The SARS-CoV-2 pandemic has resulted in shortages of both critical reagents for nucleic acid purification and highly trained staff as supply chains are strained by high demand, public health measures and frequent quarantining and isolation of staff. This created the need for alternate workflows with limited reliance on specialised reagents, equipment and staff. We present here the validation and implementation of such a workflow for preparing samples for downstream SARS-CoV-2 RT-PCR using liquid handling robots. The rapid sample preparation and inactivation technique evaluated, which included sample centrifugation and heating prior to RT-PCR, showed a 97.37% (95% CI: 92.55-99.28%) positive percent agreement and 97.30% (95% CI: 90.67-99.52%) negative percent agreement compared to nucleic acid purification-based testing. A total of 195 samples were tested as part of the validation. This method was subsequently adopted as the primary sample preparation method in the Groote Schuur Hospital Virology Diagnostic Laboratory in Cape Town, South Africa.

A diferència de la infecció per virus lliure, la transmissió del VIH de cèl·lula a cèl·lula és un mecanisme altament eficient i citopàtic degut a l'establiment de les sinapsis virològiques induïdes per l'embolcall del virus on la informació es transmet en forma de partícules virals (acompanyades de traços de membrana) des de la cèl·lula efectora a la cèl·lula diana. L'objectiu inicial va ser la caracterització dels contactes cel·lulars mediats pel VIH en cultius mixtes de cèl·lules infectades i cèl·lules no infectades en absència o presència del coreceptor apropiat. Posteriorment es va evaluar la transferència viral de cèl·lula T a cèl·lula T, un fenòmen que pot o no portar a la infecció de la cèl·lula diana. Les molècules d'adhesió tenen un paper secundari en la transmissió viral a través de la sinapsis virològica. Tot i que les cèl·lules T de memòria expressen més quantitats d'aquestes molècules que les cèl·lules T naïve, la major transferència viral observada cap a aquestes no es pot explicar per aquest fet. La trogocitosis o intercanvi de components de membrana, no permetla transferència de virus ja que només opera en el sentit invers a la transmissió tot i que podria estar jugant un paper modulador en la formació i durada dels contactes. Finalment, la transmissió viral de cèl·lula a cèl·lula desencadena la infecció de cèl·lules T CD4 quiescents, les quals presenten nivells baixos de fosforilació de les proteïnes senyalitzadores p56Lck i ZAP70 un cop han contactat amb les cèl·lules infectades, en absència de proliferació cel·lular i infecció productiva d'aquestes cèl·lules en estat en repòs, permanent així, en un estat latentment infectades. / A diferencia de la infección por virus libre, la transmisión de VIH de célula a célula es un mecanismo altamente eficiente y citopático debido al establecimiento de las sinapsis virológicas inducidas por la envuelta del virus donde la información se transmite en forma de partículas virales (acompañadas de trazos de membrana) desde la célula efectora a la célula diana. Inicialmente se caracterizó los contactos celulares mediados por el VIH en cultivos mixtos de células infectadas y células no infectadas en ausencia o presencia del coreceptor apropiado y posteriormente se evaluó la transferéncia viral. Las moleculas de adhesion juegan un papel secundario en la transmisión viral a través de la sinapsis virológica. Las células T de memoria expresan más cantidades de estas moléculas que las células T naive, aún así, la mayor transferencia viral observada hacia as células de memoria no se explica por este hecho. La trogocitosis o intercambio de componentes de membrana, no permite la transferéncia de virus ya que sólo opera en el sentido inverso a la transmisión aunque puede tener un papel modulador en la formación y durada de los contactos. Finalmente, la transmisión viral de célula a célula desencadena la infección de células T CD4 quiescentes, las cuales presentan niveles bajos de fosforilación de las proteínas p56Lck y ZAP70 un vez han contactado con las células infectadas, en ausencia de proliferación celular y infección productiva de estas células en estado de reposo, permaneciendo así, en un estado latentemente infectadas. / Unlike virus-to-cell contacts, cell-to-cell HIV transmission (from infected cells to CD4 T cells) is a highly efficient and cytopathic mechanism. Such a high efficiency relies in the formation of "virological synapses" induced by the HIV envelope glycoproteins. The particularity of this synapse as compared to the classical ones would be that information is transferred as virus particles (accompanied by other components of the cell membrane) from effector to target cells. First, the aim was to characterize T cell-T cell contacts mediated by the HIV Envelope in mixed cultures of infected and target primary cells expressing or not the appropriate coreceptor. Then we have evaluated HIV transfer, which is an early event occurring immediately after the VS formation that precedes but does not necessarily lead to transmission, a later event resulting in infection. The presence of adhesion molecules at the synaptic junction showed a secondary role in cell-to-cell HIV transfer. Despite memory CD4 T cells expressed higher levels of adhesion molecules that naïve cells, it did not explain the selectivity of HIV transfer observed towards the memory CD4 T cell subset. Trogocytosis (i.e. intercellular exchange of membrane components) operates from the uninfected towards the infected cell direction but may modulate the extent and durability of HIV-mediated T cell contacts. Finally, cell-to-cell HIV transmission triggers the infection of quiescent CD4 T cells, which showed low levels of Envelope-CD4 mediated p56Lck and ZAP-70 phosphorylation in the absence of proliferation and productive infection of target cells.

Orientador: Rejane Maria Tommasini Grotto / Banca: Dennis Armando Bertolini / Banca: Giovanni Faria Silva / Resumo: O vírus da Hepatite C (VHC) é um importante patógeno associado com doença hepática crônica sendo que alguns infectados podem desenvolver cirrose e carcinoma hepatocelular. O tratamento da hepatite C crônica visa a resposta virológica sustentada (RVS), definida como níveis de RNA viral indetectáveis no soro por seis meses depois do término do tratamento. Atualmente, a terapia padrão ouro é a combinação de interferon α peguilado e ribavirina, porém esse esquema terapêutico vem se mostrando eficaz em, apenas, 50% dos pacientes infectados com o genótipo 1, o mais prevalente no Brasil. Portanto, novas drogas mais eficazes e menos tóxicas estão sendo desenvolvidas para melhorar a assistência aos pacientes infectados pelo VHC, entre as quais merecem destaque os inibidores da serina protease NS3, a qual é uma enzima essencial para a replicação do VHC e assim um potencial alvo para novas terapias antivirais. Entretanto, a emergência de variantes resistentes é o maior obstáculo para o sucesso da terapêutica. Variantes resistentes já foram isoladas em pacientes tratados com os inibidores de protease e, estão associadas com a falência terapêutica. Porém o impacto dessas variantes resistentes em pacientes virgens de tratamento ainda não foi esclarecido e, esse tipo de informação pode avaliar o impacto dos inibidores de protease na terapia antiviral. O objetivo deste estudo foi avaliar a presença de mutações de resistência e polimorfismos genéticos na região genômica NS3 do VHC em 37 pacientes virgens de tratamento com inibidores de protease infectados com genótipo 1. RNA viral sérico foi utilizado como fonte para amplificação e seqüenciamento da região NS3 do VHC e, avaliar a presença de mutações de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Hepatitis C Virus (HCV) is an important pathogen associated with chronic hepatic disease and some infected patients can develop cirrhosis and hepatocellular carcinoma. The treatment of chronic hepatitis C aimed the sustained virological response (SVR), defined as having undetectable serum HCV RNA at the end of therapy for at least 6 months. Currently, the gold standard therapy is a combination of pegylated interferon-α and the ribavirin, however this treatment present efficacy in only 50% of patients infected with genotypes 1, the most prevalent in Brazil. Then, new drugs more effective and less toxic have been developed to improve the attendance of the HCV infected patients as the serine protease NS3 inhibitors, which is an enzyme essential to HCV replication and main target of new antiviral therapies. However, the emergence of drug resistant variants has been the major obstacle to therapeutic successful. Resistant variants have already been isolated in patients treated with protease inhibitors and, these resistant variants are associated with non response to treatment. But the impact of the resistant variants in naïve protease inhibitors patients is unclear yet and, this information can evaluate the impact of protease inhibitors in antiviral therapeutic. The goal of this study was evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV in 37 protease inhibitors-naive genotype 1 HCV infected patients. Serum viral RNA was used as source to amplification and sequencing of NS3 region of HCV and, evaluates the presence of resistance mutations and polymorphisms in this region. The results showed that only 07 (18.9%) samples presented resistant variants, the mutations... (Complete abstract click electronic access below) / Mestre

Orientador: Maria del Pilar Taboada Sotomayor / Banca: Denis Ricardo Martins de Godoi / Banca: Ronaldo Censi Faria / Resumo: O vírus Zika é um agente causador de doenças infecciosas que se espalha nos seres humanos através dos vetores do vírus, os mosquitos Aedes aegypti e Aedes albopictus. A transmissão durante a gravidez leva à microcefalia em recém-nascidos e também outros defeitos de nascimento. Os esforços para monitorar e controlar a infecção pelo Zika vírus globalmente são muito limitados. No contexto do desenvolvimento de novas metodologias de diagnóstico, a construção de sensores biológicos tem se tornado uma atividade ampla no ramo da ciência. Neste sentido eletrodos modificados com polímeros condutores e não condutores são opções viáveis, pois além de auxiliar na imobilização das biomoléculas podem melhorar a resposta analítica. Assim, realizou-se, o estudo das propriedades elétricas e morfológicas de um novo material derivado do monômero ácido 3-amino-4- hidroxibenzóico (AAHB) sobre a superfície de eletrodos de grafite de lapiseira para construção de um biossensor contendo uma sequência de oligonucleotídeo específica do Zika vírus. Foi formado um material eletroquimicamente ativo na superfície dos eletrodos de grafite de lapiseira. Analisou-se diferentes condições de eletropolimerização e comportamentos físico-químicos do monômero e foi proposto um mecanismo de formação do polímero. Vários parâmetros foram investigados para otimizar a resposta do genossensor ao substrato utilizando a voltametria de onda quadrada (VOQ). Dentre essas investigações, as melhores respostas foram obtidas, e o... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Zika virus is a causative agent of infectious diseases that spreads in humans through the virus vectors, Aedes aegyptiand Aedes albopictusmosquitoes. Transmission during pregnancy leads to microcephaly in newborns and also other birth defects. Efforts to monitor and control Zika virus infection globally are very limited. In the context of the development of new diagnostic methodologies, the construction of biological sensors has become a broad activity in the field of science. In this sense, electrodes modified with conductive and non-conductive polymers are viable options, since besides aiding in the immobilization of the biomolecules they can improve the analytical response. Thus, the study of the electrical and morphological properties of a new material derived from the 3-amino-4-hydroxybenzoic acid monomer (AHBA) on the surface of graphite graphite electrodes was carried out to construct a biosensor containing an oligonucleotide sequence specific Zika virus. An electrochemically active material was formed on the surface of the graphite graphite electrodes. It was analyzed the sedimentation conditions of electropolymerization and physicochemical behavior of the monomer and a mechanism of formation of the polymer was proposed. Several parameters were investigated to optimize the response of the genosensor to the substrate using square wave voltammetry (SWV). Among these investigations, the best responses were obtained, and the sensor showed a linear working range for th... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:23:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-15Bitstream added on 2014-06-13T19:28:48Z : No. of bitstreams: 1 zeminian_lb_me_botfm.pdf: 506409 bytes, checksum: 08bea6131bbfbe54e0327803c5f6bb14 (MD5) / Ministério da Saúde / O vírus da Hepatite C (VHC) é um importante patógeno associado com doença hepática crônica sendo que alguns infectados podem desenvolver cirrose e carcinoma hepatocelular. O tratamento da hepatite C crônica visa a resposta virológica sustentada (RVS), definida como níveis de RNA viral indetectáveis no soro por seis meses depois do término do tratamento. Atualmente, a terapia padrão ouro é a combinação de interferon α peguilado e ribavirina, porém esse esquema terapêutico vem se mostrando eficaz em, apenas, 50% dos pacientes infectados com o genótipo 1, o mais prevalente no Brasil. Portanto, novas drogas mais eficazes e menos tóxicas estão sendo desenvolvidas para melhorar a assistência aos pacientes infectados pelo VHC, entre as quais merecem destaque os inibidores da serina protease NS3, a qual é uma enzima essencial para a replicação do VHC e assim um potencial alvo para novas terapias antivirais. Entretanto, a emergência de variantes resistentes é o maior obstáculo para o sucesso da terapêutica. Variantes resistentes já foram isoladas em pacientes tratados com os inibidores de protease e, estão associadas com a falência terapêutica. Porém o impacto dessas variantes resistentes em pacientes virgens de tratamento ainda não foi esclarecido e, esse tipo de informação pode avaliar o impacto dos inibidores de protease na terapia antiviral. O objetivo deste estudo foi avaliar a presença de mutações de resistência e polimorfismos genéticos na região genômica NS3 do VHC em 37 pacientes virgens de tratamento com inibidores de protease infectados com genótipo 1. RNA viral sérico foi utilizado como fonte para amplificação e seqüenciamento da região NS3 do VHC e, avaliar a presença de mutações de... / The Hepatitis C Virus (HCV) is an important pathogen associated with chronic hepatic disease and some infected patients can develop cirrhosis and hepatocellular carcinoma. The treatment of chronic hepatitis C aimed the sustained virological response (SVR), defined as having undetectable serum HCV RNA at the end of therapy for at least 6 months. Currently, the gold standard therapy is a combination of pegylated interferon-α and the ribavirin, however this treatment present efficacy in only 50% of patients infected with genotypes 1, the most prevalent in Brazil. Then, new drugs more effective and less toxic have been developed to improve the attendance of the HCV infected patients as the serine protease NS3 inhibitors, which is an enzyme essential to HCV replication and main target of new antiviral therapies. However, the emergence of drug resistant variants has been the major obstacle to therapeutic successful. Resistant variants have already been isolated in patients treated with protease inhibitors and, these resistant variants are associated with non response to treatment. But the impact of the resistant variants in naïve protease inhibitors patients is unclear yet and, this information can evaluate the impact of protease inhibitors in antiviral therapeutic. The goal of this study was evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV in 37 protease inhibitors-naive genotype 1 HCV infected patients. Serum viral RNA was used as source to amplification and sequencing of NS3 region of HCV and, evaluates the presence of resistance mutations and polymorphisms in this region. The results showed that only 07 (18.9%) samples presented resistant variants, the mutations... (Complete abstract click electronic access below)

The use of antiretroviral therapy (ART) for HIV-1 treatment effectively suppresses viral replication if managed appropriately, but sometimes patients experience incomplete viral suppression with manifestation of either persistent low-level viremia (LLV) or discernible virological failure (VF). In the present study, potential risk factors associated with LLV and VF were investigated in a cohort of HIV-1 infected Kenyan children receiving ART. Drug resistant (DR) variants in children with or without LLV were examined using a next-generation sequencing-based HIV DR typing protocol. The potential association between HIV DR mutations (DRMs) and LLV and/or VF was then examined. To measure the potential impacts from other clinical and epidemiological confounding factors, a database comprising of epidemiological and clinical information from this patient cohort was established and sanitized for ensured accountability. Statistically significant correlations between the examined factors and LLV or VF were determined using chi-square test, Kaplan-Meier survival analysis, and Cox proportional hazard models. Of 293 examined patients, 20% had LLV and 22% of the selected patients progressed to VF with no significant association observed between LLV and VF. ART adherence during therapy, baseline CD4 counts, DRMs at LLV, WHO clinical stage, gender, ART therapy stage (1st/2nd line), ART drugs and co-morbidities were not significantly associated with LLV, whereas, the ART adherence, CD4 counts and co-infection with pneumonia was significantly associated with VF. This study highlights the factors predictive of VF, and the relevance of maintaining LLV in HIV-infected children. / February 2017

No description available.

Doctor of Philosophy(PhD) / ABSTRACT Despite the extensive quantitative and qualitative knowledge about therapeutic regimens and the molecular biology of HIV/AIDS, the eradication of HIV infection cannot be achieved with available antiretroviral regimens. HIV drug resistance remains the most challenging factor in the application of approved antiretroviral agents. Previous investigations and existing HIV/AIDS models and algorithms have not enabled the development of long-lasting and preventive drug agents. Therefore, the analysis of the dynamics of drug resistance and the development of sophisticated HIV/AIDS analytical algorithms and models are critical for the development of new, potent antiviral agents, and for the greater understanding of the evolutionary behaviours of HIV. This study presents novel computational methods for the analysis of drug-resistance dynamics, including: viral sequences, phenotypic resistance, immunological and virological responses and key clinical data, from HIV-infected patients at Royal Prince Alfred Hospital in Sydney. The lability of immunological and virological responses is analysed in the context of the evolution of antiretroviral drug-resistance mutations. A novel Bayesian algorithm is developed for the detection and classification of neutral and adaptive mutational patterns associated with HIV drug resistance. To simplify and provide insights into the multifactorial interactions between viral populations, immune-system cells, drug resistance and treatment parameters, a Bayesian graphical model of drug-resistance dynamics is developed; the model supports the exploration of the interdependent associations among these dynamics.

Doctor of Philosophy(PhD) / ABSTRACT Despite the extensive quantitative and qualitative knowledge about therapeutic regimens and the molecular biology of HIV/AIDS, the eradication of HIV infection cannot be achieved with available antiretroviral regimens. HIV drug resistance remains the most challenging factor in the application of approved antiretroviral agents. Previous investigations and existing HIV/AIDS models and algorithms have not enabled the development of long-lasting and preventive drug agents. Therefore, the analysis of the dynamics of drug resistance and the development of sophisticated HIV/AIDS analytical algorithms and models are critical for the development of new, potent antiviral agents, and for the greater understanding of the evolutionary behaviours of HIV. This study presents novel computational methods for the analysis of drug-resistance dynamics, including: viral sequences, phenotypic resistance, immunological and virological responses and key clinical data, from HIV-infected patients at Royal Prince Alfred Hospital in Sydney. The lability of immunological and virological responses is analysed in the context of the evolution of antiretroviral drug-resistance mutations. A novel Bayesian algorithm is developed for the detection and classification of neutral and adaptive mutational patterns associated with HIV drug resistance. To simplify and provide insights into the multifactorial interactions between viral populations, immune-system cells, drug resistance and treatment parameters, a Bayesian graphical model of drug-resistance dynamics is developed; the model supports the exploration of the interdependent associations among these dynamics.

Submitted by FERNANDA CRISTINA WINCKLER null (fwinckler@fmb.unesp.br) on 2016-11-30T19:00:44Z No. of bitstreams: 1 DISSERTAÇÃO FINAL 30.11.16.pdf: 468887 bytes, checksum: 372ef2c27b73313e5608616205ed1d2e (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-12-02T16:28:26Z (GMT) No. of bitstreams: 1 winckler_fc_me_bot.pdf: 468887 bytes, checksum: 372ef2c27b73313e5608616205ed1d2e (MD5) / Made available in DSpace on 2016-12-02T16:28:26Z (GMT). No. of bitstreams: 1 winckler_fc_me_bot.pdf: 468887 bytes, checksum: 372ef2c27b73313e5608616205ed1d2e (MD5) Previous issue date: 2016-10-19 / A hepatite C é uma doença infecciosa que torna-se crônica em cerca de 85% dos infectados que poderão desenvolver cirrose e carcinoma hepato celular. O tratamento antiviral em muitos dos pacientes não é eficaz, principalmente quando estes portam o genótipo 1 e fibrose avançada, a resposta inflamatória também desempenha seu papel sobre a resposta virológica sustentada (RVS) durante o tratamento com Interferon Peguilado (PegIFN) associado a Ribavirina (RBV). Nesse estudo nosso objetivo principal foi avaliar a influência da resposta inflamatória através de células e citocinas/quimiocinas sobre a resposta virológica do paciente em tratamento antiviral com terapia tripla. Incluimos pacientes com RNA VHC+, nunca tratados (naive), portadores do genótipo 1, ambos os sexos e com fibrose avançada F3 (n=6); F4 (n=21) candidatos ao tratamento em regime triplo. Os pacientes tiveram suas amostras coletadas e analizadas nas semanas 0 e 12 do tratamento e os seguintes parâmetros foram analisados: IL-2, IL-4, IL-6, IL-8, IL-10, IL-17A, TNF-α, IFN-γ, RANTES, MCP-1, MIG, IP- 10, através de citometria de fluxo (método CBA). Foram incluídos 15 voluntários saudáveis (grupo controle) e 27 pacientes que foram separados em G1(RVS) e G2 (não RVS), a taxa de RVS foi de 63%. Os pacientes com hepatite C crônica tiveram os níveis circulantes de IP10, MCP-1, MIG, RANTES, IL-8 e IL-6 mais elevados quando comparados com voluntários saudáveis, quando comparados G1xG2 os níveis de RANTES (p=0,04) e IL-6 (p=0,02) foram associadas com a RVS na semana 0, seus níveis eram mais baixos em G1, na semana 12 os níveis de RANTES (p=0,04) e IL-8 (n=0,01) foram associados com a RVS, seus níveis são mais elevados em G2, a comparação entre as semanas 0 e 12 mostrou que em G1 os níveis de IL6 (p= 0,02) e MCP-1 (p=0,001) apresentam associação com o tratamento e em G2 os parâmetros associados ao tratamento foram RANTES (p=0,05) e MCP-1 (p=0,01). Os resultados sugerem que, a citocina IL-6 e a quimiocina RANTES estão associadas com a RVS na semana 0. Na semana 12, RANTES assim como IL-8 influenciam na RVS durante terapia antiviral em regime triplo. Quando comparado semana 0 e 12 em pacientes RVS, a citocina IL-6 está associada ao tratamento. Em pacientes não RVS, RANTES esta associada ao tratamento e MCP-1 está associada ao tratamento independente da resposta obtida. / Hepatitis C is an infectious disease which becomes chronic in about 85% of infected people who can develop cirrhosis and hepatocellular carcinoma. Antiviral therapy isn’t effective in many patients, especially when these patients are genotype 1 and have advanced fibrosis, the inflammatory response also plays a role on sustained virological response (SVR) during treatment with Pegylated (PegIFN) plus Ribavirin (RBV). The aim of this study was evaluate the influence of the inflammatory response by cells and cytocines/chemokines on the virologic response of the patients under antiviral treatment with triple therapy. We included patients with HCV RNA+, naive, genotype 1, both male and female and with advanced fibrosis F3 (n=6); F4 (n=21) for triple treatment regimen. Patients had their samples collected and analyzed at weeks 0 and 12 of treatment and the following parameters were analyzed: IL- 2, IL-4, IL-6, IL-8, IL-10, IL-17A, TNF-α, IFN-γ RANTES, MCP-1, MIG, IP-10 by flow cytometry (CBA method). Control group of 15 healthy volunteers and 27 patients, who were separated into GI (SVR) and G2 (not SRV), were included, the SVR rate was 63%. Patients with chronic hepatitis C had higher circulating levels of IP10, MCP-1, MIG, RANTES, IL-8 and IL-6 compared with healthy volunteers, when G1xG2 were compared, levels of RANTES (p=0,040 and IL-6 (n=0,02) were associated with a SVR at week 0 and its levels were lower in G1; at week 12, levels of RANTES (p=0,04) and IL-8 (p=0,01) were associated with a SVR and its levels were higher in G2. The comparison between weeks 0 and 12 showed that, in G1, the IL6 levels (p = 0.02) and MCP-1 (p = 0.001) were associated with the treatment and in G2, the parameters associated with the treatment were RANTES (p = 0.05) and MCP-1 (p = 0.01). The results suggest that the cytocine IL-6 and chemokine RANTES are associated with SVR at week 0. At week 12, RANTES as well as IL-8 influence in SVR during antiviral therapy in triple regimen. When weeks 0 and 12 in patients SVR are compared, the cytocine IL-6 is associated with treatment. In non-SVR patients, RANTES is associated with treatment and MCP-1 is associated with independent treatment of the patient's response.