Luca Dori

Secondary Integrase Resistance Mutations Found in HIV-1 Minority Quasispecies in Integrase Therapy-Naive Patients Have Little or No Effect on Susceptibility to Integrase Inhibitors

ABSTRACT The goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate their in vitro effects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined.

HIV-1 dual/mixed tropic isolates show different genetic and phenotypic characteristics and response to maraviroc in vitro

Dual/mixed-tropic HIV-1 strains are predominant in a significative proportion of patients, though few information is available regarding the genetic characteristics, quasispecies composition, and susceptibility against CCR5-antagonists of the primary-isolates. For this reason, we investigated in deep details, both phenotypically and genotypically, the characteristics of 54 HIV-1 primary-isolates obtained from HIV-infected patients. Tropism was assessed by multiple-cycles phenotypic-assay on U87MG-CD4(+)-CCR5(+)-/CXCR4(+)-expressing cells. In vitro selection in PBMCs of X4-tropic viral strains following maraviroc-treatment was also performed. Phenotypic-assay reported pure R5-tropic viruses in 31 (57.4%) isolates, dual/mixed-tropic viruses in 22 (40.7%), and pure X4-tropic virus in only 1 (1.8%). Among dual/mixed-tropic isolates, 12 showed a remarkably higher replication-efficacy in CCR5-expressing cells (R5(+)/X4), and 2 in CXCR4-expressing cells (R5/X4(+)). Genotypic-tropism testing showed a correlation between PSSM-scores, geno2pheno false-positive-rate, and V3-net-charge with both CCR5-usage and syncytium-inducing ability. Moreover, specific gp120- and gp41-mutations were significantly associated with tropism and/or syncytium-inducing ability. Ultra-deep V3-pyrosequencing showed the presence of a swarm of genetically distinct species with a preference for CCR5-coreceptor not only in all pure R5-isolates, but also in 6/7 R5(+)/X4-tropic isolates. In both pure-X4 and R5/X4(+)-isolates, we observed extensive prevalence of X4-using species. In vitro selection-experiments with CCR5-inhibitor maraviroc (up to 2 months) showed no-emergence of X4-tropic variants for all R5- and R5(+)/X4-isolates tested (while X4-virus remained fully-resistant). In conclusion, our study shows that dual/mixed-tropic viruses are constituted by different species, whereby those with characteristics R5(+)/X4 are genotypically and phenotypically similar to the pure-R5 isolates; thus the use of CCR5-antagonists in patients with R5(+)/X4-tropic viruses may be a therapeutic-option that deserves further investigations.

Characterization of drug resistance mutations in naïve and ART-treated patients infected with HIV-1 in Yaounde, Cameroon.

Currently the prevalence of HIV‐1 infection in Cameroon is 5.1%, CRF02_AG subtype is responsible for about 50% of infections. Since an HIV‐1 drug resistance test is not yet available widely, accurate data on the prevalence of resistant viral strains are missing. The objective of this study was to determine HIV‐1 genetic diversity and to characterize HIV‐1 mutations conferring drug resistance among antiretroviral therapy (ART)‐naïve and ART‐treated patients. A cohort of 239 patients infected with HIV were followed‐up between January 2007 and July 2010 in Cameroon. Two hundred and sixteen plasma samples were sequenced for phylogenetic analysis and identification of drug resistance mutations in the HIV‐1 pol region. A significant genetic diversity was found: Seven pure subtypes (A1, A3, D, F1, F2, G, H), nine circulating recombinant forms (CRFs: 01_AE, 02_AG, 06cpx, 09cpx, 11cpx, 13cpx, 16cpx, 18cpx, 37cpx) and one new unique recombinant form (URF) (G/F2). The rate of transmitted drug resistance (TDR) in naïve patients was 8.2% (4/49). Around 80% of patients failing a first‐line ART harbored a virus with at least one resistance mutation to two antiretroviral (ARV) classes, and 36% of those failing a second‐line regimen carried a virus with at least one resistant mutation to three ARV classes. The high level of drug resistance observed in the cohort is alarming because this occurred as a result of only few years of treatment. Adherence to therapy, adequate education of physicians, and the appropriate use of genotypic resistance assay are critical points of intervention for the improvement of patient care. J. Med. Virol. 84:721–727, 2012.

The lowest X4 Geno2Pheno false-positive rate is associated with greater CD4 depletion in HIV-1 infected patients

Through this study we evaluated whether the HIV-1 tropism determined by genotypic analysis correlates with HIV-1 markers, such as CD4 cell count and plasma HIV-RNA. The analysis was performed on 1221 HIV-1 B-subtype infected patients with an available V3 sequence (all maraviroc naive). Of them, 532 were antiretroviral therapy (ART) naive and 689 ART experienced. Tropism determination was performed by using the geno2pheno (co-receptor) algorithm set at a false-positive rate (FPR) of 10% and 2%. Potential associations of FPR with CD4 cell count and viraemia were evaluated. Association of V3 mutations with genotypic-determined tropism was also evaluated according to different FPR ranges. About 26% of patients (either ART naive or ART experienced) were infected by X4-tropic viruses (using the classical 10% FPR cut-off). However, a significantly lower proportion of ART-naive patients had FPR ≤ 2% in comparison with ART-experienced patients (4.9% vs. 12.6%, respectively, p <0.001). The risk of advanced HIV-1 infection (with CD4 cell count ≤ 200 cells/mm(3)) was significantly greater in X4-infected patients, either ART-naive (OR (95% CI)), 4.2 (1.8-9.2); p 0.0006) or ART-experienced (2.3 (1.4-3.6); p 0.0003), with FPR set at 2% (but not at 10%). This finding was confirmed by multivariable logistic analysis. No relationship was found between viraemia and FPR ≤2%. Some X4-related mutations were significantly associated with FPR ≤2% (ART-naive patients, S11R, Y21V, G24K and G24R, p ≤0.001; ART-experienced patients, Y7K, S11R, H13Y, p ≤0.002). In conclusion, these findings show that within the context of genotypically-assessed CXCR4 tropism, FPR ≤2% defines (far better than 10%-FPR) a viral population associated with low CD4 rank, with potentially greater cytopathic effect, and with more advanced disease.

Procalcitonin is a reliable marker of severe systemic infection in neutropenic haematological patients with mucositis

Patients with neutropenia are exposed to a high risk for infections in which fever is often the unique symptom [1]. Systemic infections remain the main cause of mortality in these patients therefore, the policy for infection management is to promptly administer empirical antibiotic therapy in order to avoid the increased risk of mortality related to the treatment delay [2]. However, microbiological diagnostic tests are not sufficiently rapid, sensitive or specific to indentify the microbial causes of fever, and a considerable number of patients suffer febrile episodes over a prolonged period without a definite microbiological etiology.

Prognostic factors of long-term CD4+ count-guided interruption of antiretroviral treatment

Aim of the study was to determine predictors of the duration of antiretroviral treatment interruption in patients infected with HIV. This pilot prospective, open‐label, multicenter trial comprised 62 HIV‐seropositive subjects who decided voluntarily to interrupt therapy after two or more years of successful HAART. The primary end‐point was the time to patients being free of therapy before reaching a CD4+ cell count ≤350/µl. Fifteen of 62 patients remained in treatment interruption for more than 180 days. Patients restarting therapy had higher HIV‐DNA levels (P = 0.05), were treated more frequently with NNRTI‐drugs (P = 0.02), had a shorter period of HAART (P = 0.046), and lower CD4+ cell counts after day 14 of interruption of treatment (P = 0.04). Multivariate regression analysis showed that less than 323 baseline proviral HIV‐DNA cp/106 PBMCs and more than 564 CD4 cells/µl at day 14 after interruption were associated independently with a reduced risk of restarting treatment (P = 0.041 and P = 0.012, respectively). A score based on CD4+ cell counts at nadir, at baseline, at week 2 of treatment interruption, and on baseline HIV‐DNA values can identify patients with a prolonged period free safely of treatment. J. Med. Virol. 81:481–487, 2009.

KSHV DNA viremia correlates with low CD4+ cell count in Italian Males at the time of diagnosis of HIV infection

To evaluate the relevance and the virological and immunological markers of Kaposi sarcoma herpesvirus 8 (KSHV) viremia in Italian male patients at the time of diagnosis of infection with HIV‐1, 481 men infected with HIV were recruited consecutively. The presence of KSHV DNA was evaluated in peripheral blood mononuclear cells (PBMCs) and in plasma and correlated with demographic and viro‐immunological parameters. Seventy‐four patients had KSHV DNA detected in PBMCs. By univariate analysis, the presence of KSHV DNA was associated significantly with unprotected homosexual relationships (P = 0.003) and it was significantly higher in patients with CD4+ cell <350 (P = 0.025). By multivariate analysis, homosexual relationships were associated independently with KSHV DNA in PBMCs (OR: 3.25; 95% CI: 1.1–9.7; P = 0.035). Among the 74 patients with KSHV DNA detected in PBMCs, plasma samples from 60 were analyzed and 33 were positive for KSHV DNA. The CD4+ cell counts and percentages were significantly lower in patients with KSHV DNA in both PBMCs and plasma as compared to patients with only KSHV DNA in PBMCs (P = 0.006 and P = 0.019, respectively). Among the patients with KSHV DNA detected in PBMCs, all 13 patients with CD4+ cells count <200 had detectable levels of KSHV in their plasma. By multivariate analysis adjusted for the epidemiologic and virological parameters, low CD4+ cell count was the only independent variable associated with the presence of KSHV DNA in plasma (OR, 0.001; 95% CI: <0.001–0.001; P = 0.03). In HIV‐positive antiretroviral therapy‐naïve males, KSHV active replication as detected by KSHV DNA in plasma was associated significantly with low CD4+ cell count. J. Med. Virol. 83:384–390, 2011.

Switching of Inferred Tropism Caused by HIV during Interruption of Antiretroviral Therapy

ABSTRACT After interruption of highly active antiretroviral therapy, 15 out of 53 patients with the X4 HIV strain had a significantly larger decrease in CD4+ T cell count (P = 0.001) and shorter length of treatment interruption (P = 0.02) than patients with the R5 strain. At treatment resumption, HIV inferred tropism switched from the X4 strain to the R5 variant in 9 patients (60%). These patients had a prolonged length of treatment interruption compared to that of those who still carried the X4 strain.

Impact of HIV-1 tropism on the emergence of non-AIDS events in HIV-infected patients receiving fully suppressive antiretroviral therapy

Objective:The impact of HIV-1 tropism on the emergence of non-AIDS events was evaluated in a cohort of 116 antiretroviral therapy (ART) responder patients. Methods:The patients were followed for the emergence of hypertension, renal impairment, metabolic and bone disorders (defined as non-AIDS events) each 8 weeks at standard visits. A V3 plasma sequence genotype analysis was performed at the time of ART initiation and the geno2pheno algorithm with the results that defines the false-positive rate (FPR) was used to infer HIV tropism. The associations between the non-AIDS events and the FPR at baseline were evaluated using the &khgr;2 test for trend. A Cox-regression analysis using the counting process formulation of Andersen and Gill was performed to define whether the emergence of non-AIDS events was correlated to FPR. Results:The prevalence of at least one non-AIDS event resulted higher in patients with a FPR below 10% than in patients with a R5 virus (P = 0.033). Patients with a FPR below 5.0% most frequently developed non-AIDS events during ART (P = 0.01). A higher prevalence of patients with at least two AIDS events was found in the group of patients with a FPR below 5.0% with respect to the others (P < 0.001). At multivariate Cox-regression analysis, having an X4 virus and age were independently associated with a higher probability of non-AIDS event development. Conclusion:This study shows that an X4 virus, particularly a FPR less than 5%, is related to non-AIDS events development. Further studies are warranted to understand the mechanisms underlying this phenomenon.