Absence of hepatitis A virus transmission by high-purity solvent detergent treated coagulation factor concentrates in Scottish haemophiliacs.

Recent reports of hepatitis A virus (HAV) infection in haemophiliacs receiving high-purity solvent detergent (HP.SD) treated factor VIII concentrates have brought into question the efficacy of this virucidal method for inactivating HAV. To assess whether HAV may have been transmitted by HP.SD concentrates, we compared seroprevalence in haemophiliacs with different disease severity, sought evidence of seroconversion to HAV since introduction of HP.SD products, and directly examined concentrates for HAV RNA by PCR. Our data suggest that Scottish haemophiliacs are not being infected with HAV by HP.SD concentrates produced initially by CRTS Lille and presently by PFC Edinburgh and supplied by the Scottish National Blood Transfusion Service (SNBTS).

Genotypic variation, clinical and histological characteristics of chronic hepatitis C detected at blood donor screening.

Since blood donor screening for the hepatitis C virus (HCV) began in 1991 a large number of seropositive subjects have been detected and several reports have suggested a high prevelance of liver disease. The aim of this study was to evaluate the severity of liver disease in HCV-positive blood donors in terms of the clinical, biochemical and histological abnormalities and to investigate the relationships between these features and the mode of transmission, duration of infection and viral genotype. We evaluated 54 consecutive blood donors who were positive for HCV both on serological testing and polymerase chain reaction. Twenty-three (43%) had a history of intravenous drug abuse and 17 (31%) had received blood transfusions. In only two (4%) was no risk factor identified. The mean duration of infection in those with a clear history of HCV exposure was 12 years. Eighty-three percent were HCV genotypes 1 or 3. All had abnormal liver biopsies with chronic hepatitis and several patients had periportal or portal-portal fibrous septa, but there was none with architectural distortion or cirrhosis. There was no correlation between severity of liver disease and duration of HCV infection, mode of transmission or viral genotype. In the majority of HCV carriers detected at donor screening there is a chronic hepatitis with bridging necrosis in one third, but the degree of fibrosis is minimal and cirrhosis was not present in our patients. The long period of infection of many patients suggests that irreversible liver injury does not necessarily develop at an early stage despite persistent infection.

Frequent reinfection and reactivation of hepatitis C virus genotypes in multitransfused hemophiliacs.

The frequency and dynamics of infection with different genotypes of hepatitis C virus were investigated in a cohort of hemophiliacs repeatedly exposed to non-virus-inactivated clotting factor. Among 63 infected hemophiliacs, genotype 1 (n = 38, subtypes 1a [27] and 1b [11]) was predominant; genotypes 2a (n = 1), 2b (n = 3), 3a (n = 20), and 5a (n = 1) accounted for the remainder. This distribution was similar to that found in Scottish blood donors from whom the infected blood products were manufactured. Hemophiliacs with severe disease were more likely to be polymerase chain reaction-positive than those with moderate or mild disease. Over 10 years, changes in the circulating major genotype and serotype were observed in 9 of 29 hemophiliacs and from one subtype to another in 3, although there was no clear trend toward replacement with any particular variant. Replacement occurred after the introduction of inactivated clotting factor in 4 subjects, implicating reactivation rather than reinfection. Those coinfected with human immunodeficiency virus were more likely to show a change in genotype.

Hepatitis C virus transmission by intravenous immunoglobulin.

The polymerase chain reaction was used to detect hepatitis C virus infection in patients who had previously been reported to have developed non-A, non-B hepatitis after intravenous immunoglobulin infusion. Of the 33 patients with intravenous immunoglobulin associated non-A, non-B hepatitis studied, HCV RNA could be detected in 15 out of 17 patients (88%) who were HCV RNA negative prior to the development of non-A, non-B hepatitis after implicated intravenous immunoglobulin batches. Similarly, eight out of nine patients (89%) in whom no sample was available for polymerase chain reaction testing prior to intravenous immunoglobulin therapy, had detectable HCV RNA after intravenous immunoglobulin therapy with intravenous immunoglobulin batches implicated in non-A, non-B hepatitis transmission. Two of the three intravenous immunoglobulin preparations implicated in non-A, non-B hepatitis transmissions that were available for polymerase chain reaction testing also had detectable HCV RNA, confirming that hepatitis C virus is the implicated virus in intravenous immunoglobulin-associated non-A, non-B hepatitis.

Prevalence and epidemiological characteristics of hepatitis C in Scottish blood donors.

All blood donors in Scotland who were found to be infected with hepatitis C virus (HCV) in the first 6 months of routine testing of all donations for anti-HCV were contacted. Those who attended were counselled, a history of exposure to risk was sought, and blood was taken for alanine aminotransferase (ALT) level as a measure of liver function. The epidemiological features were then correlated with the virological findings and ALT. In the period under study between September 1991 and February 1992, 180,658 blood donors attended. The prevalence of HCV infection was 0.088%. Of the 151 donors who attended for counselling, 101 (68%) were male. Intravenous drug use was the most common risk activity (39%), followed by previous blood transfusion (15.2%), other parenteral exposure (11.2%) and heterosexual contact with a parenterally infected partner (8.6%); 29.1% of donors gave no history of possible exposure. Elevated ALT levels were found in 59%. ALT levels were higher in donors with HCV types 1 and 3 than in HCV type 2 or non-viraemic donors. The prevalence of HCV in Scottish blood donors is thus relatively low. This may relate to the effectiveness of donor selection procedures, but donors with risk activities which should debar them continue to donate. The combination of ALT and PCR appears to be useful in counselling and assessing infected donors.

Identification of genotypes of hepatitis C virus by sequence comparisons in the core, E1 and NS-5 regions.

Isolates of hepatitis C virus (HCV) show considerable nucleotide sequence variability throughout the genome. Comparisons of complete genome sequences have been used as the basis of classification of HCV into a number of genotypes that show 67 to 77% sequence similarity. In order to investigate whether sequence relationships between genotypes are equivalent in different regions of the genome, we have carried out formal sequence analysis of variants in the 5' non-coding region (5'NCR) and in the genes encoding the core protein, an envelope protein (E1) and a non-structural protein (NS-5). In the E1 region, variants grouped into a series of six major genotypes and a series of subtypes that could be matched to the phylogenetic groupings previously observed for the NS-5 region. Furthermore, core and E1 sequences showed three non-overlapping ranges of sequence similarity corresponding to those between different genotypes, subtypes and isolates previously described in NS-5. Each major genotype could also be reliably identified by sequence comparisons in the well conserved 5'NCR, although many subtypes, such as 1a/1b, 2a/2c and some of those of type 4, could not be reliably distinguished from each other in this region. These data indicate that subgenomic regions such as E1 and NS-5 contain sufficient phylogenetic information for the identification of each of the 11 or 12 known types and subtypes of HCV. No evidence was found for variants of HCV that had sequences of one genotype in the 5'NCR but of a different one in the E1 or NS-5 region. This suggests that recombination between different HCV types is rare or non-existent and does not currently pose a problem in the use of subgenomic regions in classification.

Geographical distribution of hepatitis C virus genotypes in blood donors: an international collaborative survey.

The frequency of infection with the six classified major genotypes of hepatitis C virus (HCV) was investigated in 447 infected volunteer blood donors from the following nine countries: Scotland, Finland, The Netherlands, Hungary, Australia, Egypt, Japan, Hong Kong, and Taiwan. Viral sequences in plasma from blood donors infected with HCV were amplified in the 5'-noncoding region and were typed by restriction fragment length polymorphism analysis. Electrophoresis of DNA fragments produced by cleavage with HaeIII-RsaI and ScrFI-HinfI allowed HCV types 1 (or 5), 2, 3, 4, and 6 to be identified. Further analysis with MvaI-HinfI allowed sequences of the type 5 genotype to be distinguished from sequences of the type 1 genotype. Types 1, 2, and 3 accounted for almost all infections in donors from Scotland, Finland, The Netherlands, and Australia. Types 2 and 3 were not found in the eastern European country (Hungary), where all but one of the donors were infected with type 1. Donors from Japan and Taiwan were infected only with type 1 or 2, while types 1, 2, and 6 were found in those from Hong Kong. HCV infection among Egyptians was almost always by type 4. Donors infected with HCV type 1 showed broad serological reactivity with all four antigens of the second generation Chiron RIBA-2 assay (Chiron Corporation, Emeryville, Calif.), while infection with divergent HCV genotypes elicited antibodies mainly reactive to c22-3 and c33c. Reactivities with antibodies 5-1-1 and c100-3 were infrequent and were generally weak, irrespective of the geographical origin of the donor. Because the envelope region of HCV is even more variable than the NS-4 region, it is likely that vaccines based on these proteins need to be multivalent and perhaps specifically adapted for different geographical regions.

Effect of terminal (dry) heat treatment on non-enveloped viruses in coagulation factor concentrates.

Terminal dry heat treatment effectively inactivated hepatitis A virus (HAV) and canine parvovirus added to high-purity factor VIII. After 24 h at 80 degrees C, HAV infectivity was reduced by > or = 4.3 log10 TCID50, as measured in a newly developed infectivity assay. The same reduction in virus titer was achieved after 2 h and before 6 h at 90 degrees C. Inactivation of hepatitis A virus was also seen in the freeze-drying step prior to heat treatment with an approximately 2.0 log10 reduction in titer. Similar results were obtained with a high-purity factor IX concentrate. Canine parvovirus was also inactivated at both temperatures, with residual infectivity being undetected after 48 h at 80 degrees C or 10 h at 90 degrees C. Canine parvovirus was not affected by lyophilisation. Canine parvovirus measurements by PCR did not reflect the levels of infectivity measured by the tissue-culture-based method. The addition of the terminal dry heat treatment to solvent/detergent could effectively eliminate the potential contamination of solvent/detergent-treated coagulation factor concentrates by non-lipid-enveloped viruses. However, careful evaluation for any increased induction of non-antigens for factor VIII, as a consequence of such treatment, is needed before use in patients can be recommended.

Hepatitis C virus genotypes: an investigation of type-specific differences in geographic origin and disease.

Because of the nucleotide sequence diversity of different isolates of hepatitis C virus, it has become important to clarify whether distinct genotypes of hepatitis C virus vary with respect to pathogenicity, infectivity, response to antiviral therapy and geographic clustering. We assessed nucleotide sequence variability in the 5' noncoding region of hepatitis C virus, using restriction enzymes to analyze the distribution of hepatitis C virus genotypes, in 80 patients with chronic hepatitis C virus infection. Genotypes were correlated with demographic, clinical and histological features. Thirty-seven patients were infected with type 1, 10 had type 2 and 8 had type 3, and another 23 were infected with a new distinct hepatitis C virus type now classified as type 4. Two were infected with variants whose classification are uncertain. Types 1, 2 and 3 were found in patients from the United Kingdom, southern Europe, Asia, Africa and South America. Nineteen of 23 type 4 genotype isolates were from Middle Eastern patients, compared with 0 of 37 type 1 isolates (p < 0.001). Of 21 Middle Eastern patients, 19 (90.4%) had type 4 hepatitis C virus (p = 0.001, odds ratio = 9). We found no significant difference between the mean ages or mean serum aminotransferase concentrations between the various types. Types 1, 2, 3 and 4 were found in patients with mild-to-moderate disease or severe disease. However, 21 of 29 (72.4%) patients with type 1 who underwent liver biopsy had severe chronic hepatitis, cirrhosis or hepatocellular carcinoma histologically; 8 had mild or moderate chronic hepatitis without cirrhosis (p = 0.03, odds ratio = 2.6).(ABSTRACT TRUNCATED AT 250 WORDS)

Failure to detect hepatitis A virus RNA in factor VIII concentrates using the polymerase chain reaction. Results of a preliminary study.

The transmission of hepatitis A virus (HAV) infection to recipients of some batches of Factor VIII has recently been reported. The polymerase chain reaction (PCR) was used for the detection of HAV RNA in factor VIII concentrates. Primer sequences used were derived from a consensus of published sequences in the 5' non coding region; a nested PCR was used to increase sensitivity and specificity and the resulting fragment was 151 base pairs in length. The PCR was initially validated in clinical samples and only IgM anti-HAV positive patient samples and a sample of liver tissue from a patient who required liver transplantation for fulminant hepatitis A were HAV PCR positive. Other samples tested included those that were IgG anti-HAV positive; these were found to be PCR negative. In an investigation of coagulation factor VIII concentrates by HAV PCR, 40 batches of solvent/detergent-treated high-purity concentrate from four different manufacturers, including one batch of factor VIII possibly implicated in HAV transmission, and a further 3 batches of monoclonal antibody purified factor VIII were all HAV PCR negative. Gel chromatography material, before and after use in factor VIII purification, and eluates from this material were also negative for HAV RNA. Our preliminary results therefore suggest that either the contamination of factor VIII concentrates by HAV RNA is an extremely rare event or that the PCR is insufficiently sensitive to detect an infective HAV dose since each batch of factor VIII concentrate would have been derived from a plasma pool consisting of 10,000 donations, or more and the resulting concentration of virus may be 10(2) or less.

Confirmation of hepatitis C virus antibody in blood donors.

Of 103,203 donations collected in Scotland and Northern Ireland over a 3-month period and screened for HCV antibody by Ortho or Abbott second-generation ELISAs, 340 were found repeatedly reactive. Supplementary testing with RIBA-2 resulted in 77 being classified as positive, 130 as indeterminate, and 133 as negative. PCR analysis of the positives and indeterminates indicated viraemia in 65 (84%) of the positives and 7 (5.5%) of the indeterminates. To determine if PCR analysis could be eliminated or reduced by further serological testing, all RIBA-2 positives and indeterminates were tested by UBI and Wellcozyme ELISAs and Innolia and RIBA-3 immunoblots. All RIBA-2 positives with bands to more than 1 gene product were detected in all 4 systems, but > 60% of RIBA-2 indeterminates were negative in those tests that contain either recombinant antigens or synthetic peptides derived independently from those used by Ortho/Abbott tests. A comparison of data from the 79 reactive with the core (c22) region revealed only 16 samples reactive in all 4 systems as well as Ortho and Abbott. These 16 included all 6 of the PCR positives in the 79 c22 indeterminate samples. ELISAs and immunoblots using independently derived antigens can offer a useful method of screening out nonspecific reactions in Ortho or Abbott ELISAs, hence reducing the need for PCR testing. Some caution is required as all such tests do not contain identical mixes of antigenic material.

Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region.

Hepatitis C virus (HCV) showed substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68 to 79% overall sequence similarity to one another. Phylogenetic analysis of nucleotide sequences derived from part of the gene encoding a non-structural protein (NS-5) has provided evidence for six major genotypes of HCV amongst a worldwide collection of 76 samples from HCV-infected blood donors and patients with chronic hepatitis. Many of these HCV types comprised a number of more closely related subtypes, leading to a current total of 11 genetically distinct viral populations. Phylogenetic analysis of other regions of the viral genome produced relationships between published sequences equivalent to those found in NS-5, apart from the more highly conserved 5' non-coding region in which only the six major HCV types, but not subtypes, could be differentiated. A new nomenclature for HCV variants is proposed in this communication that reflects the two-tiered nature of sequence differences between different viral isolates. The scheme classifies all known HCV variants to date, and describes criteria that would enable new variants to be assigned within the classification as they are discovered.

A comparison of polymerase chain reaction and an infectivity assay for human immunodeficiency virus type 1 titration during virus inactivation of blood components.

Three examples of human plasma-derived concentrates, intermediate-purity factors VIII and IX, and fibrinogen were spiked with tissue culture-grown human immunodeficiency virus type 1 (HIV-1) strain RF. All examples were freeze-dried and heated at 80 degrees C for 72 hours by using validated production process models. HIV-1 infectivity was measured by a syncytial infectivity assay in C8166 cells and then compared with levels determined by nested HIV polymerase chain reaction (PCR). The infectivity assay demonstrated a reduction index of at least 4.5 log10, while PCR showed an average 1.7 log10. Large amounts of HIV-1 RNA (10(5)) were still detectable by PCR in samples in which infectivity assays failed to detect any HIV-1. These data suggest that HIV-1 PCR levels do not parallel HIV-1 infectivity levels during virus-inactivation procedures involved in coagulation factor concentrate production. PCR was able to detect the RNA associated with inactivated HIV-1 particles in the factor concentrates, which allows the conclusion that PCR is not a useful test with which to monitor virus-inactivation procedures such as heating at 80 degrees C for 72 hours. This judgment contrasts with the more definite and sensitive role of PCR in diagnosing HIV-1 infection in patients in whom a positive HIV-1 PCR result correlates with active HIV-1 infection and with PCR's usefulness in monitoring virus removal.

Mapping of serotype-specific, immunodominant epitopes in the NS-4 region of hepatitis C virus (HCV): use of type-specific peptides to serologically differentiate infections with HCV types 1, 2, and 3.

The effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenicity of the NS-4 protein was investigated by epitope mapping and by enzyme-linked immunosorbent assay with branched oligopeptides. Epitope mapping of the region between amino acid residues 1679 and 1768 in the HCV polyprotein revealed two major antigenic regions (1961 to 1708 and 1710 to 1728) that were recognized by antibody elicited upon natural infection of HCV. The antigenic regions were highly variable between variants of HCV, with only 50 to 60% amino acid sequence similarity between types 1, 2, and 3. Although limited serological cross-reactivity between HCV types was detected between peptides, particularly in the first antigenic region of NS-4, type-specific reactivity formed the principal component of the natural humoral immune response to NS-4. Type-specific antibody to particular HCV types was detected in 89% of the samples from anti-HCV-positive blood donors and correlated almost exactly with genotypic analysis of HCV sequences amplified from the samples by polymerase chain reaction. Whereas almost all blood donors appeared to be infected with a single virus type (97%), a higher proportion of samples (40%) from hemophiliacs infected from transfusion of non-heat-inactivated clotting factor contained antibody to two or even all three HCV types, providing evidence that long-term exposure may lead to multiple infection with different variants of HCV.

Sequence variability in the 5' non-coding region of hepatitis C virus: identification of a new virus type and restrictions on sequence diversity.

We have analysed the pattern of nucleotide sequence variability in the 5' non-coding region (5' NCR) of geographically dispersed variants of hepatitis C virus (HCV). Phylogenetic analysis of sequences in this region indicated the existence of a new virus type, provisionally termed type 4, the identity of which was confirmed by further analysis of the more variable part of the HCV core protein coding region. The geographical distribution of HCV type 4 was distinct from that of other HCV types, it being particularly widespread in Africa and absent or rare in Europe and the Far East. Much of the variability in the 5' NCR appears to be constrained by a requirement for specific secondary structures in the viral RNA. In one of the most variable regions of the 5' NCR (positions -169 to -114), most of the nucleotide changes that are characteristic of different HCV types were covariant, with complementary substitutions at other positions. According to the proposed secondary structure of the 5' NCR, such changes preserved base pairing within a stem-loop structure, whereas the nucleotide insertions found in a proportion of 5' NCR sequences, including those of type 4, localized exclusively to the non-base-paired terminal loop. The specific nucleotide substitutions in the 5' NCR that differentiate each of the four HCV types can be detected by restriction enzyme cleavage, providing a rapid and reliable method for virus typing.

Infrequent vertical transmission of hepatitis C virus.

The rate of vertical transmission of hepatitis C virus (HCV) was determined by a combination of assays for anti-HCV antibody and the polymerase chain reaction (PCR) in 66 children born to infected mothers. Only 4 children showed evidence of infection with HCV, being positive for anti-HCV in all samples collected from 6 months to 5 years of age. All samples from the remaining 62 children were repeatedly anti-HCV-negative on screening by two second-generation antibody assays. Furthermore, samples collected at age 12 months from 30 antibody-negative children born of HCV-infected mothers were uniformly PCR-negative, showing that "seronegative" infection with HCV was rare or absent in this study group. Serologic reactivity to HCV-encoded antigens in samples from infected children was largely confined to the HCV core protein. Infection with human immunodeficiency virus in the mother was not a significant cofactor for mother-to-child transmission of HCV.