A new line method; A direct test in spinal muscular atrophy screening for DBS.

BACKGROUND: Nucleic acid-based assays provide an opportunity to screen for genetically encoded diseases like spinal muscular atrophy (SMA), before the onset of symptoms. Nowadays, such assays could be easily utilized as high-throughputs in SMA to detect a homozygous deletion of exon 7 of the survival motor neuron 1 gene (SMN1) that is responsible for >95% of SMA patients. METHODS: We developed a new line method (NLM) as a direct real time PCR test procedure without nucleic acid extraction in dried blood spots (DBS) to screen for homozygous deletion of exon 7 of the SMN1 gene. Performance of this setup was evaluated on 580 DBS newborn samples and air dried 50 DBS from whole blood including 20 samples for homozygous deletion of the SMN1 gene detected earlier with MLPA. RESULTS: We found all 580 newborn DBS samples as wild type. DBS prepared from 50 whole blood samples also including 20 affected people were correctly identified as homozygous deletions and 30 wild types of exon 7 of SMN1 as before with MLPA. When the MLPA method was taken as the gold standard, the sensitivity and specificity of the NLM test were found 100% for the detection of SMN1 exon 7 homozygous deletion. CONCLUSION: In the NLM, the total test duration has been reduced to less than 75 min without requiring any extra process such as DNA extraction step and sample plate preparation after the punching step. Thereby, newborn SMA screening with the NLM has gained an environmentally friendly feature with not requiring additional tedious steps.

Thrombophilia and Recurrent Pregnancy Loss: The Enigma Continues.

BACKGROUND Thrombophilic gene polymorphism is known to be a risk factor for recurrent pregnancy loss (RPL), but few studies have confirmed a possible role of thrombophilic genes polymorphism in RPL risk. This study was conducted to understand the relationship of the mutations of some thrombophilia-associated gene polymorphism (heterozygous/homozygous) with RPL. We compared patients with 2 abortions to patients with 3 or more abortions among Turkish women. MATERIAL AND METHODS In this study, patients previously diagnosed with habitual abortus at Obstetrics and Gynecology outpatient clinics in Turkey between 2012 and 2016 were included. In their peripheral blood, we detected factor V Leiden H1299R, prothrombin G20210A, MTHFR C677T, MTHFR A1298C, PAI-1 4G/5G, and PAI-1 4G/4G gene mutations. RESULTS In this study, we have observed statistically meaningful data (P<0.01) related to the relationship between RPL and thrombophilia-associated gene polymorphisms such as heterozygous factor V Leiden H1299R, heterozygous prothrombin G20210A, PAI-1 4G/5G, and PAI-1 4G/4G. CONCLUSIONS We found that diagnosis of thrombophilic genes polymorphism is useful to determine the causes of RPL, recognizing that this multifactorial disease can also be influenced by various acquired factors, including reproduction-associated risk factors and prolonged immobilization.

Human Papilloma Viruses and Their Genotype Distribution in Women with High Socioeconomic Status in Central Anatolia, Turkey: A Pilot Study.

BACKGROUND In the present study we retrospectively evaluated the results of outpatients who had an HPV analysis, and present objective evidence for the administration of preventive inoculation in our area. MATERIAL AND METHODS We retrospectively reviewed 532 outpatients who visited a single center between 2012 and 2016 and had an HPV infection analysis. The criteria for inclusion of patients with unhealthy cervix in the study were: erosion, chronic cervicitis, healed lacerations, hypertrophied cervix, and abnormal discharges from the cervix. RESULTS We found that 122 out of 532 patients were infected with HPV, and the rate of multiple infections was 59.0% (72/122). HR-HPV (group 1 carcinogens HPV-16 (18.9%, 23/122), HPV-18 (13.1%, 16/122), HPV- 31 (4.9%, 6/122), HPV-33 (3.3%, 4/122), HPV-35 (7.4.9%/122), HPV-39 (5.7%, 7/122), HPV-45 (5.7%, 7/122), HPV-51 (11.5%, 15/122); Group 3 LR-HPV; HPV-6 (31.1%, 38/122), HPV-11 (26.2%, 32/122), HPV-42 (9.0%, 11/122) and HPV-43 (4.9%, 6/122). In terms of linear-by-linear association test, no significant statistical difference was identified between years. The P value for HPV infection rate on year basis was P>0.05. CONCLUSIONS In this hospital-based retrospective analysis, HPV types were found to be similar to HPV types reported in developed countries. We firmly suggest that patients should be informed about the risk of HPV infection at early ages.

Porphyromonas gingivalis and Epstein-Barr Virus Are Associated With Increased Levels of Visfatin in Gingival Crevicular Fluid.

BACKGROUND: There is little clinical information on the relationship between periodontopathogens and visfatin. The purpose of this study is to determine visfatin levels in the gingival crevicular fluid (GCF) of healthy individuals and patients with periodontitis and to investigate the possible relationship between this adipokine and the presence and levels of Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescense, and Epstein-Barr virus (EBV). METHODS: Eighteen healthy individuals and 27 patients with periodontitis were included in this study. GCF and plaque samples were obtained from all individuals. Visfatin levels were analyzed by enzyme-linked immunosorbent assay, and the bacterial numbers were evaluated by the reverse transcription-polymerase chain reaction method. RESULTS: In patients with periodontitis, the visfatin levels in the GCF (mean: 84.29 ng/mL; range: 63.8 to 108.9 ng/mL) were significantly higher compared with those of the healthy individuals (mean: 38.06 ng/mL; range: 13.8 to 89.02 ng/mL) (P <0.01). There was a positive correlation between the visfatin levels and P. gingivalis (r = 0.266, P <0.05), whereas no correlation was found between visfatin levels and other microorganisms. In addition, the visfatin levels were found to be higher in individuals in whom P. gingivalis was detected than for those without P. gingivalis (P <0.01). The visfatin levels were also found to be higher in individuals in whom EBV was detected (P <0.05). CONCLUSIONS: To the best of the authors' knowledge, the present study is the first one to show the correlation of periodontopathogens and GCF visfatin levels. P. gingivalis colonization of the periodontal pockets may increase visfatin secretion. Furthermore, the presence of EBV in the plaque may be another factor that causes an increase in visfatin levels.

Efficacy of autologous mesenchymal stem cell transplantation in patients with liver cirrhosis.

BACKGROUND/AIMS: Because of several limitations and complications of liver transplantation, new alternative treatment modalities are required for patients with liver cirrhosis. Many study results encourage the use of autologous bone marrow-derived mesenchymal stem cells for liver diseases. In this study, we assessed the impact of autologous mesenchymal stem cell transplantation on liver tissue and liver chemistry. MATERIALS AND METHODS: Twenty-five patients with biopsy-proven liver cirrhosis were enrolled in the study. Patients received 1×106 autologous mesenchymal stem cells/kg via a peripheral vein. Biochemical parameters were checked monthly. Periodical radiological screening and liver biopsies before mesenchymal stem cell transplantation were performed after 6 months. Liver specimens were assessed by a pathologist. RESULTS: No side effect was observed and the mesenchymal stem cell transplantation procedure was well tolerated. Twelve patients completed the study. In 8 patients, improvements in Model for End-Stage Liver Disease (MELD) scores were observed. Serum albumin levels markedly increased in the third month. In patients with non-responder hepatitis C, HCV RNA levels both became negative after mesenchymal stem cell transplantation. Histopathological examinations of liver tissues before and at 6 months after transplantation revealed no change in liver tissue regeneration or fibrosis. However, in 5 patients, hepatitis activity index scores decreased. CONCLUSION: Autologous mesenchymal stem cell transplantation via peripheral vein is safe and feasible. Consecutive liver biopsy examinations suggested that mesenchymal stem cells could not reach the liver in a sufficient amount. Improvement in patients and clearance of HCV RNA may have occurred through immunomodulatory mediators secreted by transplanted mesenchymal stem cells, namely the "endocrine" effect.

Congenital cytomegalovirus infections and glycoprotein B genotypes in live-born infants: a prevalence study in Turkey.

BACKGROUND: Cytomegalovirus (CMV) infections are the leading cause of infectious hearing loss and central nervous system disease among children worldwide. In this study, we aimed to determine the birth prevalence of congenital CMV infection in live-born infants in Turkey. METHODS: In total, 944 consecutive live-born infants born from 926 pregnant women were included in this study. CMV-DNA was investigated in saliva samples of all newborns within the first 3 days after birth using TaqMan-based real-time PCR. RESULTS: The birth prevalence of congenital CMV infection in live-born infants was 1.91% (18/944), and all congenitally infected infants were asymptomatic at birth. The prevalence of congenital CMV infection was 16.7% (3/18) in twin pregnancies and 1.32% (12/908) in single pregnancies (p = 0.002). Genotyping analysis showed glycoprotein B-1 (gB1) to be the most frequently detected genotype at 83.3%. CONCLUSION: The study results suggest that the majority of congenital CMV infection in Turkey occurs following nonprimary maternal infection. We believe that congenital CMV infection and its long-term effects have been underestimated in our country, as infected infants are usually asymptomatic at birth.

Sequential determination of serum viral titers, virus-specific IgG antibodies, and TNF-α, IL-6, IL-10, and IFN-γ levels in patients with Crimean-Congo hemorrhagic fever.

BACKGROUND: Although there have been a number of studies on the pathogenesis of Crimean-Congo hemorrhagic fever (CCHF) recently, knowledge on this topic is still insufficient. This study aims to reveal the kinetics of serum CCHF virus (CCHFV) titers, serum levels of anti-CCHFV immunoglobulin (Ig)G, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and interferon (IFN)-γ in CCHF patients. METHODS: In total, 31 CCHF cases (11 fatal) were studied. Serum samples were obtained daily from all patients from the time of admission and continued for a 7-day hospitalization period for serologic (ELISA), virologic (real-time PCR), and cytokine (ELISA) analysis. RESULTS: The mean serum CCHFV titer at admission was 5.5E + 09 copies/mL in fatal cases and 5.7E + 08 copies/mL in survivors (p < 0.001). Compared to survivors, both the mean serum levels of IL-6 and TNF-α at admission were found to be significantly increased in fatal cases. The serum levels of IL-6, TNF-α and serum CCHFV titer at admission were significantly and positively correlated with disseminated intravascular coagulation (DIC) scores (r = 0.626, p = 0.0002; r = 0.461, p = 0.009; and r = 0.625, p = 0.003, respectively). When the data obtained from the sequential determination of CCHFV titer and levels of anti-CCHFV IgG, IL-6, TNF-α, IL-10 and IFN-γ were grouped according to the days of illness, the initial serum CCHFV titer of a fatal patient was 5.5E + 09 (copies/mL) and it was 6.1E + 09 (copies/mL) in a survivor on the 2 day of illness. While significant alterations were observed in all cytokines during the monitoring period, IL-6 levels remained consistently higher in fatal cases and TNF-α levels increased in both in fatal and non-fatal CCHF cases. CONCLUSIONS: The increased CCHFV load and higher concentrations of IL-6 and TNF-α, the presence of DIC, and the absence of CCHFV specific immunity are strongly associated with death in CCHF.

Coexistence of Epstein-Barr virus and Parvovirus B19 in tonsillar tissue samples: quantitative measurement by real-time PCR.

OBJECTIVE: In this study, we aimed to investigate the presence and copy number of six different viruses in tonsillar tissue samples removed surgically because of chronic recurrent tonsillitis or chronic obstructive tonsillar hypertrophy. METHODS: In total, 56 tissue samples (tonsillar core) collected from 44 children and 12 adults were included in this study. The presence of viruses was investigated using a new TaqMan-based quantitative real-time PCR assay. RESULTS: Of the 56 tissue samples, 67.9% (38/56) were positive for at least one of the six viruses. Epstein-Barr virus was the most frequently detected virus, being found in 53.6% (30/56), followed by human Parvovirus B19 21.4% (12/56), human adenovirus 12.5% (7/56), human Cytomegalovirus 5.4% (3/56), BK polyomavirus 1.8% (1/56), and Herpes simplex virus 1.8% (1/56). Precancerous or cancerous changes were not detected in the tonsillar tissue samples by pathologic examination, whereas lymphoid hyperplasia was observed in 24 patients. In contrast to other viruses, B19 virus was present in high copy number in tonsillar tissues. The rates of EBV and B19 virus with high copy number (>500.000 copies/ml) were higher in children than in adults, and a positive relationship was also found between the presence of EBV and the presence of B19 virus with high copy number (P=0.037). CONCLUSIONS: It is previously reported that some viral agents are associated with different chronic tonsillar pathologies. In the present study, the presence of B19 virus in tonsillar core samples was investigated quantitatively for the first time, and our data suggests that EBV infections could be associated with B19 virus infections or could facilitate B19 virus replication. However, further detailed studies are needed to clarify this observation.

Efficiency of MY09/11 consensus PCR in the detection of multiple HPV infections.

Human papillomavirus (HPV) DNA testing has become an important component of cervical cancer screening programs. In this study, we aimed to evaluate the efficiency of MY09/11 consensus polymerase chain reaction (PCR) for the detection of multiple HPV infections. For this purpose, MY09/11 PCR was compared to an original TaqMan-based type-specific real-time PCR assay, which can detect 20 different HPV types. Of the 654 samples, 34.1% (223/654) were HPV DNA positive according to at least one method. The relative sensitivities of MY09/11 PCR and type-specific PCR were 80.7% (180/223) and 97.8% (218/223), respectively. In all, 352 different HPV isolates (66 low-risk and 286 high-risk or probable high-risk types) were identified in 218 samples, but 5 samples, which were positive by consensus PCR only, could not be genotyped. The distribution of the 286 high-risk or probable high-risk HPVs were as follows: 24.5% HPV-16, 8.4% HPV-52, 7.7% HPV-51, 6.3% HPV-39, 6.3% HPV-82, 5.6% HPV-35, 5.6% HPV-58, 5.6% HPV-66, 5.2% HPV-18, 5.2% HPV-68, and 19.6% the other 8 types. A single HPV type was detected in 57.3% (125/218) of the genotyped samples, and multiple HPV types were found in the remaining 42.7% (93/218). The false-negative rates of MY09/11 PCR were found to be 17.4% in single infections, 23.3% in multiple infections, and 34.6% in multiple infections that contained 3 or more HPV types, with the condition that the low-risk types HPV-6 and HPV-11 be considered as a monotype. These data suggest that broad-range PCR assays may lead to significant data loss and that type-specific PCR assays can provide accurate and reliable results during cervical cancer screening.

Evaluation of periodontal pathogens of the mandibular third molar pericoronitis by using real time PCR.

OBJECTIVES: The aim of this study was to investigate the mandibular third molar pericoronitis flora by using real-time polymerase chain reaction (PCR). MATERIALS AND METHODS: The quantitative values of Aggregatibacter actinomycetemcomitans (Aa), Campylobacter rectus (Cr), Fusobacterium nucleatum (Fn), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Tannerella forsythia (Tf) were evaluated in comparison with the healthy third molar flora by using real time PCR. RESULTS: Aa, Cr, Pg, and Pi were not statistically significant but numerically higher than the pericoronitis group. In contrast to samples from control subjects, statistically significant higher numbers of Tf were detected in samples from pericoronitis patients. The study revealed the strong relation between risk of pericoronitis and the presence of Tf. Individuals who have Tf in their samples present with an almost eight times relative risk of pericoronitis as the individuals with an absence of Tf in their samples. CONCLUSION: Tf plays an important role in the development of clinical symptoms related to pericoronitis.

Detection of major HPVs by a new multiplex real-time PCR assay using type-specific primers.

In this study, we aimed to develop a cost-effective, practical, and sensitive method to be used for the diagnosis of HPV infections. The presence of HPV-DNA was investigated in cervical smear samples using three different methods: MY09/11 consensus PCR, TaqMan-based type-specific real-time PCR, and SYBR Green-based multiplex PCR. Of the 315 samples, 21.6% (68/315) were HPV-DNA positive by using at least one of the three methods. The relative sensitivities of MY09/11 PCR, type-specific PCR, and multiplex PCR were found to be 86.8% (59/68), 91.2% (62/68), and 91.2% (62/68), respectively. Genotyping analyses were successfully carried out in 62 of 68 HPV-DNA positive samples, and 77 isolates (8 low-risk and 69 high-risk HPV) were identified, while six samples were determined to be positive by consensus PCR only and could not be genotyped. The type distribution of the 69 high-risk HPV strains was as follows: 37.7% HPV 16, 13.0% HPV 52, 11.6% HPV 58, 7.2% HPV 18, 7.2% HPV 31, 7.2% HPV 68, 4.3% HPV 35, 4.3% HPV 39, 4.3% HPV 82, 1.4% HPV 33, and 1.4% HPV 45. Our data suggests that the diagnosis of HPV infections using only consensus PCR may lead to epidemiologically significant data loss, and that our multiplex PCR is more sensitive than consensus PCR and lower in cost than the type-specific PCR. We believe that the SYBR Green-based multiplex PCR may be useful and cost-effective for other microbiological fields. In addition, type-specific screening of HPV-DNA gives more reliable results, but it may also be used in combination with consensus PCR if the type spectrum of the test is not large enough.

[Investigation of HPV-DNA in cervical smear samples by two different methods: MY09/11 consensus PCR and type-specific real-time PCR]. / Servikal Sürüntü Örneklerinde Iki Farkli Yöntemle HPV-DNA Varliginin Arastirilmasi: MY09/11 Konsensus PCR ve Tipe Özgül Gerçek Zamanli PCR.

Cervical cancer that has been proven to be associated with human papillomavirus (HPV) is the second most common cancer in women worldwide and is a leading cause of cancer deaths in women in developing countries. Cervical cancers can be detected in the early stages by screening programs since a long latency period exists between the beginning of HPV infection and the development of cervical cancer. HPV-DNA testing is widely used throughout the world and today is an important part of cervical cancer screening programs. In this study, we analyzed the presence of HPV-DNA in 356 cervical smear samples by two different methods which are MY09/11 consensus real-time polymerase chain reaction (Rt-PCR) and type-specific Rt-PCR. All samples were also tested by type-specific PCR, regardless of consensus PCR results. PCR analysis were performed using the type- specific primers and TaqMan probes that were designed for a total of 13 different HPV types; two low risk HPV and 11 high risk HPV types. A total of 142 different isolates, 95 being high risk HPV isolates, 39 low risk HPV isolates and eight unidentified isolates, were determined in 109 (30.6%) smear samples that were defined as HPV-DNA positive by at least one of the two methods. Frequencies of detection of high risk HPV types in HPV-positive samples were as follows respectively: HPV-16; 32 (33.7%), HPV-52; 12 (12.6%), HPV-58; 11 (11.6%), HPV-18; 7 (7.4%), HPV-31; 7 (7.4%), HPV-35; 7 (7.4%), HPV-68; 6 (6.3%), HPV-33; 4 (4.2%), HPV-82; 4 (4.2%), HPV-39; 3 (3.2%) and HPV-45; 2 (2.1%). Various cytologic atypia were reported in 84 (23.6%) smear samples according to the simultaneously performed cytopathologic examination. Single HPV type was detected in 72 (71.3%) and multiple HPV types were detected in 29 (28.7%) of 101 smear samples with the exception of the unidentified isolates by type-specific RtPCR. HPV-18, HPV-33 and HPV-35 had higher detection rates of 7.4, 3.7 and 3.0 fold in mixed infections than single ones, respectively. HPV-DNA could not be detected by MY09/11 consensus primers in 24 (23.8%) of 101 cervical smear samples that were accepted as HPV-DNA positive by type-specific PCR. Thus, investigation of the presence of HPV-DNA by only consensus primers would be insufficient for the diagnosis, treatment and follow-up of HPV infections. Initial assessment of smear samples by using consensus primers and genotyping only positive samples seem to be the most practical strategy for the diagnosis and screening of HPV infections throughout the world. When this situation is taken into consideration, we think that the current prevalence data in our country and around the world must be updated by using large-scale studies that apply new generation screening and diagnostic tests.

[Investigation of West Nile virus RNA in blood donors by real-time RT-PCR]. / Kan Donörlerinde Gerçek Zamanli RT-PCR ile Bati Nil Virusu RNA'sinin Arastirilmasi

West Nile virus (WNV), a member of Flaviviridae family, is an enveloped, icosahedral symmetric RNA virus. Primary reservoir hosts of WNV are birds, but the virus can cause various infections in humans and other mammals. The most common and natural transmission way of WNV infections is mosquito bites, however, humans can be infected by different routes. The most important non-mosquito transmission route is contaminated blood and blood products. In this study, we aimed to investigate the risk of WNV transmission through blood and blood products in Ankara, Turkey. The presence of WNV RNA was investigated by in house real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in serum samples obtained from 729 healthy blood donors (mean age: 27.7 years; 711 were male), regardless of the donor's seropositivity status since the virus can be transmitted at the early stages of infection when seroconversion has not yet developed. Serum samples were collected in August-September 2009, the period when these infections are more frequent due to mosquito activity. The vast majority of donors (n= 702, 96.3%) have been inhabiting in Ankara and 569 (78%) of donors have had risk factors for arboviral infections (e.g. outdoor activity, mosquito and tick bites). WNV RNA was not detected by real-time RT-PCR analysis in any serum sample included in this study. According to the results of our study, it can be said that the risk of WNV transmission through blood and blood products is low in Ankara. However, WNV seropositivity was detected within the range of 0.56 to 2.4% among blood donors in previous studies and probable and confirmed WNV infections have been reported in our region. In addition, WNV outbreaks have emerged in some countries neighbouring Turkey recently. Thus, the risk of WNV transmission through blood and blood products should not be ignored and blood donor questionnaires should be evaluated in detail.

Antiviral activity of salivary microRNAs for ophthalmic herpes zoster.

Ophthalmic herpes zoster is a common ocular infection caused by the varicella-zoster virus (VZV). Viral mRNA transcripts play a major role in the replicative cycle of the virus and current antiviral agents have little effect in preventing and treating the complications. Therapeutic use of saliva for certain painful ocular diseases such as ophthalmic herpes zoster is a well-known public practice in our region. We thought that antiviral activity of saliva may stem from salivary microvesicles and we aimed to look for molecules with antiviral activity in these vesicles. As a possible candidate for antiviral activity, salivary microvesicles contain at least 20 microRNAs (miRNAs), small noncoding RNAs, which suppress the translation of target mRNAs. miRNAs not only participate in maintenance of normal cell functions, but are also involved in host-virus interactions and limit the replication of certain virus types. Thus, miRNA gene therapy by targeting mRNAs required for VZV survival may find a niche in the treatment of ophthalmic herpes zoster. But, how could salivary microvesicles reach into the corneal cells to demonstrate their antiviral activity. We suggest that human salivary microvesicles can be effective carriers of miRNA for corneal cells, because they contain a molecular machinery for vesicle trafficking and fusion allowing them to be endocytosed by target cells. After binding to the plasma membrane, microvesicles seem to enter into the corneal cells through the clathrin-mediated endocytosis. In the cytosol, human salivary miRNAs base-pair with specific viral mRNAs and inhibit their translation, thus limiting the replication of the virus.

Prompt administration of Crimean-Congo hemorrhagic fever (CCHF) virus hyperimmunoglobulin in patients diagnosed with CCHF and viral load monitorization by reverse transcriptase-PCR.

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes a severe disease in humans with high mortality rates. In Turkey, the number of patients with CCHF has increased since 2002. Here, we aimed to treat CCHF patients with CCHFV hyperimmunoglobulin. We prepared a CCHFV hyperimmunoglobulin product from 22 individuals who survived CCHF infection. A total of 26 CCHF patients were enrolled into this study. For CCHFV hyperimmunoglobulin administration, a Kubar Unit (KU) was defined. As a standard therapeutic approach, 400 KU of hyperimmunoglobulin were given to each patient as a single dose before viral load was detected. We used one-step real-time reverse transcriptase-PCR to monitor the viral load of CCHF patients. According to the one-step real-time PCR results, 15 patients with a viral load of 10(8) copies/mL or more were defined as high risk. In this high-risk group, the survival rate was found to be 86.6% (13/15) and 2 patients died despite CCHFV hyperimmunoglobulin administration. CCHF is a very serious and highly fatal infection, particularly for patients in the defined high-risk group. Prompt administration of CCHFV hyperimmunoglobulin might be a very promising new treatment approach, especially for high-risk individuals.

Effect of oral ribavirin treatment on the viral load and disease progression in Crimean-Congo hemorrhagic fever.

OBJECTIVES: Crimean-Congo hemorrhagic fever (CCHF) is a lethal hemorrhagic disease. There is currently no specific antiviral therapy for CCHF approved for use in humans. In this study we aimed to investigate the effect of oral ribavirin treatment on the viral load and disease progression in CCHF. METHODS: The study population was composed of patients who had a definitive diagnosis of CCHF by means of clinical presentation plus detection of viral RNA by reverse transcriptase polymerase chain reaction (RT-PCR). Ten patients who received oral ribavirin for 10 days and 40 control patients who received supportive treatment only were included in the study. Ribavirin treatment consisted of oral ribavirin 4 g/day for 4 days and then 2.4 g/day for 6 days. Viral load and hematological and biochemical laboratory parameters, which were measured daily, were analyzed. RESULTS: Mean age (37.4 vs. 45.5, p=0.285), gender (male 50% vs. 62.5%, p=0.470), days from the appearance of symptoms to admission (4.3 vs.4.4 days, p=0.922), and initial complaints were similar between the ribavirin group and the control group. Upon hospital admission, mean viral load was 8.2×108 copies/ml in the ribavirin group and 8.3×108 copies/ml in the control group (p=0.994). During follow-up, no statistically significant differences were found between the groups with regard to the decrease in viral load, the reduction in alanine aminotransferase and aspartate aminotransferase levels, and the increase in platelet count. The case-fatality rate was 20% (2/10 patients) in the ribavirin group and 15% (6/40 patients) in the control group (p=0.509). CONCLUSION: In this study, oral ribavirin treatment in CCHF patients did not affect viral load or disease progression.

[Expression of hepatitis B virus core antigen gene region in yeast cell]. / Hepatit B virusu kor antijeni gen bölgesinin mayada ekspresyonu.

In this study, the core antigen (HBcAg) gene region of hepatitis B virus (HBV) was transformed and expressed into an eukaryotic expression vector by recombinant DNA technology in order to obtain the protein used in anti-HBc tests which is being one of the most important marker for the serodiagnosis of HBV infections. For this purpose, HBV-DNA positive patient sera were used as the source of viral nucleic acids, and the primers coding HBcAg gene region have been designed. After the amplification of HBcAg gene region by polymerase chain reaction (PCR), the amplicons purified by Invisorb Spin Rapid PCR Kit" (Invitek, Germany), were cloned to pYES2.1 plasmid via the TOPO TA expression kit (Invitrogen, USA) and this plasmid was transformed to competent bacteria (TOPO 10F' Escherichia coli) by CaCl2 method. After competent bacteria were grown on LB (Lysogeny Broth) agar media supplemented with ampicillin, the plasmid "pYES2.1 + HBcAg" were isolated and transformed to Saccaromyces cerevisiae via the "S.c. EasyComp Transformation Kit" (Invitrogen, USA). Finally, the expression of HBcAg by the yeast was confirmed with the use of in house ELISA method. Since the diagnostic kits used in our country for hepatitis B serology are usually imported products, this creates a great economical burden. Thus, the experience and knowledge that builds up following such studies will help to produce our own diagnostic products using our equity.

Stability of hepatitis C virus RNA in blood samples by TaqMan real-time PCR.

The storage conditions of blood samples for reliable results are very important in hepatitis C virus (HCV) RNA amplification tests used in routine HCV analyses. According to many studies, storage conditions could affect the RNA stability for HCV RNA detection. We have studied HCV RNA stability in blood samples stored at 4 degrees C. Nineteen blood samples containing different HCV RNA levels were stored at 4 degrees C and they were then analyzed by TaqMAN real-time PCR method. HCV RNA levels remained almost stable (100%) at least for five weeks at this storage condition. However, among them, the stability period was up to 11 weeks in two of the samples. As with these findings, there was a slightly significant correlation between the positivity time and the beginning HCV RNA levels (r=0.474, P=0.040). We conclude that, blood samples can be stored at 4 degrees C for five weeks without any significant difference in detected HCV RNA level by using TaqMan real-time PCR.

TaqMan Real-Time Quantification of Epstein-Barr Virus in Severe Early Childhood Caries.

OBJECTIVES: Early childhood caries (ECC) has several risk factors and it is important stressful/painful events of childhood and immunosuppression may occur during this unique rampant caries pattern. The changes in the host immune competence by compromised cellular immune system functions can activate Epstein Barr virus (EBV). The objective of this study was to determine whether the supragingival plaque samples of severe-ECC (S-ECC) patients harbor more EBV load than the non-carious healthy children by quantitative TaqMan Real-Time polymerase chain reaction (PCR). METHODS: Sixty subjects, including 30 S-ECC patients as well as age and gender matched 30 caries-free patients were studied. The supragingival plaque samples were collected from patients by brushing their teeth for 1 minute and the toothbrush was washed in 1 ml of sterile deionized water. After viral DNA extraction, TaqMan real-time PCR assay was used to quantify EBV DNA. Dental treatments were completed for all S-ECC patients and they were called for routine controls. Only 10 treated S-ECC patients were come to the 3(rd) months' control and post-treatment viral sampling was made in the same manner. RESULTS: EBV DNA was detected 16 of 30 S-ECC patients and 6 of the healthy controls (P<.001). There was no relationship between baseline and post-treatment samples of 10 treated S-ECC patients. CONCLUSIONS: The results of the study suggest that oro-dental hygiene motives of S-ECC patients might be important contributory factor for S-ECC and EBV would not be involved in the etiopathogenesis of ECC.

Detection of Crimean-Congo hemorrhagic fever virus genome in saliva and urine.

BACKGROUND: The Crimean-Congo hemorrhagic fever (CCHF) virus is transmitted by tick bites and by contact with the blood or tissues of infected patients and livestock. This study was designed to investigate the genome of CCHF virus in saliva and urine samples of patients with CCHF. METHODS: Eight patients with laboratory-confirmed CCHF were included in the study. The diagnosis was made by detection of viral RNA in blood by real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR). Samples of saliva from six patients and samples of urine from three patients were collected at the same time as the blood samples and analyzed for viral RNA. RESULTS: The genome of CCHF virus was detected in the saliva from five of the six patients and in the urine from two of the three patients. The levels of viral load in the saliva and urine samples were similar to those in the blood samples in all but one patient, in whom higher levels were detected in blood compared to saliva or urine. CONCLUSIONS: This study shows that during human infection with CCHF virus, viral genomes are present in the saliva and urine. Further studies to isolate infectious viruses from these fluids and to study whether they represent an infectious risk are underway.