Evolução de pacientes com AIDS pós cART: Evolução clínica e laboratorial de pacientes com AIDS após 48 semanas de tratamento antirretroviral

Combination Antiretroviral Therapy (cART) aims to inhibit viral replication, delay immunodeficiency progression and improve survival in AIDS patients. The objective of this study was to compare two different schemes of cART, based on plasma viral load (VL) and CD4+ T lymphocyte count, during 48 weeks of treatment. For this purpose, 472 medical charts of a Specialized Outpatient Service were reviewed from 1998 to 2005. Out of these, 58 AIDS patients who had received a triple drug scheme as the initial treatment were included in the study and two groups were formed: Group 1 (G1): 47 individuals treated with two nucleoside reverse-transcriptase inhibitors (NRTI) and one non-nucleoside reverse-transcriptase inhibitor; Group 2 (G2): 11 patients treated with two NRTI and one protease inhibitor. In G1 and G2, 53.2% and 81.8% respectively were patients with an AIDS-defining disease. The T CD4+ lymphocyte count increased progressively up until the 24th week of treatment in all patients, while VL became undetectable in 68.1% of G1 and in 63.6% of G2. The study concluded that the evolutions of laboratory tests were similar in the two treatment groups and that both presented a favorable clinical evolution.

Avaliação da resistência primária aos inibidores de integrase em pacientes soropositivos para o HIV-1

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

Caracterização da estrutura da serino-protease NS3 em pacientes infectados com o vírus da hepatite C do genótipo 3

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Genotipagem do HIV-1 no Pará em pacientes experimentando falha terapêutica antirretroviral

Terapia supressiva antirretroviral reduz significativamente morbidades e mortalidade relacionadas ao HIV, mas a emergência de vírus resistentes pode limitar o sucesso do tratamento. Objetivou-se neste estudo descrever, em portadores de HIV/sida experimentando falha à terapia antirretroviral (TARV), no estado do Pará, a prevalência de mutações nas enzimas transcriptase reversa e protease do HIV-1 e correlacioná-las à resistência aos antirretrovirais (ARV). Foi um estudo descritivo, retrospectivo, do tipo transversal, com dados obtidos na Unidade de Referência Especializada em Doenças Infecciosas e Parasitárias Especiais de Belém do Pará, de pacientes com perfil laboratorial de falha terapêutica. A presente amostra incluiu genotipagem de cinquenta pacientes no período de janeiro de 2004 a dezembro de 2005. Os critérios de inclusão foram: adesão à terapia imediata a genotipagem, falha terapêutica, perfil de resistência viral à TARV e ser paciente da rede pública de saúde. Foram descritos aspectos demográficos da população estudada, perfil de uso de TARV previamente a genotipagem, tempo conhecido de infecção pelo HIV, perfil quantitativo de células CD4+ e de carga viral, além do teste de genotipagem realizado. A resistência encontrada predominou em pacientes residentes em Belém (72%), no sexo masculino (90%) e na faixa etária de 30 a 49 anos de idade. As maiores prevalências de mutações na transcriptase reversa do HIV-1 foram: 214F (86%), 184V (76%), 215FY (56%), 211K (48%), 219QEN, 67N e 103N (42%) cada, 41L (32%), 70R (28%) e 210W (20%). Na protease 46IL (38%), 90M (32%) e 82AFT (20%) foram as mais prevalentes dentre as mutações principais e, dentre as secundárias, 63P (74%), 93LM (52%), 10FIV (48%) e 35D (46%). Atribuiu-se estas mutações a pressão seletiva dos ARV mais utilizados: 3TC, AZT, D4T, DDI, EFZ, IDV, NFV, RTV e SQV. O uso de múltiplos esquemas ARV, favoreceu a prevalência das mutações encontradas. Houve impacto dentro das classes com 32% de resistência completa a uma classe, 22% a duas classes e 4% a três classes. Conclui-se que pacientes expostos a única TARV previamente à genotipagem comparados aos expostos a mais de uma TARV, apresentaram menor prevalência de resistência aos ARV, com possibilidade de resgate terapêutico com ARV ativos disponíveis na época que, entretanto foi a minoria.

Caracterização molecular da resistência genotípica secundária aos antirretrovirais em pacientes com Aids e prevalência de subtipos do HIV-1 nos Estados do Pará e Amazonas, Brasil: 2002 a 2006

A resistência às drogas antirretrovirais é o inevitável resultado da incompleta supressão da replicação do HIV-1. No presente estudo foi caracterizado o perfil de resistência genética aos antirretrovirais em amostras sorológicas provenientes dos estados do Amazonas e Pará, Região Norte do Brasil, no período de 2002 a 2006. Um total de 127 amostras de plasmas obtidas de pacientes HIV positivos e/ou Aids foram submetidas ao teste de resistência pelo ViroSeq^TM Genotyping System (Celera Diagnostic-Abbott, USA). Considerando a informação genética derivada das regiões da protease e/ou transcriptase reversa do HIV-1, o subtipo B foi observado em 85% dos casos; seguido por ambos subtipo F1 e forma recombinante BF1 (4,6%) e CF1 (0,8%). A mutação M184V (81,1%) foi a mais comumente observada associada aos NRTI, em indivíduos positivos com TARV no estado do Pará, e a mutação T215F/Y (56,3%) em indivíduos do estado do Amazonas. A mutação K103N foi a mais prevalente (em torno de 33,5%) para os NNRTI em ambos os estados. O perfil de mutação de resistência associado ao gene da protease mostrou a mutação minor L63P como a mais frequente em ambos os estados. O estudo revelou a importância da identificação de mutações associadas com resistência às drogas antirretrovirais para o uso em futuros esquemas terapêuticos. Os resultados deste estudo foram similares aos outros realizados em várias regiões do Brasil.

Epidemiologia molecular do Vírus da imunodeficiência humana 1 (HIV-1) no município de Macapá (Amapá), Brasil

Uma das principais características do Vírus da imunodeficiência humana (HIV) é sua grande diversidade genética. O presente trabalho tem como objetivo descrever a epidemiologia molecular do HIV-1 circulante na cidade Macapá, Amapá, assim como os fatores associados à aquisição da infecção e correlacionar estes com os subtipos virais encontrados e as informações epidemiológicas da população examinada. Amostras de sangue de 48 indivíduos portadores do HIV foram colhidas no Serviço de Assistência Especializada (SAE) da cidade de Macapá, no período de junho de 2003 a junho de 2004. Após a extração do DNA, foi realizada a amplificação de 297 pb da protease (gene pro) por meio da técnica de Nested PCR, sendo seqüenciadas, posteriormente, para a determinação dos subtipos virais. Foram identificados os subtipos B (93,7%) e F (6,3%) na cidade de Macapá, que são os mais prevalentes no Brasil, não tendo sido observada associação entre os subtipos do HV-1 circulantes nesta cidade e a preferência sexual. No entanto, uma parcela significativa da população examinada possui um baixo grau de escolaridade, característica esta que reflete a maior parcela população infectada pelo HIV no Brasil.

Perfil de mutações e resistência do Vírus da Imunodeficiência Humana (HIV) em pacientes coinfectados com o Vírus da Hepatite B (VHB)

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance

Background: Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance. Methods/Results: We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in Sao Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naive individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples. Conclusion: The developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3-5 x less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance.

Lipid levels in the second year of life among HIV-infected and HIV-exposed uninfected Latin American children

Background: Dyslipidemia is observed among older children and adults with HIV. We examined nonfasting cholesterol and triglycerides in two groups of 12-23-month-old Latin American children - HIV-infected vs. HIV-exposed but uninfected (HEU). Methods: HIV-infected and HEU children in Latin America and Jamaica were enrolled in an observational cohort. Eligibility for this analysis required having cholesterol and triglyceride results available during the second year of life. Results: HIV-infected (n = 83) children were slightly older at the time of lipid testing than the HEU (n 681). Forty percent of the HIV-infected children were on protease inhibitor-based antiretroviral therapy (ART); 41% were not on ART. There was no statistically significant difference in mean cholesterol concentrations (mg/dl) by HIV status; however, the HIV-infected children had higher mean triglyceride concentrations. The prevalence of high cholesterol (>200 mg/dl) and high triglycerides (>110 mg/dl) was higher among the HIV-infected vs. HEU. Among the HIV-infected children, mean cholesterol and triglyceride concentrations varied by ART. Children receiving no ART had a significantly lower mean cholesterol concentration. Those receiving protease inhibitor-containing ART had a significantly higher mean triglyceride concentration compared to the other two antiretroviral regimen groups. Conclusion: A greater proportion of HIV-infected children at 12-23 months have hyperlipidemia when compared to HEU children, with the highest triglyceride concentrations observed among those receiving protease inhibitor-containing ART, and the lowest cholesterol levels among those not receiving ART. Implications of these findings will require continued follow-up of HIV-infected children who initiate therapy early in life. (C) 2012 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

Lufaxin, a Novel Factor Xa Inhibitor From the Salivary Gland of the Sand Fly Lutzomyia longipalpis Blocks Protease-Activated Receptor 2 Activation and Inhibits Inflammation and Thrombosis In Vivo

Objective-Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Methods and Results-Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant approximate to 3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl3-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. Conclusion-Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events. (Arterioscler Thromb Vasc Biol. 2012;32:2185-2196.)

BF Integrase Genes of HIV-1 Circulating in Sao Paulo, Brazil, with a Recurrent Recombination Region

Although some studies have shown diversity in HIV integrase (IN) genes, none has focused particularly on the gene evolving in epidemics in the context of recombination. The IN gene in 157 HIV-1 integrase inhibitor-naive patients from the Sao Paulo State, Brazil, were sequenced tallying 128 of subtype B (23 of which were found in non-B genomes), 17 of subtype F (8 of which were found in recombinant genomes), 11 integrases were BF recombinants, and 1 from subtype C. Crucially, we found that 4 BF recombinant viruses shared a recurrent recombination breakpoint region between positions 4900 and 4924 (relative to the HXB2) that includes 2 gRNA loops, where the RT may stutter. Since these recombinants had independent phylogenetic origin, we argue that these results suggest a possible recombination hotspot not observed so far in BF CRF in particular, or in any other HIV-1 CRF in general. Additionally, 40% of the drug-naive and 45% of the drug-treated patients had at least 1 raltegravir (RAL) or elvitegravir (EVG) resistance-associated amino acid change, but no major resistance mutations were found, in line with other studies. Importantly, V151I was the most common minor resistance mutation among B, F and BF IN genes. Most codon sites of the IN genes had higher rates of synonymous substitutions (dS) indicative of a strong negative selection. Nevertheless, several codon sites mainly in the subtype B were found under positive selection. Consequently, we observed a higher genetic diversity in the B portions of the mosaics, possibly due to the more recent introduction of subtype F on top of an ongoing subtype B epidemics and a fast spread of subtype F alleles among the B population.

Pilot Studies for Development of an HIV Subtype Panel for Surveillance of Global Diversity

The continued global spread and evolution of HIV diversity pose significant challenges to diagnostics and vaccine strategies. NIAID partnered with the FDA, WRAIR, academia, and industry to form a Viral Panel Working Group to design and prepare a panel of well-characterized current and diverse HIV isolates. Plasma samples that had screened positive for HIV infection and had evidence of recently acquired infection were donated by blood centers in North and South America, Europe, and Africa. A total of 80 plasma samples were tested by quantitative nucleic acid tests, p24 antigen, EIA, and Western blot to assign a Fiebig stage indicative of approximate time from initial infection. Evaluation of viral load using FDA-cleared assays showed excellent concordance when subtype B virus was tested, but lower correlations for subtype C. Plasma samples were cocultivated with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) from normal donors to generate 30 viral isolates (50-80% success rate for samples with viral load >10,000 copies/ml), which were then expanded to 10(7)-10(9) virus copies per ml. Analysis of env sequences showed that sequences derived from cultured PBMCs were not distinguishable from those obtained from the original plasma. The pilot collection includes 30 isolates representing subtypes B, C, B/F, CRF04_cpx, and CRF02_AG. These studies will serve as a basis for the development of a comprehensive panel of highly characterized viral isolates that reflects the current dynamic and complex HIV epidemic, and will be made available through the External Quality Assurance Program Oversight Laboratory (EQAPOL).

Analysis of Transmitted Resistance to Raltegravir and Selective Pressure among HIV-1-Infected Patients on a Failing HAART in Sao Paulo, Brazil

We studied the presence of primary resistance to raltegravir (RAL), natural polymorphisms, and selection pressure on HIV-1 integrase. We found a high frequency of integrase polymorphisms related to the resistance to RAL and sequence stability. Further studies are needed to determine the importance of these polymorphisms to RAL resistance.

Influence of HAART on Alternative Reading Frame Immune Responses over the Course of HIV-1 Infection

Background: Translational errors can result in bypassing of the main viral protein reading frames and the production of alternate reading frame (ARF) or cryptic peptides. Within HIV, there are many such ARFs in both sense and the antisense directions of transcription. These ARFs have the potential to generate immunogenic peptides called cryptic epitopes (CE). Both antiretroviral drug therapy and the immune system exert a mutational pressure on HIV-1. Immune pressure exerted by ARF CD8(+) T cells on the virus has already been observed in vitro. HAART has also been described to select HIV-1 variants for drug escape mutations. Since the mutational pressure exerted on one location of the HIV-1 genome can potentially affect the 3 reading frames, we hypothesized that ARF responses would be affected by this drug pressure in vivo. Methodology/Principal findings: In this study we identified new ARFs derived from sense and antisense transcription of HIV-1. Many of these ARFs are detectable in circulating viral proteins. They are predominantly found in the HIV-1 env nucleotide region. We measured T cell responses to 199 HIV-1 CE encoded within 13 sense and 34 antisense HIV-1 ARFs. We were able to observe that these ARF responses are more frequent and of greater magnitude in chronically infected individuals compared to acutely infected patients, and in patients on HAART, the breadth of ARF responses increased. Conclusions/Significance: These results have implications for vaccine design and unveil the existence of potential new epitopes that could be included as vaccine targets.

Necrose avascular da cabeça femoral em pacientes HIV positivos: resultados iniciais do tratamento cirúrgico por substituição articular cerâmica-cerâmica

OBJETIVOS: Avaliar os resultados funcionais iniciais e o índice de complicações precoces das artroplastias totais do quadril cerâmica-cerâmica em pacientes que convivem com o HIV e apresentam osteonecrose da cabeça femoral. MÉTODO: Doze pacientes HIV+ com diagnóstico de osteonecrose da cabeça do fêmur incongruente foram avaliados através de critérios clínicos, laboratoriais, pela escala funcional WOMAC antes e após o tratamento com substituição articular. RESULTADOS: Observamos que 83,3% dos indivíduos faziam uso de inibidores de protease, 75% apresentavam dislipidemia e 66,6% síndrome lipodistrófica, a melhora na evolução no escore WOMAC foi estatisticamente significativa para seis e 12 meses de pós-operatório em comparação com o escore pré-operatório e não observamos complicações secundárias a esse procedimento. CONCLUSÃO: A artroplastia total do quadril com implante de cerâmica-cerâmica para o tratamento da necrose avascular do quadril nessa parcela da população é opção cirúrgica adequada, apresenta melhora funcional inicial significativa e baixo índice de complicação precoce.