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Background: A novel, isothermal nucleic acid amplification method, RT-LAMP, presents potential for nucleic acid amplification-based diagnostics in resource-limited settings. Low-cost HIV-1 viral load monitoring will improve access to ART for HIV-1-infected individuals present in settings where on-site viral load testing is unavailable. Aim: The aim of this dissertation was to develop an RT-LAMP HIV-1 viral load assay by combining the RT-LAMP reaction with colorimetric amplification detection by hydroxy-naphthol blue dye. Methods: Different approaches for HIV RNA extraction from patient plasma and culture supernatant were studied to obtain template for RT-LAMP. Reaction products for 4 different RT-LAMP primer sets were analysed using agarose gel electrophoresis and restriction digestion. Results: The first 3 primers sets produced persistent off-target amplification. The fourth primer set, designed against culture supernatant DU179, produced a target-specific colour change from violet to blue after 1 hour, following optimisation of amounts of Mg2SO4 and AMV RT. Further studies showed HNB detection sensitivity to template copy number. Conclusions: Initial reaction conditions pertaining to an RT-LAMP based, colorimetric HIV-1 viral load assay were established. Further work is required to determine the reaction duration at which the colour change represents a viral load of ≥1000 copies HIV RNA per ml plasma.

Introduction and aims: In the United Kingdom hepatitis B virus (HBV) and hepatitis C virus (HCV) disproportionately affect migrants and people who inject drugs (PWID). Ascertainment rates of HBV and HCV are 10-40% and screening is recommended in at risk groups. Treatment uptake for HCV in PWID is low at 2-18%, and the most effective way to increase uptake is not known. This research aims to evaluate methods to address the low ascertainment and treatment rates of HBV and HCV in these populations. Methods: A pilot observational cohort study of screening for chronic viral hepatitis in primary care. A retrospective observational cohort study into outcomes of HCV treatment in PWID. A prospective cluster randomised trial of nurse versus doctor initiated treatment for HCV in PWID and a qualitative analysis exploring the engagement with treatment for HCV of PWID. Results: Direct testing results in a greater uptake of screening than opportunistic testing in migrants in primary care (21% versus 1.9%, p = < 0.0001). PWID have SVR rates of 55%, re-infection rates of 2.4 per 100 person years, and crack cocaine use reduces over treatment (90% to 49%, p= < 0.0001). Nurse initiation of treatment does not result in a higher uptake of therapy (9.6 % versus 7.8%, p=0.53). Treatment engagement themes included the normalisation and stigmatisation of HCV and the perception of HCV treatment as a transformative process. Discussion: Direct testing for HBV and HCV appears to result in a greater uptake of testing in migrants in primary care and should be investigated in a randomised controlled trial. HCV treatment in PWID is safe and effective, and illicit drug use may reduce over treatment. Further service development is unlikely to result in a greater uptake of antiviral therapy for HCV in PWID and other options should be explored to improve treatment uptake.

Bronquiolite Viral Aguda (BVA) é uma doença de alta prevalência no Rio Grande do Sul e causa um importante número de hospitalizações em crianças menores de um ano de idade. Os pacientes com maior gravidade, nos três primeiros dias de internação, podem evoluir para insuficiência ventilatória e necessitarem oxigênio ou até ventilação mecânica, como forma de tratamento. Com o objetvo de identificar precocemente os episódios mais graves, investigaram-se fatores prognósticos através de sinais clínicos e laboratoriais, durante a hospitalização de crianças com BVA. Estudou-se uma coorte de 213 crianças menores de um ano com BVA admitidas no Hospital da Criança Santo Antônio de Porto Alegre. Esses pacientes foram seguidos desde a hospitalização, nos três dias subseqüentes, na alta hospitalar e até 30 e 60 dias após. Caracterizou-se a necessidade de oxigênio no terceiro dia ou de ventilação mecânica nos três primeiros dias de hospitalização, como critérios de gravidade. As hospitalizações ocorreram, predominantemente, nos meses de julho a setembro, e a maior parte das crianças (60%) apresentava idade inferior a quatro meses. Dentre as características sócio-econômicas estudadas, observou-se que 50% das famílias tinham renda mensal menor que três salários mínimos e 18% viviam em residências aglomeradas. Das 213 crianças acompanhadas até a alta hospitalar, 61(29%) necessitaram de oxigênio no terceiro dia, e 12(6%), ventilação mecânica nas primeiras 72 horas. Verificou-se que 17% dos pacientes apresentavam história de prematuridade, 24% estavam sendo amamentados quando hospitalizaram e 12% nunca haviam mamado no peito. Cerca de 35% das famílias referiram história de asma brônquica. As principais características clínicas na hospitalização foram: freqüência respiratória maior que 60(67%), presença de sibilos(76%) e estertores crepitantes(57%) à ausculta pulmonar e tiragem subcostal (56%). Entre os sinais investigados, assodaramse significativamente com nlaior gravidade: frequência respiratória maior que 70, enchimento capilar lento, cianose de extremidades e tiragem supraesternal. A saturação transcutânea de oxigênio da hemoglobina inferior a 91% e atelectasia ao exame radiológico de tórax, mostraram-se associadas significativamente com maior gravidade. Entre os fatores prognósticos, identificou-se o risco relativo associado a maior gravidade: idade inferior a 4 mes{~s (RRl~7), peso de nascimento inferior a 2500g (RR 2,3) e desnutrição grave(RR 2,0). Constituíram-se escalas de gravidade através de análise discriminante incluindo-se os seguintes itens: idade inferior a 3 meses, prostração, batimento de asas do nariz, freqüência respiratória maior ou igual a 70, tiragem (subcostal, intercostal e supraesternal), saturação de oxigênio menor ou igual a 90%, uso de oxigênio, internação em UTI e uso de ventilação mecânica. Através do somatório de cada um dos itens, resultaram os escores de gravidade. Os escores foram dicotomizados em menores ou iguaisl a 3 e maiores que 3 (mais graves). No seguimento após a alta hospitalar, observou-se que a Inaioria dos pacientes que compareceram apresentou episódios de sibilância e que foi elevado o número de reinternações (26% aos 60 dias). / Acute viral bronchiolitis(AVB) has a high prevalence in Rio Grande do Sul. It accounts for a high number of hospital admissions in infants. The patients with a more severe disease, in the first three days of hospitalization may develop respiratory failure and might need oxygen or mechanical ventilation . The aim of this study was to investigate prognostic factors (clinicaI signs and laboratory tests) in hospitalized infants with AVB. The research conducted was a cohort study of 213 infants with AVB who were admitted to the Hospital da Criança Santo Antônio, Porto Alegre. These patients were followed up from the admission, in the three first days, to their discharge and at 30 and 60 days after admission. Severity criteria were defined such as need of oxygen in the third day of admission or mechanical ventilation in the first three days. The admissions occurred predominantly from July to September and the majority (60%) were infants under four months of age. It was observed that 50% of the families had monthly wages below three minimum saIaries and 18% lived in crowded homes. Sixty one (29%) of the children needed oxygen in the third day of admission and 12 (6%) were put on mechanical ventilation. It was observed that 17% of the patients had a past history of prematurity, 24% were being breast fed at admission and 12% had never been breast fedo Around 35% of the families had a past history of bronchial asthma. The main clinicaI characteristics were: respiratory rate above 60 (67%), wheezes (76%), crepitations (57%) and subcostal retraction (56%). The following findings were significantly associated to severity : respiratory rate above 70 mpm, peripheric cyanosis, low capillary filling, supraesternal retraction, transcutaneous oxygen saturation below 91%, atelectasis at the chest X-ray. Among the prognostic features, some were identified as presenting high relative risk associated to severity: Age under 4 months (RR 1,7), birth weight below 2500 g (RR 2,3) and malnutrition (RR 2,0). Severity scales have been developed using discriminant analysis with the following items: age under 3 months, prostration, flaring of the alae nasi, respiratory rate above 70 mpm, retractions (subcostal, intercostal and supraesternal), transcutaneous oxygen saturation, need of oxygen, admission in intensive care unit and use of mechanical ventilation. Severity scores resulted from the addition of the value attributed to each of the items (O or 1). They were dichotomized in above (more severe) and below or equal to 3. At the follow up, after the discharge it was observed that the majority of the children who had been seen at thirty and sixty days had wheezing episodes and there was a high rate of re admissions (26% at 60 days).