Characterization of Near Full-Length Transmitted/Founder HIV-1 Subtype D and A/D Recombinant Genomes in a Heterosexual Ugandan Population (2006-2011).

Detailed characterization of transmitted HIV-1 variants in Uganda is fundamentally important to inform vaccine design, yet studies on the transmitted full-length strains of subtype D viruses are limited. Here, we amplified single genomes and characterized viruses, some of which were previously classified as subtype D by sub-genomic pol sequencing that were transmitted in Uganda between December 2006 to June 2011. Analysis of 5' and 3' half genome sequences showed 73% (19/26) of infections involved single virus transmissions, whereas 27% (7/26) of infections involved multiple variant transmissions based on predictions of a model of random virus evolution. Subtype analysis of inferred transmitted/founder viruses showed a high transmission rate of inter-subtype recombinants (69%, 20/29) involving mainly A1/D, while pure subtype D variants accounted for one-third of infections (31%, 9/29). Recombination patterns included a predominance of subtype D in the gag/pol region and a highly recombinogenic envelope gene. The signal peptide-C1 region and gp41 transmembrane domain (Tat2/Rev2 flanking region) were hotspots for A1/D recombination events. Analysis of a panel of 14 transmitted/founder molecular clones showed no difference in replication capacity between subtype D viruses (n = 3) and inter-subtype mosaic recombinants (n = 11). However, individuals infected with high replication capacity viruses had a faster CD4 T cell loss. The high transmission rate of unique inter-subtype recombinants is striking and emphasizes the extraordinary challenge for vaccine design and, in particular, for the highly variable and recombinogenic envelope gene, which is targeted by rational designs aimed to elicit broadly neutralizing antibodies.

Mutation profiling of the c.1521_1523delCTT (p.Phe508del, F508del) cystic fibrosis transmembrane conductance regulator allele using haplotype-resolved long-read next generation sequencing.

Current approaches to characterize the mutational profile of the cystic fibrosis transmembrane conductance regulator (CFTR) gene are based on targeted mutation analysis or whole gene studies derived from short-read next generation sequencing (NGS). However, these methods lack phasing capability which, in certain scenarios, can provide clinically valuable information. In the present work, we performed near-full length CFTR using Single-Molecule Real-Time Sequencing to produce haplotype-resolved data from both homozygous and heterozygous individuals for mutation c.1521_1523delCTT (p.Phe508del, F508del). This approach utilizes target enrichment of the CFTR gene using biotinylated probes, facilitates multiplexing samples in the same sequencing run, and utilizes fully-automated bioinformatics pipelines for error correction and variant calling. We show a remarkable conservation of F508del haplotype, consistent with the single gene founder effect, as well as diverse mutational profiles in non-F508del alleles. By the same method, 105 single nucleotide polymorphisms (SNPs) exhibiting invariant linkage to F508del CFTR (which better define the founder haplotype) were identified. High level homology between F508del sequences derived from heterozygotes, and those obtained from homozygous individuals, demonstrate accuracy of this method to produce haplotype resolved sequencing. The studies provide a new diagnostic technology for detailed analysis of complex CFTR alleles linked to disease severity.

Increased Frequency of Inter-Subtype HIV-1 Recombinants Identified by Near Full-Length Virus Sequencing in Rwandan Acute Transmission Cohorts.

Most studies of HIV-1 transmission have focused on subtypes B and C. In this study, we determined the genomic sequences of the transmitted founder (TF) viruses from acutely infected individuals enrolled between 2005 and 2011 into IAVI protocol C in Rwanda and have compared these isolates to viruses from more recent (2016-2019) acute/early infections in three at risk populations - MSM, high risk women (HRW), and discordant couples (DC). For the Protocol C samples, we utilized near full-length single genome (NFLG) amplification to generate 288 HIV-1 amplicons from 26 acutely infected seroconverters (SC), while for the 21 recent seroconverter samples (13 from HRW, two from DC, and six from MSM), we PCR amplified overlapping half-genomes. Using PacBio SMRT technology combined with the MDPseq workflow, we performed multiplex sequencing to obtain high accuracy sequences for each amplicon. Phylogenetic analyses indicated that the majority of recent transmitted viruses from DC and HRW clustered within those of the earlier Protocol C cohort. However, five of six sequences from the MSM cohort branched together and were greater than 97% identical. Recombination analyses revealed a high frequency (6/26; 23%) of unique inter-subtype recombination in Protocol C with 19% AC and 4% CD recombinant viruses, which contrasted with only 6.5% of recombinants defined by sequencing of the pol gene previously. The frequency of recombinants was significantly higher (12/21; 57%) in the more recent isolates, although, the five related viruses from the MSM cohort had identical recombination break points. While major drug resistance mutations were absent from Protocol C viruses, 4/21 of recent isolates exhibited transmitted nevirapine resistance. These results demonstrate the ongoing evolution and increased prevalence of recombinant and drug resistant transmitted viruses in Rwanda and highlight the importance of defining NFLG sequences to fully understand the nature of TF viruses and in particular the prevalence of unique recombinant forms (URFs) in transmission cohorts.

Utilizing Computational Machine Learning Tools to Understand Immunogenic Breadth in the Context of a CD8 T-Cell Mediated HIV Response.

Predictive models are becoming more and more commonplace as tools for candidate antigen discovery to meet the challenges of enabling epitope mapping of cohorts with diverse HLA properties. Here we build on the concept of using two key parameters, diversity metric of the HLA profile of individuals within a population and consideration of sequence diversity in the context of an individual's CD8 T-cell immune repertoire to assess the HIV proteome for defined regions of immunogenicity. Using this approach, analysis of HLA adaptation and functional immunogenicity data enabled the identification of regions within the proteome that offer significant conservation, HLA recognition within a population, low prevalence of HLA adaptation and demonstrated immunogenicity. We believe this unique and novel approach to vaccine design as a supplement to vitro functional assays, offers a bespoke pipeline for expedited and rational CD8 T-cell vaccine design for HIV and potentially other pathogens with the potential for both global and local coverage.

HIV-1 Gag-Pol Sequences from Ugandan Early Infections Reveal Sequence Variants Associated with Elevated Replication Capacity.

The ability to efficiently establish a new infection is a critical property for human immunodeficiency virus type 1 (HIV-1). Although the envelope protein of the virus plays an essential role in receptor binding and internalization of the infecting virus, the structural proteins, the polymerase and the assembly of new virions may also play a role in establishing and spreading viral infection in a new host. We examined Ugandan viruses from newly infected patients and focused on the contribution of the Gag-Pol genes to replication capacity. A panel of Gag-Pol sequences generated using single genome amplification from incident HIV-1 infections were cloned into a common HIV-1 NL4.3 pol/env backbone and the influence of Gag-Pol changes on replication capacity was monitored. Using a novel protein domain approach, we then documented diversity in the functional protein domains across the Gag-Pol region and identified differences in the Gag-p6 domain that were frequently associated with higher in vitro replication.

Resistance profile of HIV-1 quasispecies in patients under treatment failure using single molecule, real-time sequencing.

OBJECTIVE: Short-read next-generation sequencing (NGS) has been implemented to study the resistance profile of HIV as it provides a higher sensitivity than Sanger sequencing. However, short-reads only generates a consensus view of the viral population rather than a reconstruction of the viral haplotypes. In this study, we evaluated the resistance profile of HIV quasispecies in patients undergoing treatment failure using SMRT sequencing. DESIGN: Whole-pol RT-PCR was performed on viral RNA extracted from plasma samples of 38 HIV-positive individuals undergoing treatment failure, and sequenced in the RSII instrument. Error correction and viral haplotype phasing was performed with the Multilayer Directed Phasing and Sequencing (MDPSeq) algorithm. Presence of resistance mutations reported by the IAS-USA in 2017 was assessed using an in-house script. RESULTS: The SMRT sequencing-based test detected 131/134 resistance mutations previously detected using a Sanger sequencing-based test. However, the SMRT test also identified seven additional mutations present at an estimated frequency lower than 30%. The intra-host phylogenetic analysis showed that seven samples harbored at least one resistance variant at 20--80% frequency. The haplotype-resolved sequencing revealed viral diversification and selection of new resistance during suboptimal treatment, an overall trend toward selection and accumulation of new resistance mutations, as well as the co-existence of resistant and susceptible variants. CONCLUSION: Our results validate the SMRT sequencing-based test for detection of HIV drug resistance. In addition, this method unraveled the complex dynamic of HIV quasispecies during treatment failure, which might have several implications on clinical management.

Infection with multiple HIV-1 founder variants is associated with lower viral replicative capacity, faster CD4+ T cell decline and increased immune activation during acute infection.

HIV-1 transmission is associated with a severe bottleneck in which a limited number of variants from a pool of genetically diverse quasispecies establishes infection. The IAVI protocol C cohort of discordant couples, female sex workers, other heterosexuals and men who have sex with men (MSM) present varying risks of HIV infection, diverse HIV-1 subtypes and represent a unique opportunity to characterize transmitted/founder viruses (TF) where disease outcome is known. To identify the TF, the HIV-1 repertoire of 38 MSM participants' samples was sequenced close to transmission (median 21 days post infection, IQR 18-41) and assessment of multivariant infection done. Patient derived gag genes were cloned into an NL4.3 provirus to generate chimeric viruses which were characterized for replicative capacity (RC). Finally, an evaluation of how the TF virus predicted disease progression and modified the immune response at both acute and chronic HIV-1 infection was done. There was higher prevalence of multivariant infection compared with previously described heterosexual cohorts. A link was identified between multivariant infection and replicative capacity conferred by gag, whereby TF gag tended to be of lower replicative capacity in multivariant infection (p = 0.02) suggesting an overall lowering of fitness requirements during infection with multiple variants. Notwithstanding, multivariant infection was associated with rapid CD4+ T cell decline and perturbances in the CD4+ T cell and B cell compartments compared to single variant infection, which were reversible upon control of viremia. Strategies aimed at identifying and mitigating multivariant infection could contribute toward improving HIV-1 prognosis and this may involve strategies that tighten the stringency of the transmission bottleneck such as treatment of STI. Furthermore, the sequences and chimeric viruses help with TF based experimental vaccine immunogen design and can be used in functional assays to probe effective immune responses against TF.

HIV-1 variants are archived throughout infection and persist in the reservoir.

The HIV-1 reservoir consists of latently infected cells that persist despite antiretroviral therapy (ART). Elucidating the proviral genetic composition of the reservoir, particularly in the context of pre-therapy viral diversity, is therefore important to understanding reservoir formation and the persistence of latently infected cells. Here we investigate reservoir proviral variants from 13 Zambian acutely-infected individuals with additional pre-therapy sampling for a unique comparison to the ART-naïve quasispecies. We identified complete transmitted/founder (TF) viruses from seroconversion plasma samples, and additionally amplified and sequenced HIV-1 from plasma obtained one year post-infection and just prior to ART initiation. While the majority of proviral variants in the reservoir were most closely related to viral variants from the latest pre-therapy time point, we also identified reservoir proviral variants dating to or near the time of infection, and to intermediate time points between infection and treatment initiation. Reservoir proviral variants differing by five or fewer nucleotide changes from the TF virus persisted during treatment in five individuals, including proviral variants that exactly matched the TF in two individuals, one of whom had remained ART-naïve for more than six years. Proviral variants during treatment were significantly less divergent from the TF virus than plasma variants present at the last ART-naïve time point. These findings indicate that reservoir proviral variants are archived throughout infection, recapitulating much of the viral diversity that arises throughout untreated HIV-1 infection, and strategies to target and reduce the reservoir must therefore permit for the clearance of proviruses encompassing this extensive diversity.

Better Viral Control despite Higher CD4+ T Cell Activation during Acute HIV-1 Infection in Zambian Women Is Linked to the Sex Hormone Estradiol.

The influence of biological sex on disease progression in HIV-1-infected individuals has been focused on the chronic stage of infection, but little is known about how sex differences influence acute HIV-1 infection. We observed profound differences in viral load and CD4+ T cell activation from the earliest time points in men and women in a Zambian heterosexual acute infection cohort. Women exhibited a >2-fold higher rate of CD4+ T cell loss despite significantly lower viral loads (VL) than men. The importance of studying acute infection was highlighted by the observation that very early in infection, women exhibited significantly higher levels of CD4+ T cell activation, a difference that was lost over the first 3 years of infection as activation in men increased. In women, activation of CD4+ T cells in the acute phase was significantly correlated with plasma levels of 17ß-estradiol (E2). However, unlike in men, higher CD4+ T cell activation in women was not associated with higher VL. In contrast, a higher E2 level in early infection was associated with lower early and set-point VL in women. We attribute this to an inhibitory effect of estradiol on virus replication, which we were able to observe with relevant transmitted/founder viruses in vitro Thus, estradiol plays a key role in defining major differences between men and women during early HIV-1 infection by contributing to both viral control and CD4+ T cell loss, an effect that extends into the chronic phase of the disease.IMPORTANCE Previous studies have identified sex-specific differences during chronic HIV-1 infection, but little is known about sex differences in the acute phase, or how disparities in the initial response to the virus may affect disease. We demonstrate that restriction of viral load in women begins during acute infection and is maintained into chronic infection. Despite this, women exhibit more rapid CD4+ T cell loss than men. These profound differences are influenced by 17ß-estradiol, which contributes both to T cell activation and to reduced viral replication. Thus, we conclude that estradiol plays a key role in shaping responses to early HIV-1 infection that influence the chronic phase of disease.

CD8 T cells targeting adapted epitopes in chronic HIV infection promote dendritic cell maturation and CD4 T cell trans-infection.

HIV-1 frequently escapes from CD8 T cell responses via HLA-I restricted adaptation, leading to the accumulation of adapted epitopes (AE). We previously demonstrated that AE compromise CD8 T cell responses during acute infection and are associated with poor clinical outcomes. Here, we examined the impact of AE on CD8 T cell responses and their biological relevance in chronic HIV infection (CHI). In contrast to acute infection, the majority of AE are immunogenic in CHI. Longitudinal analyses from acute to CHI showed an increased frequency and magnitude of AE-specific IFNγ responses compared to NAE-specific ones. These AE-specific CD8 T cells also were more cytotoxic to CD4 T cells. In addition, AE-specific CD8 T cells expressed lower levels of PD1 and CD57, as well as higher levels of CD28, suggesting a more activated and less exhausted phenotype. During CHI, viral sequencing identified AE-encoding strains as the dominant quasispecies. Despite increased CD4 T cell cytotoxicity, CD8 T cells responding to AE promoted dendritic cell (DC) maturation and CD4 T cell trans-infection perhaps explaining why AE are predominant in CHI. Taken together, our data suggests that the emergence of AE-specific CD8 T cell responses in CHI confers a selective advantage to the virus by promoting DC-mediated CD4 T cell trans-infection.

Clustered Mutations at the Murine and Human IgH Locus Exhibit Significant Linkage Consistent with Templated Mutagenesis.

Somatic hypermutation generates a myriad of Ab mutants in Ag-specific B cells, from which high-affinity mutants are selected. Chickens, sheep, and rabbits use nontemplated point mutations and templated mutations via gene conversion to diversify their expressed Ig loci, whereas mice and humans rely solely on untemplated somatic point mutations. In this study, we demonstrate that, in addition to untemplated point mutations, templated mutagenesis readily occurs at the murine and human Ig loci. We provide two distinct lines of evidence that are not explained by the Neuberger model of somatic hypermutation: 1) across multiple data sets there is significant linkage disequilibrium between individual mutations, especially among close mutations, and 2) among those mutations, those <8 bp apart are significantly more likely to match microhomologous regions in the IgHV repertoire than predicted by the mutation profiles of somatic hypermutation. Together, this supports the role of templated mutagenesis during somatic diversification of Ag-activated B cells.

Computational comparison of availability in CTL/gag epitopes among patients with acute and chronic HIV-1 infection.

BACKGROUND: Recent studies indicate that there is selection bias for transmission of viral polymorphisms associated with higher viral fitness. Furthermore, after transmission and before a specific immune response is mounted in the recipient, the virus undergoes a number of reversions which allow an increase in their replicative capacity. These aspects, and others, affect the viral population characteristic of early acute infection. METHODS: 160 singlegag-gene amplifications were obtained by limiting-dilution RT-PCR from plasma samples of 8 ARV-naïve patients with early acute infection (<30 days, 22 days average) and 8 ARV-naive patients with approximately a year of infection (10 amplicons per patient). Sanger sequencing and NGS SMRT technology (Pacific Biosciences) were implemented to sequence the amplicons. Phylogenetic analysis was performed by using MEGA 6.06. HLA-I (A and B) typing was performed by SSOP-PCR method. The chromatograms were analyzed with Sequencher 4.10. Epitopes and immune-proteosomal cleavages prediction was performed with CBS prediction server for the 30 HLA-A and -B alleles most prevalent in our population with peptide lengths from 8 to 14 mer. Cytotoxic response prediction was performed by using IEDB Analysis Resource. RESULTS: After implementing epitope prediction analysis, we identified a total number of 325 possible viral epitopes present in two or more acute or chronic patients. 60.3% (n = 196) of them were present only in acute infection (prevalent acute epitopes) while 39.7% (n = 129) were present only in chronic infection (prevalent chronic epitopes). Within p24, the difference was equally dramatic with 59.4% (79/133) being acute epitopes (p < 0.05). This is consistent with progressive viral adaptation to immune response in time and further supported by the fact that cytotoxic responses prediction showed that acute epitopes are more likely to generate immune response than chronic epitopes. Interestingly, only 27.5% of acute epitopes match the population-level consensus sequence of the virus. CONCLUSIONS: Our results indicate that certain non-consensus viral residues might be transmitted more frequently than consensus-residues when located in immunological relevant positions (epitopes). This observation might be relevant to the rationale behind development of an effective vaccineto reduce viral reservoir and induce functional cure of HIV infection based in prevalent acute epitopes.

Balance between transmitted HLA preadapted and nonassociated polymorphisms is a major determinant of HIV-1 disease progression.

HIV-1 adapts to a new host through mutations that facilitate immune escape. Here, we evaluate the impact on viral control and disease progression of transmitted polymorphisms that were either preadapted to or nonassociated with the new host's HLA. In a cohort of 169 Zambian heterosexual transmission pairs, we found that almost one-third of possible HLA-linked target sites in the transmitted virus Gag protein are already adapted, and that this transmitted preadaptation significantly reduced early immune recognition of epitopes. Transmitted preadapted and nonassociated polymorphisms showed opposing effects on set-point VL and the balance between the two was significantly associated with higher set-point VLs in a multivariable model including other risk factors. Transmitted preadaptation was also significantly associated with faster CD4 decline (<350 cells/µl) and this association was stronger after accounting for nonassociated polymorphisms, which were linked with slower CD4 decline. Overall, the relative ratio of the two classes of polymorphisms was found to be the major determinant of CD4 decline in a multivariable model including other risk factors. This study reveals that, even before an immune response is mounted in the new host, the balance of these opposing factors can significantly influence the outcome of HIV-1 infection.

Heterosexual Transmission of Subtype C HIV-1 Selects Consensus-Like Variants without Increased Replicative Capacity or Interferon-α Resistance.

Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential.

HLA Class-II Associated HIV Polymorphisms Predict Escape from CD4+ T Cell Responses.

Antiretroviral therapy, antibody and CD8+ T cell-mediated responses targeting human immunodeficiency virus-1 (HIV-1) exert selection pressure on the virus necessitating escape; however, the ability of CD4+ T cells to exert selective pressure remains unclear. Using a computational approach on HIV gag/pol/nef sequences and HLA-II allelic data, we identified 29 HLA-II associated HIV sequence polymorphisms or adaptations (HLA-AP) in an African cohort of chronically HIV-infected individuals. Epitopes encompassing the predicted adaptation (AE) or its non-adapted (NAE) version were evaluated for immunogenicity. Using a CD8-depleted IFN-γ ELISpot assay, we determined that the magnitude of CD4+ T cell responses to the predicted epitopes in controllers was higher compared to non-controllers (p<0.0001). However, regardless of the group, the magnitude of responses to AE was lower as compared to NAE (p<0.0001). CD4+ T cell responses in patients with acute HIV infection (AHI) demonstrated poor immunogenicity towards AE as compared to NAE encoded by their transmitted founder virus. Longitudinal data in AHI off antiretroviral therapy demonstrated sequence changes that were biologically confirmed to represent CD4+ escape mutations. These data demonstrate an innovative application of HLA-associated polymorphisms to identify biologically relevant CD4+ epitopes and suggests CD4+ T cells are active participants in driving HIV evolution.

Multiplexed highly-accurate DNA sequencing of closely-related HIV-1 variants using continuous long reads from single molecule, real-time sequencing.

Single Molecule, Real-Time (SMRT) Sequencing (Pacific Biosciences, Menlo Park, CA, USA) provides the longest continuous DNA sequencing reads currently available. However, the relatively high error rate in the raw read data requires novel analysis methods to deconvolute sequences derived from complex samples. Here, we present a workflow of novel computer algorithms able to reconstruct viral variant genomes present in mixtures with an accuracy of >QV50. This approach relies exclusively on Continuous Long Reads (CLR), which are the raw reads generated during SMRT Sequencing. We successfully implement this workflow for simultaneous sequencing of mixtures containing up to forty different >9 kb HIV-1 full genomes. This was achieved using a single SMRT Cell for each mixture and desktop computing power. This novel approach opens the possibility of solving complex sequencing tasks that currently lack a solution.

Host genetic factors associated with symptomatic primary HIV infection and disease progression among Argentinean seroconverters.

BACKGROUND: Variants in HIV-coreceptor C-C chemokine receptor type 5 (CCR5) and Human leukocyte antigen (HLA) genes are the most important host genetic factors associated with HIV infection and disease progression. Our aim was to analyze the association of these genetic factors in the presence of clinical symptoms during Primary HIV Infection (PHI) and disease progression within the first year. METHODS: Seventy subjects diagnosed during PHI were studied (55 symptomatic and 15 asymptomatic). Viral load (VL) and CD4 T-cell count were evaluated. HIV progression was defined by presence of B or C events and/or CD4 T-cell counts <350 cell/mm3. CCR5 haplotypes were characterized by polymerase chain reaction and SDM-PCR-RFLP. HLA-I characterization was performed by Sequencing. RESULTS: Symptoms during PHI were significantly associated with lower frequency of CCR5-CF1 (1.8% vs. 26.7%, p = 0.006). Rapid progression was significantly associated with higher frequency of CCR5-CF2 (16.7% vs. 0%, p = 0.024) and HLA-A*11 (16.7% vs. 1.2%, p = 0.003) and lower frequency of HLA-C*3 (2.8% vs. 17.5%, p = 0.035). Higher baseline VL was significantly associated with presence of HLA-A*11, HLA-A*24, and absence of HLA-A*31 and HLA-B*57. Higher 6-month VL was significantly associated with presence of CCR5-HHE, HLA-A*24, HLA-B*53, and absence of HLA-A*31 and CCR5-CF1. Lower baseline CD4 T-cell count was significantly associated with presence of HLA-A*24/*33, HLA-B*53, CCR5-CF2 and absence of HLA-A*01/*23 and CCR5-HHA. Lower 6-month CD4 T-cell count was associated with presence of HLA-A*24 and HLA-B*53, and absence of HLA-A*01 and HLA-B*07/*39. Moreover, lower 12-month CD4 T-cell count was significantly associated with presence of HLA-A*33, HLA-B*14, HLA-C*08, CCR5-CF2, and absence of HLA-B*07 and HLA-C*07. CONCLUSION: Several host factors were significantly associated with disease progression in PHI subjects. Most results agree with previous studies performed in other groups. However, some genetic factor associations are being described for the first time, highlighting the importance of genetic studies at a local level.

Increasing trends in primary NNRTI resistance among newly HIV-1-diagnosed individuals in Buenos Aires, Argentina.

OBJECTIVE: Our objective was to estimate primary resistance in an urban setting in a developing country characterized by high antiretroviral (ARV) coverage over the diagnosed population and also by an important proportion of undiagnosed individuals, in order to determine whether any change in primary resistance occurred in the past five years. DESIGN: We carried out a multi-site resistance surveillance study according to WHO HIV resistance guidelines, using a weighted sampling technique based on annual HIV case reports per site. METHODS: Blood samples were collected from 197 drug-naive HIV-1-infected individuals diagnosed between March 2010 and August 2011 at 20 HIV voluntary counselling and testing centres in Buenos Aires. Clinical records of enrolled patients at the time of diagnosis were compiled. Viral load and CD4 counts were performed on all samples. The pol gene was sequenced and the resistance profile determined. Phylogenetic analysis was performed by neighbour-joining (NJ) trees and bootscanning analysis. RESULTS: We found that 12 (7.9%) of the 152 successfully sequenced samples harboured primary resistance mutations, of which K103N and G190A were the most prevalent. Non-nucleoside reverse transcriptase inhibitors (NNRTI) resistance mutations were largely the most prevalent (5.9%), accounting for 75% of all primary resistance and exhibiting a significant increase (p=0.0072) in prevalence during the past 10 years as compared to our previous study performed in 1997-2000 and in 2003-2005. Nucleoside reverse transcriptase inhibitor (NRTI) and protease inhibitor primary resistance were low and similar to the one previously reported. CONCLUSIONS: Levels of primary NNRTI resistance in Buenos Aires appear to be increasing in the context of a sustained ARV coverage and a high proportion of undiagnosed HIV-positive individuals.

Estimation of HIV-testing rates to maximize early diagnosis-derived benefits at the individual and population level.

BACKGROUND: In HIV infection, initiation of treatment is associated with improved clinical outcom and reduced rate of sexual transmission. However, difficulty in detecting infection in early stages impairs those benefits. We determined the minimum testing rate that maximizes benefits derived from early diagnosis. METHODS: We developed a mathematical model of HIV infection, diagnosis and treatment that allows studying both diagnosed and undiagnosed populations, as well as determining the impact of modifying time to diagnosis and testing rates. The model's external consistency was assessed by estimating time to AIDS and death in absence of treatment as well as by estimating age-dependent mortality rates during treatment, and comparing them with data previously reported from CASCADE and DHCS cohorts. RESULTS: In our model, life expectancy of patients diagnosed before 8 years post infection is the same as HIV-negative population. After this time point, age at death is significantly dependent on diagnosis delay but initiation of treatment increases life expectancy to similar levels as HIV-negative population. Early mortality during HAART is dependent on treatment CD4 threshold until 6 years post infection and becomes dependent on diagnosis delay after 6 years post infection. By modifying testing rates, we estimate that an annual testing rate of 20% leads to diagnosis of 90% of infected individuals within the first 8.2 years of infection and that current testing rate in middle-high income settings stands close to 10%. In addition, many differences between low-income and middle-high incomes can be predicted by solely modifying the diagnosis delay. CONCLUSIONS: To increase testing rate of undiagnosed HIV population by two-fold in middle-high income settings will minimize early mortality during initiation of treatment and global mortality rate as well as maximize life expectancy. Our results highlight the impact of achieving early diagnosis and the importance of strongly work on improving HIV testing rates.

Drug resistance mutations in HIV pol sequences from Argentinean patients under antiretroviral treatment: subtype, gender, and age issues.

We studied drug resistance mutations (DRMs) in 2623 pol sequences. Out of 94,828 amino acid substitutions that were detected, 8749 corresponded to nucleoside reverse transcriptase inhibitor (NRTI), 3765 to nonnucleoside reverse transcriptase inhibitor (NNRTI), and 7141 to protease inhibitor (PI) resistance-associated mutations. The most common DRMs were L10I, I54V, L90M, V82A, A71V, L10V, M46I, M184V, M41L, T215Y, D67N, L210W, K70R, N348I, V118I, K103N, Y181C, G190A, K101E, V108I, L100I, V90I, K101Q, and A98G. As expected, DRMs frequencies depended on viral genotype. The amounts of NRTI and PI resistance mutations among B and BF sequences from children were higher than among sequences from adults. The frequencies of PI and NRTI resistance mutations among B and BF sequences from adult men were higher than among sequences from women. Some of these observations can be explained in light of the available epidemiological information, but some cannot, indicating that further studies are needed to understand the antiretroviral resistance epidemics in Argentina.