Retrospective analysis of antiretroviral HIV treatment success based on medical history or guided by the reverse hybridisation LiPA HIV genotyping system.

The changes in viral load and CD4(+) count at 3 and 6 months in a group of 166 HIV-infected patients was evaluated. The new therapy was chosen based on the medical history procedures for 70 patients, and in 96 patients it was guided by the partial or complete result of the line probe assay (LiPA) HIV RT and Protease resistance tests. The absolute difference from the baseline of the log viral load at 3 and 6 months was significantly different between the two groups when adjusted for baseline viral load (P < 0.0001) and stayed significant when intention-to-treat analysis was carried out (P < 0.001). The absolute difference of the CD4(+) count was not significantly different when adjusted for baseline CD4(+) (P = 0.854, 3 months; P = 0.06, 6 months). The proportion of patients with a viral load

Assessment of a new immunoassay for serological confirmation and discrimination of human T-cell lymphotropic virus infections.

The present study evaluated a new confirmatory assay for antibodies to human T-cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) proteins performed with serum samples from various commercial sources. The new test is a line immunoassay (LIA) with a nylon membrane sensitized with the most relevant antigens of HTLVs: the envelope gp46 and gp21 as well as the gag p24 and p19 antigens, represented by either recombinant proteins or synthetic peptides. A total of 176 serum or plasma samples were tested, of which 66 were HTLV-1 positive, 72 were HTLV-2 positive, and 38 were HTLV negative; of the 38 HTLV-negative samples 23 were indeterminate by Western blotting (WB). Serially diluted samples (n = 33) from HTLV-1- and HTLV-2-infected patients were also analyzed to determine the sensitivity of the new assay. The new confirmatory assay (INNO-LIA HTLV) performed markedly better than WB assays for those samples reactive by screening. Accurate confirmation of the presence of HTLV-1 and HTLV-2 antibodies and accurate discrimination of HTLV-1 and HTLV-2 antibodies were obtained for all the HTLV-seropositive samples. Due to its enhanced specificity and sensitivity, the new assay not only improves the ability to confirm and discriminate HTLV infections but also eliminates the vast majority of WB-indeterminate and false-positive specimens.

Line probe assay for rapid detection of drug-selected mutations in the human immunodeficiency virus type 1 reverse transcriptase gene.

Upon prolonged treatment with various antiretroviral nucleoside analogs such as 3'-azido-3'-deoxythymidine, 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, (-)- beta-L-2', 3'dideoxy-3'thiacytidine and 2',3'-didehydro-3'-deoxythymidine, selection of human immunodeficiency virus type 1 (HIV-1) strains with mutations in the reverse transcriptase (RT) gene has been reported. We designed a reverse hybridization line probe assay (LiPA) for the rapid and simultaneous characterization of the following variations in the RT gene: M41 or L41; T69, N69, A69, or D69; K70 or R70; L74 or V74; V75 or T75; M184, I184, or V184; T215, Y215, or F215; and K219, Q219, or E219. Nucleotide polymorphisms for codon L41 (TTG or CTG), T69 (ACT or ACA), V75 (GTA or GTG), T215 (ACC or ACT), and Y215 (TAC or TAT) could be detected. In addition to the codons mentioned above, several third-letter polymorphisms in the direct vicinity of the target codons (E40, E42, K43, K73, D76, Q182, Y183, D185, G213, F214, and L214) were found, and specific probes were selected. In total, 48 probes were designed and applied to the LiPA test strips and optimized with a well-characterized and representative reference panel. Plasma samples from 358 HIV-infected patients were analyzed with all 48 probes. The amino acid profiles could be deduced by LiPA hybridization in an average of 92.7% of the samples for each individual codon. When combined with changes in viral load and CD4+ T-cell count, this LiPA approach proved to be useful in studying genetic resistance in follow-up samples from antiretroviral agent-treated HIV-1-infected individuals.

Mother-to-child transmission of human T-cell lymphotropic virus types I and II (HTLV-I/II) in Gabon: a prospective follow-up of 4 years.

SUMMARY: For 4 years. we determined the mode and risk of mother-to-child transmission of HTLV-I in a prospective cohort of 34 children born to seropositive mothers in Franceville, Gabon. We also determined the prevalence of antibodies to HTLV-I/II in siblings born to seropositive mothers. Antibodies to HTLV-I/II were detected by Western blot, and the proviral DNA was detected by the polymerase chain reaction (PCR). The risk of seroconversion to anti-HTLV-I for the 4 years of follow-up was 17.5 percent. Anti-HTLV-I/II and proviral DNA were only detected after age 18 months. We observed a seroprevalence rate of 15 percent among the siblings born to HTLV-I/II seropositive mothers. Furthermore, we report a case of mother-to-child transmission of HTLV-II infection in a population of HTLV-II-infected pregnant women that is emerging in Gabon. The lack of detection of HTLV-I/II proviral DNA in cord blood and amniotic fluid and, furthermore, the late seroconversion observed in the children indirectly indicate that mother-to-child transmission occurred postnatally, probably through breast milk.

Genetic diversity of the envelope glycoprotein from human immunodeficiency virus type 1 isolates of African origin.

The genetic diversity of the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) isolates was studied. HIV-1 isolates were obtained from eight countries in Africa: Djibouti, Gabon, Kenya, Senegal, Somalia, Uganda, Zaire, and Zambia. The DNA sequences encoding the complete HIV-1 envelope protein were PCR amplified and sequenced. Phylogenetic relationships among the 21 sequences from this study and the 32 previously published full-length env HIV-1 sequences were determined. Twenty of the newly sequenced African isolates could be assigned to envelope subtypes A, C, D, and G. One isolate, collected in Zambia, did not belong to any of the eight previously described subtypes and may represent a prototype sequence of its envelope subtype. The phylogenetic classification of these isolates was strongly supported by bootstrapping and the congruence of trees generated by either distance methods or maximum parsimony analysis. The data presented in this study confirm the existence of several genetic subtypes within the global HIV epidemic and broaden the genetic variability previously observed for envelope subtypes. The geographic spread of different subtypes was shown to be substantial, and the notion of cocirculation of subtypes was reinforced.

Influence of apolipoprotein E genotype on senile dementia of the Alzheimer and Lewy body types. Significance for etiological theories of Alzheimer's disease.

Alzheimer's disease (AD) is associated with an increased frequency of the apolipoprotein E type epsilon 4 allele. To address both the disease and the allele specificity of this association, we have examined the apolipoprotein E allele distribution in 255 elderly persons including those with autopsy-confirmed AD, senile dementia of the Lewy body type (SDLT), vascular dementia, Parkinson's disease (PD) or Huntington's disease and in nondemented controls either with or without coronary complications. The epsilon 4 allele frequency was increased in SDLT (0.365) and AD (0.328) as compared with controls (0.147), PD (0.098), or Huntington's chorea (0.171). Coronary disease and vascular dementia were associated with marginally higher epsilon 4 allele frequencies than in controls. In PD, amyloid beta-protein accumulated to a greater extent in those cases possessing an epsilon 4 allele than in those without. Those PD cases with dementia were not distinguished from either controls or PD cases without dementia, whether tested biochemically or by apolipoprotein E genotype. It is the comparison of the results in AD and SDLT that yielded the most significant findings. There was a 1.8-fold excess of amyloid beta-protein in AD as compared with controls, and the levels in SDLT were intermediate between those in AD and controls. In contrast, AD was discriminated from both controls and SDLT by the substantial accumulation of paired helical filament tau and phosphorylated tau (both increased more than 20-fold as compared with controls). SDLT was nevertheless characterized by an increased epsilon 4 allele frequency in the absence of significant tau pathology (at least 10-fold less than that in AD). These findings indicate that tau processing is more specifically associated with AD than is amyloid beta-protein accumulation and that presence of the epsilon 4 allele is not an etiological factor that accounts for tau pathology.

Identification of an env G subtype and heterogeneity of HIV-1 strains in the Russian Federation and Belarus.

OBJECTIVE: To identify HIV-1 envelope sequence subtypes in infected individuals from the Russian Federation and Belarus. PATIENTS: A cohort of children infected after exposure to non-sterile needles during the 1988-1989 HIV-1 epidemic in southern Russia (n = 20) and HIV-1-seropositive individuals from Russia (n = 1) and Belarus (n = 7) infected via sexual transmission. METHODS: DNA samples derived from peripheral blood mononuclear cells were analysed for their HIV-1 genotypes by the heteroduplex mobility assay (HMA). The 1.3 kilobase-pair env gene fragments encoding a portion of gp120 were amplified by nested polymerase chain reaction, cloned and sequenced. The env sequences derived from these patients were aligned and phylogenetic neighbour-joining and maximum parsimony-derived trees generated. RESULTS: The env sequences derived from eight individuals infected in Russia and Belarus belong to subtype A (one), B (four), C (two), and D (one). Sequences derived from children, infected during parenteral manipulations in southern Russia, and one mother were closely related, but highly divergent, as a group, from all prototypic strains (genetic divergence, 17.2-22.9%). However, they clustered together with env sequences of the V1525 and LBV21-7 isolates from Gabon, recently described to be members of a new HIV-1 env subtype G. CONCLUSION: Extensive heterogeneity of HIV-1 subtypes was evident in the Russian Federation and Belarus. Our data also support the existence of an HIV-1 env genetic subtype G, and such isolates are now apparently present on both the African and European continents. These variants were identified through V3 peptide enzyme-linked immunosorbent assay screening and subsequent HMA analysis. The combination of these techniques represents a model for screening HIV variants within a large population.

Design and evaluation of new, highly sensitive and specific primers for polymerase chain reaction detection of HIV-1 infected primary lymphocytes.

Primer pairs in the HIV-1 POL and ENV genes were evaluated by performing a PCR on lysed peripheral blood mononuclear cells (PBMCs) from 96 HIV-1 seropositive and 40 seronegative individuals originating from 16 different geographical localities in Africa, Europe and Haiti. A single PCR using primer pairs to the LTR, GAG and ENV regions and detection by radioactively labelled oligonucleotide probes was compared to a nested PCR scheme using newly designed POL and ENV primers which used ethidium-bromide staining of the amplified product on agarose gel. The newly designed POL nested primer pair was shown to be highly sensitive (93%) and specific (100%) for the detection of HIV-1 proviral DNA of very diverse geographical and genetic origin, including highly aberrant HIV-1 isolates. The sensitivity of the newly designed ENV primers was 68.7%, which does not differ significantly from the sensitivity of the classical primers, SK 68/69. Both ENV primers were unable to amplify two SIVcpz isolates from naturally infected chimpanzees.

Genetic analysis of HIV-1 isolates from Brazil reveals presence of two distinct genetic subtypes.

The spread of the human immunodeficiency virus type 1 (HIV-1) is by now virtually worldwide. An understanding of the genetic, biological, and immunological differences among isolates collected in different geographic locales is crucial for the development of globally effective vaccines. Here we report the genetic characteristics of 21 HIV-1 isolates from Brazil. The isolates were initially characterized using a heteroduplex mobility assay. The majority (17 of 21) were related to North American/European reference isolates of genetic subtype B. Four isolates belonged to a more recently identified genotype, termed subtype F. The subtype F sequences from Brazil are distinguishable in both gag and env from five other genetic subtypes of HIV-1 currently recognized. Like many locales, Brazil harbors more than one HIV-1 subtype.

PIP: The spread of the human immunodeficiency virus type 1 (HIV-1) is by now virtually worldwide. An understanding of the genetic, biological, and immunological differences among isolates collected in different geographic locales is crucial for the development of globally effective vaccines. The genetic characteristics of HIV-1 isolates from whole blood samples of 21 HIV-1-seropositive Brazilian patients collected during 1989 and 1990 are reported. Virus was isolated by cocultivation of patient peripheral blood mononuclear cells (PBMCs) with phytohemagglutinin (PHA)-stimulated donor PBMCs. The isolates were initially characterized using a heteroduplex mobility assay, which estimates the DNA sequence homology of a selected genomic region from different HIV-1 isolated from the migration of heteroduplexes in polyacrylamide gels. Five distinct HIV-1 envelope subtypes were identified, and a sixth subtype, termed F, was identified using isolates from Brazil and Romania. One Brazilian isolate was identified as subtype B by the more rapid relative migration of heteroduplexes. The majority (17 of 21) were related to North American/European reference isolates of genetic subtype B, whereas 4 isolates belonged to subtype F, a more recently identified genotype. The gag and env genes of several Brazilian isolates belonging to subtypes B and F were cloned and sequenced to allow a detailed analysis of their phylogenic relationships. This further established the existence of two distinct and well-separated genetic subtypes among Brazilian HIV-1 isolates. The subtype F sequences from Brazil are distinguishable in both gag and env from 5 other genetic subtypes of HIV-1 currently recognized. Like many locales, Brazil harbors more than one HIV-1 subtype.

Two antigenically distinct subtypes of human immunodeficiency virus type 1: viral genotype predicts neutralization serotype.

At least five distinct genetic subtypes (genotypes) of human immunodeficiency virus type 1 (HIV-1) have been identified by DNA sequencing. Current vaccine candidates are based on virus strains from North America and Europe that represent only one subtype. The extent to which distinct genotypes of HIV-1 correspond to antigenically distinguishable serotypes is largely unknown and may be critically important to vaccine design. Cross-neuralization studies were done with viruses and plasma from two different genotypes. Based on neutralization susceptibility, 10 primary HIV-1 isolates from Thailand and the United States were classified into one of two antigenic subtypes that correlated with viral genotype. The existence of serotypes of HIV-1 suggests that a broadly effective vaccine may have to include strains from multiple subtypes. Neutralization of these primary HIV-1 isolates differed substantially from results with laboratory strains. Future neutralization studies using primary isolates and multiple genotypes may be important for assessment of HIV-1 antigenic diversity.

Genetic relationships determined by a DNA heteroduplex mobility assay: analysis of HIV-1 env genes.

The genetic diversity of human immunodeficiency virus (HIV) is a major concern thought to impact on immunologic escape and eventual vaccine efficacy. Here, simple and rapid methods are described for the detection and estimation of genetic divergence between HIV strains on the basis of the observation that DNA heteroduplexes formed between related sequences have a reduced mobility in polyacrylamide gels proportional to their degree of divergence. Reliable phylogenetic subtypes were assigned for HIV-1 strains from around the world. Relationships between viruses were closest when derived from the same or epidemiologically linked individuals. When derived from epidemiologically unlinked individuals, the relationships between viruses in a given geographic region correlated with the length of time HIV-1 had been detected in the population and the number of strains initiating widespread infection. Heteroduplex mobility analysis thus provides a tool to expedite epidemiological investigations by assisting in the classification of HIV and is readily applicable to the screening and characterization of other infectious agents and cellular genes.

Phylogenetic analysis of gag genes from 70 international HIV-1 isolates provides evidence for multiple genotypes.

OBJECTIVE: To determine the extent of genetic variation among internationally collected HIV-1 isolates, to analyse phylogenetic relationships and the geographic distribution of different variants. DESIGN: Phylogenetic comparison of 70 HIV-1 isolates collected in 15 countries on four continents. METHODS: To sequence the complete gag genome of HIV-1 isolates, build multiple sequence alignments and construct phylogenetic trees using distance matrix methods and maximum parsimony algorithms. RESULTS: Phylogenetic tree analysis identified seven distinct genotypes. The seven genotypes were evident by both distance matrix methods and maximum parsimony analysis, and were strongly supported by bootstrap resampling of the data. The intra-genotypic gag distances averaged 7%, whereas the inter-genotypic distances averaged 14%. The geographic distribution of variants was complex. Some genotypes have apparently migrated to several continents and many areas harbor a mixture of genotypes. Related variants may cluster in certain areas, particularly isolates from a single city collected over a short time. CONCLUSIONS: The genetic variation among HIV-1 isolates is more extensive than previously appreciated. At least seven distinct HIV-1 genotypes can be identified. Diversification, migration and establishment of local, temporal 'blooms' of particular variants may all occur concomitantly.

Genetic comparison of HIV-1 isolates from Africa, Europe, and North America.

PIP: Laboratory scientists used anchored polymerase chain reaction (PCR) and DNA sequencing to compare HIV-1 isolates from countries in Africa (Ivory Coast, Gabon, Zaire, Kenya, and others), Europe (Belgium and other countries), and the US. The US isolates had the most homogenous PCR profile followed by the European pattern. There was considerable PCR primer mispairing for the African isolates, especially those from Kenya, indicating that the range of HIV-1 variation could have been rather extensive. This virus diversity could greatly affect therapy or intervention in sites in Africa with such a complex mix of variants. Nevertheless, the genetic information of these diverse isolates could bring about research leading to an anti-HIV-1 vaccine. For example, the expanded DNA sequence data base could record phylogenetic relationships, thereby, helping researchers choose prototypic variants for vaccine development. More information would allow researchers to generate new PCR primers for better discrimination of variants. They could apply PCR typing to huge sample sizes to adequately document HIV-1 variation in Africa. It could also prove invaluable as a means to determine incidence and prevalence of local variants during vaccine field trials. It can also discern the limiting criteria for HIV-1 genetic variation.^ieng

Prevalence of HTLV-I and HTLV-II infection in Gabon, Africa: comparison of the serological and PCR results.

A cluster sampling survey was performed in 1989 in Libreville, Gabon, to determine HTLV-I and HTLV-II prevalence and to compare the efficacy of polymerase chain reaction (PCR) and serology in detecting HTLV-I and HTLV-II infections. A total of 322 sera from adults were tested by ELISA and by Western blot (WB). The WB patterns were interpreted according to WHO criteria and those of the manufacturer. PCR analysis using primer pairs in the gag and pol region, with a specific probe for HTLV-I and HTLV-II, was performed on the lymphocytes of the 322 adults. In addition, 134/322 samples were re-tested with tax primers, in a second laboratory. Using WHO criteria, 8/322 (2.5%) samples were positive on WB and 25 were indeterminate; with the criteria of the kit, 26/322 (8.1%) were positive and 7 were indeterminate by WB. By PCR, 13 (4%) samples were positive, including 12 for HTLV-I (3.7%) and one for HTLV-II (0.3%). All 8 seropositive samples (by the WHO criteria) were positive by PCR, as were 4 out of 25 indeterminate samples. Only one out of 289 seronegative samples was positive by PCR. In contrast, only 12/26 positive samples by the kit criteria were confirmed by PCR. These results confirm the relatively high HTLV-I/II seroprevalence observed in Gabon. HTLV-I infection is preponderant, but HTLV-II is also present. The WHO criteria for WB give a better fit with PCR results than the kit criteria for WB. In the absence of a specific confirmatory test and based on the uncommon "seronegative" HTLV-I/II infection, the indication for PCR appears limited to the positive WB samples (to differentiate HTLV-I and II infection) and to the indeterminate WB samples.

Anti-idiotypic and anti-anti-idiotypic antibodies as highly specific tools in epitope and active site studies on human interferon-gamma.

Immunization of rabbits with F(ab')2 fragments of different monoclonal antibodies directed against human interferon-gamma yielded antisera with anti-idiotypic characteristics. Isolation of the anti-idiotypic fraction, resulting in a highly specific antiserum, allowed us to prove that out of six competing monoclonal antibodies directed against human interferon-gamma, only two really recognize the same epitope. The other monoclonal antibodies compete on the basis of steric hindrance, which is not surprising, because of the large difference in Mr between interferon-gamma and an immunoglobulin. The anti-idiotypes provided us also with a tool to study isolated epitopes on the human interferon-gamma molecule, a task which was previously not practicable. Exploration of the biological properties of these anti-idiotypes allows us to determine whether the investigated epitopes are involved in receptor binding. The production of an anti-anti-idiotypic antiserum not only proved that we generated real internal images, but also that these images conserved all of their properties, although with a decreased affinity in comparison with the original monoclonal antibody. As the former is a polyclonal antiserum, directed against a single epitope of the human interferon-gamma molecule, competition experiments yielded additional information on the relative position of three epitopes recognized by inhibiting monoclonal antibodies. These antisera will possibly open new ways for the affinity purification of interferon-gamma and perhaps for the treatment of autoimmune diseases.