Role of the line probe assay INNO-LiPA HBV DR and ultradeep pyrosequencing in detecting resistance mutations to nucleoside/nucleotide analogues in viral samples isolated from chronic hepatitis B patients.

Despite the effectiveness of nucleoside/nucleotide analogues in the treatment of chronic hepatitis B (CHB), their long-term administration is associated with the emergence of resistant hepatitis B virus (HBV) mutants. In this study, mutations resulting in antiviral resistance in HBV DNA samples isolated from 23 CHB patients (nine treatment naïve and 14 treated previously) were studied using a line probe assay (INNO-LiPA HBV DR; Innogenetics) and ultradeep pyrosequencing (UDPS) methods. Whilst the INNO-LiPA HBV DR showed no resistance mutations in HBV DNA samples from treatment-naive patients, mutations mediating lamivudine resistance were detected in three samples by UDPS. Among patients who were treated previously, 19 mutations were detected in eight samples using the INNO-LiPA HBV DR and 29 mutations were detected in 12 samples using UDPS. All mutations detected by the INNO-LiPA HBV DR were also detected by UDPS. There were no mutations that could be detected by INNO-LiPA HBV DR but not by UDPS. A total of ten mutations were detected by UDPS but not by INNO-LiPA HBV DR, and the mean frequency of these mutations was 14.7 %. It was concluded that, although INNO-LiPA HBV DR is a sensitive and practical method commonly used for the detection of resistance mutations in HBV infection, UDPS may significantly increase the detection rate of genotypic resistance in HBV at an early stage.

Molecular epidemiology of HIV in a cohort of men having sex with men from Istanbul.

In Turkey, the first HIV/AIDS case was reported in 1985. Since then the number of persons with HIV infection has increased, HIV is getting a public health problem. The aim of this study was to determine HIV-1 subtype diversity, drug resistance and gag cleavage site mutations among 20 HIV-infected men having sex with men from Istanbul, Turkey. The most prevalent subtype was found to be subtype B (50 %), but also the non-B subtypes A1, C and CRF02_AG, CRF03_AB and CRF06_cpx were found. Resistance-associated mutations were found in 6 patients (30 %) with 2/6 patients being therapy-experienced and 4/6 therapy-naïve at the time-point of analysis. In these patients, the nucleoside reverse transcriptase inhibitor (NRTI)-associated resistance mutations M41L, T215C, V75I, T69N, the non-NRTI associated mutations V106I, E138A, K103N and the protease inhibitor associated mutations Q58E and V82I were detected. Two virus strains also presented Gag cleavage site mutations. With increasing numbers of HIV-infected Turkish patients that require anti-retroviral treatment, HIV-1 drug-resistance testing is strongly recommended in order to choose the most active drug combination for therapy to achieve better clinical outcomes.

Sensitivity of rapid influenza antigen tests in the diagnosis of pandemic (H1N1)2009 compared with the standard rRT-PCR technique during the 2009 pandemic in Turkey.

The real-time reverse transcription polymerase chain reaction (rRT-PCR) technique has been used as the reference technique for the diagnosis of pandemic (H1N1)2009 virus infections. However, rapid influenza diagnostics tests (RIDTs) have been considered in the diagnosis of pandemic (H1N1)2009 by some healthcare institutions in Turkey due to their ease of use and generation of fast results. Nevertheless, their low sensitivity has caused concern during the control of the pandemic. This study aimed to determine the sensitivity of 4 different rapid tests available on the market in Turkey in the diagnosis of pandemic (H1N1)2009 infections compared to the reference rRT-PCR technique. One hundred and four patient samples that tested positive and 88 samples that tested negative for pandemic (H1N1)2009 by rRT-PCR were tested with RIDTs available on the market. The sensitivity of the rapid tests ranged from 31.7% to 50% depending on the brand of RIDT. Specificity ranged from 97.7% to 100%. Currently available RIDTs are not sensitive enough and could lead physicians to delay the treatment of patients, adversely affecting control efforts to mitigate the pandemic. Therefore, these tests should only be used for screening, and negative results should not rule out influenza. More sensitive and rapid point-of-care techniques are needed to meet the demands of point-of-care testing.

Surveillance and oseltamivir resistance of human influenza a virus in Turkey during the 2007-2008 season.

Monitoring the activity of influenza viruses is important for establishing the circulating types and for detection of the emergence of novel sub-types and antiviral resistant strains. This is the first report from Turkey on the surveillance and oseltamivir resistance of influenza viruses in 2007-2008. Five hundred twenty-four nasal swabs were tested from different geographical regions in Turkey during November 2007-April 2008. One hundred sixty-three (31%) samples were positive for influenza viruses of which 111 (68%) were influenza A, 52 (31%) influenza B using an immuno-capture ELISA. Forty isolates were selected at random from influenza A positive samples and grown in MDCK cell cultures. The supernatant of the cell cultures was used for RNA extraction followed by RT-PCR to detect the sub-types. Sub-typing revealed all samples as A/H1N1. The N1 gene segment of 30 A/H1N1 samples was sequenced in part, from the 201st to 365th residue, which included the critical region for oseltamivir resistance. Then resulting sequences were analyzed with oseltamivir sensitive and resistant strains obtained from National Center for Biotechnology Information (NCBI) GenBank by CLC Main Workbench Software. H275Y (H274Y according to N2 numbering) mutation, which is known to confer resistance to oseltamivir, was detected in 6 out of 30 (20%) H1N1 isolates from four cities (Istanbul, Bursa, Ankara, and Izmir). The D354G mutation was observed in all oseltamivir resistant H1N1 isolates but not in the oseltamivir sensitive isolates. Assay of neuraminidase activity revealed that these isolates were resistant to oseltamivir, but sensitive to zanamivir.

Mutations in influenza A virus (H5N1) and possible limited spread, Turkey, 2006.

We report mutations in influenza A virus (H5N1) strains associated with 2 outbreaks in Turkey. Four novel amino acid changes (Q447L, N556K, and R46K in RNA polymerase and S133A in hemagglutinin) were detected in virus isolates from 2 siblings who died.

Influenza and respiratory syncytial virus morbidity among 0-19 aged group in Yunus Emre Health Center.

The objective of the study was to determine the morbidity of influenza and respiratory syncytial virus (RSV) infection in the 0-19 years of age group with influenza-like illness among the outpatient cases. From 20 January to 31 March 2003 a total of 123 subjects with upper respiratory tract infection attended Yunus Emre Health Center. Ninety-one subjects fit the case definition of influenza-like illness, which consisted of acute fever of more than 38 degrees C, cough, and sore throat. After obtaining their consent, nasal swabs were taken for isolation of influenza and RSV. Of these, 10 were influenza A virus, 6 were influenza B virus and 20 were RSV. All of influenza virus A was typed as subtype H3N2. The rates of influenza virus among 5-9 and 1-4 years of age groups and of RSV among 1-4 years of age group were high. The average number of absentee days of schoolchildren with influenza was 3.33 days and of those with RSV infection was 1.43 days; this rate was calculated as 2.25 days for the influenza-like illness. Continuous surveillance and influenza vaccination for target groups are recommended for beneficial effects of reducing influenza morbidity and mortality in the community.

[The use of real-time polymerase chain reaction and enzyme immunoassay for the diagnosis of acute parvovirus B19 infections in immunosuppressed patients]. / Immünsüpresif hastalarda akut parvovirus B19 enfeksiyonunun tanisinda enzim immún yóntemi ile gerçek zamanli polimeraz zincir reaksiyonunun kullanimi.

Human parvovirus B19 (PV-B19) infection may lead to very serious clinical situations such as transient aplastic crisis in patients with hemolytic anemia, thrombocytopenia, neutropenia and transient arthritis accompanied with erythema infectiosum, especially in immunosuppressed patients. Early diagnosis of PV-B19 infection is of critical importance especially in immunosuppressed patients since the necessary precautions can be undertaken accordingly. In this study, PV-B19 IgM and IgG antibodies and viral DNA have been searched by enzyme immunoassay (ELISA) and real-time polymerase chain reaction (PCR), respectively, in 50 PV-B19 suspected immunosuppressed patients. Viral IgM, IgG and DNA positivities were detected in 7 (14%), 20 (40%) and 7 (14%) of the patients, respectively. During the first week three patients were found DNA and IgM positive but IgG negative, while four patients were found positive for the viral DNA, IgM and IgGs. The DNA copy numbers were high in all of the patients during the first week, with a gradual decrease during a seven-week follow-up period. IgM antibodies have disappeared in the sixth week in three of the patients and at the end of the seventh week in four of the patients. Although the IgG antibodies were negative in three patients in the first week, they became positive in the second week and the titers gradually increased during the following weeks. According to the results of this study, it can be concluded that, in high risk groups such as immunosuppressed patients, in addition to ELISA, real-time PCR method would be helpful for the early diagnosis of PV-B19 infections.

Frequency of common viruses in etiology of acute respiratory tract infections.

OBJECTIVE: To determine the frequency rate of C. pneumoniae, rhinovirus, respiratory syncytial virus (RSV), influenza virus, metapneumovirus, adenovirus', parainfluenza virus and coronavirus in acute respiratory tract infections in children. METHODS: One hundred nine pediatric patients having respiratory tract infections were included in this study. Real time PCR, DFA and cell culture method were used for detection of C. pneumoniae, RSV antigen and influenza virus respectively. Multiplex PCR was used for detection of other viruses. RESULTS: No C. pneumoniae DNA was detected in the samples. Virus was detected in 43 cases from larynx swabs (43/109, 39.4 %). The frequency order of the viral agents detected were as follows; rhinoviruses 14.7 %, RSV B 7.3 %, influenza A 6.4 %, metapneumovirus 3.6 %, adenovirus 3.6 %, coronavirus 0.9 %, parainfluenzavirus type 3, 0.9 %, parainfluenzavirus type 4, 0.9 % and RSV A 0.9 %. Sensitivity of the PCR and DFA methods for the diagnosis of RSV infections were detected as 100 % and 100 %, respectively. Specificity of the PCR and DFA methods for RSV infections were detected as 97 % and 100 % respectively. Sensitivity of the PCR and cell culture methods for influenzavirus infections were detected as 100 % and 100 %, respectively. Specificity of the PCR and DFA methods for RSV infections were detected as 96 % and 100 % respectively. CONCLUSIONS: Prevalence of viral agents was detected as 39.4 %. Influenza viruses and RSV were common. Metapneumovirus was also frequent (3.6 %). C. pneumoniae was not found to be a common agent for acute respiratory disease in children.