Analysis of Transmitted Resistance to Raltegravir and Selective Pressure among HIV-1-Infected Patients on a Failing HAART in São 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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fed Univ São Paulo UNIFESP, Div Infect Dis, Retrovirol Lab, São Paulo, BrazilLusiada Fdn Santos, Mol Biol Lab, Dept Med, Santos, BrazilUniv São Paulo, Div Clin Immunol & Allergy, São Paulo, BrazilFed Univ São Paulo UNIFESP, Div Infect Dis, Retrovirol Lab, São Paulo, BrazilFAPESP: 09/05712-3Web of Scienc

Techniques used to identify the Brazilian variant of HIV-1 subtype B

The purpose of the present study was to compare the sensitivity and specificity of V3 enzyme immunoassay (solid phase EIA and EIA inhibition) and restriction fragment length polymorphism (RFLP) with the DNA sequencing "gold standard" to identify the Brazilian HIV-1 variants of subtype B and B"-GWGR. Peripheral blood mononuclear cells were collected from 61 HIV-1-infected individuals attending a clinic in São Paulo. Proviral DNA was amplified and sequentially cleaved with the Fok I restriction enzyme. Plasma samples were submitted to a V3-loop biotinylated synthetic peptide EIA. Direct partial DNA sequencing of the env gene was performed on all samples. Based on EIA results, the sensitivity for detecting B-GPGR was 70%, compared to 64% for the Brazilian variant B"-GWGR while, the specificity of B-GPGR detection was 85%, compared to 88% for GWGR. The assessment of RFLP revealed 68% sensitivity and 94% specificity for the B-GPGR strain compared to 84 and 90% for the B"-GWGR variant. Moreover, direct DNA sequencing was able to detect different base sequences corresponding to amino acid sequences at the tip of the V3 loop in 22 patients. These results show a similar performance of V3 serology and RLFP in identifying the Brazilian variant GWGR. However, V3 peptide serology may give indeterminate results. Therefore, we suggest that V3 serology be used instead of DNA sequencing where resources are limited. Samples giving indeterminate results by V3 peptide serology should be analyzed by direct DNA sequencing to distinguish between B-GPGR and the Brazilian variant B"-GWGR

Frequency of coreceptor tropism in PBMC samples from HIV-1 recently infected blood donors by massively parallel sequencing: the REDS II study

BACKGROUND: The interaction of HIV-1 and target cells involves sequential binding of the viral gp120 Env protein to the CD4 receptor and a chemokine co-receptor (either CCR5 or CXCR4). CCR5 antagonists have proved to be an effective salvage therapy in patients with CCR5 using variants (R5) but not with variants capable of using CXCR4 (×4) phenotype. Thus, it is critically important to determine cellular tropism of a country’s circulating HIV strains to guide a management decision to improve treatment outcome. In this study, we report the prevalence of R5 and ×4 HIV strains in 45 proviral DNA massively parallel sequencing “MPS” data from recently infected Brazilian blood donors. METHODS: The MPS data encompassing the tropism-related V3 loop region of the HIV‐1 env gene was extracted from our recently published HIV-1 genomes sequenced by a paired-end protocol (Illumina). HIV‐1 tropism was inferred using Geno2pheno([coreceptor]) algorithm (3.5 % false-positive rate). V3 net charge and 11/25 rules were also used for coreceptor prediction. RESULTS: Among the 45 samples for which tropism were determined, 39 were exclusively R5 variants, 5 ×4 variants, and one dual-tropic or mixed (D/M) populations of R5 and ×4 viruses, corresponding to 86.7, 11.1 and 2.2 %, respectively. Thus, the proportion of all blood donors that harbor CXCR4-using virus was 13.3 % including individuals with D/M-tropic viruses. CONCLUSIONS: The presence of CCR5-tropic variants in more than 85 % of our cohort of antiretroviral-naïve blood donors with recent HIV-1 infection indicates a potential benefit of CCR5 antagonists as a therapeutic option in Brazil. Therefore, determination of viral co-receptor tropism is an important diagnostic prerequisite

Expansion in CD39(+) CD4(+) Immunoregulatory T Cells and Rarity of Th17 Cells in HTLV-1 Infected Patients Is Associated with Neurological Complications

HTLV-1 infection is associated with several inflammatory disorders, including the neurodegenerative condition HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is unclear why a minority of infected subjects develops HAM/TSP. CD4(+) T cells are the main target of infection and play a pivotal role in regulating immunity to HTLV and are hypothesized to participate in the pathogenesis of HAM/TSP. the CD39 ectonucleotidase receptor is expressed on CD4(+) T cells and based on co-expression with CD25, marks T cells with distinct regulatory (CD39(+)CD25(+)) and effector (CD39(+)CD25(-)) function. Here, we investigated the expression of CD39 on CD4(+) T cells from a cohort of HAM/TSP patients, HTLV-1 asymptomatic carriers (AC), and matched uninfected controls. the frequency of CD39(+)CD4(+) T cells was increased in HTLV-1 infected patients, regardless of clinical status. More importantly, the proportion of the immunostimulatory CD39(+)CD25(-) CD4+ T-cell subset was significantly elevated in HAM/TSP patients as compared to AC and phenotypically had lower levels of the immunoinhibitory receptor, PD-1. We saw no difference in the frequency of CD39(+)CD25(+) regulatory (Treg) cells between AC and HAM/TSP patients. However, these cells transition from being anergic to displaying a polyfunctional cytokine response following HTLV-1 infection. CD39(-)CD25(+) T cell subsets predominantly secreted the inflammatory cytokine IL-17. We found that HAM/TSP patients had significantly fewer numbers of IL-17 secreting CD4(+) T cells compared to uninfected controls. Taken together, we show that the expression of CD39 is upregulated on CD4(+) T cells HAM/TSP patients. This upregulation may play a role in the development of the proinflammatory milieu through pathways both distinct and separate among the different CD39 T cell subsets. CD39 upregulation may therefore serve as a surrogate diagnostic marker of progression and could potentially be a target for interventions to reduce the development of HAM/TSP.National Institute of Allergies and Infectious DiseasesNational Institutes of HealthUniversity of CaliforniaSan Francisco-Gladstone Institute of Virology & Immunology Center for AIDS ResearchFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)John E. Fogarty International CenterNational Center for Research ResourcesNational Institute of General Medical Sciences from the National Institutes of HealthUniv Calif San Francisco, Dept Med, Div Expt Med, San Francisco, CA 94143 USAUniv Hawaii, John A Burns Sch Med, Dept Trop Med, Hawaii Ctr AIDS, Honolulu, HI 96822 USAUniv São Paulo, Sch Med, Deparment Infect Dis, São Paulo, BrazilUniv São Paulo, Sch Med, Div Clin Immunol & Allergy, São Paulo, BrazilFuncacao Prosangue, Hemoctr São Paulo, Mol Biol Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilSan Francisco-Gladstone Institute of Virology & Immunology Center for AIDS Research: P30 AI027763FAPESP: 04/15856-9/KallasFAPESP: 2010/05845-0/KallasFAPESP: 11/12297-2/SanabaniJohn E. Fogarty International Center: D43 TW00003National Center for Research Resources: 5P20RR016467-11National Institute of General Medical Sciences from the National Institutes of Health: 8P20GM103466-11Web of Scienc

Correlation between LTR point mutations and proviral load levels among Human T cell Lymphotropic Virus type 1 (HTLV-1) asymptomatic carriers

<p>Abstract</p> <p>Background</p> <p>In vitro studies have demonstrated that deletions and point mutations introduced into each 21 bp imperfect repeat of <it>Tax</it>-responsive element (TRE) of the genuine human T-cell leukemia virus type I (HTLV-1) viral promoter abolishes <it>Tax </it>induction. Given these data, we hypothesized that similar mutations may affect the proliferation of HTLV-1i</p> <p>nfected cells and alter the proviral load (PvL). To test this hypothesis, we conducted a cross-sectional genetic analysis to compare the near-complete LTR nucleotide sequences that cover the TRE1 region in a sample of HTLV-1 asymptomatic carriers with different PvL burden.</p> <p>Methods</p> <p>A total of 94 asymptomatic HTLV-1 carriers with both sequence from the 5' long terminal repeat (LTR) and a PvL for <it>Tax </it>DNA measured using a sensitive SYBR Green real-time PCR were studied. The 94 subjects were divided into three groups based on PvL measurement: 31 low, 29 intermediate, and 34 high. In addition, each group was compared based on sex, age, and viral genotypes. In another analysis, the median PvLs between individuals infected with mutant and wild-type viruses were compared.</p> <p>Results</p> <p>Using a categorical analysis, a G232A substitution, located in domain A of the TRE-1 motif, was detected in 38.7% (12/31), 27.5% (8/29), and 61.8% (21/34) of subjects with low, intermediate, or high PvLs, respectively. A significant difference in the detection of this mutation was found between subjects with a high or low PvL and between those with a high or intermediate PvL (both <it>p </it>< 0.05), but not between subjects with a low or intermediate PvL (<it>p </it>> 0.05). This result was confirmed by a non-parametric analysis that showed strong evidence for higher PvLs among HTLV-1 positive individuals with the G232A mutation than those without this mutation (<it>p </it>< 0.03). No significant difference was found between the groups in relation to age, sex or viral subtypes (<it>p</it> > 0. 05).</p> <p>Conclusions</p> <p>The data described here show that changes in domain A of the HTLV-1 TRE-1 motif resulting in the G232A mutation may increase HTLV-1 replication in a majority of infected subjects.</p

The Use of Imatinib Mesylate as a Lifesaving Treatment of Chronic Myeloid Leukemia Relapse after Bone Marrow Transplantation

We describe the response of imatinib as lifesaving treatment of chronic myeloid leukemia (CML) relapse in seven patients who underwent allogeneic bone marrow transplantation (alloBMT) at our institution over a period of 4 years. Retrospective analysis of their medical records revealed that a mean age at transplant was 45.2 years. The median time to diagnosis was 7.4 years after transplant. At relapse, four, two, and one patients were classified as having hematologic, major molecular, and cytogenetic relapse, respectively. At imatinib initiation, five had CML in a chronic phase, while one patient was diagnosed as having accelerated phase and blast crisis. All these patients could be evaluated for the therapeutic efficacy. At a mean of follow-up of 1.9 years of therapy, all evaluable patients achieved major molecular response without compromising safety. Consistent with available data, our results indicate that imatinib is safe and effective treatment option for patients with relapse after BMT

Successful Pregnancy and Delivery in a Patient with Chronic Myeloid Leukemia while on Dasatinib Therapy

Here we report the case of an 18-year-old woman with chronic myeloid leukemia (CML) who became pregnant while undergoing treatment with dasatinib. Before pregnancy, she received imatinib mesylate therapy but could not tolerate the treatment. The regimen was then changed to dasatinib at a dose of 70 mg b.i.d. While she was in hematological remission and on dasatinib therapy, she became pregnant. The unplanned pregnancy was identified after the patient had experienced four weeks of amenorrhea. Because the patient elected to continue the pregnancy to term, dasatinib was stopped immediately. Meanwhile, CML hematological relapse occurred and then she was treated with interferon-α (IFN-α) (9 million IU/day) throughout the pregnancy without a complete hematological response. She successfully gave birth to a male baby at 33 weeks by cesarean section delivery with no sequelae or malformations. Although this experience is limited to a single patient, it provides a useful contribution for counselling patients inadvertently exposed to dasatinib during pregnancy

Surveillance programs for detection and characterization of emergent pathogens and antimicrobial resistance: results from the Division of Infectious Diseases, UNIFESP

Several epidemiological changes have occurred in the pattern of nosocomial and community acquired infectious diseases during the past 25 years. Social and demographic changes possibly related to this phenomenon include a rapid population growth, the increase in urban migration and movement across international borders by tourists and immigrants, alterations in the habitats of animals and arthropods that transmit disease, as well as the raise of patients with impaired host defense abilities. Continuous surveillance programs of emergent pathogens and antimicrobial resistance are warranted for detecting in real time new pathogens, as well as to characterize molecular mechanisms of resistance. In order to become more effective, surveillance programs of emergent pathogens should be organized as a multicenter laboratory network connected to the main public and private infection control centers. Microbiological data should be integrated to guide therapy, adapting therapy to local ecology and resistance patterns. This paper presents an overview of data generated by the Division of Infectious Diseases, Federal University of São Paulo, along with its participation in different surveillance programs of nosocomial and community acquired infectious diseases.Várias alterações epidemiológicas ocorreram no perfil das doenças infecciosas hospitalares e comunitárias nos últimos 25 anos. Mudanças sociais e demográficas possivelmente relacionadas com esse fenômeno incluem o rápido crescimento populacional, o aumento da migração urbana e deslocamento através de fronteiras internacionais por turistas e imigrantes, alterações nos habitats de animais e artrópodes que transmitem doença assim como o aumento no número de pacientes com deficiências nas respostas de defesa. Os programas contínuos de vigilância de patógenos emergentes e resistência antimicrobiana são necessários para a detecção em tempo real de novos patógenos assim como para caracterizar mecanismos moleculares de resistência. Para serem mais efetivos, os programasde vigilância dos patógenos emergentes devem ser organizados em uma rede de laboratórios multicêntricos ligados aos principais centros de controle de infecções, públicos e privados. Os dados microbiológicos devem ser integrados a guias terapêuticos adaptando práticas terapêuticas à ecologia local eaos padrões de resistência. O artigo apresenta uma revisão dos dados gerados pela Disciplina de Infectologia, Universidade Federal de São Paulo (UNIFESP), contemplando sua participação nos diferentes programas de vigilância de doenças infecciosas hospitalares e adquiridas na comunidade.Universidade Federal de São Paulo (UNIFESP) Departamento de Medicina Divisão de Doenças InfecciosasUniversidade Federal de São Paulo (UNIFESP) Departamento de Microbiologia, Imunologia e ParasitologiaUNIFESP, Depto. de Medicina Divisão de Doenças InfecciosasUNIFESP, Depto. de Microbiologia, Imunologia e ParasitologiaSciEL

Combination Random Isothermal Amplification and Nanopore Sequencing For Rapid Identification of the Causative Agent of an Outbreak

Background Outbreaks of fever of unknown origin start with nonspecific symptoms and case definition is only slowly developed and adapted, therefore, identifying the causative agent is crucial to ensure suitable treatment and control measures. As an alternative method for Polymerase Chain Reaction in molecular diagnostics diagnostic, metagenomics can be applied to identify the pathogen responsible for the outbreak through sequencing all nucleic acids present in a sample extract. Sequencing data obtained can identify new or variants of known agents. Objectives To develop a rapid and field applicable protocol to allow the identification of the causative agent of an outbreak. Study design We explored a sequencing protocol relying on multiple displacement isothermal amplification and nanopore sequencing in order to allow the identification of the causative agent in a sample. To develop the procedure, a mock sample consisting of supernatant from Zika virus tissue culture was used. Results The procedure took under seven hours including sample preparation and data analysis using an offline BLAST search. In total, 63,678 sequence files covering around 10,000 bases were extracted. BLAST search revealed the presence of Zika virus. Conclusion In conclusion, the protocol has potential for point of need sequencing to identify RNA viruses. The whole procedure was operated in a suitcase laboratory. However, the procedure is cooling chain dependent and the cost per sequencing run is still high

HIV-1 Proviral DNA Loads (as Determined by Quantitative PCR) in Patients Subjected to Structured Treatment Interruption after Antiretroviral Therapy Failure

The impact of Structured Treatment Interruption (STI) in peripheral blood mononuclear cell (PBMC) proviral reservoirs in 41 highly active antiretroviral therapy (HAART)-treated viremic individuals at baseline and 12 weeks after STI was determined using quantitative PCR (qPCR). Viral load increased 0.7 log(10) and CD4 decreased 97.5 cells/mm(3) after 12 weeks. A total of 28 of the 41 individuals showed an increased proviral load, 19 with a statistically significant increase above 10%. An increase in active viral replication is an important factor in the replenishment of the proviral reservoir even for short time periods.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo [03/11781-1