Possible relation between expression of circulating microRNA and plasma cytokine levels in cases of pulmonary tuberculosis.

INTRODUCTION: Tuberculosis (TB) is a life-threatening infection and early diagnosis is critical for treatment and prevention of transmission. There is evidence of correlation between miRNA expression and cytokine regulation during TB infection. The aim of this study was to determine the relationship between expression levels of miRNAs in plasma and cytokine levels as a potential biomarker for genetic predisposition and/or early diagnosis of TB infection. METHODOLOGY: The expression levels of 86 miRNAs were examined in plasma samples of 44 TB patients and 44 healthy controls by qRT-PCR using BioMarkTM 96.96 Dynamic Array (Fluidigm Corporation, South San Francisco, CA, USA) system. The levels of plasma TNF-α, IFN-γ, IL-1ß, IL-4, IL-6, IL-8, IL-10, and IL-12/P40 were examined with ELISA. RESULTS: We identified dysregulation of 18 miRNAs which included upregulation of miR-1, miR-7-5p, miR-9-5p, miR-10a-5p, miR-10b-5p, miR-100-5p, miR-106b-5p, miR-128-3p, miR-133a-3p, miR-143-3p, miR-193a-5p, miR-200b-3p, miR-205-5p, miR-210-3p, and miR-296-5p, and downregulation of miR-15b-5p, miR-16-5p, and miR-25-3p in plasma samples of patients with pulmonary TB (p < 0.05). A significant correlation between the expression levels of miR-1, miR-7-5p, miR-9-5p, miR-10a-5p, miR-10b-5p, miR-15b-5p, miR-100-5p, miR-143-3p, miR-193a-5p, miR-200b-3p, miR-210-3p and cytokine levels of TNF-α, IFN-γ, IL-1ß, IL-8 and IL-10 was identified (p < 0.05). CONCLUSIONS: We demonstrated that altered expression levels of plasma miRNAs consistent with immunological response have the potential to serve as non-invasive biomarkers for early diagnosis of pulmonary TB. Additional investigations with larger sample sizes will be required to confirm our findings and to determine if miRNAs can be possible targets for TB management strategies.

[Legionella Species Isolated in the Water Systems of Ships and Their Molecular Characterization]. / Gemilerin Su Sistemlerinden Izole Edilen Legionella Türleri ve Moleküler Karakterizasyonu.

Legionella species are generally found in nature and in water resources, and they are gram negative bacilli that can cause pneumonia by being transmitted from water systems to humans via aerosol or aspiration. Legionnaires' disease caused by this agent continues to be a public health problem in cruise ships. In this study, it was aimed to determine the prevalence of the colonization of Legionella species by culture method and to determine the molecular characterization of the isolated Legionella in water samples taken from the water systems of the ships docking in Mersin International Port. A total of 158 cold water samples were taken from 18 ferry and/or cargo ships docking in Mersin International Port between December 2014 and June 2015. Fifty-four of the samples were obtained from tanks, 68 from taps and 36 from shower heads. All samples were centrifuged and inoculated from the pellet onto "Buffered Coal Yeast Extract" (BCYE) (Oxoid, CM0655, UK) agar medium supplemented with iron pyrophosphate, L-cysteine and α-ketoglutarate (Oxoid, SR0110, UK). The culture plates were incubated for 10-15 days in microaerophilic environment in a desiccator at 37°C. The suspicious colonies grown in cultures were serogrouped by latex agglutination test (Oxoid, DR0800M, UK) and fluorescent antibody method (m-Tech Monoclonal Technologies, Inc., USA). For the molecular analysis of Legionella species grown in culture, DNA isolation was made from Legionella colonies and then polymerase chain reaction amplification was performed using specific primer sequences targeting the rpoB gene region of the Legionella genome. Direct DNA sequencing of rpoB gene products was performed in the "ABI PRISM 3130XL Genetic Analyzer" (Applied Biosystems, USA). The DNA sequences were typed by BLAST analysis and the determined types, and NCBI (National Center for Biotechnology Information) reference Legionella sequences were phylogenetically compared with the Neighbor-Joining comparison method by using the Mega 7 program. Legionella spp. was isolated in 18 (11.4%) of 158 samples. Of these, four (7.4%, 4/54) were detected from the tank, 11 (16.2%, 11/68) from the tap and three (8.33%, 3/36) from the shower head. After the latex agglutination test performed from the growing bacterial colonies, five (27.8%) were serogrouped as Legionella spp., four (22.2%) as Legionella pneumophila sg 5, two (11.1%, each) as L.pneumophila sg 1,L.pneumophila sg 8 and Legionella bozemanii and one (5.6%) as L.pneumophila sg 3. Two (11.1%) of the isolates grown in culture could not be serogrouped. Molecular characterization of 12 Legionella isolates could be performed. One of them was serologically serogrouped as L.bozemanii, and it was found to be 99% similar to Legionella rubrilucens when compared with NCBI Legionella sequence data in the BLAST program. One isolate that could not be differentiated by serogrouping was identified as Legionella erytra in the BLAST program after DNA sequence analysis. The remaining 10 isolates (55.6%, n= 18) were confirmed as L.pneumophilia after the comparison with reference NCBI sequences. In this study, it was determined that 11.4% of the water samples collected from the water systems of the ships docking in Mersin International Port were contaminated with Legionella species. The detected Legionella species have an important potential source of infection for the captain, ship workers and passengers travelling on the ships. In this respect, this study reveals the necessity of establishing studies to improve the risk management of Legionella in the water systems of ships.

Association of cytokine gene polymorphisms with susceptibility to cutaneous leishmaniasis in a Turkish population.

AIMS: The objective of this study was to determine the association of TNF-α -308 G/A, IFN-γ +874 T/A, IL-12B + 1188 A/C, IL-10 -1082 G/A and IL-4 -590 C/T polymorphisms with susceptibility to CL. METHODS AND RESULTS: A total of 55 CL patients and 110 controls from Sanliurfa province of Turkey were included to this study. Polymorphisms were genotyped by 'polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)' and 'amplification refractory mutation system-PCR (ARMS-PCR)' methods. A statistically significant difference was noted in the allele (P < .001, P = .002) and genotype (P < .001, P = .001,) frequencies of TNF-α -308 G/A and IL-4 -590 C/T, respectively. TNF-α 308 GG versus GA genotype (OR = 19.556 [95% CI 8.310-46.019] P < .001), GG versus GA + AA genotype (OR = 20.444 [95% CI 8.707-48.004] P < .001) and G versus A allele (OR = 6.968 [95% CI 3.903-12.440] P < .001) revealed significant association with CL. IL-4 -590 CC versus TT + CT genotype (OR = 2.049 [95% CI 1.025-4.096], P = .041) and C versus T allele (OR = 2.441 [95% CI 1.355-4.396], P = .002) revealed significant association with CL. CONCLUSION: Our study indicates that TNF-α 308 G/A and IL-4-590 C/T polymorphisms are significantly associated with susceptibility to CL. Individuals carrying A allele at TNF-α promoter -308 position and T allele at IL-4 promoter -590 position are at a higher risk for CL.

[Detection and Molecular Characterization of Human Adenoviruses from Acute Conjunctivitis Cases]. / Akut Konjunktivitli Olgularda Insan Adenovirüslerinin Belirlenmesi ve Moleküler Karakterizasyonu.

Human adenoviruses (hAdV) can cause a wide range of clinical diseases in children and adults that mainly affect respiratory, eye and gastrointestinal systems. Ocular hAdV infections have various clinical manifestations such as epidemic keratoconjunctivitis, pharyngoconjunctival fever and non-specific follicular conjunctivitis. The hAdV genotypes which can cause conjunctivitis vary according to geographic distribution. In the study, we aimed to determine the frequency of the presence of hAdV by molecular methods and to determine the types with phylogenetic analysis in conjunctival swab samples taken from patients diagnosed clinically as acute conjunctivitis. Conjunctival swab samples (n= 100) were taken from the patients with acute conjunctivitis who have admitted to Mersin University Faculty of Medicine Hospital Ophthalmology Clinic and 50 conjunctival swab samples taken from healthy individuals as a control, between September 2014-July 2017 were included in the study. Following the DNA isolation from swab samples, polimerase chain reaction (PCR) amplification was performed using specific primer sequences targeting the hexon gene region of the hAdV genome. In order to determine hAdV types, direct DNA sequence analysis of hexon gene products was performed in "ABI PRISM 3130XL Genetic Analyzer" (Applied Biosystems, Foster City, CA, USA). The obtained hAdV DNA sequences were typed by BLAST analysis and the identified genotypes were compared phylogenetically with the reference hAdV sequences of the NCBI In the study, 30 (30%, 30/100) of the swab samples of the patients with acute conjunctivitis were found positive for hAdV hexon gene PCR. The hAdV DNA was not found in the conjunctival swab samples belonging to the healthy individuals included as controls. A total 27 samples found as positive of the hexon gene PCR were genotyped by direct DNA sequence analysis. A total of 5 genotypes were identified and the most common genotypes were hAdV-8 (n= 17, 63%) and followed by hAdV-53 (n= 4, 14.8%), hAdV-4 (n= 4, 14.8%), hAdV-7 (n= 1, 3.7%) and hAdV-37 (n= 1, 3.7%). In this study, the prevalence of adenoviral conjunctivitis determined by hexon gene PCR in patients with clinical diagnosis of acute conjunctivitis was similar to the prevalence rate reported in other regions of the world. In our region, more than one type of hAdV type was associated with acute conjunctivitis. The predominant type was determined as hAdV-8 with a 63% ratio. These results will significantly contribute to the molecular epidemiology of hAdV types in conjunctivitis cases.

Female genital tuberculosis cases with distinct clinical symptoms: Four case reports.

BACKGROUND: Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. Genital TB (GTB) is a form of extrapulmonary TB that occurs more frequently in women, in whom it classically presents in association with menstrual irregularity, pregnancy loss and short and long-term sequelae especially infertility in infected women. Patients with GTB are usually young women diagnosed during workup for infertility. GTB is rare in postmenopausal women and responsible for only approximately 1% of postmenopausal bleeding. In this study, we aimed to evaluate the laboratory, clinical and demographic characteristics of female GTB cases. CASE: We presented four female GTB cases with distinct clinical symptoms. All patients have no history of TB, and no acid-fast bacilli were seen in smears prepared from the clinical materials of the patients. Histopathological examinations revealed granulomatous inflammation in all patients. CONCLUSION: In the light of the clinical features of these cases we aimed to emphasize that, female GTB must be taken into account in the patients with different clinical symptoms like postmenopausal bleeding, menometrorrhagia, infertility, and menstrual irregularities. We believe that these symptoms will be helpful for the diagnosis and treatment of female GTB.

El valor de proteínas de fase aguda y la prueba LightCycler® SeptiFast en el diagnóstico de infecciones bacterianas y virales en niños / The value of acute phase reactants and LightCycler® SeptiFast test in the diagnosis of bacterial and viral infections in pediatric patients

Introducción. Evaluamos el nivel de reactantes de fase aguda y la prueba LightCycler® SeptiFast para diferenciar infecciones bacterianas vs.virales. Métodos. Estudio prospectivo en niños febriles. Se analizaron recuento de leucocitos, proteína C-reactiva y procalcitonina en días 1, 3 y 7 de hospitalización. El día 1 se realizaron hemocultivo y radiografía de tórax. Se evaluaron dos grupos de niños que presentaron infecciones bacterianas o virales. Resultados. Se incluyeron 94 niños febriles. La temperatura media de la fiebre fue significativamente más alta en niños con infecciones bacterianas que con infecciones virales (p < 0,001). En 34 (72,3%) niños con infecciones bacterianas, el hemocultivo fue negativo. De ellos, 12 (35,2%) presentaron prueba SeptiFast positiva. No hubo resultados positivos en hemocultivos de niños con infecciones virales y todos tuvieron resultado negativo para la prueba SeptiFast. La media de proteína C-reactiva el primer día de hospitalización fue significativamente más alta en el grupo con infecciones bacterianas (p < 0,001) y en los días 3 y 7 junto con la procalcitonina fueron significativamente más altas en niños con infecciones bacterianas (p <0,001). La sensibilidad y especificidad de los leucocitos, la proteína C-reactiva y la procalcitonina fueron 63,8%, 44,7%, 74,5% y 78,7%, 68,1% y 100%, respectivamente. Las áreas bajo la curva de los leucocitos, la proteína C-reactiva y la procalcitonina fueron 0,519, 0,764 y 0,835, respectivamente. Conclusiones. Los reactantes de fase aguda, en especial procalcitonina, y la prueba LightCycler® SeptiFast podrían ayudar a diferenciar infecciones bacterianas de virales.

Introduction: This study was performed to investigate the value of acute phase reactants and LightCycler® SeptiFast test to differentiate bacterial and viral infections. Population and methods: Children with fever were enrolled to this prospective study. Peripheral white blood cell (WBC), C-reactive protein (CRP) and procalcitonin (PCT) were studied from all patients on day 1, 3 and 7. Blood culture and chest X-ray were also obtained on day 1. Blood samples for LightCycler® SeptiFast test were obtained in all patients to use them if there was uncertain diagnosis between bacterial or viral infection. The patients were divided into two groups as bacterial and viral infection. Results: A total of 94 children with fever were enrolled. The mean value of fever was significantly higher in bacterial group than viral group (p <0.001). In bacterial infection group, 34 (72.3%) patients had negative blood culture. Of those, 12 (35.2%) had positive SeptiFast test. There were no positive blood culture in patients with viral infection group and all of them had negative SeptiFast test. The mean levels of CRP on the first day of admission were significantly higher in bacterial group than viral group (p <0.001). CRP and PCT levels of day 3 and 7 were significantly higher in bacterial group (p <0.001). The sensitivity and specificity levels of WBC, CRP and PCT were 63.8%, 44.7%, 74.5% and 78.7% ,68.1% and 100%, respectively. Conclusions: We found that acute phase reactants, especially PCT, and LightCycler® SeptiFast test may help to differentiate bacterial and viral infections.

Urinary tuberculosis: a cohort of 79 adult cases.

We aimed to investigate the demographic, clinical, diagnostic, treatment and outcome features of patients with urinary tuberculosis (UTB). Patients with UTB admitted to seven separate centers across Turkey between 1995 and 2013 were retrospectively evaluated. The diagnosis of UTB was made by the presence of any clinical finding plus positivity of one of the following: (1) acid-fast bacilli (AFB) in urine, (2) isolation of Mycobacterium tuberculosis, (3) polymerase chain reaction (PCR) for M. tuberculosis, (4) histopathological evidence for TB. Seventy-nine patients (49.36% male, mean age 50.1 ± 17.4 years) were included. Mean time between onset of symptoms and clinical diagnosis was 9.7 ± 8.9 months. The most common signs and symptoms were hematuria (79.7%), sterile pyuria (67.1%), dysuria (51.9%), weakness (51.9%), fever (43%) and costovertebral tenderness (38%). Cystoscopy was performed in 59 (74.6%), bladder biopsy in 18 (22.8%), kidney biopsy in 1 (1.26%) and nephrectomy in 12 (15.2%) patients. Histopathological verification of UTB was achieved in 12 (63.1%) patients who undergone biopsy and in 100% of those undergone nephrectomy. Mycobacterium tuberculosis was isolated in the urine of 50 (63.3%) cases. Four-drug standard anti-TB treatment was the preferred regimen for 87.3% of the patients. Mean treatment duration was 10.5 ± 2.7 months. Deterioration of renal function occurred in 15 (18.9%) patients two of whom progressed to end-stage renal disease and received hemodialysis. Only one patient died after 74-day medical treatment period. Cases with UTB may present with non-specific clinical features. All diagnostic studies including radiology, cyctoscopy and histopathology are of great importance to exclude UTB and prevent renal failure.

Serum microRNAs; miR-30c-5p, miR-223-3p, miR-302c-3p and miR-17-5p could be used as novel non-invasive biomarkers for HCV-positive cirrhosis and hepatocellular carcinoma.

Recently, serum miRNAs have been evolved as possible biomarkers for different diseases including hepatocellular carcinoma and other types of cancers. Investigating certain serum miRNAs as novel non-invasive markers for early detection of HCV-positive cirrhosis and hepatocellular carcinoma (HCC). The expression profiles of 58 miRNA were analyzed in patient's plasma of chronic hepatitis C (CHC), HCV-positive cirrhosis and HCV-positive HCC and compared with control group samples. Totally 94 plasma samples; 64 patient plasma (26 CHC, 30 HCV-positive cirrhosis, 8 HCV-positive HCC) and 28 control group plasma, were included. The expression profiles of 58 miRNAs were detected for all patient and control group plasma samples by qRT-PCR using BioMarkTM 96.96 Dynamic Array (Fluidigm Corporation) system. In CHC group, expression profiles of miR-30a-5p, miR-30c-5p, miR-206 and miR-302c-3p were found significantly deregulated (p < 0.05) when compared versus control group. In HCV-positive cirrhosis group, expression profiles of miR-30c-5p, miR-223-3p, miR-302c-3p, miR-17-5p, miR-130a-3p, miR-93-5p, miR-302c-5p and miR-223-3p were found significantly deregulated (p < 0.05). In HCV-positive HCC group, expression profiles of miR-17-5p, miR-223-3p and miR-24-3p were found significant (p < 0.05). When all groups were compared versus control, miR-30c-5p, miR-223-3p, miR-302c-3p and miR-17-5p were found significantly deregulated for cirrhosis and HCC. These results imply that miR-30c-5p, miR-223-3p, miR-302c-3p and miR-17-5p could be used as novel non-invasive biomarkers of HCV-positive HCC in very early, even at cirrhosis stage of liver disease.

Human papillomavirus genotype distribution and E6/E7 oncogene expression in Turkish women with cervical cytological findings.

BACKGROUND: Infection with certain human papillomavirus (HPV) genotypes is the most important risk factor related with cervical cancer. The objective of the present study was to investigate the prevalence of HPV infection, the distribution of HPV genotypes and HPV E6/E7 oncogene mRNA expression in Turkish women with different cervical cytological findings in Mersin province, Southern Turkey. MATERIALS AND METHODS: A total of 476 cytological samples belonging to women with normal and abnormal cervical Pap smears were enrolled in the study. For the detection and genotyping assay, a PCR/direct cycle sequencing approach was used. E6/E7 mRNA expression of HPV-16, 18, 31, 33, and 45 was determined by type-specific real-time NASBA assay (NucliSENS EasyQ(®)HPV v1.1). RESULTS: Of the 476 samples, 106 (22.3%) were found to be positive for HPV DNA by PCR. The presence of HPV was significantly more common (p<0.001) in HSIL (6/8, 75%) when compared with LSIL (6/14, 42.9%), ASC-US (22/74, 29.7%) and normal cytology (72/380, 18.9%). The most prevalent genotypes were, in descending order of frequency, HPV genotype 66 (22.6%), 16 (20.8%), 6 (14.2%), 31 (11.3%), 53 (5.7%), and 83 (4.7%). HPV E6/E7 oncogene mRNA positivity (12/476, 2.5%) was lower than DNA positivity (38/476, 7.9%). CONCLUSIONS: Our data present a wide distribution of HPV genotypes in the analyzed population. HPV genotypes 66, 16, 6, 31, 53 and 83 were the predominant types and most of them were potential carcinogenic types. Because of the differences between HPV E6/E7 mRNA and DNA positivity, further studies are required to test the role of mRNA testing in the triage of women with abnormal cervical cytology or follow up of HPV DNA positive and cytology negative. These epidemiological data will be important to determine the future impact of vaccination on HPV infected women in our region.

[Determination of the frequency of human bocavirus and other respiratory viruses among 0-2 years age group children diagnosed as acute bronchiolitis]. / Bronsiyolit tanisi alan 0-2 yas grubu çocuklarda insan bokavirusu ve diger solunum viruslarinin sikliginin arastirilmasi.

Acute bronchiolitis, mostly seen in infants and younger children, is a lower respiratory tract infection frequently caused by viral agents. We aimed to determine the frequency of a broad panel of respiratory viruses including human bocavirus (HBoV) and to assess the clinical characteristics of acute bronchiolitis in a group of children under 24 months of age. A total of 62 children (45 male, 17 female; age range: 0-2 years) with the initial diagnosis of acute bronchiolitis and 33 healthy children (21 male, 12 female; age range: 0-2 years) as control group who were admitted to the Pediatrics Department of Mersin University Hospital, southern Turkey, from January to July 2010 were included in the study. Nasopharyngeal aspirates were collected from the study groups and the detection of respiratory viruses [respiratory syncytial virus (RSV) A & B; rhinovirus (RV); human metapneumovirus (hMPV) A & B; influenza virus type A [H1N1, H3N2, H1N1v], B & C; parainfluenza virus (PIV) type 1, 2, 3 & 4A/B; adenovirus (AdV); HBoV; coronavirus (CoV 229E); enterovirus (EV)], were performed by using a commercial system namely CLART®Pneumovir (Clinical Array Technology, Genomica, Spain) based on the principle of multiplex polymerase chain reaction (M-PCR) and DNA microarray. Demographic features, clinical and laboratory findings of the patients, treatment protocols and the relationship between the length of hospitalization and the viral agents determined were also evaluated. Of the 62 samples collected from bronchiolitis cases, at least one virus was detected in 52 (83.9%) and viral co-infections were detected in 31 (50%) of them. Including the co-infections, RSV was the most commonly identified virus (n= 21; 33.9%), followed by influenza A [H1N1] (n= 18; 29%), RV (n= 18; 29%), hMPV (n= 13; 21%), PIV (n= 10; 16.1%), AdV (n= 5; 8%), HBoV (n= 3; 4.8%) and EV (n= 1; 1.6%). Of the 33 samples from healthy children, at least one virus was detected in 21 (63.6%) and viral co-infections were detected in seven (21.2%) samples. Including the co-infections, the most commonly detected virus was RV (n= 10; 30.3%), followed by influenza A [H1N1] (n= 6; 18.1%), AdV (n= 6; 18.1%), RSV (n= 4; 12.1%) and PIV (n= 3; 9%), however HBoV and hMPV were not detected in the control group. The differences of demographic features (age, gender, history of atopy, exposure of smoking, length of breast-feeding, presence of school-age sibling) and frequency of virus detection (83.9% and 63.6%, respectively) between the patient and control groups were not statistically significant (p> 0.05). The most common complaints of patients on admission were cough (100%), runny nose (82.3%) and respiratory difficulty (71%), whereas fever was present in 21 (33.9%) patients. The most common findings on physical examination were prolonged expirium (98.4%), rhonchi (98.4%), rales (80.6%), tachypnea (71%) and tachycardia (67.7%). Pulmonary graphies revealed that diffuse air trappings were more common in virus-associated bronchiolitis (36/52; 69.2%) cases, on the other hand infiltrations were more common (6/10; 60%) in patients who were virus-negative (p< 0.05). The demographic features, clinical and laboratory findings, clinical severity scores, hospitalization rates and duration of hospitalizations in bronchiolitis cases did not show statistically significant differences between the viral agents (p> 0.05 for each parameter). However the rates of antibiotic and steroid use in hospitalized patients (24/34 and 5/34, respectively) were significantly higher than those of outpatients (7/28 and 0/28, respectively) (p= 0.001 and p= 0.03). Our data indicated a high rate (~84%) of respiratory viruses in children with bronchiolitis in the Mersin province and the detection of hMPV (21%) and HBoV (4.8%) only in the patient group encouraged their roles in the etiology of acute brochiolitis. It was concluded that viral etiology should be investigated in selected cases to prevent unnecessary antibiotic treatment and to initiate appropriate antiviral therapy when necessary.

[Investigation of the clonal relationship between nosocomial Acinetobacter baumannii isolates by Rep-PCR]. / Hastane enfeksiyonu etkeni olan Acinetobacter baumannii izolatlari arasindaki klonal iliskinin Rep-PCR ile arastirilmasi

Acinetobacter spp. are opportunistic bacterial pathogens primarily associated with hospital-acquired infections and the spread of multidrug resistant Acinetobacter strains is a growing problem in terms of infection control. The aim of this study was to determine the clonal relationship between strains of nosocomial Acinetobacter baumannii by using rep-PCR method. A total of 75 Acinetobacter strains isolated from various clinical samples of the hospitalized patients between October 2011-May 2012 were included in the study. Antibiotic susceptibilities of Acinetobacter isolates were investigated by Kirby-Bauer disk diffusion method according to CLSI guidelines. According to disk diffusion test, the resistance rates for piperacillin, piperacillin-tazobactam, cefepime, ceftazidime, imipenem, meropenem, gentamicin, amikacin, tetracycline, levofloxacin, ciprofloxacin and trimetoprim-sulfamethoxazole were 96%, 96%, 97.3%, 89.3%, 96%, 94.6%, 66.7%, 85.3%, 68%, 82.7%, 97.3% and 89.3% respectively. In this study, 73 (97%) strains were found resistant to three or more than three antibiotics (multidrug resistant). Rep-PCR analysis have shown the presence of eight clones, including two major clones [A (7subtypes), B (3 subtypes)] and six unique clones (C-H). Clone A was found to be the dominant type. Fifty-four (72%) of the 75 Acinetobacter strains belonged to clone A, 13 (17.3%) to clone B, two strains to clone C, D, and one of each to the other clones (E, F, G, H). Clone A was isolated from 71% (20/28), 70% (7/10) and 100% (6/6) of the samples sent from reanimation intensive care unit, surgery ward and internal diseases intensive care units, respectively. The time interval between the first and last strain was eight months. The results of this study indicated an increase in the resistance rates of Acinetobacter strains in our hospital and this increase was attributed to the clonal dissemination of the strains. Strains of the clone A were found to be dominant at the intensive care and other clinics of our hospital. It is contemplated that Acinetobacter strains were scattered as a result of cross transmission and patient transfer among clinics. The rep-PCR method which was used in this study was evaluated as a rapid, easily applicable and successful procedure for epidemiological studies. Clonal distribution of resistant strains in the hospital environment emphasizes the significance of infection control measures.

[Investigation of the presence of human metapneumovirus in patients with chronic obstructive pulmonary disease and asthma and its relationship with the attacks]. / Kronik Obstrüktif Akciger Hastaligi ve Astimi Olan Hastalarda Insan Metapnömovirus Varliginin Arastirilmasi ve Hastalik Ataklari ile Iliskisi.

Human metapneumovirus (hMPV), an enveloped RNA virus classified in Paramyxoviridae family, was first characterized in 2001 from children with acute respiratory tract infection. Recent studies have suggested hMPV to play a role in chronic obstructive pulmonary disease (COPD) and asthma attacks. The aims of this study were to investigate the frequency of hMPV in patients with COPD and asthma, its effects on the severity of the attacks and the relationship between demographical and clinical factors. A total of 123 patients, including 66 with COPD (45 were in attack and 21 were stable) and 57 with asthma (33 were in attack and 24 were under control) diagnosed according to the criteria of Global Initiative for Chronic Obstructive Lung Disease and the Global Strategy for Asthma Management and Prevention, respectively, were included in the study. Nasopharyngeal lavage samples collected from all of the patients have been evaluated for the presence of hMPV-RNA by using a reverse transcriptase-polymerase chain reaction (RT-PCR) targeting F gene region of the virus. hMPV-RNA positivity rates in patients with COPD and asthma were observed as 30.3% (20/66) and 31.6% (18/57), respectively, and the difference between the groups were not statistically significant (p= 1.00). When patients were compared according to their disease status, hMPV was detected in 31.1% (14/45) of patients with COPD attack and 28.6% of stable patients (p> 0.05). These rates were found as 36.4% (12/33) and 25% (6/24) in patients with asthma attack and controlled asthma, respectively (p> 0.05). Although the virus detection rates in patients with COPD and asthma attacks (26/78; 33.3%) were higher than the patients with stable/controlled disease (12/45; 26.7%), the difference was not found as statistically significant (p= 0.57). The detection rate of hMPV-RNA was 26.1% in patients who can be treated at home and hospital without any need of intensive care and mechanical ventilation, while this rate was 36.4% in patients with COPD attack who require intensive care and mechanical ventilation (p= 0.67). Similarly, hMPV-RNA was detected more frequently in asthma patients with moderate and severe attacks (45%) than in patients with mild attacks (23.1%); however this difference was also not statistically significant (p= 0.28). No association of hMPV-RNA detection and demographical and clinical characteristics (age, gender, medical history, smoking status, allergy, COPD severity, asthma severity, the severity of attacks, using inhaled steroid, fever) of the patients could be demonstrated (p> 0.05), except the severity of the disease in patients with asthma (p= 0.02). In conclusion, further studies with large number of cases are needed to elucidate the role of hMPV in the occurrence and severity of COPD and asthma attacks.

Detection of hepatitis B virus X gene and PreC promoter mutations from chronic hepatitis B patients in the south of Turkey.

Hepatitis B virus (HBV) infection is a global health problem with more than 2 billion infected individuals. HBV infection leads to diverse outcomes ranging from acute to chronic hepatitis, which may result in severe complications as liver cirrhosis and hepatocellular carcinoma (HCC). HBV is one of the most important human DNA viruses having strong oncogenic potential. Recently, many studies have reported on HBV X gene and PreC promoter mutations associated with HCC. In order to detect the prevalence of HBx gene and PreC promoter mutations possibly related to HCC, we have analyzed sera samples collected from 61 patients with chronic hepatitis B. We have detected TI653 mutation in 1 of 61 (1,63%), A1896 mutation in 10 of 61 (16,39%), and T1762 - A1764 dual mutation in 4 of 61 (6,55%). T1653 and T1762- A1764 dual mutations were suggested significantly related to HCC in earlier reported studies. Our findings demonstrate that HBx gene and PreC promoter mutations related to HCC are present in our region and prospective clinical chord studies would be useful for better patient management and of early diagnosis of possible HCC cases.

[Determination of the cytokine gene polymorphism and genetic susceptibility in tuberculosis patients]. / Tüberküloz hastalarinda sitokin gen polimorfizmi ve genetik yatkinligin belirlenmesi.

Tuberculosis (TB) is a complicated disease in which biological, socioeconomical and environmental factors play role. Since only 10% of the individuals infected with Mycobacterium tuberculosis develop active disease, it has been suggested that host genetic factors may influence the risk for the development of TB. In this study, we aimed to investigate the presence and role of single nucleotide polymorphisms in the gene regions responsible for cytokine production, since these factors are considered to be associated with susceptibility or resistance to disease development. Single nucleotide polymorphisms were investigated by Amplification Refractory Mutational System (ARMS) Polymerase Chain Reaction (PCR) and PCR-Restriction Fragment Length Polymorphism (RFLP) methods. The presence of single nucleotide polymorphisms were analyzed in tumor necrosis factor alpha (TNF-α) gene promoter -308 G>A (rs1800629) region, interferon gamma (IFN-γ) gene +874 T>A (rs61923114) region, interleukin (IL)-12B p40 gene 1188 A>C (rs3212227) region, IL-10 gene promoter -1082 G>A (rs1800896) region and IL-4 gene promoter -590 C>T (rs2243250) region. A total of 84 patients (71 male, 13 female; mean age: 32.57 ± 15.94 years) whose clinical samples yielded M.tuberculosis complex growth, and 110 healthy blood donors (93 male, 17 female; mean age: 29.40 ± 11.56 years) as control group were included in this study. Of the patients, 76 (90.5%) were diagnosed as pulmonary and 8 (9.5%) as extrapulmonary TB. While 79 (94.1%) patients were newly diagnosed as TB, 5 (5.9%) patients had a TB history (relapsed TB). It was detected that acid-fast bacilli (AFB) were positive in 58 (69%) patients. According to the single nucleotide polymorphism results, gene frequencies could not be compared for TNF-a gene promoter -308 G>A region since healthy controls were in Hardy-Weinberg equilibrium while the patients were not. There were no statistically significant differences in allele and genotype distribution between the patients and healthy controls in IFN-γ gene +874 T>A region, IL-12B p40 gene 1188 A>C region, IL-10 gene promoter -1082 G>A region and IL-4 gene promoter -590 C>T region (p> 0.05). There were also no statistically significant differences between AFB positive (n= 58) and negative (n= 26) patients, and AFB positive (n= 56) and negative (n= 20) pulmonary TB patients (p> 0.05). In conclusion, no statistically significant differences were found associated with the susceptibility or resistance to TB with single nucleotide polymorphisms in the gene regions responsible for cytokine production in the study population. Only some of the single nucleotide polymorphisms of the gene regions responsible for cytokine release were investigated in our study. Therefore further detailed studies to investigate the polymorphisms in the genes that control the cytokine release and receptors specific for these cytokines, should be conducted. Although this study was performed in a relatively small sized population, these findings might provide a significant contribution to the epidemiologic data about the molecular immunology of TB in Turkey.

[Detection of Helicobacter pylori and antimicrobial resistance in gastric biopsy specimens]. / Mide Biyopsi Örneklerinden Helicobacter pylori'nin Tanimlanmasi ve Antimikrobiyal Direncinin Arastirilmasi

Helicobacter pylori is reported as the etiological agent of gastritis, gastric and duodenal ulcer, gastric adenoid carcinoma and mucosa-associated lymphoid tissue lymphoma. In the diagnosis of H.pylori infections invasive (culture, histopathological examination, rapid urease test and molecular tests) and non-invasive (urea breath test, serological tests, stool culture and stool antigen/nucleic acid tests) methods may be used. Clarithromycin, amoxicillin and combination of metronidazole and protonpump inhibitor or ranitidine bismuth citrate triple treatment protocol is applied in order to treat and eradicate the infection. However, increasing rates of antibiotic resistance among H.pylori strains reduces the success of eradication therapy. The aim of this study was to investigate the presence of H.pylori in the gastric antral biopsy specimens and to determine the antimicrobial resistance of the isolates. A total of 149 gastric antral biopsy specimens obtained from patients (age range: 17-83 years; 73 were male) who admitted to Mersin University Faculty of Medicine Department of Internal Medicine Gastroenterology clinic with dyspeptic complaints were included in the study. H.pylori presence was investigated by culture, polymerase chain reaction (PCR) and urease test from gastric biopsy specimens, and H.pylori-specific antigen (HpSA) was investigated by ELISA in the stool samples of patients. Resistance to tetracycline, amoxicillin, metronidazole and levofloxacin was determined with E-test method. Clarithromycin resistance was determined both by E-test and PCR-RFLP (restriction fragment length polymorphism) methods. H.pylori was detected in 29.6% (43/145) of patients with culture, 55.2% (80/145) of patients with urease test, 57% (65/114) of patients with HpSA test and 71.3% (102/143) of patients with PCR. The sensitivity and specificity of culture, PCR, HpSA and urease tests were determined as 52.4% and 100%, 96.3% and 62.3%, 80.3% and 81.4%, 86.6% and 85.7%, respectively. According to the E-test results, resistance to clarithromycin was 18.2%, to tetracycline 9.1%, to metronidazole 45.5%, to levofloxacin 18.2% and no resistance was determined to amoxicillin. Clarithromycin resistance was searched in 94 of PCR positive 102 samples, and 17 (18.1%) of them yielded clarithromycin resistance. Of them 11 (64.7%) harbored A2144G (at 2144. nucleotide), and 6 (%35.3) harbored A2143G (at 2143. nucleotide) point mutations. In our study, PCR was determined as the most sensitive method, however due to its low specificity, the results should be confirmed with at least one of the other methods. The specificity of culture method was high, but sensitivity was found to be quite low compared with other methods. The sensitivity and specificity of urease and HpSA tests were found to be similar. In conclusion, in cases which endoscopy could not be done, non-invasive, rapid and practical HpSA method can be used in diagnosis and monitorization of the treatment. In the case of treatment failure, culture should be performed for antibiotic susceptibility testing of the isolate.

[Identification of filamentous fungi isolated from clinical samples by two different methods and their susceptibility results]. / Klinik Örneklerden Izole Edilen Filamentöz Mantarlarin Iki Farkli Yöntemle Tanimlanmasi ve Duyarlilik Sonuçlari

Molds are widely distributed in nature. Aspergillus spp. represent the most frequently observed causative agents, however less frequent pathogens Fusarium, Scedosporium and Zygomycetes have also been considered the most important causes of morbidity and mortality in profoundly immunosuppressed hosts. The aims of this study were to identify filamentous fungi isolated from clinical specimens by conventional and molecular methods, and to detect their antifungal susceptibilities. A total of 6742 clinical specimens obtained from hospitalized patients at critical units of Mersin University Medical Faculty Hospital and sent to our laboratory between April 2008-January 2010 were included in the study. The isolates were identified by classical mycological methods and polymerase chain reaction-based DNA sequencing. Susceptibilities to fluconazole and voriconazole were tested by disk diffusion method and to fluconazole, voriconazole, amfoterisin B, caspofungin and posaconazole by E-test. Filamentous fungi were isolated from 71 (1.05%) samples (13 sputum, 4 wound, 4 peritoneal fluid, 3 extrenal ear discharge, 3 abscess and one of each cerebrospinal fluid, blood, tissue biopsy, nasal swab and conjunctival swab) which belonged to 32 patients (13 female, 19 male; age range 7 months-77 years, mean age: 46.6 years). Of the patients 62.3% presented one or more risk factors such as chronic renal failure (n= 8), chronic obstructive lung disease (n= 6), malignancy (n= 6), diabetes mellitus (n= 5) and peripheral vascular disease (n= 5). Of the isolates six were identified as Aspergillus niger, six as Aspergillus flavus, five as Aspergillus fumigatus, four as Aspergillus terreus, five as Fusarium spp., two as Bipolaris spp., and one of each as Acremonium spp., Aurebasidium spp., Mucor spp., and Scedosporium spp. By conventional methods. Three isolates exhibited different identities by DNA sequencing. All Aspergillus isolates were correctly identified at species level by both methods, Other fungi were identified at genus level by conventional methods and at species level by DNA sequencing. Fluconazole minimum inhibitory concentration (MIC) values were determined as > 256 mg/L in all strains, except Scedosporium; voriconazole MIC values were < 0.38 mg/L in all Aspergillus spp. Caspofungin MIC values were > 32 mg/L for Fusarium, Scedosporium, Rhizopus and Bipolaris strains and ≤ 0.006-0.125 mg/L in all Aspergillus isolates, In three strains (Fusarium equiseti, Cylindrocarpon lichenicola and Rhizopus oryzae) posaconazole minimum inhibitory concentration (MIC) values were > 32 mg/L, however it was < 1.5 mg/L, for the other strains. Amphotericin B MIC values were > 32 mg/L for Fusarium, Scedosporium, Rhizopus and all A.terreus strains and < 2 mg/L for the others. E-test and disk diffusion test results were compatible with each other for all the antifungal agents tested. In conclusion, the identification of filamentous fungi such as Aspergillus and Fusarium spp. is easily and reliably achieved by conventional methods. Since the rate of invasive fungal infections is increasing currently, filamentous molds should be searched especially in the clinical specimens of immunocompromised patients for accurate and prompt diagnosis of such infections and to decrease the related mortality risk.

Procalcitonin is more useful than C-reactive protein in differentiation of fever in patients with sickle cell disease.

This study aimed at evaluating the value of C-reactive protein (CRP) and procalcitonin (PCT) levels in the differential diagnosis of fever in patients with sickle cell disease (SCD). The study included 86 children with SCD (group 1) and 49 controls (group 2). During the study, the patients had 114 acute episodes or routine visits to the units. They were classified as having vasoocclusive crisis with fever (group 1A), vasoocclusive crisis without fever (group 1B), and no crisis or fever (steady state, group 1C). Only patients with crises were admitted to the hospital. Patients admitted to the hospital with various clinical signs and symptoms each and every time were included in groups 1A, 1B, and 1C. Thus, a total of 114 clinical episodes were analyzed. The mean CRP levels in the 3 patient groups were significantly higher than that in the group 2, and among the patient groups, the mean CRP was significantly higher in group 1A than the other groups. The mean CRP level in group 1A and group 1B was significantly higher than that in group 1C. There were no significant differences among the 3 SCD groups in terms of the median serum PCT level; however, the median PCT level in group 1A, group 1B, and group 1C patients was significantly higher than that in group 2 patients. These data indicate that vasoocclusive disease with or without fever apparently does not significantly increase PCT levels in relation to the baseline status of children with SCD, which in turn are clearly more elevated than PCT levels of control children.

[A case of fatal disseminated infection caused by Mycobacterium bovis BCG strain and the identification of the isolate by spoligotyping]. / Mycobacterium bovis BCG susunun neden oldugu fatal disemine enfeksiyon olgusu ve izolatin spoligotiplendirme ile tanimlanmasi.

The vaccine strain Mycobacterium bovis BCG may lead to disseminated infection in patients with immune deficiency. In this report a patient who developed fatal disseminated tuberculosis caused by M. bovis BCG strain was presented. One year old male patient with the previous history of recurrent lower respiratory tract infection, was admitted to the hospital with the complaints of fever, cough and diarrhea continuing for 20 days. There was no family history of tuberculosis or history of contact with a tuberculosis case. Physical examination of the case revealed growth retardation and reticular and reticulonodular infiltration was detected in his chest X-ray. The results of sweat test, cystic fibrosis gene mutation analysis and metabolic screening tests were normal. Since fever continued and infiltrations persisted in the chest X-ray despite antibiotic therapy, PPD test was applied and acid-fast bacilli (AFB) were investigated in his gastric aspirate and stool samples for three consecutive days. PPD test was negative and no AFB were detected in the microscopic examination of the clinical samples. However, growth in Lowenstein-Jensen medium was detected in the stool sample on the 38th day of incubation. The antimycobacterial susceptibility testing performed at BACTEC MGIT (Mycobacterial Growth Indicator Tube) 960 system (Becton-Dickinson, USA) revealed that the isolate was susceptible to rifampin, isoniazid, streptomicin and ethambutol. Since the isolates did not grow at PNB (para-nitro benzoic acid) medium and niacin and nitrate activities were negative, spoligotyping (spacer oligonucleotide typing) was performed and DR loci characteristic for M. bovis BCG strain were detected. However, the patient died 2 weeks before the culture results were obtained. The effective use of mycobacteriology laboratories and cooperation between laboratory and clinics provide advantages in the early diagnosis and treatment of tuberculosis cases, decreasing the morbidity and the mortality.

[Investigation of aflatoxin M1 levels in raw and market milks in Mersin Province, Turkey]. / Mersin ilinde cig ve market sütlerinde aflatoksin M1 düzeyinin arastirilmasi.

Aflatoxin M1 (AFM1) which is produced by some Aspergillus species, mainly Aspergillus flavus, is the hydroxylated metabolite of aflatoxin B1 (AFB1) and can be found in milk and dairy products of livestock fed with contaminated feed. AFM1 is a potential threat to human health owing to its toxic and carcino- genic effects. This study was aimed to investigate the level of AFM1 in raw and ultra-high-temperature pasteurized (UHT) milk samples. A total of 137 milk specimens (39 from goats, 53 from cows, and 45 from UHT marketmilks) were included to the study, and AFM1 levels were analyzed by high performance liquid chromatography (HPLC; Aflaprep M immunoaffinity column; R-Biopharm Rhone Ltd., Glasgow) method. AFM1 positivity was detected in 14 (35.8%) goat milk samples, in 46 (86.7%) cow milk samples and 33 (73.3%) UHT milk samples, with the levels ranging from 0.0021 microg/l to 0.8666 microg/I in raw milk, and from 0.001 microg/I to 0.059 microg/I in UHT milk samples. In 10.2% (4/39) of goat milk, 73.5% (39/53) of cow milk, and 2.2% (1/45) of UHT milk samples, the toxin levels were found to be above the officially reasonable limit value (> 0.05 microg/l) recommended by Turkish Food Codex Regulation. Since AFM1 levels were found to be significantly high in the raw milk samples collected in Mersin province (located on Mediterranean part of Turkey), it was concluded that people dealing with feed, milk and dairy products should be informed about the importance of aflatoxins and the preventive measures to get protected from AFB1 and AFM1.

Detection of point mutations on 23S rRNA of Helicobacter pylori and resistance to clarithromycin with PCR-RFLP in gastric biopsy specimens in Mersin, Turkey.

BACKGROUND/AIMS: Helicobacter pylori infection has a high prevalence and is considered an important health problem in Turkey. Unfortunately, an effective treatment has not yet been found for the eradication of Helicobacter pylori infection, at least in our country. Standard therapies recommended for the eradication of Helicobacter pylori have failed in the province of Mersin, Turkey. The rate of eradication with the standard triple treatment was only 45% in the province of Mersin. It may be that Helicobacter pylori has become resistant to antibiotics. Therefore, we aimed to determine the rate of resistance to clarithromycin in the province of Mersin. METHODS: The study included 92 patients presenting with dyspepsia to the Gastroenterology Clinic of Mersin University Medical School and undergoing endoscopy. We obtained gastric biopsy specimens and investigated whether Helicobacter pylori was present and resistant to clarithromycin. We used polymerase chain reaction-restriction fragment length polymorphism to determine A2143G and A2144G mutations and resistance to clarithromycin. RESULTS: Out of 92 specimens, 37 (40.2%) had Helicobacter pylori DNA. Out of 37 specimens with Helicobacter pylori DNA, 15 (40.5%) had point mutations. Eleven specimens (29.7%) had mutations on nucleotide 2144 and 4 specimens (10.8%) had mutations on 2143. CONCLUSIONS: Taking account of the failure of the treatment regimens used to eradicate Helicobacter pylori infection in the province of Mersin, the high rate of point mutations determined in this study was not surprising and the rate of resistance to clarithromycin was an important indicator for the failure in the eradication of Helicobacter pylori infection.