Seropositivity of neurotropic infectious agents in first-episode schizophrenia patients and the relationship with positive and negative symptoms.

BACKGROUND: According to the neurodevelopmental model, schizophrenia is a disorder that occurs as a result of different etiologic factors during brain development, including viral infections. However, it is unclear whether these infections are related to the disease or whether they affect the symptom pattern. We investigated the presence of four herpes viruses (EBV, CMV, HSV-1 and HSV-2) in first-episode schizophrenia patients and compared seropositive with seronegative patients and healthy volunteers to reveal the etiological role of viral agents on schizophrenia symptoms. SUBJECTS AND METHODS: Ninety-two first-episode patients who met the DSM-IV diagnostic criteria for schizophreniform disorder were included the study, along with 88 healthy volunteers. The presence of the four herpes viruses was investigated with serological methods (ELISA) in both groups. Positive and negative symptoms were evaluated with Scale for the Assessment of Negative Symptoms (SANS) and the Scale for the Assessment of Positive Symptoms (SAPS). RESULTS: There was no difference between the patient and control groups in terms of seropositivity of the four viruses. We found that SANS scores of HSV-1 and CMV seropositive schizophrenia patients were significantly higher than the scores of patients with seronegative schizophrenia. No difference was found in SAPS scores. CONCLUSIONS: The results suggest a role of HSV and CMV infections in negative symptoms. This supports the hypothesis that viruses do not directly give rise to schizophrenia, but patients who were previously been infected with these viral agents may be prone to schizophrenia, and some of the symptom patterns may be related to different agents.

Preservative solution for skeletal muscle biopsy samples.

CONTEXT: Muscle biopsy samples must be frozen with liquid nitrogen immediately after excision and maintained at -80°C until analysis. Because of this requirement for tissue processing, patients with neuromuscular diseases often have to travel to centers with on-site muscle pathology laboratories for muscle biopsy sample excision to ensure that samples are properly preserved. AIM: Here, we developed a preservative solution and examined its protectiveness on striated muscle tissues for a minimum of the length of time that would be required to reach a specific muscle pathology laboratory. MATERIALS AND METHODS: A preservative solution called Kurt-Ozcan (KO) solution was prepared. Eight healthy Sprague-Dawley rats were sacrificed; striated muscle tissue samples were collected and divided into six different groups. Muscle tissue samples were separated into groups for morphological, enzyme histochemical, molecular, and biochemical analysis. STATISTICAL METHOD USED: Chi-square and Kruskal Wallis tests. RESULTS: Samples kept in the KO and University of Wisconsin (UW) solutions exhibited very good morphological scores at 3, 6, and 18 hours, but artificial changes were observed at 24 hours. Similar findings were observed for the evaluated enzyme activities. There were no differences between the control group and the samples kept in the KO or UW solution at 3, 6, and 18 hours for morphological, enzyme histochemical, and biochemical features. The messenger ribonucleic acid (mRNA) of ß-actin gene was protected up to 6 hours in the KO and UW solutions. CONCLUSION: The KO solution protects the morphological, enzyme histochemical, and biochemical features of striated muscle tissue of healthy rats for 18 hours and preserves the mRNA for 6 hours.

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.

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.

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.

First molecular characterization of a Turkish orf virus strain from a human based on a partial B2L sequence.

Cases of orf virus infection in human in Turkey have been reported for many years. Scab material from a man was found positive by PCR using pan-parapox-specific primers for parapoxvirus infection. The amplicon was purified and sequenced. The present study provides for the first time a phylogenetic analysis of parapoxviruses from Turkey. The partial B2L gene sequence of a Turkish orf virus from a human presented here may be useful for characterization of parapoxvirus infections in Turkey based on the phylogenetic analysis studies.

[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.

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.

[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.

[Progressive multifocal leukoencephalopathy in an elderly patient with no obvious immunosuppression]. / Belirlenebilen bir immünyetmezligi olmayan yasli bir hastada gelisen progresif multifokal lökoensefalopati.

Progressive multifocal leukoencephalopathy (PML) which is a severe demyelinating disease of the central nervous system (CNS), is caused by a human polyomavirus known as JC virus (JCV). PML is seen primarily in immunosuppressed (AIDS, organ transplant or malignancy) patients. In this report, a case of PML that developed in a 75-years-old female patient with no known immunosuppression was presented. The patient was admitted to the emergency department with complaints of headache and burning sensation in head. Cerebrospinal fluid (CSF) examination revealed increase in lymphocytic cells. Since lesions compatible with tuberculoma were detected in brain tissue by magnetic resonance imaging, antituberculous therapy initiated empirically. The disease exhibited a progressive course and all the serological, molecular, microbiological and biochemical tests performed in blood and CSF failed to identify the causative agent. Pathological and immunohistochemical examination of the brain biopsy specimens demonstrated demyelinating disease. Brain biopsy, CSF, serum and urine specimens were investigated by real-time polymerase chain reaction specific for JCV and JCV-DNA was detected in the urine samples. Follow-up visits of the patient indicated a progressive course. In conclusion, after ruling out the other primary causes, JCV should be investigated in patients with demyelinating CNS disease even in the absence of significant immunosuppressive condition. Elderly patients should be considered in the risk group for demyelinating disease of CNS due to JCV.

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.

Plasma levels of trace elements have an implication on interferon treatment of children with chronic hepatitis B infection.

This study evaluated the plasma levels of trace elements in children with chronic hepatitis B virus (HBV) infection and assessed whether they can be a factor that affects the response to interferon alpha (IFN-alpha) treatment. The study included 35 cases (ten girls, 25 boys) aged 3-13 years with chronic HBV infection and the control group. Plasma levels of copper (Cu), manganese (Mn), molybdenum (Mo), selenium (Se), and zinc (Zn) were measured before IFN-alpha treatment and biochemical, virological, and histopathologic response to treatment were assessed. Children were followed for at least 15 months. Although plasma Cu levels showed no difference between the groups, Mn, Mo, Se, and Zn levels were significantly lower in the study group before treatment. Fourteen cases (40%) showed biochemical response; 17 (48.6%) showed virological response; 16 (47.6%) showed histopathologic response, and ten (28.6%) showed response according to all three parameters. Plasma Cu and Mn levels of patients with triple response showed no difference; but Mo, Se, and Zn levels were significantly lower (p < 0.001) in the study group. No difference was observed between responders and nonresponders (p > 0.05). Plasma levels of Mn, Mo, Se, and Zn are lower in children with chronic HBV infection compared to healthy children. The pretreatment levels of these elements did not show difference between responders and nonresponders to IFN-alpha.

Stress-related Epstein-Barr virus reactivation.

Epstein-Barr virus (EBV) remains latent in 90% of the patients following primary infection. The infection might be reactivated due to various stress factors. We, therefore, examined the levels of stress hormones (epinephrine, norepinephrine and cortisol), viral capsid antigen (VCA) immunoglobulin Ig G, VCA IgM, EBV early antigen IgG, Epstein-Barr nuclear antigen (EBNA) IgG, EBNA IgM antibody screening tests and performed EBV polymerized chain reaction (PCR) test and EBV DNA PCR in 100 draftees on their first day of recruitment and at the end of 1 month. Examination of the initial samples revealed that 94 (94%) subjects previously had EBV infection and 6 (6%) were seronegative. Second samples obtained at the end of first month showed that 7 (7.4%) reactivations occurred in 94 subjects who previously had EBV infection (P < 0.001). Two out of six (33.3%) who were initially seronegative had acute infection (P = 0.289). There was no significant difference between the median values of the levels of stress hormones in the initial and second serum and plasma samples. There was a significant difference between the rates of acute infection and reactivation among subjects with elevated cortisol and epinephrine levels in the second samples compared to subjects with normal levels (P < 0.001). No significant difference was determined between the first and second sample hormone levels of all nine subjects whose EBV-DNA turned positive. Routine examinations might not reveal any specific findings since EBV infection often has an asymptomatic course. EBV reactivations should always be kept in mind in patients subject to such stressful conditions.

[Investigation of the presence and subgroups of adenoviruses in nasopharyngeal samples of military recruits with respiratory tract infections]. / Solunum yolu enfeksiyonu olan askerlerin nazofarengeal örneklerinde adenovirus varliginin ve alt gruplarinin arastirilmasi.

Adenoviruses (AdV) are important pathogens primarily associated to respiratory infections of children and military staff even though it is also associated to many clinical manifestations, such as cystitis, conjunctivitis, diarrhea, hepatitis, myocarditis, and encephalitis. The goals of this study were to detect and type acute respiratory disease associated AdV isolates among military trainees in a selected region without an evidence of an outbreak. Throat swab samples were obtained during February 2006-March 2006 period, from 180 military male trainees aged 20-29, who were presented with respiratory tract symptoms and an oral temperature of > or = 38.0 degrees C. All specimens were tested by HEp-2 cell culture and real-time TaqMan PCR with AdV specific primers and probes. Positive cell culture results, presented as AdV-specific cytopathic effects, were confirmed by real-time polymerase chain reaction (PCR). AdV subgroup differentiation were performed using conventional PCR assays with the primer set specific for subgroup B, C or E. Subgroup specific PCR products were restricted with Mspl enzyme in order to check whether they were specific or not. AdV positivity was detected in 8 (4.4%) samples by cell culture and in 9 (5.0%) by the real-time PCR. All culture positive samples were also positive by real-time PCR. Eight of the nine real-time PCR-positive specimens were found to be in the subgroup E (this group contains only AdV type 4) and the results were confirmed with restriction enzyme analysis. One isolate could not be typed with the available primers. These data indicated that both real-time TaqMan PCR and restriction enzyme analysis provide sensitive and specific tools for AdV detection and subgroup differentiation for throat swab specimens. It can be concluded that since the prevalence of AdV infections was low in the study group, AdV infections were not considered as a vaccine requiring health problem in Turkish armed forces, however, larger scale studies were needed to reach a more precise conclusion.

[Investigation of viral nucleic acids in middle-ear effusion specimens from children with acute otitis media]. / Akut otitis media'li çocuklardan alinan orta kulak efüzyonu örneklerinde viral nükleik asit varliginin arastirilmasi.

Acute otitis media with effusion (OME) is one of the major causes of antibiotic use, indication for operation and hearing loss in children. In two third of the cases the etiologic agents are bacteria. Nonetheless, increasing numbers of reports have implicated viruses as etiologic agents that may have some effect on prognosis of OME. The aim of this study was to investigate the presence of nucleic acids of respiratory syncytial virus (RSV) type A and B, influenza type A virus, adenovirus, cytomegalovirus (CMV), herpes simplex virus type-1 (HSV-1), and enteroviruses in the middle ear effusion specimens from children with otitis media by TaqMan real-time PCR. As a result, 18 of 30 (60%) OME samples were found positive in terms of viral nucleic acids by real-time PCR. RSV-A was detected in nine samples (30%), CMV in 3 (10%) samples and HSV-1 in 1 (3.3%) sample. In five of the samples two viruses were detected in the same sample (three were positive for adenovirus and RSV-A, and two were positive for CMV and RSV-A). Our data have supported the importance of viruses as etiologic agents of OME. Additionally, it was thought that TaqMan real-time PCR may be used as a reliable and rapid method for the detection of viruses in the middle ear effusion samples.

Nosocomial outbreak of Sphingomonas paucimobilis bacteremia in a hemato/oncology unit.

Nosocomial Sphingomonas paucimobilis infections can arise from contaminated water and the contaminated hands of hospital staff. Within a 1-month period, we isolated six S. paucimobilis strains, including four from blood cultures of four patients and two from hospital environment specimens including tap water and a bathtub in a hemato/oncology unit. We described here these strains' molecular epidemiological analyses by pulsed-field gel electrophoresis (PFGE) and antibiotic susceptibilities by E-test. Although clinical and environmental isolates yielded three different antibiotic resistances and PFGE patterns, all four clinical strains had an identical pattern by both methods. Thus, the isolated clinical strain clone could be traced neither to health care workers nor to environmental samples. It was concluded that S. paucimobilis strains can cause outbreaks in hemato/oncology units. We did not demonstrate genetic relatedness between clinical and environmental isolates by PFGE, but did find PFGE a useful identification technique for epidemiological investigation.

Viral load as a predictor of outcome in Crimean-Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever (CCHF) is a potentially fatal disease affecting multiple organ systems. To determine the association between viral load and severity of CCHF infection, quantitative measurement of CCHF virus was performed using 1-step reverse-transcriptase polymerase chain reaction for 36 patients with CCHF infection. Viral loads ranged from 1.1x10(3) copies/mL to > or = 9.9x10(9) copies/mL. Nine (25%) of 36 patients died. In 8 of the 9 patients with fatal outcomes, viral loads were detected that were > or = 1x10(9) copies/mL, whereas in 25 of the 26 patients with nonfatal outcomes, viral loads were detected that were < 1x10(9) copies/mL (P<.001). A viral load > or = 1x10(9) RNA copies/mL can be considered to predict a fatal outcome with a positive predictive value of 80%, with 88.9% sensitivity and 92.6% specificity. We suggest that viral load is a measure of the severity of CCHF.

Rapid and quantitative detection of Crimean-Congo hemorrhagic fever virus by one-step real-time reverse transcriptase-PCR.

In this article, the development of a new TaqMan-based one-step real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection and quantification of Crimean-Congo hemorrhagic fever virus (CCHFV) RNA is described. Selected oligos targeting the highly conserved S region of CCHFV were designed by using our oligo design and analysis software, Oligoware 1.0. None of the primer sequences showed genomic cross-reactivity with other viruses or cells in a BLAST (NCBI) search analysis. The sensitivity and specificity of the primers and the probe were tested using 18 serum samples from patients from East Anatolian who were suspected of having CCHFV, including 2 samples that had already been confirmed to be positive for CCHFV. Among the 16 previously unconfirmed samples, 5 were positive by TaqMan-based one-step real-time RT-PCR and 1 was positive by non-nested RT-PCR, and these results were confirmed with DNA sequencing analysis. The 2 previously confirmed CCHFV RNA samples were also positive by both TaqMan-based one-step real-time RT-PCR and non-nested RT-PCR tests. To ensure the quantitative reproducibility of TaqMan-based one-step real-time RT-PCR, the procedure was repeated several times and the same results were obtained (SD = 0.84 [maximum value]). The developed assay was able to sensitively quantify the concentration of CCHFV RNA, which ranged from 10(2) to 10(7) copies/ml per reaction, using plasmid standards generated from the CCHFV RNA (correlation coefficiency = 0.989). The results of the one-step real-time RT-PCR assay were more sensitive than those of the non-nested RT-PCR assay. It can be concluded that our one-step real-time RT-PCR assay is a reliable, reproducible, specific, sensitive and simple tool for the detection and quantification of CCHFV.