Ayhan Kubar

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.

Salivary infectious agents and periodontal disease status

BACKGROUND AND OBJECTIVES The potential of salivary microorganisms to diagnose periodontal disease and to guide periodontal treatment is a research topic of current interest. This study aimed to determine whether the salivary counts of periodontopathic microbes correlated with the periodontal pocket counts of the same infectious agents, and whether the salivary counts of the test infectious agents could distinguish among individuals with periodontal health and various types of periodontal disease. MATERIAL AND METHODS The study included 150 systemically healthy adults, of whom 37 were periodontally healthy, 31 had gingivitis, 46 had chronic periodontitis and 36 had aggressive periodontitis. Each study subject contributed microbial samples from the two deepest periodontal pockets of the dentition and from whole saliva. Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Epstein-Barr virus were identified using the TaqMan real-time PCR methodology. Statistical analysis was performed using the Mann-Whitney U-test and the receiver operating characteristic statistics. RESULTS C. rectus, F. nucleatum, P. gingivalis, P. intermedia and T. forsythia occurred with significantly higher copy-counts in salivary samples from patients with gingivitis, chronic periodontitis and aggressive periodontitis than from periodontally healthy individuals. A. actinomycetemcomitans only showed higher salivary copy-counts in subjects with aggressive periodontitis compared with subjects with healthy periodontium, and the salivary copy-counts of Epstein-Barr virus did not reveal any significant difference among the four subject groups studied. The diagnostic sensitivity for periodontitis was 89.19 for P. gingivalis and for T. forsythia and 86.49 for P. intermedia, with specificities ranging from 83.78 to 94.59. The optimal copy-counts per mL saliva for identifying periodontitis were 40,000 for P. gingivalis, 700,000 for T. forsythia and 910,000 for P. intermedia. CONCLUSION Salivary copy-counts of P. gingivalis, T. forsythia and P. intermedia appear to have the potential to identify the presence of periodontitis, whereas the salivary level of the other test infectious agents may possess little or no diagnostic utility. Longitudinal studies are warranted to determine the ability of salivary copy-counts of major periodontopathic bacteria to predict future periodontal breakdown.

Healing of artificial fenestration defects by seeding of fibroblast-like cells derived from regenerated periodontal ligament in a dog : a preliminary study

The purpose of this study was to assess the seeding of fibroblast-like cells to promote periodontal healing in artificial fenestration defects in a dog. Fibroblast-like cells were cultured by incubating regenerated periodontal ligament tissue, that had been surgically taken, underneath a Teflon membrane. Fenestration defects were surgically induced on the maxillary canine and first molar teeth at a spacing of 5 to 5 mm. Passage 4 cells (2 x 10(5) cells) in autologous blood coagulum were placed on root surfaces in two defects; the remaining two defects were used as controls. Healing was evaluated histomorphometrically on postoperative day 42. The main periodontal healing pattern consisted of connective tissue adaptation in three of the four specimens including one control, with cementum formation at 9-12%; one control specimen that exhibited 100% cementum formation. New bone formation was greater in the cell-seeding group (84%) compared with control (39%). In the cell-seeding group, one specimen exhibited total regeneration of bone (100%); however, the connective tissue located between newly formed bone and the root surface was observed to adapt to the dentin surface, with limited cementum formation. Seeding of cells from periodontal ligament may be promising to promote periodontal regeneration, but needs to be investigated in further studies.

Assessment of periodontal healing by seeding of fibroblast-like cells derived from regenerated periodontal ligament in artificial furcation defects in a dog: a pilot study.

The regeneration of periodontal supporting tissues lost as a result of disease could be accomplished by repopulating the exposed root surfaces with cells originating from periodontal ligament. Thus, we aimed to assess the seeding of cells derived from regenerated periodontal ligament (RPL) to promote the regeneration in artificial furcation defects of a dog. The fibroblast-like cells were obtained by incubating the explants of RPL tissue taken under a teflon (E-PTFE) membrane. Class II furcation defects were induced on the second and fourth mandibular premolars. Control defects were also included on the contralateral side. A suspension of the fourth passage cells (2 x 10(5) cells) in 0.5 mL of autologous blood coagulum was placed over each furcation area. The healing was histomorphometrically evaluated at the 42nd day postoperatively and expressed as percentage. The healing by new connective tissue attachment with cementum formation was found 75% in the cell-seeding defects whereas, it was 71% in controls. Bone formation was found to fill 51% of furcation defects; however, it was 35% of the defects in the control sites. In this pilot study, we suggested that regeneration of furcation defects by cell-seeding technique may be useful, but further studies are needed to determine the outcome of the procedure.

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.

A preliminary study to evaluate the effect of intravenous ribavirin treatment on survival rates in Crimean-Congo hemorrhagic fever.

CrimeaneCongo Hemorrhagic Fever (CCHF) is a serious infection with mortality rates of 15e70%. It has been an endemic disease in Turkey and large outbreaks have been seen during spring and summer seasons since 2002. Effective therapy of CCHF is not exactly described yet. Oral ribavirin has been the most applied therapy of choice in clinical practices, but the effect of this therapy on the outcome of the disease is controversial. We present here the efficacy of i.v. ribavirin therapy on survival rate among the patients with severe CCHF. This case-control study was conducted in Ankara Numune Education and Research Hospital and Sivas Cumhuriyet University Hospital between May and August 2006. Patients with suspected cases of CCHF and having severe disease were applied i.v. ribavirin therapy immediately at admission after informed consent was taken. Because the results of diagnostic tests were obtained a few days later, i.v. ribavirin therapy was initiated before laboratory confirmation. According to the laboratory test results, confirmed cases were included in the study. Patients suspected of having CCHF were selected as those who had clinical signs and symptoms, epidemiological risk factors and laboratory findings of CCHF. Confirmed cases were defined as suspected cases with positive serum RT-PCR test and/or positive serum serological test results for CCHF specific IgM by ELISA. Severe cases were defined as having at least one of the findings from splenomegaly, INR 1.4 or impaired consciousness which had been found as independent risk factors for mortality of the patients with CCHF in the study of Bakir et al. Nine patients with severe CCHF were given i.v. ribavirin immediately at admission to the hospital. Intravenous ribavirin (Virazole , Valeant Pharmaceuticals International) was supplied by Ministry of Health of Turkey. The dosage and duration of the therapy were applied according to the recommendations of the World Health Organization (WHO). It was used at the dosage of 17 mg/kg i.v. loading dose, then 17 mg/kg every 6 h for 4 days, and then 8 mg/kg every 8 h for 6 days. Sixteen severe cases of the confirmed CCHF who didn’t receive i.v. ribavirin were included in control group. Totally 25 confirmed CCHF cases with severe disease were included in the study. Nine patients receiving i.v. ribavirin in addition to supportive treatment were included in the i.v. ribavirin group while 16 patients receiving only supportive treatment were included in the control group. There were no statistically significant differences between the two groups for demographic characteristics and laboratory test results. The efficacy of i.v. ribavirin therapy was evaluated with respect to some parameters between the ribavirin group and the control group (Table 1). No statistically significant differences between the two groups were determined for fatality rate, mean duration of hospitalization and the need for transfusion of blood or blood products. Adverse effects associated with i.v. ribavirin were detected in three patients, but it didn’t need discontinuation of the therapy. Allergic maculopapular rash developed in one patient who recovered after antihistaminic therapy. Two patients had nausea and vomiting due to i.v. ribavirin and those symptoms resolved with symptomatic treatment. Ribavirin has in vitro activity against CCHF virus. It is well-absorbed from the gastrointestinal tract. Oral formulation of ribavirin is more easily available and cheaper than intravenous formulation. However, gastrointestinal tract bleeding, impaired consciousness or vomiting affects oral intake and absorption of the drug. Some of the successful results were reported with oral ribavirin. Mardani et al. from Iran reported the largest series of patients with CCHF. In this historical cohort study, the efficacy of oral ribavirin in confirmed cases of CCHF had been

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.

Periodontitis lesions are the main source of salivary cytomegalovirus.

BACKGROUND Herpesviruses play causal or cooperative roles in childhood infections, tumorigenesis, ulcerogenesis, and periodontitis. Saliva is a common vehicle of herpesvirus horizontal transmission, but the source of salivary herpesviruses remains obscure. To evaluate the significance of periodontal disease in shedding of oral herpesviruses, this study determined the genome-copy counts of human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in whole saliva of subjects with periodontitis, gingivitis, or no natural teeth. METHODS Whole saliva was collected from 14 periodontitis patients, 15 gingivitis patients and 13 complete denture wearers. The study subjects were systemically healthy and had not received periodontal treatment in the past 3 months. Real-time TaqMan polymerase chain reaction was used to determine the salivary load of HCMV and EBV. RESULTS Salivary HCMV was detected in seven (50%) periodontitis patients, but not in any gingivitis or edentulous subjects (P < 0.001). Salivary EBV was detected in 11 (79%) periodontitis patients, in five (33%) gingivitis patients, and in seven (54%) edentulous subjects (P = 0.076). Salivary samples showed copy counts of HCMV in the range of 3.3 x 10(3)-4.2 x 10(4)/ml and of EBV in the range of 3.6 x 10(2)-1.6 x 10(9)/ml. CONCLUSIONS HCMV and EBV are commonly present in the saliva of periodontitis patients. Periodontitis lesions of systemically healthy subjects seem to constitute the main origin of salivary HCMV, but do not comprise the sole source of salivary EBV.

Laboratory diagnosis of enteroviral infections of the central nervous system by using a nested RT-polymerase chain reaction (PCR) assay

Enteroviruses are the most common pathogens identified in infants hospitalized for suspected aseptic meningitis. Rapid detection of enterovirus infection is essential in taking the decision for treatment with antiviral agents and applying infection control measures in hospitalized pediatric patients. The purpose of this study was to compare the results of conventional virus isolation with those of enteroviral RNA detection by reverse transcription (RT)-PCR method in identical specimens from cases of suspected aseptic meningitis. Cerebrospinal fluid (CSF) samples were collected for viral examination from 68 pediatric patients with suspected aseptic meningitis from 1999 to 2002. These samples were inoculated in HeLa, Hep-2 and RD cell culture. The viral RNA was investigated by in-house RT-PCR method. The isolated viruses were typed by neutralization test. 36 of the 68 specimens were detected to be enterovirus positive by culture method, while 43 of them yielded positive results when RT-PCR method is used. Discrepancies occurred between the two methods in 15 specimens. While 11 specimens were positive by RT-PCR, these are found to be culture-negative. The isolated viruses were typed as Echovirus 30 (n: 30), Group B coxsackievirus (n: 5) and one isolate could not be typed by neutralization. Because of higher sensitivity and rapidity of RT-PCR, it is superior (p = 0.016) to virus culture of CSF for the diagnosis of enterovirus meningitis. Although the clinical usefulness of viral culture from CSF is limited, the final laboratory identification needs cultural techniques.

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

BackgroundAlthough 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.MethodsIn 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.ResultsThe 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.ConclusionsThe 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.