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.

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.

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.

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.

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.

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.

[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.

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.

[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.