[Determination of Whole Genome Sequence and Resistance Genes in Hypermucoviscous Klebsiella pneumoniae with Long-Read Sequencing Technology]. / Hipermukoviskoz Klebsiella pneumoniae Izolatlarinin Uzun Okuma Yapan Dizileme Teknolojisi ile Tüm Genom Dizisinin ve Direnç Genlerinin Belirlenmesi.

Numerous virulence factors are present in the hypervirulent/hypermucoviscous Klebsiella pneumoniae, which primarily causes community-acquired infections. In recent years, hypervirulent/hypermucoviscous K.pneumoniae has acquired resistance genes and has been linked to healthcare-associated infections. The aims of this study were to determine whole genome sequencing of hypermucoviscous K.pneumoniae that induces healthcare-associated bloodstream infection utilizing the Oxford Nanopore (MinION) platform, to identify resistance genes using various databases, and to compare the database results. K.pneumoniae isolates that were sent to the Dokuz Eylül University Research and Training Hospital Center Laboratory and were isolated from blood cultures were included in this study between January 2018 and December 2020. K.pneumoniae isolates were identified using the automated VITEK-2 system. The disc diffusion method was used to characterize the antimicrobial resistance profile, and the string test was used to assess the hypermucoviscous phenotype. By using specific primers blaOXA-48, blaNDM, blaKPC, blaIMP, and blaVIM, carbapenem resistance genes were investigated using the PCR method. To ascertain clonal relatedness among hypermucoviscous K.pneumoniae isolates, PFGE was used. The whole genome sequencing of five K.pneumoniae strains with different origins was determined by Oxford Nanopore (MinION) technology. Using the ResFinder, CARD and BacWGSTdb databases, resistance genes were examined. Capsule regulation genes were analyzed with the BacWGSTdb database. Resistance genes on the plasmid were discovered using the ResFinder database after plasmid analyses were carried out using the PLSDB and PlasmidFinder databases. A total of 244 K.pneumoniae isolates were included in the study. Ten isolates were found to be hypermucoviscous. A carbapenem-resistant hypermucoviscous K.pneumoniae isolate was discovered to carry the blaOXA-48. Five hypermucoviscous isolates of which whole genome sequences were determined had blaSHV types, oqxB, oqxA, and fosA genes on the chromosome. Capsule regulation genes rcsA, rcsB were detected in all five isolates, and rmpA/rmpA2 genes caused hypermucoviscous phenotype was detected in two of the five isolates. In the plasmid analysis, IncFIB (K) pCAV1099- 14, Col (pHAD28), IncFIA (HI1)-IncR, Col (pHAD28), Col440, IncFIB (pNDM_Mar)-IncHI1 (pNDM_MAR), IncL, IncFIB (K)-IncHI1 (K), IncFIB (K), IncR, IncFIB (pNDM_Mar)-IncHI1 (pNDM_MAR)-IncR, repB plasmids were identified. Resistance genes; aac(3)-IId, aph(6)-Id, aph(3")-Ib qnrB1, sul2, dfrA14, blaTEM-1A, blaCTX-M-15, armA, msr(E), mph(E), catB3, blaOXA-1, aac(6')-Ib-cr, catA1 and blaOXA-48 were detected on the plasmid. In the bioinformatic analyzes, it was determined that two study isolates with hypermucoviscous phenotype had various plasmids and carried many resistance genes on these plasmids. Various resistance and virulence genes are spread through plasmids and the number of resistant hypermucoviscous K.pneumoniae is increasing day by day. Appropriate infection control strategies should be implemented to prevent healthcare-associated infections and outbreaks caused by antimicrobial-resistant hypermucoviscous K.pneumoniae. For this purpose, resistance monitoring of antimicrobials used in the treatment of K.pneumoniae infections should be performed. There were some differences in the results of the three databases used to detect resistance genes in this study. For this reason, it is important to analyze different databases and compare the database results in studies while performing whole genome sequencing. Further studies and updates using different databases will increase the compatibility between the databases.

First molecular detection and identification of Leishmania species in small wild rodents from Turkey.

Leishmaniasis is a parasitic disease infecting animals and humans. Two clinical forms (Visceral and cutaneous leishmaniasis) and four species are reported to be present in Turkey. Several studies have investigated canine and human leishmaniasis in Turkey but no study was performed to screen the infection among wild rodents, so far. The present study aims to investigate the role of small wild rodents as reservoir animals for Leishmania spp. in different regions of Turkey. Formalin-preserved tissue samples (spleen, liver, lung) of 712 rodents from 30 provinces were screened for the presence of Leishmania spp. DNA. Before DNA extraction, tissues were dried, rehydrated, and homogenated. Leishmania screening in rodent tissues and species determination was performed with a combination of real-time kDNA and ITS1 polymerase chain reaction protocols. Eight (1.12%) out of 712 animals were found to be positive for Leishmania spp. DNA and species typing revealed five L. infantum, two L. tropica and one L. major among positives. Leishmania major and L. infantum DNA were detected in Apodemus spp. from Zonguldak province located in the Western Black Sea Region, while L. tropica DNA was found in Meriones sp. and Gerbillus dasyurus from Adana and Hatay provinces located in Eastern Mediterranean Region of Turkey. The present study is first to report natural infection of L. infantum, L. major and L. tropica in small wild rodents in Turkey, suggesting their possible roles as reservoirs. Further studies are needed for planning epidemiological studies and also for developing rodent control measures in risky endemic areas to break the transmission cycle.

Seroprevalence and social determinants of varicella in Turkey.

BACKGROUND: In Turkey, varicella vaccine was introduced into routine childhood immunization in 2013, with a single dose administered to children aged 12 months. However, there is limited information on the morbidity (incidence and seroprevalence), mortality and burden of disease of varicella in the overall Turkish population. AIM: To determine varicella seroprevalence and its social determinants in Manisa Province, Turkey in children aged > 2 years before single-dose varicella vaccination was introduced in 2013. METHODS: The presence of anti varicella-zoster virus IgG antibodies was determined using enzyme-linked immunosorbent assay in serum samples collected from 1250 participants. RESULTS: The overall seroprevalence was 92.8% and the seroprevalence was > 90% among all age groups except 2-9 years (55.7%). Seroprevalence was significantly associated with family size, annual per capita equivalent income, number of people per room and education level. After adjusting by age, only education level remained significantly associated with seroprevalence, reflecting the early age effect. CONCLUSION: High seroprevalance depends on natural exposure to the infectious agent itself and is not associated with social determinants. High vaccine coverage should be maintained for effective varicella control and switching to a 2-dose schedule may also be considered to reduce the number and size of outbreaks in the Turkish population.

[Optimization of ELISA and immunoblot methods for the detection of IgG antibodies against old world hantaviruses in wild rodents]. / Yabani kemiricilerde Eski Dünya hantavirus IgG antikorlarinin saptanmasi için ELISA ve immünoblot yöntemlerinin optimizasyonu.

Hantaviruses infect humans via inhalation of viral particles in infected rodents' secretions such as saliva, urine and faeces or via direct contact with infected rodents. The rodent species that are known as the carriers of Dobrava (DOBV), Puumala (PUUV), Saaremaa (SAAV), Tula (TULV) and Seoul (SEOV) viruses are found in our country. The presence of specific antibodies against hantaviruses have been demonstrated in rodents collected from Black Sea and Aegean Regions of Turkey in 2004 for the first time. The first hantavirus-related hemorrhagic fever with renal syndrome (HFRS) cases were reported in Black Sea region in 2009. The determination of the hantavirus prevalence in wild life and rodent populations in the field is crucial for the information about hantavirus-related cases and to clarify the state of risk. There is no commercial product optimized for the screening of rodent serum samples in terms of HFRS agents like DOBV and PUUV that are widely seen in Eurasia as well as Turkey. In this study, the antigens belonging to the commercial enzyme-linked immunoassay (ELISA) and immunoblot tests that are produced for the screening of human sera were used for the development of antibody screening tests against hantavirus in rodent sera and were optimized. The most appropriate serum and conjugate dilutions were determined for the optimization of ELISA (Anti-Hantavirus Pool ELISA; Euroimmun, Germany) and immunoblot (Euroline Anti-Hanta Profile 1 strips; Euroimmun, Germany) methods. Optimized ELISA method was used for the screening and optimized immunoblot method was used for the confirmation. A total of 84 wild rodent sera that belonged to Apodemus and Microtus species were evaluated with this procedure and the cut-off value, sensitivity and specificity of optimized ELISA method were determined. For the optimization of ELISA 1/50, 1/100 and 1/200 serum dilutions and 1/10.000, 1/20.000 and 1/40.000 conjugate dilutions were tested. For the optimization of immunoblot, 1/50 and 1/100 serum dilutions and 1/5.000 and 1/10.000 conjugate dilutions were tested. The horseradish peroxidase conjugated goat anti-mouse IgG for ELISA and the alkaline phosphatase conjugated goat anti-mouse IgG for immunoblot were used. We followed the manufacturer's recommendations for the incubation parameters, substrate and the number of washes. 1/50 serum dilution and 1/10.000 conjugate dilution for ELISA and 1/100 serum dilution and 1/5.000 conjugate dilution for immunoblot were determined as optimal concentrations. By using the optimized ELISA, 26.2% (22/84) of rodents were found positive for hantavirus antibodies according the determined cut-off value (OD(450/620): 0.325). By using immunoblot as a confirmatory test, 20 out of 22 ELISA positive samples could be studied because of the insufficient amount of sera and 17 of them was found positive in terms of DOBV antibodies. Of these rodents 11 were Apodemus flavicollis, three were Apodemus agrarius, two were Microtus guentheri and one was Apodemus sylvaticus. When the results of ELISA were compared to immunoblot results, the optimized ELISA's sensitivity and specificity were found as 100% and 95%, respectively. In this study, a method that can be used in the screening of rodent sera was constituted which uses commercial antigens that can be provided easily, gives fast and reliable results. Similar serological methods optimized for different types of rodents are of great importance for the realization of active follow-up and monitoring of the studies in the field.

Borrelia miyamotoi in wild rodents from four different regions of Turkey.

Borrelia miyamotoi is a tick-borne zoonotic agent that causes hard tick-borne relapsing fever, an emerging disease in humans. Some small mammalian and bird species are reported to be reservoirs of B. miyamotoi. This study aims to examine Borrelia species present in rodents captured from rural areas of Turkey. Blood samples of rodents were initially screened with Borrelia 16S rRNA qPCR. The Borrelia flaB gene was subsequently amplified by conventional PCR, after which all positive samples were sequenced. Borrelia miyamotoi was observed in nine out of 536 blood samples (1.7%) collected from wild rodents. Phylogenetic analysis showed that all positive samples belonged to the European genotype clade of B. miyamotoi. PCR positivity was 5.3%, 3.7%, and 1.8% in Apodemus uralensis, Apodemus flavicollis, and Myodes glareolus, respectively. Borrelia burgdorferi sensu lato that causes Lyme borreliosis in humans could not be detected in the rodents. In this study, presence of B. miyamotoi DNA is reported for the first time in rodents in Turkey.

[Unnecessary test repeats in viral hepatitis serology]. / Viral hepatit serolojisinde gereksiz test tekrari.

Inappropriate laboratory testing constitutes a vast burden regarding the cost and the workload for health care system. Unnecessary repetition of tests is one of the most common reasons accounting for inappropriate laboratory utilization. A wide spectrum of tests are utilized to diagnose hepatitis A and B infections in Turkey because of the high seroprevalence rates. The present research aimed to determine the rate of unnecessary repeat testing of anti-HAV total and anti-HBc used in serological diagnosis of hepatitis A and B infections and to discuss the possible solutions for this problem. Laboratory records of the serology laboratory of Dokuz Eylul University Hospital, dating between May 2002 - May 2005, were evaluated retrospectively. Unnecessary repeat testing was defined as the repetition of anti-HAV total and/or anti-HBc for the patients positive for one or both of the tests. During the study period of three years, a total of 10.658 anti-HAV total and 13.047 anti-HBc were tested of which 8.550 (80%) and 4.959 (38%) were found positive, respectively. Out of the positive results, 1.197 (14%) anti-HAV total and 904 (18%) anti-HBc tests were detected to be unnecessarily repeated tests. The estimated cost of a total of 2101 unnecessarily repeated tests was found approximately 17.000 US Dollars. Our data display that considerable amount of unnecessary testing takes place in serological testing of viral hepatitis. Prevention of inappropriate laboratory requests by clinicians would help to diminish the work load in serology laboratories and the cost both for the hospitals and the country.

HLA genotypes in Turkish patients with myasthenia gravis: comparison with multiple sclerosis patients on the basis of clinical subtypes and demographic features.

The nature and intensity of the association of myasthenia gravis (MG) with distinct human leukocyte antigens (HLA) haplotypes differ between ethnic populations. The aims of the present study were to examine the relationship between HLA class I and II haplotypes and MG; to show the HLA associations with various MG subsets; and to investigate the association between MG and clinical subgroups of multiple sclerosis (MS) regarding HLA haplotypes. A total of 66 patients with MG were enrolled onto the study. The mean age at onset was 42.01 years. A total of 122 clinically definite MS patients and 188 healthy subjects were examined as control groups. The present study clearly showed associations with HLA-DR3, -B8, -A1, and -A2 in MG. In patients with early-onset MG, associations with HLA-DR3, -B8, and -A2 were stronger. When compared with MS, in the MG group, there was still a strong association with -B8, -DR3, and -A1. In subgroup analysis, there was no difference between MG and primary progressive MS patients. On the basis of the presence of anti-AChR antibodies, there was a statistically significant association with HLA-DR3. On the basis of presence of thymoma, no HLA allele showed clear associations in MG patients with thymoma. This is the first study to examine the relationship between HLA haplotypes and MG in the Turkish population and to compare MG with another autoimmune disease, MS, on the basis of the HLA haplotypes. Further investigations with a larger population are required to explain this finding.

Pregnenolone protects the PC-12 cell line against amyloid beta peptide toxicity but its sulfate ester does not.

Pregnenolone (P), the main precursor of the steroids, and its sulfate ester, pregnenolone sulfate (PS), are the major neurosteroids produced in the neural tissue. Many neuroendocrinological studies stressed the neuroprotective role of neurosteroids although it has been suggested that the inhibition of P and PS synthesis can delay neuronal cell death. The potential roles of P and PS in vital neuronal functions and in amyloid beta peptide (Abeta) toxicity are not clearly identified. This work aims to investigate the effects of P and PS on cell viability and Abeta peptide toxicity in a concentration and exposure time-dependent manner in rat PC-12 cells. The cells were treated with 20muM Abeta peptide 25-35 and variable concentrations of P and PS ranging from 0.5muM to 100muM. To examine the effects of steroid treatment on Abeta peptide toxicity, 0.5muM (low) and 50muM (high) neurosteroids were used. The cell viability and lactate dehydrogenase release of cells were evaluated after 24, 48 and 72h. Morphological changes of cells were also examined. The treatment with higher than 1muM concentrations of P and PS significantly decreased the cell viability comparing to untreated cells. At lower concentrations, P and PS had no toxic actions until 72h. The Abeta treatment resulted in a significant decrease in cell viability comparing to untreated cells. P showed a dose-dependent protective effect against Abeta peptide in PC-12 cells. But its sulfate ester did not have the same effect on Abeta peptide toxicity, even it significantly decreased cell viability in Abeta-treated cells. Consequently, the discrepant effects of P and PS on Abeta peptide toxicity may provide insight on the pathogenesis of Alzheimer's disease.