Selda Erensoy

Electrochemical DNA biosensor for the detection of TT and Hepatitis B virus from PCR amplified real samples by using methylene blue.

DNA biosensors based on nucleic acid hybridization processes are rapidly being developed towards the goal of rapid and inexpensive diagnosis of genetic and infectious diseases. Electrochemical transducers are often being used for detecting the DNA hybridization event, due to their high sensitivity, small dimensions, low cost, and compatibility with microfabrication technology. In this study, an electrochemical biosensor for the voltammetric detection of DNA sequences related to the Hepatitis B virus (HBV) and TT virus (TTV) from polymerase chain reaction (PCR) amplified real samples is described for the first time. The biosensor relies on the immobilization of the 21- or 24-mer single stranded oligonucleotides (probe) related to the HBV and TTV sequences and hybridization of these oligonucleotides with their complementary sequences (target) at carbon paste electrode (CPE). The extent of hybridization between the probe and target sequences was determined by using square wave voltammetry (SWV) with moving average baseline correction and methylene blue (MB) as the hybridization indicator. As a result of the interaction between MB and the bound guanine bases of hybrid at CPE surface, the MB signal decreased, when it was compared with the MB signal, which was observed with probe modified CPE. The difference between the MB signals, obtained from the hybrid modified and the probe modified CPE is used to detect the DNA sequences of the infectious diseases from PCR amplified real samples. Numerous factors affecting the target hybridization and indicator binding reactions are optimized to maximize the sensitivity.

Diagnosis of hepatitis C virus (HCV) infection and laboratory monitoring of its therapy

BACKGROUND Just after the identification and characterization of hepatitis C virus (HCV) in 1989, tests for the detection of HCV antibodies or HCV RNA in serum were developed. The enzyme-linked immunosorbent assays (ELISAs) and confirmatory/supplemental analytical antibody tests were improved in sensitivity and specificity with the development of further generations of these assays. Application of molecular tests for detecting, quantifying, and characterization of the infecting virus became very important in management of HCV infection. OBJECTIVE AND DESIGN This review summarizes the assays developed for the diagnosis and management of HCV infection. Strategies for the diagnosis and monitoring with the advantages and disadvantages of the assays based on the setting and goal are discussed according to data in the literature and our experience. RESULTS Specific laboratory diagnostic tests for hepatitis C virus infection may be discussed under two titles: (i) Serological antibody tests which detect anti-HCV in serum or plasma; (ii) Molecular tests which detect HCV RNA genome, investigate viral load, and determine the characteristics of the genome. Strategies in different laboratory settings which screen populations with different HCV prevalences vary. CONCLUSIONS Anti-HCV positive result in a low-risk setting such as blood banks should be confirmed with an analytical antibody test. Then a HCV RNA test should be performed on serum of the person with a positive or indeterminate confirmatory test result. On the contrary, anti-HCV positive test result in high-risk population or a situation where HCV infection is suspected, it is likely to be true positive and confirmation with HCV RNA test will be significant. Quantitative HCV RNA test and genotyping should be performed if therapy is considered.

Low-dose intradermal versus intramuscular administration of recombinant hepatitis B vaccine: a comparison of immunogenicity in infants and preschool children

Two hundred infants and two hundred preschool children were randomly assigned to receive either 10 micrograms of recombinant hepatitis B vaccine (GenHevac B) intramuscularly (i.m.) or 2 micrograms intradermally (ID) in the deltoid region at 0, 1 and 6 months. Antibody to hepatitis B surface antigen (anti-HBs) was tested eight weeks after the third vaccine dose. Standard dose i.m. and low-dose ID administration of recombinant hepatitis B vaccine produced comparable rates of anti-HBs equal to or higher than 10 mIU ml-1 in infants (98% and 94%, respectively) and preschool children (98% and 100%, respectively). Although i.m. vaccination produced higher anti-HBs concentrations than ID vaccination both in infants (geometric mean titre-GMT, 935 versus 621 mIU ml-1) and preschool children (GMT, 1393 versus 804 mIU ml-1), the differences were not statistically significant (p > 0.05). The preschool children tended to have higher anti-HBs concentrations than the infants. No clinically serious adverse effects were observed in both vaccine groups; however, induration and hyperpigmentation at the injection site were more often seen in the study population that was vaccinated intradermally. We conclude that intradermal administration of 2 micrograms recombinant hepatitis B vaccine is safe and effective in infants and preschool children, and may be an acceptable, less expensive alternative to full-dose i.m. vaccination for mass immunization, especially in developing countries.

TT Virus Infection and Genotype Distribution in Blood Donors and a Group of Patients from Turkey

Abstract.Background: TT virus (TTV) DNA has been found in a large proportion of patients with different forms of non-A-G hepatitis, however the clinical importance is unclear. We aimed to determine the genotypes of TTV isolates foung in blood donors and different patient groups from the western part of Turkey. Materials and Methods: TT DNA was investigated in serum samples of 91 volunteer blood donors (BD), 105 thalassemia (TH) patients, ten patients with fulminant hepatitis (FH) and 16 hemodialysis (HD) patients by heminested PCR using primers NG059, NG061 and NG063 from the ORF1 region. 39 isolates were genotyped by analyzing the partial sequence of ORF1. Results: TTV DNA was found in 75% of HD, 80% of FH, 61% of TH patients and in 51.6% of BD. Among the sequenced isolates, 14 (35.9%) belonged to genotype 1 (G1) and 25 (64.1%) belonged to genotype 2 (G2). Among the G2 sequences, 22 were grouped as G2c. Conclusion: TTV infection was common in the population studied, even with moderately sensitive primers. G2 was the major genotype of the studied population without any significant differences in distribution between various patient groups and BD.

Low-dose intradermal administration of recombinant hepatitis B vaccine in children: 5-year follow-up study.

Several studies have documented the efficacy of low-dose intradermal administration of hepatitis B vaccine. However, little is known about the duration of protection provided by low-dose intradermal administration of hepatitis B vaccine. This study reports results from a 5-year follow up period of 200 healthy children (100 infants and 100 preschool children) immunized intradermally with 2 microg doses of recombinant hepatitis B vaccine (GenHevac B) at months 0,1, and 6. In the 8th week after the third vaccine dose, 97% of the children developed anti-HBs antibodies higher than or equal to 10 mlU ml(-1), and the antiHBs geometric mean titre (GMT) was 676 mlU ml(-1). In month 18 and year 5, the anti-HBs GMT decreased to approximately one-third (220 mlU ml(-1)) and one-tenth (68 mlU ml(-1)) of the initial levels, respectively. However, 87% of the children had protective levels of anti-HBs (> or =10 mlU ml(-1)) after 5 years. Among 156 children followed for 5 years, none became positive for anti-HBc and/or HbsAg. Seven children who were seronegative after 5 years developed anti-HBs antibodies higher than 1000 mlU ml(-1) after an additional 10 microg intramuscular hepatitis B vaccine. Persistent immunologic memory over periods of 5 years or more is evident, the anamnestic antibody response to a booster dose of vaccine, even in these children who have lost antibody. We conclude that intradermal administration of 2 microg recombinant hepatitis B vaccine provides long-term protection against hepatitis B virus in infants and preschool children.

Investigation of herpes simplex virus DNA in pityriasis rosea by polymerase chain reaction

Background  Pityriasis rosea (PR) is an acute, inflammatory disease of unknown cause. Clinical and experimental findings indicate an infectious etiology of PR. Our purpose is to examine the skin lesions and blood samples of PR patients by polymerase chain reaction (PCR) for the presence of HSV type 1 and 2 DNA.

Screening for human immunodeficiency virus type 1 and 2 in a Turkish blood donor population

OBJECTIVES To determine the prevalence of human immunodeficiency virus-1 and -2 infection in voluntary blood donors at a university hospital in the third largest city of Turkey and to evaluate the HIV testing strategy for notifying blood donors. METHODS Between July 1995 and August 1997, 36,373 voluntary blood donors who met the criteria for donating blood were tested for the presence of HIV-1 and -2 antibodies by using an automated enzyme-linked fluorescent immunoassay. Repeatedly reactive samples were subjected to a different enzyme-linked immunosorbent assay (ELISA) and a line immunoassay (LIA) for the detection of antibodies. RESULTS Of the 36,373 samples tested 72 were found to be repeatedly reactive or borderline by the first screening enzyme immunoassay (EIA). None of the 72 samples was reactive by the second EIA. These samples were further tested by LIA: 64 were negative on the line immunoassay and 8 were indeterminate. Three of eight donors who had indeterminate results by LIA were tested for HIV-1 DNA by polymerase chain reaction (PCR) and were found to be negative. One additional donor with an indeterminate LIA was found to be negative by EIA and LIA during the 6-month follow-up period. CONCLUSION Donor questioning, repeat EIA testing, LIA testing, and HIV-1 DNA analysis did not confirm evidence for HIV infection among this blood donor population. Blood donor notification of test results according to the World Health Organization (WHO) strategy III was found to be an appropriate approach.

Comparison of different polymerase chain reaction methods for detection of herpes simplex virus types 1 and 2 encephalitis

Herpes simplex virus (HSV) has been recognized as a relatively common pathogen affecting the central nervous system because of its neurotropic nature. HSV types 1 (HSV-1) and 2 (HSV-2) can cause life-threatening infections of the central nervous system. HSV encephalitis has been reported to be mainly due to HSV-1, whereas most cases of aseptic meningitis are due to HSV-2 [1]. The main reasons for identifying the causative organisms in cases of aseptic meningitis and encephalitis are (a) to provide a rational basis for chemotherapy and prognosis and (b) to limit the number of unnecessary investigations [2]. Rapid detection of HSV DNA in clinical samples can be critical for the diagnosis of HSV-mediated etiologies and may allow early initiation of effective patient management with acyclovir [3]. Following the introduction of molecular biological methods into virological diagnostics, polymerase chain reaction (PCR) has become the method of choice for the laboratory diagnosis of herpes simplex encephalitis (HSE) [4]. The aim of our study was to compare the performance of our in-house HSV PCR method with that of the commercially available LightCycler (LC) HIV 1/2 realtime PCR assay (Roche Diagnostics, Indianapolis, IN, USA) and the RoboGene HSV-1 and RoboGene HSV-2 detection kits (Roboscreen, Leipzig, Germany). Thirty cerebrospinal fluid (CSF) samples that were sent to the virology laboratory for HSV DNA testing were included in the study. In addition to these clinical specimens, nine lyophilized samples from the 2004 Herpes Simplex Virus Proficiency Programme of the Quality Control for Molecular Diagnostics (QCMD, Glasgow, Scotland) were tested. The proficiency panel consisted of lyophilized samples that were positive for HSV at a range of concentrations (from 2.4×10 to 1.2×10 Geq/ml) and samples that were negative for HSV. DNA was extracted from CSF samples using a commercial extraction kit (RTP DNA/RNAVirus Mini Kit; Invitek, Berlin, Germany). The in-house PCR used in our laboratory is based on amplification of the 148-bp region of the glycoprotein B (gB) gene using the sense primer (nt 1359– 1378) 5′-GCA TCG TCG AGG AGG TGG AC, the antisense primer (nt 1487–1507) 5′-TTG AAG CGG TCG GCG GCG TA-3′ [5], and gB prob biotin-5′ GGC GAC TTT GTG TAC ATG TCC CCG TTT TAC GGC TAC CGG-3′. For detecting the products amplified by the inhouse gB PCR, the dig-detection kit (Roche Diagnostics) was used. When a CSF sample was gB PCR positive for differentiating HSV-1 and HSV-2 by sequencing, a second amplification was carried out. This second PCR amplified a 179-bp fragment of the HSV polymerase (pol) gene using primers 5′-ATC AAC TTC GAC TGG CCC TTC-3′ and 5′CCG TAC ATG TCG ATG TTC ACC-3′. The Big Dye Terminator DNA sequencing kit (Applied Biosystems, Foster City, CA, USA) was used with the ABI Prism 310 Genetic Analyser (Perkin Elmer, Boston, MA, USA) for sequencing the pol gene. Sequences obtained from the positive samples were compared with the sequences representing HSV-1 and -2 in the database of GenBank/ EMBL/DDJB (accession numbers M16321 and M12356). Eur J Clin Microbiol Infect Dis (2006) 25:669–671 DOI 10.1007/s10096-006-0202-3

The association between Cytomegalovirus co-infection with Pneumocystis pneumonia and mortality in immunocompromised non-HIV patients

Impact of Cytomegalovirus (CMV) co‐infection pneumonia in non‐HIV patients with Pneumocystis jirovecii pneumonia (PCP) is unclear.

Laboratuvar tasarımı bir HBV DNA kantitasyon protokolü rutin kullanıma uygun olabilir mi? – Ege Üniversitesi Klinik Viroloji Laboratuvarı deneyimi

Amac: Hepatoselluler karsinoma ve fatal karaciger hasarina yol acmasi nedeniyle ulkemizde de onemli bir halk sagligi sorunu olan kronik Hepatit B enfeksiyonunun izleminde Hepatit B virus (HBV) DNA kantitasyonu onemli bir gostergedir. Klinik viroloji laboratuvarlarinda HBV DNA kantitasyonu icin kullanilan bircok farkli yontem ve sistemler mevcuttur. Bu calismanin amaci laboratuvar tasarimi gercek zamanli bir PCR protokolun Ege Universitesi Tip Fakultesi Hastanesi Tibbi Mikrobiyoloji Anabilim Dali Viroloji/Molekuler Biyoloji laboratuvarinda ABI Prism 7500 (PE Biosystems) icin uygunlugunun degerlendirilmesi ve rutin kullanilan HBV DNA test COBAS AmpliPrep-COBAS TaqMan 48 (CAP-CTM; Roche, Branchburg, NJ) HBV DNA testi ile karsilastirilmasidir. Gerec ve Yontem: HBV DNA saptanmasina yonelik gelistirilmis, laboratuvar tasarimi, Taqman teknolojisine dayanan, bir gercek zamanli test Ege Universitesi Tip Fakultesi Hastanesi Tibbi Mikrobiyoloji Anabilim Dali Viroloji/Molekuler Biyoloji laboratuvarinda denendi. Rutin HBV DNA kantitasyonu amaciyla gonderilen 332 ornek calismaya alindi ve kullanilmakta olan HBV DNA test COBAS AmpliPrep-COBAS TaqMan 48 HBV DNA testi ile karsilastirildi. Bulgular: 332 ornegin 176sinin sonuclari protokolun dinamik araligi icinde idi. Dinamik aralik icinde olan 176 ornegin kantitatif sonuclari uyumlu idi. 106 ornek her iki sistem ile negatif bulundu. Sonuc: Laboratuvar tasarimli HBV DNA kantitatif protokolunun klinik viroloji laboratuvarinda uygulanabilir, rutin tani ve klinik pratikte HBV ile infekte hastalarin izlenmesi icin uygun, ucuz, genis dinamik aralik saglayan bir yontem oldugu sonucuna varildi.