anti-HCV Positive Patients Research Articles

Detection of Serum HCV RNA by Polymerase Chain Reaction in Patients with Chronic Hepatitis C

Hepatitis C virus (HCV) RNA was isolated from the sera of 15 anti-HCV positive patients. cDNAs were synthesized by reverse transcription and amplified by two round nested polymerase chain reaction (PCR). HCV infection was confirmed by the detection of 256 base pairs long DNA fragments on the 2% agarose gels in the electrophoresis of PCR products with the positive and negative controls. 10 of 15 infection were confirmed by this molecular technique.

Response to interferon of anti-HCV positive chronic hepatitis patients: The impact of quantitative HCV-RNA RT-PCR: [formula omitted] Dept. of Gastroenterol Molinette Hosp. Torino, 2Div. of Med. “Crespi”, Niguarda Hosp. Milano, Italy and 3Roche Mol. Div. Basel, Switzerland

Response to interferon of anti-HCV positive chronic hepatitis patients: The impact of quantitative HCV-RNA RT-PCR: [formula omitted] Dept. of Gastroenterol Molinette Hosp. Torino, 2Div. of Med. “Crespi”, Niguarda Hosp. Milano, Italy and 3Roche Mol. Div. Basel, Switzerland

HCV viremia in hemodialysis patients: detection by a DNA enzyme immunoassay for amplified HCV sequences.

The aim of this study was the detection of HCV viremia in both anti-HCV antibody positive and negative hemodialysis patients. Sera from 75 patients on extracorporeal blood purification in the same dialysis unit were analyzed. Anti-HCV antibodies were detected using a 2nd-generation ELISA assay and in all positive cases a RIBA 3rd-generation test was performed. HCV-RNA was tested by a reverse transcription-nested polymerase chain reaction (RT-PCR) assay with primers located in the 5' region. PCR products were analyzed by a nonradioactive hybridation assay. The presence of anti-HCV antibodies was detected in 30 (40%) patients by means of ELISA II test; 28 of them were RIBA III positive and two indeterminate. Twenty-four of the 30 HCV Ab ELISA II positive patients (80%) were HCV-RNA positive (23 RIBA III positive and 1 indeterminate). Six anti-HCV Ab ELISA II positive patients tested negative for HCV-RNA (20%); 5 of these patients were also positive for anti-HCV antibodies with a RIBA III test and 1 was indeterminate. None of the anti-HCV negative patients was HCR-RNA positive. In two cases we documented the disappearance of viremia after an acute HCV infection, with the persistence of antibody reactivity. In conclusion, anti-HCV antibody positive hemodialysis patients should be considered as potentially infectious.

Long interval between HCV infection and development of hepatocellular carcinoma

A high prevalence of HCV infection has been reported in patients with hepatocellular carcinoma. The progression from acute transfusion-associated hepatitis to hepatic cirrhosis and hepatocellular carcinoma has been suggested in several studies to be very long. We have investigated the prevalence of anti-HCV and the interval between HCV infection and hepatocellular carcinoma among 191 consecutive patients with cirrhosis and liver-cell carcinoma. Serum samples from 191 patients with cirrhosis and hepatocellular carcinoma, consecutively diagnosed in our hospital between 1988 and 1993, were tested for serological markers of HBV and HCV infection. One hundred and forty-eight patients (77.5%; 95% confidence interval (c.i): 76% to 80%) were anti-HCV positive by 2nd generation enzyme immunoassay (confirmed by 2nd generation recombinant immunoblot assay) and 152 patients (79.5%; 95% c.i: 76% to 80%) were anti-HCV positive by 3rd generation enzyme immunoassay, while only 14 (7.4%; 95% c.i: 5% to 10%) were HBsAg positive. Of the 29 anti-HCV positive patients with previous transfusion, the interval between the date of blood transfusion and the diagnosis of hepatic cirrhosis was 24 +/- 12.5 years and that of hepatocellular carcinoma was 26.8 +/- 12.4 years. These results confirm the high prevalence of HCV infection in patients with hepatocellular carcinoma and the slow sequential progression from HCV infection through cirrhosis and hepatocellular carcinoma.

Liver cytosolic 1 antigen-antibody system in type 2 autoimmune hepatitis and hepatitis C virus infection.

Within the multiform liver/kidney microsomal (LKM) family, a subgroup of sera that reacts with a liver cytosolic (LC) protein has been isolated and the new antigen-antibody system is called LC1. Unlike LKM antibody type 1 (anti-LKM1), anti-LC1 is said to be unrelated to hepatitis C virus (HCV) infection and has therefore been proposed as a marker of 'true' autoimmune hepatitis type 2. Altogether 100 LKM1 positive sera were tested by immunodiffusion (ID). Twenty five gave a precipitation line with human liver cytosol; 17 of the 25 also reacted with rat liver cytosol. Thirteen of the 25 sera were anti-HCV positive by second generation ELISA: anti-HCV positive patients were significantly older (p < 0.001) and tended to have less active disease. No difference in anti-LC1 titre or ID immunoreactivity was found between anti-LC1/anti-HCV positive and anti-LC1/anti-HCV negative cases. In Western blotting experiments, 14 of 24 ID positive sera recognised a 58 kD protein of the human cytosolic fraction and 11 gave a similar reactivity when tested with human microsomes, suggesting the presence of the LC1 target antigen also in the microsomal preparation. Western blotting reactivity was similar for both anti-HCV positive and negative sera. These data confirm the existence of the LC1 antigen-antibody system that partially overlaps with LKM1, and that it is an additional marker of juvenile autoimmune hepatitis type 2. It does not, however, discriminate between patients with and without HCV infection.

Efficacy and tolerance of α-2b interferon therapy on HCV infection of hemodialyzed patients

A high frequency (25%) of anti-hepatitis C virus (HCV) antibodies is observed in French hemodialyzed patients; this is associated with detectable viremia in 85% and results in chronic hepatitis in more than 90%. We conducted a pilot study to examine the tolerance and efficacy of alpha-2b Interferon therapy upon HCV infection in hemodialyzed patients. Nineteen anti-HCV positive hemodialyzed patients were given a standard alpha-2b interferon regimen (3 megaunits subcutaneously three times weekly, following each hemodialysis) over six months as a treatment of biopsy-proven chronic hepatitis (N = 16) or acute hepatitis (N = 3). Thirteen of these 19 had increased levels of aminotransferase at the time of treatment. Serum HCV RNA was tested qualitatively and quantitatively by the polymerase chain reaction and the bDNA test, respectively, at the beginning and at the end of antiviral treatment, and a third time at least six months after the end of therapy (mean follow-up 18 +/- 9 months). HCV genotype was determined in the 15 patients who had detectable HCV RNA before treatment. The biological response (long-term response, relapse or non-response) was defined as usual according to the serum aminotransferase levels during therapy and at least six months after. A post-treatment liver biopsy, allowing comparison with semiquantitative pathological scores, was performed in 14 patients. Only one of the 19 treated patients did not complete therapy because of poor tolerance, while 18 of the 19 fairly tolerated a complete six month course of alpha-2b interferon.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatitis C infection and viremia in Dutch Hemophilia patients

Serum samples from 316 patients visiting the Dutch National Hemophilia Center were collected from 1979 to 1993 and stored at -30 degrees C. Patients were placed into three different groups: 1) patients ever treated with large pool non-hepatitis C virus (HCV)-safe concentrate (n = 179); 2) patients treated with cryoprecipitate (n = 125); and 3) patients treated exclusively with HCV-save concentrate (n = 12). In order to examine the prevalence of HCV infection in the different treatment groups serum samples were tested retrospectively for anti-HCV antibody using second generation enzyme-linked immunosorbent assay (ELISA) and recombinant immunoblot assay (RIBA-2). Significant differences in the prevalence of HCV infection were found between these 3 groups (group 1: 99%, group 2: 66%, group 3: 0%). The safety of currently administered clotting products is demonstrated in 57 patients who remained without HCV markers between 1989 and 1993. To examine the natural course of HCV infection fresh-frozen plasma samples were obtained recently from a subgroup of 277 hemophilia patients for HCV-RNA detection by a well-validated cDNA-PCR assay. In contrast to other reports, no evidence was found for seronegative HCV carriers. None of 52 patients without anti-HCV had detectable HCV-RNA. Of 225 patients with anti-HCV, 182 (81%) were HCV-RNA positive. None of 39 anti-HCV positive patients with a negative HCV-RNA reaction had serum alanine aminotransferase (ALT) levels above 50 U/l, whereas 44% of HCV-RNA positive patients had persistently elevated ATL levels above 50 U/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of hepatitis C virus RNA in the cell fraction of saliva before and after oral surgery

The presence of hepatitis C virus (HCV) RNA in serum, whole saliva, and saliva from the submaxillary glands was investigated before and after oral surgery. The presence of HCV RNA (positive and negative-strand RNA) was determined in serum and saliva by a nested polymerase chain reaction in 26 anti-HCV positive patients, of whom 11 were coinfected with human immunodeficiency virus-1. Oral surgery was carried out on five occasions on four of the patients. HCV RNA was detected in the sera of 23 of 26 (88%) patients, and in the saliva of 4 of the 23 (17%) of the viremic patients. In all four cases, HCV RNA was detected only in the cell fraction derived from centrifugation of whole saliva. Negative-stranded HCV RNA was not detected. At one of five occasions of oral surgery, HCV RNA was detected in saliva sampled immediately after surgery, but not before or 24 hours after surgery. The results suggest that HCV is present in saliva in less than 25% of HCV viremic persons. The presence of the virus in saliva is restricted to the cell fraction. Thus, saliva may serve as a possible, but low, nonparenteral transmission route of HCV. Contamination of saliva by blood during and after oral surgery may result in an increased risk of viral exposure. Except for trauma caused by sharp instruments during surgery, this might contribute to the higher HCV seropositivity found among dentists.

Hepatitis B and D genomes in hepatitis B surface antigen negative patients with chronic hepatitis C.

Hepatitis B and hepatitis D viral genomes were tested by nested polymerase chain reaction in the serum and liver of 69 hepatitis B surface antigen (HBsAg) negative, anti-hepatitis C virus (HCV) positive patients (47 with HCV RNA and 22 without HCV RNA). Serum hepatitis B virus (HBV) DNA-was detected in 49% of the patients with HCV-RNA and in 64% of those without HCV-RNA. Furthermore, intrahepatic HBV-DNA was found in four of five (80%) of the biopsies analysed. Delta genome was found in 72% and 73%, respectively, of the anti-HCV positive patients with or without HCV-RNA. In addition, intrahepatic delta virus genome was detected in another four liver biopsies studied. In the group of patients with HCV-RNA, the simultaneous presence of hepatitis B and D genomes was statistically higher in transfused patients than in drug addicts, or in those with an unknown infection route (P < 0.001). These results show a high percentage of B and D genomes in HBsAg negative patients with anti-HCV, irrespective of the presence or absence of the HCV genome. However, the clinical implications of this finding should be examined in future studies.

Detection of hepatitis C virus-RNA by polymerase chain reaction in dental surgeries.

The mean prevalence of anti-hepatitis C virus (HCV) in Italy is 0.87%. It reaches 2% in Campania, Southern Italy. Approximately 50% of community acquired non-A, non-B (NANB) hepatitis cannot be associated with known parenteral exposure. A recent Italian study has shown that the only demonstrable risk factor in 9% of acute C/NANB hepatitis is dental treatment. There are no data on direct contamination by HCV of dental surgeries. Possible environmental contamination by HCV-RNA was investigated in dental surgeries after treatment of anti-HCV and HCV-RNA positive patients. Thirty-five anti-HCV and HCV-RNA positive patients with chronic hepatitis underwent dental treatment and were enrolled in this study. Eight had chronic persistent hepatitis (CPH), 23 chronic active hepatitis (CAH), and 4 cirrhosis. A total of 328 samples collected from instruments and surfaces were tested after dental treatment of 35 anti-HCV positive patients. The presence of HCV-RNA was determined by polymerase chain reaction (PCR) to evaluate contamination of instruments and surfaces in dental surgeries. Twenty (6.1%) out of 328 collected samples were positive for HCV-RNA. The positive samples were from work benches (two), air turbine handpieces (one), holders (four), suction units (one), forceps (four), dental mirrors (two), and burs (six). Our data indicate that there is extensive contamination by HCV of dental surgeries after treatment of anti-HCV patients and that if sterilisation and disinfection are inadequate there is the possible risk of transmission to susceptible individuals.

Transplantation of kidneys from donors with hepatitis C antibody into recipients with pre-transplantation anti-HCV

Hepatitis C virus (HCV) is transmitted by organ transplantation. Consequently, several organ procurement organizations have imposed a moratorium on use of organs from anti-HCV positive donors. Because of the inadequate supply of cadaver kidneys for transplantation, we adopted a policy to transplant kidneys from anti-HCV donors into anti-HCV positive recipients. During the period between March 1990 and December 1992, 24 anti-HCV positive dialysis patients received a kidney from anti-HCV positive donors (group I) and 40 anti-HCV positive patients received a kidney from anti-HCV negative donors (group II). We compared the prevalence of liver disease, anti-HCV, HCV RNA, graft and patient survival between groups. Pre-transplantation 17 of 24 (71%) patients in group I and 31 of 40 (79%) of patients in group II had serum HCV RNA. Post-transplantation follow-up was 26 +/- 8 months and 30 +/- 10 months in groups I and II, respectively. During follow-up, elevated ALT levels were present in 7 of 24 (29%) and 16 of 40 (40%) of patients in groups I and II, respectively (P > 0.05). Post-transplantation, all patients in both groups retained anti-HCV. The prevalence of HCV RNA post-transplantation was 22 of 23 (96%) patients in group I and 30 of 39 (77%) of patients in group II (P > 0.05). Graft and patient survival in group I (96% and 100%, respectively) were not significantly different from those in group II (93% and 98%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

The Localization of HCV and the Expression of Fas Antigen in the Liver of HCV-related Chronic Liver Disease.

We have investigated the localization of HCV in the liver and the expression of Fas antigen, involved in hepatocellular death through apoptosis, to examine the relation between HCV infection and Fas antigen cellular expression. The localization of HCV was investigated by in situ hybridization (ISH), and the expression of Fas antigen was determined by immunohistochemistry using an anti-Fas monoclonal antibody, in 20 surgically-resected liver specimens from anti-HCV positive patients. The Methylgreen/Pyronin Y technique clearly showed well-preserved tissue RNA in 8 of 20 livers. ISH determined the co-localization of signals for plus and minus strands of HCV-RNA in the cytoplasm of hepatocytes. Fas antigen was more strongly expressed on the plasma membrane of the periportal hepatocytes than on the plasma membrane of the centrilobular hepatocytes, even when HCV-RNA was found to be widely distributed in the hepatocytes throughout the liver lobule. Furthermore, Fas antigen positive hepatocytes were frequently observed close to the area of piecemeal necrosis associated with a high incidence of apoptosis. The results suggested that the expression of Fas antigen does not occur by HCV infection alone, but probably requires several other factors.

Risk Factors for Hepatitis C Virus Infection in Canadian Patients with Chronic Type C Hepatitis

Previous reports from the United States indicate that as many as 40% of patients with chronic hepatitis C virus (HCV) have no identifiable risk factor for HCV infection. To determine whether the same is true of Canadian patients with chronic HCV the records of 89 anti-HCV positive patients referred to the authors' tertiary care centre for evaluation of liver disease were reviewed. Each patient had been specifically asked about the following risk factors: previous blood transfusions; intravenous drug abuse; homosexual activity; sexual promiscuity (multiple sexual partners or a history of sexually transmitted diseases); tattoos made with nonsterile techniques; and ear piercing using nonsterile techniques. The results of the study revealed that 76 of 89 patients (85%) had at least one risk factor for HCV exposure, 38 (43%) had only one risk factor, 19 (21%) had two, 12 (14%) had three and the remaining three patients (3%) had four. The most common risk factor was a history of intravenous drug abuse (30 of 89 patients, 34%) followed by sexual promiscuity (28, 32%), previous blood transfusions (21, 24%), tattoos (17, 19%), homosexual contacts (seven, 8%) and ear piercing (five, 6%). Contrary to a recent report identifying sexual contact as an independent risk factor for HCV infection, only four cases (5%) were found where sexual promiscuity was identified as the only risk factor. In conclusion, these findings indicate that a possible source of HCV infection can be identified in a large majority of Canadians referred to an urban centre with chronic HCV infection.

Hepatocellular expression of HLA-A, B, C molecules predicts primary response to interferon in patients with chronic hepatitis C.

Primary response rate to alpha-interferon (IFN) is about 50% in patients with chronic hepatitis C. Criteria for predicting a positive primary response are lacking. HLA-A,B,C molecule expression is known to be stimulated by viral infections. In 36 consecutive interferon-treated anti-HCV positive patients with an available frozen liver biopsy sample, the predictive value of liver HLA-A,B,C expression, and of histologic, clinical, and biochemical parameters was evaluated. Response to treatment was defined by normalization of transaminases, and disappearance of serum HCV-RNA within 3 months. According to these criteria, 17 patients were classified as nonresponders and 19 were classified as responders. The pattern of HLA-A,B,C hepatocellular positivity varied from normal (negative or occasional faint staining of hepatocellular membranes) to diffuse, strong "honeycomb" positivity. The highest scores of positivity were found in nonresponder patients. The discriminant capacity of HLA-A,B,C scores of positivity was compared with clinical, biochemical and histologic parameters by discriminant analysis. HLA-A,B,C expression was found to be the main discriminant parameter, in addition to alkaline phosphate (ALP) and gamma-glutamyl-transpeptidase (GGT) which added little additional information. The higher hepatocellular expression of class I MHC molecules in nonresponder cases may reflect a different viral effect on hepatocytes, which is induced by different HCV genotypes or levels of viremia. From a clinical point of view, the pretreatment HLA-A,B,C pattern of positivity represents a powerful tool in the selection of patients for interferon treatment.

Hepatitis C virus infection among elderly patients in a geriatric hospital

The aim of the study was to determine the prevalence of anti-hepatitis C virus (HCV) antibodies and HCV viremia among elderly patients in a geriatric hospital in Jerusalem, Israel. Serum samples from 273 patients were analyzed for the presence of anti-HCV antibodies. Serum samples of anti-HCV positive patients were analyzed for HCV RNA after amplification with the polymerase chain reaction (PCR). The samples were also analyzed for the presence of HBsAg and the HBsAg positive samples were analyzed for the presence of hepatitis B virus (HBV) DNA. Anti-HCV antibodies were found in 5 patients (1.8%). HCV RNA was detected in one of the 5 patients. HbsAg was found in 2 patients (0.7%). None had detectable HBV DNA. The findings of the study indicate that seropositivity for hepatitis C virus is relatively common among the elderly population studied. Most of those patients are probably not HCV carriers.

A twelve year natural history of hepatitis C virus infection in hemodialyzed patients

A prospective non-A, non-B follow-up program, implemented in a hepatitis B surface antigen-free dialysis unit, enabled us to report on the natural history of hepatitis C virus (HCV) infection in hemodialyzed patients between 1980 and 1992. For this program, every patient was prospectively monitored every two weeks for alanine amino transferase (ALT) activity, and every month for gammaglutamyl transpeptidase (GGT) activity and systematic collection of frozen sera. Sequences of stored sera from 217 patients were repeatedly tested for anti-HCV antibodies using second generation assays. Eighty-six of the 217 patients (39.6%), including 61 of the 67 patients with non-A, non-B hepatitis (91%), had HCV infection repeatedly evidenced by positive ELISA in all, and confirmed by RIBA in 84 of 86 (97.5%). In addition, 19 out of 23 patients (82.6%) were positive for HCV RNA by the polymerase chain reaction (PCR). Of the 86 anti-HCV positive patients, 41 had previously acquired HCV infection, and 45 seroconverted during chronic dialysis. Of these, all but one patient developed hepatitis with raised ALT activity which lasted for at least six months in all. Only 29 of 45 patients (64.5%) had a history of blood transfusion. Seventy-eight of the 86 patients (91%) who were followed up for one to 11.5 years (median 5) retained anti-HCV for several years. Nineteen liver biopsies performed in 16 patients showed chronic active hepatitis in 8 (50%) and hepatocellular carcinoma without cirrhosis in one patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Displacement of hepatitis B virus by hepatitis C virus as the cause of continuing chronic hepatitis

Background/Aims: It has been shown that hepatitis C virus (HCV) superinfection may suppress hepatitis B virus (HBV) leading to hepatitis B surface antigen (HBsAg) clearance and that hepatitis may persist after HBsAg clearance in a few patients. The role of HCV in continuing hepatitis after termination of chronic HBsAg antigenemia remains to be explored in a series of patients. Methods: HCV markers were studied using second generation enzyme immunoassay and polymerase chain reaction with reverse transcription and were compared between 41 patients with persistent alanine aminotransferase (ALT) elevation (hepatitis group) and 82 age/sex-matched patients with normal ALT (control group) after HBsAg clearance. Results: Twenty-six (63%) of the 41 hepatitis group patients were seropositive for antibodies to HCV (anti-HCV) compared with only 4 (5%) of 82 controls (P < 0.0001). Six patients of the hepatitis group and 1 control had an episode of acute hepatitis C with seroconversion of anti-HCV 1–68 months before HBsAg clearance. Of those seropositive for anti-HCV, serum HBV DNA was not detectable, and serum HCV RNA was detected in 23 (88.5%) of the 26 hepatitis patients but none of the 4 controls (P < 0.001). Liver biopsy in 6 anti-HCV positive patients with continuing hepatitis showed features compatible with chronic hepatitis C. HCV RNA, but not HBV DNA, was detected in liver tissues of these 6 patients. Conclusions: The results suggest that HCV may usurp the role of HBV in chronic hepatitis and act as the major cause of continuing hepatitis or ALT elevation after HBV/HBsAg clearance.

Hepatitis C: Description of a highly sensitive method for clinical detection of viral RNA

To improve the sensitivity of hepatitis C virus RNA (HCV RNA) detection in serum by ‘nested’ polymerase chain reaction (PCR), primers belonging to 5' non-coding (5'NC) regions were used to compare the classical phenol/chloroform technique by using the proteinase K and silica gel technique with guanidinum thiocyanate. The silica gel technique was found to be more efficient and sensitive for the extraction and purification of viral RNA from serum samples. The silica gel technique also avoids contact with hazardous volatile chemicals like phenol and chloroform and provides a better protection for viral RNA. Furthermore, the RNA detection sensitivity was greatly improved by modifying the buffer for reverse transcription and PCR. Using silica gel extraction, and the modified buffer, viral RNA was detected in 699 sera from anti-HCV second generation ELISA positive patients. These sera were distributed in second generation RIBA confirmed, indeterminate and non confirmed groups with PCR positive rates of 71.4%, 45.7% and 15.8%, respectively. Two out of 227 ELISA negative patients showed the presence of HCV RNA in serum. An association between the presence of antibodies against a determined viral peptide and viremia was not detected.

Disease Activity of Hepatitis C Correlates with Single-stage Polymerase Chain Reaction Detection of Hepatitis C Virus

Hepatitis C virus (HCV) RNA was amplified and detected in the serum of 17 anti-HCV antibody positive patients using a single-stage (30 cycles) polymerase chain reaction (PCR). Specific amplification, targeted to the C-100 protein (anti-HCV) region, was confirmed by direct sequencing of the PCR product. Single-stage PCR detected the virus in 11 patients. Polymerase chain reaction-positive patients demonstrated a significantly higher histologic activity index (10.3 + 1.2 standard error of mean [SEM] than those testing negative (5.8 + 1.5 SEM, P < .05). Nine of 11 PCR-positive patients exhibited a rise in alanine transaminase (ALT) values within 1 month of assay, compared with only 1 of 6 PCR-negative patients. The correlation between rising ALT levels and PCR positivity was significant (P < .01). Direct sequencing revealed mutability in all cases, some of which resulted in amino acid substitutions. The authors concluded that HCV detection using single-stage PCR correlates with biochemical and histologic features of disease activity. Mutability is likely an important feature of HCV pathobiology and may significantly affect detection methods.

Detection of serum HCV-RNA by PCR in anti-HCV positive patients

Detection of serum HCV-RNA by PCR in anti-HCV positive patients