Laboratory testing and rapid HIV assays: applications for HIV surveillance in hard-to-reach populations.

Most HIV surveillance has been performed through serologic surveys in relatively stable, accessible populations. Similar surveillance, with or without counseling and testing, in populations that are hard-to-reach, presents logistical challenges, including the selection of laboratory testing strategy and algorithm. The advent of rapid serologic assays for HIV now allows for on-site testing, including confirmatory testing, and rapid provision of test results and counseling. The possibility of only a single contact makes repeat sampling, which current diagnostic testing recommendations include, difficult. To address the logistical complexities in surveillance in hard-to-reach populations and the increased availability of rapid tests, we propose adapting the testing strategies for HIV of the World Health Organization/the joint United Nations Programme on HIV/AIDS in order to facilitate this surveillance, including, where carried out, the provision of test results back to individuals. The choice of enzyme-linked immunosorbent assay (ELISA) versus rapid testing for these settings is discussed, as is the choice of specimen--blood, oral fluid, or urine. Three appendices summarize: (1) test algorithms for the various testing strategies; (2) advantages and disadvantages of ELISA and of rapid test formats, and (3) the characteristics and status of currently available rapid HIV tests. We also discuss the potential application of the recently developed 'detuned' methodology for estimating HIV incidence in hard-to-reach populations.

Predominance of HIV-1 subtype A and D infections in Uganda.

To better characterize the virus isolates associated with the HIV-1 epidemic in Uganda, 100 specimens from HIV-1-infected persons were randomly selected from each of two periods from late 1994 to late 1997. The 200 specimens were classified into HIV-1 subtypes by sequence- based phylogenetic analysis of the envelope (env) gp41 region; 98 (49%) were classified as env subtype A, 96 (48%) as D, 5 (2.5%) as C, and 1 was not classified as a known env subtype. Demographic characteristics of persons infected with the two principal HIV-1 subtypes, A and D, were very similar, and the proportion of either subtype did not differ significantly between early and later periods. Our systematic characterization of the HIV-1 epidemic in Uganda over an almost 3-year period documented that the distribution and degree of genetic diversity of the HIV subtypes A and D are very similar and did not change appreciably over that time.

The HIV epidemic in the Amazon Basin is driven by prototypic and recombinant HIV-1 subtypes B and F.

This paper describes genetic subtypes of HIV-1 found in blood samples from 31 HIV-1-infected people who visited the Counseling and Testing AIDS Center of Instituto de Medicina Tropical in Manaus, Brazil. Manaus, the main city in Brazil's Amazon Basin, is also the closest urban connection for more than 100,000 Indians living in the rain forests of this region. Although to date there is no evidence of increased incidence of HIV-1 infection among the indigenous population, our understanding of both the prevalence and nature of the epidemic in the region as a whole is limited. From the 31 samples analyzed by C2V3 sequencing, we found almost equal proportions of HIV-1 strains belonging to subtype B (n = 16; 51.6%) and subtype F (n = 15; 48.4%), a finding that differs from results from previous studies conducted in urban areas of southeastern Brazil. We also observed the presence of the GWGR amino-acid sequence in the critical tetra-peptide crown of the env V3 loop in the HIV-1 subtype B samples analyzed. Among these samples, we also found 14 mosaic genomes (45.16%) in which different combinations of subtypes B, C, and F were identified between the p24 gag, pro, and env regions. Our data support the hypothesis that the Amazonian HIV-1 infections linked to the urban epidemic in southeastern Brazil. The genetic diversity and the prevalence of mosaic genomes among the isolates in our study confirm an integral role of recombination in the complex Brazilian epidemic.

Genetic characterization and phylogenetic analysis of HIV-1 subtype C from Uganda.

To better understand the emergence of subtype C and its potential impact on vaccine efforts in Uganda, we have characterized subtype C sequences from Uganda (n = 13), Zimbabwe (n = 11), Mozambique (n = 5), South Africa (n = 4), and India (n = 3). Phylogenetic analysis of subtype C sequences in the env gp41 gene region revealed multiple subclusters within subtype C. Further, while most Ugandan specimen subclustered together, other subclusters did not reflect a clear geographic location. The nucleotide divergence within the Ugandan subset was 8.2% (6.1-9.8%) compared with 9.5% (2.5-15%) for the other subtype C gp41 sequences. The protein sequence alignment revealed marked sequence conservation of major immunodominant epitopes within the gp41 region.

Analysis of genetic variability within the immunodominant epitopes of envelope gp41 from human immunodeficiency virus type 1 (HIV-1) group M and its impact on HIV-1 antibody detection.

The serodiagnosis of human immunodeficiency virus type 1 (HIV-1) infection primarily relies on the detection of antibodies, most of which are directed against the immunodominant regions (IDR) of HIV-1 structural proteins. Among these, the N-terminal region of gp41 contains cluster I (amino acids [aa] 580 to 623), comprising the cytotoxic T-lymphocyte epitope (AVERYLKDQQLL) and the cysteine loop (CSGKLIC), and cluster II (aa 646 to 682), comprising an ectodomain region (ELDKWA). To delineate the epitope diversity within clusters I and II and to determine whether the diversity affects serologic detection by U.S. Food and Drug Administration (FDA)-licensed enzyme immunoassay (EIA) kits, gp41 Env sequences from 247 seropositive persons infected with HIV-1 group M, subtypes A (n = 42), B (n = 62), B' (n = 13), C (n = 38), D (n = 41), E (n = 18), F (n = 27), and G (n = 6), and 6 HIV-1-infected but persistently seronegative (HIPS) persons were analyzed. While all IDR were highly conserved among both seropositive and HIPS persons, minor amino acid substitutions (<20% for any one residue, mostly conservative) were observed for all subtypes, except for B', in comparison with the consensus sequence for each subtype. Most importantly, none of the observed substitutions among the group M plasma specimens affected antibody detection, since all specimens (n = 152) tested positive with all five FDA-licensed EIA kits. Furthermore, all specimens reacted with a group M consensus gp41 peptide (WGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNASW), and high degrees of cross-reactivity (>80%) were observed with an HIV-1 group N peptide, an HIV-1 group O peptide, and a peptide derived from the homologous region of gp41 from simian immunodeficiency virus from chimpanzee (SIVcpz). Taken together, these data indicate that the minor substitutions observed within the IDR of gp41 of HIV-1 group M subtypes do not affect antibody recognition and that all HIV-1-seropositive specimens containing the observed substitutions react with the FDA-licensed EIA kits regardless of viral genotype and geographic origin.

Construction of a selectable nef-defective live-attenuated human immunodeficiency virus expressing Escherichia coli gpt gene.

We have developed a replication-competent human immunodeficiency virus (HIV) carrying a selective marker that can be used in vivo. This recombinant virus (Z6 Delta nef gpt) was generated by replacing the 5' half of the HIV nef gene with the Escherichia coli guanine phosphoribosyl transferase gene (gpt). This new vector can express the gpt product on infection and works as a positive selective marker for mycophenolic acid (MPA) resistance, a potent immunosuppressive drug used in organ rejection therapy. Conversely, gpt expression also served as a negative selectable marker, since its intracellular expression induces host-cell susceptibility to 6-thioxantine (6-TX), a nucleotide analog that is toxic to the infected cell under these conditions. In this manner, we could suppress the recombinant virus replication through 6-TX selection in both transformed cells and primary human peripheral blood mononuclear cells (PBMCs), suggesting the vector's potential as a model for a new live-attenuated vaccine approach against HIV.

Identification of a window period for susceptibility to dual infection with two distinct human immunodeficiency virus type 2 isolates in a Macaca nemestrina (pig-tailed macaque) model.

The potential to establish dual retroviral infections was investigated in this study. Groups of macaques infected with human immunodeficiency virus type 2 (HIV-2) isolate (either GB122 or CDC77618) were exposed to the other virus at 2, 4, 8, 12, 14, or 72 weeks after primary inoculation. Dual infections were established in macaques simultaneously exposed to both viruses. In other groups, secondary infections were observed only if challenge occurred at early intervals after primary infection but before a full seroconversion. Polymerase chain reaction and virus-isolation data demonstrated that challenges at 8, 12, 14, or 72 weeks after infection with the initial isolate failed to result in a dual infection. Anti-HIV-2 serologic titers, CD4 levels, virus burden, and the ability to superinfect peripheral blood mononuclear cells in vitro were not correlated with susceptibility to or protection from secondary challenges in this investigation. These findings demonstrate a window period for susceptibility to dual infection and indicate that protection from retroviral infection may be achievable.

Detection of phylogenetically diverse human immunodeficiency virus type 1 groups M and O from plasma by using highly sensitive and specific generic primers.

The high degree of genetic diversity within human immunodeficiency virus type 1 (HIV-1), which includes two major groups, M (major) and O (outlier), and various env subtypes within group M (subtypes A to J), has made designing assays that will detect all known HIV-1 strains difficult. We have developed a generic primer set based on the conserved immunodominant region of transmembrane protein gp41 that can reliably amplify as few as 10 copies/PCR of viral DNA from near-full-length clones representing group M subtypes A to H (subtypes I and J were not available). The assay is highly sensitive in detecting plasma viral RNA from HIV-1 strains of diverse geographic origins representing different subtypes of HIV-1 group M as well as HIV-1 group O. Of the 253 group M plasma specimens (subtypes A, 68 specimens; B, 71; C, 19; D, 27; E, 23; F, 33; and G, 12), 250 (98.8%) were amplified by using the gp41 M/O primer set. More importantly, all 32 (100%) group O plasma samples were also amplified with these primers. In vitro spiking experiments further revealed that the assay could reliably detect as few as 25 copies/ml of viral RNA and gave positive signals in HIV-1-seropositive specimens with plasma copy numbers below the limits of detection by all commercially available viral load assays. In addition, analysis of five seroconversion panels indicated that the assay is highly sensitive for early detection of plasma viremia during the "window period." Thus, the highly sensitive assay will be useful for early detection of HIV-1 in clinical specimens from all known HIV-1 infections, regardless of their genotypes and geographic origins.

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.

Dual and recombinant infections: an integral part of the HIV-1 epidemic in Brazil.

We systematically evaluated multiple and recombinant infections in an HIV-infected population selected for vaccine trials. Seventy-nine HIV-1 infected persons in a clinical cohort study in Rio de Janeiro, Brazil, were evaluated for 1 year. A combination of molecular screening assays and DNA sequencing showed 3 dual infections (3.8%), 6 recombinant infections (7.6%), and 70 (88.6%) infections involving single viral subtypes. In the three dual infections, we identified HIV-1 subtypes F and B, F and D, and B and D; in contrast, the single and recombinant infections involved only HIV-1 subtypes B and F. The recombinants had five distinct B/F mosaic patterns: Bgag-p17/Bgag-p24/Fpol/Benv, Fgag-p17/Bgag-p24/Fpol/Fenv, Bgag-p17/B-Fgag-p24/Fpol/Fenv, Bgag-p17/B-Fgag-p24/Fpol/Benv, and Fgag-p17/B-Fgag-p24/Fpol/Fenv. No association was found between dual or recombinant infections and demographic or clinical variables. These findings indicate that dual and recombinant infections are emerging as an integral part of the HIV/AIDS epidemic in Brazil and emphasize the heterogenous character of epidemics emerging in countries where multiple viral subtypes coexist.

Genetic analysis of human immunodeficiency virus in Abidjan, Ivory Coast reveals predominance of HIV type 1 subtype A and introduction of subtype G.

To better understand the molecular epidemiology of HIV genetic diversity in Abidjan, Ivory Coast, we performed a genetic analysis of 170 HIV-1-seropositive specimens representing newly diagnosed tuberculosis patients (n = 143) and women monitored in a mother-to-child transmission cohort study (n = 27). Preliminary screening with RFLP presumptively classified 162 (95.3%) of these as subtype A. The envelope region of 108 specimens was subtyped by sequence analysis: 102 (94.4%) were subtype A, 2 (1.9%) were subtype D, and 4 (3.7%) were subtype G. Subtyping gag and env regions of the genome suggested that five of the six nonsubtype A isolates exhibited a potentially mosaic structure. A comparative phylogenetic analysis of HIV-1 subtype A C2V3 from 27 Ivory Coast and 21 Ugandan sequences revealed a striking clustering among Ivory Coast variants, and an independent segregation from Ugandan subtype A. Despite independent clustering with other subtype A specimens, limited variability of the V3 loop apex was observed; the globally predominant V3 motif, GPGQ, represented 90.1% of the HIV-1 strains. This study demonstrates that clade A is the predominant HIV-1 subtype in HIV-seropositive individuals in Abidjan, Ivory Coast and that these strains are phylogenetically distinct from other subtype A strains observed in East Africa.

Genetic variation and susceptibilities to protease inhibitors among subtype B and F isolates in Brazil.

The genetic variation of the human immunodeficiency virus type 1 (HIV-1) protease gene (prt) permits the classification of HIV-1 strains into five distinct protease subtypes, which follow the gag subtyping patterns. The susceptibilities of non-B-subtype strains to protease inhibitors (PIs) and other antiretroviral drugs remain largely unknown. Subtype F is the main non-B strain contributing to the Brazilian epidemic, accounting for 15 to 20% of these infections. In this work, we report the findings on 81 isolates from PI-naive Brazilian patients collected between 1993 and 1997. In addition, the relevant PI resistance mutations and their phenotypes were determined in vitro for 15 of these patients (B = 9 and F = 6). Among these, the subtype F samples evidenced high sensitivities in vitro to ritonavir and indinavir, with MICs at which 50 and 90% of the isolates are inhibited similar to those of both the Brazilian and the U.S. subtype B isolates. Analysis of the 81 Brazilian prt sequences demonstrated that the subtype F consensus sequence differs from the U.S. and Brazilian subtype B consensus in eight positions (I15V, E35D, M36I, R41K, R57K, Q61N, L63P, and L89M). The frequency of critical PI resistance substitutions (amino acid changes D30N, V82A/F/T, I84V, N88D, and L90M) among Brazilian isolates is very low (mean, 2.5%), and the associated secondary substitutions (amino acid positions 10L, 20K, 36M, 46M, 48G, 54I, 63P, 71A, and 77A) are infrequent. These observations document the relative rarity of resistance to PIs in the treatment of patients infected with HIV-1 subtype F in South America.

Introduction of HIV-2 and multiple HIV-1 subtypes to Lebanon.

HIV genetic variability, phylogenetic relationships, and transmission dynamics were analyzed in 26 HIV-infected patients from Lebanon. Twenty-five specimens were identified as HIV-1 and one as HIV-2 subtype B. The 25 strains were classified into six env-C2-V3 HIV-1 subtypes: B (n = 10), A (n = 11), C (n = 1), D (n = 1), G (n = 1), and unclassifiable. Potential recombinants combining parts of viral regions from different subtypes Aenv/Dpol/Agag, Genv/Apol, and the unclassifiable-subtype(env)/unclassifiable-subtype(pol)/Agag were found in three patients. Epidemiologic analysis of travel histories and behavioral risks indicated that HIV-1 and HIV-2 subtypes reflected HIV strains prevalent in countries visited by patients or their sex partners. Spread of complex HIV-subtype distribution patterns to regions where HIV is not endemic may be more common than previously thought. Blood screening for both HIV-1 and HIV-2 in Lebanon is recommended to protect the blood supply. HIV subtype data provide information for vaccine development.

Optimizing the delivery of HIV counseling and testing services: the Uganda experience using rapid HIV antibody test algorithms.

The AIDS Information Center (AIC) was established in Kampala, Uganda in 1990 in response to increasing interest by members of the general public who wished to know their HIV serostatus. By 1996, >300,000 clients had been seen. HIV serologic testing was performed at a central laboratory and results reported back to AIC after 2 weeks. Approximately 25% of clients failed to learn their HIV serostatus as a result of failure to return or late arrival of results. To address these issues, AIC carried out an evaluation of 3 rapid HIV assays, Sero-Strip, SeroCard, and Capillus, against a standard criterion to identify a testing algorithm that could be used as an on-site confirmatory testing strategy. The study was carried out over a period of 5 working days and 325 clients were seen. An algorithm was identified, which gave no indeterminate results with unambiguously positive or negative specimens, which was 100% sensitive and specific, and which could be integrated with minimal disruption into existing counseling procedures. All clients left AIC knowing their HIV serostatus and having spent <2 hours at the Center. The results of this evaluation demonstrate that "same-day" results can be provided in counseling and testing settings without compromising the quality of counseling or the accuracy of HIV testing.

PIP: An evaluation conducted at the AIDS Information Center in Kampala, Uganda, demonstrated that same-day HIV results can be provided in counseling and testing centers without compromising service quality. The Center was established in 1990 in response to widespread public interest in HIV serodiagnosis. By 1996, more than 300,000 clients had been tested. However, 25% of these clients never received their result because of failure to report back to the Center after 2 weeks (the time required for results to be returned from an off-site laboratory) or late arrival of results. To address this problem, the Center evaluated three rapid HIV assays (Sero-Strip, SeroCard, and Capillus) against a standard criterion to identify a testing algorithm that could be used as an on-site confirmatory testing strategy. 325 clients were enrolled in the 5-day evaluation. Individually, all three rapid tests performed well when compared with the standard criterion result. The resulting algorithm (a combination of Capillus as the screening assay and SeroCard as a supplementary assay for initially positive specimens and Multispot as a tie-breaker assay) gave no indeterminate results, was 100% sensitive and specific, and could be integrated easily into existing counseling protocols. The entire process (registration, test decision counseling, phlebotomy, laboratory testing, prevention counseling, and post-test counseling) took an average of 2 hours to complete.

The development and evaluation of a probe hybridization method for subtyping HIV type 1 infection in Uganda.

We developed a method for large-scale screening of HIV-1 genotypic variation based on DNA probe hybridization. Nested PCR amplifications were performed to generate fragments in the env C2-V3 region and also in the gp41 region, which encompasses the immunodominant domain. The proviral DNA sequences were derived from 68 samples and phylogenetically analyzed. For comparison, the C2-V3 fragment was used in DNA probe hybridization to rapidly determine the infecting HIV subtype. The hybridizing probes were designed on the basis of the two most prevalent subtypes in Uganda, A and D. The results were compared to evaluate the feasibility of using this hybridization method for large-scale genotypic screening. Sequence analysis of the 68 amplified PCR fragments showed that 39 were subtype A and 29 were subtype D. The results of DNA hybridization to the amplified products with A and D subtype-specific probes were more than 90% concordant with the subtypes determined by sequence analysis. Our findings suggest that probe hybridization with subtype-specific probes is effective for large-scale screening of HIV-infected populations. Application of this method will significantly reduce the time needed for large, population-based investigations.

A molecular epidemiologic survey of HIV in Uganda. HIV Variant Working Group.

OBJECTIVE: Previous data, based on a small sampling of convenience, reported subtypes A, B, C, D, and G in Uganda, but neither the extent nor the proportion of these subtypes could be evaluated. To establish correctly the prevalence and distribution of HIV-1 subtypes, we analysed viral clades in 739 HIV-1-seropositive specimens from different areas of Uganda. METHODS: Blood specimens from 1100 patients were collected in five districts of Uganda. Within this collection, 929 HIV-1-seroreactive samples underwent analysis of viral DNA, and 739 were selected for further subtyping in env or pol regions. RESULTS: Using a combination of subtype A- and D-specific probes to C2-V3 region and DNA sequencing, HIV-1 env subtypes were determined in 594 specimens: 341 were of subtype A (57.4%), 250 of subtype D (42.1%), and three of subtype C (0.5%). Sixty-two samples showed reactivity with both probes, suggesting potential mixed infections, cross-reactivity to probes, or possibly other subtypes. Subsequent sequence analysis of 19 randomly selected specimens revealed subtypes A (n = 4), D (n = 12), and C (n = 3). Sequence analysis of the 27 samples chosen from the remaining 83 samples, which could be amplified only with viral gp41 or protease gene primers, classified them as subtypes A (n = 13) and D (n = 14). No significant clinical, demographic, or geographic differences were found between HIV-1 infections with viruses of subtypes A and D, despite considerable genetic diversity within these clades. CONCLUSIONS: This is the first major population-based study of the prevalent HIV-1 strains in an African country selected for vaccine trials. The subtyping methods we describe should be of use to investigators seeking to conduct large-scale screening for HIV variants in other populations.

Horizontal and vertical transmission of human immunodeficiency virus type 1 dual infections caused by viruses of subtypes B and C.

This article describes a case of horizontal (heterosexual) and subsequent vertical (mother to infant) transmission of 2 human immunodeficiency viruses type 1 (HIV-1) subtypes. Dual infection in a husband, his wife, and their child was initially detected by use of a restriction fragment length polymorphism assay of the proviral protease in peripheral blood mononuclear cells. The simultaneous presence of highly similar sets of HIV-1 subtypes B and C infecting the 3 family members was confirmed by DNA sequence analysis of pol, gag, and env genes. These data, together with available epidemiologic information, may indicate that the husband's high-risk sexual behavior was the source of dual infections. Because his wife did not report such activities, it was likely that he passed HIV-1 strains to his spouse, who subsequently transmitted them to their child.

Surveillance for human immunodeficiency virus type 1 group O infections in the United States.

BACKGROUND: Reports that the human immunodeficiency virus type 1 (HIV-1) group O variants are not reliably detected by some commercial diagnostic tests have raised concerns about the sensitivity of existing screening tests, especially with regard to blood safety. Although it is unlikely that these divergent strains are prevalent in North America, systematic, continuous surveillance is needed to monitor the potential spread of HIV variants into that region. STUDY DESIGN AND METHODS: Stored serum samples (n = 1072) from both high- and low-risk population groups at several sites in the United States and Puerto Rico were tested by peptide enzyme immunoassays specific for the prototypic HIV-1 group O strains, MVP5180 and ANT70. RESULTS: None of the 1072 samples examined had peptide reactivity that was consistent with HIV-1 group O infection. CONCLUSION: While no evidence of specific HIV-1 group O (MVP5180 or ANT70) infection was found in this study, the sensitivity of current tests has not been fully evaluated against the wide range of genetic variation of HIV. Therefore, it is important to continue active surveillance for HIV-1 and HIV type 2 strains, to characterize any divergent strains, and to judiciously modify tests to correct for any deficiencies in sensitivity.