[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.

[Clinical impact of hepatitis C virus genomic variations]. / Hepatit C virusu genomik varyasyonlari ve klinige etkileri.

Hepatitis C virus (HCV) is a globally-dispersed agent of chronic hepatitis with a significant public health threat, affecting over 110 million individuals throughout the world. The increased risk for chronicity after exposure and the lack of a protective vaccine make HCV is a leading infectious cause of cirrhosis, liver failure requiring transplantation and hepatocellular carcinoma. The replicative process and infection dynamics in the host enable HCV to generate an array of closely-related but non-identical genetic variants known as quasispecies in the infected individuals. Pathogenesis and outcome in HCV infections are directly affected by the virus genetic heterogeneity, reflected as the emergence of quasispecies in infected individuals. The evolution of these highly-diverse viral populations in the host directly influences the disease course, via providing a pool of variants capable of resuming viral replication under extrinsic and/or intrinsic selective pressures. Viral quasispecies go through several alterations during the course of the infection, and provide a background for the selection of escape mutants from the host humoral and cell-mediated immune responses and antiviral treatment. Supported by the robust next generation sequencing techniques, recent studies have provided significant insights on the genomic diversity and progression as well as on the origin and the epidemiology of HCV. This review provides an overview of the mechanisms of HCV genetic variability, and the interactions with the host, that affects clinical disease, covering viral and host determinants of humoral and cell-mediated immune responses, alterations during the early and late stages of the infection and disease progression leading to chronicity. In addition, current findings in virus evolution and epidemiology were briefly interpreted from the inter-species and population perspectives. The impact of viral genomic heterogeneity on antiviral treatment in the era of direct-acting agents is also discussed, along with an overview of current methods employed for the characterization of viral diversity.

Phylogenetic analysis of HCV-4d in Turkey: the curious case of Kayseri province.

In Turkey, genotype 1, especially type 1b virus, causes approximately 90% of these infections, while types 2, 3, and 4 exist, albeit in low prevalences and are due to relatively recent and limited introductions. Two recent reports from Kayseri, a relatively large city in Central Anatolia, indicated unusually high prevalence for type 4 infections in the province reaching a 35% among patients admitted to hospitals for treatment of chronic hepatitis C. In this study, the origin, the demographic history, and the dynamic of the epidemic of unusual HCV genotype 4d in Turkey by using Bayesian coalescent-based method were investigated. A gene flow migration approach was also used to describe the synchronous geographical dispersal and genetic diversification of this unusual genotype in Kayseri province. The Turkish clade had a tMRCA of 44 years corresponding to the year 1967 and seems to have a different origin being completely segregated from the European one. Gene flow migration analysis indicated that Kayseri province appeared to be the epicenter of HCV-4d epidemic, exporting the infections. The demographic history of HCV-4d showed that the epidemic started in 1970s year then following a slow exponential growth until 1980s. The Turkish monophyletic clade suggests a segregate circulation of the epidemic in this region mostly due to unsafe parenteral medical procedures (with drug addiction playing a relatively negligible role).

[Molecular detection of Helicobacter pylori vacA and cagA genes in gastric tissue specimens of patients with peptic ulcer disease and non-ulcer dyspepsia]. / Peptik Ülserli ve Ülser Olmayan Dispepsili Hastalarin Mide Doku Örneklerinde Helicobacter pylori vacA ve cagA Genlerinin Moleküler Yöntemlerle Belirlenmesi.

Helicobacter pylori can colonize the gastric mucosa and is considered as a risk factor for chronic active gastritis, peptic ulcer, gastric adenocarcinoma and primary gastric lymphoma. Among its various virulence factors, vacuolating cytotoxin encoded by vacA and cytotoxin-associated toxin encoded by cagA gene play an important role. The aims of this study were the detection of H.pylori vacA s and m genotypes, investigation of the association between vacA genotypes and cagA gene presence, and evaluation of the correlation between those factors and the clinical diagnosis. Gastric tissue specimens of patients who were clinically diagnosed as peptic ulcer disease (PUD) and non-ulcer dyspepsia (NUD) were included in the study. A total of 29 patients (age range: 18-74 years, mean age: 47.8 ± 13.6 years; 19 were female) without any familial relationship were evaluated. Thirteen (44.8%) of the patients were diagnosed clinically as PUD, while 16 (55.2%) as NUD. All of the patients' gastric tissue samples obtained by endoscopy were urease positive. H.pylori DNA was extracted from the tissue specimens by proteinase-K, phenol-chloroform-isoamyl alcohol method and vacA s, m1, m2 and cagA regions were identified by polymerase chain reaction (PCR) using four different primer sets. In addition, DNA sequencing was performed for the protected 785 base-pairs region of vacA m gene in all of the samples, and the sequences were aligned with Gene-Bank sequences, creating a phylogenetic tree. The distribution of vacA genotypes between 29 H.pylori positive patients were found as; s1m1 (n= 16), s1m2 (n= 6) and s2m2 (n= 7), while 19 patients yielded positive results for cagA gene. CagA positivity was detected in all of the 16 patients harboring s1m1 genotype, and 13 of those were the patients diagnosed as PUD (p= 0.008). Genotyping data achieved by phylogenetic analysis of the vacA m region were compatible with m genotypes identified by PCR. In conclusion, we detected a significant relationship between PUD and vacA s1m1 and cagA positivity. It was also determined that PCR would be a reliable, simpler and cheaper alternative to nucleotide sequencing for the identification of H.pylori vacA m genotypes.

New generation IQ-200 automated urine microscopy analyzer compared with KOVA cell chamber.

OBJECTIVE: The examination of the urine remains to be one of the most commonly performed tests in laboratory practice. Currently, laboratories also need to accredit their urine diagnostics by comparing their measurement methods to acceptable references. In this study we compared particle counts obtained by new generation automated technique, image capture analysis (IQ-200) with those of a standardized chamber counts. DESIGN AND METHODS: The same 258 urine samples from different departments of a hospital assayed by IQ-200 were analyzed in parallel with the KOVA cell chamber system. Clinically significant discrepancy results (positive vs. negative) for red blood cell (RBC) and white blood cell (WBC) were also compared with those obtained by dipstick testing. RESULTS: There was a good agreement between the automated system and sediment microscopy for RBCs, WBCs, and squamous epithelial cells (SCs) (r=0.90; r=0.80; r=0.72, respectively: P<0.001). The IQ-200 was more sensitive for determining RBCs, WBCs, and SCs than other formed elements. CONCLUSIONS: IQ-200 can perform accurate quantification of microscopic element in urine. However, automated techniques are not completely free of error. Therefore, by adopting an appropriate algorithm and combining the results with stript analysis and other laboratory tests allows further reduction of clinically important errors.

Identification of the nucleotide substitutions in 62 SARS-CoV-2 sequences from Turkey.

A previously unknown coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been shown to cause coronavirus disease 2019 (COVID-19) pandemic. The first case of COVID-19 in Turkey has been declared in March 11th, 2020 and from there on, more than 150,000 people in the country have been diagnosed with the disease. In this study, 62 viral sequences from Turkey, which have been uploaded to GISAID database, were analyzed by means of their nucleotide substitutions in comparison to the reference SARS-CoV-2 genome from Wuhan. Our results indicate that the viral isolates from Turkey harbor some common mutations with the viral strains from Europe, Oceania, North America and Asia. When the mutations were evaluated, C3037T, C14408T and A23403G were found to be the most common nucleotide substitutions among the viral isolates in Turkey, which are mostly seen as linked mutations and are part of a haplotype observed high in Europe.

Changes on Hepatitis C virus genotype distribution in Western Turkey: Evaluation of twelve-year data.

BACKGROUND/AIMS: Hepatitis C virus (HCV) prevalence is 1% in Turkey with genotype 1 being the predominant type traditionally. However unique geographical location of Turkey and increasing human migration in the region influences the epidemiology of the infection. The aim of this study was to determine the changes in distribution of HCV genotypes and risk factors. MATERIALS AND METHODS: In this retrospective single-center study, HCV genotyping results of 558 patients were evaluated in between 2005 and 2016.Three different HCV genotyping assays were used during the 12-year study period;restriction fragment length polymorphism (RFLP), Abbott Real Time HCV Genotype II and Bosphore HCV genotyping kit. RESULTS: The most prevalent HCV genotype was genotype 1 detected in 88.4% of the patients followed by genotype 3 (5.2%),genotype 4 (2.9%),genotype 2 (2.1%), mixed genotypes (1.1%) and genotype 5 (0.3%).Genotype 1a showed an increasing prevalence.There were 19 patients (3.4%) either of foreign nationalities or Turkish citizens living abroad. Genotype 3 was the most common type among these patients which 10.3% had intravenous drug use history.Syrian migrant population differed in terms of HCV genotypes.Genotype 5 detected in two Syrian patients, which is the first report of HCV type 5 in Western Turkey. Among the HCV genotype 4 infected patients, 31.3% were Syrians. CONCLUSION: Our study showed that although genotype 1b dominance continues, the distribution and prevalence of HCV genotypes are changing in our region mainly due to migration and increase in the frequency of patients with non-traditional risk factors such as intravenous drug use. Monitoring the epidemiology of HCV genotypes may provide guidance in treatment decisions.

Migrants rescued on the Mediterranean Sea route: nutritional, psychological status and infectious disease control.

INTRODUCTION: North Africa has become a key migratory hub where a large number of migrants attempt the journey by sea from the Libyan coastline to the south of Europe. In this humanitarian disaster scenario, the Mediterranean route has been one of the most used by illegal boats. METHODOLOGY: In this report, the state of physical and psychological health of a cluster of Eritrean migrants, escaped from Libya and rescued in the Mediterranean Sea after a shipwreck, was described by epidemiological, clinical and laboratory investigations. RESULTS: Data suggest that despite the majority  of the migrants being apparently in good health upon a syndromic surveillance approach, most of them suffered a decline in psychological status as well as severe malnutrition. The emergence of infectious diseases, related to poor living conditions during the journey, is not a rare event. CONCLUSION: The present report highlights the risks of failures of the syndromic medical approach in the setting of the extremely challenging migration route and underlines migrant frailties consequent to a prolonged journey and long period of detention. These stressors, which can degrade the initial health condition of traveling migrants, can lead to a premature "exhausted migrant effect" that should be carefully investigated in order to avoid the early emergence of diseases related to frailty.

The inappropriate use of HbA1c testing to monitor glycemia: is there evidence in laboratory data?

RATIONALE, AIMS AND OBJECTIVES: Diabetes is a chronic illness and have relatively high prevalence. Glycemic control is fundamental to the management of diabetes. Hemoglobin A1c (HbA1c) is a commonly used laboratory test to monitor glycemia and to manage diabetes. This study aimed to assess the appropriateness of the frequency of HbA1c test order with respect to the commonly approved guidelines for monitoring glycaemia of patients. METHODS: To assess the rate of inappropriate test orders, laboratory records of HbA1c tests ordered between 2002 and 2004 were evaluated. Inappropriate orders were defined as any order for a given patient that takes place within a 29- or 89-day-period following the previous HbA1c order. The effects of various parameters, like ordering clinics, the first HbA1c level, or the on-line availability of test results on test ordering were evaluated. RESULTS: Evaluation of test intervals showed that 10.3% of all orders and 33.8% of the inpatients' orders were performed within 29 days, 35.5% of all orders and 55% of the inpatients' orders were within 89 days. CONCLUSIONS: Inappropriate laboratory utilization of HbA1c testing is very common especially in the inpatient clinics. We think that the application of the guidelines may decrease unnecessary health expenditure.

[Investigation of transplacental transmission of TT virus in mother--newborn pairs]. / Anne--Yenidogan ciftlerinde TT virusun transplasental Geçisinin arastirilmasi.

TT virus (TTV) is widespread throughout the world and can be detected in 50-95% of healthy individuals. However, in chronic hepatitis C virus (HCV) infected patients coinfected with TTV, histological activity indices were higher than patients with HCV infection alone. There are studies which indicate that TTV can cause aplastic anemia and thrombocytopenia. While TTV is known to be transmitted through blood transfusions and by fecal-oral route, published information on transplasental transmission is controversial. The aims of this study were to detect the frequency of TTV infection among healthy pregnant women and to search whether TTV is transmitted transplasentally during pregnancy. For this purpose, plasma samples collected from 54 women and their newborns were investigated by polymerase chain reaction (PCR), and viral loads were determined for infected newborns, and their respective mothers. Also, ten mothers whose newborns were tested negative for TTV-DNA were enrolled in viral load tests for comparison. TTV-DNA was detected in 49 (90.7%) of 54 women. Of 49 newborns whose mothers were infected with TTV, only 4 (8.2%) were found positive for TTV-DNA. There was no statistically significant difference in TTV-DNA loads between mothers who transmitted the virus and those who did not (Mann-Whitney U analysis Z=-0.071, P=0.944). As a result, transplasental transmission of TTV is possible but occurs in low frequency and independent of viral load.

Is there any association between hepatitis G virus (HGV), other hepatitis viruses (HBV, HCV) and Behçet's disease?

Infectious agents, especially viruses, have been implicated in the pathogenesis of Behçet's disease (BD). The aim of this study was to determine whether BD is associated with hepatitis viruses. In this study, the serological markers of hepatitis (HBsAg, anti-HBs, anti-HBc and anti-HCV) and viral nucleic acid (HGV-RNA) were studied in the sera of 35 patients, all of whom fulfilled the diagnostic criteria of the International Study Group for BD, and the results were compared with those of 36 healthy controls. The prevalences of HBsAg, anti-HBs, anti-HBc in BD patients were 2.9%, 45.7%, and 31.4%, respectively, which were not significantly different from those in healthy controls. None of the subjects in either group were found to be positive for anti-HCV. HGV-RNA was detected in two patients with BD and in none of the healthy controls. In conclusion, BD does not seem to be associated with hepatitis viral infections including hepatitis B, C, or G.

Hospital-acquired infections in elderly patients: results of a West Anatolian University Hospital surveillance.

The objective of this study was to determine the frequency and the pattern of hospital-acquired infections (HAIs) in elderly (age over 65) patients, using routine surveillance data collected by the infection control committee in Dokuz Eylül University Hospital. In this study, 199 elderly patients diagnosed with HAIs in the years of 1999-2000 were included. During this period, 22.7% of all patients who had HAI were over 65 years old. The incidence rate of HAI in elderly patients was 1.2%, increasing with age. The most common types of HAIs were surgical site infections, septicemia, lower respiratory tract infections and urinary tract infections.

Beta-lactam antibiotic resistance in aerobic commensal fecal flora of newborns.

BACKGROUND: The purpose of the present paper was to prospectively determine the rate of beta-lactam antibiotic resistance in commensal fecal flora of newborns and the risk factors leading to this colonization. METHODS: One hundred and eighteen newborns in the neonatal intensive care unit (NICU) group (n = 38), the neonatal ward group (n = 36), and the control group (n = 44) were enrolled. Three or four stool samples were obtained from each infant, 15 days apart. Bacterial growth in Eozin Methylene Blue (EMB) agar + 10 microg ampicillin/mL was considered to be ampicillin-resistant bacteria, and antibiotic susceptibility and extended spectrum beta-lactamases (ESBL) production was investigated in those bacteria. RESULTS: Colonization with ampicillin-resistant commensal fecal flora microorganisms was determined in 75.2% of 367 stool samples. Klebsiella spp. and Escherichia coli were identified in 59% and 41% of the samples, respectively. The lowest rate of ampicillin-resistant bacterial colonization was determined in the NICU group. Microorganisms producing ESBL were identified in 33.7% of 367 stool samples. Fifty-one and 73 of ampicillin-resistant E. coli and Klebsiella spp. isolates were determined to produce ESBL, respectively. There was no difference with respect to colonization with ESBL-producing microorganisms between the three groups. When risk factors related to colonization with ESBL-producing microorganisms in stool samples were evaluated through the whole study period, very low birthweight, vaginal delivery, infant antibiotic usage, maternal antibiotic usage, male sex and premature rupture of membranes were determined as risk factors, while feeding with nasogastric tube was identified as a protective factor. When the risk factors related to colonization with ESBL-producing bacteria in stool samples after discharge from the hospital were evaluated, failure to feed breast milk was determined as the only risk factor. CONCLUSIONS: To decrease mortality and morbidity due to infection caused by resistant microorganisms colonized in the intestine flora of the infant, protection of normal non-pathogenic bacterial colonization is important. This can be provided by feeding neonates with breast milk, application of infection control measures efficiently, and limitation of antibiotic usage only to strict clinical indications.

Faculty Development Program in Dokuz Eylül School of Medicine: In the process of curriculum change from traditional to PBL.

INTRODUCTION: In Dokuz Eylül School of Medicine (DESM) a faculty development program is being carried out by the "Trainers' Training Committee?. DESM made a fundamental change in its curriculum from traditional to Problem-based Learning (PBL) in 1997. This was the first implementation of a PBL curriculum in Turkey. Faculty development activities were initiated in the same year. This paper describes the faculty development activities with a special emphasis on PBL courses. PROGRAM DESCRIPTION: Between 1997-2000 27 four-day long PBL courses were held for 343 participants. The curriculum consisted of PBL philosophy, PBL steps, role of the tutor and students in PBL process, effective case design, assessment principles and group dynamics. PBL simulations enabled the participants to play the roles of both tutors and students. Process evaluation: At the end of the program most of the participants stated that length of the program, content, training methods and the course organization was appropriate. The majority of the participants (89.5%) found the program very useful. PBL steps, PBL practices and PBL philosophy were found as the most useful sessions. DISCUSSION: These courses gave medical staff the opportunity to develop their understanding of PBL methodology and theory. PBL courses and continuous educational activities such as weekly tutor meetings are being held and new courses on advanced tutoring skills are being planned for the near future in DESM.