Current HIV Research (v.14, #4)

Formulation Development, Physicochemical Characterization and In Vitro-In Vivo Drug Release of Vaginal Films by Kajal Ghosal, Bipad Taran Hazra, Benoy Brata Bhowmik, Sabu Thomas (295-306).
Purpose: The purpose of this study was formulation and optimization of vaginal film formulation containing abacavir (ABC), a potent nucleoside reverse transcriptase inhibitor.
Methods: Vaginal films were prepared by solvent evaporation method using hydroxypropyl methylcellulose (HPMC) blended with polyvinyl pyrrolidone (PVP). Various physicochemical parameters of the prepared films such as drug content, thickness, tensile strength, percentage elongation at break, drug polymer interaction, swelling capacity, folding endurance, bio-adhesion, pH, and moisture content were evaluated with morphological studies. In vitro release study and in vivo release study were also performed.
Results: Films exhibited favorable physicochemical properties. The in vitro study showed that HPMC-PVP combination can control the release of abacavir through vaginal films with higher amount of PVP in the formulation resulting in an enhanced drug release rate. During the in vivo study in rabbits, systemic absorption of the drug was noted and the films remained intact for long in vagina without causing any sort of irritations.
Conclusion: Thus, in a nutshell, the findings of our experimental work indicate that such films can be considered as a novel drug carrier system for the treatment of AIDS and other sexually transmitted diseases (STDs), and are suitable for local as well as systemic effects.

Current Approaches in Computational Drug Resistance Prediction in HIV by Mona Riemenschneider, Dominik Heider (307-315).
Background: Today a broad range of antiretroviral drug regimens are applicable for the successful suppression of virus replication in human immunodeficiency virus (HIV) infected people. However, there still remains an obstacle in therapy: the high mutation rate of the HI virus under drug pressure leads to resistant variants causing failure of permanent and effective treatment. Therefore, resistance testing is therefore inevitable to administer appropriate antiviral drugs to infected patients.
Methods: By means of current high-throughput sequencing technologies, computational models have recently constituted important assistance in drug resistance prediction and can guide the choice of medical treatment. Several machine learning algorithms, e.g. support-vector machines, random forests, as well as statistical methods have been already applied to genotypic data and structural information to predict drug resistance.
Results: In this review, we provide an overview of existing approaches in computational drug resistance prediction in HIV. We further highlight the challenges and limitations of current methods, e.g. time complexity and prediction of non-B subtypes.
Conclusion: Moreover, we give a perspective on multi-label and multi-instance classification techniques that potentially tackle the problem of cross-resistances among drugs.

Persistent But Not Replicating HIV-1 Cell-Associated DNA in Semen of Long-Term ART Experienced Men by Amandine Gagneux-Brunon, Dorsaf Nasri, Rachel Terrasse, Karine Sauné, Marie-Claude Gagnieu, Anne Frésard, Olivier Delezay, Jacques Izopet, Bruno Pozzetto, Frédéric Lucht, Thomas Bourlet (316-323).
Background: The semen of HIV-1 infected men represents the main vector of HIV-1 spread following sexual transmission of cell-free or cell-associated virions.
Objective: The present study aimed to assess the impact of HAART on HIV-1 RNA/DNA and on inflammatory environment in the semen of long-term HAART-experienced men.
Methods: Forty-five paired samples of semen and blood were obtained from 37 consenting men, 10 untreated and 27 under HAART. Blood and seminal HIV RNA and DNA loads were quantified by the Abbott RealTime m2000rt assay and an inhouse real-time PCR protocol, respectively. Tat/rev/nef intra-cellular mRNA was tested by qualitative PCR. Interleukin (IL)- 1?, IL-2, IL-6, IL-7, IL-8, IL-10, GM-CSF and TNF? were quantified in 20 paired samples by Bio-plex® assay.
Results: No semen was found HIV RNA positive in men under HAART. Twenty-six percent of semen samples from HAART-experienced men remained positive for HIV DNA. Seminal HIV DNA was significantly associated with the duration of infection and the HIV DNA load in blood. No seminal mononuclear cells were found positive for intracellular HIV RNA in HAART experienced men. All the tested chemokines exhibited significantly higher concentration in semen than in blood in both treated and untreated men. No effect of HAART on cytokines/chimiokines loads was observed.
Conclusion: These results demonstrate the efficacy of HAART on the reduction of seminal RNA HIV-1 loads despite the persistence of local inflammation. Moreover, in our hands the seminal cell-associated virus reservoir was not reactivated in an inflammatory environment was not productive and its reactivation seems unlikely.

Lower CSF A? is Associated with HAND in HIV-Infected Adults with a Family History of Dementia by Pariya. L. Fazeli, David J. Moore, Donald R. Franklin, Anya Umlauf, Robert K. Heaton, Ann C. Collier, Christina M. Marra, David B. Clifford, Benjamin B. Gelman, Ned C. Sacktor, Susan Morgello, David M. Simpson, John A. McCutchan, Igor Grant, Scott L. Letendre (324-330).
Background: Both family history of dementia (FHD) and lower levels of A?-42 are indepentently associated with worse neurocognitive functioning in HIVinfected patients.
Objective: To examine the relationships between cerebrospinal fluid (CSF) A?-42 and FHD with HIV-associated neurocognitive disorders (HAND).
Methods: One hundred eighty-three HIV+ adults underwent neuropsychological and neuromedical assessments, and determination of CSF A?-42 concentration and FHD (defined as a self-reported first or second-degree relative with a dementia diagnosis). Univariate analyses and multivariable logistic regressions were used.
Results: FHD was not associated with HAND (p = 0.24); however, CSF A?-42 levels were lower (p = 0.03) in the HAND group, but were not associated with FHD (p = 0.89). Multivariable models showed a main effect of CSF A?-42 (p = 0.03) and a trend-level (p = 0.06) interaction between FHD and CSF A?-42, such that lower CSF A?-42 was associated with HAND in those with FHD (p < 0.01) compared to those without FHD (p = 0.83). An analysis in those with follow-up data showed that higher baseline CSF A?-42 was associated with lower risk of neurocognitive decline (p = 0.02). While we did not find an FHD X CSF A?-42 interaction (p = 0.83), when analyses were stratified by FHD, lower CSF A?-42 was associated at the trend-level with neurocognitive decline in the FHD group (p = 0.08) compared to the no FHD group (p = 0.15).
Conclusion: FHD moderates the relationship between of CSF A?-42 and HAND. The findings highlight the complexities in interpreting the relationships between biomarkers of age-related neurodegeneration and HAND.

Blood Myeloid Dendritic Cells and slanDC in Antiretroviral Therapy- Suppressed HIV-Infected Patients by Miriam Lichtner, Raffaella Rossi, Serena Vita, Stefano Savinelli, Marco Iannetta, Claudia Mascia, Raffaella Marocco, Camilla Ajassa, Fabio Mengoni, Laura Scorzolini, Claudio Maria Mastroianni, Vincenzo Vullo (331-339).
Myeloid dendritic cells (mDCs) play a complex role in HIV infection regardless of viral replication. The aim of our study was to analyse mDCs in long term antiretroviral therapy (ART)- suppressed HIV-infected patients. We evaluated the numbers of mDCs and slanDC in the context of different degree of CD4+ T cell recovery, persistent T cell activation (as HLA-DR+/CD38+ expression) and monocyte-macrophage activation assessed in terms of circulating levels of both sCD14 and sCD163. We enrolled 72 aviremic patients under effective ART and 34 healthy donors (HD). Patients were divided into two groups on the bases of ?CD4, indicating the difference between the value of CD4 at the time of sampling and CD4 nadir. Higher levels of mDCs and slanDC were found in patients with ?CD4>200/mmc in comparison to HD. In those patients also an increased level of sCD14 was found, whereas sCD163 seemed to be at normal levels. An augmentation of activated CD4 T lymphocytes was found, although less pronounced in patients with ?CD4<200/mmc. In conclusion, our findings showed an expansion of mDCs with a shift to inflammatory slanDC that could sustain both microbial translocation and HIV latency in CD4 T cells.

Chemokines SNPs in HIV-1+ Patients and Healthy Controls from Northeast Brazil: Association with Protection against HIV-1 Infection by Ronaldo Celerino da Silva, Antonio Victor Campos Coelho, Luiz Cl&#225;udio Arraes, Lucas Andr&#233; Cavalcanti Brand&#227;o, Rafael Lima Guimar&#227;es, Sergio Crovella (340-345).
Background: HIV-1 virus is known to infect the host mainly through CD4+ T-lymphocyte cells, by interactions among the viral envelope proteins, CD4 receptor and HIV-1 coreceptors, such as chemokines receptors. Variations in the genes encoding HIV-1 coreceptors and their natural ligands have been shown to modify HIV-1 infection susceptibility and disease progression.
Methods and Results: We analysed the distribution of SNPs in chemokines (CCL3, CCL4, CCL5, CXCL12) and chemokine receptor (CXCR6) genes, in 268 HIV-1 infected patients (HIV-1+) and 221 healthy controls from Northeast Brazil, and their possible connection with susceptibility to HIV-1 infection. The genotyping were performed through allele specific fluorogenic probes using real time PCR. We observed that the T alleles and AT genotype of rs1719153 CCL4 SNP were more frequent in healthy controls (19.8% and 35.0%, respectively) than in HIV-1+ patients (T allele: 14.1%; OR=0.67; 95%CI=0.47-0.95; p-value=0.020; and AT genotype: 24.4%; OR=0.61; 95%CI=0.40- 0.93; p-value=0.021) after correcting for age and sex. The rs1719134 (CCL3) and rs1719153 (CCL4) SNPs presented linkage disequilibrium (D'=0.83). The AT haplotype frequency was increased in healthy controls (17.3%) in relation to HIV-1+ patients (11.0%; OR=0.62; 95%CI=0.42-0.93; p-value=0.020).
Conclusion: Since our results revealed an increased frequency of alleles and genotypes of CCL3/CCL4 SNPs and haplotype (CCL3-CCL4) among healthy controls, we suggest that these variations might have a potential protective role against HIV-1 infection.

Disease Progression in HIV Late Presenters: the Role of HIV Clinical Indicator Diseases Prior to HIV Diagnosis by Viola Guardigni, Mario Luca Morieri, Daniela Segala, Laura Sighinolfi (346-353).
Background: Late diagnosis represents a major challenge in the control of HIV epidemics. The rate of disease progression is higher among late presenters. In Europe, HIV Clinical Indicator Diseases (CIDs) have been proposed to improve early diagnosis.
Objectives: Our observational study evaluated the presence of these HIV CIDs prior to HIV diagnosis among a population of late presenters and assessed its correlation to disease progression.
Method: A retrospective cohort study was conducted in HIV late presenters diagnosed from 2007 to 2013 at University Hospital of Ferrara (Italy). Hazard Ratios (H.R.s) for disease progression (new AIDS-events and death) were estimated by Cox proportional hazard model.
Results: We analysed 77 patients and we found that those with CIDs prior to HIV diagnosis (22%) had a 2.8 fold higher rate of disease progression compared to those without HIV CIDs (H.R. 2.82; 95% CI 1.21-6.53; P 0.02). Other factors associated with disease progression were AIDS presentation, HCV coinfection and Haemoglobin levels, with H.R.s of 3.14 (95%CI 1.23-7.99), 2.95 (95% CI 1.14-7.61) and 0.74 (95% CI 0.60-0.91), respectively.
Conclusion: HIV CIDs confer a higher risk for disease progression even after adjustment for these confounding factors. Evaluation of previous HIV CIDs at HIV diagnosis could be an additional tool to identify and better manage HIV late presenters with higher risks of disease progression.

Assessment of the Accuracy of Whole Blood/Serum Rapid Point-of-Care HIV Three Dot Test for Oral Fluid Specimens by N. Rakesh, Shakilla Shetty, S. Sujatha, Shivani Sharma, Ankit Saxena (354-359).
Background: Saliva rapid point of care HIV tests have proven advantages over blood-based HIV tests in terms of quality, rapidity and convenience.
Aim: To assess the sensitivity, specificity and reproducibility of saliva samples using the serum/ whole blood rapid test and to compare it with serum specimens.
Material & Methods: 52 seropositive and 52 seronegative patients were included in the study. Stimulated and unstimulated saliva samples were collected and tested using the serum/ whole blood signal HIV THREE DOT rapid test (span diagnostics).
Results: The sensitivity, specificity, positive predictive value and negative predictive value of the test was found to be 100% for saliva samples.
Conclusion: Saliva samples can be used as a substitute to serum/whole blood for HIV testing. It can be done using the serum/whole blood kits which are cheaper and readily available thus broadening the reach of testing programs in resource limited settings.

Prevalence of Drug Resistance Associated Mutations Among the Anti Retroviral Therapy Exposed HIV-1 Infected Individuals in Manipur, Northeast India by Adhikarimayum Lakhikumar Sharma, Thiyam Ramsing Singh, Khuraijam Ranjana Devi, Lisam Shanjukumar Singh (360-370).
Background: Manipur is one of the highest HIV prevalence states of India because of its geographical location at the international border near the golden triangle of South-East Asia, but no study on drug resistance associated mutations (DRAMs) has been reported yet.
Objective: A population-based study on DRAMs of HIV-1 among the anti-retroviral therapy (ART) exposed HIV-1 infected individuals of Manipur was conducted.
Methods: 110 HIV-1 positive individuals who had initially exposed to first line anti-HIV drugs were recruited for the surveillance of DRAMs. Reverse transcriptase and protease genes of HIV-1 were amplified, sequenced and analyzed.
Results: Significant prevalence of DRAMs of HIV-1 was found among the ART exposed HIV-1 infected individuals of Manipur. The results revealed that 37%, 29% and 7% individuals harbor HIV-1 strains mutated at the target sites of nonnucleoside reverse transcriptase inhibitors, nucleoside reverse transcriptase inhibitors and protease inhibitors respectively. Predominant DRAMs at RT genes were M184V, T215Y, M41L and V108I and H221Y while at PR genes were M46I and I47V. Among the high risk groups, intravenous drug users have the highest number of DRAMs followed by heterosexual individuals. Analysis of viral subtype based on pol gene revealed 83% subtype C, 11.8% recombinant forms and 5.2% subtype B.
Conclusion: DRAMs at the target sites of reverse transcriptase inhibitors are high and these were found to have developed resistance to the primary ART drugs that are used in Manipur. The findings of this study will help the clinicians to guide patients during the course of ART treatment regimes.