Riza Durmaz

Prevalence of rotavirus genotypes in children younger than 5 years of age before the introduction of a universal rotavirus vaccination program: report of rotavirus surveillance in Turkey.

Background Group A rotaviruses are the most common causative agent of acute gastroenteritis among children less than 5 years of age throughout the world. This sentinel surveillance study was aimed to obtain baseline data on the rotavirus G and P genotypes across Turkey before the introduction of a universal rotavirus vaccination program. Methods Rotavirus antigen-positive samples were collected from 2102 children less than 5 years of age who attended hospitals participating in the Turkish Rotavirus Surveillance Network. Rotavirus antigen was detected in the laboratories of participating hospitals by commercial serological tests such as latex agglutination, immunochromatographic test or enzyme immunoassay. Rotavirus G and P genotypes were determined by reverse transcription polymerase chain reaction (RT-PCR) using consensus primers detecting the VP7 and VP4 genes, followed by semi-nested type-specific multiplex PCR. Results RT-PCR found rotavirus RNA in 1644 (78.2%) of the samples tested. The highest rate of rotavirus positivity (38.7%) was observed among children in the 13 to 24 month age group, followed by children in the age group of 25 to 36 months (28.3%). A total of eight different G types, six different P types, and 42 different G–P combinations were obtained. Four common G types (G1, G2, G3, and G9) and two common P types (P[8] and P[4]) accounted for 95.1% and 98.8% of the strains, respectively. G9P[8] was the most common G/P combination found in 40.5% of the strains followed by G1P[8] (21.6%), G2P[8] (9.3%), G2P[4] (6.5%), G3P[8] (3.5%), and finally, G4P[8] (3.4%). These six common genotypes included 83.7% of the strains tested in this study. The rate of uncommon genotypes was 14%. Conclusion The majority of the strains analyzed belonged to the G1–G4 and G9 genotypes, suggesting high coverage of current rotavirus vaccines. This study also demonstrates a dramatic increase in G9 genotype across the country.

Distribution of nontuberculous Mycobacteria strains.

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

Molecular characterisation and control of Acinetobacter baumannii isolates resistant to multi-drugs emerging in inter-intensive care units

BackgroundA nosocomial outbreak of Acinetobacter baumannii (AB) infections occurred among intensive care units (ICU) (surgery, medical, cardiovascular surgery, coronary unit) of Recep Tayyip Erdogan University Medical School (Rize, Turkey) between January 2011 and May 2012. The identification of isolates and clonal relation among them were investigated by molecular techniques.MethodsA total of 109 AB isolates were obtained from 64 clinical materials from 54 ICU patients and 3 from the hands of healthcare workers (HCWs) of 42 environmental samples. The isolates were identified by 16S rDNA sequencing and OXA- specific PCR. The clonal relation between isolates was investigated by PFGE methods using ApaI restriction enzyme.ResultsAll isolates were determined as AB by 16S rDNA sequencing and OXA-spesific PCR. While the blaOXA-51-like gene was amplified in all isolates, the blaOXA-23-like gene was amplified from 103 isolates. The PFGE pattern generated 9 pulsotypes and showed that the isolates from patients, HCWs, and the environment were genetically related. In 7 of these pulsotypes, there were 107 strains (98%) showing similar PFGE profiles that cannot be distinguished from each other, ranging from 2 to 53. The remaining 2 pulsotypes were comprised of strains closely associated with the main cluster. Two major groups were discovered with similarity coefficient of 85% and above. The first group consisted of 97 strains that are similar to each other at 92.7% rate, and the second group consisted of 12 strains that are 100% identical.ConclusionsThe common utilization of the blood gas device among ICU was the reason for the contamination. AB strains can remain stable for a long period of time, although due to the disinfection procedures applied in hospitals, there is a small chance that the same clone might reappear and cause another epidemic. For that reason, the resistance profiles of the strains must be continuously followed with amplification-based methods, and these methods should be used to support the PFGE method in the short term.

Prevalence of Neisseria meningitidis carriage: a small-scale survey in Istanbul, Turkey

INTRODUCTION The human nasopharynx is the main reservoir of Neisseria meningitidis, and asymptomatic carriage is common. N. meningitidis one of the common causes of bacterial meningitis in Turkey, especially after the implementation of the national immunization program that includes conjugated pneumococcal and Haemophilus influenzae type b vaccines. The purpose of this study was to evaluate the prevalence of meningococcal carriage and determine the leading serogroup, which may help authorities to adapt appropriate meningococal vaccine into the national immunization programme. METHODOLOGY The prevalence of oropharyngeal carriage of N. meningitidis in 1,000 healthy subjects, 0-79 years of age, was investigated. Oropharyngeal swabs were collected during an 18-month period. Samples obtained were inoculated onto Thayer-Martin agar. The API-NH test and VITEK-MS system were used for identification of colonies. Multiplex real-time polymerase chain reaction assay was used to determine serogroups with serogroup-specific genes. RESULTS N. meningitidis was isolated from 6 of 1,000 subjects (0.6%). Meningoccocal carriers were between 21 and 40 years of age. All isolates were serogrouped as B, except one that did not survive on subculture. N. lactamica was isolated from 13 of 1,000 subjects (1.3%). CONCLUSIONS Carriage rate of meningococci in our study was relatively low. However, we detected that serogroup B was the leading strain in meningococcal carriage in Istanbul; choosing an appropriate meningococcal vaccine containing serogroup B should therefore be considered. High absolute humidity throughout the year in Istanbul may explain the low prevalence of carriage in our study. This should be verified with a multicenter national survey.

Molecular characterization of vancomycin-resistant Enterococcus faecium strains isolated from carriage and clinical samples in a tertiary hospital, Turkey.

This study aimed to determine the presence of vancomycin resistance (vanA and vanB) and virulence genes (esp, asa1, gelE, ace, hyl, cylA, cpd and ebpA) in vancomycin-resistant Enterococcus faecium (VREfm) strains and to analyse the clonal relationships among the strains. E. faecium strains were identified from rectal and clinical specimens by biochemical tests and the API-20 Strep kit. Susceptibility testing was performed using disc-diffusion and broth-dilution methods. PFGE was used for molecular typing of the VREfm strains. The vancomycin resistance and virulence genes were amplified by two-step multiplex PCR. All 55 VREfm isolates were resistant to penicillin G, ampicillin and high-level gentamicin but were susceptible to quinupristin/dalfopristin and linezolid. Multiplex PCR analysis indicated that all isolates harboured vanA and that 41 (75 %) were positive for virulence genes. The esp gene was the most common virulence factor and was detected in nine (41 %) invasive and 32 (96.7 %) non-invasive isolates. Multiple virulence genes were observed only in two non-invasive isolates; one harboured esp and ebpA and the other harboured esp, ebpA, asa1, gelE and cpd. PFGE typing yielded 16 different types, seven of which were clusters with two to 14 strains each. The clustering rates of the rectal swab, blood and urine isolates were 72.7 %, 61.5 % and 87.5 %, respectively. The genetic similarity observed among the VREfm isolates indicated cross-transmission in the hospital. Further studies on the virulence factors present in the strains might provide insight into the acquisition of these traits and their contribution to increased prevalence of VREfm.

Pulsed-field gel electrophoresis characterization of Bordetella pertussis clinical isolates circulating in Turkey in 2001–2009

Our purpose was to evaluate the natural epidemiological history of circulating Bordetella pertussis clinical isolates in Turkey, comparing isolates by means of pulsed-field gel electrophoresis (PFGE) profiles according to years, geographic regions, vaccination status, and demographic characteristics. We analyzed genotypically a collection of 92 clinical isolates recovered during the period 2001–2009 at the National Pertussis Reference Laboratory by PFGE. A total of 61 genotypes were identified among the 92 isolates. Fifteen of 61 genotypes were a cluster including 46 isolates, and the remaining 46 genotypes were unique. The clustering rate was 50% (46/92). The size of the cluster varied from 2 to 14 clinical isolates. There was no association between clustering rates and age, gender, or quarterly season. The clustering rate was significantly higher in 2006. When the isolates were grouped according to similarity coefficient higher than 85%, 89 (96.7%) of the 92 isolates were clonally related. There was one major group including 65.2% of the isolates mainly observed. This is the first study on the molecular characterization of B. pertussis isolates in Turkey. We consider that this study lays a good foundation for further monitoring of the circulating B. pertussis clinical isolates in Turkey.

Prevalence and diversity of rotavirus A genotypes cirulating in Turkey during a 2‐year sentinel surveillance period, 2014‐2016

Human rotavirus A (RVA) is the main etiological agent of watery diarrhea among children under 5 years of age worldwide. The aims of this study were to investigate the prevalence and diversity of RVA genotypes circulating in Turkey during a 2‐year sentinel surveillance study. A total of 1639 rotavirus antigen‐positive stool samples were obtained from children younger than 5 years of age hospitalized with acute gastroenteritis. Rotavirus G and P genotypes were determined by reverse transcription polymerase chain reaction (RT‐PCR) with consensus primers for the VP7 and VP4 genes, followed by semi‐nested type‐specific multiplex PCR. Rotavirus RNA was detected in 1396 (85.3%) of the samples tested. The highest detection rate (38.2%) was obtained among children in the 0‐12 months age group, followed by children in the 13‐24 months age group (36.2%). The most prevalent genotype was G1P[8] (24.6%) followed by G3P[8] (19.6%), G9P[8] (12.2%), G2P[4] (9.5%), G2P[8] (6.5%), and G4P[8] (4.8%). The proportions of uncommon and mixed genotypes were 21.5% and 1.14%, respectively. The large number of genotypes observed, including common, uncommon, and mixed types, indicates a high heterogeneity of RVA strains circulating in Turkey. The current study also exhibited dramatic fluctuations on the prevalences of the common genotypes, with increases in G3 and G1 and decreases in G9 and G2 from 2014‐2016.

Towards measles elimination: Phylogenetic analysis of measles viruses in Turkey (2012–2013) and identification of genotype D8

Molecular characterization of different measles virus (MV) strains is essential to combat the disease. Sixty measles MV strains were obtained from throat swabs or urine of patients in Turkey between 2012 and 2013 and characterized. MV RNA sequences (n = 60) were analysed for 456 nucleotides representing hypervariable domain of the nucleoprotein (N) gene. Of the 60 strains analysed 53 were the D8 genotype, 6 were B3, 1 was D4, and 1 was A. This report describes MV genotype D8 that was involved in a measles outbreak in Turkey. Sequences of most genotype D8 strains (n = 51) were identical to the sequence of variant D8‐Frankfurt‐Main, which has been associated with outbreaks throughout Europe. Despite the lack of epidemiologic information, a phylogenetic analysis suggested that the genotype D8 MV may have been brought to Turkey from elsewhere. Phylogenetic and epidemiological findings suggested that strains identified in tourists and associated with importation included one strain of genotype D8, one strain of genotype B3, and one strain of genotype D4. These findings from the 2012 to 2013 outbreak in Turkey confirm that pockets of unimmunised individuals are making the country susceptible to measles outbreaks. To prevent further outbreaks, deliberate and sustained effort must be made to reach, and immunise susceptible age groups. Towards measles elimination process, continued molecular surveillance of measles strains in Turkey will help identify transmission patterns of virus and evaluate vaccination efforts. J. Med. Virol. 88:1867–1873, 2016.

High-Level Genetic Diversity among Invasive Streptococcus pneumoniae Isolates in Turkey

This study obtained information on the serotypes and molecular typing characteristics of Streptococcus pneumoniae strains causing invasive diseases in Turkey. Sixty-eight S. pneumoniae isolates causing invasive pneumococcal diseases were collected from different regions of Turkey from 2009 to 2011. The isolates were characterized by performing multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and capsular serotyping, and 25 different serotypes were identified. Serotypes 19F, 23F, 1, 14, and 7F were common and accounted for 52.9% of all the serotypes. In addition, 54 different PFGE profiles (pulsotypes) were observed. Twenty-three of the 68 (33.8%) isolates were clustered into 9 pulsotypes. MLST analysis yielded 36 sequence types, of which 12 (33.3%) were novel. A comparison of results with the global pneumococcal MLST database by performing eBURST analysis showed that our strains belonged to 20 different clonal complexes and 5 singletons. In addition, we identified 4 new alleles: 2 gdh, 1 xpt, and 1 ddl. Thus, the results of this study highlighted a high level of diversity among pneumococcal isolates. In addition, the study identified a case of possible capsular switching.

Metagenomics Analysis of Bacterial Population of Tympanosclerotic Plaques and Cholesteatomas

Objective Chronic otitis media can cause cholesteatomas or tympanosclerosis; however, the pathophysiology of such conditions is not completely known. The aim was to identify a bacterial genome that might be present in tympanosclerotic plaques and cholesteatomas using sequence analysis of the gene responsible for the transcription of 16 ribosomal RNA (rRNA). Study Design Metagenomics analysis of the samples. Setting Samples were collected and evaluated at tertiary care centers. Subjects and Methods Sixty-five tympanosclerotic plaques and 37 cholesteatomas were evaluated. The polymerase chain reaction (PCR) was performed using primers designed for the amplification of the gene responsible for the transcription of bacterial 16 rRNA. The PCR-positive samples were sequenced via Sanger method, and 46 selected samples were analyzed with next-generation sequencing (NGS). Results Sanger sequencing revealed the presence of bacterial genomes in a total of 18 of the 102 samples tested. Sequencing of these genomes indicated the presence of Alloiococcus otitis, Staphylococcus aureus, Achromobacter xylosoxidans, Escherichia coli, Staphylococcus sciuri, Staphylococcus caprae, Parvimonas spp., and Bacillus sp. in the tested samples. The NGS showed 1 or more different bacterial genomes in 44 (95.7%) of the 46 samples tested. Predominately, genome of Clostridiales (27 samples), Staphylococcaceae (24 samples), Peptoniphilaceae (12 samples), and Turicella otitidis (9 samples) were identified. Conclusion The middle ear is inhabited by a diverse microbial community than that previously known. With the use of molecular biology, it has become easier to identify the bacterial genomes and improve our understanding of the role of middle ear microbiota in the pathogenesis of chronic inflammatory ear diseases.