J.F Peutherer

Hepatitis C quantification and sequencing in blood products, haemophiliacs, and drug users

The polymerase chain reaction (PCR) detected specific hepatitis C viral (HCV) RNA sequences in plasma from 15 of 21 haemophiliacs (12 HCV-antibody positive) and 7 of 27 intravenous drug users (13 HCV-antibody positive). Quantification of RNA-positive samples showed high levels of HCV (10(5) to 10(6) copies of RNA/ml) in infected patients. HCV was more frequently found in haemophiliacs infected with human immunodeficiency virus (11/11 HIV-positive and 4/10 HIV-negative patients). HCV-RNA was detected in all batches of commercially available factor VIII tested and in low concentrations in some pools of plasma donations from volunteers. Factor VIII, manufactured from volunteer donations, was uniformly negative by PCR. Phylogenetic analysis of viral sequences showed two distinct groups: one was associated with intravenous drug users and the other with haemophiliacs infected with Scottish factor VIII preparations. Both were distinct from sequences found in commercially available factor VIII.

HLA HAPLOTYPE A1 B8 DR3 AS A RISK FACTOR FOR HIV-RELATED DISEASE

Of 32 patients exposed to a single batch of factor VIII contaminated with human immunodeficiency virus (HIV), 18 became antibody positive. Serial T cell subset analyses over the succeeding four years have shown a progressive decline in circulating T4 cells in those 18 but no change in the 14 who remain seronegative. 2 of the seroconverters have died and a further 7 have symptoms attributable to HIV infection. In the group as a whole, the HLA haplotype A1 B8 DR3 was weakly associated with an increased risk of seroconversion on exposure to the virus while, in those who seroconverted, it was strongly associated with a rapid decline in T4 cells and development of HIV-related symptoms within four years of infection.

Abnormalities of circulating lymphocyte subsets in haemophiliacs in an AIDS-free population

Markers of the immune system were examined in 47 patients with haemophilia A and B who had been treated exclusively with blood products from a population apparently free from acquired immunodeficiency syndrome (AIDS). In haemophilia A the absolute number of T helper cells was depressed, resulting in a reduction in the helper/suppressor ratio in about half the patients. The serum IgG and IgA concentrations were raised and the serum IgG correlated with serum alanine aminotransferase. In haemophilia B, the helper/suppressor ratio was also depressed but this was attributable to a slight increase in the mean suppressor cell number and a slight decrease in the helper cells. These observations suggest that the abnormalities result from transfusion of foreign proteins and not from a specific infective agent and, further, that individuals may differ in susceptibility to the induced disturbances of immunity.

Use of several second generation serological assays to determine the true prevalence of hepatitis C virus infection in haemophiliacs treated with non‐virus inactivated factor VIII and IX concentrates

Summary To investigate the prevalence of hepatitis C virus infection in two risk groups, stored serum samples from treated haemophiliacs and intravenous drug users were tested for anti‐HCV by both anti‐C‐100 based and second generation ELISAs (Abbott and Ortho) followed by testing in two confirmatory immunoblot assays that incorporate core as well as other non‐structural antigens (Innogenetics LIA and Chiron RIBA‐HCV test). Clear evidence of HCV infection was found in all but one of 78 haemophiliacs treated with non‐virus inactivated clotting factor concentrates, but in none exposed only to super dry heat‐treated concentrates. Only four samples gave rise to conflicting serological results between the four tests, two of these occurred in patients with advanced HIV related disease and almost certainly reflected loss of humoral immunity associated with disease progression, and the others occurred in the only two patients tested who were chronic carriers of hepatitis B infection and may reflect an interaction between the two viruses. Comparison of anti‐C‐100 versus second generation tests in immunocompetent drug users revealed a false negative rate of 20% using C‐100 alone, indicating the advantage of using second generation assays for detection of past or current HCV infection. Of all of the antigens used in the confirmatory assay, positive sera showed strongest and most frequent reactivity with the C22 and C33c proteins (Ortho RIBA).

Determinants of HIV disease progression: six-year longitudinal study in the Edinburgh haemophilia/HIV cohort

Markers of immune function present before infection may determine the subsequent course of disease in HIV-infected individuals. In 1983, we measured immune function in a group of haemophiliacs in Edinburgh. In 1984, 18 of these patients became infected with HIV-1 from contaminated factor VIII. We have followed-up these patients since their seroconversion. The rate of disease progression, as assessed by the appearance or not of AIDS symptoms or signs within five years of seroconversion, was related both to the concentration of total plasma IgM before exposure to infection and to the pattern of specific IgM and IgA anti-HIV response around the time of IgG seroconversion. Disease progression also correlated with concentrations of plasma interleukin-2 receptor (a marker of lymphocyte activation) and with the number and percentage of circulating DR + ve (activated) T cells. Our findings show that the extent of host immune reactivity, which may be genetically determined, is a powerful factor in the pathogenesis of HIV-associated disease.

First coitus before menarche and risk of sexually transmitted disease.

The prevalence of sexually transmitted disease (STD), pelvic inflammatory disease (PID), and cervical cancer, and the relation between these conditions were studied in 2111 Ethiopian women. Early sexual activity was associated with an increase in prevalence rates of STD and PID; possible aetiological factors include physical and immunological immaturity of the female genital tract and the number of sexual partners.

HIV antigen and antibody detection: variable responses to infection in the Edinburgh haemophiliac cohort

Sequential serum samples from 18 haemophiliac patients exposed simultaneously to human immunodeficiency virus type 1 (HIV 1) in early 1984 were tested retrospectively for serological markers of infection. Assay for total antibodies to HIV established that the time to seroconversion might be as long as 110 days after exposure to contaminated factor VIII; serum samples were also tested by Western blotting, by enzyme linked immunosorbent assay (ELISA) for specific antibodies to envelope and core proteins, and for p24 antigen by two assay systems during the two years after infection. The studies showed that five of the 12 patients for whom serum samples obtained between exposure and seroconversion were available had transient p24 antigenaemia. Although amounts of total antibody to HIV and of antibodies to envelope proteins rose continuously during the two years of the study, amounts of antibody to the core protein were variable and tended to decline in patients who became symptomatic. Two patients had persistent p24 antigenaemia that began four months after seroconversion; these patients remained asymptomatic. One patient who developed the acquired immune deficiency syndrome (AIDS) had transient antigenaemia at the time of seroconversion but failed to show any antigen for the rest of the study; progression to AIDS was accompanied by an increase in antibodies to envelope proteins. Much of the variability in the course of infection with HIV must represent the differences in the susceptibility of the patients to infection.

Genetic heterogeneity of HIV type 1 subtypes in Kimpese, rural Democratic Republic of Congo

A relatively low and stable seroprevalence of HIV-1 was previously reported among pregnant women attending for antenatal care between 1988 and 1993 in Kimpese, a rural town in the Democratic Republic of Congo (DRC, formerly Zaire). To characterize the HIV-1 subtypes circulating in this area, we have examined a 330-bp fragment of the p17 region of the gag gene of HIV-1 strains obtained from 70 patients (55 mothers, 15 children), of whom 61 were epidemiologically unlinked. Phylogenetic analyses revealed the existence of at least seven HIV-1 subtypes within the Kimpese region. Among the 61 epidemiologically unlinked patients, subtype A was predominant and found in 29 (47.5%) individuals. Other subtypes cocirculating in this rural part of DRC include subtypes C (1.6%), D (9.8%), F (3.2%), G (6.5%), H (21.3%), and J (4.9%). Sequences from four patients did not cluster with any of the currently documented HIV-1 subtypes, in analyses of fragments of both the gag (247 to 330 bp, 197 bp, and 310 bp) and env (340 bp) genes. Overall, comparisons of the gag(p17) gene regions revealed high pairwise divergences (mean, 19.9%; range, 1 to 46%). This level of gag(p17) gene variation in the DRC is considerably greater than previously appreciated. These results are relevant for the molecular epidemiology of HIV-1 in Africa and for the design of a future vaccine against HIV-1 in this region.

The Epidemiology of HIV Infection in Edinburgh Related to the Injecting of Drugs: An Historical Perspective and New Insight Regarding the Past Incidence of HIV Infection Derived from Retrospective HIV Antibody Testing of Stored Samples of Serum

The pattern of sudden explosive outbreaks of HIV infection among drug users has been seen in several countries but is as yet incompletely understood. The epidemic of injecting drugs in Edinburgh was associated with at least four overlapping epidemics of blood-borne viruses (hepatitis B, C, D and HIV). Only hepatitis B was initially recognized, being followed by HIV and latterly hepatitis C. Retrospective HIV testing of stored samples of serum from clinically diagnosed patients with HIV has allowed the HIV epidemic to be delineated and more accurate seroconversion dates identified for most of the patients. There is evidence to suggest that the explosive drug-related Edinburgh HIV epidemic may have been self-terminating and that the epidemic in male drug users preceded that in female drug users by around 3 months. We suggest that the self-terminating nature of this epidemic may have been related to changes in drug injecting behaviour or to varying infectivity of the virus. This latter possibility should be explored in future studies of HIV transmission.

Immunological studies in HIV seronegative haemophiliacs: relationships to blood product therapy

Immunological studies were performed on a group of 44 haemophilia A and 15 haemophilia B patients who were treated exclusively with blood products manufactured by the Scottish National Blood Transfusion Service (SNBTS). All patients were HIV seronegative throughout the study.