Tevhide Ziver

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.

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.

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.

Problems encountered in conventional HIV 1/2 Algorithms: lack of necessity for immunoblot assays to confirm repeated ELISA reactive results

Background The use of conventional (serologically based) HIV 1/2 diagnostic algorithms has become controversial in recent years. Objectives Sera from patients who underwent verification tests were evaluated because repeated ELISA-reactive results demonstrated a HIV1+HIV2 positive band pattern. Methods The line immunoassay (LIA) test was used for repeated HIV enzyme immunoassays (EIA)-reactive sera in patients at three centers. The Bio-Rad Geenius™ HIV 1/2 and the HIV-1 RNA tests were used. HIV-1 and RNA HIV-2 were investigated using PCR. Results LIA was used to evaluate 3,224 out of 10,591 samples with repeated ELISA reactivity (30%). We found that 32 (1%) of the sera, along with HIV1 bands and HIV2 gp36 bands, were positive. Only 28 of the 32 verified serum samples with gp36 bands were repeated, and no gp36 band positivity was detected using the Bio-Rad Geenius™ HIV-1/2 confirmatory assay in these serum samples. The HIV-2 proviral DNAs were also negative. Therefore, we excluded the possibility of HIV1+2 co-infection. All samples from the 32 patients were positive for HIV-1 RNA. Conclusion Our findings highlight the need to exclude confirmatory tests like the LIA test from the current diagnostic HIV algorithm and replace it with rapid HIV-1 and HIV-2 confirmatory immunochromotographic tests.

The Prevalence of Cyclospora cayetanensis and Cryptosporidium spp. in Turkish patients infected with HIV-1

Opportunistic infections such as cryptosporidiosis and cyclosporiasis are commonly encountered in patients with acquired immunodeficiency syndrome (AIDS).We investigated the existence of opportunistic protozoans that significantly affect the quality of life in HIV-1 infected patients using conventional and molecular methods. The study group comprised 115 HIV-1 positive patients. In the identification of Cyclospora cayetanensis and Cryptosporidium, the formol-ether precipitation method was used and smears were evaluated in optical microscope by staining modified Ziehl-Neelsen (ZN). The primers and probes used for PCR were Heat shock protein 70 for C. cayetanensis and the oocysts wall protein for Cryptosporidium spp.. Cyclospora and Cryptosporidium spp. oocysts were detected in one and two patients, respectively, by staining, whereas we detected C. cayetanensis in three patients out of 115 (2.6%) by PCR, and Cryptosporidium spp. in a further three patients (2.6%). C. cayetensis was detected in patients with CD4 counts of 64 cells/μm, 182 cells/μm and 287 cells/μm, respectively. Cryptosporidium spp. was detected in patients with CD4 counts of 176 cells/μm, 241 cells/μm and 669 cells/μm. As conclusion, PCR method is faster and more sensitive than microscopic methods and to screen intestinal pathogens routinely in patients infected with HIV should not be neglected in developing countries like Turkey.

Relationship between aneurysm and microorganism: Is Helicobacter pylori a primer agent or has an affinity to the tissue?

1 Department of Medical Microbiology, Cerrahpasa Faculty of Medicine, Istanbul University, Istanbul, Turkey. 2 Thoracic and Cardiovascular Surgery Training and Research Hospital, Istanbul, Turkey. 3 Medical Faculty, Sifa University, Izmir, Turkey. 4 Faculty of Engineering, Koc University, Istanbul, Turkey. 5 Department of Microbiology, Forensic Sciences Institute, Istanbul University, Istanbul, Turkey.

Entamoeba histolytica Lectin Antigen from Stool Specimens by ELISA Method: 3-Year Data

Amaç: Bu çalışmada Ocak 2007-Aralık 2009 tarihleri arasında Fakültemiz Seroloji/ELISA Laboratuvarı’na, gastroenterit klinik bulgularıyla dışkı örneği gönderilen ve amip antijen testi istenen olgularda antijen pozitifliğinin dağılımı ve demografik verilerle ilişkisinin retrospektif olarak değerlendirilmesi amaçlanmıştır. Yöntemler: Çeşitli poliklinik ve servislerden, Seroloji/ELISA Laboratuvarı’na gönderilen dışkı örneklerinden ELISA yöntemi (Ridascreen Entamoeba [c1701], R-Biopharm, Almanya) ile 260 kd’luk Entamoeba histolytica Galveya GalNAc-spesifik lektin antijeni araştırılmıştır. Bulgular: Antijen testi istenen 476 dışkı örneğinin 33’ünde (%7) amip antijeni pozitif bulunmuştur. Bu olguların 11’inin (%33) 4150 yaş grubundan olduğu belirlenmiştir. Antijen pozitifliği en fazla 14 (%42) olguda sonbahar aylarında saptanmıştır. Sonuçlar: Direkt mikroskopinin duyarlılığının düşük olmasından dolayı, amebiyaz şüphesi olan hastalarda tanıyı doğrulamak için, ELISA ile antijen tayini yönteminin kullanılması, verilecek tedavinin belirlenmesi veya gereksiz tedavinin önlenmesi açısından uygun olacaktır. Klimik Dergisi 2011; 24(3): 150-3.

The diagnostic role of indirect fluorescence antibody in csytic echinococcus and the role of western blot in following-up patients with csytic echinococcus after surgery

Cystic echinococcosis (CE) is a complicated zoonotic infection with diagnosis and treatment. In this study, we aimed to determine the diagnostic performance parameters of Indirect Fluorescence Antibody (IFA) method and also the usability of Western Blot (WB) method for following up CE cases by detecting the antigenic patterns. Cases diagnosed as CE in General Surgery Department of Cerahpasa Faculty of medicine and a selected control group were included in this case-control and cross-sectional study between January 2010 and December 2010. Laboratory studies were performed in Serology/ELISA laboratory of Medical Microbiology Department. Clinically, radiologically and serologically (IHA, ELISA) diagnosed 110 patients with CE, and 80 healthy control group (HCG) individuals were included in the study. Echinococcus granulosus specific antigen IgG test was applied by IFA method in CE cases and HCG. E. granulosus specific antigen patterns were also are detected with WB method in CE cases and HCG. According to the results, performance parameters of IFA test in CE diagnosis were calculated as 100, 93, 95, 100 and 94% for sensitivity, specificity, positive predictive value, negative predictive value and Kappa values, respectively. Ninety six cases were detected positive with WB method. p7 and other accompanying bands were detected in 50 out of this 96 cases and only p39 band was detected in 45 cases. In conclusion, using IFA method in the diagnosis of CE which is a complicated infection disease can be effective in the presence of an appropriate microscope and experienced expert and we also suggest the use of WB method can be useful especially in parasitic treatment or after spontaneous cyst calcification in post-operative period.

Confirmatory Test Results in Anti-HIV-Positive Patients: Evaluation of Five-Year Data

Amaç: Seroloji/ELISA Laboratuvarına HIV infeksiyonu kuşkusuyla ya da kan donörü taraması veya ameliyat öncesi serolojik tarama amacıyla anti-HIV-1/2 testi yaptırmak üzere başvuran ya da kanları gönderilen olguların, ELISA ve Western-Blot (WB) test sonuçlarını ve özellikle HIV’e spesifi k seropozitif band dağılımlarını retrospektif olarak irdelemeyi amaçladık. Yöntemler: Anti-HIV-1/2 antikorları rutin olarak ELISA yöntemiyle ve ikinci bir yöntem olarak mikropartikül enzim immünoessey (MEIA) ile araştırılmıştır. Anti-HIV-pozitif bulunan serumların doğrulaması WB yöntemiyle yapılmıştır. Bulgular: Retrospektif olarak beş yıllık dönemde incelenen 85 881 olgunun 84 164 (%98)’ünü, kan donörü ve ameliyat öncesi rutin serolojik inceleme istenenler; 1717 (%2)’sini ise HIV infeksiyonu kuşkusu olanlar oluşturmaktadır. Kan donörü olan ve ameliyat öncesi taranan 84 164 kişinin 5 (%0.006)’i anti-HIV-1/2 pozitif, bu beş seropozitif hastanın da biri WB pozitif bulunmuştur. HIV infeksiyonu kuşkulu 1717 kişinin 72 (%4.2)’sinde anti-HIV-1/2 seropozitifl iği belirlenirken, WB bu 72 kişinin 56’sında HIV-1 ile ilişkili, birinde ise HIV-2 yönünden pozitif olarak belirlenmiştir. Sonuçlar: Merkezimizden beş yıllık dönemde retrospektif olarak elde ettiğimiz anti-HIV-1/2 ve WB seroprevalans sonuçları, ülkemizden bildirilen diğer çalışmalarınkine yakın oranlarda bulunmuş; HIV’le ilişkili bulaşma yollarının dağılımı da dünyadan ve ülkemizden bildirilenlerinkine uyum göstermiştir. Bu retrospektif değerlendirmede ülkemizde bildirimi nadir olan bir HIV-2 olgusunun da yer alması, özellikle küreselleşmenin yansımalarından dolayı HIV-1 pandemisinin yanı sıra HIV-2 infeksiyonlarına karşı da dikkatli olunmasını işaret etmektedir. Klimik Dergisi 2010; 23(2): 51-4.