Immunogenicity of personalized dendritic-cell therapy in HIV-1 infected individuals under suppressive antiretroviral treatment: interim analysis from a phase II clinical trial.

BACKGROUND: We developed a personalized Monocyte-Derived Dendritic-cell Therapy (MDDCT) for HIV-infected individuals on suppressive antiretroviral treatment and evaluated HIV-specific T-cell responses. METHODS: PBMCs were obtained from 10 HIV+ individuals enrolled in trial NCT02961829. Monocytes were differentiated into DCs using IFN-α and GM-CSF. After sequencing each patient's HIV-1 Gag and determining HLA profiles, autologous Gag peptides were selected based on the predicted individual immunogenicity and used to pulse MDDCs. Three doses of the MDDCT were administered every 15 days. To assess immunogenicity, patients' cells were stimulated in vitro with autologous peptides, and intracellular IL-2, TNF, and interferon-gamma (IFN-γ) production were measured in CD4+ and CD8+ T-cells. RESULTS: The protocol of ex-vivo treatment with IFN-α and GM-CSF was able to induce maturation of MDDCs, as well as to preserve their viability for reinfusion. MDDCT administration was associated with increased expression of IL-2 in CD4+ and CD8+ T-cells at 15 and/or 30 days after the first MDDCT administration. Moreover, intracellular TNF and IFN-γ expression was significantly increased in CD4+ T-cells. The number of candidates that increased in vitro the cytokine levels in CD4+ and CD8+ T cells upon stimulation with Gag peptides from baseline to day 15 and from baseline to day 30 and day 120 after MDDCT was significant as compared to Gag unstimulated response. This was accompanied by an increasing trend in the frequency of polyfunctional T-cells over time, which was visible when considering both cells expressing two and three out of the three cytokines examined. CONCLUSIONS: MDDC had a mature profile, and this MDDCT promoted in-vitro T-cell immune responses in HIV-infected patients undergoing long-term suppressive antiretroviral treatment. Trial registration NCT02961829: (Multi Interventional Study Exploring HIV-1 Residual Replication: a Step Towards HIV-1 Eradication and Sterilizing Cure, https://www.clinicaltrials.gov/ct2/show/NCT02961829 , posted November 11th, 2016).

HCV genotype profile in Brazil of mono-infected and HIV co-infected individuals: A survey representative of an entire country.

INTRODUCTION: To be eligible for government-provided treatment in Brazil, all HCV-infected individuals are required to be genotyped shortly after diagnosis. We describe the HCV genotype (G) profiles by geographic region, gender, age and HIV co-infection. METHODS: We assessed 29,071 genotypes collected from HCV-infected individuals from March 2016 to March 2018 (Abbott Real-Time HCV Genotype). We randomly selected 12,336 samples for HIV co-infection testing using an EIA rapid test kit (TR DPP HIV 1/2 Bio-Manguinhos). Descriptive statistical analyses were performed using R. RESULTS: Overall, HCV genotype distribution was 40.9% G1A, 30.2% G1B, 23.8% G3, 3.8% G2, 0.7% G4, 0.1% G5 and 0.6% with multiples genotypes. G1A prevalence was 44.4% among males and 35.8% among females. G1B and G2 were more prevalent in older individuals than G1A and G3. G3 was more prevalent in the South region. Of samples tested for HIV co-infection, 15% were HIV+. Median age among HCV/HIV co-infected individuals was 50 years old compared to 57 years old among mono-infected individuals. Distinct HCV genotype prevalence between HCV/HIV co-infected and HCV mono-infected individuals were respectively: G1A 60.6% versus 37.8%, G1B 15.2% versus 32.9%, and G3 18.9% versus 24.7%. G4 was detected among co-infected young men (3.5% versus 0.2% among mono-infected). CONCLUSION: The increasing prevalence of G3, as inferred by the younger ages of the HCV-infected individuals, poses an extra challenge with regards to disease progression. Distinct genotypical profiles between HCV mono-infection and HCV/HIV co-infection warrant future research in order to better understand and help mitigate HCV chains of transmission.

Laboratory surrogate markers of residual HIV replication among distinct groups of individuals under antiretroviral therapy.

BACKGROUND: Residual HIV-1 replication among individuals under antiretroviral therapy (ART) relates to HIV micro-inflammation. OBJECTIVES: To determine the levels of residual HIV replication markers among distinct subgroups of antiretroviral-treated individuals. METHODS: One hundred sixteen patients were distributed into 5 treatment groups: first-line suppressive ART with a non-nucleoside analog reverse-transcriptase inhibitor (NNRTI) (n = 26), first-line suppressive ART with boosted protease inhibitors (PI-r) (n = 25), salvage therapy using PI-r (n = 27), salvage therapy with PI-r and raltegravir (n = 22) and virologic failure (n = 16). Episomal and total DNA quantitation was evaluated. ELISA was used for HIV antibody and LPS quantitation. RESULTS: Episomal DNA was positive in 26% to 38% of individuals under suppressive ART, and it was higher among individuals experiencing ART virologic failure (p = 0.04). The HIV proviral load was higher among patients with detectable episomal DNA (p = 0.01). Individuals receiving initial PI-r treatment presented lower HIV antibody (p = 0.027) and LPS (p = 0.029) levels than individuals receiving NNRTI. There was a negative correlation between episomal DNA quantitation and the duration of suppressive ART (p = 0.04), CD4+ T-cell count (p = 0.08), and CD8+ T-cell count (p = 0.07). CONCLUSIONS: Residual HIV replication has been inferred among individuals under suppressive ART according to episomal DNA detection. Residual replication may decrease with longer periods of suppressive ART and higher levels of CD4+ and CD8+ T cells. The relationship between episomal DNA and total DNA suggests there is a replenishment of the proviral reservoir with impacts on HIV persistence. Lower antibody and LPS levels among patients with initial PI-r ART suggest these regimens may more effectively suppress HIV and have a higher capacity to decrease the HIV antigenic component.

Modulation of epigenetic factors during the early stages of HIV-1 infection in CD4+ T cells in vitro.

Several studies have related epigenetic mechanisms to HIV-1 latency. However, the epigenetic modifications of the host cell genome involved in the early stages of HIV-1 infection remain unclear. This study aimed to investigate epigenetic factors that are regulated at the beginning of HIV-1 infection in activated and resting CD4+ T cells. We analyzed the gene expression of 84 epigenetic targets, global DNA methylation, and HIV-1 replication kinetics for 36 h after infecting CD4+ T cells obtained from the blood of twelve healthy donors. The epigenetic targets aurora kinase B (AURKB), aurora kinase C (AURKC) and DNA methyltransferase 3B (DNMT3B), and the global DNA methylation profile are regulated during HIV-1 replication in CD4+ T cells, and this regulation can be influenced by the activation state of the cell at the time of infection. Approaches that affect the expression of these epigenetic targets could help current strategies to suppress HIV-1 replication.

Insights about minority HIV-1 strains in transmitted drug resistance mutation dynamics and disease progression.

Objectives: The presence of minority transmitted drug resistance mutations was assessed using ultra-deep sequencing and correlated with disease progression among recently HIV-1-infected individuals from Brazil. Methods: Samples at baseline during recent infection and 1 year after the establishment of the infection were analysed. Viral RNA and proviral DNA from 25 individuals were subjected to ultra-deep sequencing of the reverse transcriptase and protease regions of HIV-1. Results: Viral strains carrying transmitted drug resistance mutations were detected in 9 out of the 25 patients, for all major antiretroviral classes, ranging from one to five mutations per patient. Ultra-deep sequencing detected strains with frequencies as low as 1.6% and only strains with frequencies >20% were detected by population plasma sequencing (three patients). Transmitted drug resistance strains with frequencies <14.8% did not persist upon established infection. The presence of transmitted drug resistance mutations was negatively correlated with the viral load and with CD4+ T cell count decay. Conclusions: Transmitted drug resistance mutations representing small percentages of the viral population do not persist during infection because they are negatively selected in the first year after HIV-1 seroconversion.

Uniquely altered transcripts are associated with immune preservation in HIV infection.

The mechanisms underlying host HIV control hold much promise in the search for a functional HIV cure. We investigated the host genomic signatures in elite controllers or rapid progressors following recent infection and the correlates of immune reconstitution during combination antiretroviral therapy. We characterized the HIV-specific longitudinal host transcriptional response of peripheral blood mononuclear cells from elite controllers, rapid progressors, immune responders and non-responders using a RT-qPCR array in a cohort of recently HIV-infected Brazilian individuals. The elite controllers expressed unique transcripts early in infection that were closely associated with specialized cross-presentation between XCR1+ DCs and antigen-specific CD8+ T cells (XCL1). The natural suppression of HIV was also associated with the highly functional co-expression of cytokines and chemokines (CCL2, TNF and IL-10) concomitant with the maintenance of important anti-inflammatory and anticoagulant properties (Antithrombin III). Immune responders exhibited exclusively upregulated mRNAs possibly related to stem cell mobilization before combination antiretroviral therapy (neutrophil elastase). Our longitudinal approach to gene expression permitted us to discover previously unrecognized determinants that contribute to natural or antiretroviral-mediated HIV-1 immune control.

Correction: Plasma Metabolomics Biosignature According to HIV Stage of Infection, Pace of Disease Progression, Viremia Level and Immunological Response to Treatment.

[This corrects the article DOI: 10.1371/journal.pone.0161920.].

Plasma Metabolomics Biosignature According to HIV Stage of Infection, Pace of Disease Progression, Viremia Level and Immunological Response to Treatment.

BACKGROUND: We evaluated plasma samples HIV-infected individuals with different phenotypic profile among five HIV-infected elite controllers and five rapid progressors after recent HIV infection and one year later and from 10 individuals subjected to antiretroviral therapy, five of whom were immunological non-responders (INR), before and after one year of antiretroviral treatment compared to 175 samples from HIV-negative patients. A targeted quantitative tandem mass spectrometry metabolomics approach was used in order to determine plasma metabolomics biosignature that may relate to HIV infection, pace of HIV disease progression, and immunological response to treatment. RESULTS: Twenty-five unique metabolites were identified, including five metabolites that could distinguish rapid progressors and INRs at baseline. Severe deregulation in acylcarnitine and sphingomyelin metabolism compatible with mitochondrial deficiencies was observed. ß-oxidation and sphingosine-1-phosphate-phosphatase-1 activity were down-regulated, whereas acyl-alkyl-containing phosphatidylcholines and alkylglyceronephosphate synthase levels were elevated in INRs. Evidence that elite controllers harbor an inborn error of metabolism (late-onset multiple acyl-coenzyme A dehydrogenase deficiency [MADD]) was detected. CONCLUSIONS: Blood-based markers from metabolomics show a very high accuracy of discriminating HIV infection between varieties of controls and have the ability to predict rapid disease progression or poor antiretroviral immunological response. These metabolites can be used as biomarkers of HIV natural evolution or treatment response and provide insight into the mechanisms of the disease.

High prevalence and incidence of HIV-1 in a counseling and testing center in the city of Itajaí, Brazil

ABSTRACT Itajaí is a port city in southern Brazil with one of the highest incidence and mortality rates from AIDS in the country. The prevalence and incidence of HIV infection were investigated in 1085 of 3196 new HIV-1 infection cases evaluated in the counseling and testing center of Itajaí from January 2002 to August 2008. Recent infections were assessed using the BED(tm), and polregion sequencing was performed in 76 samples. The prevalence ranged from 3.08% to 6.17% among women and from 10.26% to 17.36% among men. A total of 17% of infections were classified as recent, with annual incidence varying from 1.6% to 4.8 per 100 patient/year among women and from 2.05% to 8.5 per 100 patient/year among men. Pol sequences were obtained from 38 randomly recent infections selected individuals: 71% were infected by subtype C, 24% B, 2% D, and 2% F1. Among 38 subjects with established infection, 76% were subtype C, and 24% B. Transmitted drug resistance was detected in 18.4% of recent infection subjects (7.8% to nucleoside analog reverse-transcriptase inhibitors, 5.2% to non-nucleoside reverse-transcriptase inhibitors, and 5.2% protease inhibitors) and 5.2% of subjects with established infection had nucleoside analog reverse-transcriptase inhibitors resistance. The high prevalence and incidence of HIV infection in this region is unprecedented in studies involving cases evaluated in the counseling and testing centers in Brazil.

The Virological and Immunological Characteristics of the HIV-1-Infected Population in Brazil: From Initial Diagnosis to Impact of Antiretroviral Use.

BACKGROUND: Immunological and virological status of HIV-infected individuals entering the Brazilian public system over time was analyzed. We evaluated the impact of ART on virological, immunological and antiretroviral resistance over time. METHODS: CD4+ T cell counts, viral loads and genotypes from patients over 13 years old from 2001-2011 were analyzed according to demographic data. We compared groups using parametric t-tests and linear regression analysis in the R statistical software language. RESULTS: Mean baseline CD4+ T cell counts varied from 348 (2003) to 389 (2009) and was higher among women (p = 1.1 x 10(-8)), lower in older patients (p< 1 x 10(-8)) and lower in less developed regions (p = 1.864 x 10(-5)). Percentage of treated patients with undetectable viral loads increased linearly from 46% (2001) to 77% (2011), was lower among women (p = 2.851 x 10(-6)), younger ages (p = 1 x 10(-3)), and in less developed regions (p = 1.782 x 10(-4)). NRTI acquired resistance was 86% in 2001-3 and decreased over time. NNRTI resistance increased from 2001-3(50%) to 2006-9 (60%), PI resistance decreased from 2001-3 (60%) to 2009 (40%), and 3-class resistance was stable over time around 25%. Subtype prevalence comprised B (75.3%), B/F recombinants (12.2%), C (5.7%), F (5.3%) and B/C recombinants (1.5%), with regional variations. Three-class resistance was 26.5% among Bs, 22.4% among Fs and 17.2% among Cs. CONCLUSIONS: HIV diagnosis occurs late, especially among elderly Brazilians. Younger individuals need special attention due to poor virological response to treatment. Antiretroviral Resistance profile is subtype related.

High prevalence and incidence of HIV-1 in a counseling and testing center in the city of Itajaí, Brazil.

Itajaí is a port city in southern Brazil with one of the highest incidence and mortality rates from AIDS in the country. The prevalence and incidence of HIV infection were investigated in 1085 of 3196 new HIV-1 infection cases evaluated in the counseling and testing center of Itajaí from January 2002 to August 2008. Recent infections were assessed using the BED™, and pol region sequencing was performed in 76 samples. The prevalence ranged from 3.08% to 6.17% among women and from 10.26% to 17.36% among men. A total of 17% of infections were classified as recent, with annual incidence varying from 1.6% to 4.8 per 100 patient/year among women and from 2.05% to 8.5 per 100 patient/year among men. Pol sequences were obtained from 38 randomly recent infections selected individuals: 71% were infected by subtype C, 24% B, 2% D, and 2% F1. Among 38 subjects with established infection, 76% were subtype C, and 24% B. Transmitted drug resistance was detected in 18.4% of recent infection subjects (7.8% to nucleoside analog reverse-transcriptase inhibitors, 5.2% to non-nucleoside reverse-transcriptase inhibitors, and 5.2% protease inhibitors) and 5.2% of subjects with established infection had nucleoside analog reverse-transcriptase inhibitors resistance. The high prevalence and incidence of HIV infection in this region is unprecedented in studies involving cases evaluated in the counseling and testing centers in Brazil.

In vivo HIV-1 hypermutation and viral loads among antiretroviral-naive Brazilian patients.

Hypermutation alludes to an excessive number of specific guanine-to-adenine (G- >A) substitutions in proviral DNA and this phenomenon is attributed to the catalytic activity of cellular APOBECs. Population studies relating hypermutation and the progression of infection by human immunodeficiency virus type 1 (HIV-1) have been performed to elucidate the effect of hypermutation on the natural course of HIV-1 infection. However, the many different approaches employed to assess hypermutation in nucleotide sequences render the comparison of results difficult. This study selected 157 treatment-naive patients and sought to correlate the hypermutation level of the proviral sequences in clinical samples with demographic variables, HIV-1 RNA viral load, and the level of CD4(+) T cells. Nested touchdown polymerase chain reaction (PCR) was performed with specific primers to detect hypermutation in the region of HIV-1 integrase, and the amplified sequences were run in agarose gels with HA-Yellow. The analysis of gel migration patterns using the k-means clustering method was validated by its agreement with the results obtained with the software Hypermut. Hypermutation was found in 31.2% of the investigated samples, and a correlation was observed between higher hypermutation levels and higher viral load levels. These findings suggest a high frequency of hypermutation detection in a Brazilian cohort, which can reflect a particular characteristic of this population, but also can result from the method approach by aiming at hypermutation-sensitive sites. Furthermore, we found that hypermutation events are pervasive during HIV-1 infection as a consequence of high viral replication, reflecting its role during disease progression.

Homogenous HIV-1 subtype B quasispecies in Brazilian men and women recently infected via heterosexual transmission.

HIV has extraordinary genetic mutability, both among individuals and at the population level. However, studies of primary HIV-1 infection and serum-converters indicate that the viral population is homogeneous at the sequence level, which suggests clonal HIV transmission. It remains unclear whether this feature applies to the female population. Ten single genome amplification sequences were generated from ten individuals (five females) with recent heterosexually acquired HIV infection as determined by the serologic testing algorithm for recent HIV seroconversion. Intra-individual genetic diversity was equally low in both genders (<2 %), with mean and median variations of 0.8 and 0 %, respectively. All of the subjects were infected with clade B. Three subjects (two females) appeared to be infected by two related viral populations, and four subjects harbored non-R5 strains. Our results support the hypothesis of clonal selection for sexual transmission of HIV-1 in both genders. Future studies that generate a larger number of clones, preferably by next generation deep sequencing, are needed to confirm these results.

Analysis of HIV-1 protease gene reveals frequent multiple infections followed by recombination among drug treated individuals living in São Paulo and Santos, Brazil.

The present study investigated the prevalence of HIV-1 multiple infections in a population composed by 47 patients under HAART failure and enrolled at the National DST/AIDS, Program, Ministry of Health, Brazil.Detection of multiple infections was done using a previously published RFLP assay for the HIV-1 protease gene, which is able of distinguishing between infections caused by a single or multiple HIV-1 subtypes. Samples with multiple infections were cloned, and sequence data submitted to phylogenetic analysis. We were able to identify 17 HIV-1 multiple infections out of 47 samples. Multiple infections were mostly composed by a mixture of recombinant viruses (94%), with only one case in which protease gene pure subtypes B and F were recovered. This is the first study that reports the prevalence of multiple infections and intersubtype recombinants in a population undergoing HAART in Brazil. Based on the data there was a steep increase of multiple infections after the introduction of the combined antiretroviral therapy in Brazil. Cases of multiple infections may be associated with HIV-1 genetic diversity through recombination allowing for the generation of viruses showing a combination of resistance mutations.

Faster HIV-1 disease progression among Brazilian individuals recently infected with CXCR4-utilizing strains.

INTRODUCTION: Primary HIV infection is usually caused by R5 viruses, and there is an association between the emergence of CCXR4-utilizing strains and faster disease progression. We characterized HIV-1 from a cohort of recently infected individuals in Brazil, predicted the virus's co-receptor use based on the env genotype and attempted to correlate virus profiles with disease progression. METHODS: A total of 72 recently infected HIV patients were recruited based on the Serologic Testing Algorithm for Recent HIV Seroconversion and were followed every three to four months for up to 78 weeks. The HIV-1 V3 region was characterized by sequencing nine to twelve weeks after enrollment. Disease progression was characterized by CD4+ T-cell count decline to levels consistently below 350 cells/µL. RESULTS: Twelve out of 72 individuals (17%) were predicted to harbor CXCR4-utilizing strains; a baseline CD4<350 was more frequent among these individuals (p = 0.03). Fifty-seven individuals that were predicted to have CCR5-utilizing viruses and 10 individuals having CXCR4-utilizing strains presented with baseline CD4>350; after 78 weeks, 33 individuals with CCR5 strains and one individual with CXCR4 strains had CD4>350 (p = 0.001). There was no association between CD4 decline and demographic characteristics or HIV-1 subtype. CONCLUSIONS: Our findings confirm the presence of strains with higher in vitro pathogenicity during early HIV infection, suggesting that even among recently infected individuals, rapid progression may be a consequence of the early emergence of CXCR4-utilizing strains. Characterizing the HIV-1 V3 region by sequencing may be useful in predicting disease progression and guiding treatment initiation decisions.

Prevalence, incidence density, and genotype distribution of GB virus C infection in a cohort of recently HIV-1-infected subjects in Sao Paulo, Brazil.

BACKGROUND: The results of previous studies elsewhere have indicated that GB virus C (GBV-C) infection is frequent in patients infected with the human immunodeficiency virus type 1 (HIV-1) due to similar transmission routes of both viruses. The aim of this study was to determine the prevalence, incidence density and genotypic characteristics of GBV-C in this population. METHODOLOGY/PRINCIPAL FINDINGS: The study population included 233 patients from a cohort primarily comprised of homosexual men recently infected with HIV-1 in São Paulo, Brazil. The presence of GBV-C RNA was determined in plasma samples by reverse transcriptase-nested polymerase chain reaction and quantified by real-time PCR. GBV-C genotypes were determined by direct sequencing. HIV viral load, CD4+ T lymphocyte and CD8+ T lymphocyte count were also tested in all patients. The overall prevalence of GBV-C infection was 0.23 (95% CI: 0.18 to 0.29) in the study group. There was no significant difference between patients with and without GBV-C infection and Glycoprotein E2 antibody presence regarding age, sex, HIV-1 viral load, CD4+ and CD8+T cell counts and treatment with antiretroviral drugs. An inverse correlation was observed between GBV-C and HIV-1 loads at enrollment and after one year. Also, a positive but not significant correlation was observed between GBV-C load and CD4+ T lymphocyte. Phylogenetic analysis of the GBV-C isolates revealed the presence of genotype 1 and genotype 2, these sub classified into subtype 2a and 2b. CONCLUSION/SIGNIFICANCE: GBV-C infection is common in recently HIV -1 infected patients in Sao Paulo, Brazil and the predominant genotype is 2b. This study provides the first report of the GBV-C prevalence at the time of diagnosis of HIV-1 and the incidence density of GBV-C infection in one year.

The detection of in vivo and in vitro HIV type 1 B/F profiles in Brazil using a real-time PCR assay for five HIV type 1 genomic regions.

We sought to determine the frequency and profile of HIV-1 BF recombinants in vitro and in vivo. Laboratory HIV-1 strains from subtypes B and F were cocultured and evaluated. Clinical samples from the city of Santos, Brazil, where the first HIV-1 B/F circulating recombinant forms (CRF) were described, were also assessed. Five real-time PCR assays were developed to equally amplify subtypes B and F, and subtype-specific probes were developed and optimized. To validate the PCR systems, clinical samples from Santos were sequenced and phylogenetically analyzed. The real-time PCR assays were performed on these samples and on the supernatant of an in vitro competition assay to assess emergent recombinant strains. Out of 157 clinical samples, 62.1% were defined as subtype B, 3.0% were subtype F, 16.7% presented the CRF28_BF profile, and 13.6% of the samples presented the CRF29_BF profile. The specificity and sensitivity in the discrimination assay for this sample panel were 93% and 92%, respectively. The HIV that emerged from the coinfected cell culture closely resembled the CRF28_BF profile. The first-described CRFs are still fixed in this geographic region of Brazil, and the in vitro emerging strains detected by real-time PCR suggest that in addition to the shaping of recombinant strains by immune selection, viral structures may also play an important role in emerging CRFs.

Estimating HIV-1 incidence using the serologic testing algorithm for recent HIV infections at HIV counseling and testing centers in the city of São Paulo, Brazil.

The network of HIV counseling and testing centers in São Paulo, Brazil is a major source of data used to build epidemiological profiles of the client population. We examined HIV-1 incidence from November 2000 to April 2001, comparing epidemiological and socio-behavioral data of recently-infected individuals with those with long-standing infection. A less sensitive ELISA was employed to identify recent infection. The overall incidence of HIV-1 infection was 0.53/100/year (95% CI: 0.31-0.85/100/year): 0.77/100/year for males (95% CI: 0.42-1.27/100/year) and 0.22/100/ year (95% CI: 0.05-0.59/100/year) for females. Overall HIV-1 prevalence was 3.2% (95% CI: 2.8-3.7%), being 4.0% among males (95% CI: 3.3-4.7%) and 2.1% among females (95% CI: 1.6-2.8%). Recent infections accounted for 15% of the total (95% CI: 10.2-20.8%). Recent infection correlated with being younger and male (p = 0.019). Therefore, recent infection was more common among younger males and older females.

Estimating HIV-1 incidence using the serologic testing algorithm for recent HIV infections at HIV counseling and testing centers in the city of São Paulo, Brazil

The network of HIV counseling and testing centers in São Paulo, Brazil is a major source of data used to build epidemiological profiles of the client population. We examined HIV-1 incidence from November 2000 to April 2001, comparing epidemiological and socio-behavioral data of recently-infected individuals with those with long-standing infection. A less sensitive ELISA was employed to identify recent infection. The overall incidence of HIV-1 infection was 0.53/100/year (95 percent CI: 0.31-0.85/100/year): 0.77/100/year for males (95 percent CI: 0.42-1.27/100/year) and 0.22/100/ year (95 percent CI: 0.05-0.59/100/year) for females. Overall HIV-1 prevalence was 3.2 percent (95 percent CI: 2.8-3.7 percent), being 4.0 percent among males (95 percent CI: 3.3-4.7 percent) and 2.1 percent among females (95 percent CI: 1.6-2.8 percent). Recent infections accounted for 15 percent of the total (95 percent CI: 10.2-20.8 percent). Recent infection correlated with being younger and male (p = 0.019). Therefore, recent infection was more common among younger males and older females.