[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.

Detection of cytomegalovirus and Helicobacter pylori DNA in arterial walls with grade III atherosclerosis by PCR.

It has been suggested that some microorganisms may play a role in the etiology or progression of atherosclerotic plaques. The purpose of this study was to assess for the presence of Helicobacter pylori and cytomegalovirus (CMV) DNA using polymerase chain reaction (PCR) technique in vascular-wall specimens obtained during autopsy. Four to 5 mm long samples from 3 different vascular wall specimens (coronary, carotid and abdominal aortas) of 30 patients (23 male, 7 female) were taken for pathologic and microbiologic investigations during autopsy. H. pylori DNA was found in 48.2% atherosclerotic and 19.6% non-atherosclerotic vascular wall specimens, whereas CMV DNA was found in 37.9% atherosclerotic and 32.7% non-atherosclerotic vascular wall specimens. In terms of CMV DNA detection, no statistically significant differences between the atherosclerotic and non-atherosclerotic groups were present (P > 0.05). However, there was a statistically significant difference between the atherosclerosis and non-atherosclerotic groups in terms of H. pylori DNA in coronary and abdominal aorta arteries (p = 0.016 and p = 0.0029 respectively) but not in carotid arteries (p = 1.00). In conclusion, the correlation between H. pylori and atherosclerosis could be suggested. These finding warrant further investigation regarding the role of H. pylori in atherosclerosis.

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.

Performance of the immunoglobulin G avidity and enzyme immunoassay IgG/IgM screening tests for differentiation of the clinical spectrum of toxoplasmosis.

Toxoplasmosis has been well known as an important human infection to consider especially in pregnant women. Although many serologic methods are available, the diagnosis of toxoplasmosis can be extremely difficult. The presence of increased levels of Toxoplasma-specific IgG antibodies indicates an infection, but it does not differentiate between a recent and past infection. The purpose of our study was to compare the performance of the ELISA T. gondii IgG/IgM test, a widely used enzyme-linked immunosorbent assay, to the ELISA IgG avidity method. One hundred and four serum samples (from 38 males and 66 females) were tested and evaluated from symptomatic patients (chorioretinitis, lymphadenopathy), and from women in their first trimester of pregnancy who were suspected of having toxoplasmosis. The high IgG avidity and ELISA IgG antibody levels were in agreement for 51 of the specimens (49.0%). Thirty-eight discrepant (borderline) results from the IgG avidity method were positive for IgM (3 specimens) and IgG (37 specimens). Interestingly, out of the eight serum samples that were positive for both IgG and IgM antibodies, two samples were low IgG avidity, and three samples were borderline. There was no statistically significant relation observed between the results of the IgG avidity method and the ELISA IgG test, and the IgG avidity method and ELISA IgM test (chi2 = 1.987; p = 0.370 and chi2 = 2.152; p = 0.341, respectively). The IgG avidity method was considered easy to perform and an acceptable approach for the differentiation of discrepant results (recent/chronic) and for the current detection of T. gondii antibodies. We concluded that the determination of IgG avidity is a helpful tool for the diagnosis of the ocular form of toxoplasmosis and it is a safe method for screening this disease in the first trimester of pregnancy.

An outbreak of respiratory infection due to respiratory syncytial virus subgroup B in Ankara, Turkey.

During the outbreak, from 16 January 2002 to 3 March 2002, nasopharyngeal secretions obtained from 35 pediatric patients under 2 years of age and suffering from acute respiratory disease were tested by VIDAS respiratory syncytial virus (RSV) assay (an automated enzyme-linked fluorescent immunoassay) and reverse transcription- polymerase chain reaction (RT-PCR). RSV antigen was detected in 16 specimens by VIDAS RSV assay, and 15 of these were confirmed by the RT-PCR. A total of 18 samples were found to be positive by RT-PCR. RSV subgroup B was identified by further restriction fragment length polymorphism analysis using AvaII and BglII endonucleases in 17 of 18 (94%) RT-PCR positive samples. These findings indicated that RSV subgroup B was highly dominant during an outbreak of RSV infection among children in Ankara. To our knowledge, this is the first outbreak due to dominant RSV subgroup B documented in Turkey.

Rapid and quantitative detection of mumps virus RNA by one-step real-time RT-PCR.

We developed a new TaqMan-based one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for detection and quantification of mumps virus RNA. Oligos targeting the matrix protein gene of mumps virus were designed by using our oligo designing and analyzing software, Oligoware 1.0. Oligos's specificity was tested with 5 strains (4 laboratory isolated and 1 Jeryl Lynn strain) of mumps virus. The suggested TaqMan-based one-step real-time RT-PCR assay correctly detected the 4 laboratory-isolated strains and 1 Jeryl Lynn strain. To confirm the specificity of the TaqMan PCR assay, parainfluenza type 1, 2, 3 strains, sendai virus, and measles virus (vaccine strain) were tested, and no cross-reactivity was observed between mumps and tested strains. In addition, a BLAST (NCBI) search showed no genomic cross-reactivity with other viruses or cells. Testing of the assay's reproducibility was repeated several times, and the same results were achieved. The new assay was able to quantify the concentrations of mumps virus gene ranging from 10(1) to 10(8) copies per reaction sensitively with generated plasmid standards. In addition, it was shown that a significant correlation (R2 = 0.9564) between genome number as determined by one-step real-time RT-PCR and the corresponding number of plaque in paired samples was found with regression analysis. The results of one-step real-time RT-PCR assay also corresponded well to those of nested PCR. We conclude that our one-step real-time RT-PCR assay is a reliable, specific, and sensitive tool for the diagnosis of mumps virus. We consider that these results come from highly conserved primers and probe set that were designed with Oligoware 1.0.

High prevalence of transfusion-transmitted virus infection in patients with chronic liver diseases in an endemic area of hepatitis B and C virus.

OBJECTIVE: To determine the prevalence and clinical impact of transfusion-transmitted virus (TTV) DNA in patients with chronic liver diseases in the Southeast Anatolia region of Turkey where hepatitis B and C viral infections are endemic. SUBJECTS AND METHODS: Patients diagnosed with chronic liver disease by clinical, biochemical and histologic means were enrolled in the study. Serum samples of 60 patients (19 males, 41 females) with chronic liver diseases, and of 45 healthy volunteer blood donors as a control group were collected. The chronic liver disease group consisted of 11 patients with hepatitis B, 44 with hepatitis C and 5 with chronic liver disease of unknown etiology. Presence of TTV DNA was investigated by the polymerase chain reaction. Using a scoring system histological grading of inflammation and staging of fibrosis were performed only in the chronic hepatitis C group. RESULTS: TTV DNA was detected in 47 (78%) patients with chronic liver disease and 5 (11%) volunteers in the control group. The difference was statistically significant (p < 0.001). Ten of the 11 (91%) patients with hepatitis B, 32 of 44 (73%) of those with hepatitis C-related chronic liver disease, and 5 of 5 (100%) of the patients with cryptogenic liver disease were positive for TTV DNA. CONCLUSION: TTV is highly prevalent in patients with chronic liver diseases in Southeast Anatolia, Turkey but no pathogenic effect attributable to TTV infection was detected.

Resistance patterns of Mycobacterium tuberculosis complex isolates in the Turkish Army from 1998 to 2000.

The aim of this retrospective study was to investigate susceptibility rates of Mycobacterium tuberculosis complex (MTBC) isolates against streptomycin, rifampicin, isoniazid, and ethambutol between January 1998 and December 2000 in the Turkish Army. Specimens collected from patients were cultured both conventionally and radiometrically. Differentiation of MTBC bacteria from Mycobacteria other than tuberculosis bacilli was made by the BACTEC p-nitro-alpha-acetyl-amino-beta-hydroxypropiophenone test. Susceptibility testing of MTBC isolates was performed using the BACTEC radiometric susceptibility assay for mycobacteria. Most of the specimens originated from respiratory system. A total of 98 isolates in 1998, 123 isolates in 1999, and 84 isolates in 2000 were obtained and identified as MTBC using the radiometric BACTEC TB460 system. Initial resistance was most frequent to isoniazid followed by ethambutol, streptomycin , and rifampicin in this study period. The differences between resistance rates were not statistically significant on an annual basis. None of these isolates was resistant to all four antimycobacterial agents. Although resistance rates of our isolates were not as high as previously reported by some authors from Turkey and there was no significant difference between the annual susceptibility rates, routine screening of antituberculosis drug susceptibility should be continued to control the resistance development and its spread.

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.