HIV-1 tropism determination using a phenotypic Env recombinant viral assay highlights overestimation of CXCR4-usage by genotypic prediction algorithms for CRRF01_AE and CRF02_AG

Background: Human Immunodeficiency virus type-1 (HIV) entry into target cells involves binding of the viral envelope (Env) to CD4 and a coreceptor, mainly CCR5 or CXCR4. The only currently licensed HIV entry inhibitor, maraviroc, targets CCR5, and the presence of CXCX4-using strains must be excluded prior to treatment. Co-receptor usage can be assessed by phenotypic assays or through genotypic prediction. Here we compared the performance of a phenotypic Env-Recombinant Viral Assay (RVA) to the two most widely used genotypic prediction algorithms, Geno2Pheno([coreceptor]) and webPSSM. Methods: Co-receptor tropism of samples from 73 subtype B and 219 non-B infections was measured phenotypically using a luciferase-tagged, NL4-3-based, RVA targeting Env. In parallel, tropism was inferred genotypically from the corresponding V3-loop sequences using Geno2Pheno([coreceptor]) (5-20% FPR) and webPSSM-R5X4. For discordant samples, phenotypic outcome was retested using co-receptor antagonists or the validated Trofile (R) Enhanced-Sensitivity-Tropism-Assay. Results: The lower detection limit of the RVA was 2.5% and 5% for X4 and R5 minority variants respectively. A phenotype/genotype result was obtained for 210 samples. Overall, concordance of phenotypic results with Geno2Pheno([coreceptor]) was 85.2% and concordance with webPSSM was 79.5%. For subtype B, concordance with Geno2pheno([coreceptor]) was 94.4% and concordance with webPSSM was 79.6%. High concordance of genotypic tools with phenotypic outcome was seen for subtype C (90% for both tools). Main discordances involved CRF01_AE and CRF02_AG for both algorithms (CRF01_AE: 35.9% discordances with Geno2Pheno([coreceptor]) and 28.2% with webPSSM; CRF02_AG: 20.7% for both algorithms). Genotypic prediction overestimated CXCR4-usage for both CRFs. For webPSSM, 40% discordance was observed for subtype A. Conclusions: Phenotypic assays remain the most accurate for most non-B subtypes and new subtype-specific rules should be developed for non-B subtypes, as research studies more and more draw conclusions from genotypically-inferred tropism, and to avoid unnecessarily precluding patients with limited treatment options from receiving maraviroc or other entry inhibitors

Differences in Reversion of Resistance Mutations to Wild-Type under Structured Treatment Interruption and Related Increase in Replication Capacity

The CPCRA 064 study examined the effect of structured treatment interruption (STI) of up to 4 months followed by salvage treatment in patients failing therapy with multi-drug resistant HIV. We examined the relationship between the reversion rate of major reverse transcriptase (RT) resistance-associated mutations and change in viral replication capacity (RC). The dataset included 90 patients with RC and genotypic data from virus samples collected at 0 (baseline), 2 and 4 months of STI.Rapid shift towards wild-type RC was observed during the first 2 months of STI. Median RC increased from 47.5% at baseline to 86.0% at 2 months and to 97.5% at 4 months. Between baseline and 2 months of STI, T215F had the fastest rate of reversion (41%) and the reversion of E44D and T69D was associated with the largest changes in RC. Among the most prevalent RT mutations, M184V had the fastest rate of reversion from baseline to 2 months (40%), and its reversion was associated with the largest increase in RC. Most rates of reversion increased between 2 months and 4 months, but the change in RC was more limited as it was already close to 100%. The highest frequency of concurrent reversion was found for L100I and K103N. Mutagenesis tree models showed that M184V, when present, was overall the first mutation to revert among all the RT mutations reported in the study.Longitudinal analysis of combined phenotypic and genotypic data during STI showed a large amount of variability in prevalence and reversion rates to wild-type codons among the RT resistance-associated mutations. The rate of reversion of these mutations may depend on the extent of RC increase as well as the co-occurring reversion of other mutations belonging to the same mutational pathway

Determinação do tropismo viral por ensaios genotípicos e fenotípicos em pacientes brasileiros infectados por HIV-1

The clinical application of CCR5 antagonists involves first determining the coreceptor usage by the infecting viral strain. Bioinformatics programs that predict coreceptor usage could provide an alternative method to screen candidates for treatment with CCR5 antagonists, particularly in countries with limited financial resources. Thus, the present study aims to identify the best approach using bioinformatics tools for determining HIV-1 coreceptor usage in clinical practice. Proviral DNA sequences and Trofile results from 99 HIV-1-infected subjects under clinical monitoring were analyzed in this study. Based on the Trofile results, the viral variants present were 81.1% R5, 21.4% R5X4 and 1.8% X4. Determination of tropism using a Geno2pheno[coreceptor] analysis with a false positive rate of 10% gave the most suitable performance in this sampling: the R5 and X4 strains were found at frequencies of 78.5% and 28.4%, respectively, and there was 78.6% concordance between the phenotypic and genotypic results. Further studies are needed to clarify how genetic diversity amongst virus strains affects bioinformatics-driven approaches for determining tropism. Although this strategy could be useful for screening patients in developing countries, some limitations remain that restrict the wider application of coreceptor usage tests in clinical practice.A aplicação clínica dos antagonistas de CCR5 envolve em primeiro lugar determinar o uso de co-receptor pela cepa viral infectante. Programas de bioinformática que prevêem o uso co-receptor poderiam fornecer um método alternativo para selecionar candidatos para o tratamento com os antagonistas do CCR5, particularmente em países com poucos recursos financeiros. Assim, o presente estudo teve por objetivo identificar a melhor abordagem utilizando ferramentas de bioinformática para determinar qual o tipo de co-receptor do HIV-1 que poderia ser usado na prática clínica. Sequências de DNA proviral e Trofile resultados a partir de 99 pacientes infectados pelo HIV-1 sob monitorização clínica foram avaliadas. Com base nos resultados do Teste Trofile, as variantes virais presentes eram R5 (81,1%), R5X4 (21,4%) e X4 (1,8%). Determinação do tropismo pela análise do Geno2pheno, com taxa de falso positivos de 10% apresentou desempenho mais adequado para esta amostragem: as cepas R5 e X4 foram encontradas em frequências de 78,5% e 28,4%, respectivamente, e foi de 78,6% a concordância entre os resultados fenotípicos e genotípicos. Mais estudos são necessários para esclarecer como a diversidade genética entre as cepas do vírus afeta abordagens baseadas na determinação do tropismo pelas ferramentas de bioinformática. Embora esta estratégia possa ser útil para o rastreio de pacientes em países em desenvolvimento, permanecem algumas limitações que restringem a aplicação mais ampla para utilização de testes de co-receptor na prática clínica

Active Methamphetamine Use is Associated with Transmitted Drug Resis-tance to Non-Nucleoside Reverse Transcriptase Inhibitors in Individuals with HIV Infection of Unknown Duration

BackgroundFrequent methamphetamine use among recently HIV infected individuals is associated with transmitted drug resistance (TDR) to non-nucleoside reverse transcriptase inhibitors (NNRTI); however, the reversion time of TDR to drug susceptible HIV may exceed 3 years. We assessed whether recreational substance use is associated with detectable TDR among individuals newly diagnosed with HIV infection of unknown duration.DesignCross-sectional analysis.MethodsSubjects were enrolled at the University California, San Diego Early Intervention Program. Demographic, clinical and substance use data were collected using structured interviews. Genotypic resistance testing was performed using GeneSeq, Monogram Biosciences. We analyzed the association between substance use and TDR using bivariate analyses and the corresponding transmission networks using phylogenetic models.ResultsBetween April 2004 and July 2006, 115 individuals with genotype data were enrolled. The prevalence of alcohol, marijuana and methamphetamine use were 98%, 71% and 64% respectively. Only active methamphetamine use in the 30 days prior to HIV diagnosis was independently associated with TDR to NNRTI (OR: 6.6; p=0.002).ConclusionDespite not knowing the duration of their HIV infection, individuals reporting active methamphetamine use in the 30 days prior to HIV diagnosis are at an increased risk of having HIV strains that are resistant to NNRTI

Drug resistance and viral tropism in HIV-1 subtype C-infected patients in KwaZulu-Natal, South Africa: implications for future treatment options

Article approval pendingDrug resistance poses a significant challenge for the successful application of highly active antiretroviral therapy (HAART) globally. Furthermore, emergence of HIV-1 isolates that preferentially use CXCR4 as a coreceptor for cell entry, either as a consequence of natural viral evolution or HAART use, may compromise the efficacy of CCR5 antagonists as alternative antiviral therapy

HIV-1 Tropism Testing in Subjects Achieving Undetectable HIV-1 RNA : Diagnostic Accuracy, Viral Evolution and Compartmentalization

Technically, HIV-1 tropism can be evaluated in plasma or peripheral blood mononuclear cells (PBMCs). However, only tropism testing of plasma HIV-1 has been validated as a tool to predict virological response to CCR5 antagonists in clinical trials. The preferable tropism testing strategy in subjects with undetectable HIV-1 viremia, in whom plasma tropism testing is not feasible, remains uncertain. We designed a proof-of-concept study including 30 chronically HIV-1-infected individuals who achieved HIV-1 RNA <50 copies/mL during at least 2 years after first-line ART initiation. First, we determined the diagnostic accuracy of 454 and population sequencing of gp120 V3-loops in plasma and PBMCs, as well as of MT-2 assays before ART initiation. The Enhanced Sensitivity Trofile Assay (ESTA) was used as the technical reference standard. 454 sequencing of plasma viruses provided the highest agreement with ESTA. The accuracy of 454 sequencing decreased in PBMCs due to reduced specificity. Population sequencing in plasma and PBMCs was slightly less accurate than plasma 454 sequencing, being less sensitive but more specific. MT-2 assays had low sensitivity but 100% specificity. Then, we used optimized 454 sequence data to investigate viral evolution in PBMCs during viremia suppression and only found evolution of R5 viruses in one subject. No de novo CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized. The absence of tropism shifts during viremia suppression suggests that, when available, testing of stored plasma samples is generally safe and informative, provided that HIV-1 suppression is maintained. Tropism testing in PBMCs may not necessarily produce equivalent biological results to plasma, because the structure of viral populations and the diagnostic performance of tropism assays may sometimes vary between compartments. Thereby, proviral DNA tropism testing should be specifically validated in clinical trials before it can be applied to routine clinical decision-making

HIV Tropism and Decreased Risk of Breast Cancer

During the first two decades of the U.S. AIDS epidemic, and unlike some malignancies, breast cancer risk was significantly lower for women with human immunodeficiency virus (HIV) infection compared to the general population. This deficit in HIV-associated breast cancer could not be attributed to differences in survival, immune deficiency, childbearing or other breast cancer risk factors. HIV infects mononuclear immune cells by binding to the CD4 molecule and to CCR5 or CXCR4 chemokine coreceptors. Neoplastic breast cells commonly express CXCR4 but not CCR5. In vitro, binding HIV envelope protein to CXCR4 has been shown to induce apoptosis of neoplastic breast cells. Based on these observations, we hypothesized that breast cancer risk would be lower among women with CXCR4-tropic HIV infection.We conducted a breast cancer nested case-control study among women who participated in the WIHS and HERS HIV cohort studies with longitudinally collected risk factor data and plasma. Cases were HIV-infected women (mean age 46 years) who had stored plasma collected within 24 months of breast cancer diagnosis and an HIV viral load≥500 copies/mL. Three HIV-infected control women, without breast cancer, were matched to each case based on age and plasma collection date. CXCR4-tropism was determined by a phenotypic tropism assay. Odds ratios (OR) and 95% confidence intervals (CI) for breast cancer were estimated by exact conditional logistic regression. Two (9%) of 23 breast cancer cases had CXCR4-tropic HIV, compared to 19 (28%) of 69 matched controls. Breast cancer risk was significantly and independently reduced with CXCR4 tropism (adjusted odds ratio, 0.10, 95% CI 0.002-0.84) and with menopause (adjusted odds ratio, 0.08, 95% CI 0.001-0.83). Adjustment for CD4+ cell count, HIV viral load, and use of antiretroviral therapy did not attenuate the association between infection with CXCR4-tropic HIV and breast cancer.Low breast cancer risk with HIV is specifically linked to CXCR4-using variants of HIV. These variants are thought to exclusively bind to and signal through a receptor that is commonly expressed on hyperplastic and neoplastic breast duct cells. Additional studies are needed to confirm these observations and to understand how CXCR4 might reduce breast cancer risk

GRAPE’S LEATHER AND SEED EXTRACT (VITIS VINIFERA) IMPROVING THE FUNCTION OF WISTAR RATS’ MOTOR (RATTUS NORVEGICUS) ISCHEMIC STROKE MODEL

Background. Grape peel and seed extract (Vitis vinifera), that has resveratrol, is one of many antioxidants that can pass through blood brain barrier and can induce release neurotrophic factor that contribute in ERK 1/2 pathway mechanism in post stroke. Objective. To prove that grape peel and seed extract can regenerate neuron in brain functional Methods. True experimental design with five groups in this research. The five groups are negative control, positive control, grape peel and seed extract 50mg/KgBW, 100mg/KgBW, and 200mg/KgBW. rats are given grape peel and seed extract in variable dose to know how extract’s effect in neuron repairment. The repairment is monitored from ladder rung walking test score. Results. Range average score ladder rung walking test post stroke dan post treatment group N, K, Ra, Rb, dan Rc, were 0 ± 0, 0.001028933 ± 0.011664445, 0.123214286 ± 0.019834983, 0.064744427 ± 0.024296721, 0.03781401 ± 0.006888803. Statistical test used Annova significantly p;0,001.Dose 50mg/KgBW is effective in repairing neuron. Conclusion. Grape’s leather and Seed extract 50 mg/kgBW can improve neuron regeneration on animal model

BRAIN NEURON REGENERATION IN POST-STROKE REHABILITATION TREATMENT USING GRAPE PEEL AND SEED EXTRACT (Vitis vinifera) IN INDUCING ERK1 / 2 PATHWAY

Background and aims. Ischemic stroke can cause hypoxia in brain tissue and damage the neuron. The defense ability and plasticity is activated by ERK1/2 pathway. Grape peel and seed extract (Vitis vinifera can induce release neurotrophic factor that contribute in ERK1/2 pathway mechanism. This research aiming to prove that grape peel and seed extract can regenerate neuron in anatomical and functional view. Method. This research use true experimental design with sample in this research is 20 male 8-10 weeks old wistar strain rats which are induced stroke by internal and external carotid artery occlusion method. There are five groups in this research, negative control, positive control, grape peel and seed extract 50mg/KgBW, 100mg/KgBW, and 200mg/KgBW. After reassurement that rat suffer from stroke with MMP9, cylinder test and ladder rung walking test, rats are given grape peel and seed extract in variable dose to know how extract’s effect in neuron repairment. Results. The repairment is monitored from four parameters, namely cylinder test score, ladder rung walking test score, extensive infarct volume, and number of damaged neuron. Conclusion. The result is 50mg/KgBW is effective in repairing neuron. It is seen from the result of improvement in four parameters explained above