A total of 347 pol gene sequences from 88 Tuscan and 259 Apulian subjects (including 52 non-Italians and 9 children) were analyzed phylogenetically. Forty-four (12.6%) non-B subtypes were found, including 3.4% C, 1.4% F1, 0.8% G, and 0.3% each for J and A pure subtypes, and 3.7% CRF02_AG, 1.4% CRF01_AE, 0.6% BF, and 0.3% CRF06-cpx recombinant forms. An additional sample close-matched the pol gene of an unique recombinant form (URF AGK 99GR303). The non-B subtypes were from 40 adults and 4 children; 12 of these 44 patients were epidemiologically linked. Thirty-three of the 44 non-B viruses pertained to non-Italian immigrants and 11 to Italians, signifying that 63.4% immigrants and 3.7% Italians harbored non-B subtypes. The overall frequency of non-B subtypes was higher in Tuscany than in Apulia (18.1% vs. 10.8%). Moreover, 6.1% and 3.0% non-B subtypes were found among Italians from Florence and Apulia, respectively, while 52.1% and 72.4% of immigrants living in Tuscany and Apulia harbored non-B subtypes. Women infected by means of sexual contact prevailed among non-Italian adults; the majority of Italians were males and admitted high-risk sexual behavior. Four Italians had a history of extensive travel in countries of high endemicity. Social and epidemiological changes are responsible for an increasing circulation of non-B subtypes in Italy. Although non-B subtypes principally infect non-Italian patients, in Italy they can no longer be considered exclusively restricted to subjects from endemic areas.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 The influence of antiretroviral therapy on co-receptor tropism remains controversial. To 25 verify if co-receptor tropism shift was affected by HAART, the evolution of proviral DNA V3 26 genotype after 12 months of a new antiretroviral regimen was compared between 27 responder and non-responder patients. 28Baseline blood samples were collected from 36 patients infected with HIV-1 subtype-B (18 29 naïve and 18 experienced) for virus isolation and env V3 genotyping from plasma HIV-1 30 RNA and PBMC DNA. DNA V3 genotyping was repeated after 12 months from initiating 31 HAART. WebPSSM was used for categorizing V3 sequences into X4 or R5; for analysis 32 purposes, dual/mixed viruses were considered as X4. 33From the 10 (28%) patients changing their proviral DNA V3 genotype during therapy, six 34shifted from R5-to-X4 and four from X4-to-R5. The lack of reaching virological suppression 35 was not associated with an X4-to-R5 (p=0.25) or R5-to-X4 (p=0.14) shift; time-to-viral 36 suppression and CD4 increase were similar in both groups. No association was found 37 between tropism shift and patient baseline characteristics including age, sex, CDC stage, 38 CD4 count, viral load, exposure and length of previous HAART, enfuvirtide use in the new 39 regimen, number of reverse-transcriptase and protease resistance-associated mutations. 40Conversely, CD4 nadir was correlated to emergence of X4 virus in proviral DNA (mean 41 27.2±30.6 in R5-to-X4 shifting patients vs 161.6±150.6 in non-shifting patients, p=0.02). 42The occurrence of a tropism shift in both directions was independent of HAART use, 43 irrespective of its efficacy. The CD4 count nadir was the only baseline characteristic able 44 to predict an R5-to-X4 viral shift. 45

HIV coreceptor tropism (CTR) testing is a prerequisite for prescribing a coreceptor antagonist. CTR is increasingly deduced by analyzing the V3 loop sequence of gp120. We investigated the impact of mutations outside V3 on CTR as determined by the enhanced-sensitivity Trofile assay (ESTA). Paired ESTA and gp120 sequencing (population sequencing; from codon 32 of the conserved C1 to the variable V5 domains) were obtained from 60 antiretroviral treatment (ART)-naïve patients (15 with AIDS) infected with subtype B HIV-1. For gp120 sequence analysis, nucleotide mixtures were considered when the second highest electropherogram peak was >25%; sequences were translated into all possible permutations and classified as X4, dual/mixed (DM), and R5 based on coincident ESTA results. ESTA identified R5 and DM viruses in 72 and 28% of patients, respectively; no pure X4 was labeled. Forty percent of AIDS patients had R5 strains. Thirty-two positions, mostly outside V3, were significantly (P < 0.05) different between R5 and DM sequences. According to multivariate analysis, amino acid changes at 9 and 7 positions within the C1 to C4 and V1 to V5 regions, respectively, maintained a statistical significance, as did the net charge of V3 and C4. When analyzing only R5 sequences, 6 positions in the variable regions were found which, along with the V4 net charge, were significantly different for sequences from early-and end-stage disease patients. This study identifies specific amino acid changes outside V3 which contribute to CTR. Extending the analysis to include pure X4 and increasing the sample size would be desirable to define gp120 variables/changes which should be included in predictive algorithms.Based on coreceptor usage, HIV-1 isolates currently are classified as R5 tropic (using only the CCR5 receptor to enter cells), X4 tropic (using only CXCR4), and dual/mixed (DM) (able to use both coreceptor types). Maraviroc (MVC), the only currently licensed CCR5 antagonist, is ineffective in patients harboring non-R5 strains (28), therefore its prescription requires that the presence of R5-tropic virus is ascertained.Until recently, the only recommended method for tropism determination was by phenotypic assay (Trofile; Monogram Biosciences), which has been extensively used to provide tropism information in MVC clinical trials (6, 11). Trofile is a recombinant virus assay in which a pseudovirus is generated from the full-length env gene amplified from the patient's virus population and subsequently used to infect U87 cell lines expressing either the CXCR4 or CCR5 receptor on their surfaces. A new version of the test (enhanced-sensitivity Trofile assay [ESTA]) with a 0.3% sensitivity for X4 variants (24) was made available in 2008; however, the Trofile assay is not suitable for routine patient care. In fact, the assay is expensive and labor-intensive and is performed in only a single reference laboratory in the United States. Genotypic methods represent a more feasible alternative due to their greater accessibility, lower cost, and fast...

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Objectives Fostemsavir, a novel attachment inhibitor targeting the HIV-1 gp120, has demonstrated wide in vitro activity. However, the high rate of HIV gp120 substitutions could jeopardize its efficacy. We investigated envelope (env) substitutions at positions associated with resistance to fostemsavir in patients with a new HIV-1 diagnosis according to HIV subtype and tropism. Methods Gp120 sequences from 409 subjects were retrospectively analysed and the presence of the L116P, A204D, S375H/M/T, M426L, M434I and M475I mutations was evaluated. Other amino acid changes at the same positions were also recorded. The variability at each amino acid position was evaluated using Shannon entropy. Results The frequency of mutations was: S375T (13.2%); M426L (6.8%); M434I (2.9%); M475I (2.7%); S375H (1.0%)/M (0.8%) and L116P (0.31%). Statistically significant differences were found at positions 375 (R5/non-R5 strains and B/non-B subtypes) and 426 (B/non-B subtypes); post hoc analysis revealed that significance for position 375 was steered by S375T while for position 426 significance was governed by unusual substitutions, in particular M426R (B/non-B, P &lt; 0.00001). The variability of env constant domains appeared to be more relevant in the non-B virus population. Conclusions In conclusion, gp120 substitutions were detected in different subtypes and in both R5 and non-R5 variants. Despite the great variability of gp120, the frequency of mutations was low overall and the predominant substitution was S375T, the role of which in reducing fostemsavir efficacy is less substantial.