Pelin Yuksel

Are isolated anti-HBc blood donors in high risk group? The detection of HBV DNA in isolated anti-HBc cases with nucleic acid amplification test (NAT) based on transcription-mediated amplification (TMA) and HBV discrimination

AIM Hepatitis B virus (HBV) can be transmitted by blood transfusions even so using serological tests having high sensitivity and specificity. We aimed to detect HBV DNA in isolated Anti-HBc donors and show if they have transfusion risk or not. METHOD We investigated Anti-Hbc and Anti-HBs in serum samples of 12858 HBs-Ag negative blood donors who were applied to the Turkish Redcrescent between June 2007 and October 2008 by the Micro ELISA kit (Hepanostica ultra HBsAg, Bio Meriux, France). Anti-HBc and Anti-Hbs positive cases were omitted. We used Procleix ultrio (Chiro, USA) test kit (Chiron Tigris automated instrument was used) based TMA (Transcription-Mediated Amplification) for NAT study in Anti-HBc positive and Anti-HBs negative plasma samples. The discrimination of HBV in NAT positive samples were performed by Procleix Discrimination (Chiro, USA) test. RESULTS 2748 (21.4%) Anti-HBc seropositivity were detected in 12852 HBsAg(-) serum samples. 23.5% Anti-HBs negativity was detected in 2748 Anti-HBc positive serum samples. On the other hand, 5.1% isolated Anti-Hbc positivity [HBsAg(-), Anti-HBc(+), Anti-HBs(-)] were detected in 12852 HBsAg(-) serum samples. 0.091% and 0.047% HBV-DNA positivity were detected in isolated Anti-HBc positive plasma samples and HBsAg(-) plasma samples, respectively. CONCLUSION As a result, even we have detected one HBV transmission in every 2142 blood transfusion by HBsAg screening tests; we suggest that it is not necessary to add additional tests to detect isolated Anti-HBc for routine purposes in Blood Banking. The reasons are higher negativity rates (99%) of isolated Anti-HBc serum samples and the rejection of blood donors with Anti-HBc positivity.

Antimicrobial resistance of Helicobacter pylori strains to five antibiotics, including levofloxacin, in Northwestern Turkey

INTRODUCTION Antibiotic resistance is the main factor that affects the efficacy of current therapeutic regimens against Helicobacter pylori. This study aimed to determine the rates of resistance to efficacy clarithromycin, amoxicillin, tetracycline, levofloxacin and metronidazole among H. pylori strains isolated from Turkish patients with dyspepsia. METHODS H. pylori was cultured from corpus and antrum biopsies that were collected from patients with dyspeptic symptoms, and the antimicrobial susceptibility of H. pylori was determined using the E-test (clarithromycin, amoxicillin, tetracycline, metronidazole and levofloxacin) according to the EUCAST breakpoints. Point mutations in the 23S rRNA gene of clarithromycin-resistant strains were investigated using real-time PCR. RESULTS A total of 98 H. pylori strains were isolated, all of which were susceptible to amoxicillin and tetracycline. Of these strains, 36.7% (36/98) were resistant to clarithromycin, 35.5% (34/98) were resistant to metronidazole, and 29.5% (29/98) were resistant to levofloxacin. Multiple resistance was detected in 19.3% of the isolates. The A2143G and A2144G point mutations in the 23S rRNA-encoding gene were found in all 36 (100%) of the clarithromycin-resistant strains. Additionally, the levofloxacin MIC values increased to 32 mg/L in our H. pylori strains. Finally, among the clarithromycin-resistant strains, 27.2% were resistant to levofloxacin, and 45.4% were resistant to metronidazole. CONCLUSIONS We conclude that treatment failure after clarithromycin- or levofloxacin-based triple therapy is not surprising and that metronidazole is not a reliable agent for the eradication of H. pylori infection in Turkey.

The detection of occult HBV infection in patients with HBsAg negative pattern by real-time PCR method

AIM Diagnostic problems may be encountered in Hepatitis B virus (HBV) infections by serological tests and HBV DNA can be detectable in plasma and liver tissue while the HBsAg test is negative. This situation can be defined as occult or isolated Anti-HBc infections. Occult HBV infections may be divided into two categories by using hepatitis markers. One of them being that all hepatitis markers are negative and the other situation is having Anti-HBc +/- and Anti-HBs+patterns. These situations can be seen in isolated Anti-HBc cases. METHOD In this study, we aimed to detect the ratio of occult HBV infections by investigating HBV DNA in four different groups. These groups are: (1) 20 isolated Anti-HBc positive individuals, (2) 23 individuals naturally immune to HBV infection, (3) 20 individuals with seronegative hepatitis markers and high ALT levels, and (4) 23 vaccinated individuals against HBV. In order to detect HBV DNA the real-time PCR kit (QIAGEN, Artus HBV RG PCR Kit, Germany) with high analytical sensitivity (≤3.8IU/ml) was used. RESULTS The reliability of the molecular methods was assessed by increasing the quantitation standards of internal, external and also positive controls. No HBV DNA was detected in any of the 86 individuals consisting of four study groups. CONCLUSION In conclusion, we did not detect occult HBV infection in our four study groups by using a high sensitivity real-time (RT) PCR method, while occult HBV infections with various frequencies were detected in other large, serial international studies in which highly sensitive analytical molecular methods were used. Although we also used a high standard molecular kit to detect occult HBV infections, we suggest that the reason for the absence of detection of occult HBV infections may be due to the small number of cases included in this study. However, it was assumed that the use of a nucleic acid amplification technology (NAT) with high analytical sensitivity in blood banks to prevent HBV transmission by blood transfusion is controversial due to both costs and diagnostic efficacy and for this reason we suggest that it will be useful to perform large serial studies regarding occult HBV infections in the future.

New approaches to in vitro diagnosis of hepatitis C infection a reason for post transfusion hepatitis: Diagnostic value of determination of hepatitis C virus core antigen

In between the dates of February 2008-March 2009, by applying to Istanbul University CTF Microbiology and Clinical Microbiology Basic Sciences Branch and Duzen laboratories, 123 cases, where HCV RNA and anti-HCV positivity are identified with molecular (real-time PCR) and serologic (ELISA) methods as a positive control group, and 48 cases where HCV RNA and anti-HCV negativity are identified as a negative control group are established. The values of sensitivity, specificity, positive and negative approximation of recently developed HCV Core Ag (Abbott Diagnostics, Germany) kit are determined successively as 94.3%, 97.9%, 99.1%, 87%, 95.3% and 88%. Although the new HCV Ag assay is clearly not sensitive enough to replace HCV NAT it may serve as a valuable tool in the HCV diagnostic algorithm as it is able to pick up a great majority of anti-HCV and HCV RNA positive samples, thus allowing a timely and less expensive serological diagnosis of an active HCV infection. This may be an advantage for labs that do not have access to PCR easily.

Isolation and diagnosis of Helicobacter pylori by a new method: microcapillary culture.

AIM To investigate the performance of the microcapillary culture method (MCM) in Helicobacter pylori (H. pylori) isolation and diagnosis. METHODS Microcapillary culture (MC), classical culture (CC), rapid urease (CLO) test, and histopathologic examination (HE) were performed with biopsy samples. Homogenized biopsy samples were loaded into capillary tubes and incubated for 48 h at 37 °C without providing a microaerophilic environment. Additionally, three or four loops of the homogenized sample were inoculated in a ready-to-use selective medium (Becton Dickinson, Helicobacter Agar, Modified) specific for the isolation of H. pylori and incubated at 37 °C in a microaerophilic atmosphere provided by CampyGen (Becton Dickinson, GasPack). Bacteria reproducing in microcapillary tubes were evaluated in an inverted microscope and also were evaluated after performing a CC with the content. Results obtained by CC, CLO test, and HE were compared with those of MC. The diagnostic performances of the methods used in this study were evaluated for specificity, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and CI. RESULTS H. pylori was found positive by CLO test + HE and/or CC culture in 26 patient antrum and corpus biopsy samples. In 25 (25/26) patient biopsy samples, H. pylori was isolated by MCM, whereas in only 14 (14/26) patient biopsy samples, H. pylori was isolated by CC. CLO test and HE were found positive in 17 (17/26) patient biopsy samples. Comparing the results of the isolation of H. pylori by MCM, CC, CLO test, and HE, the sensitivity of the MCM was found as 96%, the specificity as 80%, the PPV as 83%, the NPV as 95%, and the 95%CI as 0.76 (χ (2) = 31.51, P < 0.01) whereas the sensitivity of the CC was found as 54% (χ (2) = 19.15, P < 0.01), and the sensitivity of the CLO test and HE were found as 65% (χ (2) = 25.26, P < 0.01). CONCLUSION This new microcapillary cultivation method for H. pylori has high diagnostic sensitivity compared with CC, HE, and CLO tests.

The cytokine response in THP-1 (monocyte) and HL-60 (neutrophil-differentiated) cells infected with different genotypes of Helicobacter pylori strains.

BACKGROUND/AIMS Helicobacter pylori (H. pylori) is a microaerophilic bacterium related with peptic ulcer and gastric cancer. Its virulence factors include cytotoxin-associated gene A (CagA) and vacuolating cytotoxin gene A (VacA) proteins. Cytokine release inducted by H. pylori colonization has an important role in pathogenesis of H. pylori. The severity of gastric pathologies depends on the H. pylori genotypes found in different geographical regions. We aimed to determine the relationship between different H. pylori genotypes and their effects on the cytokine release levels. MATERIALS AND METHODS ureC, cagA, vacAs1/s2, vacAm1/m2, and blood group antigen-binding adhesion protein A2 (babA2) virulence related genes were investigated in 21 H. pylori strains. Genotyping of 21 strains were made due to the presence of cagA, vacAs1/s2, vacAm1/m2, and babA2 genes. The H. pylori strains were cultured together with THP-1 and neutrophil-differentiated Human promyelocytic leukemia cells (HL-60) cells. The levels of cytokines interleukin (IL)-1β, IL-6, IL-8, IL-12, tumor necrosis factor-alpha (TNF-α), and IL-10 in these cells were measured after co-culturing with H. pylori strains. RESULTS The following five different genotypes were detected: Genotype1: cagA and vacAs1m2; Genotype2: cagA and vacAs1m1; Genotype3: cagA, vacAs1m2, and babA2; Genotype4: vacAs2m2; and Genotype5: cagA and vacAs2m2. All these genotypes significantly induced the levels of IL-1β, IL-6, IL-8, IL 10, and TNF-α in THP-1 cells. Genotype 5 caused higher amounts of IL-1β, IL-6, TNF-α, and IL-10, whereas genotype 1 induced the highest levels of IL-8. In neutrophil-differentiated HL-60 cells, genotype 4 increased IL-6 levels and genotype 3 and 4 elevated IL-8 levels significantly. CONCLUSION These results suggested that cytokine response of the host varies depending on the specific immune response of the host against different H. pylori strains.

A contribution to the Torrey et al.'s “cat ownershipinchildhood is a significant risk factor for laterdeveloping schizophrenia” study

Torrey et al. (2015) recently published a third study reporting that cat ownership in childhood is a significant risk factor for later developing schizophrenia. In their paper they urged others “to collect data on cat and pet ownership” to try and replicate the findings from their three studies. Apparently unknown to Torrey et al., we have published such a study (Yuksel et al., 2010). As shown in Table 1 and reported in our paper: “The prevalence of close cat contact in the individuals with schizophrenia (59%) compared to the patient controls (6%) or healthy controls [blood donors] (9%) is one of the most striking findings from this study (p b .0001).” However, when we included the toxoplasma IgG for each patient with schizophrenia in amultivariate logistic regression along with age, gender, education and cat contact, the results suggested that “toxoplasmosis has no effect on the risk of schizophrenia in Turkey.” Thus, the strong relationship between cat ownership in childhood and later schizophrenia may be caused by another infectious agent carried by cats.

Neopterin and Soluble CD14 Levels as Indicators of Immune Activation in Cases with Indeterminate Pattern and True Positive HIV-1 Infection

Background We aimed to evaluate the roles of the plasma immune activation biomarkers neopterin and soluble CD14 (sCD14) in the indirect assessment of the immune activation status of patients with the indeterminate HIV-1 (IHIV-1) pattern and a true HIV-1-positive infection (PCG). Methods This cross-sectional and descriptive study included eighty-eight patients with the IHIV-1 pattern, 100 patients in the PCG, and 100 people in a healthy control group (HCG). Neopterin and sCD14 levels were determined by competitive and sandwich ELISA methods, respectively. Results Mean neopterin and sCD14 levels among those with the IHIV-1 pattern were significantly lower than among the PCG (p < 0.001 and p = 0.001, respectively), but they were similiar to those in the HCG (p = 0.57 and p = 0.66, respectively. Mean neopterin and sCD14 levels among the PCG were found to be significantly higher than among those with the IHIV-1 pattern (p < 0.001 and p = 0.001, respectively) and among those in the HCG (p = 0.001, p < 0.001, respectively). Neopterin did not have adequate predictive value for identifying those in the PCG (area under the curve [AUC] = 0.534; 95% CI, 0.463–0.605; p = 0.4256); sCD14 also had poor predictive value but high specificity (100%) for identifying those in the PCG (AUC = 0.627; 95% CI, 0.556–0.694; p = 0.0036). Conclusions While low levels of these two biomarkers were detected among those with the IHIV-1 pattern, they were found in high levels among those in the PCG. These two markers obviously cannot be used as a sceening test because they have low sensitivies. Taken together, we suggest that neopterin and sCD14 may be helpful because they both have high specificity (92%-100%) as indirect non-specific markers for predicting the immune activation status of individuals, whether or not they have true positive HIV-1.

Patterns of EPIYA motifs among cagA-positive Helicobacter pylori strains: a case–control study in a Turkish population with Eurasian geographical features

Geographical variation in the frequency of various gastroduodenal pathologies was shown to be related to the geographical diversity of H. pylori CagA Glu-Pro-Ile-Tyr-Ala (EPIYA) patterns. We examined the EPIYA patterns of H. pylori and the association of EPIYA patterns with gastric cancer (GC) for the first time, to the best of our knowledge, in Turkey. The patient group (PG) contained 60 patients [38 GC and 22 duodenal ulcer (DU) patients]. The control group (CG) was 110 individuals [94 gastritis patients and 16 persons with a normal gastrointestinal system (NGIS)]. Specific primers were used for the detection of cagA including empty-site-positive and EPIYA-A, -B, -C, -D PCR. Bands of EPIYA-A, -B, -C were confirmed by DNA sequencing. One hundred and forty-two (83.5 %) strains [60 in the PG (38 GC, 22 DU), 82 in the CG (72 gastritis, 10 NGIS)] were positive for the cagA gene. EPIYA-C with multiple repeats was detected in 34 (23.9 %) strains, and 22 (64.7 %) were from GC patients. EPIYA-C with one repeat was detected in 89 (62.7 %) strains, and 54 (60.7 %) were from gastritis patients. EPIYT was detected in 10 strains, and EPIYA-D was not detected. The number of EPIYA-C with multiple repeats was significantly higher for the PG than for the CG (P < 0.0001). In GC patients, the number of EPIYA-C with multiple repeats was significantly higher than one repeat (P < 0.0001). In conclusion, our study showed that multiple EPIYA-C repeats increases the GC risk by 30.6-fold and the DU risk by 8.9-fold versus the CG. This indicates that Western-type H. pylori strains in Turkey have similar EPIYA motifs to those of neighbouring countries and Western populations.

Diagnostic performance of the RT-qPCR method targeting 85B mRNA in the diagnosis of pulmonary Mycobacterium tuberculosis infection

BACKGROUND Several nucleic acid amplification techniques (IS6110, 16S rRNA, and 85B mRNA) were developed for the rapid, direct detection of Mycobacterium tuberculosis. We aimed to assess the diagnostic performance of 85B mRNA-based RT-qPCR by comparing with the real-time PCR COBAS TaqMan MTB Kit while using the BACTEC MGIT 960 method as the gold standard. METHODS 60 patients with confirmed pulmonary TB and 60 individuals without TB were included as the study and control groups, respectively. Sputum specimens were cultured using LJ and BACTEC MGIT 960 systems. Extracted DNA was used for COBAS PCR in a CONAS TaqMan 48 analyzer. 85B mRNA detection was performed by RT-qPCR. RESULTS The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of COBAS TaqMan MTB Test were detected as 93.3%, 83.3%, 84.8%, 92.6%, and 88.3%, respectively. The same diagnostic parameters of RT-qPCR were: 98.3%, 95.0%, 95.2%, 98.3%, and 96.7%, respectively. According to the binary logistic regression analysis, RT-qPCR (OR: 19,924, p<0.001) was identified as the more optimal test. CONCLUSION RT-qPCR targeting the 85B gene of M. tuberculosis seems to be a more useful and rapid technique than DNA-based methods for detecting live M. tuberculosis bacilli from sputum specimens.