Saban Cavuslu

Analytic sensitivities of hybrid-capture, consensus and type-specific polymerase chain reactions for the detection of human papillomavirus type 16 DNA

Human papillomavirus type 16 (HPV‐16) DNA is detected commonly in cervical carcinomas; in this study, we have determined the analytical sensitivities of Hybrid Capture, HPV‐consensus PCR, and three HPV‐16‐specific polymerase chain reactions (PCRs) for the detection of HPV‐16 DNA. Samples investigated included a cervical cancer cell line, cervical scrapes from 20 patients attending colposcopy clinics, and buccal swabs from eight immunosuppressed children. HPV‐16 E7 and E5‐nested PCRs [Cavuslu et al. (1996): Journal of Virological Methods, in press] produced positive signals from samples containing fewer than ten HPV‐16 genomes per reaction. HPV‐consensus PCR [Manos et al. (1989): Cancer Cells 7:209–214] and HPV‐16 PCR using primers of van den Brule et al. [(1990): Journal of Clinical Microbiology 25:2739–2743] were of intermediate sensitivity (i.e., produced positive signals from samples containing 250 and 2,500 HPV‐16 genoms/reaction, respectively) and Hybrid Capture could detect just 50,000 HPV‐16 genomes/reaction. Highest rates of positivity for cervical samples were detected with HPV‐16 E7 or E5‐nested PCRs [50% (10 of 20 samples) and 60% (12 of 20 samples) positive, respectively], intermediate rates with HPV‐consensus PCR and PCRs using the primers of van den Brule et al. [both 35% (7 of 20 samples)], and lowest rates of positivity [25% (5 of 20 samples)] with Hybrid Capture. None of eight buccal swab samples from immunosuppressed children were positive by Hybrid Capture, yet three (37.5%) were positive by HPV‐16 E5‐nested PCR. These data indicate that HPV‐16 type‐specific PCRs should be used for the investigation of specimens that may contain low amounts of HPV‐16 DNA.

Cutaneous Anthrax in Turkey: A Review of 32 Cases

Anthrax, caused by the Gram-positive, rod-shaped, spore-forming bacterium Bacillus anthracis, is rarely seen in industrialized nations but is common in developing countries. Cutaneous anthrax accounts for 95% of cases and usually develops on exposed sites. This study reviews the clinical and laboratory findings of 32 patients diagnosed with cutaneous anthrax over a 4-y period in the eastern part of Turkey. All patients had a history of direct contact with infected animals. The patients, aged 6-72 y, comprised 17 (53%) males and 15 (47%) females. The most frequent localization site of skin lesions was the hands and fingers (31 patients), whereas the suborbital part of the face was invaded in 1 patient. The diagnosis was made as a result of typical clinical lesions, direct microscopy or bacterial isolation. All but 2 patients were successfully treated with penicillin; these other 2 patients were treated initially with sulbactam-ampicillin. All patients, including the patient with suborbital anthrax, were cured.

HBV, HCV and HIV seroprevalence among blood donors in Istanbul, Turkey: how effective are the changes in the national blood transfusion policies?

The national blood transfusion policies have been changed significantly in recent years in Turkey. The purpose of this study was to determine the prevalence of HBV, HCV, and HIV in blood donors at the Red Crescent Center in Istanbul and to evaluate the effect of changes in the national blood transfusion policies on the prevalence of these infections. The screening results of 72695 blood donations at the Red Crescent Center in Istanbul between January and December 2007 were evaluated retrospectively. HBsAg, anti-HCV, and anti-HIV-1/2 were screened by microparticle enzyme immunoassay (MEIA) method. Samples found to be positive for anti-HIV 1/2 and anti-HCV were confirmed by Inno-Lia HCV Ab III and Inno-Lia HIV I/II Score, respectively. The seropositivity rates for HBsAg, anti-HCV, and anti-HIV-1/2 were determined as 1.76%, 0.07%, and 0.008%, respectively. Compared to the previously published data from Red Crescent Centers in Turkey, it was found that HBV and HCV seroprevalances decreased and HIV seroprevalance increased in recent years. In conclusion, we believe that the drop in HBV and HCV prevalence rates are likely multifactorial and may have resulted from more diligent donor questioning upon screening, a higher level of public awareness on viral hepatitis as well as the expansion of HBV vaccination coverage in Turkey. Another factor to contribute to the decreased prevalence of HCV stems from the use of more sensitive confirmation testing on all reactive results, thereby eliminating a fair amount of false positive cases. Despite similar transmission routes, the increase in HIV prevalence in contrast to HBV and HCV may be linked to the increase in AIDS cases in Turkey in recent years.

Relationship between human papillomavirus infection and overexpression of p53 protein in cervical carcinomas and lymph node metastases.

Overexpression of p53 protein is common in cervical carcinoma. We investigated archival biopsies from 26 cervical cancer patients (24 with available lymph nodes) to determine the relationship between p53 overexpression and HPV infection at the cervix and lymph nodes. Twelve cervical carcinoma patients had p53 protein in cervical biopsies detectable by immunohistochemistry using monoclonal antibody DO‐1, and 22 were positive for HPV DNA in polymerase chain reaction assays (16 contained HPV‐16; 3, HPV‐18; and, 3 HPV‐X). Seven cervical cancer patients had one or more lymph nodes positive for p53 protein, and all but one of these were concordantly p53 positive at the cervix. However, detection of p53 protein in cervical biopsies was predictive neither of the expression of p53 at draining lymph nodes (P > 0.1) nor of the occurrence of metastases (P > 0.1). Fourteen patients were positive at one or more lymph nodes for HPV DNA. Cervical positivity for HPV DNA was associated significantly with concordant HPV positivity at the lymph nodes (P = 0.039) and was predictive of metastases (P = 0.019). There was no association between positivity for p53 and for HPV DNA at primary cervical carcinomas or at the lymph nodes (all P > 0.1). We conclude that, although detectable p53 protein is a common feature of cervical carcinomas, it is not predictive of metastases and is independent of HPV infection. J. Med. Virol. 53:111–117, 1997.

Detection of human papillomavirus type-16 DNA utilising microtitre-plate based amplification reactions and a solid-phase enzyme-immunoassay detection system

The development of a nested polymerase chain reaction (PCR) assay to detect low concentrations of human papillomavirus type-16 (HPV-16) DNA for epidemiological studies is described. The PCR utilises primers located in the E5 open reading frame, has an analytical sensitivity of 4 HPV-16 genomes and does not produce amplicons from other common genital HPVs (types-6, -11, -18, -31 and 33). This assay was carried out in 96-well plates utilising internal primers labelled with dinitrophenol (DNP) and biotin so that amplicons can be captured onto streptavidincoated plates and detected using an alkaline phosphatase-labelled monoclonal antibody to DNP. The assay was effective for detecting HPV-16 DNA in plasmids, cell-lines and, both freshly collected or archival (formalin-fixed/paraffin embedded) clinical specimens. This system is therefore suitable for epidemiological studies to identify individuals infected with HPV-16 DNA in episomal form who may be at increased risk of developing anogenital carcinomas.

The significance of repeat testing in Turkish blood donors screened with HBV, HCV and HIV immunoassays and the importance of S/CO ratios in the interpretation of HCV/HIV screening test results and as a determinant for further confirmatory testing

The purpose of this study was to investigate the intra‐assay correlations amongst initial reactive and repeat screening results used in enzyme immunoassays (EIAs) for hepatitis B virus (HBV), hepatitis C virus (HCV) and HIV in blood donors. This study evaluated the value of using the power of the signal to cut‐off (S/CO) ratio index for confirming anti‐HCV/HIV reactive screening results, thereby touching upon the utility of S/CO indices in determining whether further confirmatory testing was necessary. Screening test results of the 72 695 blood donors were evaluated over a 1‐year period. Correlation analysis among each initial test and retests was done by Pearson r test. Appropriate S/CO values to determine the need of the confirmation testing was investigated by ROC analyses. EIA intra‐assay correlations were of statistical significance and were determined as follows: 0·948 for anti‐HCV, 0·827 for anti‐HIV and 0·948 for HBsAg. The threshold S/CO ratio values which predicted more than 95% of the confirmation test result were 3·8 for HCV and 5·6 for HIV. We were able to demonstrate a strong level of intra‐assay correlation amongst EIAs, thereby eliminating the need for repetition of the screening test. Hence, we suggest that repeat screening should only be limited to HBV and HIV tests with low EIA S/CO ratios. Thus, using the power of the S/CO ratio in determining the need for HCV confirmation testing can be a cost‐effective measure, especially if the S/CO value is ≥3·8.

Leptospirosis in Istanbul, Turkey: a wide spectrum in clinical course and complications.

Patients with high fever and multiorgan involvement were investigated for the determination of frequency, clinical course and complications of leptospirosis in Istanbul. Leptospirosis was determined in 22 cases among the 35 hospitalized patients that were prediagnosed as leptospirosis according to ‘Probable Leptospirosis Diagnosis and Follow-up’ form. Among the leptospirosis cases 19 were male and 16 were military staff. Mean age was 35.6 y. Dark field examination (DFE), latex agglutination test (LAG), ELISA IgM, leptospirosis culture (LC) and microscopic agglutination test (MAT) were performed to confirm the diagnoses. The most frequent initial symptoms and findings were fever, fatigue, headache, nausea-vomiting and increased muscle sensitivity. Jaundice was noted only in 2 cases. A 74-y-old female patient died after the recurrence of the disease with severe rhabdomyolysis and pulmonary failure. Sagittal sinus thrombosis, perimyocarditis and chronic renal failure were major complications in another 3 patients. ELISA IgM, LC, DFE, LAG and MAT tests were positive in 68, 72, 82, 100 and 100% of the patients, respectively. As a conclusion, diagnosis of leptospirosis is usually overlooked. Clinical awareness, use of probable leptospirosis diagnosis forms and the application of different laboratory methods in the diagnosis of suspected cases may offer the chance to diagnose the leptospirosis accurately.

Detection of human papillomavirus type 16 in microtitre plate based immuno-enzymatic assays: use to determine E5 gene expression in cervical carcinomas

BACKGROUND E5-based nested polymerase chain reaction (PCR) assays and a PCR-enzyme immunoassay (EIA) to detect human papillomavirus type 16 (HPV-16) DNA have been developed. These assays were designed to detect small amounts of HPV-16 DNA for epidemiological studies of subclinical infection. OBJECTIVES The E5 gene of HPV-16 may be lost in some cell lines derived from cervical carcinomas. The aim of this study was to determine if, and how frequently, E5 gene loss occurs in biopsy samples from patients with cervical lesions. STUDY DESIGN Sixteen HPV-16 (E7) DNA positive and five HPV-16 DNA negative cervical lesions (nineteen cervical carcinomas, two cervical intraepithelial neoplasias) were investigated by E5 nested PCR and EIA. RESULTS Overall, 15 of the 16 (93.75%) HPV-16 E7 positive samples were positive for HPV-16 E5 DNA: 14 of 16 (87.5%) were positive by E5 PCR and 15 of 16 (93.75%) were positive by E5 PCR, nested PCR and by PCR-EIA. One of 14 HPV-16 (E7) DNA positive cervical carcinomas was negative for E5 DNA in all three assays. CONCLUSION Loss of the HPV-16 E5 open reading frame (ORF) is a rare event in HPV-16 positive cervical carcinomas and was detected in just one of 14 (7.1%) cases.

Anicteric leptospirosis and renal involvement.

We read the clinical study report entitled ‘‘Acute renal failure: a common manifestation of leptospirosis’’ by Cetin et al. with great interest. We work in another teaching and research hospital serving the same city as the medical center in which the cases described in the article were observed. We prospectively investigated leptospirosis in patients applying to the GATA Haydarpasa Training and Research Hospital with fever and multiorgan involvement between February 2004 and October 2004 (over a 9-month period), and leptospirosis was determined in 39 cases within that time frame. Dark field examination, macroagglutination, culture, ELISA IgM, and microagglutination tests aimed toward leptospirosis were performed, and leptospirosis was diagnosed when at least two tests resulted positive. While only three cases were icteric course leptospirosis (Weil’s disease), 36 (92%) were determined to be anicteric course leptospirosis. This finding was in agreement with the information in the literature to the effect that 90% of leptospirosis cases are anicteric. Urinary abnormalities (leukocyturia and/or hematuria and/or proteinuria) were determined in 26 of the 39 cases diagnosed as leptospirosis (66%), similar to Cetin et al.’s findings. Acute renal failure (ARF), an indicator of poor prognosis, was determined in eight cases. Hyperbilirubinemia was present in only two (25%) of the cases in which ARF occurred. This finding suggests that the classic idea that severe leptospirosis equals icteric leptospirosis may not always be accurate. A severe clinical course was observed in 10 of our leptospirosis cases. ARF was present in eight of these. All responded to supportive therapy and crystallized penicillin (12–24 MIU/day) or doxycycline (200 mg/day b.i.d.). Hemodialysis therapy was administered to only one patient (a 20-year-old male), monitored for 9 months, who was diagnosed with end-stage renal disease due to lack of improvement in kidney functions and was included in a regular hemodialysis program for 4 h, three days a week. Apart from Cetin et al.’s reports and the case we report here, leptospirosis has not been described as a complication leading to ESRD in the literature. It was regarded as particularly interesting that our case had no previous kidney pathology. Two cases presenting with anicteric leptospirosis died. One of these died due to ‘‘pulmonary involvement Address correspondence to Dr. Vedat Turhan, GATA Haydarpasa Egitim Hastanesi İnfeksiyon Hastaliklari ve Klinik, Mikrobiyoloji Servisi, Kadikoy-Istanbul 34668, Turkey; Fax: +90-216-348-7880; E-mail: vedatturhan@yahoo.com

Leptospirosis is still a mortal infection in even today's world

Dear Sir, We read with a great interest the article written by Peric et al. [1] in which 270 leptospirosis cases from eastern Croatia have been reported. This study includes 1 of the largest series of leptospirosis cases. The retrospective data have been collected from the records of Osijek University between the y 1969 and 2003. Interestingly, no lethal outcome has been reported among the 270 leptospirosis cases. The mortality rate of leptospirosis was about 40% in the past and due to improved respiratory support facilities and haemodialysis, it has been reported between 5% and 17% in recent y [2 5]. Risk of mortality increases especially in patients with icteric course or pulmonary involvement [6]. Two patients died among the 44 leptospirosis cases followed in our hospital during 2004 2005 (unpublished data). Pulmonary involvement was prominent in these 2 patients who presented unicteric course of leptospirosis. Leptospirosis could only be diagnosed post mortem in 1 of the patients. It is surprising that no lethal outcome has been reported by Peric et al. in their large series of leptospirosis cases from the records of the last 34 y. The reasons for this discrepancy may be that severe leptospirosis cases might have been admitted or transported to another centre, or severe cases might have died before the leptospirosis specific antibodies became positive. Another reason might be that the patients could have been treated with penicillins, doxycyclines or cephalosporins, empirically, when they were admitted to the health care centre. This might have improved the course of the disease and prevented a lethal outcome. High variability in the mortality rate of leptospirosis may also be due to the absence of standard diagnostic criteria.