[Viral Respiratory Tract Infection Agents Detected Between Years 2019-2021, COVID-19, and Co-Infections]. / 2019-2021 Yillari Arasinda Saptanan Viral Solunum Yolu Enfeksiyonu Etkenleri, COVID-19 ve Ko-Enfeksiyonlar.

Respiratory tract infections are a major cause of morbidity and mortality at all ages and are seen as a very important public health problem all over the world. In this study, we aimed to evaluate the effect of the pandemic on the epidemiological and seasonal characteristics of the agents by analyzing the respiratory viral infection agents, viral co-infections and associations with Coronavirus diseases-2019 (COVID-19) studied by multiplex polymerase chain reaction (PCR) test in the molecular microbiology laboratory in a three-year period, including the one-year period before the pandemic. Between March 2019 and December 2021, 8825 respiratory tract specimens accepted to the molecular microbiology laboratory with respiratory tract multiplex PCR test requests were included in the study. In addition, severe acute respiratory syndrome (SARS-CoV-2) PCR test results of the patients with positive results with respiratory tract multiplex PCR test, which were studied within ± 3 days, were evaluated retrospectively. Respiratory viral pathogens were detected using FTD Respiratory Pathogens 21 kit (Fast Tract Diagnostics, Siemens Healthineers Company). Two different kits based on real-time reverse transcription PCR were used for SARS-CoV-2 RNA detection in different periods. According to our results, at least one viral agent was detected in 2156 (24.4%) of a total of 8825 samples and a single agent was detected in 1843 (85.5%) of these. The distribution of viruses in the samples with a single agent was determined as RV, RSV A/B, HCoVs, AdV, flu A virus, MPV A/B, PIV 1-4, flu B virus, EV, BoV and PeV, in order of frequency. Multiple agents were found in 313 (14.5%) of these 2156 samples. They were found to be two agents in 291 samples, three in 21 samples and four in one sample. When the SARS-CoV-2 PCR test results of the patients who had positive results with respiratory tract multiplex PCR and who were studied within ± 3 days were evaluated retrospectively, SARS-CoV-2 RNA was detected in 45 (3.5%) of 1277 samples in which at least one agent was detected. In four of these patients, SARS-CoV-2 was found together with multiple agents. Consequently, there was a sharp decrease in the prevalence of all viral agents during the pandemic period. It was evaluated that besides the COVID-19 infection, the restrictions applied during the pandemic period were also effective in this situation.

Impact of non-pharmaceutical interventions on circulating respiratory viruses during the COVID-19 pandemic in Turkey.

BACKGROUND: Non-pharmaceutical interventions (NPIs) applied to limit the SARS-CoV-2 pandemic also affect the circulation and seasonal characteristics of other respiratory viruses. OBJECTIVES: Assess the impact of NPIs on the spread and seasonal characteristics of non-SARS-CoV-2 respiratory viruses and examine viral respiratory co-infections. DESIGN: Retrospective cohort SETTING: Single center in Turkey. PATIENTS AND METHODS: Syndromic multiplex viral polymerase chain reaction (mPCR) panel results of patients admitted to the Ankara Bilkent City Hospital with symptoms of acute respiratory tract infection between April 1, 2020 and October 30, 2022 were evaluated. Two study periods before and after 1 July 2021, when the restrictions were discontinued, were statistically analyzed and compared to determine the effect of NPIs on circulating respiratory viruses. MAIN OUTCOME MEASURES: Prevalence of respiratory viruses as determined by syndromic mPCR panel. SAMPLE SIZE: 11300 patient samples were evaluated. RESULTS: At least one respiratory tract virus was detected in 6250 (55.3%) patients. Of these, at least one respiratory virus was detected in 5% in the first period (between April 1, 2020 and June 30, 2021, when NPIs were applied), and in 95% in the second period (between July 1, 2021 and October 30, 2022, when NPIs were relaxed). After the removal of NPIs, there was a statistically significant increase in hRV/EV, RSV-A/B, Flu A/H3, hBoV, hMPV, PIV-1, PIV-4, hCoV-OC43, PIV-2 and hCoV-NL63 (P<.05). In the 2020-2021 season, when strict NPIs were applied, all respiratory viruses evaluated did not have the usual seasonal peak and there were no seasonal influenza epidemics during this period. CONCLUSIONS: NPIs resulted in a dramatic decrease in the prevalence of respiratory viruses and notable disruption of seasonal characteristics. LIMITATIONS: Single-center study and retrospective. CONFLICT OF INTEREST: None.

Comparison of clinical characteristics of wild-type SARS-CoV-2 and Omicron.

OBJECTIVE: This study aimed to investigate the effect of mutations by comparing wild-type SARS-CoV-2 and Omicron regarding clinical features in patients with COVID-19. It also aimed to assess whether SARS-CoV-2 cycle threshold value could predict COVID-19 severity. METHODS: A total of 960 wild-type and 411 Omicron variant patients with positive results in SARS-CoV-2 real-time reverse transcriptase polymerase chain reaction test from oropharyngeal and/or nasopharyngeal samples during their hospital admissions were included in this retrospective study. The reference symptoms of the patients were obtained from the hospital database. The correlation between chest computed tomography findings and the "cycle threshold" of patients with wild-type SARS-CoV-2 was assessed. RESULTS: Cough, fever, shortness of breath, loss of taste and smell, and diarrhea were found to be statistically significantly higher (p=0.001; 0.001; 0.001; 0.001; and 0.006; respectively) in the wild-type cohort, while in the Omicron cohort, sore throat and headache were found to be statistically significantly higher (p=0.001 and 0.003, respectively). An inverse relationship was found between chest computed tomography findings and viral load. CONCLUSION: This study revealed that the Omicron variant tended to infect predominantly the upper respiratory tract and showed decreased lung infectivity, and the disease progressed with a milder clinical course. Therefore, the study showed that the tropism of the virus was changed and the viral phenotype was affected. It was also found that SARS-CoV-2 viral load did not predict COVID-19 severity in patients with wild-type SARS-CoV-2.

A Genetically Engineered Biofilm Material for SARS-CoV-2 Capturing and Isolation.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuously infecting people all around the world since its outbreak in 2019. Studies for numerous infection detection strategies are continuing. The sensitivity of detection methods is crucial to separate people with mild infections from people who are asymptomatic. In this sense, a strategy that would help to capture and isolate the SARS-CoV-2 virus prior to tests can be effective and beneficial. To this extent, genetically engineered biomaterials grounding from the biofilm protein of Escherichia coli are beneficial due to their robustness and adaptability to various application areas. Through functionalizing the E. coli biofilm protein, diverse properties can be attained such as enzyme display, nanoparticle production, and medical implant structures. Here, E. coli species are employed to express major curli protein CsgA and Griffithsin (GRFT) as fusion proteins, through a complex formation using SpyTag and SpyCatcher domains. In this study, a complex system with a CsgA scaffold harboring the affinity of GRFT against Spike protein to capture and isolate SARS-CoV-2 virus is successfully developed. It is shown that the hybrid recombinant protein can dramatically increase the sensitivity of currently available lateral flow assays for Sars-CoV-2 diagnostics.

Secondary bacterial infections of the respiratory tract in COVID-19 patients.

INTRODUCTION: Secondary Bacterial Infections (SBIs) of the respiratory system are one of the biggest medical concerns in patients undergoing hospitalization with a diagnosis of COVID-19. This study aims to provide relevant data for the initiation of appropriate empirical treatment after examining the etiology and antimicrobial resistance of SBIs in COVID-19 patients under care in the Intensive Care Units (ICUs) in the largest pandemic hospital of our country. METHODOLOGY: Between March 16, 2020 and December 31, 2021, 56,993 COVID patients were hospitalized, of which 7684 were admitted to ICUs. A total of 1513 patients diagnosed with SBIs have been included in this study. During the course of the study, demographic data, clinical course, etiology and antimicrobial resistance data of all patients were collected. RESULTS: The most common causative agents of SBIs were inferred as Acinetobacter baumanii (35.1%), Staphylococcus aureus (15.2%), Klebsiella pneumoniae (12.3%) and Pseudomonas aeruginosa (10.4%). The isolation rates of carbapenem-resistant and colistin-resistant A. baumannii, K. pneumoniae and P. aeruginosa were 83.7%; 42.7%, 79.2%, and 5.6%, 42.7%, 1.7%, respectively. Acinetobacter pittii clustering was seen in one of the ICUs in the hospital. Multidrug resistant 92 (5.4%) Corynebacterium striatum isolates were also found as a causative agent with increasing frequency during the study period. CONCLUSIONS: SBI of the respiratory system is one of the major complications in patients hospitalized with COVID-19. The antimicrobial resistance rates of the isolated bacteria are generally high, which indicates that more accurate use of antibacterial agents is necessary for SBIs in patients hospitalized with COVID-19 diagnosis.

SARS-CoV-2 is not found in the sweat of COVID-19 positive patients.

BACKGROUND: As the SARS-CoV-2 virus made a pandemic all over the world, its transmission routes became significant. Transmission from human to human is known, but other possible routes are not determined well. AIMS: This study aimed to reveal the presence of SARS-CoV-2 virus in sweat. METHODS: This prospective study was conducted in a tertiary care education and training hospital. Fifty patients were included in this study. Skin disinfection was done with an alcohol-based solution. Swabs for RT-PCR (real-time reverse transcriptase polymerase chain reaction) were taken from forehead and axilla skin after sweating patients for 30 min. After collection of sweat, swabs were placed into 2 ml of sterile viral transport medium, then transported quickly to the microbiology laboratory. RESULTS: No SARS-CoV-2 virus was detected in RT-PCR of forehead and axilla swabs. CONCLUSION: This study showed that there is no transmission of SARS-CoV-2 virus via sweat. However, general precautions must be taken while doing interventional procedures.

[Investigation of the Relationship of Systemic Immune-Inflammation Index, C-Reactive Protein and Interleukin-6 with Viral Dynamics in Patients with COVID-19]. / COVID-19 Tanili Hastalarda Sistemik Immün-Enflamasyon Indeksi, C-Reaktif Protein ve Interlökin-6'nin Viral Dinamik ile Iliskisinin Arastirilmasi.

Coronaviruses are enveloped, positivepolarity, single-stranded RNA viruses that can cause respiratory and gastrointestinal tract infections, less likely to cause infections with hepatic, neurological and nephrotic involvement. A novel coronavirus termed as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in the city of Wuhan, China, and caused an outbreak of unusual viral pneumonia at the end of 2019. This study aimed to reveal the relationship between systemic immune-inflammation index (SII), C-reactive protein (CRP) and interleukin-6 (IL-6) and viral dynamics in COVID-19 patients. This retrospective, single-center study was conducted in Ankara City Hospital from April 1 to May 31, 2020. A total of 338 hospitalized patients who had positive results in SARS-CoV-2 reverse transcrytase polymerase chain reaction (RT-PCR) test from nasopharyngeal and oropharyngeal samples during their hospital admission were included in this study. Patients were divided into three groups according to their ward/intensive care unit, intubation and mortality situation and their clinical data were evaluated. Correlation analysis was performed to determine the relationship between viral dynamics and laboratory parameters such as SII, the neutrophil-to-lymphocyte ratio (NLR), the lymphocyte-to-CRP ratio (LCR), the lymphocyte-to-monocyte ratio (LMR), the platelet-to-lymphocyte ratio (PLR), CRP, IL-6 ferritin, albumin levels and lymphocyte count. Advanced age, low Ct value, increase in IL-6, increase in SII, decrease in albumin, increase in ferritin, decrease in lymphocyte count, increase in NLR, decrease in LCR, decrease in LMR, increase in PLR and increase in CRP levels were found statistically significantly different in all three groups (p<0.001; p= 0.02; p<0.001; p<0.001; p<0.001; p<0.001; p<0.001; p<0.001; p<0.001; p<0.001; p<0.001; p<0.001, respectively). Statistical analysis revealed a significant negative correlation between serum IL-6, NLR, LCR and CRP values with Ct values (p<0.01, r= -0.233; p= 0.021, r= -0.126; p=0.004, r= -0.156 and p= 0.011, r= -0.138, respectively) and a significant positive correlation between Ct values and lymphocyte count and albumin levels (p= 0.005; r= 0.151 and p= 0.050; r= 0.106, respectively). Severe progression was observed in patients with advanced age, low Ct value, high IL-6 levels, high SII, hypoalbuminemia, high ferritin levels, lymphopenia, high NLR, low LCR, low LMR, high PLR and high CRP. In these patients hospitalization in intensive care unit, intubation and mortality were found to be higher. High levels of IL-6, NLR, LCR and CRP, lymphopenia and hypoalbuminemia were associated with low PCR Ct values.

The effect of real-time polymerase chain reaction cycle threshold values on perinatal outcomes of pregnant women with COVID-19.

OBJECTIVE: To evaluate the effect of cycle threshold (Ct) values on the pregnancy outcomes of women with coronavirus disease 2019 (COVID-19). MATERIALS AND METHODS: This prospective cohort study was conducted on pregnant women with COVID-19. A real-time polymerase chain reaction (RT-PCR) assay of a nasopharyngeal and oropharyngeal specimen was used for the diagnosis. Initial Ct values for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RT-PCR tests were recorded. 22.9 was the 50th percentile Ct value of the study population. The study population was divided into two groups based on their Ct values: (1) Cases with Higher Ct values (Ct > 22.9)(n = 50) and (2) Cases with lower Ct values (Ct ≤ 22.9)(n = 55). Demographic features, clinical characteristics, disease progression, laboratory test results and pregnancy outcomes were compared between the groups. A receiver operating characteristic (ROC) curve was used to assess the performance of Ct values in predicting obstetric complications. RESULTS: Obstetric complication rate was significantly higher in cases with lower Ct values (p < .001). A significantly lower lymphocyte count together with higher ESR, procalcitonin and IL-6 values were observed in the cases with lower Ct values (p > .05). Additionally, a significantly higher NICU admission rate and longer hospital stays were present in the cases with lower Ct values (p > .05). The value in ROC curves with the best balance of sensitivity/specificity was 22.5 (85.7% sensitivity, 63.6% specificity). CONCLUSION: Lower Ct values may be associated with an increased rate of obstetric complications in pregnant women with COVID-19. Physicians should be cautious in the management of cases with Ct levels below 22.5.

Comparing ICU admission rates of mild/moderate COVID-19 patients treated with hydroxychloroquine, favipiravir, and hydroxychloroquine plus favipiravir.

BACKGROUND: In this study, we aimed to compare the intensive care unit (ICU) admission rate of hospitalized mild/moderate COVID-19 patients treated with hydroxychloroquine (HCQ), favipiravir, and HCQ plus favipiravir. METHODS: Single center retrospective designed observational study conducted in Ankara City Hospital. Patients who were hospitalized between March 15, 2020 and June 1, 2020 in COVID-19 inpatient clinics with laboratory confirmed diagnosis of COVID-19 were included in the study. An inverse probability of treatment weighting (IPTW) for multiple treatment groups approach was used to balance the differences in several variables on admission. RESULTS: Among 2441 patients hospitalized with diagnosis of COVID-19 during the study period, 824 were eligible for the analysis. Median age of patients was 42 (18-93 years). Among all, 347 (43.2%) of the patients had mild disease, 470 (56.8%) had pneumonia. Propensity scores ranged from 0.1841 to 0.9381 in the HCQ group, from 0.03643 to 0.29885 in the favipiravir group, and from 0.03542 to 0.56184 in the HCQ plus favipiravir group. After IPTW for multiple treatment groups was applied, all the covariates in the planned propensity score had weighted standardized effect sizes below 10% which were ranged from 0.005 to 0.092. Multivariate analysis of treatment effect (adjusted effect of treatment) was indicated that there is no statistically significant difference between HCQ, favipiravir, and HCQ plus favipiravir treatment. After using combination of SMOTE and Bootstrap resampling approach, we found no statistically significant difference between HCQ and HCQ plus favipiravir groups in terms of ICU admission. However, compared with the HCQ group, ICU admission rate was statistically significantly higher in the favipiravir group. We obtained the similar results after the sensitivity analysis. CONCLUSIONS: HCQ with or without favipiravir treatment is associated with reduced risk of ICU admission compared to favipiravir alone in mild to moderate COVID-19 adult patients.

Molecular Characterization of Carbapenem-Resistant Acinetobacter baumannii Blood Culture Isolates from Three Hospitals in Turkey.

We aimed to investigate the clonal relationships, common sequence types, and carbapenemase genes in 177 non-repetitive blood culture isolates of Acinetobacter baumannii collected from patients at three university hospitals in Turkey in 2016. Molecular epidemiological characteristics of the isolates were examined using pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) (Pasteur scheme-cpn60, fusA, gltA, pyrG, recA, rplB, and rpoB). Multiplex PCR was used to investigate the carbapenemase genes, including blaOXA-23-like, blaOXA-24-like, blaOXA-48-like, blaOXA-58-like, blaIMP, blaVIM, and blaNDM. PFGE genotyping yielded 92 pulsotypes with a clustering ratio of 69.7%. As per a ≥85% similarity coefficient, 159 (90.9%) isolates were found to be clonally related. The blaOXA-23-like and blaOXA-58-like genes were identified in 100% and 28.2% of the isolates, respectively. The blaNDM gene was identified in two isolates. The MLST analysis included 54 isolates with different pulsotypes, and 29 sequence types (STs). Most of the isolates (n = 36) belonged to the clonal complex (CC)2, one isolate belonged to CC1, and one isolate belonged to CC164. Sixteen new STs (ST1235-ST1250) were identified. Identifying both global ST2 and a large number of new STs, revealed high genetic diversity in A. baumannii isolates in the study population.

Assessment of SARS-CoV-2 in the Cerumen of COVID-19-Positive Patients.

OBJECTIVE: To evaluate the presence of SARS-CoV-2 virus in the cerumen of patients with COVID-19. METHODS: A prospective study was conducted in a tertiary care pandemic hospital. Sixty COVID-19 patients with cerumen in their external auditory canals were included in the study. Swabs were taken from the external auditory canal of the patients by an experienced otolaryngologist with the test swab. Sampling was done by rotating the sample swab 360° 10 times in each external auditory canal for a total of 20 times. After collection, swabs were placed into 2 mL of the sterile viral transport medium (various manufacturers), then transported and tested as soon as possible after collection. RESULTS: SARS-CoV-2 was not detected in the cerumen polymerase chain reaction (PCR) samples of any of the 60 patients with positive nasopharyngeal/oropharyngeal swabs. CONCLUSION: Cerumen cleaning is one of the most common procedures performed by otolaryngologists, and care should be taken during the procedure or due to the possibility of infection from the resulting contaminants. The cerumen contains the secretions of the glands in the external auditory canal and may contain certain pathogens that are actively found in the body. The presence of hepatitis B virus in the cerumen was examined and isolated in the cerumen. In our study, the presence of SARS-CoV-2 virus in the cerumen was evaluated in SARS-CoV-2 PCR-positive patients. SARS-CoV-2 virus was not detected in the cerumen samples of any of the patients.

[The Identification of Acinetobacter baumannii Blood Isolates by MALDI-TOF MS, ARDRA and blaOXA-51-like Gene-Specific Real-Time PCR]. / Acinetobacter baumannii Kan Izolatlarinin MALDI-TOF MS, ARDRA ve blaOXA-51-benzeri Gene Özgül Gerçek Zamanli Polimeraz Zincir Reaksiyonu ile Tanimlanmasi.

The Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) complex consists of phenotypically very similar nosocomial species; A.baumannii, Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter seifertii and Acinetobacter djikshoorniae and one environmental species A.calcoaceticus. The rapid and accurate identification of the members of Acb complex is critical as these nosocomial pathogens can show differences in antimicrobial susceptibility and clinical outcomes. The conventional phenotypic methods are slow, unreliable and less efficient for the differentiation of Acb complex species, including the A.baumannii species within the Acb complex. Although various molecular methods are available, such as amplified ribosomal DNA restriction analysis (ARDRA) and blaOXA-51-like gene specific PCR, they are usually inconvenient for the routine diagnostic laboratories. Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) offers an opportunity for rapid, cost-effective and convenient bacterial identification in routine diagnostic procedures conducted in clinical laboratory. In this study, we aimed to evaluate the diagnosis performance of MALDI-TOF MS system to identify blood isolates of A.baumannii. A total of 180 nonduplicate carbapenem resistant Acb complex (strain numbers; TR1-TR60) and A.baumannii (TR61-TR180) blood isolates were collected from the intensive care units of the three university hospitals in Turkey from January 2016 to December 2016. All isolates were evaluated by using blaOXA-51-like gene specific real time (Rt-PCR) analysis, ARDRA (restriction enzymes-AluI, CfoI, MboI, MspI, RsaI) method and MALDI-TOF MS (VITEK® MS, bioMérieux, France) system. All the strains except TR10, TR31, TR35 and TR52 were identified as A.baumannii by ARDRA method. Out of 177 of all the isolates, presence of blaOXA-51-like gene was found except for TR10, TR31 and TR52 isolates. However, TR31 without the presence of blaOXA-51-like gene was identified as A.pittii using the ARDRA. Totally 176 isolates which were identified as A.baumannii by both of the methods, ARDRA and Rt-PCR- blaOXA-51-like, were accepted as a reference for the evaluation of the diagnosis performance capacity of the MALDI-TOF MS. Overall, for all 176 isolates tested, the sensitivity obtained with the MALDI-TOF MS were 99.4% with 75% specificity. The accuracy value of the method was determined as 98.9% for the identification of A.baumannii to the species level. MALDI-TOF MS is increasingly used in diagnostic microbiology for the routine identification of bacteria to the genus, species or subspecies level with high rates of sensitivity and specificity. In future, by expanding the database, MALDI-TOF MS system would possibly become the ideal method for routine diagnostic laboratories that could potentially identify more species and even determine some characteristics of antimicrobial resistance and virulence determinants.

Role of efflux pump and OprD porin expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates.

INTRODUCTION: In recent years, the prevalence of multidrug-resistant P. aeruginosa has remarkably increased. Thus, we wanted to investigate the carbapenem resistance mechanisms and clonal relationship among 80 carbapenem-resistant P. aeruginosa strains. METHODOLOGY: Carbapenemase production was detected using the Modified Hodge Test (MHT), EDTA combined disc method (ECD), and PCR. Expression levels of efflux and porin genes were mesured by real-time reverse transcription PCR. Clonal relationship of the isolates was investigated by pulsed-field gel electrophoresis (PFGE). RESULTS: Carbapenemase production was detected in 7.5% of the isolates with MHT/ECD tests and in 11.3% of the isolates with PCR. Although the specificity of MHT/ECD was high, the sensitivitivity was low. oprD downregulation and mexB, mexY, and mexD overexpression were demonstrated in 55%, 16.3%, 2.5%, and 2.5% of the isolates, respectively. Multiple carbapenem resistance mechanisms were found in nearly a quarter of the isolates. PFGE typing of the 80 P. aeruginosa isolates yielded 61 different patterns. A total of 29 isolates (36.3%) were classified in 10 clusters, containing 2 to 7 strains. We could not find a strict relationship between PFGE profile and carbapenem resistance mechanisms. CONCLUSIONS: Although oprD downregulation and MexAB-OprM overexpression were the most common mechanisms, carbapenem resistance was associated with multiple mechanisms in the study. MHT/ECD tests should not be used alone for investigation of carbapenemase production in P. aeruginosa. Rapid tests with high sensitivity and specificity should be developed for the detection of carbapenemase production in P. aeruginosa.

Comparison of a novel real-time PCR method (RTA) and Artus RG for the quantification of HBV DNA and HCV RNA.

INTRODUCTION: The purpose of this study was to evaluate and compare two manual isolation and real-time (RT) polymerase chain reaction (PCR) kits (RTA RT-PCR with RTA isolation kit and Artus RG RT-PCR with QIAamp isolation kit) for molecular diagnosis of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. METHODOLOGY: The study was conducted on 121 and 54 clinical samples for the detection of HBV DNA and HCV RNA, respectively, with an additional 8 HCV RNA external quality control samples. RESULTS: Though a high correlation was observed between the two kits for the HBV DNA (r = 0.955, p = 0.001) and HCV RNA quantifications (r = 0.828, p = 0.001), discordant results were found in nine of the HBV DNA and in six of the HCV RNA samples. The mean difference between the two systems was found to be 0.4 log IU/mL in Quality Control for Molecular Diagnostics (QCMD) HCV RNA samples by Bland-Altman analysis. CONCLUSIONS: Although there was a high correlation between HBV DNA and HCV RNA tests according to the results of the study, the RTA system requires improvement for the determination of HCV RNA.

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.

[Detection of resistance mutations in chronic hepatitis B patients receiving antiviral therapy for over one year]. / Bir yildan uzun süreli antiviral ilaç kullanan kronik hepatit B hastalarinda direnç mutasyonlarinin saptanmasi

Primary mutations conferring hepatitis B virus (HBV) antiviral resistance and associated secondary/compensatory mutations were investigated in this study by DNA sequencing (SEQ) and two commercial Line Probe Assays (LiPAs) (Inno-Lipa HBV DRv2 and Inno-Lipa HBV DRv3, Innogenetics, Belgium). A total of 71 subjects under follow-up for chronic hepatitis B and receiving lamivudine (LVD) therapy for one year or longer at the Hacettepe University Faculty of Medicine, Department of Internal Medicine, Gastroenterology Unit were included in the study with informed consent. Male to female ratio and mean age was noted as 47/24 and 43 ± 15.8 (age range: 13-71) years, respectively. Twenty samples with low HBV DNA levels (mean: 204.6 IU/ml) could not be interpreted by SEQ due to insufficient amplification. All samples with a positive consensus PCR were further analysed via LiPAs, as directed by the manufacturer. Thus a total of 51 and 56 samples could be evaluated via SEQ and LiPA assays, respectively. In 13 of the 51 (25.5%) samples by SEQ and in 9 of 56 (16%) samples by LiPAs, primary and compensatory mutations associated with resistance were identified. Multiple mutations that comprise L180M + M204I in four and L180M + M204V in one sample and single mutations (M204I) in three samples were identified by SEQ. In one sample which had multiple mutations associated with LVD resistance single mutations (S202G, N236T) associated with entecavir resistance and in two other such samples mutations associated with adefovir resistance were detected by SEQ. Also, in three samples amino acid substitution at position rt215 (QÆS) as alone and in one sample with multiple mutations were observed via SEQ. In five of nine samples primary and compensatory multiple mutations (L180M + M204I in one sample, L80I + L180M + M204I in two samples, L80I/V + M204I in one sample) and primary single mutations associated with LVD resistance (M204I/V) in four samples were detected by Inno-Lipa HBV DRv2. Two different mutations (G202, ILFM184) were observed in two samples with multiple mutations associated LVD resistance via Inno-Lipa HBV DRv3. However, mutation at position rt184 was evaluated as a weak positive. Any mutation associated with adefovir resistance was not detected by LiPA. As a result, SEQ and LiPAs displayed comparable performances for the detection of HBV drug resistance mutations. We suggested that for the evaluation of discordant results, it should be better to test consecutive serum samples.

[Investigation of interleukin-10, tumor necrosis factor-alpha and interferon-gamma expression in experimental model of pulmonary aspergillosis]. / Deneysel Pulmoner Aspergilloz Modelinde Interlökin-10, Tümör Nekroz Faktörü-alfa ve Interferon-gama Ifadelenmesinin Arastirilmasi

Pulmonary aspergillosis which is an important opportunistic infection in neutropenic patients, is usually caused by Aspergillus fumigatus. Since the pathogenesis of disease is not well understood, the main proposed mechanism is thought to be cell-mediated immunity and cytokine response. The aim of this study was to investigate the local production of cytokines in the lung tissues of rats with experimentally developed aspergillosis, by reverse transcriptase-polymerase chain reaction (RT-PCR). A total of 33 Wistar albino type rats were included in the study with the consent of Experimental Animal Ethics Committee. Twenty-five of the rats were infected with A.fumigatus by intratracheal way, while 8 animals were used as controls. The presence of A.fumigatus in the lung tissues of infected rats was confirmed with the use of quantitative culture and histologic staining methods. RNA isolation from the lung tissue samples of both groups were performed by a commercial kit (Qiagen, Germany). After obtaining complementary DNAs from the genomic RNAs, in-house qualitative and quantitative (real-time) PCR methods were used to amplify the target regions for interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-?) and interferon-gamma (IFN-?) by using specific primers (Tib Molbiol, Germany). Mean mRNA levels achieved by real-time PCR for IL-10, TNF-? and IFN-? in aspergillosis group were 6.5 x 106 copies/ml, 7.9 x 105 copies/ml and 2.2 x 103 copies/ml, respectively, while those values in control group were 4.3 x 102 copies/ml, 5.6 x 103 copies/ml and 1.3 x 102 copies/ml, respectively. Our data indicated that rat model of aspergillosis was associated with significantly increased expression of mRNA encoding IL-10 and TNF-? than controls (p< 0.05), however there was no statistically significant difference between the groups with respect to IFN-? expression (p= 0.53). In conclusion, the production of proinflammatory cytokines which mediate the influx of phagocytic cells might account for the localization of Aspergillus infection to the upper respiratory tract. The up-regulation of the expression of the immunomodulatory cytokine TNF-? and IL-10 in lung tissue from infected rats might be important to limit the extent of local tissue destruction, but might also account for the fact that infected rats are generally unable to clear the infection spontaneously.

Performance of various commercial assays for the detection of Toscana virus antibodies.

INTRODUCTION: Sandfly fever virus (SFV) serotypes sandfly fever Naples virus, sandfly fever Sicilian virus, and sandfly fever Cyprus virus cause febrile diseases, whereas Toscana virus (TOSV) is responsible for aseptic meningoencephalitis. Diagnosis and surveillance of TOSV depend heavily on virus serology, and various commercial assays utilizing various antigen sources and formats have been available. The aim of this study was to perform comparative evaluation of commercially available serological assays for anti-TOSV immunoglobulins. MATERIALS AND METHODS: A collection of 120 sera from healthy blood donors from an endemic region, previously identified to be reactive for antibodies against various SFV serotypes by indirect immunofluorescence test (IIFT), was reevaluated for IgG/IgM via IIFT, enzyme-linked immunosorbent assay, and an immunoblot assay manufactured by Euroimmun, Diesse, and Mikrogen, respectively. Virus neutralization test (VNT) was performed for 99 sera using standard TOSV, sandfly fever Sicilian virus, and sandfly fever Naples virus strains. RESULTS: A total of 89 samples (74.2%) were reactive for TOSV IgG in at least one of the commercial assays, and 31 samples (31.3%) were reactive in VNT for various SFV serotypes. Average percentage agreements among commercial assays and between VNT and the commercial assays were noted as 57.8% and 62.6%, respectively. No significant correlation between assay results and VNT titers was observed. SFV IgM antibodies were detected in a total of eight samples (6.7%) via IIFT, which were nonreactive in enzyme-linked immunosorbent assay and VNT. DISCUSSION: Commercial diagnostic immunoassays displayed slight to fair agreement for TOSV IgG as assessed via kappa and percentage agreement values. The results could only be confirmed via virus neutralization in a portion of the samples, and overall agreement between the commercial assays and VNT was slight. Commercial assays such as immunoblot can be used in addition to VNT for confirmation of TOSV exposure.

[Investigation of West Nile virus seroprevalence in Hacettepe University Hospital blood donors and confirmation of the positive results by plaque reduction neutralization test]. / Hacettepe Üniversitesi Hastanesi Kan Donörlerinde Bati Nil Virusu Seroprevalansinin Arastirilmasi ve Pozitif Sonuçlarin Plak Redüksiyon Nötralizasyon Testi ile Dogrulanmasi

West Nile virus (WNV), a member of Flaviviridae family, is an enveloped, icosahedral, single-stranded positive-sense RNA virus. WNV is transmitted to humans by infected mosquitoe (especially Culex spp.) bites and cause a variety of clinical outcomes, ranging from asymptomatic infection to severe meningoencephalitis. The aims of this study were to determine and confirm the WNV seroprevalence in a chosen healthy population and to provide epidemiological data for Turkey about the recent status of the infection at our region. A total of 1200 serum samples collected from blood donors (400 were female, 800 were male; age range: 18-61 years, mean age: 37.8) who were admitted to Hacettepe University Hospital Blood Donation Center between April to December 2009, were included to the study. The presence of WNV IgG antibodies were screened by ELISA (Euroimmun, Germany), and the positive samples have been further investigated by WNV IgG avidity test in order to estimate the time of encounter to the virus. Indirect immunofluorescence antibody (IFA) test (Euroimmun, Germany) and plaque reduction neutralization test (PRNT) which is accepted as the reference method, were performed for the confirmation of WNV IgG positive results. Nineteen (1.6%) of the samples yielded WNV IgG positivity with ELISA, and all of which were IgGs with high avidities (Avidity index values were between 67.8-99.2%). Eight of 19 (42.1%) WNV ELISA IgG positive donors, had risk factors such as joining outdoor activities, contact with mosquitos and ticks and consuming raw milk and milk products. Of 19 samples that were taken into confirmation tests, 15 (78.9%) were found positive with IFA, and 10 (52.6%) were found positive with PRNT. WNV antibody positivity of 10 samples were then confirmed by PRNT, however eight samples which were found positive with both ELISA and IFA yielded negative results with PRNT. This data might indicate that ELISA and IFA methods in which virus-infected cells were used as substrates, have detected non-neutralizing antibodies against viral nucleocapsid antigens rather than the neutralizing antibodies detected against envelope glycoproteins by PRNT method. One sample which yielded low positive result only by ELISA test has been evaluated as a specificity issue of the test. As a result, the positivity rate (19/1200; 1.6%) of WNV IgG detected by ELISA in blood donors, has been confirmed as 0.8% (10/1200) by a gold standard method, PRNT. The data of this study indicated that the prevalence of WNV infections, although low in our region, deserves attention to be considered in surveillance and control programs related to WNV in Turkey.