Human Immunodeficiency Virus Type 1 Subtype Research Articles

Genetic subtypes of human immunodeficiency virus type 1 (HIV-1) in Istanbul, Turkey

Epidemiological surveillance of HIV-1 subtypes is an important and ongoing element of preparation for global antiviral interventions. To assess the molecular epidemiology of HIV-1 in Istanbul, Turkey. 27 HIV/AIDS patients were investigated. Data on age, sex, country of birth, and HIV acquisition route were collected. Following amplification with PCR the sequences of the gp41 region of the env gene were determined using a 310 DNA sequencer (ABI prism, Foster City, USA) and phylogenetically analyzed. Among the 27 patients (26 adults and 1 infant), 22 were male, born in Turkey, and 20 infected through heterosexual contact. Two patients acquired the virus through blood and/or blood transfusion and one infant by vertical transmission. The distribution of the subtypes was as follows: four were subtype A, 19 subtype B, one subtype C, one subtype D, and two subtype F1. According to our results, although the B subtype is still predominant, non-B subtypes are also present, even though the number of registered HIV/AIDS patients is low. These are the first subtyped HIV-1 strains in Turkey where a low level of HIV prevalence has been observed since the first reported case in 1985. These findings and Turkey's specific geographic localization indicate the need for a nationwide surveillance to detect all subtypes including the new recombinant ones.

Real-Time PCR Quantification of Genital Shedding of Herpes Simplex Virus (HSV) and Human Immunodeficiency Virus (HIV) in Women Coinfected with HSV and HIV

The accuracy and usefulness of laboratory-developed real-time PCR procedures using a Light Cycler instrument (Roche Diagnostics) for detecting and quantifying human immunodeficiency virus type 1 (HIV-1) RNA and DNA as well as herpes simplex virus type 1 (HSV-1)/HSV-2 DNA in cervicovaginal secretions from women coinfected with HIV and HSV were evaluated. For HIV-1, the use of the NEC152 and NEC131 primer set and the NEC-LTR probe in the long terminal repeat gene allowed us to detect accurately the majority of HIV-1 subtypes of group M circulating in sub-Saharan Africa, including subtypes A, B, C, D, and G as well as circulating recombinant forms 02 and 11. The detection threshold of real-time PCR for HIV in cervicovaginal lavage samples was 5 copies per assay for both RNA and DNA; the intra- and interassay coefficients of variation of C(T) values were 1.30% and 0.69% (HIV-1 RNA) and 1.84% and 0.67% (HIV-1 DNA), respectively. Real-time PCR for HSV using primers and probe targeting the HSV DNA polymerase gene allowed both detection and quantification of HSV DNA and also differentiation between HSV-1 and HSV-2 genotypes. The detection threshold of real-time PCR for HSV was 5 copies per assay; the intra- and interassay coefficients of variation of C(T) values were 0.96% and 1.49%, respectively. Both manual and automated silica-based procedures were appropriate for combined extraction of HIV and HSV genomes from female genital secretions. Taken together, these findings indicate that real-time PCR may be used as a unique nucleic acid amplification procedure to detect and quantify HIV and HSV genomes in cervicovaginal secretions and thus to assess at reduced costs the genital shedding of both viruses in women included in intervention studies.

Dynamic evolution of the human immunodeficiency virus type 1 pathogenic factor, Nef.

The human immunodeficiency virus type 1 (HIV-1) early gene product Nef is a multifunctional protein that alters numerous pathways of T-cell function, including endocytosis, signal transduction, vesicular trafficking, and immune modulation, and is a major determinant of pathogenesis. Individual Nef functions include PAK-2 activation, CD4 downregulation, major histocompatibility complex (MHC) class I downregulation, and enhancement of viral particle infectivity. How Nef accomplishes its multiple tasks presents a difficult problem of mechanistic analysis because of the complications associated with multiple, overlapping functional domains in the context of significant sequence variability. To address these issues we determined the conservation of each Nef residue based on 1,643 subtype B Nef sequences. Mutational analysis based on conservative substitutions and Nef sequence data allowed us to search for amino acids on the surface of Nef that are specifically required for PAK-2 activation. We found residues 85, 89, and 191 to be highly significant determinants for Nef's PAK-2 activation function but functionally unlinked to CD4 and MHC class I downregulation or enhancement of infectivity. These residues are not conserved across HIV-1 subtypes but are confined to separate sets of surface elements within a subtype. Thus, L85/H89/F191 and F85/F89/R191 are dominant in subtype B and subtype E or C, respectively. Our results provide support for developing subtype-specific interventions in HIV-1 disease.

Development of a candidate DNA/MVA HIV-1 subtype C vaccine for India

Development of a vaccine against human immunodeficiency virus type-1 (HIV-1) is the mainstay for controlling the AIDS pandemic. An ideal HIV vaccine should induce neutralizing antibodies, CD4+ helper T cells, and CD8+ cytotoxic T cells. While the induction of broadly neutralizing antibodies remains a highly challenging goal, there are a number of technologies capable of inducing potent cell-mediated responses in animal models, which are now starting to be tested in humans. Naked DNA immunization is one of them. The present study focuses on the stimulation cell-mediated and humoral immune responses by recombinant DNA–MVA vaccines, the areas where this technology might assist either alone or as a part of more complex vaccine formulations in the HIV vaccine development. Candidate recombinant DNA–MVA vaccine formulations expressing the human immunodeficiency virus-1 env and gagprotease genes from HIV-1 Indian subtype C were constructed and characterized. A high level of expression of the respective recombinant MVA (rMVA) constructs was demonstrated in BHK-21 cells followed by the robust humoral as well as cell mediated immune (CMI) responses in terms of magnitude and breadth. The response to a single inoculation of the rDNA vaccine was boosted efficiently by rMVA in BALB/c mice. This is the first reported candidate HIV-1 DNA/MVA vaccine employing the Indian subtype C sequences and constitutes a part of a vaccine scheduled to enter a preclinical non-human primate evaluation in India.

Molecular analysis and phylogenetic characterization of HIV in Iran

The rate of human immunodeficiency virus type 1 (HIV-1) infection in Iran has increased dramatically in the last few years. While the earliest cases were found in hemophiliacs, intravenous drug users are now fueling the outbreak. In this study, both the 122 clones of HIV-1 gag p17 and the 131 clones of env V1-V5 region were obtained from 61 HIV-1 seropositives belonging to these two groups in Iran. HIV-1 subtyping and phylogenetic analysis was done by heteroduplex mobility assays (HMA) and multiple clone sequencing. The result indicated all hemophiliacs are infected with HIV-1 subtype B and all intravenous drug users are infected with HIV-1 subtype A. Since intravenous drug abuse is the major transmission route in Iran, HIV-1 subtype A is likely to be the dominant viral subtype circulating in the country. The analysis of genetic distances showed subtype B viruses in Iran to be twice as heterogeneous as the subtype A viruses. In conclusion, this first molecular study of HIV-1 genotypes in Iran suggests two parallel outbreaks in distinct high-risk populations and may offer clues to the origin and spread of infection in Iran.

Screening for CD8 cytotoxic T lymphocytes specific for Gag of human immunodeficiency virus type 1 subtype B′ Henan isolate from China and identification of novel epitopes restricted by the HLA-A2 and HLA-A11 alleles

The human immunodeficiency virus type 1 (HIV-1) epidemic in China is increasing rapidly at an irrepressible rate. It is caused by HIV-1 subtype B' in central China. After the full-length genome sequencing of the Henan isolate was performed, the definition of optimal cytotoxic T-lymphocyte (CTL) epitopes across the Henan isolate genome has become crucial for vaccine design. In this study, by using ELISPOT assays with synthetic peptides corresponding to the sequence of the Henan isolate, the identification and analysis of Gag-specific CTL responses among 28 treated and 26 untreated infected paid blood donors (PBDs) from the Henan and Hubei provinces of China are presented. These studies focused on CTL responses restricted by the human leukocyte antigen (HLA)-A2 and -A11 molecules, two of the most prominent HLA-A alleles in the Chinese population. The results suggested that, in the subgroup analysis, the magnitude of response in the infected treated subgroup [median, 93 spot-forming cells (SFCs) per 10(6) peripheral blood mononuclear cells (PBMCs)] was significantly lower than that in the chronically infected untreated subgroup (median, 221 SFCs per 10(6) PBMCs), and HLA-A2-restricted treated PBDs had a response of a much higher frequency and magnitude than that of HLA-A11-restricted treated PBDs. Moreover, some novel peptides restricted by the HLA-A2 and -A11 molecules were identified.

Human immunodeficiency virus type 1 (HIV-1) circulating recombinant form 02_AG (CRF02_AG) has a higher in vitro replicative capacity than its parental subtypes A and G

Human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) 02_AG is the predominant subtype in Cameroon, even more prevalent than the parental subtypes A and G. An important question that needs to be addressed is whether recombination in HIV-1 infection can lead to the emergence of viruses with biological advantages. The replicative capacity was investigated in peripheral blood mononuclear cells (PBMCs) of 13 R5-tropic primary HIV-1 isolates, including 5 CRF02_AG, 4 subtype A, and 4 subtype G viruses. HIV-1 subtype identity was defined by phylogeny either of the full-length genome or analysis of a combination of segments of the gag, pro, pol, and env genes followed by recombination breakpoint analysis. All viruses were grown on PBMCs for 11 days and culture supernatant was analyzed for reverse transcriptase (RT) activity and p24 production. On day 11 post-infection, CRF02_AG strains had a 1.4-1.9 times higher RT activity and reached a significantly higher level of p24 production than the parental subtypes A and G. Furthermore, the replication rate as measured by p24 production was 1.4 times higher for CRF02_AG strains compared to the subtypes A and G. This study suggests that the recombination event that led to CRF02_AG resulted in a variant with a better replicative capacity than its progenitors. This adaptation could contribute to the broader spread of HIV-1 CRF02_AG leading to its predominance in West Central Africa compared to the lower prevalence of its parental subtypes A and G.

Identification of C/EBP Binding Sites Within the Clade C LTR

Human immunodeficiency virus type 1 (HIV-1) has been transmitted worldwide and regional viral clades have been designated as subtype A through K. Subtype C, which is concentrated in southeast Asia and sub-Saharan Africa, is the most prevalent subtype worldwide. To date, no studies have examined the role of CCAAT/enhancer binding proteins (C/EBP) in LTR-directed viral gene expression in the clade C LTR. Within clade B viruses, two functional C/EBP sites upstream of the TATA box have been shown to be required for efficient viral replication in cells of monocyte/macrophage lineage. In order to assess the role of the C/EBP sites within the subtype C viral LTR, 211 HIV-1 subtype C LTR sequences were collected and aligned via the Clustal V method. From these analyses, three potential C/EBP sites were identified: two upstream binding sites and one downstream binding site. Interestingly, the putative downstream site was highly conserved between clades B and C, suggesting the presence of a functionally important cis-acting element that has yet to be characterized. Electrophoretic mobility shift analyses demonstrated that two of the three sites within the HIV-1 subytpe C were able to bind C/EBP factors. Additional studies focused on examining relative binding affinities of naturally occurring variants of these two sites. Future studies will examine the roles of these sites in the regulation of LTR activity. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Predominance of Three NF-κB Binding Sites in the Long Terminal Repeat Region of HIV Type 1 Subtype C Isolates from Zambia

Human immunodeficiency virus type-1 (HIV-1) is a leading cause of mortality and morbidity in the world, with almost 46 million people infected globally. HIV-1 subtype C accounts for 55% of these infections. In Zambia, the majority of HIV-1 infections are subtype C. However, to its north most countries have non-subtype C as the most predominant HIV-1 subtype while to its south most of them are predominantly subtype C. The aim of this study was to determine the subtype distribution and to analyze the long terminal repeat (LTR) region of HIV-1 isolates from the northern part of Zambia. We amplified as well as directly sequenced the LTR, gag, and env regions of 78 HIV-1 peripheral blood samples from adult Zambians. Our results show 95% (74/78) of our isolates were HIV-1 subtype C. Furthermore, of the subtype C samples analyzed across the LTR, 61% (25/41) carried 3 NF-kappaB signature binding site sequences.

Use of sequence data generated in the Bayer Tru Gene genotyping assay to recognize and characterize non-subtype-b human immunodeficiency virus type 1 strains.

Human immunodeficiency virus type 1 (HIV-1) protease (PR) and reverse transcriptase (RT) gene sequences obtained during antiretroviral resistance testing with a commercial genotyping assay (Tru Gene; Bayer Corp.) were analyzed to assess the utility of these data for detecting and characterizing non-subtype-B HIV-1 strains. A total of 125 viral sequences obtained from patients believed to have acquired their HIV-1 infection in Africa were analyzed, of which 121 were determined to belong to non-B subtypes. Utilizing Tru Gene sequence data alone, 92 (76%) of these viruses could be subtyped by conventional phylogenetic analysis. The addition of supplemental RT sequence data enabled a further 28 (23.1%) viruses to be classified, while one (0.9%) sample could not be classified conclusively. Two internet-accessible databases that generate HIV-1 subtypes from PR and RT sequences (HIV-SEQ and Geno 2 Pheno) were also evaluated, and both achieved 88% concordance (106/120) with phylogenetic analysis. Non-subtype-B and B-subtype HIV-1 sequences could be readily discriminated by tallying silent polymorphisms listed on the Tru Gene research report. The mean number of silent polymorphisms in the non-B HIV-1 sequences identified in this study was 58.3 (95% confidence interval [CI], 41.1 to 75.5), compared with 20.7 (95% CI, 9.9 to 31.5) for the four subtype B viruses in the study cohort and 118 case-matched B-subtype controls. Sequence data generated in the Tru Gene HIV-1 genotyping assay could, therefore, provide a ready means of tracking the prevalence and identity of non-B subtypes in HIV-1-infected populations undergoing routine antiretroviral resistance testing.

Simultaneous Visual Detection of Multiple Viral Amplicons by Dipstick Assay

A sensitive, simple, and instrument-independent method for the visual detection and identification of multiple nucleic acid amplicons by dipstick has been developed. This method is based on nucleic acid hybridization on the dipstick membrane and a signal amplification system to allow visual detection. With hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) as model analytes, it is demonstrated that the visual dipstick test combined with multiplex reverse transcription (RT)-PCR for the amplification of viral nucleic acid provides a specific and sensitive detection method. The RT-PCR products were detected by the dipstick with an efficiency similar to that of a complex, expensive, and instrument-dependent method based on fluorogenic oligonucleotide probes. The detection limits of the dipstick combined with multiplex RT-PCR were 50, 125, and 500 IU/ml for HBV DNA, HCV RNA, and HIV-1 RNA, respectively. The dipstick assay detected with similar efficiencies amplicons derived from strains of HBV genotypes A through F, HCV genotypes 1 to 6, and HIV-1 subtypes A through H as well as CRF02 circulating recombinant forms of HIV-1. Analysis of 295 clinical samples and 19 pools of 10 plasma specimens from blood donors revealed that multiplex dipstick detection was reproducible, sensitive, and specific. The visual dipstick detection of multiple amplicons thus provides an attractive alternative to complex, instrument-dependent detection methods currently in use for nucleic acid testing. This new and sensitive method for nucleic acid detection should increase the availability of genomic screening in resource-limited settings and its applicability to near-patient testing.

Cytolytic T Lymphocytes (CTLs) from HIV‐1 Subtype C–Infected Indian Patients Recognize CTL Epitopes from a Conserved Immunodominant Region of HIV‐1 Gag and Nef

Analysis of the human immunodeficiency virus type 1 (HIV-1) cytolytic T lymphocyte (CTL) epitopes recognized by the targeted population is critical for HIV-1 vaccine design. Peripheral blood mononuclear cells from 47 Indian subjects at different stages of HIV-1 infection were tested for HIV-1 Gag-, Nef-, and Env-specific T cell responses by interferon (IFN)- gamma enzyme-linked immunospot (ELISPOT) assay, using pools of overlapping peptides. The Gag and Nef antigens were targeted by 83% and 36% of responders. Five immunodominant regions, 4 in Gag and 1 in Nef, were identified in the study; these regions are conserved across clades, including the African subtype C clade. Three antigenic regions were also found to be recognized by CTLs of the study participants. These regions were not identified as immunodominant regions in studies performed in Africa, which highlights the importance of differential clustering of responses within HIV-1 subtype C. Twenty-six putative epitopes--15 Gag (10 in p24 and 5 in p17), 10 Nef, and 1 Env (gp 41)--were predicted using a combination of peptide matrix ELISPOT assay and CTL epitope-prediction software. Ninety percent of the predicted epitopes were clustered in the conserved immunodominant regions of the Gag and Nef antigens. Of 26 predicted epitopes, 8 were promiscuous, 3 of which were highly conserved across clades. Three Gag and 4 Nef epitopes were novel. The identification of conserved epitopes will be important in the planning of an HIV-1 vaccine strategy for subtype C-affected regions.

Low CD4 T Cell Counts before HIV‐1 Seroconversion Do Not Affect Disease Progression in Ethiopian Factory Workers

Human immunodeficiency virus type 1 (HIV-1)-uninfected Ethiopians have lower CD4 T cell counts than do other populations in Africa and industrialized countries. We studied whether this unique immunological profile results in shorter survival times in HIV-1-infected Ethiopians. Data from an open cohort study of 149 HIV-1-infected factory workers in Ethiopia for 1997-2002 were used. To estimate survival times, a continuous-time Markov model was designed on the basis of CD4 T cell counts and World Health Organization clinical staging. By use of a random-effects model, decline in CD4 T cell counts was compared between HIV-1-infected Ethiopian and Dutch individuals. Median survival times were in the range of 9.1-13.7 years, depending on the approach used. This range is similar to that for populations in industrialized countries before the advent of antiretroviral therapy. Ethiopians had a lower annual decline in CD4 T cell counts than did Dutch individuals, which remained when groups with similar CD4 T cell count categories were compared. Moreover, the slower decline in CD4 T cell counts was not due merely to lower HIV-1 RNA loads or an absence of syncytium-inducing/X4 HIV-1 subtype C strains in Ethiopians. Low baseline CD4 T cell counts do not imply shorter survival times in Ethiopians than in other populations, presumably because of a slower decline in CD4 T cell counts.

Characterization of Human Immunodeficiency Virus Type 1 (HIV-1) Envelope Variation and Neutralizing Antibody Responses during Transmission of HIV-1 Subtype B

We analyzed neutralization sensitivity and genetic variation of transmitted subtype B human immunodeficiency virus type 1 (HIV-1) in eight recently infected men who have sex with men and the virus from the six subjects who infected them. In contrast to reports of heterosexual transmission of subtype C HIV-1, in which the transmitted virus appears to be more neutralization sensitive, we demonstrate that in our study population, relatively few phenotypic changes in neutralization sensitivity or genotypic changes in envelope occurred during transmission of subtype B HIV-1. We suggest that limited genetic variation within the infecting host reduces the likelihood of selective transmission of neutralization-sensitive HIV.

Vpu-mediated CD4 down-regulation and degradation is conserved among highly divergent SIV cpz strains

Human immunodeficiency virus type 1 (HIV-1) along with simian immunodeficiency viruses from chimpanzees (SIV cpz) and three species of Old World monkeys from the genus Cercopithecus have been shown to encode a Vpu protein. To date, the functional characterization of Vpu has been limited to a small number of subtype B and more recently subtype C Vpu proteins. Using a recently developed VpuEGFP reporter system, we have shown that the subtype B and C Vpus are capable of preventing CD4 from being expressed on the cell surface. Using the same reporter system, we report here on the expression and functional analysis of Vpu protein from four SIV cpz isolates (CAM13, ANT, TAN1, and GAB1). All four SIV Vpu fusion proteins were efficiently expressed and prevented CD4 expression on the cell surface and induced CD4 degradation. This was surprising as three of the SIV cpz Vpu fusion proteins had only one canonical casein kinase II (CK-II) site (CAM13, ANT, TAN1) while previous studies with laboratory adapted HXB2 had indicated that both CK-II sites were required for CD4 degradation. Both ANT and TAN1 Vpu sequences encoded five consecutive negatively charged amino acids residues following the only CKII site ( SAIEEDEE for ANT; SGVEEDEE for TAN1). We thus explored the possibility that this stretch of negatively charged amino acids might substitute for the lack of second CK-II site. Substitution of the aspartic acid at position 61 and glutamic acid at position 63 in the SIV cpz ANT Vpu within with lysine residues abolished the ability of this protein to down-modulate cell surface expression of CD4. Similarly, change of a serine to an alanine residue following the single consensus CK-II site of the CAM13 Vpu ( SGNESDGGEEE) abolished CD4-down-regulation, suggesting that this serine was phosphorylated in the absence of a canonical CK-II site. Our results indicate that the serine was required, suggesting that this serine was phosphorylated by CK-II or possibly another cellular kinase. Taken together, these results show for the first time that Vpu proteins from SIV cpz isolates, although quite diverse in sequence and predicted secondary structure from the HIV-1 subtype B protein, are capable of down-regulating CD4, which is one of the major functions of the HIV-1 protein.

Human immunodeficiency virus type 1 (HIV-1) genotyping in Rio de Janeiro, Brazil: assessing subtype and drug-resistance associated mutations in HIV-1 infected individuals failing highly active antiretroviral therapy

In order to assess the human immunodeficiency virus type 1 (HIV-1) drug resistance mutation profiles and evaluate the distribution of the genetic subtypes in the state of Rio de Janeiro, Brazil, blood samples from 547 HIV-1 infected patients failing antiretroviral (ARV) therapy, were collected during the years 2002 and 2003 to perform the viral resistance genotyping at the Renageno Laboratory from Rio de Janeiro (Oswaldo Cruz Foundation). Viral resistance genotyping was performed using ViroSeq Genotyping System (Celera Diagnostic-Abbott, US). The HIV-1 subtyping based on polymerase (pol) gene sequences (protease and reverse transcriptase-RT regions) was as follows: subtype B (91.2%), subtype F (4.9%), and B/F viral recombinant forms (3.3%). The subtype C was identified in two patients (0.4%) and the recombinant CRF_02/AG virus was found infecting one patient (0.2%). The HIV-1 genotyping profile associated to the reverse transcriptase inhibitors has shown a high frequency of the M184V mutation followed by the timidine-associated mutations. The K103N mutation was the most prevalent to the non-nucleoside RT inhibitor and the resistance associated to protease inhibitor showed the minor mutations L63P, L10F/R, and A71V as the more prevalent. A large proportion of subtype B was observed in HIV-1 treated patients from Rio de Janeiro. In addition, we have identified the circulation of drug-resistant HIV-1 subtype C and are presenting the first report of the occurrence of an African recombinant CRF_02/AG virus in Rio de Janeiro, Brazil. A clear association between HIV-1 subtypes and protease resistance mutations was observed in this study. The maintenance of resistance genotyping programs for HIV-1 failing patients is important to the management of ARV therapies and to attempt and monitor the HIV-1 subtype prevalence in Brazil.

Analysis of Full-Length Human Immunodeficiency Virus Type 1 Genome Reveals a Variable Spectrum of Subtypes B and F Recombinants in São Paulo, Brazil

Recombination is one of the major mechanisms contributing to human immunodeficiency virus type 1 (HIV-1) variability. Analysis of pol gene sequences of 215 HIV-1 samples from São Paulo, Brazil classified 189 sequences as subtype B (87.9%), 8 sequences as subtype F (3.7%), and 18 sequences (8.4%) as B/F recombinants. After the analysis of the pol gene, a subset of six recombinant samples composed of sequences with a related recombinant pol structure was selected for full-length genome analysis to identify a possible circulating recombinant form. According to full-length genome analysis, recombination was higher in gag, protease, reverse transcriptase, integrase, and vif. Identification of many distinct recombinant forms and the absence of an identifiable HIV-1 circulating recombinant form suggest that a high frequency of dual infections between HIV-1 subtypes B and F is occurring in São Paulo, Brazil.

Failure of Serial Human Immunodeficiency Virus Type 1 DNA Polymerase Chain Reactions to Identify Human Immunodeficiency Virus Type 1 Clade A/G

The most commonly used test to screen for human immunodeficiency virus type 1 (HIV-1) infection in HIV-exposed infants in the United States is HIV-1 qualitative DNA polymerase chain reaction (PCR). However, the commercially available HIV-1 DNA PCR lack optimal sensitivity to detect non-subtype B subtypes of HIV-1. We report here HIV-1 infection in a West African infant that went undetected by serial HIV-1 DNA PCR tests.

Evaluation of Two Commercially Available, Inexpensive Alternative Assays Used for Assessing Viral Load in a Cohort of Human Immunodeficiency Virus Type 1 Subtype C-Infected Patients from South Africa

Although human immunodeficiency virus type 1 (HIV-1) RNA is the acknowledged "gold standard" marker for monitoring disease activity in patients receiving highly active antiretroviral therapy (HAART), it remains unaffordable in resource-constrained settings. The present study investigated two commercially available kits for the detection of HIV-1 viral load markers as more affordable alternatives to HIV-1 RNA quantitation. The greatly improved heat-denatured, signal-boosted HiSens HIV-1 p24 Ag Ultra kit (Perkin-Elmer) and the ExaVir Load Quantitative HIV-RT kit (Cavidi Tech AB) were compared with the Amplicor HIV-1 Monitor (version 1.5) assay (Roche Molecular Systems Inc.). A total of 117 samples containing HIV-1 subtype C were analyzed by all three methodologies. Eighty-nine of these samples represented serial measurements from 20 patients receiving HAART. The remaining samples analyzed were from a group of treatment-naive patients. The association between the p24 antigen assay and the RNA assay was fairly strong (R(2) = 0.686). The association between the reverse transcriptase (RT) quantitation assay and the RNA assay was strong (R(2) = 0.810). Both alternative assays seemed most useful for the serial monitoring of patients receiving HAART (n = 89 plasma samples from 20 patients), as all assays showed a statistically significant downward trend over time, with the trend being either linear or curvilinear. In addition, all three assays showed negative correlations with the CD4 count (CD4 count versus RNA load, r = -0.336 and P = 0.001; CD4 count versus p24 antigen level, r = -0.541 and P < 0.0001; CD4 count versus RT level, r = -0.358 and P = 0.0006). Still of major concern are both the lack of sensitivity and the wide degrees of variability of both assays. However, both assays provide a less expensive alternative to the Roche viral load assay and demonstrate the same trends during treatment.

Evaluation of a modified version of Heteroduplex Mobility Assay for rapid screening of HIV-1 isolates in epidemics characterized by mono/dual clade predominance

The geographical distribution of human immunodeficiency virus type 1 (HIV-1) subtypes show, with the exception of some African Countries, a specific pattern. However, the significant phenomenon of migration to Western Countries, coupled to inter-ethnic blending, may result in a constant introduction and spread of novel subtypes and/or recombinant forms in previously homogeneous HIV-1 epidemics. The need to identify and trace these events prompted the development of a rapid and specific bio-molecular tool for the HIV-1 screening, based on the well-established Heteroduplex Mobility Assay (HMA). This modified version of HMA (rHMA) has been designed to detect, by a short electrophoretic analysis, HIV-1 isolates remarkably divergent form the local predominant clade, for subsequent more accurate genetic and phylogenetic analyses. The method has been validated for both C2-V5 region of env gene and the p24-p7 region of the gag gene, by proof-of-concept experiments performed on a panel of reference standards representing the globally most prevalent HIV-1 subtypes, and applied to screen Italian and Ugandan field isolates. The rHMA experimental conditions identified in this study have been shown to be specific and reliable for both sub-genomic regions of each subtype used. In the context of nationwide monitoring programs, the rHMA may represent a powerful tool for the HIV-1 molecular surveillance in both developed and developing countries, particularly those characterized by mono/dual-clade HIV-1 epidemic, which is relevant for epidemiological studies and for the development of preventive and therapeutic strategies.