SEN Virus Infection in Egyptian Patients With Chronic Hepatitis C and Patients Undergoing Hemodialysis

Maisa Omar, PhD; Samah Saad El-Din, PhD; Nevine Fam, MD; Manal Diab, MD; Mohamed Shemis, PhD; Manar Raafat, MD; Moataz Seyam, MD; Moataz Hssan, MD; Afkar Badawy, PhD; Maha Akl, PhD; Mohamed Saber, PhD

Disclosures

Medscape J Med. 2008;10(12):290 

In This Article

Patients and Methods

Patients and serum samples: The study group consisted of 119 patients who were consecutively examined and followed up at Theodor Bilharz Research Institute in Giza, Egypt. Seventy-four patients had HCV-related chronic liver disease, and 45 patients had uremia and were undergoing maintenance hemodialysis. The chronic liver disease group comprised 49 males and 25 females, with a mean age (standard deviation) of 46 ± 11 years (range, 13-72 years).

Diagnosis of chronic HCV infection was based on the following criteria: 1) detection of HCV RNA or continuous positivity for antibody to HCV (anti-HCV) in serum for more than 6 months; 2) absence of detectable hepatitis B surface antigen; and 3) exclusion of other causes of chronic liver disease. Diagnosis of chronic liver disease was based on prolonged elevation of serum alanine aminotransferase (ALT) levels for more than 6 months. Liver cirrhosis was diagnosed by histopathologic examination or characteristic clinical signs of advanced liver disease. Hepatocellular carcinoma was diagnosed by histopathologic examination of liver biopsy samples or imaging studies and by serum alpha-fetoprotein levels greater than 400 ng/mL. Serum samples were collected from all patients at the time of clinical evaluation and were stored at -70°C until further testing. Eleven patients were receiving alpha-interferon/ribavirin combination therapy for 24 weeks; their serum samples were available before and 12 weeks after they had received this treatment.

The hemodialysis group comprised 38 men and 7 women; their age ranged from 34 to 72 years (mean ± standard deviation, 56 ± 9 years). The duration of hemodialysis ranged from 0.5 to 16 years. A history of blood transfusion was recorded for 34 (66.7%) patients. Maintenance hemodialysis had been performed 3 times a week by using disposable dialyzers with standard acetate dialysate. Serum samples were collected from all patients before the dialysis session and were stored at -70°C.

Twenty-eight healthy volunteers who had no clinical, virologic, or biochemical signs of liver disease provided serum samples and served as a control group.

Methods: All patients were subjected to full clinical assessment, with special emphasis on symptoms and signs of chronic liver disease. Abdominal ultrasonography and upper endoscopy were performed. Liver biopsy specimens were taken from 48 patients for histopathologic examination.

Laboratory tests: Liver biochemical tests, including ALT, aspartate aminotransferase, and total bilirubin, were done by using an autoanalyzer. Commercially available enzyme-linked immunosorbent assays were used to measure serum alpha-fetoprotein levels (DiaMetra, Milano, Italy), hepatitis B surface antigen, antihepatitis B core antibody (DiaSorin, Saluggia,Italy), and third-generation anti-HCV antibody (version 4 from the same laboratory); HCV serotyping (serotypes 1 to 6) was also done (Murex-Biotech Ltd, Dartford, United Kingdom). HCV RNA and hepatitis G virus RNA were detected by means of qualitative reverse transcriptase polymerase chain reaction (RT-PCR) with primers in the 5' noncoding region, as reported elsewhere.[17,18] TTV DNA was detected by nested PCR using primers as designated by Takahashi and colleagues.[19]

Detection of SEN virus DNA by PCR: Total DNA was extracted from 100 µL of serum, as previously described by Boom and colleagues.[20] The extracted DNA was resuspended in 60 µL TE buffer. The oligonucleotide primers used were according to the method of Tanaka and colleagues.[4] For PCR, 50 µL of reaction mixture containing 10 µL of the DNA sample, 1X PCR buffer (10 mM Tris-h ydrochloride [pH, 9.0], 50 mM potassium chloride, 1.5 mM magnesium chloride, 0.01% gelatin, and 0.1% TritonX-100), 200 µM of each deoxyribonucleotide triphosphate, 20 pmol of each primer (sense primer for SEN virus-D, 5'-GTAACTTTGCGGTCAACTGCC-3'; sense primer for SEN virus-H, 5'-GGTGCCCCTWGTYAGTTGGCGGTT-3' [W = A or T]; universal antisense primer, 5'-CCTCGGTTKSAAAKGTYTGATAGT-3' [K = G or T, S = C or G, and Y= C or T]), and 1.5 U of Taq DNA polymerase were amplified in a thermal cycler (PTC- 200,MJ Research, St Bruno, Quebec, Canada) for 40 cycles. Each cycle consisted of denaturation at 95°C for 60 seconds, primer annealing at 55°C for 30 seconds, and extension at 72°C for 60 seconds, with a final extension step at 72°C for 10 minutes.[8] The amplified products (231 base pairs for SEN virus-D and 230 base pairs for SEN virus-H) were separated by using 3% agarose gel electrophoresis, stained with 0.3 µg/mL ethidium bromide, and visualized by using an ultraviolet transilluminator.

All methods were performed according to the principles outlined in the Declaration of Helsinki.[21] Institutional review board approval was obtained.

Statistical analysis: Numeric data were compared by using a Mann-Whitney U test, and categorical data were compared by using a chi-square test. A P value of .05 or less was considered to represent a statistically significant difference. Data analysis was performed using by SPSS software, version 10 (SPSS Inc, Chicago, Illinois).

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