogens. The epithelial response regulated by host-microorganism interaction may change due to histological and physiological differences between sterile and nonsterile sites. In this study we examined the presence of a difference between bactericidal capacities of urinary and oral epithelial cells against different strains of Escherichia coli. Oral epithelial cells were collected from unstimulated saliva and uroepithelial cells were collected from urine of eight healthy volunteers. E. coli K12 and O75 strains and epithelial cells were prepared for the experiment, added to microplates and incubated for one hour. The growth of bacteria was detected by the quantitative colony counting method. Antibacterial effects of the oral and urinary epithelial cells against E. coli K12 and O75 strains were 49.8%, 34.7%, 45.7% and 29.2%, respectively. As a result, it was detected that the antibacterial activity of oral epithelial cells to E. coli K12 and O75 were higher than urinary epithelial cells.

Epidemiological, demographic, and molecular characteristics of laboratory-confirmed pandemic influenza A (H1N1) virus infection in Turkey, May 15-November 30, 2009.

A total of 19,973 clinical specimens obtained from suspected cases of pandemic influenza A virus infection were analyzed by real-time reverse transcription-polymerase chain reaction. Mutations in hemagglutinin (HA) gene and alteration at position 275 in neuraminidase (NA) gene of the randomly selected 29 isolates were detected by sequencing analysis. The virus RNA was detected in 47.3% of the clinical specimens. The pandemic flu cases increased from the 42nd week and peaked in the 46th week of 2009. This intensity continued to the end of the study period. Pandemic flu mainly affected children in the 5-14 year age group, without any gender predominance. The analyzed strains had >98.9% homology with vaccine strains and with each other. More than 37% of the isolates had mutation at position D222E/N on HA gene. There was no isolate harbored mutation at the position H275Y of the NA gene, indicating that the virus isolates currently circulating in Turkey are sensitive to oseltamivir.