A. Arzu Sayiner

YMDD motif variants in inactive hepatitis B carriers detected by Inno-Lipa HBV DR assay

Introduction:  Mutations of hepatitis B virus (HBV) polymerase, especially occurring at the highly conserved YMDD region, are related to resistance to lamivudine. Although these mutations are frequently secondary to lamivudine use, they can also occur naturally. The aim of the present study was to determine the prevalence of YMDD variants that exist naturally in patients who are inactive HBV carriers.

Naturally occurring MHR variants in Turkish patients infected with hepatitis B virus.

Major B‐cell epitopes are located at the major hydrophilic region (MHR) of hepatitis B virus (HBV) surface antigen (HBsAg). The genotypes, subtypes, and naturally occurring amino acid (aa) substitutions of MHR were analyzed in 81 Turkish adult patients (41 inactive HBsAg carriers and 40 patients with chronic hepatitis B) by direct sequencing of the S gene fragment. All the isolates were genotype D according to the phylogenetic analysis. The most common HBsAg subtype was ayw2, followed by ayw3 while one isolate specified ayw4 by encoding Leu127. MHR variants were detected in 22 of the 81 (27.2%) isolates. The prevalence was significantly higher in the chronic hepatitis B group (42.5%) compared to inactive HBsAg carriers (12.2%). Twenty‐two samples had a total of 26 amino acid substitutions involving 14 positions. The majority of the patients had a single variation. Most of the amino acid substitutions were located at the HBs1 region of the MHR, while 9 of the 26 were in the classic “a” determinant (aa 124–147). When samples with “a” variants were evaluated by two different commercial HBsAg tests, only the isolate with Ser143Leu variation had a decreased reactivity in the assay using monoclonal antibodies for capture and detection. In conclusion, the findings of the study was in accordance with previous studies showing HBV genotype and subtype homogeneity (genotype D/ayw) in Turkey. Naturally occurring MHR and “a” determinant variants were common, especially among chronic hepatitis B patients. The influence of detected “a” variants on diagnostic assays was limited. J. Med. Virol. 80:405–410, 2008.

A nucleic acid-based electrochemical biosensor for the detection of influenza B virus from PCR samples using gold nanoparticle-adsorbed disposable graphite electrode and Meldola's blue as an intercalator†

In the presented study, a novel method is introduced that demonstrates the electrochemical detection of influenza B virus based on DNA hybridisation. The detection utilised gold nanoparticles (AuNPs) and Meldolas Blue (MDB), which is utilised as an intercalator label. The developed methodology, combined with a disposable pencil graphite electrode (PGE) and differential pulse voltammetry (DPV), was performed using both synthetic oligonucleotides and polymerase chain reaction (PCR) amplicons. The electrochemical oxidation response of guanine (approximately +0.1 V) and the voltammetric reduction signal of MDB (approximately −0.2 V) were measured before and after hybridisation reactions between a single strand DNA probe and its complementary target strain (synthetic target or denatured PCR samples). Before the immobilisation of the synthetic DNA probe of influenza type B virus, the transducer surface was interacted with AuNPs solution using a simple wet adsorption method. AuNP immobilisation was confirmed with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) to characterise the recognition surface of the genosensor. After the interaction between the PGE and AuNPs, a thiol-linked DNA probe was immobilised onto the nanoparticle-covered surface. When hybridisation occurred between the probe and its synthetic targets or specific PCR products, the highest MDB signal was observed. The probes were also challenged with equal quantities of non-complementary DNA at the PGE surface for the determination of biosensor selectivity. AuNP-coated electrodes showed high sensitivity and selectivity, specifically in real samples for the detection of the hybridisation reaction. The results obtained in the presented study indicated that the electrode surface area could be enhanced with AuNPs. The detection limit of the genosensor was found to be 54 picomoles for the synthetic target and 3.3 × 107 molecules for the real samples (PCR) in 30 μL of sample volume. Future prospects and analytical performance of the sensor is briefly discussed.

Multicentre evaluation of central nervous system infections due to Flavi and Phleboviruses in Turkey.

OBJECTIVES Flavi- and Phleboviruses associated with central nervous system (CNS) infections including West Nile Virus (WNV), Tick-borne Encephalitis Virus (TBEV) and Toscana Virus (TOSV) cause significant morbidity and mortality in humans. In this study, the impact of these agents have been investigated in CNS infections at referral hospitals in two provinces in Turkey, where circulation of these viruses have previously been recognized. METHODS In the study, 258 samples from 126 individuals from Ankara and 113 samples from 108 individuals from Izmir provinces collected in 2010 were included. Viral RNAs were investigated by multiple genus and strain specific primers. Commercial serological assays were employed in screening and reactive results were evaluated with additional assays and by plaque reduction neutralization assay. RESULTS Two cases of WNV CNS infections, 14 cases of TOSV infections and one TBEV-exposed individual were identified via serological testing. WNV infections in 61 and 56-year old individuals from Ankara presented with fever and encephalitis without skin rash and residual neurologic damage. TOSV-associated cases from both provinces mainly displayed signs of meningitis. TOSV exposure was documented for the first time from Izmir. CONCLUSIONS WNV, TBEV and TOSV infections must be considered in cases of meningoencephalitis of unknown etiology in Turkey.

A 5-year study of adenoviruses causing conjunctivitis in Izmir, Turkey.

Adenoviruses are a common cause of conjunctivitis. Genotypes are diverse and differ according to population and geographical distribution of the virus. There is limited data regarding ocular adenoviral infections and genotype distribution in Turkey. This study aimed to determine the adenovirus genotypes and their epidemiological features among patients with conjunctivitis between 2006 and 2010, in Izmir, Turkey. Adenoviral DNA was detected by PCR in 213 of 488 (44%) of the ocular samples collected from patients with viral conjunctivitis during the 5‐year study period. Of these, 101 (47%) were randomly chosen and genotyped by sequence analysis. Seven genotypes were identified, including 3, 4, 8, 11, 19, 37, and 53. Genotype 8 and 4 were the dominant types detected in 67 (66.3%) and 25 (24.7%) of the samples, respectively. Other five genotypes (3, 11, 19, 37, 53) were detected in 9 (8.9%) samples. Genotype and seasonal differences observed throughout the study. Human adenoviruse (HAdV)‐8 was the most frequent type, except 2008. The prevalence of genotype 4 increased starting from 2006, became dominant in 2008 and decreased in the following years. The peak season was mostly spring months, although it was possible to detect positive samples throughout the year. In conclusion, genotype 8 followed by genotype 4 was the most frequent adenoviral types causing conjunctivitis during the 5‐year study period. Findings suggest that there is a slow shift between genotypes throughout the years. J. Med. Virol. 87:472–477, 2015.

Fecal calprotectin: can be used to distinguish between bacterial and viral gastroenteritis in children?

OBJECTIVE Fecal calprotectin is used as a good indicator of intestinal mucosal inflammation. The aim of this study is to evaluate the diagnostic value of fecal calprotectin (f-CP) for the etiology of acute gastroenteritis in children. MATERIALS AND METHODS All patients presenting with acute diarrhea (<18 years) who had 3 or more soft or watery stools per day were enrolled in this study. Stool microscopic examination and cultures for bacteria and parasites were performed. Polymerase chain reaction test was also applied to stool samples for viruses (Rotavirus, Adenovirus, Norwalk, and Astrovirus). The level of f-CP was carried out by using enzyme-linked immunosorbent assay test. RESULTS Eighty-four patients with diarrhea were enrolled. The f-CP level was higher in patients with microscopic examination positive (n=17) (median with interquartile range, 1610.0 [908.8-2100] mg/L) than in patients with microscopic examination negative (n=67) (123.8 [25.0-406.3] mg/L) (P<.001). Concentrations of f-CP in patients with stool culture positive (1870.0 [822.5-2100] mg/L) were significantly elevated compared with the concentrations of the patient with virus detected in stool (95.0 [21.3-240.9] mg/L) (P<.001). In the diagnosis for bacterial acute gastroenteritis, the area under the receiver operating characteristic curve for f-CP was 0.867 (95% confidence interval, 0.763-0.971), sensitivity was 88.9%, and specificity was 76.0% if the threshold was taken as 710 mg/L. CONCLUSION We conclude that f-CP, which is useful, valuable, noninvasive, easily and rapidly measured laboratory test along with simple microscopic examination of stool, can be used as an indicator of intestinal inflammation and to distinguish the bacterial gastroenteritis from the viral gastroenteritis.

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.

EBV Serolojik Tanısında Atipik Profil Sorunu

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis. Burkitts lymphoma, nasopharyngeal carcinoma and post-transplant lymphoproliferative diseases are also associated with EBV. Diagnosis is frequently based on detection of specific antibodies. Using three parameters (anti VCA-IgM, anti VCA-IgG and anti EBNA-1 IgG), it is possible to define infection status and diagnose acute or past infection. However, sometimes the detection of atypical serological profiles makes it difficult to interpret these results. This study aims to evaluate the serological profiles of patient sera suspected of EBV infection and to determine atypical profiles. Sera of 2749 patients were analyzed between January 2014 and August 2016, in the Dokuz Eylul University Hospital Central Laboratory and evaluated retrospectively. Serum samples were tested for EBV VCA IgM and EBV VCA IgG antibodies with immunofluorescence test (Euroimmun, Germany), EBNA-1 IgG antibodies with enzyme immunoassay (Euroimmun, Germany). Medical files of the patients with two or more samples and have an atypical profile were reviewed. Patients were grouped as no EBV infection, acute infection, past infection and atypical serologic profile according to three routine laboratory assays (VCA IgG, VCA IgM and EBNA-1 IgG). Out of 2794 subjects 1334 (48.5%) were female and 1415 (51.5%) were male, with mean age 30 (< 1-89 years, median value: 27). The distribution of the results was; 72.5% past infection, 10.9% absence of EBV infection and 5.2% acute infection and 11.4% showed atypical serologic profile. Among the atypical profiles, isolated VCA-IgG positivity was the most frequent pattern detected in 7.9% which is followed by 2.7% of the cases with all three markers positive and 0.8% with isolated EBNA-1 IgG positivity. Off the patients, 72.5% were seropositive for EBV and this result is consistent with the seroprevalence studies previously conducted in Turkey. The rate of atypical profiles was 11.4% which is close to the result (15%) of another study performed in Izmir. Nearly one third of the patients with atypical serological profile had an immune disorder and it was possible to reach a conclusion only among half of the patients during serological follow-up. This study points out that clinical diagnosis and serologic follow-up is important for the interpretation of the atypical profiles.