Comparison of ELISA with shell vial cell culture method for the detection of human rotavirus in fecal specimens.

The aim of the study was to compare an enzyme immunoassay method with shell vial cell culture method for detection of rotavirus in fecal specimens. In addition, the correlation between laboratory results and clinical scores of patients with gastroenteritis was evaluated. A total of 219 fecal specimens from children (ages 3 weeks to 5 years) with acute gastroenteritis submitted to pediatric emergency room were evaluated by both ELISA and shell vial cell culture. A Vesikari score was used for assessing the severity of the illness. Among 219 stool samples tested, 107 (48.9%) were determined to be positive. Two specimens were positive by shell vial cell culture method while they were ELISA negative. According to these results the calculated sensitivity, specificity, PPV, and NPV of ELISA were 98.1%, 100%, 100%, and 98.2%, respectively. The mean severity score for the 107 episodes of rotavirus diarrhoea was 11.0 +/- 3.6 compared to 4.5 +/- 1.9 for the 112 episodes of non-rotavirus diarrhea in the same population. Our study indicates that ELISA, which is easier to perform, faster and cheaper than cell culture methods may be suitable for routine diagnosis of rotavirus infections. The severity of rotavirus positive gastroenteritis was significantly higher than that of rotavirus negative patients.

Comparison of the "ProDect BCS RV CHIP" assay with the combination of shell vial cell culture and immunofluorescence antibody test for the detection of respiratory viruses.

In the present study, a multiplex reverse transcriptase polymerase chain reaction combined with a chip hybridization assay (ProDect BCS RV CHIP) was evaluated as an alternative to the combination of immunofluorescent antibody test and shell vial cell culture considered as gold standard for the detection of respiratory viruses. Among 100 specimens, 40 were positive using the combination of immunofluorescent antibody test and shell vial cell culture assay in which 9 of them were infected by two different viruses (27 parainfluenza virus type 3, 10 adenovirus, 9 respiratory syncytial virus, 2 influenza type B, and 1 influenza type A). ProDect BCS RV CHIP detected only 10 positive specimens in which one of them was infected by two different viruses (5 respiratory syncytial virus, 3 parainfluenza virus type 3, 2 adenovirus, and 1 influenza virus type B). The sensitivity, specificity, PPV, NPV, and diagnostic accuracy of ProDect BCS RV CHIP were 25.0%, 100%, 100%, 66.6%, and 70.0%, respectively, compared to the combination of shell vial cell culture and immunofluorescent antibody test. As a result, the specificity of ProDect BCS RV CHIP is high, however, the sensitivity (25%) of the assay is not sufficient for routine laboratory use.

Comparison of Brucella immunoglobulin M and G flow assays with serum agglutination and 2-mercaptoethanol tests in the diagnosis of brucellosis.

The diagnostic value of Brucella IgM/IgG flow assays was evaluated in comparison with serum agglutination and 2-mercaptoethanol tests by testing a selection of serum samples submitted to the laboratory because of clinical suspicion of brucellosis. All 39 admission and 11 follow-up samples that agglutinated in the serum agglutination test tested positive in the flow assay, whereas all 20 serum agglutination negative samples with clinical suspicion of brucellosis, 23 control samples from healthy individuals and 20 control samples from cases with chronic hepatitis tested negative in the flow assay. The Brucella IgM and IgG flow assays were slightly more sensitive than the agglutination tests in discriminating between specific IgM and IgG antibodies. The Brucella IgM and IgG flow assays are easy-to-perform and quick assays that can be used for the diagnosis of brucellosis. The flow assays are very useful, especially in rural settings where brucellosis is widespread and where well-equipped laboratories to perform the laboratory tests are not readily available.

[Evaluation of INNO-LiPA mycobacteria, INNO-LiPA Mycobacteria v2 and hsp65 sequencing for identification of the clinical nontuberculous mycobacterial isolates]. / Tuberküloz dili mikobakteri izolatlarinin tanimlanmasinda INNO-LiPA mycobacteria, INNO-LiPA mycobacteria v2 ve hsp65 dizi analizinin karsilastirilmasi.

In this study, polymerase chain reaction (PCR) reverse hybridization based line probe assays, INNO-LiPA Mycobacteria and INNO-LiPA Mycobacteria v2 (Innogenetics, Ghent, Belgium) and partial sequencing of hsp65 gene were evaluated for the identification of 29 clinical mycobacterial isolates. Unique hsp65 sequences identified during the study were deposited in EMBL (European Molecular Biology Laboratory) under accession numbers AY379074, AY379077, AY379075 and AY553874. All mycobacterial isolates were identified at the genus level on both LiPA assays, whereas 8 of 9 different known mycobacteria species (M. kansasii, M. gordonae, M. avium, M. intracellulare, M. chelonae, M. abscessus, M. fortuitum and M. peregrinum) were identified at species level with LiPA Mycobacteria v2. A clear correlation was found between the results of identifications obtained by the both LiPA assays, which targeted the 16S-23S rRNA ITS region, and DNA sequencing, which targeted the hsp65 gene. In conclusion, although LiPA Mycobacteria v2 could not identify all mycobacteria species, it may be especially useful for routine work in clinical laboratories, which are not capable of carrying out DNA sequencing.

[Investigation of Epstein-Barr virus DNA and RNA in tissues of patients with lymphoma]. / Lenfomali hastalarin dokularinda EpsteinBbarr virus DNA ve RNA'sinin arastirilmasi.

Relation between Epstein-Barr virus (EBV) and nasopharyngeal carsinoma, Burkitt's lymphoma, and lymphomas in immunosupressed patients have been shown previously in different studies. The same relationship was also shown in Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) by some researchers. The aim of this study was to demonstrate EBV nucleic acids in tissue sections of adult patients with lymphoma. The presence of EBV encoded RNA (EBER) were investigated with in situ hybridization and EBV-DNA with PCR method in 29 formalin-fixed paraffin-embedded tissue sections (19 lymph nodes, the others being gastric, orbital, skin, salivary gland, testicle, small intestinal, tongue root, bone marrow and gingival tissues) of 8 patients with HL and 21 patients with NHL who were followed-up in Haematology Clinics of our university hospital. EBER and EBV-DNA positivity rates were found as follows respectively; 50% (n: 4) and 37.5% (n: 3) of 8 HL patients, and 23.8% (n: 5) and 47.6% (n: 10) of 21 NHL patients. In total evaluation EBER and/or EBV-DNA were positive in 5 of 8 (62.5%) HL, and 12 of 21 (57.1%) NHL tissue sections. There was no significant difference in EBER and EBV-DNA positivity between HL and NHL groups. As a result, our study emphasize a possible EBV related aetiology in HL and NHL.

Genotyping of rifampin-resistant Mycobacterium tuberculosis isolates from western Turkey.

BACKGROUND: Although the rate of multiple drug resistance is high, there is no published data on the transmission rate of drug-resistant strains of Mycobacterium tuberculosis in the Aegean region of western Turkey that are based on molecular methods. METHODS: IS6110 and pTBN12 restriction fragment length polymorphism (RFLP) methods were used for typing M. tuberculosis strains isolated from 26 sputum samples from 26 patients. RESULTS: Nineteen of the rifampin-resistant isolates (73.1%) contained 6 to 11 copies of IS6110. Eighteen different IS6110 DNA fingerprint patterns were observed in the 26 rifampin-resistant isolates. Twenty-three of the 26 rifampin-resistant isolates were also resistant to isoniazid. When evaluated together, both methods yielded 21 (80.9%) different banding patterns and the level of clustering was 34.6%. The average number per pattern was 1.23 (26/21). CONCLUSIONS: IS6110 fingerprinting suggests that the rifampin-resistant isolates obtained from the Aegean region had a relatively high clustering rate and were clonally related. These findings showed that the rifampin-resistant isolates are actively transmitted between patients. Urgent measures should be taken to prevent the spread of these resistant strains.

Assessment of Chlamydia trachomatis prevalence by cell culture and transcription-mediated amplification in symptomatic women.

OBJECTIVE: The frequency of Chlamydia trachomatis in women with mucopurulent discharge was determined by a cell culture technique and a transcription-mediated amplification (TMA) assay in endocervical swab specimens. SUBJECTS AND METHODS: Endocervical swab specimens were obtained from 116 symptomatic patients with genitourinary complaints or abdominal pain. All of the women were married, with an age range of between 19 and 44 (median 29) years. The cell culture assay was used in all specimens. For 75 specimens the TMA assay was also performed. RESULTS: Positive cell culture test results were obtained in 6 (5.2%) patients. Among 75 specimens, 2 were positive by both TMA and culture assays, while 1 specimen was positive only by the culture assay. Of those positive for C. trachomatis, 5 were in the 19- to 25-year age group, and 1 was in the >25-year age group. All of the patients with positive results were of low socioeconomic status. CONCLUSIONS: This study revealed a relatively low rate of C. trachomatis infections in symptomatic married women in Turkey. A commercial TMA assay failed to identify all positive patients, in contrast to a 'gold standard' culture assay used in patients having such infections.

[Current approaches to the clinical virologic diagnosis of viral respiratory tract infections]. / Viral solunum yolu enfeksiyonlarinin klinik virolojik tanisina güncel yaklasim.

Respiratory viruses (respiratory syncytial virus, influenza virus type A and B, parainfluenza virus and adenovirus) can cause a wide variety of human disease. Viral respiratory diseases in adults and children cause significant morbidity and mortality. Proper and rapid diagnosis of these etiological agents is necessary for the appropriate use of effective antiviral drugs and to decrease unnecessary antibiotic therapy. Many virology laboratories detect respiratory viruses by inoculating conventional cell culture tubes with respiratory samples and then examining for cytopathic effect or hemadsorption. However, this procedure can require many days or even weeks for viral detection and identification, providing culture results to clinicians in a period of time that may not be clinically useful. Laboratory diagnosis of respiratory virus infections has become easier and more rapid with the use of immunofluorescent antibody tests, shell vial culture methods, and molecular biological assays. In this review article, the specificities and sensitivities of these assays were compared on the basis of recent studies, and the favorable ones for the routine diagnosis were updated.

[Determination of rifampin resistance in Mycobacterium tuberculosis isolates by the RNA/RNA mismatch method]. / Mycobacterium tuberculosis izolatlarinda rifampin direncinin RNA/RNA uyumsuz birlesme (mismatch) yöntemi ile saptanmasi.

Rifampin is one of the most potent antituberculosis drugs and therefore, rifampin resistance leads to high clinical relapse rates. Detection of rifampin resistance could be an indication of multidrug resistance. In the recent years several molecular methods have been developed to evaluate the mutations in rpoB gene for the detection of rifampin resistance. The aim of the present study was to evaluate the performance of the RNA/RNA Mismatch Assay for detection of the mutations in the rpoB gene in 20 M. tuberculosis isolates which were determined as resistant to rifampin by agar proportion method. While RNA/RNA Mismatch Assay detected the mutations in the rpoB region in 16 of 20 (80%) M. tuberculosis isolates, the remaining four isolates yielded no band pattern indicating resistance. However, there may be situations where interpretation of the results is difficult in RNA/RNA Mismatch Assay which is already cheaper than DNA sequencing and other molecular methods. In conclusion, if the RNA/RNA Mismatch Assay can be optimized, it can be used for the rapid detection of rifampin resistance.

Anti-HTLV-I/II Seroprevalence in Healthy Blood Donors in Izmir, Turkey.

Human T-cell lymphotropic virus type I (HTLV-I) is the first human retrovirus to be associated with malignant disease-namely, adult T-cell leukemia/lymphoma. HTLV-I has also been associated with several diseases. HTLV-I has a worldwide distribution with major endemic foci in the Caribbean and Southern Japan. HTLV-II is a closely related retrovirus that shares considerable genomic homology with HTLV-I but has not been proven to be a pathogen. Major routes of transmission are blood transfusion, breast milk and sexual activity. In this study, we examined the seroprevalance of HTLV-I/II among healthy blood donors attended to Ege University Hospital in Izmir. 913 healthy blood donors were examined for the presence of anti-HTLV-I/II antibody in their sera. Serum specimens were tested with an enzyme immunoassay (EIA) (Organon Teknika, Vironostika HTLV-I/II Microelisa System, Holland). All of the 913 healthy blood donors were seronegative with EIA. These findings indicate that screening of blood donors for HTLV I/II is not necessary at present time.

[Comparison of standard proportion dilution test and Etest for Mycobacterium tuberculosis sensitivity to ofloxacin and ciprofloxacin]. / Mycobacterium tuberculosis kökenlerinde ofloksasin ve siprofloksasin duyarliliginin saptanmasinda standart proporsiyon dilüsyon ile etest yönteminin karsilastirlmasi.

The antibacterial activities of ofloxacin and ciprofloxacin on 100 clinical isolates of Mycobacterium tuberculosis were determined by using standard proportion dilution method and Etest. When 98 of 100 M. tuberculosis isolates were susceptible to ofloxacin and ciprofloxacin, two were resistant to both of the drugs. The correlation between E test and standard proportion dilution method was 100%.

Evaluation of Brucella dipstick assay for the diagnosis of acute brucellosis.

The diagnostic value of the dipstick assay was evaluated by comparison with Rose Bengal (RB), serum aglutination tests (SAT) and 2 mercaptoethanol test (2-ME) on consecutive serum samples submitted because of suspicion of brucellosis. Serum samples of 232 patients with suspected brucellosis that were submitted for laboratory confirmation were included in the study. Twelve out of 232 serum samples were detected as positive with the dipstick assay. All of these 12 patients had positive RB and SAT results. In 16 RB positive samples dipstick test was negative. Fifteen of these samples had insignificant (titer<1/160) or borderline (titer 1/160) SAT results and the clinical symptoms of these patients were consistent with chronic brucellosis rather than acute or recent-onset brucellosis. Dipstick assay is an easy-to-perform assay that can be used for the diagnosis of acute brucellosis especially in rural areas where brucellosis is widespread and in settings where well-equipped laboratories are not available.

Characterization of rpoB mutations in rifampin-resistant clinical isolates of Mycobacterium tuberculosis from Turkey by DNA sequencing and line probe assay.

Mutations in an 81-bp region of the rpoB gene associated with rifampin resistance were studied in 41 rifampin-resistant clinical strains of Mycobacterium tuberculosis isolated in Turkey. Fourteen different rpoB alleles, three of which had not been reported before, were found. A reverse hybridization-based line probe assay (the Inno-LiPA Rif.TB test) for rapid detection of the mutations was evaluated with these isolates. Rifampin resistance was correctly identified in 23 of 41 isolates (56.1%) with the kit's R probes specific for these mutations. Seventeen of 41 isolates (41.5%) yielded hybridization patterns, with at least one negative signal obtained with the S probes for the wild type. One isolate was identified as rifampin sensitive by the line probe assay. The rate of concordance of the results of the line probe assay with the results of the in vitro susceptibility test was high (97.6%). These results demonstrate that the line probe assay kit may be useful for the rapid diagnosis of rifampin-resistant tuberculosis.

Mycobacterium tuberculosis infection and laboratory diagnosis in solid-organ transplant recipients.

Tuberculosis (TB) is an unusual infection in transplant recipients. We evaluated (i) the frequency of TB, (ii) the duration to develop the TB infection, and (iii) clinical consequences, in 380 solid-organ recipients from January 1995 to December 2000. A total of 10 (2.63%) patients (eight renal, two liver transplant recipients) were found to have post-transplantation TB. The frequency of TB in this patient population is 8.5-fold higher than the prevalance in the general Turkish population. Tuberculosis developed within 2-33 months after transplantation, with a median of 15 months. In all of these 10 patients, Mycobacterium tuberculosis (MTB) was isolated from the culture. All the patients continued to have low dose immunosuppressive treatment, and also quadriple antituberculosis treatment [isoniazid (INH), rifampin (RIF), pyrazinamide (PRZ) and ethambutol (ETB)] has been given. The two recipients had died of disseminated form of TB. Relapse was detected in one patient 6 months after the completion of the treatment. As post-transplant TB infection develops mostly within the first year after transplantation, clinicians should be more careful for early and fast diagnosis and treatment should be started immediately.

[Polymerase chain reaction for Chlamydia trachomatis in urine specimens from patients with mucopurulent genital discharge]. / Mukopürülan genital akintisi olan hastalarin idrar örneklerinde Chlamydia trachomatis'in polimeraz zincir reaksiyonu ile arastirilmasi.

The aim of this study was to investigate the DNA of Chlamydia trachomatis by polymerase chain reaction (PCR) in the first-void urine samples of patients with mucopurulent genital discharge and to compare the results with the urethral/endocervical swab culture method. First-void urine samples from 56 patients (46 female, 14 male) and urethral swab samples from 14 male patients were tested by PCR. Additionally, shell-vial culture method was performed for the urethral/endocervical swab samples which were collected from 46 female and 14 male patients. Four (2 females, 2 males) of the patients (7.1%) showed positive results by both of the methods. In five (8.9%) of the urine samples, internal control tests were found negative, indicating the presence of amplification inhibitors, and the culture results of these patients were also negative. Since the PCR method (Cobas Amplicor CT, Roche Diagnostic Systems, NJ, USA) which was used in the study included internal control programme to identify inhibitors in urine, the sensitivity was improved. As a result, the perfect (100%) correlation between culture and PCR methods, lead to the conclusion that PCR is a rapid and reliable method for the detection of C. trachomatis in urine samples, however more detailed studies are necessary related to the sensitivity and specificity of PCR method.