CA-IX-Expressing Small Extracellular Vesicles (sEVs) Are Released by Melanoma Cells under Hypoxia and in the Blood of Advanced Melanoma Patients.

Cutaneous melanoma is a highly aggressive skin cancer, with poor prognosis. The tumor microenvironment is characterized by areas of hypoxia. Carbonic anhydrase IX (CA-IX) is a marker of tumor hypoxia and its expression is regulated by hypoxia-inducible factor-1 (HIF-1). CA-IX has been found to be highly expressed in invasive melanomas. In this study, we investigated the effects of hypoxia on the release of small extracellular vesicles (sEVs) in two melanoma in vitro models. We demonstrated that melanoma cells release sEVs under both normoxic and hypoxic conditions, but only hypoxia-induced sEVs express CA-IX mRNA and protein. Moreover, we optimized an ELISA assay to provide evidence for CA-IX protein expression on the membranes of the sEVs. These CA-IX-positive sEVs may be exploited as potential biomarkers for liquid biopsy.

Isolation of extracellular vesicles improves the detection of mutant DNA from plasma of metastatic melanoma patients.

Detection of BRAFV600E within cell free tumor DNA (ctDNA) is emerging as a promising means to improve patients' stratification or enable BRAF inhibitor (BRAFi) therapeutic monitoring in a minimally invasive manner. Here, we investigated whether extracellular vesicle-(EV)-associated-DNA (EV-DNA) has value as an alternative source of circulating BRAFV600E. To do so, we identified a clinical practice-compatible protocol for the isolation of EV-DNA and assessed BRAF gene status on plasma samples from metastatic melanoma patients at the beginning and during BRAFi therapy. This protocol uses a peptide with high affinity for EVs and it has been found to recover more mutant DNA from plasma than standard ultracentrifugation. Molecular analyses revealed that mutant DNA is largely unprotected from nuclease digestion, interacting with the outer side of the EV membrane or directly with the peptide. When used on clinical samples, we found that the protocol improves the detection of BRAFV600E gene copies in comparison to the reference protocol for ctDNA isolation. Taken together, these findings indicate that EVs are a promising source of mutant DNA and should be considered for the development of next-generation liquid biopsy approaches.

When Less Is More: Specific Capture and Analysis of Tumor Exosomes in Plasma Increases the Sensitivity of Liquid Biopsy for Comprehensive Detection of Multiple Androgen Receptor Phenotypes in Advanced Prostate Cancer Patients.

We evaluated the advantages and the reliability of novel protocols for the enrichment of tumor extracellular vesicles (EVs), enabling a blood-based test for the noninvasive parallel profiling of multiple androgen receptor (AR) gene alterations. Three clinically relevant AR variants related to response/resistance to standard-of-care treatments (AR-V7 transcript, AR T878A point mutation and AR gene amplification) were evaluated by digital PCR in 15 samples from patients affected by Castration-Resistant Prostate Cancer (CRPC). Plasma was processed to obtain circulating RNA and DNA using protocols based on tumor EVs enrichment through immuno-affinity and peptide-affinity compared to generic extraction kits. Our results showed that immuno-affinity enrichment prior to RNA extraction clearly outperforms the generic isolation method in the detection of AR-V7, also allowing for a distinction between responder (R) and non-responder (NR) patients. The T878A mutation was detected, overall, in nine out of 15 samples and no approach alone was able to reveal mutations in all harboring samples, showing that the employed methods complement each other. AR amplification was detected in the majority of CRPC samples analysed using either cell-free DNA (cfDNA) or exosome isolation kits (80%). We demonstrated that selective isolation of a subset of circulating exosomes enriched for tumor origin, rather than analysis of total plasma exosomes, or total plasma nucleic acids, increases sensitivity and specificity for the detection of specific alterations.

TM9SF4 expression in tumor tissues: a novel diagnostic biomarker for gastrointestinal tumors.

BACKGROUND: The identification of novel biomarkers for the early detection and monitoring of gastric (GC) and colorectal cancer (CRC) is of paramount importance. TM9SF4 is a newly described V-ATPase interacting protein involved in the malignant progression of cancer cells. While TM9SF4 expression pattern and cellular localization have been described in in vitro in tumor cell lines of different histotypes, its expression in gastrointestinal tumor tissues has never been investigated. METHODS: In this study, we detected by immunohistochemistry (IHC) in tumor and surrounding healthy tissues TM9SF4, in comparison with clinically adopted biomarkers CEA and CA 19-9 to evaluate TM9SF4 potential as a novel tissue marker for early detection and monitoring of GC and CRC cancers. RESULTS: The expression of TM9SF4, CEA and CA 19-9 was evaluated in samples from 108 cancer patients (68 with GC and 40 CRC) and in healthy tissues from 20 non-cancer patients. Our results clearly suggest that TM9SF4 expression was significantly increased in GC and CRC samples and significantly correlated to disease stage in both cancer types. CONCLUSIONS: We propose TM9SF4 as highly specific cancer biomarker, exploitable for disease detection and staging of gastrointestinal cancers patients, with tumor tissue levels of expression outperforming those of clinically adopted markers such as CEA and CA 19-9.

Social capital components and social support of persons with multiple sclerosis: a systematic review of the literature from 2000 to 2018.

Purpose: To identify experiences of persons with multiple sclerosis (MS) in terms of social capital and its components (i.e., social networks, trust, and interpersonal relationships) and social support based on the current scientific knowledge.Methods: Systematic literature review was conducted through PubMed, Scopus, Web of Science, ProQuest, and PsycINFO. Included articles were published from 2000 to 2018 and met specific selection criteria. Screening of records determined eligible studies for inclusion to data extraction and synthesis process.Results: A total of 551 abstracts were screened, of which 34 studies met all selection criteria. The themes that emerged referred to the impact of physical and cognitive impairments on social functioning, stigma, psychosocial, emotional and mental challenges, association of quality of life with social capital components and social support, and contribution of social support to improvement of social functioning and health of persons with MS. Persons with MS face a series of issues regarding social support and social capital-related components, primarily facing psychological difficulties, difficulties with making and maintaining interpersonal relationships, and limitations for participating in social and daily activities due to the symptoms of MS, particularly fatigue.Conclusion: It appears that the ability to seek and maintain social relationships and to participate in social and daily activities is important for persons with MS. This has an impact on their quality of life, as well as on their health functioning, however issues around mobility and stigmatization of their condition hinder their social functioning.

Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocapture approaches.

Clinical implementation of exosome based diagnostic and therapeutic applications is still limited by the lack of standardized technologies that integrate efficient isolation of exosomes with comprehensive detection of relevant biomarkers. Conventional methods for exosome isolation based on their physical properties such as size and density (filtration, ultracentrifugation or density gradient), or relying on their differential solubility (chemical precipitation) are established primarily for processing of cell supernatants and later adjusted to complex biological samples such as plasma. Though still representing gold standard in the field, these methods are clearly suboptimal for processing of routine clinical samples and have intrinsic limits that impair their use in biomarker discovery and development of novel diagnostics. Immunoisolation (IA) offers unique advantages for the recovery of exosomes from complex and viscous fluids, in terms of increased efficiency and specificity of exosome capture, integrity and selective origin of isolated vesicles. We have evaluated several commercially available solutions for immunoplate- and immunobead-based affinity isolation and have further optimized protocols to decrease non-specific binding due to exosomes complexity and matrix contaminants. In order to identify best molecular targets for total exosome capture from diverse biological sources, as well as for selective enrichment in populations of interest (e.g. tumor derived exosomes) several exosome displayed proteins and respective antibodies have been evaluated for plate and bead functionalisation. Moreover, we have optimized and directly implemented downstream steps allowing on-line quantification and characterization of bound exosome markers, namely proteins and RNAs. Thus assembled assays enabled rapid overall quantification and validation of specific exosome associated targets in/on plasma exosomes, with multifold increased yield and enrichment ratio over benchmarking technologies. Assays directly coupling selective immobilization of exosomes to a solid phase and their immune- and or molecular profiling through conventional ELISA and PCR analysis, resulted in easy-to-elaborate, quantitative readouts, with high low-end sensitivity and dynamic range, low costs and hands-on time, minimal sample handling and downscaling of a working plasma volumes to as few as 100 µl.

Comparison of serum exosome isolation methods for microRNA profiling.

OBJECTIVES: Exosomes are small membrane bound vesicles secreted by most cell types. Exosomes contain various functional proteins, mRNAs and microRNAs (miRNAs) that could be used for diagnostic and therapeutic purposes. Currently, a standard method for serum exosome isolation is differential ultracentrifugation, but a search for alternative, less time-consuming and labour extensive exosomal isolation method for use in clinical settings is ongoing. The effect of serum exosome isolation method on obtained miRNA profile is not yet clear. The aim of this study was to determine to which extent selected exosome isolation methods influence the serum exosomal miRNA profile. DESIGN AND METHODS: Exosomes were isolated from blood serum of healthy individuals by ultracentrifugation and ExoQuick Precipitation methods. The expression profile of 375 miRNAs was determined by real time PCR using Exiqon miRCURY LNA™ microRNA Human panel I assays. RESULTS: Although a strong correlation of exosomal miRNA profiles was observed between the two isolation methods, distinct clusters of miRNA levels between the used methods were identified. The detected levels of two miRNAs, miR-92a and miR-486-5p, were significantly influenced by the exosome isolation method used. CONCLUSIONS: Both exosome isolation methods are suitable for serum exosomal miRNA profiling. Differences found in miRNA patterns between the two methods indicate that the observed exosomal miRNA profile is slightly affected by the extracellular vesicle isolation method.

Exosomes as intercellular signaling organelles involved in health and disease: basic science and clinical applications.

Cell to cell communication is essential for the coordination and proper organization of different cell types in multicellular systems. Cells exchange information through a multitude of mechanisms such as secreted growth factors and chemokines, small molecules (peptides, ions, bioactive lipids and nucleotides), cell-cell contact and the secretion of extracellular matrix components. Over the last few years, however, a considerable amount of experimental evidence has demonstrated the occurrence of a sophisticated method of cell communication based on the release of specialized membranous nano-sized vesicles termed exosomes. Exosome biogenesis involves the endosomal compartment, the multivesicular bodies (MVB), which contain internal vesicles packed with an extraordinary set of molecules including enzymes, cytokines, nucleic acids and different bioactive compounds. In response to stimuli, MVB fuse with the plasma membrane and vesicles are released in the extracellular space where they can interact with neighboring cells and directly induce a signaling pathway or affect the cellular phenotype through the transfer of new receptors or even genetic material. This review will focus on exosomes as intercellular signaling organelles involved in a number of physiological as well as pathological processes and their potential use in clinical diagnostics and therapeutics.

Exosomes: the ideal nanovectors for biodelivery.

Nanomedicine aims to exploit the improved and often novel physical, chemical, and biological properties of materials at the nanometric scale, possibly with the highest level of biomimetism, an approach that simulates what occurs in nature. Although extracellularly released vesicles include both microvesicles (MVs) and exosomes, only exosomes have the size that may be considered suitable for potential use in nanomedicine. In fact, recent reports have shown that exosomes are able to interact with target cells within an organ or at a distance using different mechanisms. Much is yet to be understood about exosomes, and currently, we are looking at the visible top of an iceberg, with most of what we have to understand on these nanovesicles still under the sea. In fact, we know that exosomes released by normal cells always trigger positive effects, whereas those released by cells in pathological condition, such as tumor or infected cells, may induce undesired, dangerous, and mostly unknown effects, but we cannot exclude the possibility that exosomes may also be detrimental for the body in normal conditions. However, whether we consider extracellular vesicles as a whole, thus including MVs, it appears that even in normal conditions, extracellular vesicles may lead to unwanted effects, depending on gender and age. This review aims to critically emphasize existing data in the literature that support the possible roles of exosomes in both diagnostic and therapeutic scopes.

Serious fatal and nonfatal non-AIDS-defining illnesses in Europe.

BACKGROUND: Little is known about the incidence and risk factors for serious non-AIDS-defining events. METHODS: The incidence of non-AIDS events (malignancies, end-stage renal disease, liver failure, pancreatitis, cardiovascular disease), and AIDS after January 1, 2001, was calculated; Poisson regression was used to investigate factors associated with non-AIDS and AIDS. RESULTS: Among 12,844 patients, 1058 were diagnosed with a non-AIDS event [incidence 1.77 per 100 person-years of follow-up; 95% confidence interval (CI): 1.66 to 1.87]; 462 patients (43.7%) died. The incidence of AIDS (1025 diagnoses; 339 deaths, 33.1%) was 1.72 per 100 person-years of follow-up (1.61 to 1.83). After adjustment, older age [incidence rate ratio (IRR): 1.71 per 10 years older, 95% CI: 1.60 to 1.83], diabetes (IRR: 1.49, 95% CI: 1.22 to 1.82) and hypertension (IRR: 1.63, 95% CI: 1.43 to 1.87) were associated with non-AIDS events. Compared with patients without an event, there was a 4-fold increased risk of death after an AIDS event (relative hazard: 4.14; 95% CI 3.47 to 4.94) and almost a 7-fold increased risk of death after a non-AIDS event (relative hazard: 6.72; 95% CI: 5.61 to 8.05). CONCLUSIONS: Non-AIDS events were common in the combination antiretroviral therapy era and associated with considerably mortality. Evidence on the impact of modifying immunodeficiency and lifestyle-related factors on the risk of non-AIDS events in HIV-infected persons is an important but unmet research need.

Proton dynamics in cancer.

Cancer remains a leading cause of death in the world today. Despite decades of research to identify novel therapeutic approaches, durable regressions of metastatic disease are still scanty and survival benefits often negligible. While the current strategy is mostly converging on target-therapies aimed at selectively affecting altered molecular pathways in tumor cells, evidences are in parallel pointing to cell metabolism as a potential Achilles' heel of cancer, to be disrupted for achieving therapeutic benefit. Critical differences in the metabolism of tumor versus normal cells, which include abnormal glycolysis, high lactic acid production, protons accumulation and reversed intra-extracellular pH gradients, make tumor site a hostile microenvironment where only cancer cells can proliferate and survive. Inhibiting these pathways by blocking proton pumps and transporters may deprive cancer cells of a key mechanism of detoxification and thus represent a novel strategy for a pleiotropic and multifaceted suppression of cancer cell growth.Research groups scattered all over the world have recently started to investigate various aspects of proton dynamics in cancer cells with quite encouraging preliminary results. The intent of unifying investigators involved in this research line led to the formation of the "International Society for Proton Dynamics in Cancer" (ISPDC) in January 2010. This is the manifesto of the newly formed society where both basic and clinical investigators are called to foster translational research and stimulate interdisciplinary collaboration for the development of more specific and less toxic therapeutic strategies based on proton dynamics in tumor cell biology.

Does less frequent routine monitoring of patients on a stable, fully suppressed cART regimen lead to an increased risk of treatment failure?

OBJECTIVE: To investigate whether HIV-infected patients on a stable and fully suppressive combination antiretroviral therapy (cART) regimen could safely be monitored less often than the current recommendations of every 3 months. DESIGN: Two thousand two hundred and forty patients from the EuroSIDA study who maintained a stable and fully suppressed cART regimen for 1 year were included in the analysis. METHODS: Risk of treatment failure, defined by viral rebound, fall in CD4 cell count, development of new AIDS-defining illness, serious opportunistic infection or death, in the 12 months following a year of a stable and fully suppressed regimen was assessed. RESULTS: One hundred thirty-one (6%) patients experienced treatment failure in the 12 months following a year of stable therapy, viral rebound occurred in 99 (4.6%) patients. After 3, 6 and 12 months, patients had a 0.3% [95% confidence interval (CI) 0.1-0.5], 2.2% (95% CI 1.6-2.8) and 6.0% (95% CI 5.0-7.0) risk of treatment failure, respectively. Patients who spent more than 80% of their time on cART with fully suppressed viraemia prior to baseline had a 38% reduced risk of treatment failure, hazard ratio 0.62 (95% CI 0.42-0.90, P = 0.01). CONCLUSION: Patients who have responded well to cART and are on a well tolerated and durably fully suppressive cART regimen have a low chance of experiencing treatment failure in the next 3-6 months. Therefore, in this subgroup of otherwise healthy patients, it maybe reasonable to extend visit intervals to 6 months, with cost and time savings to both the treating clinics and the patients.

Incidence of abacavir hypersensitivity reactions in euroSIDA.

BACKGROUND: The aim of the study was to investigate the incidence of abacavir-related hypersensitivity reaction (HSR) and associated deaths in EuroSIDA HIV-1-infected patients. METHODS: Poisson regression models were developed to compare incidence of abacavir discontinuation according to the line of therapy within which abacavir was received, geographical regions, calendar time and drug formulation (abacavir/lamivudine combination tablet versus abacavir as a single drug or abacavir/zidovudine/lamivudine combination). RESULTS: Of 3,278 patients that started abacavir, 2,101 (64.1%) discontinued. Of these, 167 (5.1%) discontinued abacavir within 3 months due to HSR with an incidence of 22.1 (95% confidence interval [CI] 18.7-25.4) per 100 person-years of follow-up. After adjustment for gender, prior AIDS, hepatitis C serostatus, baseline CD4+ T-cell count, region and calendar time, HSR incidence was significantly higher in those starting abacavir in a first-line regimen compared with second-line (incidence rate ratio [IRR] 2.04 [95% CI 1.24-3.38]; P=0.005). There was no significant difference between regions. HSR incidence from 2005 onwards was significantly lower compared with 1999-2000 (IRR 0.54 [95% CI 0.32-0.92]; P=0.024). There was a lower observed incidence in patients starting abacavir/lamivudine compared with other formulations (IRR 0.33 [95% CI 0.13-0.88]; P=0.027), however, available data were limited. CONCLUSIONS: Incidence of abacavir-related HSR is higher in patients starting abacavir in first-line therapy, which could indicate increased over-diagnosis. HSR incidence has decreased in recent years, which might reflect the wider availability of genetic screening and improved awareness of symptoms. There were no reported deaths due to abacavir HSR.

Sex issues in HIV-1-infected persons during highly active antiretroviral therapy: a systematic review.

BACKGROUND: Since the introduction of highly active antiretroviral therapy (HAART), morbidity and mortality rates have sharply decreased among HIV-infected patients. Studies of possible differences between men and women in the course of HIV infection give conflicting results. The objective of this study was to assess sex differences during HAART. METHODS: A literature search by using the MEDLINE database between March 2002 and February 2007 was performed to identify all published studies on the sex-specific differences on the impact of HAART. All articles with measures of effect (preferably adjusted odds ratio, relative risk or hazard ratio with 95% CI) of sex on viroimmunological and clinical parameters during HAART were included. Five different topics of interest in our research were selected: time of initiation of HAART, adherence, viroimmunological response, clinical response and adverse reactions during HAART. RESULTS: US data report an initiation of HAART at an earlier disease stage in men compared with women. After initiation of HAART, most authors do not report any viroimmunological difference, although a few clinical studies showed a significantly better virological response in women compared with men. Nevertheless, women were more likely to be less adherent to antiretrovirals and to have non-structured treatment interruptions than men. This is likely to be related to the higher number of adverse reactions they experience during HAART. Finally, discordant opinions with regard to clinical benefits during HAART exist, but recent clinical and observational trials suggest a better clinical outcome for women. CONCLUSIONS: We found little evidence of sex differences during antiretroviral treatment. Nevertheless, most of these studies were underpowered to detect sex differences and had limited follow-up at 6 or 12 months. Design of new gender-sensitive clinical trials with both prolonged follow-up and sample size representative of the current HIV prevalence among women are strongly needed to detect the likely sex differences of antiretroviral agents during HIV infection.

Limited but increasing use of treatment for hepatitis C across Europe in patients coinfected with HIV and hepatitis C.

Uptake of hepatitis C (HCV) treatment in HIV-coinfected patients is not well described. Of 2356 HCV-seropositive patients, 180 (7.6%) started HCV treatment with interferon-based therapies. In multivariate Poisson-regression models, there was a 38% increase per year in the incidence of starting HCV treatment (95% CI 26 - 51%, p<0.0001); this increased from 3.9 per 1000 person-years follow-up (PYFU) before 1998 (95% CI 1.6 - 6.1) to 32.6 per 1000 PYFU at/after 2004 (95% CI 22.5 - 42.7). Although prescription of HCV therapy is increasing in HIV-coinfected patients, it remains infrequent and variable across regions of Europe.

Relationship between antiretrovirals used as part of a cART regimen and CD4 cell count increases in patients with suppressed viremia.

BACKGROUND: It is unknown if the CD4 cell count response differs according to antiretroviral drugs used in combination antiretroviral therapy (cART) in patients with maximal virological suppression [viral load (VL) < 50 copies/ml]. OBJECTIVES: To compare the change in CD4 cell count over consecutive measurements with VL < 50 copies/ml at both time-points according to nucleoside backbones and other antiretrovirals used. METHODS: Generalized linear models, accounting for multiple measurements within patients, were used to compare CD4 cell count changes after adjustment for antiretrovirals, time from starting cART, age, CD4 at first VL < 50 copies/ml, prior antiretroviral treatment, and change in CD4 cell count since starting cART. RESULTS: We studied 28418 instances of VL < 50 copies/ml in 4041 patients. The mean annual change in CD4 cell count was +45.5/microl [95% confidence interval (CI) +39.4 to +51.6/microl). Comparing two drug nucleoside backbones, there was a lower annual change in CD4 cell count for zidovudine/lamivudine (n = 13038; -15.4/microl; P = 0.012) and for those on tenofovir (n = 1809; -27.3/microl; P = 0.029) compared to lamivudine/stavudine (n = 7339). Compared to the boosted-protease inhibitor regimen (n = 5915), use of an abacavir-based triple-nucleoside regimen was associated with a lower annual change in CD4 cell count (n = 2504 pairs; -26.1/microl; P = 0.011). CONCLUSIONS: A nucleoside backbone of zidovudine/lamivudine or any tenofovir-based backbone was associated with significantly poorer increases in CD4 cell count compared to a nucleoside backbone of stavudine/lamivudine, as was an abacavir-based triple nucleoside regimen compared to a boosted protease inhibitor regimen. Long-term studies are needed to determine whether the differences in immunological response seen here translate into differences in the risk of clinical disease.

Decline in esophageal candidiasis and use of antimycotics in European patients with HIV.

BACKGROUND: Esophageal candidiasis (EC) remains one of the most common AIDS defining illnesses in patients with human immunodeficiency virus (HIV) in the era of highly active antiretroviral therapy (HAART), but little is known about factors associated with EC after starting HAART. OBJECTIVES: To describe changes in the use of antimycotic medication, the incidence of EC and factors associated with EC before and after starting HAART. METHODS: Patients from EuroSIDA, a pan-European longitudinal, prospective observational study. Generalized linear models and poisson regression models were used to investigate the relationships. RESULTS: A total of 9,873 patients did not have EC at recruitment, subsequently 537 (15.8%) developed EC. The proportion of patients taking any antimycotic dropped from 18% at January 1995 to 2% at January 2004 (p < 0.0001); the duration of treatment declined from 10 to 3 months over the same period (p < 0.0001). There was a 32% annual decline in the incidence of EC (95% CI 30-35%, p < 0.0001). There was a significant annual decline in the incidence of EC pre-HAART in time-updated, adjusted models, (incidence rate ratio (IRR) 0.80, 95% CI 0.76-0.85, p < 0.0001) but not post-HAART (IRR 0.97; 95% CI 0.90-1.06, p= 0.54). Older patients and those with low CD4 counts had the greatest incidence of EC in the post-HAART era. CONCLUSIONS: There has been a marked decline in the incidence of EC between 1994 and 2004. This was accompanied by a decline in markers associated with fungal disease, including use of antimycotics and a decline in duration of treatment.

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 10(4)-10(5) 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.

Clinical outcome after 4 years follow-up of HIV-seropositive subjects with incomplete virologic or immunologic response to HAART.

The duration of the clinical, virologic, and immunologic response to HAART, is not well defined. In this observational multi-center study 2,143 patients were enrolled classified according to virologic suppression (<500 cp/ml) and immune recovery (>100 CD4+ cells/mul from baseline) at month 12 of HAART as complete responders, virologic only responders, immunologic only responders and non-responders. Kaplan Meyer curves, multivariate and politomous regression analysis were used. Complete responders patients were 781 (36.4%), immunologic only responders 441 (20.6%), virologic only responders 336 (15.7%), and non-responders 585 (27.3%). Using multivariate analysis, being antiretroviral-naive increased the probability of having both a virologic only or a complete response and reduced the probability of an immunologic only response (P < 0.001 for all tests). Older age was associated directly with a virologic only response and inversely associated with an immunologic only response (P = 0.027 and P = 0.035, respectively). Using politomous analysis, patients baseline HIV-RNA level more than 5 log cp/ml had a 1.9-fold higher probability of an immunologic response than of a complete response (P = 0.001). After 4 years, the clinical progression rate was six times greater in non-responders, 1.9 times greater in virologic only responders, and 2.3 times greater in immunologic only responders than for responders. However, patients with virologic only response or with immunologic only response had a significantly reduced risk for clinical progression than non-responders (P < 0.001). After 4 years of HAART, the risk of clinical progression in patients with immunologic only or virologic only response is low but still higher than in complete responder patients.