Antibodies to synthetic peptides from the pre-S1 and pre-S2 regions of one subtype of the hepatitis B virus (HBV) envelope protein recognize all HBV subtypes.

Immunodominant B and T cell epitopes have been demonstrated recently on the preS1 and PreS2 regions of the hepatitis B virus (HBV) envelope protein. Synthetic peptide analogs corresponding to the preS2 region elicit virus-neutralizing antibodies and protect chimpanzees against HBV infection. Antibodies raised by immunization with peptides derived from the preS1 sequence block the site involved in HBV attachment to cell receptors, and are expected to be virus-neutralizing. Results presented here show that antisera raised against synthetic peptide analogs carrying the immunodominant epitope of the preS1 and preS2 sequence, respectively, and corresponding to two HBV subtypes, adw2 and ayw, each recognized preS1 and preS2 specific epitopes on all serological subtypes of the HBV envelope protein. Thus, the sequence variability within the preS1 and preS2 regions does not represent an impediment to the development of synthetic peptide or genetically engineered hepatitis B preS immunogens for worldwide immunization.

A prospective study of HIV-2 prevalence in France.

A seroepidemiological surveillance study to investigate the spread of HIV-2 infection in France was organized in the last semester of 1987 on 100 114 blood donors and 10 004 selected people. Sera were simultaneously tested for antibodies to HIV-1 and to HIV-2 by enzyme-linked immunosorbent assay (ELISA) and were confirmed by specific Western blot. A spot test using synthetic peptides was also applied when a sample was reactive by ELISA HIV-1 and HIV-2 and was confirmed as HIV-1 by Western blot. A total of 30 blood donors were confirmed as HIV-1 positive (0.030%) and no sample was confirmed as HIV-2 positive. In the selected population, the prevalence of HIV-infected people was 7.1% for HIV-1 and 0.09% for HIV-2. Eight of the nine HIV-2 cases were directly or indirectly connected to West Africa. Only one HIV-2 case had no known contact with these countries; he has been transfused and is under investigation. Except for this possible HIV-2 infection in a blood recipient, no HIV-2 seropositivity was found in at-risk groups (homosexuals, drug addicts and haemophiliacs). At present, HIV-2 does not seem to be widely spread in France. Seroepidemiological surveillance will be continued.

Increasing diversity of HIV-1M serotypes in French blood donors over a 10-year period (1985-1995). Retrovirus Study Group of the French Society of Blood Transfusion.

OBJECTIVE: Phylogenetic analysis of gene sequences of HIV-1 has led to the classification of isolates into a major group (M) of viruses, itself divided into subtypes (A to I), and a minor group (O) of rare isolates. Subtype B viruses are the most prevalent in Western countries but little is known about the dynamics of diffusion of the other subtypes in these regions. The prevalence of B subtypes and non-B subtypes in French blood donors between 1985 and 1995 was evaluated. METHODS: A retrospective study was conducted in 490 blood donors, identified as positive for antibody to HIV-1, by twelve French blood banks between 1985 and 1995. Serological subtyping was performed with a subtype-specific enzyme immunoassay, the reliability for genotyping of which has been demonstrated previously. RESULTS: Of 450 typable samples, 48 (10.7%) were non-B subtypes. Non-B reactive samples were found in all of the regions. An increasing prevalence of individuals infected by non-B viruses was observed, from approximately 4% in the early period to more than 20% in 1994-1995 (P = 0.0004). Non-B viruses did not appear to be restricted to patients with direct or indirect epidemiological links to non-European populations. CONCLUSION: We observed an increasing diversity of HIV-1 strains in the population of blood donors residing in France. This stresses the necessity to broaden the surveillance of HIV-1 diversity in order to improve measures to prevent HIV-1 infections.

Progression to AIDS in the majority of asymptomatic HIV-infected people.

Sixty-eight asymptomatic HIV-seropositive people with a CD4 lymphocyte count above 400/mm3 at the first examination were followed up every year over a 3-year period, by monitoring the biological markers of AIDS (CD4 lymphocyte decrease, loss of anti-p24 or anti-p17 antibodies, positive p24 antigenemia, increase of erythrocyte sedimentation rate, and of serum levels of immunoglobulin G. immunoglobulin A, neopterin and beta 2-microglobulin). The percentages of subjects positive for at least one marker at the first, second, third and fourth examinations were 66, 88, 94 and 97%, respectively. The increase in the number of markers with time was significant (chi-square test; P less than 0.001). This increase suggests a progression to AIDS in the majority of asymptomatic seropositive subjects, even those without a decreased CD4 lymphocyte count.

Seroepidemiology of HTLV-I/II in universal screening of blood donations in France.

OBJECTIVE: To evaluate the serological and epidemiological characteristics of HTLV-I/II-positive blood donors in continental France during the first 6 months of universal screening of blood donations (n = 1,816,927). METHOD: A collaborative investigation of all confirmed anti-HTLV-I/II-positive samples reported by blood transfusion centres was performed. Seventy-three out of 77 reported samples were retested at two reference laboratories. Epidemiological data on risk factors were compiled. RESULTS: Of the 73 retested samples, 66 were confirmed to be HTLV-I-positive and one to be HTLV-II-positive; six samples were designated false-positive, mainly because of non-specific reactivity to recombinant gp21 in Western blot. The overall prevalence of HTLV-I/II in continental France is 0.039 per thousand. The main risk factor identified for HTLV-I infection was directly (origin) or indirectly (heterosexual contact) linked to endemicity in the Caribbean. The cost per case of avoided contamination in the 6-month period of this study was 1.36 million French francs. CONCLUSIONS: Sixty-two per cent of HTLV-I/II-infected blood donations would not have been discarded through the previous targeted HTLV screening or through other mandatory tests, including anti-hepatitis B core. To avoid false-positive results, we propose a new algorithm of diagnosis.

Frequency and prognostic importance of thrombocytopenia in symptom-free HIV-infected individuals: a 5-year prospective study.

The exact frequency of HIV-associated thrombocytopenia (TCP), defined as platelet count less than 150 x 10(9)/1, was studied in 435 symptom-free HIV-seropositive individuals. At the baseline control, 23 (5.5%) had TCP. TCP individuals had a significantly lower mean CD4 lymphocyte count than the non-TCP individuals. During a mean follow-up of 30 months, 79 out of the 435 individuals (18%) had TCP at least once. During the study period, only 1% of our patients had a platelet count less than 50 x 10(9)/l. TCP was more frequent in intravenous drug users than in other risk groups. A spontaneous normalization of platelet count was observed in more than 50% of TCP individuals.

Follow-up of subjects with isolated and persistent anti-core (anti-p24 or anti-p17) antibodies to HIV.

Systematic screening of blood donations by enzyme-linked immunosorbent assay (ELISA) for HIV antibodies carries a false-positive rate: the sera involved react in Western blot to core antigens (p24 or p17) but reactivity to envelope is absent. We studied 22 subjects with persistent and isolated anti-core reactivities; 75 HIV seropositive patients were controls. The epidemiological data and the follow-up and biological tests performed in these two populations argue that donors with persistent and isolated anti-core antibodies are not seroconverting for HIV. We conclude: (1) that verification of all anti-HIV ELISA-positive sera by Western blot is essential and that the presence of at least once anti-envelope (gp120 or gp41) antibody is indispensable for the diagnosis of HIV infection; (2) that the solitary anti-p24 or anti-p17 bands observed on Western blot are false-positive. There is no evidence that donors with such reactivities are HIV-infected.

DNA amplification of HIV-1 in seropositive individuals and in seronegative at-risk individuals.

We assessed the HIV-1 status of seropositive and seronegative at-risk individuals by the polymerase chain reaction. Fifty-four out of 55 HIV-1-seropositive samples scored positive. However, HIV-1 proviral DNA was not detected in 16 seronegative homosexuals, 20 seronegative polytransfused haemophiliacs and 20 seronegative thalassaemic children, 20 individuals with isolated and persistent anti-core antibodies and 74 seronegative blood donors. These data indicate that positive HIV-1 DNA is likely to be an exceptional phenomenon in HIV-seronegative people.

Clinical and biological features in the 12 months preceding onset of AIDS in HIV-infected subjects.

Biological markers of HIV infection were studied in 17 asymptomatic HIV seropositive subjects in the 12 months preceding the onset of the disease. No single marker of HIV infection preceded the development of AIDS. Therefore, the clinical care of asymptomatic seropositive subjects should include a number of tests to evaluate HIV activity and immune suppression.

No evidence of HIV-1 infection in seronegative hemophiliacs and in seronegative partners of seropositive hemophiliacs through polymerase chain reaction (PCR) and anti-NEF serology.

We prospectively studied a well-characterized cohort including 60 seronegative hemophiliacs or von Willebrand's disease patients, 6 seronegative female sexual partners of seropositive hemophiliacs, 59 seropositive hemophiliacs or von Willebrand's disease patients and 2 seropositive partners of seropositive hemophiliacs (used as positive controls), and 117 seronegative low risk individuals (used as negative controls). PCR assay, performed in peripheral blood mononuclear cells using three primer pairs in the gag, pol, LTR regions, showed no positive results in the 60 seronegative patients, in the 6 seronegative partners of seropositive patients and in the 117 seronegative low risk individuals, while PCR was positive with at least one primer pair in 53 (87%) of 61 seropositive patients. Anti-nef serology (Western-blot) was negative in seronegative patients, in seronegative partners of seropositive patients and positive in 58% out of the seropositive individuals. These results strongly suggest an absence of HIV-1 infection in individuals with a lastingly negative HIV serology.

Complete nucleotide sequences of six hepatitis B viral genomes encoding the surface antigen subtypes ayw4, adw4q-, and adrq- and their phylogenetic classification.

The complete nucleotide sequences of six hepatitis B viral (HBV) genomes were determined by dideoxy chain termination sequencing of ten overlapping nucleotide fragments obtained by the polymerase chain reaction. Four of the genomes belonged to the two genomic groups E and F of HBV which have been previously identified by us on the basis of sequence divergences within the S gene. Genomic group E encodes the HBsAg subtype ayw4, group F adw4q-. The other two genomes were of Pacific origin within group C and encoded adrq-. The relationship of these complete human HBV genomes to 21 that have been previously published, together with one chimpanzee virus and four rodent hepadnaviral genomes, was investigated by constructing a phylogenetic tree utilizing a combination of distance matrix and approximate parsimonious methods. Thereby, the previously demonstrated segregation of human HBV strains into six genomic groups was confirmed. Both of the representatives of the groups E and F were found to differ by 8.1-13.6% and by 12.8-15.5% from the genomes of the other genomic groups and by 1.5 and 3.7% from each other. Since they differed by more than 8% from the genomes in the other groups, the limit originally used to define HBV, genomic groups their status as new genomic groups was confirmed. The two Pacific group C strains were found to differ by 2.7% from each other and by 4.1 to 5.4% from other group C genomes, suggesting that they diverged early from the other group C genomes. According to both the overall similarity and the phylogenetic dendrogram the F strains formed the most divergent cluster of HBV genomes favoring the concept that they represented the original HBV strains of the New World. The next split in the dendrogram segregated the A, D, E and the chimpanzee strains from the Asian B and C strains. Information on the nucleotide sequences and their encoded products of HBV strains of different genomic groups will provide a basis to understand biological variations of the HBV infection in different parts of the world.

Antibodies to hepatitis C virus by second generation test in hemodialyzed patients.

To assess the prevalence and the incidence of hepatitis C virus (HCV) in a dialysis unit, we prospectively tested for anti-HCV in chronic hemodialysis patients and staff members since January 1989, using a first generation assay. Incidence was nil in staff and low in patients (3.7% in 89, 1% in 90), and prevalence was 30% in patients. In January 1991 blood samples from 115 patients were tested by first (EL1) and second generation (EL2) ELISA (Ortho Diagnostic System). Positive subjects were tested by a RIBA-2 confirmation test. Fifty-three patients were negative by all tests. Positive tests were observed in 62 patients (54%) including 36 positive in EL1 and EL2, and 26 only by EL2. All positive patients were reactive by RIBA-2 but nine were classified undetermined (only one positive band). In five patients reactivity of antibodies to 5-1-1 and C-100-3 gradually declined during the study. Second generation tests gave a better correlation with time on dialysis and blood transfusion. We conclude that second generation tests for HCV are more accurate for estimating true prevalence of HCV infection in hemodialysis units.

The use of labeled fusion protein for detection of B19 parvovirus IgM antibodies by an immunocapture test.

A new anti-B19 IgM ELISA was developed taking advantage of antibody-capture with biotinylated fusion protein as antigen. Specificity was examined using serum IgM antibody positive for rubella, hepatitis B core antigen, cytomegalovirus and Epstein-Barr virus as well as with sera positive for rheumatoid factors or antinuclear antibodies. The specificity was found to be 96%. Of one hundred serum samples compared using the new ELISA or the standard MACRIA tests for the presence of B19 IgM, 88 gave the same results. Fifty-three were negative and 35 were positive. Six sera were ELISA-negative MACRIA-positive, and six MACRIA-negative ELISA-positive. Thus, the ELISA gave 90% agreement with MACRIA. In a clinical study with 725 sera from suspected B19 infections, 161 (22%) were found positive by ELISA. The positive sera were from patients suffering from arthritis (35%), rash (35%), acute or chronic erythroblastopenia (21%), pancytopenia (5%), vascular purpura (2%) and lymphadenopathy (2%). A series of serum specimens obtained from two-B19 infected individuals were also studied. The IgM antibody became undetectable after four months.

Human parvovirus and thalassaemia.

The human parvovirus (HPV) is responsible for aplastic crises in patients with chronic haemolytic anaemia. We describe the cases of four children with aplastic crises in various types of thalassaemia (alpha and beta thalassaemias, major and intermediate forms). In all four patients, specific anti-human parvovirus IgM was detected in their serum, thereby indicating recent infection.

Prevention of hepatitis B in hemodialysis patients using hepatitis B immunoglobulin. A controlled study.

In a controlled study, the protection effect of hepatitis B immune globulin (HBIG) was evaluated in patients hemodialyzed for less than one month in two collaborating units. Fifteen randomly selected patients received HBIG at five to eight week intervals throughout the study, and 13 other control patients received no immunoglobulin. During a follow-up period of 14 to 30 months, none of the HBIG-treated and 12 of the control patients developed evidence of exposure to virus B hepatitis, including 10 with HBs Ag antigenemia (p is less than 0.001): five of these remained persistently antigen positive. Evidence of non-B hepatitis was found in 8 HBIG-treated and in 3 non-treated patients. Only two HBIG-treated patients developed active antibodies against hepatitis B surface antigen. Thus, HBIG seems effective in preventing hepatitis B in hemodialysis patients, provided the interval between two injections is not greater than two months. However, prolonged administration of HBIG may impair passive-active immunization to hepatitis B virus.

Hepatitis B vaccine: randomized trial of immunogenicity in hemodialysis patients.

In order to determine whether reinforced vaccinations improve the immune response among uremic patients, three vaccination schedules with hepatitis B surface antigen vaccine (Institut Pasteur Production) were compared. A total of 215 hemodialysis patients treated in HBV free units were randomly allocated to Group I (3 injections of 1 ml), Group II (3 injections of 2 ml) and Group III (4 injections of 1 ml). Immune response was evaluated in 204 patients. The percentages of responders within 12 months after the first injection (greater than = 10 mIU/ml on 2 successive blood specimens) were: 45.6%, 75.0% and 69.4% in Group I, Group II and Group III respectively. The geometric mean peak values of anti-HBs observed 6 months after the first injection among the responders were: 60, 192 and 268 mIU/ml respectively. One month after a booster dose given to 182 patients 14 months after the first injection, anti-HBs levels were 144, 1123, 524 mIU/ml respectively, and the frequency of patients with an anti-HBs titer greater than = 50 mIU/ml was 68%, 82% and 75% respectively. These results led us to discard the use of Protocol I for these immuno-depressed patients while it is quite satisfactory in healthy subjects; they also show that Protocol II and III give better results than Protocol I, but that they cannot be statistically differentiated. We conclude that response rates and anti HBs antibody titers can be significantly improved in chronic hemodialysis patients with reinforced vaccination protocols.

Screening of viral markers for HIV HBV and HCV infections in blood donors in France and residual risk of viral transmission by blood transfusion.

The first part of this article presents the results of screening tests for antibodies to human immunodeficiency virus (HIV) and hepatitis C virus (HCV) and for hepatitis B surface antigen (HBsAg) from 1986 to 1996. The second part presents the most recent

[Anti-HIV antibody screening kits and the difficulties of serology. Retrovirus Working Group of the Société Française de la Transfusion Sanguine]. / Trousses de dépistage des anticorps anti-VIH et les difficultés de la sérologie. Groupe de travail "Rétrovirus" de la SFTS.

The combined HIV1 + HIV2 assays allow to screen simultaneously the subjects infected by HIV1 or by HIV2 (About 80 HIV1 for 1 HIV2 in blood donations in France). An improvement of both sensitivity and specificity was obtained by using artificial proteins which have been selected for having the most immuno-dominant epitopes. The sensitivity is defined by the study of samples from recent and very recent seroconverters and the specificity by testing 2000 unselected blood donors. All the early seroconversions must be recognized as positive and less than 0.5% false positive results must be found in blood donors. HIV1 variants, temporarily named sub-type O have brought a new difficulty to the HIV serology, due to a weak homology, especially in "env" domains, between these variants and the reference HIV1 strains. Subjects living in France and infected by this HIV1 variant seem rare and the screening assays which miss some of these infected individuals seem capable to modify their reagents in order to recognize all of them.

[HIV seropositivity in blood donations: prevalence, residual risk and epidemiology. The "Retrovirus" Work Group of the French Society of Blood Transfusion]. / Séropositivité VIH dans les dons de sang: prévalence, risque résiduel et épidémiologie. Le Groupe de Travail "Rétrovirus" de la Société Française de Transfusion Sanguine.

As it has been observed since the beginning of anti-HIV screening in blood donations, the rate of seropositive donations has been still decreasing during these 4 last years: 0.090/1000 in 1991 to 0.042/1000 in 1994. This decrease has been observed in blood donations from first-time donors (0.38 to 0.17/1000) and from regular donors (0.060 to 0.030/1000). However, the residual risk from cellular components, such as it is estimated, seems to be stable. The proportion of blood donations positive for both anti-HIV and anti-HBc was constantly decreasing from year to year: 63% between 1985 and 1988 and 25% in 1994. The repartition of seropositive donors into sex, ages and at-risk groups during this recent period was the same as previously observed.

[Reevaluation of the sensitivity of screening assays for anti-HIV antibodies. The Retrovirus Work Group of the SFTS. French Society of Blood Transfusion]. / Réévaluation de la sensibilité des trousses de dépistage des anticorps anti-HIV. Le Groupe de Travail Rétrovirus de la SFTS. Société Française de Transfusion Sanguine.

A comparative evaluation of the sensitivity of anti-HIV screening assays has been recently performed with a selected panel including all the actually known difficulties of HIV serology: Very recent seroconversions (per-seroconversions) and recent HIV1/M seroconversions, HIV1/M seropositive with a subtype different from B, HIV1 group 0 and HIV2. The criteria of sensitivity were to recognize as positive all the seropositive samples and at least 2 of the 8 per-seroconversions. The 18 combined ELISA fulfilled these criteria, except for one assay with a deficient HIV2 sensitivity. However, all these assays do not have the same level of sensitivity. The most important differences were observed with the pre-seroconversion samples, the number of positive results on such samples varying from 2 to 8. The capacity of a screening assay to early detect an HIV-infected subject is a supplementary criterium to retain, particularly in blood transfusion screening, in order to shorten the "window" period.