Contribution of transgender sex workers to the complexity of the HIV-1 epidemic in the metropolitan area of Milan

Objectives: Transgender people are disproportionately affected by the HIV-1 epidemic. We evaluated the origin of HIV-1 variants carried by South American transgenders living in Milan by combining accurate phylogenetic methods and epidemiological data. Methods: We collected 156 HIV-1 pol sequences obtained from transgender patients engaged in sex work (TSWs) followed between 1999 and 2015 at L. Sacco Hospital, Milan, Italy. Phylogenetic analyses were conducted by HIV-TRACE, MrBayes, MacClade and Beast programs. Reference sequences were retrieved from Los Alamos and local databases. Last negative testing or proxy data from clinical records of infected individuals were used to investigate the country of infection. Results: Among South American TSWs, the most represented HIV-1 subtypes were B (70.5%), F1 (12.8%) and C (4.4%). Gene flow migrations of B subtype indicated significant fluxes from TSWs to Italians (21.3%) belonging to all risk groups (26.4% to heterosexuals (HEs), 18.9% to men who have sex with men (MSM), 15.1% to injecting drug users). The largest proportion of bidirectional fluxes were observed between Italians and TSWs (24.6%). For F1 subtype, bidirectional viral fluxes involved TSWs and Italians (7.1% and 14.3%), and a similar proportion of fluxes linked TSWs and Italian HEs or MSM (both 15.8%). Significant fluxes were detected from Italians to TSWs for subtype C involving both MSM (30%) and HEs (40%). Country of HIV-1 acquisition was identified for 72 subjects; overall, the largest proportion of patients with B subtype (73.5%) acquired HIV-1 infection in South America. Conclusions: Our results indicated that South American transgenders largely contribute to the heterogeneity of HIV-1 variants in our country. The high number of clusters based on all subtypes indicated numerous transmission chains in which TSWs were constantly intermixed with HEs and MSM. Our results strongly advocate interventions to facilitate prevention, diagnosis and HIV-1 care continuum among transgender people

Changing patterns in HIV-1 non-B clade prevalence and diversity in Italy over three decades

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Local epidemics gone viral: Evolution and diffusion of the Italian HIV-1 recombinant form CRF60_BC

The molecular epidemiology of HIV-1 in Italy is becoming increasingly complex, mainly due to the spread of non-B subtypes and the emergence of new recombinant forms. We previously characterized the outbreak of the first Italian circulating recombinant form (CRF60_BC), occurring among young MSM living in Apulia between the years 2009 and 2011. Here we show a 5-year follow-up surveillance to trace the evolution of CRF60_BC and to investigate its further spread in Italy. We collected additional sequences and clinical data from patients harboring CRF60_BC, enrolled at the Infectious Diseases Clinic of the University of Bari. In addition to the 24 previously identified sequences, we retrieved 27 CRF60_BC sequences from patients residing in Apulia, whose epidemiological and clinical features did not differ from those of the initial outbreak, i.e., the Italian origin, young age at HIV diagnosis (median: 24 years; range: 18-37), MSM risk factor (23/25, 92%) and recent infection (from 2008 to 2017). Sequence analysis revealed a growing overall nucleotide diversity, with few nucleotide changes that were fixed over time. Twenty-seven additional sequences were detected across Italy, spanning multiple distant regions. Using a BLAST search, we also identified a CRF60_BC sequence isolated in United Kingdom in 2013. Three patients harbored a unique second generation recombinant form in which CRF60_BC was one of the parental strains. Our data show that CRF60_BC gained epidemic importance, spreading among young MSM in multiple Italian regions and increasing its population size in few years, as the number of sequences identified so far has triplicated since our first report. The observed further divergence of CRF60_BC is likely due to evolutionary bottlenecks and host adaptation during transmission chains. Of note, we detected three second-generation recombinants, further supporting a widespread circulation of CRF60_BC and the increasing complexity of the HIV-1 epidemic in Italy

Trends and correlates of HIV-1 resistance among subjects failing an antiretroviral treatment over the 2003-2012 decade in Italy

BACKGROUND: Despite a substantial reduction in virological failures following introduction of new potent antiretroviral therapies in the latest years, drug resistance remains a limitation for the control of HIV-1 infection. We evaluated trends and correlates of resistance in treatment failing patients in a comprehensive database over a time period of relevant changes in prescription attitudes and treatment guidelines. METHODS: We analyzed 6,796 HIV-1 pol sequences from 49 centres stored in the Italian ARCA database during the 2003-2012 period. Patients (n = 5,246) with viremia > 200 copies/mL received a genotypic test while on treatment. Mutations were identified from IAS-USA 2013 tables. Class resistance was evaluated according to antiretroviral regimens in use at failure. Time trends and correlates of resistance were analyzed by Cochran-Armitage test and logistic regression models. RESULTS: The use of NRTI backbone regimens slightly decreased from 99.7% in 2003-2004 to 97.4% in 2010-2012. NNRTI-based combinations dropped from 46.7% to 24.1%. PI-containing regimens rose from 56.6% to 81.7%, with an increase of boosted PI from 36.5% to 68.9% overtime. In the same reference periods, Resistance to NRTIs, NNRTIs and PIs declined from 79.1% to 40.8%, from 77.8% to 53.8% and from 59.8% to 18.9%, respectively (p < .0001 for all comparisons). Dual NRTI +\u2009NNRTI and NRTI + PI resistance decreased from 56.4% to 33.3% and from 36.1% to 10.5%, respectively. Reduced risk of resistance over time periods was confirmed by a multivariate analysis. CONCLUSIONS: Mutations associated with NRTIs, NNRTIs and PIs at treatment failure declined overtime regardless of specific class combinations and epidemiological characteristics of treated population. This is likely due to the improvement of HIV treatment, including both last generation drug combinations and prescription guidelines

Impact of social determinants on antiretroviral therapy access and outcomes entering the era of universal treatment for people living with HIV in Italy

Background: Social determinants are known to be a driving force of health inequalities, even in high income countries. Aim of our study was to determine if these factors can limit antiretroviral therapy (ART) access, outcome and retention in care of people living with HIV (PLHIV) in Italy. Methods: All ART naïve HIV+ patients (pts) of Italian nationality enrolled in the ICONA Cohort from 2002 to 2016 were included. The association of socio-demographic characteristics (age, sex, risk factor for HIV infection, educational level, occupational status and residency area) with time to: ART initiation (from the first positive anti-HIV test), ART regimen discontinuation, and first HIV-RNA &lt; 50 cp/mL, were evaluated by Cox regression analysis, Kaplan Meier method and log-rank test. Results: A total of 8023 HIV+ pts (82% males, median age at first pos anti-HIV test 36 years, IQR: 29-44) were included: 6214 (77.5%) started ART during the study period. Women, people who inject drugs (PWID) and residents in Southern Italy presented the lowest levels of education and the highest rate of unemployment compared to other groups. Females, pts aged &gt; 50 yrs., unemployed vs employed, and people with lower educational levels presented the lowest CD4 count at ART initiation compared to other groups. The overall median time to ART initiation was 0.6 years (yrs) (IQR 0.1-3.7), with a significant decrease over time [2002-2006 = 3.3 yrs. (0.2-9.4); 2007-2011 = 1.0 yrs. (0.1-3.9); 2012-2016 = 0.2 yrs. (0.1-2.1), p &lt; 0.001]. By multivariate analysis, females (p &lt; 0.01) and PWID (p &lt; 0.001), presented a longer time to ART initiation, while older people (p &lt; 0.001), people with higher educational levels (p &lt; 0.001), unemployed (p = 0.02) and students (p &lt; 0.001) were more likely to initiate ART. Moreover, PWID, unemployed vs stable employed, and pts. with lower educational levels showed a lower 1-year probability of achieving HIV-RNA suppression, while females, older patients, men who have sex with men (MSM), unemployed had higher 1-year risk of first-line ART discontinuation. Conclusions: Despite median time to ART start decreased from 2002 to 2016, socio-demographic factors still contribute to disparities in ART initiation, outcome and durability

Molecular and phylogenetic analysis of HIV-1 variants circulating in Italy

<p>Abstract</p> <p>Objective</p> <p>The continuous identification of HIV-1 non-B subtypes and recombinant forms in Italy indicates the need of constant molecular epidemiology survey of genetic forms circulating and transmitted in the resident population.</p> <p>Methods</p> <p>The distribution of HIV-1 subtypes has been evaluated in 25 seropositive individuals residing in Italy, most of whom were infected through a sexual route during the 1995–2005 period. Each sample has been characterized by detailed molecular and phylogenetic analyses.</p> <p>Results</p> <p>18 of the 25 samples were positive at HIV-1 PCR amplification. Three samples showed a nucleotide divergence compatible with a non-B subtype classification. The phylogenetic analysis, performed on both HIV-1 <it>env </it>and <it>gag </it>regions, confirms the molecular sub-typing prediction, given that 1 sample falls into the C subtype and 2 into the G subtype. The B subtype isolates show high levels of <it>intra</it>-subtype nucleotide divergence, compatible with a long-lasting epidemic and a progressive HIV-1 molecular diversification.</p> <p>Conclusion</p> <p>The Italian HIV-1 epidemic is still mostly attributable to the B subtype, regardless the transmission route, which shows an increasing nucleotide heterogeneity. Heterosexual transmission and the interracial blending, however, are slowly introducing novel HIV-1 subtypes. Therefore, a molecular monitoring is needed to follow the constant evolution of the HIV-1 epidemic.</p

Recombination analysis and structure prediction show correlation between breakpoint clusters and RNA hairpins in the pol gene of human immunodeficiency virus type 1 unique recombinant forms

Recombination is recognized as a primary force in human immunodeficiency virus type 1 (HIV-1) evolution, increasing viral diversity through reshuffling of genomic portions. The strand-switching activity of reverse transcriptase is required to complete HIV-1 replication and can occur randomly throughout the genome, leading to viral recombination. Some recombination hotspots have been identified and found to correlate with RNA structure or sequence features. The aim of this study was to evaluate the presence of recombination hotspots in the pol gene of HIV-1 and to assess their correlation with the underlying RNA structure. Analysis of the recombination pattern and breakpoint distribution in a group of unique recombinant forms (URFs) detected two recombination hotspots in the pol region. Two stable and conserved hairpins were consistently predicted corresponding to the identified hotspots using six different RNA-folding algorithms on the URF parental strains. These findings suggest that such hairpins may play a role in the higher recombination rates detected at these positions

A21 HIV-1 sub-subtype F1 outbreak among MSM in Belgium

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Cellular HIV-1 DNA Levels in Drug Sensitive Strains Are Equivalent to Those in Drug Resistant Strains in Newly-Diagnosed Patients in Europe

Background HIV-1 genotypic drug resistance is an important threat to the success of antiretroviral therapy and transmitted resistance has reached 9% prevalence in Europe. Studies have demonstrated that HIV-1 DNA load in peripheral blood mononuclear cells (PBMC) have a predictive value for disease progression, independently of CD4 counts and plasma viral load. Methodology/Principal Findings Molecular-beacon-based real-time PCR was used to measure HIV-1 second template switch (STS) DNA in PBMC in newly-diagnosed HIV-1 patients across Europe. These patients were representative for the HIV-1 epidemic in the participating countries and were carrying either drug-resistant or sensitive viral strains. The assay design was improved from a previous version to specifically detect M-group HIV-1 and human CCR5 alleles. The findings resulted in a median of 3.32 log10HIV-1copies/106PBMC and demonstrated for the first time no correlation between cellular HIV-1 DNA load and transmitted drug-resistance. A weak association between cellular HIV-1 DNA levels with plasma viral RNA load and CD4+T-cell counts was also reconfirmed. Co-receptor tropism for 91% of samples, whether or not they conferred resistance, was CCR5. A comparison of pol sequences derived from RNA and DNA, resulted in a high similarity between the two. Conclusions/Significance An improved molecular-beacon-based real-time PCR assay is reported for the measurement of HIV-1 DNA in PBMC and has investigated the association between cellular HIV-1 DNA levels and transmitted resistance to antiretroviral therapy in newly-diagnosed patients from across Europe. The findings show no correlation between these two parameters, suggesting that transmitted resistance does not impact disease progression in HIV-1 infected individuals. The CCR5 co-receptor tropism predominance implies that both resistant and non-resistant strains behave similarly in early infection. Furth

Prevalence of transmitted nucleoside analogue-resistant HIV-1 strains and pre-existing mutations in pol reverse transcriptase and protease region : outcome after treatment in recently infected individuals

We retrospectively studied 38 Italian recently HIV-1-infected subjects who seroconverted from 1994 to 1997 to investigate: (i) the prevalence of nucleoside reverse transcriptase inhibitors (NRTI)-related mutations at primary infection; (ii) the proportion of naturally occurring mutations in reverse transcriptase (RT) and protease regions of patients naive for non-nucleoside RT inhibitors (NNRTIs) and protease inhibitors (PIs); (iii) the drug-susceptibility to NRTIs and PIs in subjects with NRTI- and/or PI-related mutations; and (iv) the outcome of seroconverters treated with various NRTIs or NRTI/PI regimens. Baseline HIV-1 plasma viraemia and absolute CD4 count at baseline could not be used to distinguish patients with NRTI- and/or PI-related pre-existing mutations from those with wild-type virus (P = 0.693 and P = 0.542, respectively). The frequency of zidovudine-related mutations was 21% in the study period. The response to treatment was not significantly different in subjects with or without genotypic zidovudine-related mutations at primary infection (P = 0.744 for HIV-1 RNA and P = 0.102 for CD4 cells). Some natural variation (2.6%) was present within regions 98-108 and 179-190 of RT involved in NNRTI resistance. The high natural polymorphism in the protease region present in our patients was similar to that reported by others. In our study some PI-associated substitutions, thought to be compensatory in protease enzymatic function, could confer intermediate to high PI-resistance. As discrepancies between genotypic and phenotypic results may exist in recent seroconverters, our data suggest that the role of transmitted NRTI- and PI-resistant variants remain to be fully elucidated in vivo