Introdução: A AIDS (Síndrome da Imunodeficiência Adquirida) é caracterizada por uma disfunção grave no sistema imunológico causada por uma infecção por HIV (Human Immunodeficiency Virus). A quantificação da viremia (carga viral) é uma ferramenta muito útil no monitoramento dos pacientes infectados pelo HIV, sendo um marcador de progressão da doença e eficácia do tratamento. A estimativa incorreta da carga viral pode levar à decisão terapêutica equivocada, portanto métodos acurados de quantificação se fazem necessários. Diversas técnicas comerciais estão disponíveis para a quantificação da carga viral do HIV-1: a maioria destas se baseiam na detecção de ácidos nucléicos e outras na detecção de enzimas e antígenos. O grau de automação varia nas diferentes técnicas assim como os procedimentos de isolamento, amplificação e detecção. A correlação e a concordância entre estas técnicas têm sido estudadas e há relatos de discordância entre os valores de carga viral produzidos por diferentes métodos. O conhecimento sobre o efeito das variações entre as técnicas se faz necessário para assegurar a interpretação adequada dos resultados. A interpretação dos resultados correta é particularmente importante quando estes estão próximos a pontos de corte utilizados para definições de rebote viral e falha virológica. Objetivos: O objetivo deste estudo é comparar as técnicas de PCR (Cobas AmpliPrep TaqMan HIV-1 v2.0) e b-DNA (Siemens Versant HIV-1 RNA 3.0) para quantificação do HIV-1. Métodos: 1000 amostras recebidas no HIV/GUM Research Laboratory do Chelsea and Westminster Hospital para quantificação da carga viral do HIV-1 durante os meses de Dezembro de 2009 e Janeiro de 2010 foram testadas pelos métodos de PCR (Cobas AmpliPrep TaqMan HIV-1 v2.0) e b-DNA (Siemens Versant HIV-1 RNA 3.0). Resultados: Uma superquantificação sistemática foi observada nos resultados testados por PCR. Esta superquantificação ficou evidente nos resultados entre 50 e 250 cópias. Uma concordância elevada foi observada na análise dos pontos de corte de 500 e 1000 copias/mL. Uma correlação linear forte foi observada entre estas técnicas na análise das amostras que obtiveram resultados dentro do limite comum de detecção de ambas as técnicas, porém o nível de concordância foi insatisfatório. Conclusão: A superquantificação observada nos resultados obtidos pelo Cobas AmpliPrep TaqMan HIV- 1 v2.0 em relação ao bDNA Siemens Versant HIV-1 RNA 3.0 é provavelmente o resultado de uma sensibilidade aumentada desta técnica. Nós recomendamos cautela quando resultados de duas metodologias diferentes são comparados, especialmente quando se comparam metodologias convencionais com aquelas baseadas em PCR em tempo real. / Introduction: AIDS (Acquired Immunodeficiency Syndrome) is characterised by a severe immune dysfunction caused by the HIV (Human Immunodeficiency Virus). The HIV viral load quantification is an essential tool to monitor HIV-infected patients. The HIV quantification is a disease progression marker and it is a key indicator in treatment efficacy. Inaccurate viral RNA values may subsequently lead to inappropriate treatment decisions hence accurate quantification methods are necessary. Several different methodologies are available to quantify the HIV viral load: a number of them are based on nucleic acid detection and others in detection of enzymes and antigens. Automation is also variable among these methods in addition to differences in isolation, amplification and detection. Several studies have been carried out to evaluate their correlation and agreement and some have evidenced discordant viral load values assessed by different assays. The knowledge about these differences should be taken in to account when analysing viral load results, particularly when low-level viraemia is concerned or those close to endpoints employed for definition of virological failure. Objectives: In this study, two methods to quantify viral load are evaluated: one is based on real-time PCR (AmpliPrep TaqMan HIV-1 v2.0) and the other is based on branched-DNA technology (Siemens Versant HIV-1 RNA 3.0). Methods: 1000 plasma samples received at the HIV/GUM Research Laboratory within Chelsea and Westminster Hospital for HIV-1 viral load quantification between December 2009 and January 2010 were tested by both Cobas AmpliPrep TaqMan HIV-1 v2.0 and Siemens Versant HIV-1 RNA 3.0 methods. Results: Results obtained show that Cobas AmpliPrep TaqMan HIV-1 v2.0 PCR systematically overquantifies the viral loads results when compared to bDNA Siemens Versant HIV-1 RNA 3.0. Conclusion: The overquantification by Cobas AmpliPrep TaqMan HIV-1 v2.0 over bDNA Siemens Versant HIV-1 RNA 3.0 is likely to be a result of its increased sensitivity. We recommend caution when comparing results from different methodologies, especially when a conventional assay and a real-time PCR assay are concerned.

Introdução: A AIDS (Síndrome da Imunodeficiência Adquirida) é caracterizada por uma disfunção grave no sistema imunológico causada por uma infecção por HIV (Human Immunodeficiency Virus). A quantificação da viremia (carga viral) é uma ferramenta muito útil no monitoramento dos pacientes infectados pelo HIV, sendo um marcador de progressão da doença e eficácia do tratamento. A estimativa incorreta da carga viral pode levar à decisão terapêutica equivocada, portanto métodos acurados de quantificação se fazem necessários. Diversas técnicas comerciais estão disponíveis para a quantificação da carga viral do HIV-1: a maioria destas se baseiam na detecção de ácidos nucléicos e outras na detecção de enzimas e antígenos. O grau de automação varia nas diferentes técnicas assim como os procedimentos de isolamento, amplificação e detecção. A correlação e a concordância entre estas técnicas têm sido estudadas e há relatos de discordância entre os valores de carga viral produzidos por diferentes métodos. O conhecimento sobre o efeito das variações entre as técnicas se faz necessário para assegurar a interpretação adequada dos resultados. A interpretação dos resultados correta é particularmente importante quando estes estão próximos a pontos de corte utilizados para definições de rebote viral e falha virológica. Objetivos: O objetivo deste estudo é comparar as técnicas de PCR (Cobas AmpliPrep TaqMan HIV-1 v2.0) e b-DNA (Siemens Versant HIV-1 RNA 3.0) para quantificação do HIV-1. Métodos: 1000 amostras recebidas no HIV/GUM Research Laboratory do Chelsea and Westminster Hospital para quantificação da carga viral do HIV-1 durante os meses de Dezembro de 2009 e Janeiro de 2010 foram testadas pelos métodos de PCR (Cobas AmpliPrep TaqMan HIV-1 v2.0) e b-DNA (Siemens Versant HIV-1 RNA 3.0). Resultados: Uma superquantificação sistemática foi observada nos resultados testados por PCR. Esta superquantificação ficou evidente nos resultados entre 50 e 250 cópias. Uma concordância elevada foi observada na análise dos pontos de corte de 500 e 1000 copias/mL. Uma correlação linear forte foi observada entre estas técnicas na análise das amostras que obtiveram resultados dentro do limite comum de detecção de ambas as técnicas, porém o nível de concordância foi insatisfatório. Conclusão: A superquantificação observada nos resultados obtidos pelo Cobas AmpliPrep TaqMan HIV- 1 v2.0 em relação ao bDNA Siemens Versant HIV-1 RNA 3.0 é provavelmente o resultado de uma sensibilidade aumentada desta técnica. Nós recomendamos cautela quando resultados de duas metodologias diferentes são comparados, especialmente quando se comparam metodologias convencionais com aquelas baseadas em PCR em tempo real. / Introduction: AIDS (Acquired Immunodeficiency Syndrome) is characterised by a severe immune dysfunction caused by the HIV (Human Immunodeficiency Virus). The HIV viral load quantification is an essential tool to monitor HIV-infected patients. The HIV quantification is a disease progression marker and it is a key indicator in treatment efficacy. Inaccurate viral RNA values may subsequently lead to inappropriate treatment decisions hence accurate quantification methods are necessary. Several different methodologies are available to quantify the HIV viral load: a number of them are based on nucleic acid detection and others in detection of enzymes and antigens. Automation is also variable among these methods in addition to differences in isolation, amplification and detection. Several studies have been carried out to evaluate their correlation and agreement and some have evidenced discordant viral load values assessed by different assays. The knowledge about these differences should be taken in to account when analysing viral load results, particularly when low-level viraemia is concerned or those close to endpoints employed for definition of virological failure. Objectives: In this study, two methods to quantify viral load are evaluated: one is based on real-time PCR (AmpliPrep TaqMan HIV-1 v2.0) and the other is based on branched-DNA technology (Siemens Versant HIV-1 RNA 3.0). Methods: 1000 plasma samples received at the HIV/GUM Research Laboratory within Chelsea and Westminster Hospital for HIV-1 viral load quantification between December 2009 and January 2010 were tested by both Cobas AmpliPrep TaqMan HIV-1 v2.0 and Siemens Versant HIV-1 RNA 3.0 methods. Results: Results obtained show that Cobas AmpliPrep TaqMan HIV-1 v2.0 PCR systematically overquantifies the viral loads results when compared to bDNA Siemens Versant HIV-1 RNA 3.0. Conclusion: The overquantification by Cobas AmpliPrep TaqMan HIV-1 v2.0 over bDNA Siemens Versant HIV-1 RNA 3.0 is likely to be a result of its increased sensitivity. We recommend caution when comparing results from different methodologies, especially when a conventional assay and a real-time PCR assay are concerned.

Les entérovirus humains (EV) représentent la première cause des méningites aseptiques chez l'enfant. Le diagnostic de certitude repose sur la détection génomique de l'entérovirus par RT-PCR dans des échantillons de liquide céphalorachidien (LCR) est recommandée pour le diagnostic. La fièvre sans point d'appel et les maladies de type "sepsis" sont également des affections fréquentes chez les nourrissons (0 à 2 ans), qui peuvent être la conséquence d'une infection virale, en particulier à EV. Actuellement, le diagnostic des EV dans le sang est rarement établi dans la pratique courante.Les données concernant 1) l’existence d’une réplication de l’EV dans les leucocytes sanguins, 2) le niveau de charge virale de l’EV dans le sang et les échantillons de LCR et sa corrélation éventuelle avec l’intensité du processus inflammatoire réactionnel sont fragmentaires. Dans la première partie de cette thèse, l'objectif était d'évaluer la détection des EV par PCR dans des échantillons de sang de nouveau-nés, de nourrissons et d'enfants hospitalisés pour une fièvre isolée, un sepsis ou un syndrome méningé. Nous avons mené une étude observationnelle prospective multicentrique nationale (étude BLEDI) dans 35 départements de pédiatrie au cours de la période estivo-automnale (augmentation des circulations d'EV) en 2015-2016. Nos résultats ont montré que le taux de détection des EV dans le sang était significativement plus élevé que dans le LCR chez les nouveaux-nés et les nourrissons hospitalisés pour une fièvre isolée, un sepsis ou un syndrome méningé.Ces données ouvrent des perspectives pour un nouvel algorithme de diagnostic des fièvres inexpliquées chez les enfants âgés de moins de 2 ans. Dans le second volet de cette thèse, nous avons étudié de manière prospective la charge virale d'EV dans le sang et dans des échantillons de LCR de patients infectés. Nos résultats ont montré que la charge virale d'EV dans le sang variait selon le groupe d'âge, la présentation clinique et le type d'EV. Trois profils de cinétique d'infection comparant la charge virale dans le sang et le LCR ont été envisagés et sont en cours d'étude. Enfin, le dernier axe était de déterminer s'il existait une réplication de l’EV dans les leucocytes sanguins. Nous avons montré à partir d'échantillons de sang infectés in vitro par EV que la réplication de ces virus variait selon les génotypes d'EV. / Human enteroviruses (EV) are the most frequent cause of paediatric aseptic meningitis. Detection of enterovirus by PCR in cerebrospinal fluid (CSF) specimens is recommended for diagnosis. Fever without source and sepsis-like diseases are frequent affections in infants (0 to 2 years) which can be the consequence of a viral infection in particular to EV. At present, the daily routin diagnosis of EV in the blood is rarely performed. The concerning data 1) existence of EV replication in the blood leukocytes, 2) the EV viral load level in blood and CSF specimens and its possible relation with the intensity of the associated inflammatory process are fragmented. In the first part of this PhD thesis, the aim was to assess detection of enterovirus by PCR in blood specimens of newbrons, infants, and children with fever without source, sepsis-like disease or suspected meningitis. We did a prospective, multicentre, observational study (BLEDI study) at 35 paediatric departements during the seasonal period of increased EV circulation in 2015-2016. Our results showed that detection of EV was significantly higher in the blood than in the CSF from newborns and infants admitted with fever without source or sepsis-like disease. These data open up the perspectives for a new diagnostic algorithm of febrile illness in patients aged 2 years or younger. We also explored prospectively EV viral load in blood and in CSF specimens of infected patients. Our results showed that EV viral load in the blood varied by age group, clinical presentation and EV type. Three profiles of infection kinetics comparing EV viral load in the blood and the CSF have been considered and are under study. Finally, we explored the hypothesis of an EV replication in the blood leukocytes. We showed from blood samples infected in vitro by EV that the replication of these viruses varied by EV genotypes.

Hantaviruses are the causative agents of hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and of hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Transmission to humans usually occurs by inhalation of aerosolized virus-contaminated rodent excreta. To date, human-to-human transmission has only been described for the Andes hantavirus. The mode of transmission of Andes hantavirus is not yet known, but transmission through saliva has been suggested. In Sweden, we have one hantavirus that is pathogenic to humans, Puumala virus (PUUV), which is endemic in Central and Northern Europe. It induces a relatively mild form of HFRS, also called nephropathia epidemica (NE). The rodent reservoir is the bank vole (Myodes glareolus). The mechanism behind the pathogenesis of hantavirus is complex and probably involves both virus-mediated and host-mediated mechanisms. The aim of this project was to investigate the transmission mechanisms and pathogenesis of hantavirus disease in humans. In our first study, we described the largest outbreak of PUUV so far in Sweden. We investigated factors that might be important for causing the outbreak, and suggested that a peak in the bank vole population together with concurrent extreme weather conditions most probably contributed to the outbreak. Our next studies concentrated on human-to-human transmission of hantaviruses. We found PUUV RNA in saliva from PUUV-infected patients, suggesting that there is PUUV in the saliva of infected humans, although no person-to person transmission appears to occur with PUUV.  In the studies that followed, we showed that human saliva and human salivary components could inhibit hantavirus replication. We also found PUUV-specific IgA in the saliva of PUUV-infected patients, which might prevent person-to-person transmission of the virus.  In the final study, we focused on the pathogenesis of NE. One hundred five patients were included in a prospective study.  They were divided into a group with mild disease and a group with moderate or severe disease. We found that the immune response had a dual role in disease development. It was partly responsible for development of severe disease, with significantly higher amounts of neutrophils in severely ill patients, but it was also protective against severe disease, because patients with mild disease had higher levels of PUUV-specific IgG. In conclusion, a peak in the bank vole population in combination with extreme weather will increase the risk of human infection, PUUV RNA is present in saliva, PUUV-specific IgA and salivary components inhibit person-to-person transmission of PUUV, and the immune response is important for the pathogenesis of PUUV and the severity of the disease. / Hantavirus är en grupp av virus som finns hos gnagare som bär på viruset utan att själva bli märkbart sjuka. Varje hantavirus har anpassat sig till sin egen art av gnagare som de infekterar (kallas virusets reservoar). Hantaviruset kan överföras till människor från gnagare och kallas då för en zoonos eftersom detsprids från djur till människa. I människa orsakar hantavirus blödarfeber med njurpåverkan i Eurasien och blödarfeber med med hjärt och lungpåverkan i Nord- och Sydamerika. I Sverige har vi bara ett hantavirus som är sjukdomsframkallande hos människor, Puumala-viruset som även finns i delar av övriga Europa. Det framkallar en relativt mild form av blödarfeber, som kallas sorkfeber eller Nephropathia epidemica. Puumala-virusets reservoar är skogssorken (Myodes glareolus). Människor smittas oftast av hantavirus när de andas in infekterat damm som innehåller utsöndringar (avföring, urin eller saliv) från gnagare som har torkat in och sedan blivit luftburet. Vad man vet hittills så finns det bara ett hantavirus som smittar från person till person, för övriga hantavirus är människan en ”dead end”. Det virus som kan smitta från person till person heter Andes hantavirus och finns i Sydamerika. Andes hantavirus har en mus som reservoar från vilken människor kan smittas, sedan har smittan i vissa fall förts vidare från människa till människa, som tur är har dessa utbrott gått att stoppa. Fastän utbrotten har varit små har många personer dött, eftersom dödligheten är så hög, ungefär 30-40% av de diagnostiserade fallen dör. Hur Andes hantavirus överförs från människa till människa är inte känt men överföring genom saliv har föreslagits. Hur viruset ger upphov till sjukdom hos människa är inte klarlagt. Studier talar för att mekanismen bakom sjukdomsutvecklingen (den så kallade patogenesen) hos hantavirusorsakade blödarfebrar är komplex. Sannolikt beror patogenesen både på egenskaper hos viruset och värden d.v.s. människan som är smittad av viruset. Vårt mål med detta projekt var att undersöka vad som hindrar överföring av Puumala hantavirus från människa till människa och att undersöka hur virusinfektionen påverkar sjukdomsutvecklingen hos människan. I vår första studie beskrev vi det största utbrottet av sorkfeber hittills i Sverige och vi undersökte faktorer som kan ha orsakat utbrottet. Vi föreslog att en topp i skogssorkpopulationen samtidigt med extremt varmt väder troligen bidrog till utbrottet. Utbrottet skedde i december och det extremt varma vädret medförde att snön smälte bort. Sorkarna bor vanligtvis under snön på vintern, vi tror att frånvaro av snötäcke fick sorkarna att söka sig till byggnader för att söka skydd och där kom i kontakt med människor. Våra efterföljande studier fokuserade på överföring av hantavirus från människa till människa. Vi hittade Puumala-virusets arvsmassa (RNA) i saliv från sorkfeberpatienter, vilket tyder på att det finns Puumala-virus i saliven hos infekterade människor, även om ingen överföring från person till person verkar inträffa. I efterföljande studier visade vi att mänsklig saliv och mänskliga salivkomponenter minskar hantavirus smittsamhet. Vi fann också Puumala-virusspecifika IgA-antikroppar i saliven från sorkfeberpatienter, vilket kan förhindra överföring från person till person. I den sista studien fokuserade vi på patogenesen hos människor efter hantavirusinfektion. 105 patienter ingick i en prospektiv studie och delades in i en grupp med mild sjukdom och en grupp med måttlig/svår sjukdom. Vi hittade en dubbel roll hos immunsvaret för sjukdomsutvecklingen. Immunsvaret var delvis ansvarig för utveckling av svår sjukdom med betydligt högre mängd neutrofiler hos svårt sjuka patienter, men det var också skyddande mot allvarlig sjukdom, eftersom patienter med en mild sjukdom hade högre nivåer av Puumalavirusspecifika IgG-antikroppar. Detta talar för att behandling med IgG-antikroppar specifikt riktade mot hantavirus skulle kunna vara effektiv hos hantavirusinfekterade patienter. Sammanfattningsvis; en topp i skogssorkspopulationen i kombination med extremt väder ökar risken för infektion hos människor; Puumala-virus arvsmassa (RNA) finns i saliv; Puumala-virusspecifika IgA-antikroppar och salivkomponenter hämmar överföring av Puumalavirus från person till person; immunsvaret är viktigt för Puumala-virus patogenes och sjukdomens svårighetsgrad.

INTRODUÇÃO: Pacientes em terapia anti-retroviral podem apresentar escapes transitórios de viremia plasmática (blip), porém os preditores desse evento e seu impacto sobre a incidência de falência virológica são ainda controversos na literatura. Neste estudo de coorte estimou-se a incidência de blip e de falência virológica e investigaram-se possíveis preditores de tais desfechos. Blip foi definido como carga viral superior a 50 cópias/mL com subseqüente supressão da viremia plasmática e falência virológica como duas medidas consecutivas de carga viral plasmática superiores a 50 cópias/mL. Adicionalmente, pesquisou-se, por ocasião desses eventos, a presença de mutações genotípicas de HIV capazes de conferir resistência aos anti-retrovirais e as concentrações plasmáticas de inibidores não nucleosídicos da transcriptase reversa e inibidores da protease, comparando-as com o relato dos participantes sobre adesão à medicação. MÉTODOS: 350 participantes infectados pelo HIV (250 homens e 100 mulheres) foram selecionados no Serviço de Extensão ao Atendimento de Pacientes com HIV/Aids Casa da Aids do Hospital das Clínicas da FMUSP, São Paulo, Brasil. Na admissão ao estudo e trimestralmente, ao longo de 78 semanas, foram coletadas informações sobre dados sóciodemográficos, forma presumida de aquisição do vírus, uso de e adesão a medicações anti-retrovirais, ocorrência de outras comorbidades, bem como uso de álcool e de drogas ilícitas. Investigaram-se fatores potencialmente associados à incidência dos desfechos de interesse, tais como ocorrência de outras doenças, exposição a imunizações e falha na adesão a práticas de sexo mais seguro. Amostras de sangue periférico foram coletadas a cada visita para determinação de carga viral plasmática por RT-PCR ultrassensível, e contagem de linfócitos T CD4+ por citometria de fluxo. Nos indivíduos que apresentaram os desfechos de interesse do estudo, procedeu-se ao seqüenciamento dos genes da transcriptase reversa e da protease de HIV e à dosagem plasmática dos anti-retrovirais por método de Cromatografia Líquida de Alta Performance. As incidências de blip e falência virológica foram estimadas e os fatores associados a ambos investigados em modelo de regressão logística múltipla. RESULTADOS: As incidências de blip e falência virológica foram 9,4 e 4,2/100 pessoas-ano, respectivamente. Três indivíduos apresentaram falência virológica precedidos por blip. À análise multivariada, a não adesão às praticas de sexo mais seguro no mês precedente se mostrou independentemente associada à ocorrência de blip (OR 24,64, IC 95% 4,40 137,88, p<0,001) e de falência virológica (OR 24,69, IC 95% 4,20 145,18, p<0,001). Adicionalmente, observou-se que a exposição prévia a maior número de esquemas anti-retrovirais foi preditora dos eventos blip (OR 1,82, IC 95% 1,41 2,36, p<0,001) e falência virológica (OR 1,67, IC 95% 1,19 2,35, p=0,003). A ocorrência de blip não se associou ao desenvolvimento posterior de falência virológica. Um maior número de mutações conferidoras de resistência medicamentosa foi identificado no momento de falência virológica, quando comparado ao momento de blip, com predomínio de mutações no gene da transcriptase reversa, refletindo o maior uso desses fármacos. Das 122 concentrações plasmáticas de anti-retrovirais analisadas em 120 amostras, 84 estavam em níveis terapêuticos adequados. Porém, tais resultados apresentaram apenas 69% de concordância com a adesão auto-referida à medicação. Este estudo mostra que apresentar blip em uma medida isolada pode ser um evento benigno; por outro lado, falência virológica pode ser conseqüente a acúmulo de mutações conferidoras de resistência a pelo menos um dos anti-retrovirais em uso, podendo comprometer a eficácia do esquema terapêutico utilizado. Ambos os desfechos mostraram-se mais incidentes na população multiexperimentada à terapêutica, que, portanto, merece atenção particular. Uma importante contribuição deste estudo foi a avaliação da dosagem plasmática dos antiretrovirais, método simples e de baixo custo, que, implantado na rotina laboratorial, pode contribuir para o monitoramento da adesão aos antiretrovirais e reduzir a demanda por testes genotípicos / BACKGROUND: HIV-1-infected patients under antiretroviral therapy may present intermittent viremia (blip); however, predictors of this outcome and its influence on the incidence of virologic failure remain controversial in the literature. The aim of this study is to estimate the incidence of blip and virologic failure in a cohort of patients under stable antiretroviral therapy and to investigate their associated factors. Blip was defined as a plasma HIVRNA load above 50 copies/mL followed by a subsequent value below 50 copies/mL. Virologic failure was defined as two consecutives measures of viral load above 50 copies/mL. Moreover, at time of occurrence of these outcomes, HIV genotyping assays were performed in search of drug resistance-associated mutations, and plasma concentrations of nonnucleoside reverse transcriptase and protease inhibitors assessed and compared with self-reported adhrence to therapy. METHODS: 350 subjects (250 male and 100 female) were enrolled at the HIV Clinic, School of Medicine, University of São Paulo, Brazil and followed for 78 weeks. At baseline and in 3-month interval follow-up visits we collected sociodemographic data and information on presumed mode of HIV acquisition, use of and adherence to antiretrovirals, comorbidities and use of alcohol and illicit drugs. Additionally, patients were questioned about potential predictors of the outcomes, including occurrence of other diseases, immunizations and risky sexual behavior. Blood samples were drawn for assessment of HIV plasma viral loads, using ultrasensitive RT-PCR, and T CD4+ cell counts by flow cytometry. Individuals who presented blip and/or virologic failure were submitted to HIV genotyping assays and assessment of antiretroviral plasma concentrations by high-performance liquid chromatography. Incidences of blip and virological failure were estimated and associated factors investigated, using a multiple logistic regression model. RESULTS: The incidence of blip and of virologic failure were 9.4/100 and 4.2/100 person-years, respectively. Three individuals presented virologic failure after blip episodes. On multivariate analysis, non-adherence to safer sex measures in the previous month was shown independently associated with the occurrence of blip (OR 24.64, 95%CI 4.40 137.88, p<0.001) and virologic failure (OR 24.69, 95%CI 4.20 145.18, p<0.001). In addition, history of multiple exposures to antiretroviral regimens was also a predictor of blip (OR 1.82, 95%CI 1.41 2.36, p<0.001) and virologic failure (OR 1.67, 95%CI 1.19 2.35, p<0.001). Blips were not predictive of virologic failure. A larger number of HIV mutations were identified at time of virologic failure, as compared to blip episodes, with mutations detected predominantly in the reverse transcriptase (RT) gene, probably due to larger exposure to RT inhibitors. Eighty-four out of 122 assessments of antiretroviral plasma concentrations analyzed in 120 samples resulted in the therapeutic range. However, these results were concordant with self-reported adherence to therapy in 69% of cases only. This study shows that a single blip episode may be considered benign, whereas virologic failure could result from accumulation of HIV drug resistance-associated mutations that may impair the efficacy of therapy. Both study outcomes occurred more frequently among patients with larger exposure to antiretrovirals, and therefore they should be monitored in this regard. An important contribution of this study concerns the assessment of antiretroviral plasma concentrations, a simple and low cost laboratory tool. Incorporated routinely in patient follow-up, it would help monitoring adherence to therapy and reduce the need for HIV genotyping assays

INTRODUÇÃO: Pacientes em terapia anti-retroviral podem apresentar escapes transitórios de viremia plasmática (blip), porém os preditores desse evento e seu impacto sobre a incidência de falência virológica são ainda controversos na literatura. Neste estudo de coorte estimou-se a incidência de blip e de falência virológica e investigaram-se possíveis preditores de tais desfechos. Blip foi definido como carga viral superior a 50 cópias/mL com subseqüente supressão da viremia plasmática e falência virológica como duas medidas consecutivas de carga viral plasmática superiores a 50 cópias/mL. Adicionalmente, pesquisou-se, por ocasião desses eventos, a presença de mutações genotípicas de HIV capazes de conferir resistência aos anti-retrovirais e as concentrações plasmáticas de inibidores não nucleosídicos da transcriptase reversa e inibidores da protease, comparando-as com o relato dos participantes sobre adesão à medicação. MÉTODOS: 350 participantes infectados pelo HIV (250 homens e 100 mulheres) foram selecionados no Serviço de Extensão ao Atendimento de Pacientes com HIV/Aids Casa da Aids do Hospital das Clínicas da FMUSP, São Paulo, Brasil. Na admissão ao estudo e trimestralmente, ao longo de 78 semanas, foram coletadas informações sobre dados sóciodemográficos, forma presumida de aquisição do vírus, uso de e adesão a medicações anti-retrovirais, ocorrência de outras comorbidades, bem como uso de álcool e de drogas ilícitas. Investigaram-se fatores potencialmente associados à incidência dos desfechos de interesse, tais como ocorrência de outras doenças, exposição a imunizações e falha na adesão a práticas de sexo mais seguro. Amostras de sangue periférico foram coletadas a cada visita para determinação de carga viral plasmática por RT-PCR ultrassensível, e contagem de linfócitos T CD4+ por citometria de fluxo. Nos indivíduos que apresentaram os desfechos de interesse do estudo, procedeu-se ao seqüenciamento dos genes da transcriptase reversa e da protease de HIV e à dosagem plasmática dos anti-retrovirais por método de Cromatografia Líquida de Alta Performance. As incidências de blip e falência virológica foram estimadas e os fatores associados a ambos investigados em modelo de regressão logística múltipla. RESULTADOS: As incidências de blip e falência virológica foram 9,4 e 4,2/100 pessoas-ano, respectivamente. Três indivíduos apresentaram falência virológica precedidos por blip. À análise multivariada, a não adesão às praticas de sexo mais seguro no mês precedente se mostrou independentemente associada à ocorrência de blip (OR 24,64, IC 95% 4,40 137,88, p<0,001) e de falência virológica (OR 24,69, IC 95% 4,20 145,18, p<0,001). Adicionalmente, observou-se que a exposição prévia a maior número de esquemas anti-retrovirais foi preditora dos eventos blip (OR 1,82, IC 95% 1,41 2,36, p<0,001) e falência virológica (OR 1,67, IC 95% 1,19 2,35, p=0,003). A ocorrência de blip não se associou ao desenvolvimento posterior de falência virológica. Um maior número de mutações conferidoras de resistência medicamentosa foi identificado no momento de falência virológica, quando comparado ao momento de blip, com predomínio de mutações no gene da transcriptase reversa, refletindo o maior uso desses fármacos. Das 122 concentrações plasmáticas de anti-retrovirais analisadas em 120 amostras, 84 estavam em níveis terapêuticos adequados. Porém, tais resultados apresentaram apenas 69% de concordância com a adesão auto-referida à medicação. Este estudo mostra que apresentar blip em uma medida isolada pode ser um evento benigno; por outro lado, falência virológica pode ser conseqüente a acúmulo de mutações conferidoras de resistência a pelo menos um dos anti-retrovirais em uso, podendo comprometer a eficácia do esquema terapêutico utilizado. Ambos os desfechos mostraram-se mais incidentes na população multiexperimentada à terapêutica, que, portanto, merece atenção particular. Uma importante contribuição deste estudo foi a avaliação da dosagem plasmática dos antiretrovirais, método simples e de baixo custo, que, implantado na rotina laboratorial, pode contribuir para o monitoramento da adesão aos antiretrovirais e reduzir a demanda por testes genotípicos / BACKGROUND: HIV-1-infected patients under antiretroviral therapy may present intermittent viremia (blip); however, predictors of this outcome and its influence on the incidence of virologic failure remain controversial in the literature. The aim of this study is to estimate the incidence of blip and virologic failure in a cohort of patients under stable antiretroviral therapy and to investigate their associated factors. Blip was defined as a plasma HIVRNA load above 50 copies/mL followed by a subsequent value below 50 copies/mL. Virologic failure was defined as two consecutives measures of viral load above 50 copies/mL. Moreover, at time of occurrence of these outcomes, HIV genotyping assays were performed in search of drug resistance-associated mutations, and plasma concentrations of nonnucleoside reverse transcriptase and protease inhibitors assessed and compared with self-reported adhrence to therapy. METHODS: 350 subjects (250 male and 100 female) were enrolled at the HIV Clinic, School of Medicine, University of São Paulo, Brazil and followed for 78 weeks. At baseline and in 3-month interval follow-up visits we collected sociodemographic data and information on presumed mode of HIV acquisition, use of and adherence to antiretrovirals, comorbidities and use of alcohol and illicit drugs. Additionally, patients were questioned about potential predictors of the outcomes, including occurrence of other diseases, immunizations and risky sexual behavior. Blood samples were drawn for assessment of HIV plasma viral loads, using ultrasensitive RT-PCR, and T CD4+ cell counts by flow cytometry. Individuals who presented blip and/or virologic failure were submitted to HIV genotyping assays and assessment of antiretroviral plasma concentrations by high-performance liquid chromatography. Incidences of blip and virological failure were estimated and associated factors investigated, using a multiple logistic regression model. RESULTS: The incidence of blip and of virologic failure were 9.4/100 and 4.2/100 person-years, respectively. Three individuals presented virologic failure after blip episodes. On multivariate analysis, non-adherence to safer sex measures in the previous month was shown independently associated with the occurrence of blip (OR 24.64, 95%CI 4.40 137.88, p<0.001) and virologic failure (OR 24.69, 95%CI 4.20 145.18, p<0.001). In addition, history of multiple exposures to antiretroviral regimens was also a predictor of blip (OR 1.82, 95%CI 1.41 2.36, p<0.001) and virologic failure (OR 1.67, 95%CI 1.19 2.35, p<0.001). Blips were not predictive of virologic failure. A larger number of HIV mutations were identified at time of virologic failure, as compared to blip episodes, with mutations detected predominantly in the reverse transcriptase (RT) gene, probably due to larger exposure to RT inhibitors. Eighty-four out of 122 assessments of antiretroviral plasma concentrations analyzed in 120 samples resulted in the therapeutic range. However, these results were concordant with self-reported adherence to therapy in 69% of cases only. This study shows that a single blip episode may be considered benign, whereas virologic failure could result from accumulation of HIV drug resistance-associated mutations that may impair the efficacy of therapy. Both study outcomes occurred more frequently among patients with larger exposure to antiretrovirals, and therefore they should be monitored in this regard. An important contribution of this study concerns the assessment of antiretroviral plasma concentrations, a simple and low cost laboratory tool. Incorporated routinely in patient follow-up, it would help monitoring adherence to therapy and reduce the need for HIV genotyping assays

L’objectif d’un traitement antirétroviral (ARV) est d’obtenir une charge virale VIH plasmatiqueindétectable afin de réduire la morbi-mortalité associée au VIH.La résistance du virus aux ARV est un facteur de risque d’échec virologique (EV). Les testsgénotypiques de résistance par séquençage classique (méthode de Sanger) ne permettent pas dedétecter les virus porteurs de mutations de résistance (MR) présents à taux minoritairereprésentant moins de 20% de la population virale plasmatique. Dans notre étude (cohorteANRS CO3), l’utilisation de l’ultra-deep sequencing (UDS) a permis de détecter 1.4 fois plusde MR avant traitement et 1.3 fois plus à l’EV en comparaison à la technique de séquençageclassique, confirmant la haute sensibilité de l’UDS pour la détection des MR. Les MRminoritaires détectées uniquement par UDS augmentaient la résistance génotypique du virus autraitement ARV chez 4% des patients à l’initiation du traitement et 21% des patients à l’EV.Les conséquences des épisodes de charges virales de faible niveau persistantes (CVF) entre 50et 200 copies/ml (CVF50-200) ne sont pas clairement établies du fait de l’insuffisance dedonnées dans la littérature. Dans nos études de cohortes (cohortes ANRS CO3 et ART-CC), 4-9% des patients ont présenté au moins un épisode de CVF50-200. La survenue d’une CVF50-200 était associée à un risque plus de 2 fois plus élevé d’EV>200 copies/ml, quels que soient ladurée de la CVF, l’historique de traitement ou le traitement ARV lors de la CVF50-200(régimes comportant des INNTI ou des IP/r). La survenue d’une CVF n’était pas associée à lasurvenue d’un évènement classant SIDA ni au décès, avec toutefois un suivi médian de 3 ans. / The goal of antiretroviral therapy (ART) is to reach undetectable plasma HIV viral load in orderto reduce HIV-related morbidity and mortality.The presence of ART-resistant virus can compromise the efficiency of these treatments,resulting in virological failure (VF). Standard genotyping by Sanger sequencing (SS) usedcurrently in clinical practice cannot detect low-frequency viral variants harbouring drugresistance associated mutations (DRM) representing less than 20% of the viral population. Inour study, the use of ultra-deep sequencing (UDS) allowed us to detect 1.4-fold more DRMsbefore ART and 1.3-fold more DRMs at VF compared to SS, confirming the high sensitivity ofUDS for the detection of DRMs. The low-frequency DMRs detected only by UDS modified thegenotypic resistance of the virus to the prescribed treatment for 4% of the patients before ARTinitiation and for 21% of the patients at VF.The impact of persistent low-level viremia (LLV) between 50 and 200 copies/ml (LLV50-200)remains uncertain due to the lack of controlled comparison data. In our cohort studies (ANRSCO3 and ART-CC cohorts), 4-9% of HIV-infected patients experienced at least one episode ofLLV50-200. LLV50-200 was strongly associated with a twice higher risk of VF>200copies/ml,independently of the duration of LLV, the history of treatment or the type of ART regimen atLLV (NNRTI or PI-based regimens). LLV was not associated with AIDS event or death witha median follow-up of only 3 years.

Le virus de l'immunodéficience humaine (VIH) est à l’origine d’une infection chronique, elle-même responsable du développement du syndrome d'immunodéficience acquise (SIDA), un état de grande vulnérabilité où le corps humain est à la merci d’infections opportunistes pouvant s’avérer fatales. Aujourd’hui, 30 ans après la découverte du virus, même si aucun vaccin n’a réussi à contrôler la pandémie, la situation s’est grandement améliorée. Conséquemment à l’arrivée de traitements antirétroviraux hautement actifs (HAART) à la fin des années 1990, la mortalité associée au VIH/SIDA a diminué et un plus grand nombre de personnes vivent maintenant avec l'infection. La présente thèse avait pour objectif d’aborder trois situations problématiques, en dépit de l’efficacité reconnue des HAART, plus particulièrement la faible charge virale persistante (LLV) et sa relation avec l’échec virologique, ainsi que les effets de certains antirétroviraux (ARV) sur les fonctions rénale et hépatique. Les objectifs précis étaient donc les suivants : 1) étudier le risque d’échec virologique à long terme chez les patients sous HAART dont la charge virale est indétectable comparativement aux patients affichant une LLV persistante; 2) évaluer sur le long terme la perte de fonction rénale associée à la prise de ténofovir (TDF) 3) étudier sur le long terme l'hyperbilirubinémie associée à la prise d’atazanavir (ATV) et ses autres déterminants possibles. Afin d’atteindre les trois objectifs susmentionnés, une cohorte de 2 416 patients atteints du VIH/SIDA, suivis depuis juillet 1977 et résidant à Montréal, a été utilisée. Pour le premier objectif, les résultats obtenus ont montré un risque accru d’échec virologique établi à >1000 copies/ml d’ARN VIH chez tous les patients qui présentaient une LLV persistante de différentes catégories durant aussi peu que 6 mois. En effet, on a observé qu’une LLV de 50-199 copies/ml persistant pendant six mois doublait le risque d’échec virologique (Hazard ratio (HR)=2,22, Intervalle de confiance (CI) 95 %:1,60–3,09). Ces résultats pourraient modifier la façon dont on aborde actuellement la gestion des patients affichant une LLV, et plus particulièrement une LLV de 50-199 copies/ml, pour laquelle aucune recommandation clinique n’a encore été formulée en raison du manque de données. Pour le deuxième objectif, on a observé une augmentation du risque de perte de fonction rénale de l’ordre de 63 % (HR=1,63; 95% CI:1,26–2,10) chez les patients sous TDF comparativement aux patients traités avec d’autres ARV. La perte de fonction rénale directement attribuable à la prise de TDF, indique que cette perte est survenue au cours des premières années de l’exposition du patient au médicament. D’une perspective à long terme, cette perte est considérée comme modérée. Enfin, pour ce qui est du troisième objectif, on a constaté que l’incidence cumulative d’hyperbilirubinémie était très élevée chez les patients sous ATV, mais que cette dernière pouvait régresser lorsque l’on mettait fin au traitement. L’hyperbilirubinémie à long terme observée avec la prise d’ATV n’a été associée à aucun effet néfaste pour la santé. Dans l’ensemble, la présente thèse a permis de mieux comprendre les trois situations problématiques susmentionnées, qui font actuellement l’objet de débats au sein de la communauté scientifique, et d’éclairer sous un jour nouveau la gestion des patients séropositifs sous traitement médicamenteux. / Human immonudeficiency virus (HIV) is a virus causing a chronic infection responsible for Acquired Immunodeficiency Syndrome (AIDS), a state of vulnerability of the body where different opportunistic infections will ultimately be fatal. About 30 years after the discovery of the virus, even if no vaccine is available to control the pandemia, situation has changed for the best. With the arrival of highly active anti-retroviral therapy (HAART) in the late 90's, a reduction in HIV/AIDS mortality rate and growing number of persons living with the infection were observed. The overall objective of this thesis was to address three problematic situations, despite recognised HAART efficacy, especially low-level viremia (LLV) and its relationship with virologic failure, and the impacts of certain antiretrovirals (ARV) on kidney and hepatic functions. The specific objectives were: 1) to study the risk of virologic failure in long-term perspective in undetectable patients under HAART in comparison to patients with persistent LLV; 2) to evaluate the long-term loss of kidney function related to tenofovir (TDF) exposure 3) to evaluate long-term hyperbilirubinemia related to atazanavir (ATV) exposure and other possible determinants. In order to address the three specific objectives, a cohort of patients 2416 living with HIV/AIDS followed in Montreal since July 1977 was used. For the first objective, analyses and results shown an increased risk of virological failure defined as >1000 copies/mL of HIV RNA, for all categories of persistent LLV as soon as 6 months of persistent duration. Persistent LLV of 50-199 copies/mL for 6 months doubled the risk of virologic failure (Hazard ratio (HR)=2,22, Confidence interval (CI) 95%: 1,60-3,09). The results shed new light for the management of patients with LLV, especially for LLV of 50-199 copies/mL, for which no clinical recommendation is currently available due to a lack of data. For the second objective, an increased risk of loss of kidney function of 63% (HR=1.63; 95% CI:1.26–2.10) associated to TDF exposure in comparison to patients taking other ARV was observed. The cumulative eGFR loss directly attribuable to TDF also shown that this loss occured during the first years of exposure. This loss was mild in a long-term perspective. For the third objective, it has been shown that the cumulative incidence of hyperbilirubinemia in ATV users was very high and that regression was possible if ATV exposure was ended. Long-term hyperbilirubinemia related to ATV use was not associated with adverse health outcome. Overall, this thesis allowed a better understanding of these three problematics currently debated in scientific literature and shed new lights on management of HIV positive patients under therapy.

Le virus de l'immunodéficience humaine (VIH) est à l’origine d’une infection chronique, elle-même responsable du développement du syndrome d'immunodéficience acquise (SIDA), un état de grande vulnérabilité où le corps humain est à la merci d’infections opportunistes pouvant s’avérer fatales. Aujourd’hui, 30 ans après la découverte du virus, même si aucun vaccin n’a réussi à contrôler la pandémie, la situation s’est grandement améliorée. Conséquemment à l’arrivée de traitements antirétroviraux hautement actifs (HAART) à la fin des années 1990, la mortalité associée au VIH/SIDA a diminué et un plus grand nombre de personnes vivent maintenant avec l'infection. La présente thèse avait pour objectif d’aborder trois situations problématiques, en dépit de l’efficacité reconnue des HAART, plus particulièrement la faible charge virale persistante (LLV) et sa relation avec l’échec virologique, ainsi que les effets de certains antirétroviraux (ARV) sur les fonctions rénale et hépatique. Les objectifs précis étaient donc les suivants : 1) étudier le risque d’échec virologique à long terme chez les patients sous HAART dont la charge virale est indétectable comparativement aux patients affichant une LLV persistante; 2) évaluer sur le long terme la perte de fonction rénale associée à la prise de ténofovir (TDF) 3) étudier sur le long terme l'hyperbilirubinémie associée à la prise d’atazanavir (ATV) et ses autres déterminants possibles. Afin d’atteindre les trois objectifs susmentionnés, une cohorte de 2 416 patients atteints du VIH/SIDA, suivis depuis juillet 1977 et résidant à Montréal, a été utilisée. Pour le premier objectif, les résultats obtenus ont montré un risque accru d’échec virologique établi à >1000 copies/ml d’ARN VIH chez tous les patients qui présentaient une LLV persistante de différentes catégories durant aussi peu que 6 mois. En effet, on a observé qu’une LLV de 50-199 copies/ml persistant pendant six mois doublait le risque d’échec virologique (Hazard ratio (HR)=2,22, Intervalle de confiance (CI) 95 %:1,60–3,09). Ces résultats pourraient modifier la façon dont on aborde actuellement la gestion des patients affichant une LLV, et plus particulièrement une LLV de 50-199 copies/ml, pour laquelle aucune recommandation clinique n’a encore été formulée en raison du manque de données. Pour le deuxième objectif, on a observé une augmentation du risque de perte de fonction rénale de l’ordre de 63 % (HR=1,63; 95% CI:1,26–2,10) chez les patients sous TDF comparativement aux patients traités avec d’autres ARV. La perte de fonction rénale directement attribuable à la prise de TDF, indique que cette perte est survenue au cours des premières années de l’exposition du patient au médicament. D’une perspective à long terme, cette perte est considérée comme modérée. Enfin, pour ce qui est du troisième objectif, on a constaté que l’incidence cumulative d’hyperbilirubinémie était très élevée chez les patients sous ATV, mais que cette dernière pouvait régresser lorsque l’on mettait fin au traitement. L’hyperbilirubinémie à long terme observée avec la prise d’ATV n’a été associée à aucun effet néfaste pour la santé. Dans l’ensemble, la présente thèse a permis de mieux comprendre les trois situations problématiques susmentionnées, qui font actuellement l’objet de débats au sein de la communauté scientifique, et d’éclairer sous un jour nouveau la gestion des patients séropositifs sous traitement médicamenteux. / Human immonudeficiency virus (HIV) is a virus causing a chronic infection responsible for Acquired Immunodeficiency Syndrome (AIDS), a state of vulnerability of the body where different opportunistic infections will ultimately be fatal. About 30 years after the discovery of the virus, even if no vaccine is available to control the pandemia, situation has changed for the best. With the arrival of highly active anti-retroviral therapy (HAART) in the late 90's, a reduction in HIV/AIDS mortality rate and growing number of persons living with the infection were observed. The overall objective of this thesis was to address three problematic situations, despite recognised HAART efficacy, especially low-level viremia (LLV) and its relationship with virologic failure, and the impacts of certain antiretrovirals (ARV) on kidney and hepatic functions. The specific objectives were: 1) to study the risk of virologic failure in long-term perspective in undetectable patients under HAART in comparison to patients with persistent LLV; 2) to evaluate the long-term loss of kidney function related to tenofovir (TDF) exposure 3) to evaluate long-term hyperbilirubinemia related to atazanavir (ATV) exposure and other possible determinants. In order to address the three specific objectives, a cohort of patients 2416 living with HIV/AIDS followed in Montreal since July 1977 was used. For the first objective, analyses and results shown an increased risk of virological failure defined as >1000 copies/mL of HIV RNA, for all categories of persistent LLV as soon as 6 months of persistent duration. Persistent LLV of 50-199 copies/mL for 6 months doubled the risk of virologic failure (Hazard ratio (HR)=2,22, Confidence interval (CI) 95%: 1,60-3,09). The results shed new light for the management of patients with LLV, especially for LLV of 50-199 copies/mL, for which no clinical recommendation is currently available due to a lack of data. For the second objective, an increased risk of loss of kidney function of 63% (HR=1.63; 95% CI:1.26–2.10) associated to TDF exposure in comparison to patients taking other ARV was observed. The cumulative eGFR loss directly attribuable to TDF also shown that this loss occured during the first years of exposure. This loss was mild in a long-term perspective. For the third objective, it has been shown that the cumulative incidence of hyperbilirubinemia in ATV users was very high and that regression was possible if ATV exposure was ended. Long-term hyperbilirubinemia related to ATV use was not associated with adverse health outcome. Overall, this thesis allowed a better understanding of these three problematics currently debated in scientific literature and shed new lights on management of HIV positive patients under therapy.