Chiara Riva

Transmission of Drug-Resistant HIV-1 Is Stabilizing in Europe

The SPREAD Programme investigated prospectively the time trend from September 2002 through December 2005 of transmitted drug resistance (TDR) among 2793 patients in 20 European countries and in Israel with newly diagnosed human immunodeficiency virus type 1 (HIV-1) infection. The overall prevalence of TDR was 8.4% (225 of 2687 patients; 95% confidence interval [CI], 7.4%-9.5%), the prevalence of nucleoside reverse-transcriptase inhibitor (NRTI) resistance was 4.7% (125 of 2687 patients; 95% CI, 3.9%-5.5%), the prevalence of nonucleoside reverse-transcriptase inhibitor (NNRTI) resistance was 2.3% (62 of 2687 patients; 95% CI, 1.8%-2.9%), and the prevalence of protease inhibitor (PI) resistance was 2.9% (79 of 2687 patients; 95% CI, 2.4%-3.6%). There was no time trend in the overall TDR or in NRTI resistance, but there was a statistically significant decrease in PI resistance (P = .04) and in NNRTI resistance after an initial increase (P = .02). We found that TDR appears to be stabilizing in Europe, consistent with recent reports of decreasing drug resistance and improved viral suppression in patients treated for HIV-1 infection.

increasing Prevalence of Non-clade B Hiv-1 Strains in Heterosexual Men and Women, as Monitored by Analysis of Reverse Transcriptase and Protease Sequences

Objective: We evaluated the prevalence of HIV‐1 non‐clade B over time in a formerly clade B‐restricted area. Protease and reverse transcriptase regions of the pol gene were used for phylogenetic and recombination analysis and for clade assignment to HIV‐1 A‐D, F‐H, J, and K strains of the M group. Methods: The pol gene of 349 HIV‐1 patients belonging to the Italian Cohort Naive for Antiretrovirals (ICONA) were genotypically analyzed to study the prevalence of antiretroviral‐associated resistance mutations. All HIV‐1 pol sequences and 32 HIV reference strains were analyzed, including the reference strains for the major HIV‐1 subtypes. The non‐clade B sequences according to the HIV‐1 Subtyping Tool program were further studied by a bootscan analysis (SimPlot) to investigate the likelihood of recombination between subtypes. Results: Phylogenetic analysis detected 19 of 349 (5.4%) non‐clade B subtypes. The proportions of patients carrying non‐clade B virus before and after 1997 were 1.9% and 8.4%, respectively (p = .008). Among whites, heterosexual infection and female gender were significantly associated with the presence of non‐clade B subtypes (p = .001 and .005, respectively). Non‐clade B HIV‐1 was harbored by 14.5% of the heterosexuals who were found to be HIV‐1 positive after 1997, 60% of whom were women. Bootscan analysis identified four strains as F, two as A, one as C, one as G, and 11 (57.9 %) as non‐clade B recombinant subtypes. Conclusion: Detection of HIV‐1 subtypes and intersubtype recombinants in a previously clade B‐homogeneous area indicates that the HIV‐1 epidemic is evolving in Italy and that heterosexuals and women are at increased risk of infection with nonclade B HIV‐1 subtypes. Sequences inferred from the pol gene yield to establish the subtype of circulating HIV‐1 strains. As a consequence, genotyping of pol gene for testing resistance to antiretrovirals warrants concomitant surveillance of non‐clade B subtypes.

Immunologic characterization of children vertically infected with human immunodeficiency virus, with slow or rapid disease progression

Cytokine production of unstimulated and mitogen-stimulated peripheral blood mononuclear cells of 31 children vertically infected with human immunodeficiency virus type 1 (HIV) and with different patterns of disease progression was evaluated to establish possible correlations between the immunologic and the clinical findings. Production of interferon gamma and interleukin-2 (type 1 cytokines), and of interleukin-4 and interleukin-10 (type 2 cytokines), was analyzed in seven symptom-free patients (Centers for Disease Control and Prevention class P-1B), 10 patients with mild symptoms (class P-2A), and 14 patients with severe symptoms (class P-2B-F). Cytokine production was compared with that of 10 age- and sex-matched control subjects who were seronegative for HIV. The HIV-infected patients produced significantly fewer type 1 cytokines and significantly more type 2 cytokines than the uninfected control subjects. No differences in the production of interferon gamma and interleukin-2 were detected among the different clinical categories of HIV-infected patients. In contrast, interleukin-4 production was augmented in the patients with class P-2A (p < 0.05) and class P-2B-F HIV infection (p < 0.03), in comparison with the children with class P-1B infection. The increase in interleukin-4 production was paralleled by an increase in the number of children with hyperimmunoglobulinemia E in each of the clinical groups (0% in class P-1B; 40% in class P-2A; and 71% in class P-2 B-F infection). Similarly, interleukin-10 production was increased both in patients with class P-2A and in those with class P-2B-F infection, in comparison with the children with class P-1B disease (p < 0.006 and < 0.04, respectively). These data indicate (1) that vertically acquired HIV infection results in decreased production of type 1 cytokines and in increased production of type 2 cytokines, and (2) that an increased production of type 2 cytokines correlates with hyperimmunoglobulinemia E and is present in, and may be characteristic of, the symptomatic phases of childhood HIV infection.

Vpr and HIV-1 disease progression: R77Q mutation is associated with long-term control of HIV-1 infection in different groups of patients.

Background:Vpr (viral protein R) is a 96 amino acids soluble protein that is expressed late during viral replication. Recent studies have focused on the role of a mutation at position 77 that might be associated with the condition of long-term non-progression, but data are still controversial. Patients and methods:Fifteen long-term non-progressors (LTNP), 19 therapy-naive HIV-1-infected patients with progressive disease (Pr), 23 HIV-1-infected patients receiving sub-optimal therapy with dual nucleoside [nucleoside reverse transcriptase inhibitor (NRTI)] therapy but efficiently controlling viral replication (STP) and 19 antiretroviral therapy multi-experienced patients with actively replicating virus (MEP) were analysed. HIV-RNA was extracted from plasma samples, the Vpr region was amplified, cloned and sequenced. The Pol gene was amplified, directly sequenced and analysed using Sequence Navigator software. Results:A significantly higher prevalence of the R77Q mutation was evidenced both in LTNP (86.7%) and STP (73.9%) in comparison with Pr (42.1%) and MEP (42.1%), (P = 0.007). Comparing groups of patients with progressive disease (Pr + MEP) and groups with non-progressive disease (LTNP + STP) the probability of harbouring the R77Q mutation was significantly higher in non-progressors (odds ratio, 5.16; P = 0.001). Conclusions:Our results support the hypothesis of the association of R77Q mutation in the Vpr gene with delayed progression of HIV-1 disease. R77Q does not seem to be linked to a particular viral strain but might be associated to immunologic selection. The R77Q mutation might reduce CD4+ T-cell depletion possibly affecting T-cell survival in vivo by altering the pro-apoptotic activity of Vpr.

Evidence for type 2 cytokine production and lymphocyte activation in the early phases of HIV-1 infection

ObjectiveTo analyse changes in cytokine production in vitro and T-Iymphocyte immunophenotype in the early phases of HIV-1 infection. Design and methodsMitogen-stimulated in vitro production of interferon (IFN)-γ, interleukin (IL)-2 (type 1 cytokines), IL-4, and IL-10 (type 2 cytokines) and surface expression of activation and non-activation markers were evaluated in 11 individuals HIV-infected for >3 but <12 months (seroconverters). The data were compared to those obtained in 33 asymptomatic HIV-positive individuals infected >3 years previously and who were stratified according to CD4+ lymphocyte count (group 1: >500 × 106/l, group 2: <500 × 106/l CD4 cells) and in 12 HIV-seronegative healthy controls. ResultsWe observed that the early phase of HIV infection is characterized by (1) reduced mitogen-stimulated IL-2 and IFN-γ production, (2) increased mitogen-stimulated IL-4 and IL-10 production, (3) a relative decrease in CD4+ and CD4+CD7− as well as an increase in CD4+CD7-CD57+ Iymphocytes, and (4) a relative increase in CD8+, CD8+CD38+ and CD8+CD57+ T lymphocytes. In addition, during a 6-month follow-up of six seroconverters we observed a dynamic pattern of changes of these parameters in most individuals, with a resulting profile similar to that observed in group 1 HIV-positive patients. ConclusionThe early phase of HIV infection is immunologically characterized by type 2 cytokine secretion and alterations in the expression of phenotypic markers, and closely resembles the more advanced phases of HIV infection. These immunologic alterations are temporally limited by the successive return to a more normal profile. Thus, HIV infection is an immunological complex dynamic process even in its earliest phases.

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

HIV‐1 non‐B subtypes have recently entered Western Europe following immigration from other regions. The distribution of non‐B clades and their association with demographic factors, over the entire course of the HIV‐1 epidemic, have not been fully investigated in Italy.

Evaluation of two commercial kits for the detection of genotypic drug resistance on a panel of HIV type 1 subtypes A through J.

We compared the two commercially available sequencing kits for HIV-1 drug resistance testing, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA, U.S.A.) and the TRUGENE HIV-1 Genotyping Kit (Visible Genetics, Inc., Toronto, Ontario, Canada), with our in-house genotyping system. Fifteen viral isolates from African patients (6 treated and 9 untreated) covering a panel of HIV-1 subtypes A through J and 7 plasma samples from Belgian and African patients (2 treated and 5 untreated) were tested. All the samples could be amplified and sequenced by the three systems; however, for all systems, alternative amplification/sequencing primers had to be used for some samples belonging to subtype B as well as to other subtypes. The consensus sequence was partially derived from only one strand for the in-house system and for the ViroSeq Genotyping System. The TRUGENE HIV-1 Genotyping Kit scored the highest number of ambiguities, followed by the ViroSeq Genotyping System and the in-house system. For 11 samples, these differences in reporting mixtures affected 14 resistance-related positions, which altered the interpretation toward protease inhibitors for 2 samples when using version 1.2 RetroGram software (Virology Networks, Utrecht, The Netherlands). All three systems were able to sequence diluted samples with a viral load down to 10 3 or 10 4 RNA copies/ml. Our data therefore suggest that the performance of amplification and sequencing primers must be improved to allow fast and reliable resistance testing for all HIV-1 subtypes.

Prevalence of multiple dideoxynucleoside analogue resistance (MddNR) in a multicenter cohort of HIV-1-infected Italian patients with virologic failure.

We evaluated the prevalence of both Q151M and 6-bp insert at position 69 of RT region responsible for multiple dideoxynucleoside analogue-resistant (MddNR) HIV-1 variants in 177 patients who failed to respond to combination therapy. Patients had received protease inhibitors (PI) and/or nonnucleoside reverse transcriptase inhibitors (NNRTIs) after a long-term experience with nucleoside reverse transcriptase inhibitors (NRTIs) (including zidovudine monotherapy). Two of 177 patients (1.1%) showed the specific complex of Q151M mutation, while 4 (2.3%) had the 69 6-bp insert. Mutations that belong to the 151 set in the absence of the pivotal Q151M substitution were detected in as many as 3.9% of the patients. One patient exhibited a 69S [VG] insert that has not been previously phenotypically characterized. This HIV-1 isolate had high levels of resistance to all NRTIs except stavudine. MddNR is an emerging problem after sequential therapy with this class of compounds among HIV-1-infected patients. Either didanosine (ddI) or zidovudine (ZDV) monotherapy allowed the emergence of MddNR variants containing Q151M complex. Monotherapy with ZDV and ddI or subsequent treatments with various NRTI combinations were the common background in the patients with the 69 insert. The overall prevalence of MddNR (3.4%) in Italy is comparable with that observed in several other European countries (3.4%-6.5%). These data suggest that patients failed by NRTI regimens should be analyzed for the presence of both patterns of MddNR.

Association between serum values of C-reactive protein and cytokine production in whole blood of patients with Type 2 diabetes

Diabetes mellitus accelerates atherosclerotic processes, and it is known that inflammation plays a key role in atherosclerosis. The aim of the present study was to evaluate in patients with Type 2 diabetes whether serum levels of CRP (C-reactive protein) are associated with cytokine production in whole blood. A total of 89 outpatients with Type 2 diabetes were enrolled, and blood pressure, body mass index, fasting blood glucose, glycated haemoglobin, cholesterol, triacylglycerols (triglycerides) and hs-CRP (high-sensitivity CRP) were measured. IL-6 (interleukin-6), IL-1beta (interleukin-1beta) and TNF-alpha (tumour necrosis factor-alpha) were measured before and after 24 h of incubation of whole blood with LPS (lipopolysaccharide) or saline. The basal values of IL-1beta, IL-6 and TNF-alpha were low and were not significantly related to hs-CRP levels. A univariate analysis showed that the level of IL-1beta and IL-6, obtained after 24 h of incubation of whole blood with LPS, increased significantly with increasing levels of hs-CRP and, after adjusting for potential confounders, IL-1beta still remained statistically significant. In our sample of patients with Type 2 diabetes, there was no association between serum hs-CRP levels and basal levels of IL-6, IL-1beta and TNF-alpha. Conversely, a significant association was observed between serum hs-CRP levels and IL-1beta and IL-6 production after 24 h of incubation of whole blood with LPS. In conclusion, our data suggest that patients with Type 2 diabetes and high hs-CRP levels may have an enhanced reactivity in response to specific stimuli that produce different interleukins, with possible implications in inflammatory atherosclerotic processes.

Treatment-associated polymorphisms in protease are significantly associated with higher viral load and lower CD4 count in newly diagnosed drug-naive HIV-1 infected patients

BackgroundThe effect of drug resistance transmission on disease progression in the newly infected patient is not well understood. Major drug resistance mutations severely impair viral fitness in a drug free environment, and therefore are expected to revert quickly. Compensatory mutations, often already polymorphic in wild-type viruses, do not tend to revert after transmission. While compensatory mutations increase fitness during treatment, their presence may also modulate viral fitness and virulence in absence of therapy and major resistance mutations. We previously designed a modeling technique that quantifies genotypic footprints of in vivo treatment selective pressure, including both drug resistance mutations and polymorphic compensatory mutations, through the quantitative description of a fitness landscape from virus genetic sequences.ResultsGenotypic correlates of viral load and CD4 cell count were evaluated in subtype B sequences from recently diagnosed treatment-naive patients enrolled in the SPREAD programme. The association of surveillance drug resistance mutations, reported compensatory mutations and fitness estimated from drug selective pressure fitness landscapes with baseline viral load and CD4 cell count was evaluated using regression techniques. Protease genotypic variability estimated to increase fitness during treatment was associated with higher viral load and lower CD4 cell counts also in treatment-naive patients, which could primarily be attributed to well-known compensatory mutations at highly polymorphic positions. By contrast, treatment-related mutations in reverse transcriptase could not explain viral load or CD4 cell count variability.ConclusionsThese results suggest that polymorphic compensatory mutations in protease, reported to be selected during treatment, may improve the replicative capacity of HIV-1 even in absence of drug selective pressure or major resistance mutations. The presence of this polymorphic variation may either reflect a history of drug selective pressure, i.e. transmission from a treated patient, or merely be a result of diversity in wild-type virus. Our findings suggest that transmitted drug resistance has the potential to contribute to faster disease progression in the newly infected host and to shape the HIV-1 epidemic at a population level.