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.

In vitro measurement of cytotoxic T cell activity does not predict clinical progression in paediatric HIV disease--two case studies.

Cytotoxic T cells are believed to be an important immune response in HIV infection, both in the initial response to viraemia, and in controlling HIV replication and maintaining clinical stability. We report here the detailed findings in two vertically infected children, from the Edinburgh perinatal cohort. Both were clinically stable for the first 7 years of life. One had vigorous HIV-specific cytotoxic T lymphocyte (CTL) responses, and non-lytic suppression, measured in vitro, while the second had no CTL activity against HIV. Despite her HIV-specific immunity, the first child had a declining CD4 count, and a high and fluctuating viral load, whereas the second child maintained a stable CD4 count, a low viral load and had a virus which could not be cultured in peripheral blood mononuclear cells (PBMC) in vitro. The first child subsequently progressed to AIDS and has now died, while the second remains clinically well. More detailed investigations showed the clinically stable child to be heterozygous for the CCR5 receptor, and to be HLA-B49--both of which markers have been associated with slow HIV disease progression. These findings question the role of CTL in maintaining stable HIV disease, and stress the need for immunological investigations to be considered in the light of the genetic make-up of the patient. They may also reflect a different immunopathogenesis of HIV disease in children compared with adults.

The molecular epidemiology of human immunodeficiency virus type 1 in six cities in Britain and Ireland.

We have sequenced the p17 coding regions of the gag gene from 211 patients infected either through injecting drug use (IDU) or by sexual intercourse between men from six cities in Scotland, N. England, N. Ireland, and the Republic of Ireland. All sequences were of subtype B. Phylogenetic analysis revealed substantial heterogeneity in the sequences from homosexual men. In contrast, sequence from over 80% of IDUs formed a relatively tight cluster, distinct both from those of published isolates and of the gay men. There was no large-scale clustering of sequences by city in either risk group, although a number of close associations between pairs of individuals were observed. From the known date of the HIV-1 epidemic among IDUs in Edinburgh, the rate of sequence divergence at synonymous sites is estimated to be about 0.8%. On this basis we estimate the date of divergence of the sequences among homosexual men to be about 1975, which may correspond to the origin of the B subtype epidemic.

HIV-induced cytopathology and viral load in a pentamidine-treated lymphocytic cell line.

Preincubation of CD4 lymphocytes with pentamidine isethionate at a concentration of 1.5 mg/L then removal from incubation medium prior to addition of HIV-1, or incubation of cells with the drug and virus simultaneously, increased HIV-1 DNA load but reduced p24 antigen release. The number of syncytia generated was not affected by the presence of pentamidine. The extent of balloon degeneration of cells was greater, however, although this was not associated with a discernable increase in cellular necrosis or reduction in cell viability. This suggests that drug-treatment resulted in an increased load of intracytoplasmic (but not necessarily integrated) forms of HIV- 1; this may explain the lower levels of antigen produced and also the balloon degeneration of treated cells, a phenomenon previously observed with other retroviruses.

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.

Hepatitis B serology and DNA detection in multitransfused haemophiliacs and factor VIII and IX concentrates.

To assess the effect of HIV infection and the introduction of virus-inactivated concentrates, we conducted a retrospective 20-year longitudinal study of hepatitis B virus (HBV) serology and look for HBV DNA in recent serum samples of 63 multiply transfused haemophiliacs. Of 63 haemophiliacs, 51 had evidence of previous HBV infection and 12 vaccinees had anti-HBs only. Of 40 HIV-negative, two had persistent HBsAg but all were HBV DNA negative. All 23 HIV-positive were HBsAg-negative. Loss of anti-HBc(46% vs. 17.5%) and anti-HBs (32% vs. 14%) was more commonly seen in HIV-infected compared with noninfected individuals. One HIV-positive individual had HBV DNA detectable by PCR. Restrospective testing demonstrated that re-emergence was associated with loss of anti-HBs and advanced HIV infection (CD4<50 × 10(6-1) L CDC II), although eight other with CDC IV disease were HBV DNA negative. Forty-three batches of concentrates produced between 1965 and 1992 from both commercial and volunteer donors and subjected to different donor screening and virus inactivation methods were negative for HBV DNA. Some of these may have been infectious for HBV and therefore being negative for HBV may not equate with noninfectivity. We conclude that both HIV-positive and -negative haemophiliacs have lost protective antibodies against HBV since 1984 and that virus replication may re-emerge at least in the HIV-positive group. These observations may have implications for the management of their chronic liver disease and the risk of infection of sexual partners and medical attendants.

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.

Cytotoxic T lymphocyte activity in children infected with HIV.

Of the Edinburgh cohort of approximately 130 children born to HIV-infected women, 9 are infected and alive. This article describes results from the first 18 months of a natural history study of seven of these, and two adopted children, studying the CD8 T cell-mediated cytotoxicity against HIV proteins (Gag, Tat, Pol, and Env), over time, and relating it to clinical progression and viral activity. Autologous EBV cell lines infected with vaccinia-HIV constructs were used as target cells, and bulk-cultured peripheral blood mononuclear cells as effector cells. The children ranged in age from 0 to 93 months, with six of the nine showing CTL activity to one or more HIV proteins. The specificity of the response was directed against Tat in the younger children, switching to Pol, then Gag or Env. Preliminary analysis of virological data showed no association between CTL and virus activity. The children with CTLs tended to be well clinically, but the cohort needs to be studied longer before conclusions can be made about CTL activity and HIV disease progression. Cytotoxic T lymphocyte activity has also been observed in two children diagnosed as HIV uninfected. These results show the importance of looking at CTL specificity, and may have implications in vaccine design.

Human immunodeficiency virus and the brain: investigation of virus load and neuropathologic changes in pre-AIDS subjects.

Brain tissue was examined for evidence of human immunodeficiency virus (HIV) infection in 23 intravenous drug users who died suddenly some years after seroconversion but while still in presymptomatic stages of infection. None showed giant cell encephalitis, but 14 showed T cell lymphocytic leptomeningitis and 3 showed other significant neuropathologic features. Quantitative polymerase chain reaction for HIV was applied to 13 of the 23 with negative results in 6 and very low positive results in the other 7, a finding consistent with contamination by residual infected blood in the brain tissue. This contrasted with findings in AIDS-infected tissue, in which substantial amounts of provirus were found. It is concluded that significant infection in brain tissue does not occur in presymptomatic stages of HIV infection and that invasion of the central nervous system may be delayed until the transition to symptomatic AIDS.

Analysis of a new hepatitis C virus type and its phylogenetic relationship to existing variants.

Sequences obtained in the 5' non-coding region (5'NCR) of hepatitis C virus (HCV) were obtained from Scottish blood donors and compared with previously published HCV sequences. Phylogenetic analysis revealed the existence of three distinct groups of sequences; two of these corresponded to the recently described HCV types 1 and 2 variants, while viral sequences detected in around a third of the blood donors formed a separate phylogenetic group that probably represents infection with a novel virus species. Nucleotide sequences of this latter group differed from all previously published 5'NCR sequence variants by at least 9%. This new virus type also differed considerably from previously published variants in other regions of the viral genome (core, NS-3 and NS-5), with corrected nucleotide distances of 15, 43 and 49% respectively from the prototype HCV-1 sequence. Formal phylogenetic analysis of each of the coding regions confirmed that HCV type 1 variants could be clearly differentiated into regional variants (Far East and U.S.A./European), in contrast to the clearly overlapping geographical distributions of the main HCV types in U.K. blood donors. We discuss the evidence for and against the hypothesis that the three main phylogenetic groups identified in this study represent separate species of HCV.

Prevalence of HIV among childbearing women and women having termination of pregnancy: multidisciplinary steering group study.

OBJECTIVE: To determine the prevalence of HIV among pregnant women, in particular those whose behaviour or that of their partners put them at "low risk" of infection. DESIGN: Voluntary named or anonymous HIV testing of pregnant women during 21 months (November 1988 to July 1990). SUBJECTS AND SETTING: All women who planned to continue their pregnancy and attended clinics serving the antenatal populations of Edinburgh and Dundee. All women admitted for termination of pregnancy to gynaecology wards serving the pregnant populations of Dundee and outlying rural areas. MAIN OUTCOME MEASURES: Period prevalence of HIV antibody positivity. RESULTS: 91% of antenatal clinic attenders and 97% of women having termination of pregnancy agreed to HIV testing on a named or anonymous basis. HIV period prevalences for antenatal clinic attenders and women having termination of pregnancy tested in Dundee were 0.13% and 0.85% respectively, and for antenatal clinic attenders tested in Edinburgh 0.26%. For those at "low risk" rates for antenatal clinic attenders and women having termination of pregnancy in Dundee were 0.11% and 0.13%, and for antenatal clinic attenders in Edinburgh 0.02%. In Dundee HIV prevalence among women having a termination of pregnancy (0.85%) was significantly greater than that among antenatal clinic attenders (0.13%). CONCLUSIONS: HIV infection is undoubtedly occurring among women at "low risk," and it is clear that a policy of selective testing of those at only "high risk" is inadequate for pregnant women living in areas of high prevalence such as Edinburgh and Dundee. Moreover, when studying pregnant populations in such areas there is the need to include those having a termination of pregnancy.

PIP: This study sought to determine the prevalence of HIV among pregnant women, in particular those whose behavior or that of their partners put them at low-risk for infection. This was a voluntary named or anonymous HIV testing of pregnant women between November 1988-July 1990 among all women who planned to continue their pregnancies and attended clinics serving antenatal populations in Edinburgh and Dundee and those women admitted for termination of pregnancy to gynecology wards serving the pregnant populations of Dundee and outlying rural areas. 91% of the antenatal clinic attenders and 97% of women having pregnancy termination agreed to HIV testing on either a named or anonymous basis. HIV period prevalences for antenatal clinic attenders and women having pregnancy termination tested in Dundee were 0.13% and 0.85% respectively, and for antenatal clinic attenders tested in Edinburgh 0.26%. For those at low-risk, the rates for antenatal clinic attenders and women having pregnancy termination in Dundee were 0.11% and 0.13%, and for antenatal clinic attenders in Edinburgh 0.02%. In Dundee, HIV prevalence among women having a termination of pregnancy (0.95%) was significantly greater than that among antenatal clinic attenders (0.13%). HIV infection is undoubtedly occurring among low-risk women and it is clear that a policy of selective testing of those only at high-risk is inadequate for pregnant women living in areas of high prevalence such as Edinburgh and Dundee. Moreover, when studying pregnant populations in such areas, there is a need to include those undergoing pregnancy termination.

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.

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).

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. Of the haemophilia A patients 14 (32%) had CD4+ lymphocyte subset counts less than or equal to 0.5 x 10(9)/l, compared with one (6%) haemophilia B patient and four (8%) controls. The percentage of activated T cells was greater than 5% in 19/33 (57%) with haemophilia A, 5/9 (55%) haemophilia B and 14/50 (28%) of control subjects. beta 2 microglobulin values greater than or equal to 2.0 mg/l were observed in 19 (43%) haemophilia A and four (26%) haemophilia B patients, compared with one (2%) control. No significant increases in serum interleukin-2 receptor concentrations were observed in 15 haemophilia A and one haemophilia B patients. Significantly elevated levels of IgG, IgM and IgA were observed in the haemophilia A group, but elevation of immunoglobulins was restricted to the IgG class in the haemophilia B group. Of the haemophilia A patients 16/30 (53%) and 6/11 (54%) haemophilia B patients had depression of cell-mediated immunity (CMI) as assessed by delayed-type hypersensitivity responses to intradermally injected recall antigens. There was no correlation between factor VIII or factor IX usage and changes in lymphocyte subsets, beta 2 microglobulin, and immunoglobulin levels. There was, however, a strong correlation between annual factor VIII usage and the degree of depression of CMI for those with haemophilia A but not for those with haemophilia B. No correlation between alterations in the immune parameters and disturbance of liver function tests was observed in either haemophilia A or haemophilia B patients. We conclude that alloantigen or non-HIV viral exposure due to repeated administration of factor concentrates brings about alterations in the immune response, and that these changes are more marked following exposure to intermediate purity factor VIII compared with factor IX concentrate.

Comparison of hepatitis B virus subtyping of d/y determinants by radioimmunoprecipitation assay and the polymerase chain reaction.

Using a double polymerase chain reaction a method was devised for detecting and subtyping hepatitis B virus DNA in serum samples. Primers from the S-gene were selected from the sequence analyses of five HBV HBsAg subtypes, to amplify HBV DNA and subtype for y specific DNA. Thirty-eight samples were subtyped for d and y determinants by radioimmunoprecipitation assay (RIPA) and the polymerase chain reaction (PCR). Subtyping by PCR and RIPA was in agreement in 100% of subtype y samples and 83.3% of subtype d, giving an overall correlation of 92.1%. As a third comparison, 12 amplified samples were digested by the restriction enzyme Sau 3A, which differentiates between subtypes y and d. The digest results agreed with PCR in 83.3% of the samples. In addition, we compared our standard phenol/chloroform extraction against a rapid one step method. The phenol/chloroform stage was found to be essential for the removal of nucleases and polymerase inhibitors present in sera.

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.

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.

Five year prospective study of HIV infection in the Edinburgh haemophiliac cohort.

OBJECTIVE: To identify measures of immune state that reflect the course of HIV related disease in order to predict deterioration of symptoms and assess response to treatment. DESIGN: Five year longitudinal clinical and laboratory study. SETTING: Regional haemophilia centre, university virology laboratory, and Medical Research Council laboratory. PATIENTS: 32 Patients with haemophilia A exposed to a single batch of HIV contaminated factor VIII concentrate from the Scottish National Blood Transfusion Service in 1984 who were followed up regularly in Edinburgh (31) or abroad (one). MAIN OUTCOME MEASURES: Counts of circulating T cell subsets (CD4 and CD8); plasma beta 2 microglobulin, neopterin, and IgA concentrations; and delayed type hypersensitivity to multiple skin test antigens. RESULTS: 18 Patients who seroconverted after exposure had received significantly more contaminated factor VIII than the 14 who did not (mean 43 (range 9-109) v 15 (3-30) phials, p less than 0.01). The two groups were not distinguishable by other criteria before exposure. The group that seroconverted subsequently showed a progressive fall in mean circulating CD4 lymphocytes and an increase in plasma beta 2 microglobulin and neopterin concentrations. From 1987 patients in this group also showed an increase in mean circulating CD8 lymphocytes and in plasma IgA concentration, neither of which was seen in patients who did not seroconvert. Patients with HIV antibody who developed Centers for Disease Control category IV symptoms within five years after infection showed more extreme changes in all measures, except CD8 lymphocyte count, than those whose symptoms remained in categories II and III. Skin test reactivity declined to barely detectable levels in all patients positive for HIV antibody. CONCLUSIONS: Serial estimates of circulating CD4 lymphocytes and of plasma beta 2 microglobulin concentration are the most reliable measures of disease progression; of these, beta 2 microglobulin concentration seems to be the better predictor of impending serious symptoms. High IgA concentrations reflect rather than predict disease state. Individual variation in most measures is such that a wide range of measurements should be used in assessing the effects of trial treatment in HIV infected patients without symptoms.