[Hepatitis C; a new virus for an old disease].
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Hepacivirus↗
Biomedical subjects
H W Reesink
Publications and source records attributed to H W Reesink.
Prevention of Yersinia enterocolitica growth in red-blood-cell concentrates.
In response to concern about Yersinia enterocolitica contamination of blood products, we have studied the effects on Y enterocolitica growth of holding whole blood at 22 degrees C for 20 h and then removing leucocytes. Thirty pools of three bags of blood were inoculated with Y enterocolitica (2 x 10(1)-3 x 10(4) colony-forming units/ml). One bag in each pool was processed to red-blood-cell concentrate after 6 h at 4 degrees C (RBC); the other two were held at 22 degrees C for 20 h before processing to buffy-coat-depleted RBC (BCd-RBC). One of these bags was then depleted of leucocytes by filtration (Ld-RBC). All bags were stored at 4 degrees C for 5 weeks. RBC bags showed Y enterocolitica growth after the shortest storage times, followed by BCd-RBC then Ld-RBC (p less than 0.03-0.001). We recommend that whole blood should be held at 22 degrees C to make use of inherent bactericidal activity; leucocytes should then be removed.
Blood Bactericidal Activity↗
Sensitivity of an anti-HCV core peptide ELISA.
A newly developed antibody assay based on a synthetic peptide of 15 amino acids derived from the core region of the hepatitis C virus (HCV) genome was evaluated in serum and plasma panels of (A) 225 haemophiliacs and (B) 44 patients with chronic non-A, non-B (NANB) hepatitis, and in (C) sequential serum samples of 9 patients with transfusion transmitted HCV infection. The new anti-core peptide ELISA was compared with the anti-C100 ELISA. For confirmation of HCV infection, samples were tested in a 4-antigen recombinant immunoblot assay (4-RIBA) and samples of panels B and C were also assayed in cDNA-polymerase chain reaction (PCR). In two panels with a high prevalence of HCV infection (88.4 and 70.5% in haemophilia and NANB hepatitis patients, respectively), the sensitivity of the anti-core peptide ELISA did not differ significantly from the sensitivity of the anti-C100 ELISA. The sensitivity of the new assay as compared with the anti-C100 assay was found to be 0.84 [95% confidence interval (CI): 0.78-0.89] versus 0.92 (95% CI: 0.87-0.95) in haemophilia patients and 0.71 (95% CI: 0.52-0.86) versus 0.84 (95% CI: 0.66-0.95) in NANB hepatitis patients. In sequential serum samples of patients with transfusion-transmitted HCV infection antibodies to the core peptide (in 6/9 patients) appeared later than antibodies to C100 (in 7/9 patients): 168 (range: 70-322) and 143 (range: 59-365) days after transfusion, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Patterns of serological markers in transfusion-transmitted hepatitis C virus infection using second-generation HCV assays.
A semiautomated dot blot assay and cDNA polymerase chain reaction (PCR) were used to study longitudinal anti-hepatitis C virus (HCV) recognition patterns in relation to presence of HCV-RNA in transfusion recipients and their infectious donors. In 9 recipients, 4 different patterns of HCV infection were observed: (A) persistent HCV carriage accompanied by chronic hepatitis in 6, (B) acute resolved hepatitis, but persistent HCV replication in one, and (C) continuous HCV replication without hepatitis in one and (D) acute resolved hepatitis with clearance of infection in one. This last self-limited infection was characterized by the disappearance of HCV-RNA as well as anti-HCV reactivity. In contrast, antibody reactivity persisted in 7 of 8 patients with chronic HCV infection who could be followed until 1990. Seven of the 9 recipients developed antibodies to all recombinant peptides in dot blot assay; one became positive for anti-C33 and anti-core and one developed anti-core only. The sequence of appearance of antibodies differed among individual patients. In 7 patients with full anti-HCV recognition patterns, the sequence of events was (mean and limits in days after transfusion): onset of hepatitis at day 50 (22-74), seroconversion of anti-C33 at day 91 (59-129), anti-core at day 133 (54-203), and anti-C100 at day 143 (59-365). The incorporation of C33 and core proteins, in addition to C100, in the second generation anti-HCV ELISA enhanced the detection rate in the HCV-infected transfusion recipients from 7/9 (78%) to 9/9 (100%).(ABSTRACT TRUNCATED AT 250 WORDS)
Biomarkers↗
HCV and blood transfusion.
Posttransfusion hepatitis remains a threat to transfusion therapy. Testing for increased ALT levels has been used in an attempt to reduce this risk. Presence of the infectious agent, hepatitis C virus (HCV), appears to be a much more sensitive criterion. Stored serum samples from transfusion blood as well as recipients of transfusion were tested by ELISA, RIBA and PCR for the presence of HCV. The results show that RIBA and PCR are about equally sensitive and are able to detect HCV positivity in many sera that might have been otherwise transfused. Routine screening for the presence of virus will dramatically reduce the danger of hepatitis infection to transfusion patients.
Blotting, Western↗
Detection of hepatitis C viral RNA sequences in fresh and paraffin-embedded liver biopsy specimens of non-A, non-B hepatitis patients.
In this study methods of HCV-RNA detection in fresh frozen and formalin-fixed, paraffin-embedded liver biopsies are described. Of 22 untreated chronic non-A, non-B hepatitis patients and 6 control patients, a plasma sample and part of a liver biopsy were freshly frozen for hepatitis C virus (HCV) cDNA-PCR. From 16 of the same non-A, non-B hepatitis patients and from 5 of the same control patients formalin-fixed, paraffin-embedded liver tissue from the same biopsy was available also for HCV cDNA-PCR. In 13 of 22 non-A, non-B hepatitis patients HCV-RNA could be detected in plasma as well as in liver tissue. In the other 9 non-A, non-B hepatitis patients and in 6 control patients, no HCV-RNA was detectable in either plasma or liver tissue. The comparison between HCV cDNA-PCR results in fresh frozen versus formalin-fixed, paraffin-embedded liver biopsies showed that although detection of HCV-RNA in both correlated 100% the quantity of HCV-RNA was lower in the formalin-fixed, paraffin-embedded liver biopsies of 5 of 8 patients for whom end-point dilution titration of liver RNA was performed. We conclude that using the procedures described HCV-RNA can be reliably detected in both fresh-frozen and formalin-fixed, paraffin-embedded liver biopsies and that HCV cDNA-PCR in liver tissue may become an important assay, especially for monitoring anti-viral therapy.
Biopsy↗
Should all platelet concentrates issued be leukocyte-poor?
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Blood Component Transfusion↗
Comparison of five different filters for the removal of leukocytes from red cell concentrates.
The leukocyte depletion capacity and performance of 5 filters designed for filtration of red cell concentrates (RCC) were compared by counting leukocytes, measuring red cell volumes and by histological examination of the filters after use. To eliminate interdonor differences, 5 buffy-coat-poor RCC were pooled (in each of 10 experiments) and subsequently split up into the original bags. The RCC were passed over the Cellselect filter, a column filled with cellulose acetate, and over flat-bed polyester filters: the Cellselect Optima, the Pall RC 50, the Leukostop and the Sepacell R-500. The filtration was shortest with the Pall RC 50 (p less than 0.001 compared to the other 4 filters). Leukocyte removal was most effective with the cellulose acetate filter (p less than 0.01 compared to the other 4 filters) followed by the Cellselect Optima polyester filter (p less than 0.02 compared to the remaining 3 filters). Residual leukocytes did not exceed 50 x 10(6) for any brand of filter. Red cell recovery was similar for all 5 filters with mean values from 86.1 to 89.2%. The leukocyte numbers, counted in Türk's solution or in propidium iodide, gave comparable values in hemocytometers applying light microscopy or fluorescent microscopy, respectively. Histological examination showed that lymphocytes were mainly removed by trapping, whereas granulocytes showed a variable pattern: adhesion in presence of platelets or trapping.
Blood Component Removal↗
Early antihepatitis C virus response with second-generation C200/C22 ELISA.
Detection of early antibody to hepatitis C virus (HCV) by a new second-generation C200/C22 anti-HCV enzyme-linked immunosorbent assay (ELISA) and a four-antigen recombinant immunoblot assay (4-RIBA) was compared with the first-generation anti-HCV C100 ELISA using sequential serum samples of 9 recipients who were infected with HCV, as detected by polymerase chain reaction after transfusion of blood products. Within 26 weeks after transfusion, 9/9 (100%) recipients seroconverted with C200/22 ELISA, and 6/9 (67%) seroconverted with C100 ELISA. Compared with C100 ELISA, C200/C22 ELISA seroconversion occurred simultaneously in 3 cases, 5-6 weeks earlier in 3 other cases, and 20 weeks earlier in 1 case. Seven of 9 (78%) recipients became positive, and 2/9 (22%) became indeterminate with 4-RIBA. In 8 cases with clinical posttransfusion hepatitis non-A, non-B (PTH-NANB), anti-HCV C200/C22 ELISA seroconversion occurred 2-17 (mean 6) weeks after the onset of hepatitis. In 6 cases of PTH-NANB, anti-HCV C100 ELISA seroconversion occurred 2-26 (mean 9) weeks after the onset of hepatitis. It is concluded that the second-generation C200/C22 ELISA is more sensitive than the C100 ELISA for the detection of antibody during early HCV infection. Indeterminate 4-RIBA results are found in the early phase of HCV infection.
Enzyme-Linked Immunosorbent Assay↗
Enhanced sensitivity of a second generation ELISA for antibody to hepatitis C virus.
A second generation ELISA for combined detection of antibodies to three hepatitis C virus (HCV) recombinant proteins, i.e. C100, C33c and core, was compared with a first generation anti-HCV ELISA in which only antibodies to C100 are detected. The results of the ELISAs were evaluated in 225 haemophilia patients (panel A) and 44 patients with non-A, non-B (NANB) hepatitis (panel B). HCV infection was established by cDNA-polymerase chain reaction (PCR) (in panel B only) and by studying the anti-HCV reaction patterns in 4 separate ELISAs for detection of antibodies to the recombinant proteins C100, C33c, core and a combination of two synthetic peptides sp67/65 derived from the C100 region. The sensitivity for the detection of HCV infection had increased from 0.92[95% confidence interval (CI): 0.87-0.95] to 1.00 (95% CI: 0.89-1.00) in haemophiliacs and from 0.84 (95% CI: 0.66-0.95) to 1.00 (95% CI: 0.89-1.00) in NANB hepatitis patients when the second generation ELISA was used instead of the first generation ELISA. Concurrently the chance of a false negative result was reduced in panel A and B from 0.37 to 0 and from 0.28 to 0, respectively. Analysis of anti-HCV reaction patterns revealed that 172 of 206 (83.5%) anti-HCV ELISA-reactive haemophilia patients had antibodies to all 4 antigens tested. In the NANB hepatitis patients 18 of 31 (58.1%) anti-HCV ELISA-reactive subjects reacted with 4 antigens. In the PCR tested panel of NANB hepatitis patients 2 subjects who showed antibody reactivity to only one antigen and 5 patients with reactivity to 2 antigens were PCR-positive.(ABSTRACT TRUNCATED AT 250 WORDS)
Antigens, Viral↗
Blood donor screening or 'over-screening': how far to go in avoiding transmission of infectious agents?
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Blood Donors↗
Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus.
We have noticed that suboptimal specimen processing and storage conditions may cause false-negative results in the detection of hepatitis C virus (HCV) RNA in plasma or serum. To establish the influence of specimen handling in a serological laboratory on the rate of detection of HCV RNA by the cDNA polymerase chain reaction (cDNA-PCR), we tested routine serum samples and fresh-frozen plasma samples from the same bleeding from confirmed anti-HCV-positive blood donors. When primers from the NS3/NS4 region were used, HCV RNA was detected in fresh-frozen plasma from 67% of the donors, whereas positive results were obtained with only 50% of the serum samples that had been subjected to routine serological procedures. Analysis of the same samples with primers from the highly conserved 5'-terminal region (5'-TR) revealed an HCV RNA detection rate of 92% for both the routine and the fresh-frozen samples. However, the yield of the amplification product in routine samples was strongly reduced compared with that in fresh-frozen plasma. Comparison of both primer sets for cDNA-PCR showed that the 5'-TR primer set was 10- to 100-fold more effective in detecting HCV RNA. We also analyzed the effect of storage of whole EDTA-blood and serum at room temperature and at 4 degrees C on the yield of the amplification product. A rapid decline in detectable HCV RNA of 3 to 4 log units was observed within 14 days when whole blood and serum were stored at room temperature. By contrast, no perceptible reduction in the cDNA-PCR signal was found in freshly prepared serum stored at 4 degrees C.
Base Sequence↗
Antiviral effect of prolonged intermittent lymphoblastoid alpha interferon treatment in chronic hepatitis B.
In a European multicentre study 40 patients with HBeAg positive chronic hepatitis B virus (HBV) infection were treated with 5 mega units of lymphoblastoid alpha-interferon daily according to the following regimen: a four week primer course, four weeks of rest and a second course lasting 16 to 30 weeks. After 52 weeks of follow up, a response (HBeAg seroconversion and HBV-DNA negativity) was observed in 22 patients (55%). HBsAg seroconversion occurred in five patients (12.5%). One patient exhibited a relapse for serum HBeAg and HBV-DNA after cessation of treatment. According to a response prediction model, the observed response rate was not related to the selection of patients likely to respond. The initial interferon course induced a reduction of the serum HBV-DNA and HBeAg levels of 87% and 18%, respectively, leading to a significantly lower level of viral replication activity at the start of the second longterm course compared with baseline. After 24 weeks of follow up (week 16 of the second course), 19 (48%) patients exhibited a response, 13 (32%) a partial response (HBeAg < 50% of initial level or HBV-DNA negative) and 8 (20%) no response. For eight of the 13 partial responders treatment was stopped at week 24 and viral replication rebounded to pretreatment values. In the last five partial responders prolongation of therapy up to week 38 led to a definite response and HBsAg seroconversion in three of the five patients. The results of this study suggest that a short primer course and prolongation of therapy may help to enhance the response rate of alpha-interferon therapy for chronic hepatitis type B.
Adolescent↗
Disappearance of hepatitis C virus RNA in plasma during interferon alpha-2B treatment in hemophilia patients.
To establish the effect of interferon alpha-2B (IFN-alpha) treatment on hepatitis C virus (HCV) viremia, rather than monitor the alanine aminotransferase (ALAT) values we measured HCV-RNA by cDNA-polymerase chain reaction (cDNA-PCR) in plasma before and during IFN-alpha treatment. Eight hemophilia patients with chronic hepatitis C were treated with IFN-alpha for 24 weeks: 5 MU daily for 2 weeks, 2.5 MU daily for 4 weeks, and 1.5 MU three times a week for 18 weeks. HCV-RNA, as measured by cDNA-PCR, was present in all patients before treatment. After 24 weeks of treatment HCV-RNA was no longer detectable in three of eight (37.5%) patients, whereas only one of eight (12.5%) patients showed complete ALAT normalization. In three of eight patients a transient response to IFN-alpha was seen, with renewed HCV-RNA detection after dose reduction. HCV-RNA measurement by cDNA-PCR appeared to be more sensitive in detecting relapse than ALAT measurement.
Alanine Transaminase↗
New developments in hepatitis C.
Since the detection of hepatitis B virus (HBV) in the 1960s and hepatitis A virus in the 1970s, a considerable proportion of infections of (probably viral) hepatitis could not be classified. About 90% of transfusion-related hepatitis was identified as non-A/non-B. In 1988 investigators from the Chiron Company (USA) detected the non-A, non-B agent and named it hepatitis C virus (HCV). An anti-HCV antibody assay (ELISA) and subsequently confirmation tests (immunoblot and polymerase chain reaction) were developed. HCV infection results in a chronic carrier state of the virus in about 80%. Almost all HCV carriers have, irrespective of their liver function tests, histologic signs of chronic hepatitis and/or liver cirrhosis. Chronic HCV infection is, like HBV, also associated with the development of hepatocellular carcinoma. Most HCV carriers are infected by parenteral routes (intravenous drug use, blood transfusion, tattooing). Intravenous drug users and haemophilia patients have the highest risk (80-90%) of becoming infected. Sexual and perinatal transmission does not play an important role in spreading the infection. Antiviral therapy (alpha-interferon) in patients with chronic hepatitis C will normalize liver function tests in about 25% of the cases, but it is unclear if the HCV carrier state will disappear and if liver cirrhosis will be prevented. At present no specific immunoglobulin or vaccine preparations are available to prevent the HCV infection.
Carrier State↗
[Are there still indications for plasma transfusions?].
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Acquired Immunodeficiency Syndrome↗
Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay.
A new four-antigen recombinant immunoblot assay (4-RIBA) for confirmation of hepatitis C virus (HCV) C-100 enzyme-linked immunosorbent assay (ELISA) reactivity was tested in stored serum samples (1984-86) of blood donors and recipients and compared with results from polymerase chain reaction (PCR) analysis of fresh (1990) plasma samples in donors and recipients from the original study. Of 37 HCV C-100 ELISA-positive blood products, 8 were 4-RIBA positive, of which 7 were implicated in post-transfusion non-A, non-B hepatitis (PT-NANBH) and/or PCR confirmed recipient HCV infection. Of 9 recipients with PT-NANBH, 8 were reactive in 4-RIBA (6 positive and 2 indeterminate). With fresh plasma samples, 3 donors and 6 recipients who were 4-RIBA positive were also PCR positive. 4 4-RIBA indeterminate and 78 4-RIBA negative samples of donors and recipients were PCR negative. Of 6 4-RIBA positive recipients, 5 were PCR positive four to six years later. 1.6% of the 383 recipients became chronically infected with HCV. The new 4-RIBA represents a candidate confirmation test to discriminate between infective and non-infective HCV C-100 ELISA-positive blood donors.
Base Sequence↗
Analysis of genomic variability of hepatitis C virus.
The anti-C100-enzyme-linked immunosorbent assay, the new four-antigen antibody recombinant immunoblot assay, and detection of viral RNA sequences by copy DNA-polymerase chain reaction were used to establish the course of hepatitis C virus (HCV) infection in recipients of HCV-infected blood products. Different patterns of infection were observed: (1) persistent HCV infection with and without chronic hepatitis, and with acute resolved hepatitis; and (2) acute resolved hepatitis with clearance of HCV. In order to determine whether different infection- and anti-HCV recognition patterns are correlated to differences in viral nucleotide sequences, we compared sequences in the NS3 region between isolates from recipient(s) and their infective donors. Based on these comparisons we conclude that in The Netherlands two types of molecular variants circulate; one resembling the Japanese prototype isolate JH1, and the other the HCV-1 isolate from the U.S.A. The difference in sequence homology between the two types is approximately 24%. Comparison of sequences of donors and involved recipients determined in isolates prepared from blood samples four years after transfusion revealed that viral RNA sequences are strongly conserved (greater than 96.8%) in the NS3 region. These data indicate that the observed differences in anti-HCV immune response patterns between recipients are more a reflection of their immune reactivity than of divergence of viral strains.
Cardiac Surgical Procedures↗