Detection of TT virus (TTV) by three frequently-used PCR methods targeting different regions of viral genome in children with cryptogenic hepatitis, chronic B hepatitis and hbs carriers.

This study was designed so that three sensitive and widely-used polymerase chain reaction (PCR) methods for the detection of TT virus or Torque Teno virus (TTV) would be simultaneously applied to a large number of subjects to evaluate performances of the various PCR protocols with different genotype sensitivities. Sera were collected from 92 children admitted to Hacettepe University Ihsan Dogramaci Children's Hospital Pediatric Gastroenterology Unit (17 cryptogenic chronic hepatitis, 17 asymptomatic HBs carriers, 18 chronic HBV patients and 40 healthy children). TTV DNA was detected via nested N22, nested 3'-UTR and 5'-UTR PCRs for all samples. Differences in TTV D N A detection prevalences were n o t statistically significantbetween the study groups with all TTV DNA and liver enzyme levels. A significant agreement between PCR methods that target UTR was observed. TTV detection rate increased with age, suggesting a non-parenteral, environmental exposure to the virus for the study population.

[Investigation of anti-R7V antibodies in HIV-infected patients under highly active antiretroviral therapy]. / Yüksek düzeyde aktif antiretroviral tedavi alan HIV ile enfekte olgularda anti-R7V antikorlarinin arastirilmasi.

R7V is a seven-aminoacid peptide epitope derived from cellular beta-2 microglobulin, present on human immunodeficiency virus (HIV) virion surface in patients with HIV infection. Antibodies against R7V peptide have the property of neutralizing all strains of HIV, unrelated to genotype, phenotype, or geographical origin of the virus, even in the presence of anti-retroviral drug resistance. Patients that mount an anti-R7V antibody response have been shown to be slow or non-progressors and this epitope has been considered for vaccine and/or therapeutic uses. In this study, HIV-infected patients under highly active anti-retroviral therapy (HAART) at Hacettepe University Faculty of Medicine, Department of Internal Medicine, Division of Infectious Diseases, were evaluated for the presence of anti-R7V antibodies. Thirty-three HIV positive patients and 10 healthy controls were enrolled to the study. For HIV-infected patients, determination of viral load and CD4+ T lymphocyte counts were performed by a commercial real-time PCR assay and flow cytometry, respectively. Anti-R7V antibodies were detected from serum samples by a commercial ELISA (Anti-R7V ELISA, Ivagen, France) test. Three HIV infected patients (3/33, 9.1%) displayed anti-R7V antibodies whereas the remaining 30 (90.9%) patients and all controls were interpreted as negative. No statistically significant difference was detected for HIV-RNA levels and CD4+ T lymphocyte counts between anti-R7V positive and negative patients (p= 0.871 and p= 0.287, respectively). These results indicate the presence of anti-R7V antibodies in our study population with HIV infection. No correlation with the presence of anti-R7V and disease progression were displayed in this study. Clinical impact of anti-R7V antibody assays for the management of HIV-infected patients will be revealed in the near future with the help of advanced studies.

[Detection and typing of human papilloma virus by polymerase chain reaction and hybridization assay in cervical samples with cytological abnormalities]. / Sitolojik atipi izlenen servikal örneklerde insan papilloma virusunun polimeraz zincir reaksiyonu ve hibridizasyon yöntemleriyle saptanmasi ve tiplendirilmesi.

Certain mucosa-tropic human papillomavirus (HPV) types are associated with carcinoma of the uterine cervix or its precursor lesions. In addition to cytological screening, early diagnosis and treatment of cervical carcinoma rely on sensitive detection and typing of HPV isolates. In this study, HPV detection and typing were performed in the cervical samples of patients with abnormal cytological evaluation. Forty randomly-selected cervical samples that comprise 18 ASC-US (Aypical Squamous Cells of Undetermined Significance), four AG-US (Aypical Glandular cells of Undetermined Significance), one ASC-H (Atypical Squamous Cells-can not exclude HSIL), one HSIL (High-grade Intraepithelial Lesion), 14 LSIL (Low-grade Intraepithelial Lesion), one adenocarcinoma and one squamous cell carcinoma, obtained by a commercial liquid-based cytology system (ThinPrep Pap Smear Method, Cytyc, USA), were included to the study. HPV-DNA detection were accomplished by L1 in-house polymerase chain reaction (PCR) performed using MY09/11 and GP5/6 primers along with a commercial real-time PCR (HeliosisTM HPV LC PCR Kit; Metis Biotechnology, Turkey) that detects HPV infections and HPV-16 via melting curve analysis. A commercial PCR-array hybridization test (Rapid HPV Genotyping MacroArray; HybriBio Inc, Hong Kong) that can identify 21 low and high risk HPV types was employed for typing. Viral DNA was detected in 35% (14/40) and 57.5% (23/40) of the samples by MY09/11 and GP5/6 primers, respectively. All in-house PCR positive samples were also positive in the real-time PCR assay. PCR-array hybridization assay provided typing results in 95.6% (22/23) of the PCR positive samples while one LSIL sample could not be typed by any of the methods used. High risk HPV types 16, 18, 31, 45, 52, 56, 58, 59,68 (65.8%); probable high risk type 53 (13.2%), low risk types 6, 42 and 81 (21%) were identified out of a total of 38 HPV isolates. Multiple infections with more than one HPV type were identified in 45.5% (10/22) of positive samples. High/probable high risk types were detected in all single infections and all low risk isolates were present in multiple infections. HPV-16 was identified in 31.8% (7/22) by real-time PCR and in 45.5% (10/23) of positive samples by PCR-array hybridization assay. HPV-16 was observed to be the most frequently detected type (10/22, 45.5%), followed by types 53 and 81 (5/22, 22.7%); 68 (4/22, 18.2%); type 58 (3/22; 13.6%); types 31, 42 and 59 (2/22; 9.1%) and others (1/22, 4.5%). As a result our data have indicated the abundance of high risk HPV isolates and infections with multiple HPV types in that specific area.

Testicular persistence of Parvovirus B19: evidence for preferential infection of germ cell tumors.

Human Parvovirus B19 has previously been implicated in the pathogenesis of testicular germ cell tumors, but this could not have been confirmed. This study was designed to investigate the testicular persistence of Parvovirus B19 and possible associations with germ cell tumors. Paraffin-embedded or fresh tissues from 36 germ cell tumors, 20 germ cell aplasias, 26 normal testicular tissues, 20 liver tissues, and 20 spleen tissues were evaluated by two different molecular assays: a nested PCR for Parvovirus B19 capsid genes and a commercial quantitative real-time PCR. Positive results were further confirmed by another commercial real-time PCR assay. Viral DNA was detected in 3 of 36 (8.3%) germ cell tumors, but not in other groups. Viral loads observed in all positive samples were less than 20 IU/reaction, suggesting very low levels of viral replication or latency. These results either directly or indirectly imply the involvement of Parvovirus B19 with testicular germ cell tumors. Viral persistence in normal testis, germ cell aplasia tissues, or hepatic/splenic tissues was not observed in this study.

[Human papilloma virus DNA in cervical samples with cytological abnormalities and typing of the virus]. / Sitolojik olarak anomali saptanan serviks örneklerinde insan papilloma virus DNA'sinin arastirilmasi ve virusun tiplendirilmesi.

Human Papillomavirus (HPV) infections and cervical carcinoma which is associated with certain types of the virus have been worldwide public health issue. Early diagnosis of HPV infections and the detection of viral genotypes are important for the successful treatment and prevention of cervical carcinoma. This study has been designed as a preliminary study to estimate HPV type distribution in cervical samples with cytologic abnormalities in our country. A total of 35 cervical samples which were evaluated by a commercial liquid-based cytological system, were included to the study. The presence of HPV-DNA has been searched with nested polymerase chain reaction (PCR) by using consensus primer sets of MY09/11 and GP5/6 that target L1 region of the viral genome. HPV typing was performed by direct sequencing of the amplicons. In cytologic evaluation, 14 samples were diagnosed as ASC-US (Aypical squamous cells of undetermined significance), three were ASC-H (Atypical squamous cells-cannot exclude HSIL), five were HSIL (High-grade intraepithelial lesion), seven were LSIL (Low-grade intraepithelial lesion), four were LSIL+suspected HSIL, one was AG-US (Aypical glandular cells of undetermined significance) and one was atypical cells of undefined nature. HPV-DNA was detected in 28 of the 35 (80%) samples, and sequence analysis revealed high-risk HPV types (type 16, 18, 31, 33, 45, 56, 59) in 22 (78.6%) samples, probable high-risk types (type 53) in two (7.1%) samples and low-risk types (type 6, 54, 72, 81) in four (14.3%) samples. HPV type 16 emerged as the most frequently-detected type, comprising 50% (14/18) of all samples; followed by type 18 in 10.7% (3/28) and type 53 in 7.1% (2/28) of the samples. As a result, although the number of cervical samples were relatively low, the preliminary data obtained with this study revealed the HPV type distribution, however more detailed studies are needed to elucidate the epidemiology of HPV infections in Turkey.

Seroprevalence of West Nile virus and tick-borne encephalitis virus in southeastern Turkey: first evidence for tick-borne encephalitis virus infections.

West Nile virus (WNV) and tick-borne encephalitis virus (TBEV) are among the medically important Flaviviruses that cause significant morbidity and mortality in humans. In this study, seroprevalence of WNV and TBEV in sera from two state medical hospitals from the southeastern part of Turkey was investigated. One hundred eighty-one serum samples were evaluated for WNV immunoglobulin G (IgG) by an indirect immunofluorescence test (IIFT) and for IgG antibodies against TBEV by a commercial enzyme-linked immunosorbent assay (ELISA) kit with enhanced sensitivity and specificity. Sera positive for WNV IgG were further analyzed by plaque reduction neutralization assay (PRNA). TBEV IgM was also investigated by ELISA in all seroreactive samples. Of 181 sera, 29 (16%) were positive for WNV IgG by IIFT and 17 of 179 (9.5%) were confirmed by PRNA. Nineteen of 181 (10.5%) sera were detected to have TBEV IgG. Mean titer of TBEV IgG was 43.0 RU/mL (median, 33.9 RU/mL; cutoff: 20 RU/mL). Four samples with WNV IgG antibodies were also positive for TBEV IgG antibodies. TBEV IgM was detected in 9 of 39 (23%) of all seroreactive sera, where IgM positivity were accompanied by IgG for 6 samples. These results suggest the presence of possible human WNV and TBEV infections in southeastern Turkey where vector activity have previously been detected.

West Nile virus studies in the Sanliurfa Province of Turkey.

We attempted to isolate West Nile virus from mosquitoes collected in the field for the first time in Turkey. A total of 6,457 mosquito specimens from Culex pipiens Linnaeus, Ochlerotatus caspius (Pallas) and Aedes spp. species were included in this study. Culex pipiens samples made up 56% of the total species, O. caspius 24% and Aedes spp 20%. There were no positive results after studying mosquito samples using Real-time PCR, VecTest, and Vero cell culture. In serological tests of 181 human serum samples, 29 (16%) were found to be West Nile positive. On the basis of these results, we intend to collect more mosquito samples especially from those areas from which positive serum samples were obtained.

[The colorful clinical spectrum of cerebral toxoplasmosis in five HIV positive cases: what comes out of Pandora's box?]. / HIV pozitif bes olguda serebral toksoplazmozisin renkli klinigi: Pandora'nin kutusundan çikanlar.

In patients with human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS), the most common cause of focal intracranial lesion is Toxoplasma gondii infection. T. gondii encephalitis is an easily and effectively treatable disease, with promising outcomes. T. gondii has the potential to form a focal infection niche anywhere in the central nervous system, thus allowing for a colorful clinical picture. In this report, we attempted to present five HIV/AIDS cases with central nervous system toxoplasmosis demonstrating five different neurological presentations. The ages, gender and clinical findings of the patients who were admitted to our Infectious Diseases Clinics were as follows; 35 years old male patient with delirium, 49 years old male patient with focal dystony, 32 years old female patient with facial paralysis and monoparalysis, 53 years old male patient with Wernicke syndrome, 32 years old male patient with epilepsy. Cerebral toxoplasmosis were diagnosed by clinical findings and imaging techniques. The patients were treated with trimetoprim-sulfametoxazol (TMP-SMZ) and haloperidol, only TMP-SMZ, clindamycin and daraprim, TMP-SMZ and levotiracetam, TMP-SMZ and phenytoin, respectively, with recovery in neurological and radiological symptoms. In conclusion, until proven otherwise, HIV/AIDS patients presenting with focal neurological complaints should be accepted as having central nervous system toxoplasmosis.

Transfusion-transmitted virus prevalence in Turkish patients with thalassemia.

In hematology patients on chronic transfusion regimes, liver diseases are frequent, and mostly related to the agents transmitted by blood products and concominant iron deposition in liver. Besides hepatitis B (HBV) and C (HCV) viruses, new viral agents like hepatitis G virus (HGV) and TorqueTeno virus (TTV) are identified in these patients, although their association with any pathology or disease is not yet proved. In the present work, the authors studied the clinical importance of TTV in Turkish multitransfused patients with thalassemia. Forty-six healthy and 57 thalassemic patients were enrolled in the study. TTV was detected in serum samples by 3'-UTR nested PCR. Transaminase and ferritin levels, hepatitis B and C virus markers and number of transfusions were interpreted for possible association with TTV infection. As a result, TTV was detected in 63% of the thalassemia and 54% of the control patients. Prevalence of TTV infection, clinical features, laboratory data, and annual transfusion numbers of TTV-positive and -negative patients were not observed to be statistically significant. In conclusion, in Turkish patients with thalassemia, TTV infection cannot be considered as a risk factor for liver disease.

[Detection of TT virus DNA by nested-PCR method in non A-E hepatitis cases]. / A-E disi hepatit olgularinda tt virus dna'sinin nested-pcr yöntemiyle saptanmasi.

TT virus (TTV) is the unique single stranded circular DNA virus, isolated from humans. Viral DNA is shown to be present not only in patients with hepatitis of unknown etiology but also in apparently healthy populations. TTV's role as a causative agent for non A-E hepatitis is widely questioned. In this study, 23 non A-E hepatitis patients (mean age: 46.8 yrs), 21 chronic hepatitis B (CHB) patients (mean age: 41.0 yrs), 28 chronic hepatitis C (CHC) patients (mean age: 48.1yrs) have been investigated, together with 90 healthy blood donors (mean age: 33.4 yrs), for the presence of TTV-DNA by nested polymerase chain reaction (PCR). Two sets of primers targeting different regions (N22 and NCR) of the viral genome were combined to enhance the detection sensitivity. TTV-DNA was detected in 10 (43.5%) of non A-E hepatitis, 10 (35.7%) of CHC, 4 (19.1%) of CHB patients, and 16 of studied 52 (30.8%) blood donors, by N22-PCR. Viral DNA positivity rates by using NCR-PCR were found as follows for the groups respectively; 65.2% (15/23), 50% (14/28), 42.9% (9/21) and 57.8% (52/90). The differences of TTV-DNA positivity rates between study groups were found statistically insignificant, when data from each set of primer were compared (p>0.05). However, the positivity rate of non A-E hepatitis group (91.3%) was significantly higher than CHC (64.2%), CHB (47.6%), and control (57.8%) groups, when the detection sensitivities of both sets were combined (p=0.002 and p=0.046, respectively). The study also revealed that the prevalence of TTV might be underestimated if appropriate molecular methods were not employed. DNA sequencing and genotyping are required to establish TTV's role as a causative agent of non A-E hepatitis and to identify certain genotypes that might have a role in the pathogenesis of hepatitis.

Cytotoxicity of eluates from a gamma-ray-polymerized poly(methyl methacrylate).

The purpose of this study was to evaluate the cytotoxicity of gamma-ray-polymerized poly(methyl methacrylate) (PMMA). A total of 32 disk-shaped PMMA specimens were polymerized by gamma-irradiation with 1 Mrad for 4 h (Group 1), 2 Mrad for 8 h (Group 2), 3 Mrad for 12 h (Group 3), and thermally polymerized (Group 4). Four- and 6-day eluates of the specimens were prepared in Eagle's Minimal Essential Medium (EMEM) without Fetal Calf Serum (FCS). The eluates and EMEM supplemented by 20% FCS were placed into Vero (green African monkey kidney) cell cultures, and incubated at 37 degrees C for 24, 48, and 72 h. EMEM kept at 37 degrees C for 4 and 6 days was also tested up to 72 h, and served as controls. After each incubation period, the number of viable cells were counted and stained at the termination of the experiments for histological evaluation. The number of viable cells for Group 1 was slightly lower than that of other groups after 24 h. The time-dependent increase in cells exposed to Group 3 eluates was comparable with the control group. There was a dose-dependent effect on cell response for gamma-ray-polymerized specimens. The number of viable cells and the morphological appearance of cells in all groups were similar. Eluates from PMMA polymerized by low doses of gamma-ray with reduced polymerization periods have early inhibitory effects on cell response. Higher doses of gamma-irradiation lead to better cellular response, and therefore, may be future candidates for polymerization of PMMA.

Infective agents in fixed human cadavers: a brief review and suggested guidelines.

Cadavers remain a principal teaching tool for anatomists and medical educators teaching gross anatomy. Infectious pathogens in cadavers that present particular risks include Mycobacterium tuberculosis, hepatitis B and C, the AIDS virus HIV, and prions that cause transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker syndrome (GSS). It is often claimed that fixatives are effective in inactivation of these agents. Unfortunately cadavers, even though they are fixed, may still pose infection hazards to those who handle them. Specific safety precautions are necessary to avoid accidental disease transmission from cadavers before and during dissection and to decontaminate the local environment afterward. In this brief review, we describe the infectious pathogens that can be detected in cadavers and suggest safety guidelines for the protection of all who handle cadavers against infectious hazards.

Viral studies in the cerebrospinal fluid in subacute sclerosing panencephalitis.

OBJECTIVES: The pathogenesis of subacute sclerosing panencephalitis (SSPE), and particularly, the cause of measles virus (MV) reactivation following a latent period after primary measles infection is unknown. The hypothesis of other viruses contributing to the pathogenesis of SSPE by affecting the in vivo state of MV was investigated. METHODS: We examined the cerebrospinal fluid of SSPE patients (n=43) for DNA or RNA and antibodies against HSV type 1 and 2, EBV, CMV, VZV, Hepatitis B, Hepatitis C, JC virus, human herpesvirus (HHV)-6, HHV-7, HHV-8, HTLV-1, and HTLV-2. We compared the findings with those of patients with other neurological disorders (n=39). RESULTS: CMV DNA and HSV type 1 IgG were found more frequently in SSPE patients. Other positive results were at similar incidence in SSPE and control groups. The clinical features of SSPE cases with and without positive viral tests did not differ from each other. CONCLUSION: These data do not support a specific role for these agents in SSPE, but imply that the passage of some viruses to the CNS and local antibody synthesis may be facilitated by inflammation. The persistence or reactivation of MV in SSPE may be related to other factors pertaining to the host or environment.