Use of pre-ART laboratory screening to identify renal, hepatic and haematological abnormalities in Côte d'Ivoire.

BACKGROUND: High demand for HIV-services and extensive clinical guidelines force health systems in low-resource settings to dedicate resources to service delivery at the expense of other priorities. Simplifying services may reduce the burden on health systems and pre-antiretroviral therapy (ART) laboratory screening is among the services under consideration for simplification. METHODS: We assessed the frequencies of conditions linked to ART toxicities among 34,994 adult, ART-naïve patients with specimens referred to the RETRO-CI laboratory in Abidjan, Côte d'Ivoire between 1998 and 2017. Screening included tests for serum creatinine, alanine aminotransferase (ALT) and haemoglobin (Hb) to identify renal dysfunction (estimated glomerular filtration rate < 50 mL/min), hepatic abnormalities (ALT > 5× upper limit of normal) and severe anaemia (Hb < 6.5 g/dL), respectively. We considered screening results across four eras and identified factors associated with the conditions in question. RESULTS: The prevalence of renal dysfunction, hepatic abnormalities and severe anaemia were largely unchanged over time and just 8.4% of patients had any of the three conditions. Key factors associated with renal dysfunction and severe anaemia were age > 50 years (adjusted odds ratio (aOR): 2.53; 95% confidence interval (CI): 2.19-2.92; P < 0.001) and CD4 < 100 cells/µl (aOR: 2.57; 95% CI: 2.30-2.88; P < 0.001). CONCLUSION: The relative infrequency of conditions linked to toxicity in Côte d'Ivoire supports the notion that simplification of pre-ART laboratory screening may be undertaken with limited negative impact on identification of adverse events. Targeted screening may be a feasible strategy to balance detection of conditions associated with ART toxicities with simplification of services.

CONTEXTE: La forte demande de services VIH et les directives cliniques détaillées obligent les systèmes de santé des pays à faibles ressources à consacrer des ressources à la prestation de services au détriment d'autres priorités. La simplification des services peut réduire la charge pesant sur les systèmes de santé et les analyses de laboratoire avant la thérapie antirétrovirale (ART) fait partie des services envisagés pour la simplification. MÉTHODES: Nous avons évalué la fréquence des conditions liées aux toxicités dues à l'ART chez 34.994 patients adultes naïfs pour l'ART avec des échantillons référés au laboratoire RETRO-CI à Abidjan, en Côte d'Ivoire entre 1998 et 2017. Les analyses comprenaient les tests de créatinine sérique, d'alanine aminotransférase (ALT) et d'hémoglobine (Hb) pour identifier respectivement la dysfonction rénale (débit de filtration glomérulaire estimé <50 mL/min), les anomalies hépatiques (ALT >5x la limite supérieure normale) et l'anémie sévère (Hb <6,5 g/dL). Nous avons examiné les résultats des analyses sur quatre époques et identifié les conditions associées aux conditions en question. RÉSULTATS: La prévalence de la dysfonction rénale, des anomalies hépatiques et de l'anémie sévère est restée largement inchangée au fil du temps et seulement 8,4% des patients présentaient l'une des trois conditions. Les facteurs clés associés à la dysfonction rénale et à l'anémie sévère étaient l'âge >50 ans (odds ratio ajusté (aOR): 2,53; intervalle de confiance (IC) à 95%: 2,19 à 2,92; p <0,001) et les CD4 <100 cellules/µl (aOR: 2,57; IC95%: 2,30 à 2,88; P < 0,001). CONCLUSION: La relativement faible fréquence des conditions liées à la toxicité en Côte d'Ivoire soutient la notion selon laquelle une simplification des analyses de laboratoire pré-ART peut être entreprise avec un impact négatif limité sur l'identification des événements adverses. Le ciblage des analyses peut être une stratégie réalisable pour aligner la détection des conditions associées aux toxicités ART à la simplification des services.

Protease sequences from HIV-1 group M subtypes A-H reveal distinct amino acid mutation patterns associated with protease resistance in protease inhibitor-naive individuals worldwide. HIV Variant Working Group.

BACKGROUND: Although numerous mutations that confer resistance to protease inhibitors (PRI) have been mapped for HIV-1 subtype B, little is known about such substitutions for the non-B viruses, which globally cause the most infections. OBJECTIVES: To determine the prevalence of PRI-associated mutations in PRI-naive individuals worldwide. DESIGN: Using the polymerase chain reaction, protease sequences were amplified from 301 individuals infected with HIV-1 subtypes A (79), B (95), B' (19), C (12), D (26), A/E (23), F (26), A/G (11), and H (3) and unclassifiable HIV-1 (7). Amplified DNA was directly sequenced and translated to amino acids to analyze PRI-associated major and accessory mutations. RESULTS: Of the 301 sequences, 85% contained at least one codon change giving substitution at 10, 20, 30, 36, 46, 63, 71, 77, or 82 associated with PRI resistance; the frequency of these substitutions was higher among non-B (91%) than B (75%) viruses (P < 0.0005). Of these, 25% carried dual and triple substitutions. Two major drug resistance-conferring mutations, either 20M or 30N, were identified in only three specimens, whereas drug resistance accessory mutations were found in 252 isolates. These mutations gave distinct prevalence patterns for subtype B, 63P (62%) > 77I (19%) > 10I/V/R (6%) = 361 (6%) = 71T/V (6%) > 20R (2%), and non-B strains, 36I (83%) > 63P (17%) > 10I/V/R (13%) > 20R (10%) > 77I (2%), which differed statistically at positions 20, 36, 63, 71, and 77. CONCLUSIONS: The high prevalence of PRI-associated substitutions represent natural polymorphisms occurring in PRI-naive patients infected with HIV-1 strains of subtypes A-H. The significance of distinct mutation patterns identified for subtype B and non-B strains warrants further clinical evaluation. A global HIV-1 protease database is fundamental for the investigation of novel PRI.

Genotypic subtypes of HIV-1 in Cameroon.

OBJECTIVE: The only two HIV-1 strains (ANT70 and MVP5180) reported to date from Cameroon are members of the outlier clade (group O). In this study, we assessed the prevalence of group O viruses and other HIV-1 subtypes in Cameroon. DESIGN: A phylogenetic analysis of 18 HIV-1 strains isolated from seropositive individuals from Yaoundé and Douala, Cameroon. METHODS: A 900 base-pair fragment of the env gene coding for V3, V4, V5, and the beginning of gp41 of 17 out of 18 HIV-1 isolates from Cameroon was amplified, cloned and sequenced using polymerase chain reaction. A phylogenetic tree was constructed. RESULTS: The overall env nucleotide sequence divergence among the Cameroon isolates ranged from 6.1 to 27.5%. In a phylogenetic tree, six subtypes were identified when compared with 23 reference strains of different geographic origin. Of these 17 Cameroonian strains, 11 (61%) were of subtype A of which the interpatient distances at the sequence level varied from 6.1% to 18.3% (average, 11.9%). Three (17%) strains were of subtype F, and the other three strains (6% each) belonged to subtypes B, E and H, respectively. The remaining isolate was classified as belonging to group O, on the basis of the sequence of part of the pol gene. A very broad spectrum of different tetrameric amino-acid sequences was observed at the apex of the V3 loop. Eleven strains contained the tetrameric globally predominant GPGQ sequence at the tip of the V3 motif. Two strains had the GPGR sequence typical of the American and European HIV-1 strains. The remaining tetrameric sequences included GPGS, GSGQ, GRGQ, and GLGR. CONCLUSION: These findings on a limited number of viruses suggest extensive env gene diversity of HIV-1 strains from Cameroon, and could have implications for vaccine development in Africa.

Cross-neutralizing antibodies to HIV-1ANT70 and HIV-1IIIB in sera of African and Belgian HIV-1-infected individuals.

OBJECTIVES: To determine the neutralizing antibody patterns to HIV-1ANT70 (ANT70) and HIV-1IIIB (IIIB) in human sera obtained from HIV-1-infected individuals from different African countries and Belgium. Second, to correlate the presence of neutralizing antibodies in sera and their ability to bind to synthetic peptides derived from eight different HIV-1 V3 loop sequences. DESIGN AND METHODS: Forty sera from Belgium and 88 obtained from seven countries in Africa were tested for their ability to neutralize ANT70 (one of the most genetically divergent HIV-1 isolates documented), and IIIB. Sera found to cross-neutralize both viruses were further challenged with four HIV-1 field isolates. All sera were tested on a panel of V3 loop peptides obtained from different HIV-1 genotypes. RESULTS: Four patterns of sera were identified, including 33 (26%) sera not neutralizing any of the isolates, seven (5%) sera neutralizing only ANT70, 45 (35%) sera neutralizing only IIIB, and 43 (34%) sera cross-neutralizing both isolates. Sera capable of cross-neutralizing both ANT70 and IIIB consistently neutralized other field isolates tested, with a remarkable similarity in neutralizing antibody titre. A significantly higher number of sera cross-neutralizing both ANT70 and IIIB compared with sera lacking neutralizing antibodies, reacted simultaneously in enzyme-linked immunosorbent assays (ELISA) with three or more V3 loop peptides belonging to HIV-1 strains of different genotypes. However, none of the sera cross-neutralizing ANT70 and IIIB were reactive in ELISA with the ANT70 V3 loop peptide. CONCLUSION: These results suggest that despite pronounced genomic variation of the HIV-1ANT70 isolate, there are strongly conserved neutralizing epitopes situated outside the V3 loop that are shared by other HIV-1 isolates. These findings suggest that genetic variation might be surmountable in the design of a polyvalent HIV vaccine, if neutralizing antibodies are found to be correlates of protection in HIV infection.

Antibodies to V3 loop peptides derived from chimpanzee lentiviruses and the divergent HIV-1ANT-70 isolate in human sera from different geographic regions.

OBJECTIVE: To study the spread of antibodies to V3 loop peptides of two chimpanzee lentiviruses and the divergent HIV-1ANT-70 isolate (group O) in human sera from different geographic regions, and to compare this with reactions to peptides from known North American (subtype B) and Zaïrean (subtype D) strains. METHODS: A total 2495 HIV-1-antibody-positive sera from nine countries were tested by enzyme-linked immunosorbent assay for antibodies to the V3 loop of 10 HIV/SIV isolates (including MN, SF2, HXB2, RF, MAL, ELI, Z6 and ANT-70 for HIV-1, and cpz-gab and cpz-ant for SIV). RESULTS: In each country, the highest prevalences were observed against the MN peptide (58.8-91.7%). Seroreactivity to other peptides from subtype B were generally lower. Prevalences of antibodies to V3 peptides derived from Zaïrean strains belonging to subtype D were generally lower than to subtype B. Relative high prevalences of sera reactive with the SIVcpz-gab V3 peptide were observed. The lowest rates were seen in Brazil (4.2%) and Belgium (25.7%). Among the African countries, the prevalence rates varied between 30.1 and 67.6%. Prevalence to the V3 loop derived from the SIVcpz-ant strains was much lower. Prevalence of sera reactive to the ANT-70 V3 loop peptide was very low, and the highest rates were observed in Cameroon (10.2%), Niger (6%) and Gabon (4.6%). Only the sera reactive to the ANT-70 V3 loop peptide from Cameroon and Gabon were confirmed on a specific HIVANT-70 Western blot (i.e., presence of antibodies to the envelope protein gp 120). CONCLUSIONS: The extent to which different V3 peptide reactivity patterns reflect the circulation of different HIV-1 strains in a particular population is not yet clear. However, V3 peptide serology using the very specific V3 peptide of the HIVANT-70 is a good indicator of the very aberrant group O in a particular population.

Lack of correlation between V3-loop peptide enzyme immunoassay serologic subtyping and genetic sequencing.

OBJECTIVE: To compare the performance of V3-loop peptide enzyme immunoassay (PEIA) methodologies from four different laboratories for subtyping HIV-1, and to determine the causes for the lack of correlation between V3-loop PEIA serotyping and subtyping by sequencing. MATERIALS AND METHODS: Synthetic peptides derived from the amino-acid consensus sequences of the V3-loop of group M strains representing genetic subtypes A-F as well as reference strains were evaluated in PEIA by four different laboratories for their ability to accurately determine the subtype in a panel of 85 sera obtained from persons infected with known HIV-1 subtypes (28 subtype A, 34 subtype B, four subtype C, 10 subtype D, seven subtype F, one each of subtype H and G). Furthermore, the V3 loop of the corresponding virus was compared with the V3 loop of the peptides used in PEIA. RESULTS: The correlation between HIV-1 subtyping by sequencing and V3-loop PEIA from the different laboratories varied considerably for the different HIV-1 subtypes: subtype A (46-68%), B (38-85%), C (75-100%), D (29-50%), and F (17-57%). A 70% agreement between PEIA and sequencing subtypes was observed for samples with the concordant presence of the same octameric sequences in the V3 loop of the virus and the V3 loop of the peptide used in PEIA; however, only 42% of specimens with different V3-loop octameric viral and peptide sequences yielded concordant results in V3-loop serotyping and genetic subtyping. CONCLUSION: Our results indicate that V3-loop PEIA methodologies used in different laboratories correlate poorly with genetic subtyping, and that their accuracy to predict HIV-1 subtypes in sera of Belgian individuals infected with different HIV-1 subtypes (A, B, C, D, F, G and H) vary considerably. The poor correlation between serotyping and genetic subtyping was partly due to the simultaneous occurrence of subtype-specific octameric sequences at the tip of the V3 loop of viruses belonging to different genetic subtypes.

Cervicovaginal anti-HIV antibodies in HIV-seronegative female sex workers in Abidjan, Côte d'Ivoire.

OBJECTIVE: To detect anti-HIV antibodies in cervicovaginal secretions of HIV-seronegative female sex workers and to evaluate whether the presence of these antibodies is associated with increased sexual exposure. METHODS: A cross-sectional study was carried out at a confidential clinic for female sex workers in Abidjan, Côte d'Ivoire. The participants were 342 HIV-seronegative female sex workers in whom a cervicovaginal lavage was collected. The main outcome measures were the detection of antibodies to HIV-1 in cervicovaginal lavages using an in-house and a commercial (Seradyn Sentinel; Calypte Biomedical Corporation, Berkeley, California, USA) enzyme immunoassay; the detection of semen in cervicovaginal lavages; and the assessment of epidemiological and biological markers of sexual exposure to HIV. RESULTS: Cervicovaginal anti-HIV antibodies were detected in 7.3 and 29.8% of women using in-house enzyme-linked immunosorbent assay (ELISA) and Seradyn Sentinel respectively. All cervicovaginal secretions found to be positive by in-house ELISA were also positive by Seradyn Sentinel. In a minority of women, ranging from 2.9% by in-house ELISA to 12.3% by Seradyn Sentinel, the anti-HIV antibodies were present in vaginal fluids that did not contain semen. Sexual exposure to HIV was similar in women with anti-HIV antibodies in their semen-free cervicovaginal secretions compared with women without anti-HIV antibodies in their cervicovaginal secretions. CONCLUSIONS: Cervicovaginal HIV-specific antibodies were detected in a minority of sexually exposed HIV-seronegative female sex workers in Abidjan. The lack of association between increased sexual exposure to HIV and presence of cervicovaginal HIV-specific antibodies suggests that the production of genital HIV-specific antibodies in exposed seronegative women depends on the ability of individual women to mount specific mucosal immunity to HIV antigens, the determinants of which are currently unknown.

Interpatient genetic variability of HIV-1 group O.

OBJECTIVE: To analyse the genetic and phylogenetic characteristics of HIV-1 group O viruses. MATERIALS AND METHODS: The env gene, encoding the gp160 glycoprotein, and a partial p24-encoding gag gene fragment of a Cameroonian (CA9) and a Gabonese (VI686) HIV-1 group O virus, isolated from cultured peripheral blood mononuclear cells of symptomatic patients, were sequenced, aligned with other representatives of group O for which the same region has been documented, and genetically and phylogenetically analysed. RESULTS: Phylogenetic analysis of the env gene (gp160) revealed that CA9, VI686, ANT70, and four Ha strains formed a separate cluster, which was supported by 100% of all bootstrap trees. In addition, these seven isolates were part of the same clade in the p24 phylogeny. VAU and MVP5180 may represent two other subtypes. CONCLUSION: We have characterized two group O viruses, originating from Cameroon and Gabon, which show a close evolutionary relationship to ANT70 and four Ha strains based on the entire env gene, suggestive of a first group O subgroup, tentatively named the HIV-1 group O env ANT70 clade or subtype.

Evaluation of HIV serial and parallel serologic testing algorithms in Abidjan, Côte d'Ivoire.

OBJECTIVE: To evaluate HIV serologic testing algorithms based on a combination of three enzyme linked immunosorbent assays (ELISA) for the confirmation of HIV infection in Abidjan, Côte d'Ivoire, where HIV-2 and HIV-1 non-B subtypes are prevalent. METHODS: A total of 1069 human sera with known serologic status, in addition to a seroconversion and low titer antibody panel were initially tested by six ELISA to determine the sensitivity, specificity and delta values of the assays. On the basis of the performance of the assays, three ELISA (Enzygnost, ICE 1.0.2, and Vironostika) were selected for use in a parallel and serial testing algorithm in analyzing 8283 consecutively collected sera. In the parallel testing algorithm, sera concordantly reactive or non-reactive by Enzygnost and ICE 1.0.2 were considered as true positive or true negative, respectively. In the serial algorithm, sera reactive by Enzygnost were retested by ICE 1.0.2. Sera with discordant results were tested by Vironostika, and the results was considered definitive. All reactive sera, plus a random sample of negative sera were tested for confirmation by Peptilav. In addition, a random sample of reactive sera was tested by Western blot. RESULTS: All ELISA had 100% sensitivity; specificities ranged from 96.8 to 100%. Positive and negative delta values of the ELISA were high (range, 6.89 to 46.07 and -2.05 to -5.75, respectively). Of the 8283 sera, 2054 were considered true positives and were correctly classified by the parallel testing algorithm (sensitivity, 100%). Of the 6229 true negative sera, 6226 were negative by the parallel testing algorithm (specificity, 99.95%). The sensitivity of the serial algorithm was 99.96%, and specificity was 99.95%. None of the 250 concordant ELISA-negative sera in the algorithm that were randomly tested in Peptilav was positive; similarly, all of the 103 concordant ELISA-positive sera were confirmed by Western blot. The three-ELISA algorithm resulted in reagent cost-savings of at least 50% compared with the Peptilav-based algorithm. CONCLUSION: These results suggest that a combination of ELISA using different principles or antigens in a serial or parallel algorithm is an efficient and cost-effective alternative to the standard algorithm in areas where HIV-1 and HIV-2 are prevalent.

Lack of protection against HIV-1 infection among women with HIV-2 infection.

OBJECTIVE: To assess whether HIV-2 infection protects against HIV-1 infection by comparing the rate of HIV-1 seroconversion among HIV-negative and HIV-2-seropositive women followed in a cohort study in Abidjan, Côte d'Ivoire. DESIGN: Prospective cohort study METHODS: HIV seroconversion was assessed in 266 HIV-seronegative, 129 HIV-1-seropositive, and 127 HIV-2-seropositive women participating in a closed cohort study of mother-to-child transmission of HIV conducted during 1990-1994. Participants were seen every 6 months, and blood samples were obtained. All blood samples were screened for HIV antibodies by enzyme immunoassay (EIA) and confirmed by line immunoassay (LIA) and Western blot. Among women who were HIV-seronegative at enrolment, seroconversion was defined as new EIA-reactivity confirmed on LIA and Western blot. Among HIV-1- or HIV-2-seropositive women, seroconversion to dual reactivity was defined as new dual reactivity on the LIA that was confirmed by reactivity on both HIV-1- and HIV-2-monospecific EIA. RESULTS: Five HIV-seronegative women became HIV-1-seropositive [seroconversion rate, 1.1 per 100 person-years; 95% confidence interval (CI), 0.3-2.5), and none became HIV-2-seropositive. No HIV-1-seropositive women became HIV-1/2 dually reactive, whereas six HIV-2-seropositive women acquired HIV-1 seroreactivity and thus became HIV-1/2 dually reactive (seroconversion rate 2.9 per 100 person-years; 95% CI, 1.1-6.3). HIV-2-seropositive women were more likely to acquire HIV-1 seroreactivity than were HIV-seronegative women (rate ratio, 2.7; 95% CI, 0.7-11.2), but this difference was not statistically significant (P>0.15). CONCLUSION: HIV-2 infection does not appear to protect against HIV-1 infection.

Improved detection of HIV-2 proviral DNA in dually seroreactive individuals by PCR.

OBJECTIVE: To improve the detection rate of HIV-2 proviral DNA in primary uncultured peripheral blood mononuclear cells (PBMC) of HIV-2-seroreactive and HIV-1-HIV-2 dually seroreactive individuals. MATERIALS AND METHODS: Two newly designed HIV-2 PCR primer pairs in the long terminal repeat (LTR) gag and gag-pol regions and a previously described env and LTR HIV-2 PCR primer pairs were tested on samples from 66 confirmed HIV-2-seropositive individuals (The Gambia, 40; Côte d'Ivoire, 17; Guinea-Bissau, nine), 209 dually seroreactive individuals (The Gambia, 82; Côte d'Ivoire, 127), 24 genetically characterized isolated HIV-1 strains (group M subtypes A-H and group O), one simian immunodeficiency virus (SIV) strain cpz, 10 HIV-2 isolates (subtype A, B and unidentified), two SIVsm isolates, and 10 seronegative samples. RESULTS: All HIV-2 primers evaluated showed 100% specificity since there was no amplification observed with 24 HIV-1, one SIVcpz and 10 seronegative samples. One single copy of the HIV-2 genome could be detected with all outer primer pairs as well as all inner primer pairs on one PCR round used. Sensitivity of primers (at least one of the four primer pairs was positive) to HIV-2-seropositive samples was 100% (all nine) in Guinea-Bissau, 71% (12/17) in Côte d'Ivoire, 100% (all 20) in Gambian AIDS patients, and 85% (17/20) in Gambian pregnant women. Doubling the PBMC of dually seroreactive individuals from 7.5 x 10(4) to 1.5 x 10(5) in the PCR revealed the presence of both HIV-1 and 2 proviral DNA in 72% (92/127) in Côte d'Ivoire and 72% (59/82) in The Gambia. By doubling the number of PBMC, HIV-2 detection in dually seroreactive individuals by PCR was increased from 65 to 77% in Côte d'Ivoire and from 67 to 83% in The Gambia. CONCLUSIONS: The use of 1.5 x 10(5) primary uncultured PBMC and the newly designed HIV-2 primer pairs allowed us to document the highest percentage (72%) ever reported of HIV-1-HIV-2 dual infections amongst HIV-1-HIV-2 dually seroreactive individuals in Côte d'Ivoire and The Gambia. Improved detection of HIV-2 proviral DNA, rather than exposure to both viruses, infection with only one virus, or infection with a unique third virus containing epitopes common to both HIV-1 and HIV-2, contributes to a more accurate monitoring of the prevalence of HIV-1-HIV-2 dual infections.

Assessing eosinophil count as a marker of immune activation among human immunodeficiency virus-infected persons in sub-Saharan Africa.

In 611 human immunodeficiency virus-infected persons who had not yet begun to receive antiretroviral therapy, we evaluated the linear association between absolute eosinophil count (as a surrogate for immune response to helminthic infection) and CD4+ T cell count, and between absolute eosinophil count and log virus load. Overall, no significant correlations were observed between eosinophil count and CD4+ T cell count, or between eosinophil count and log virus load.

Hepatitis C virus genotyping by means of 5'-UR/core line probe assays and molecular analysis of untypeable samples.

To test the theoretical possibility of 5'-UR mistyping between hepatitis C virus subtypes 1a and 1b, we combined a 5'-UR/Core line probe assay (LiPA) with a nested PCR system and retested 183 sera, previously genotyped as type 1a or 1b and originating mainly from Western Europe. Eight percent of these were found to be wrongly subtyped. Based on this method, 3 additional subtypes of type 1 were discovered (1d-1f). Randomly selected European type 2 sera (n = 18) were tested with a similar type 2 5'-UR/Core LiPA. They were unexpectedly found to belong to subtype 2c in the majority of cases. Among serum samples originating from South-East Asia, several additional genotypes (7a, 7c, 7d, and 9a) were detected which had 5'-UR sequence motifs indistinguishable from genotype 1. Based on 13,203 pairwise comparisons in the 340-bp NS5B region, classification into types, subtypes, and isolates was obtained in 99.8% of all cases by using the phylogenetic border value of 0.328 for subtypes/types and 0.127 for isolates/subtypes; and evidence for a 10th major type of HCV was provided. Combination of all available HCV sequence data from the 447-bp Core/E1 region and the NS5B 340-bp and 222-bp regions provided evidence for the existence of 10 types, including 50 subtypes. Previously, extensive studies involving genotypes 1a, 1b, 2a, and 2b indicated the importance of HCV subtyping in interferon treatment and progression of chronic liver disease. The herein described expansion in the number of HCV types and subtypes should help improve diagnosis, treatment and possibly prophylaxis of hepatitis C liver disease.

The neutralization relationship of HIV type 1, HIV type 2, and SIVcpz is reflected in the genetic diversity that distinguishes them.

Neutralizing antibody (NA) patterns in the sera of individuals naturally infected with human immunodeficiency virus (HIV) type 1, HIV-2, and the simian immunodeficiency virus (SIVcpz) to their homologous and heterologous isolates were determined in a peripheral blood mononuclear cell-based neutralization assay. We examined the role of the V3 loop of HIV-1 and SIVcpz in neutralization and the cross-reactivities among them. Cross-neutralization by sera of humans and chimpanzees naturally infected, respectively, with HIV-1 and SIVcpz isolates was more extensive than the infrequent and low-titer cross-neutralizations observed between HIV-1 and HIV-2. Neutralization of 9 of the 16 HIV-1 isolates by 9 of 10 HIV-2 and all 3 SIVcpz antibody-positive sera were weak and sporadic (titer, 1:10-1:160). Twelve of 15 HIV-1 sera neutralized the 2 SIVcpz isolates with titers of 1:10-1:320 but only sporadically neutralized the 6 HIV-2 isolates (titers: 1:10-1:20). The majority of HIV-1 and SIVcpz sera bound to the V3 peptides although their binding capacity did not readily reflect their neutralizing capacity. The HIV-2 sera did not or only weakly bound to the V3 peptides. These results suggest that HIV-1 and SIVcpz share some structural and functional similarities that set them apart from HIV-2.

Quantification of RNA in HIV type 1 subtypes D and G by NucliSens and Amplicor assays in Abidjan, Ivory Coast.

We have compared the performance of the NucliSens and the standard and modified HIV Monitor assays to quantify HIV-1 RNA plasma viral load in 12 tuberculosis patients infected with HIV-1 env subtype D (n = 3) and env subtype G (n = 9) in Ivory Coast. RNA was quantified in all nine subtype G specimens by the modified Amplicor HIV Monitor (mean, 4.6 log10 copies/ml; range, 3.1-6.3 log10/ml), in seven specimens by NucliSens (mean, 4.4 log10 copies/ml; range, 2.7-5.5 log10 copies/ml), and in 6 specimens by the standard Amplicor HIV Monitor assay (mean, 4.2 log10 copies/ml; range, 3.5-5.0 log10 copies/ml). All three subtype D samples were amplified by both the modified Amplicor HIV Monitor (mean, 4.5 log10 copies/ml; range, 3.8-5.1 log10 copies/ml) and NucliSens (mean, 3.8 log10 copies/ml; range, 2.8-5.0 log10 copies/ml); two samples were quantified by the standard Amplicor HIV Monitor assay (mean, 3.0 log10 copies/ml; range, 2.4-3.6 log10 copies/ml). Our preliminary results suggest that the modified Amplicor HIV Monitor can accurately quantify HIV-1 RNA viral load in persons infected with subtype D and G strains.

Presence of diverse human immunodeficiency virus type 1 viral variants in Cameroon.

Phylogenetic analysis of the gp41 region of 123 HIV-1-seropositive specimens from Cameroon showed that 89 were subtype A (71% of these sequences were IbNg-like), 12 (10%) were subtype D, 11 (9%) were subtype G, 5 (4%; closely related to subtype F2) were subtype F, 1 was subtype H, 2 (1.6%) remained unclassifiable, while 3 were group O. Further analysis of the two unclassifiable specimens in gag(p24), pol(prot), and env (C2V3 or gp41) showed that one (98CM19) was a complex mosaic between subtype A in p24 and subtype J prot, and unclassifiable in env (C2V3 or gp41). The second, 98CM63, clustered distinctly from all known subtypes in p24, prot, C2V3, or gp41. 98CM63 clustered with a specimen from Cyprus and these two geographically and epidemiologically unlinked specimens, with their distinct clustering pattern, may represent a new subcluster of subtype A. In conclusion, these findings confirm the high HIV-1 genetic variability and further suggest the continuous appearance of new viral strains in this population.

Dual infection with human immunodeficiency virus type 1 and type 2: impact on HIV type 1 viral load and immune activation markers in HIV-seropositive female sex workers in Abidjan, Ivory Coast.

To determine the impact of dual infection with HIV-1 and HIV-2 on HIV-1 viral load and markers of immune activation among HIV-seropositive FSWs in Abidjan, we analyzed blood samples obtained from consenting HIV-seropositive FSWs attending a confidential clinic between September 1996 and June 1997 in Abidjan. Among HIV-1 and HIV-2 dually seropositive FSWs, polymerase chain reaction (PCR) testing with HIV-1 and HIV-2 primers was used to differentiate between FSWs who were PCR positive only for HIV-1 and those positive for both HIV-1 and HIV-2 (dually infected). Of the 203 FSWs, 151 (74%) were HIV-1 seropositive only (median age, 26 years), 4 (2%) were HIV-2 seropositive, and 48 (24%) were dually seropositive (median age, 30 years). Of the 48 dually seropositive FSWs, 33 (69%) were dually infected and 15 (31%) were dually seropositive. Median CD4+ T cell counts per microliter were not significantly different among the three groups (525 for HIV-1 positive only, 502 for dually infected, and 416 for dually seropositive) (p = 0.14). Median viral load (log10 copies/ml) was not significantly different among the HIV-1-only FSWs (4.8 log10 copies/ml) compared with the 32 dually infected FSWs (4.6 log10 copies/ml) and 14 dually seropositive FSWs (4.7 log10 copies/ml; p = 0.95). Median levels of HLA-DR immune activation were increased in both CD4+ and CD8+ T cells for the dually infected (n = 27) FSWs compared with those infected with HIV-1 only (n = 123) (p = 0.019 and p = 0.01, respectively). Dual infection does not appear to influence levels of HIV-1 viral load in vivo. However, levels of HLA-DR are higher among FSWs dually infected with HIV-1 and HIV-2 than among those infected with HIV-1 only.

Intrapatient variability of HIV type 1 group O ANT70 during a 10-year follow-up.

HIV-1 ANT70 is the first HIV-1 group O virus isolate obtained from a 25-year-old Cameroonian woman, who seroconverted in March 1987. This individual has remained asymptomatic and clinically healthy (clinical stage WHO 1, CDC II) even though she did not receive any antiretroviral therapy for HIV-1 before 97 months post-seroconversion. CD4+ T cell counts declined steadily to 200/microl at 70 months postseroconversion. The HIV-1 ANT70 nucleotide and amino acid sequence diversity of the V3C3-encoding env fragment within this individual was followed over a 10-year period. RT-PCR, cloning, sequencing, and genetic analyses were performed on eight plasma follow-up samples. Extensive increasing intra- and intersample variation was observed. This is the first long-term (>10 years) follow-up of the genetic variability of an HIV-1 group O-infected individual. As the course of the disease in the HIV-1 ANT70-infected woman was similar in many aspects to that of group M-infected individuals, it remains to be elucidated whether the changes observed in the V3 loop are critical for disease progression.

Evaluation of different V3 peptides in an enzyme immunoassay for specific HIV type 1 group O antibody detection.

Strategies to discriminate group O from group M infections need to be improved. We have developed and evaluated an HIV-1 group O V3 peptide-based enzyme immunoassay (PEIA) for specific HIV-1 group O antibody detection among HIV-1-infected patients. Synthetic peptides, derived from the amino acid sequences of the V3 loop of 15 different group O strains and 7 group O consensus sequences, were evaluated in a PEIA against a panel of genetically confirmed group O (n = 33), group M (n = 90), and HIV-1 antibody-negative sera (n = 17). The best-performing PEIA(s) were then used to screen 134 sera of European and 336 sera of Cameroonian origin for the presence of anti-HIV-1 group O antibodies. The reactivity of reference ("gold standard") sera to individual peptides in the PEIA resulted in the selection of five different peptides with sensitivities (sens), specificities (spec), and test efficiencies (TEs) in the range of 90 to 100%. Improvement of the PEIA was obtained with simultaneous reactivity of at least two different peptides in separate wells of an ELISA plate, together with stringent criteria for positivity. We were able to select seven peptide combinations each with a sens, spec, and TE of 96.9, 100, and 99.2%, respectively. None of the 134 European and 4 (1.2%) of the 336 Cameroonian samples sera were group O positive in the optimized HIV-1 group O PEIA; this was confirmed by the repeated presence of reactives, in agreement with the present knowledge of group O infection distribution. Finally, we were able to develop a strategy with a higher TE (99.2%) than the previously used ANT-70 (98.5%) and ANT-70/MVP5180 (95.7%). Our results show that optimal specificity rather than optimal sensitivity makes the V3 PEIA a sufficiently accurate epidemiological tool to be useful in estimating specifically group O infection among HIV-1-infected patients.

Predominance of human immunodeficiency virus type 2 subtype B in Abidjan, Ivory Coast.

We analyzed the genetic variability and phylogenetic relationships among 28 HIV-2 strains collected from patients enrolled in an HIV epidemiologic study in Abidjan, Ivory Coast, during 1995-1996. Although both subtype A (n = 8; 29%) and subtype B (n = 20; 71%) were present in this sampling, the majority of infections were caused by subtype B viruses. These findings contrasted with the reported predominance of HIV-2 subtype A in other African countries. The broad genetic diversity identified among protease gene sequences for HIV-2 subtype A (6%; range 3-15%) and subtype B (7%; range, 2-12%), and their presence in Abidjan during the 1980s, document a long coexistence of two viral subtypes in Ivory Coast. Our data indicate that viruses of subtypes A and B have contributed to the HIV-2 epidemic in Ivory Coast.