Massimo Andreoni

Gender differences in clinical progression of HIV-1-infected individuals during long-term highly active antiretroviral therapy

Objective: To assess gender differences in the long-term clinical, virological and immunological outcomes during highly active antiretroviral therapy (HAART). Methods: This longitudinal observational multicentre study followed 2460 HIV-infected patients who had begun a protease inhibitor-based regimen for a median period of 43 months. Outcome measures were virological suppression (< 500 copies/ml), confirmed virological rebound after suppression, and death or new AIDS-defining illness (ADI). Results: At baseline, 690 female patients (28.0%) had significantly lower age, higher prevalence of heterosexual contact and lower prevalence of intravenous drug use as risk factors for HIV infection compared with males. Furthermore, females had a lower number of AIDS-defining illnesses, higher CD4 cell counts and lower viral loads. No gender differences were reported in terms of proportion of patients achieving viral suppression or exhibiting rebound after achieving viral suppression. Female patients experienced reduced clinical progression during follow-up compared with males (P = 0.008) by Kaplan–Meier analysis; however this difference was not significant in an adjusted analysis. In a multivariate model, the interaction between gender and risk factor for HIV or viral load showed that female drug users and female patients with a baseline HIV RNA viral load of 104–105 copies/ml had a favourable clinical outcome compared with males (P = 0.035 and P = 0.015, respectively). Conclusion: No differences were found between genders in terms of virological and immunological outcomes during long-term HAART. Nevertheless, a lower risk of clinical progression was reported among female patients with intermediate baseline viral load than in males.

HCV Genotypes Are Differently Prone to the Development of Resistance to Linear and Macrocyclic Protease Inhibitors

Background Because of the extreme genetic variability of hepatitis C virus (HCV), we analyzed whether specific HCV-genotypes are differently prone to develop resistance to linear and macrocyclic protease-inhibitors (PIs). Methods The study includes 1568 NS3-protease sequences, isolated from PI-naive patients infected with HCV-genotypes 1a (N = 621), 1b (N = 474), 2 (N = 72), 3 (N = 268), 4 (N = 54) 5 (N = 6), and 6 (N = 73). Genetic-barrier was calculated as the sum of nucleotide-transitions (score = 1) and/or nucleotide-transversions (score = 2.5) required for drug-resistance-mutations emergence. Forty-three mutations associated with PIs-resistance were analyzed (36A/M/L/G-41R-43S/V-54A/S/V-55A-Q80K/R/L/H/G-109K-138T-155K/Q/T/I/M/S/G/L-156T/V/G/S-158I-168A/H/T/V/E/I/G/N/Y-170A/T-175L). Structural analyses on NS3-protease and on putative RNA-models have been also performed. Results Overall, NS3-protease was moderately conserved, with 85/181 (47.0%) amino-acids showing <1% variability. The catalytic-triad (H57-D81-S139) and 6/13 resistance-associated positions (Q41-F43-R109-R155-A156-V158) were fully conserved (variability <1%). Structural-analysis highlighted that most of the NS3-residues involved in drug-stabilization were highly conserved, while 7 PI-resistance residues, together with selected residues located in proximity of the PI-binding pocket, were highly variable among HCV-genotypes. Four resistance-mutations (80K/G-36L-175L) were found as natural polymorphisms in selected genotypes (80K present in 41.6% HCV-1a, 100% of HCV-5 and 20.6% HCV-6; 80G present in 94.4% HCV-2; 36L present in 100% HCV-3-5 and >94% HCV-2-4; 175L present in 100% HCV-1a-3-5 and >97% HCV-2-4). Furthermore, HCV-3 specifically showed non-conservative polymorphisms (R123T-D168Q) at two drug-interacting positions. Regardless of HCV-genotype, 13 PIs resistance-mutations were associated with low genetic-barrier, requiring only 1 nucleotide-substitution (41R-43S/V-54A-55A-80R-156V/T: score = 1; 54S-138T-156S/G-168E/H: score = 2.5). By contrast, by using HCV-1b as reference genotype, nucleotide-heterogeneity led to a lower genetic-barrier for the development of some drug-resistance-mutations in HCV-1a (36M-155G/I/K/M/S/T-170T), HCV-2 (36M-80K-155G/I/K/S/T-170T), HCV-3 (155G/I/K/M/S/T-170T), HCV-4-6 (155I/S/L), and HCV-5 (80G-155G/I/K/M/S/T). Conclusions The high degree of HCV genetic variability makes HCV-genotypes, and even subtypes, differently prone to the development of PIs resistance-mutations. Overall, this can account for different responsiveness of HCV-genotypes to PIs, with important clinical implications in tailoring individualized and appropriate regimens.

Rhodococcus equi Infection in HIV-positive Subjects: A Retrospective Analysis of 24 Cases

Rhodococcus equi causes a rare infection in immunocompromised hosts. We describe 24 cases of infection in patients with AIDS-related complex (ARC)/acquired immunodeficiency syndrome (AIDS). Pneumonia was always the first manifestation of R. equi infection, but extrapulmonary involvement was also observed. The main sources of bacteria were sputum, bronchial washings and blood. The strains isolated were mainly susceptible to erythromycin, vancomycin, teicoplanin, rifampicin, imipenem and aminoglycosides. Initial treatment should involve an intravenously administered antibiotic combination therapy including imipenem or vancomycin or teicoplanin, followed by orally administered maintenance combination therapy. Drug combinations should be investigated for serum bactericidal activity in vitro. Surgery does not increase survival time and should only be performed in cases that do not respond to antibiotic treatment. Presumptive risks of infection (contact with horses or farm dust, or cohabiting with people affected by R. equi infection) were present in more than 50% of patients. This finding, and the frequency of bacteria in the sputum, are not sufficient proof of transmission between humans, but do suggest the need for respiratory isolation of patients affected by R. equi pneumonia.

NK cell activity controls human herpesvirus 8 latent infection and is restored upon highly active antiretroviral therapy in AIDS patients with regressing Kaposi's sarcoma.

Kaposis sarcoma (KS) develops upon reactivation of human herpesvirus 8 (HHV8) infection and virus dissemination to blood and tissue cells, including endothelial and KS spindle cells where the virus is mostly present in a latent form. However, this may likely require the presence of compromised host immune responses and/or the evasion of infected cells from the host immune response.In this regard, mechanisms of evasion of productively infected cells from both CTL and NK cell responses, and resistance of latently infected cells from specific CTL, have already been shown. Here we show that cells which are latently infected by HHV8 are indeed efficiently lysed by NK cells from individuals with a normal immune response. Notably, NK cell‐mediated immunity was found to be significantly reduced in AIDS patients with progressing KS as compared to both HIV‐negative patients with indolent classic KS or normal blood donors. However, it was restored after treatment with the highly active antiretroviral therapy (HAART) in AIDS‐KS patients, that showed regression and clearance of HHV8 from PBMC. By contrast, AIDS‐KS patients with a more aggressive disease and no clinicalresponse had persistent HHV8 viremia associated with reduced NK cell cytotoxicity. These results suggest a key role for NK cells in the control of HHV8 latent infection, KS development, and in disease remission upon HAART.

Prevalence and risk factors for human herpesvirus 8 infection in northern Cameroon.

Background: The modes of transmission of HHV‐8 are still unclear. Goal: To evaluate the distribution and transmission of HHV‐8 infection. Design: Serosurvey conducted in a Cameroon hospital among 292 persons, including children (5‐10 years), adolescents (15‐20 years), and adults (30‐40 years). Antibodies against lytic and latent antigens to HHV‐8 were detected by immunofluorescence assay; antibodies against Epstein‐Barr virus viral antigens were detected by enzyme‐linked immunoabsorbent assay. Results: The prevalence of HHV‐8 antilytic antibodies remained stable and was 39.8% among children, 51.5% among adolescents, and 61.8% among adults. Epstein‐Barr virus seroprevalence was high among children, and remained stable among adolescents and adults. A history of sexually transmitted diseases was an independent determinant of HHV‐8 infection (adjusted odds ratio 2.47; 95% CI 1.09‐4.91). Conclusion: The high prevalence of HHV‐8 infection among children indicates nonsexual modes of transmission in Cameroon, with sexual transmission occurring among adolescents and adults.

A seroprevalence study of human herpesvirus type 8 (HHV8) in eastern and Central Africa and in the Mediterranean area

Human herpes virus type 8 (HHV8) is the major determinant of Kaposis sarcoma (KS), a neoplasm with wide geographic variations in incidence rates. To assess the prevalence of HHV8 infection among populations with differing rates of KS, we used sera from 1402 persons (Central Africa: Cameroon, n = 293, age range: 5–40; eastern Africa: Uganda, n = 315, age range: 1–64: Mediterranean area: Egypt, n = 236, age range: 13–19: Italy, blood donors n = 134, age range: 20–67: Italy. HIV seroconverters n = 424, age range: 16–65). Serum samples were tested for antibodies to lytic and latent antigens of HHV8 using two immunofluorescence assays. HHV8 prevalence was evaluated according to geographic area, gender and age groups. Overall, the highest prevalence of HHV8 lytic antigens (47.5%) was recorded among children and adults in Africa. Approximately 40% of children and adolescents from Egypt and of Italian HIV-positive persons (39.9%) were HHV8 seropositive. In eastern and Central Africa and in Egypt, no differences emerged between males and females for both types of HHV8 antibodies. Conversely, Italian females were at lower HHV8 risk than their male counterparts. Moreover the prevalence of HHV8 infection tended to increase with age. This investigation partially confirms that HHV8 infection mirrors incidence rates of KS. The high prevalence of HHV8 infection in newborns, children and adolescents in Egypt, in eastern and in Central Africa strongly suggests the existence of transmission modes other than sexual.

Identification of cytotoxic T lymphocyte epitopes of human herpesvirus 8

The human herpesvirus 8 (HHV‐8) is a human γ2‐herpesvirus that is implicated in the development of Kaposis sarcoma (KS), primary effusion lymphoma and Castelmans disease. Since the responses of cytotoxic T lymphocytes (CTL) play a key role in the control of herpesvirus infection, it is important to identify and to characterize the CTL target epitopes of HHV‐8 viral antigens. In this study, using peptide‐binding motifs, we selected potential human leucocyte antigen (HLA)‐A2‐binding peptides from kaposin A and glycoprotein H (gH), that are latent and lytic HHV‐8 antigens, respectively. HLA‐A2‐binding peptides were tested for their capacity to induce CTL responses in HHV‐8‐negative healthy donors. By this approach, we found that the majority of individuals responded to two HHV‐8‐derived CTL epitopes, namely, VLLNGWRWRL (amino acids 16–25), which derives from kaposin A, and FLNWQNLLNV (amino acids 59–68), which derives from gH. In addition, memory CTL responses to these epitopes were detected in disease‐free individuals infected by HHV‐8 demonstrating that the two epitopes are relevant targets of CTL‐mediated immunity in vivo. The identified epitopes may be investigated for the development of immunotherapeutic strategies against HHV‐8‐associated malignancies.

Calibrated Real-Time PCR Assay for Quantitation of Human Herpesvirus 8 DNA in Biological Fluids

ABSTRACT Accurate laboratory tests for the diagnosis of active human herpesvirus 8 (HHV-8) infection are becoming essential to study the pathogenesis of HHV-8-associated tumors and for the clinical management of HHV-8-infected individuals. We have developed a highly sensitive, calibrated quantitative real-time PCR assay for the measurement of cell-free HHV-8 DNA in body fluids, based on the addition of a synthetic DNA calibrator prior to DNA extraction. The calibrator controls each sample for the presence of PCR inhibitors, determines a cutoff value of sensitivity for negative samples, and normalizes positive samples for the efficiency of DNA recovery. The assay shows a wide dynamic range of detection (between 1 and 106 viral genome equivalents/reaction) and a high degree of accuracy even in the presence of high amounts (up to 1 μg) of human genomic DNA. Moreover, the assay has a very high sensitivity (lower detection limit, 10 genome equivalents/ml) and a high degree of reproducibility and repeatability with a coefficient of variation (CV) of <15 and 23%, respectively. Furthermore, the use of the calibrator improves the accuracy of quantitation and decreases the intersample variability (CV, 9 and 6%, respectively). The sensitivity and specificity of the assay were tested with a series of clinical specimens obtained from patients affected by various HHV-8-related diseases, as well as from a wide number of controls. In conclusion, our calibrated real-time PCR assay provides a reliable high-throughput method for quantitation of HHV-8 DNA in clinical and laboratory specimens.

Residual viraemia in subjects with chronic HIV infection and viral load < 50 copies/ml: the impact of highly active antiretroviral therapy.

Objective:To determine factors associated with < 2.5 copies/ml plasma HIV RNA in subjects treated with highly active antiretroviral therapy (HAART) and with viraemia < 50 copies/ml. Design:Cross-sectional analysis of 84 HIV-positive patients taking HAART with plasma HIV RNA < 50 copies/ml for at least 6 months and no history of virological failure. Methods:Current HAART therapy was based on a non-nucleoside reverse transcriptase inhibitor (NNRTI) in 66%, a protease inhibitor in 26% and nucleoside reverse transcriptase inhibitors in 7%. Viraemia levels were measured using a modified ultrasensitive Roche Amplicor HIV-1 Monitor test able to quantify plasma HIV RNA to a lower limit of 2.5 copies /ml; proviral DNA was measured with a real-time polymerase chain reaction assay. Analysis of variance and multiple logistic regression analysis were utilized to test for associations between residual replication and other variables. Results:Residual HIV viraemia > 2.5 copies/ml was found in 50% of subjects; 94% of subjects had detectable proviral DNA (≥ 20 copies/106 peripheral blood mononuclear cells) and 21% had archived mutations. Usage of a NNRTI-based HAART was the only independent predictor of viral suppression below the cut-off value of the modified ultrasensitive assay. Conclusions:In our population, NNRTI-based HAART seems to have a stronger impact on residual replication than protease inhibitor-based HAART. This finding may be considered in therapeutic decisions such as the choice of initial HAART regimen and the interruption or simplification of treatment.

Reliability and Clinical Relevance of the HIV-1 Drug Resistance Test in Patients With Low Viremia Levels

BACKGROUND We evaluated reliability and clinical usefulness of genotypic resistance testing (GRT) in patients for whom combination antiretroviral therapy (cART) was unsuccessful with viremia levels 50-1000 copies/mL, for whom GRT is generally not recommended by current guidelines. METHODS The genotyping success rate was evaluated in 12 828 human immunodeficiency virus type 1 (HIV-1) plasma samples with viremia >50 copies/mL, tested using the commercial ViroSeq HIV-1 Genotyping System or a homemade system. Phylogenetic analysis was performed to test the reliability and reproducibility of the GRT at low-level viremia (LLV). Drug resistance was evaluated in 3895 samples from 2200 patients for whom treatment was unsuccessful (viremia >50 copies/mL) by considering the resistance mutations paneled in the 2013 International Antiviral Society list. RESULTS Overall, the success rate of amplification/sequencing was 96.4%. Viremia levels of 50-200 and 201-500 copies/mL afforded success rates of 67.2% and 88.1%, respectively, reaching 93.2% at 501-1000 copies/mL and ≥97.3% above 1000 copies/mL. A high homology among sequences belonging to the same subject for 96.4% of patients analyzed was found. The overall resistance prevalence was 74%. Drug resistance was commonly found also at LLV. In particular, by stratifying for different viremia ranges, detection of resistance was as follows: 50-200 copies/mL = 52.8%; 201-500 = 70%; 501-1000 = 74%; 1001-10 000 = 86.1%; 10 001-100 000 = 76.7%; and >100 000 = 63% (P < .001). Similar bell-shaped results were found when the GRT analysis was restricted to 2008-2012, although at a slightly lower prevalence. CONCLUSIONS In patients failing cART with LLV, HIV-1 genotyping provides reliable and reproducible results that are informative about emerging drug resistance.