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.

In the last few years, engagement in medical care among individuals living with HIV has become a major priority among HIV medical providers and public health researchers. Engagement in medical care is an important concept as it involves the process of linking newly diagnosed individuals into medical care and retaining those individuals in care throughout the course of their infection. Although there have been major advances in the management of HIV, like the advent of Highly Active Antiretroviral Therapy, morbidity and mortality due to HIV cannot be fully reduced if the individual does not optimally retain in care. Retention in HIV medical care has become an emerging topic in HIV research, but there still remains a scarce amount of research on how to properly define retention, understand its predictors, and how it impacts HIV outcomes. The purpose of this dissertation was to evaluate retention in HIV medical care among individuals diagnosed with HIV and seeking care at an urban infectious disease clinic in Kentucky. The three specific aims of this dissertation were to: (1) compare methods in measuring retention in HIV medical care; (2) determine the predictors of poor retention in care and assess the effect of non-HIV related comorbidities have on retention over time; and (3) determine the impact early retention to medical care has on time to viral load suppression and rebound among individuals initiating Highly Active Antiretroviral Therapy. A retrospective cohort study was conducted employing a medical chart review, and patients who sought HIV care at the Bluegrass Care Clinic between January 1st 2003 and May 1st 2011 were eligible for the study. There were 1,358 patients included in the study and these individuals were followed until December 31st, 2011. The results suggested that individuals living with HIV should seek care at least once every six months (visit constancy) and that only 48.6% of the study population obtained optimal retention over time. Over time the rate of retention decreased among the study sample and those with optimal retention were more likely to suppress their viral loads compared to poor retainers.

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Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-06T18:47:19Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertacao-BrenoAlbuquerque.pdf: 1989114 bytes, checksum: 513b1650c4c44605e3bda0afef185321 (MD5) / Made available in DSpace on 2017-04-06T18:47:19Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertacao-BrenoAlbuquerque.pdf: 1989114 bytes, checksum: 513b1650c4c44605e3bda0afef185321 (MD5) Previous issue date: 2012 / O Papilomavírus bovino(BPV) é o agente etiológico da papilomatosebovina. Esta apresenta lesões que normalmente são benignas e tendem a regredir, porém podem progredir a uma neoplasia. Muitas metodologias utilizadas para detecção de BPV se mostram inespecíficas e apresentam reações cruzadas com outros organismos relacionados. No entanto, a reação quantitativa em tempo real emcadeia da polimerase (qPCR) é uma ferramenta de destaque na detecção, tipificação e quantificação de nucleotídeos e vem sendo utilizada na clínica para avaliar carga viral. O objetivo do trabalho foi desenvolver um novo protocolo de detecção, tipificação e quantificação de BPV através daqPCR. Foram desenhados cinco pares de primers, que possuem como alvo uma região conservada do genoma viral (gene L1) de diferentes BPVs. A seletividade dos primers foi testada in vitroe DNA extraído de células MDBK não infectadas foram utilizados como controle negativo. A técnica de qPCR permitiu detectar, tipificar e quantificar material viral dos BPVs 1, 2, 4, 5 e 6. O limiar relativo da detecção foi de 4fg de DNA,emtorno de 30-40 cópias de DNA/μL. Dos cinco pares de primers produzidos, quatro apresentaram mesmo perfil térmico durante a qPCR (qPCRBPV2, 4, 5 e 6), permitindo em um único procedimento detectar e tipificar os quatro tipos virais. A distinção das amostrasfoi realizada através da análise de meltingque permitiu tipificá-las. Através da metodologia desenvolvida foi observado que em lesões cutâneas de bovinos infectados com BPV a carga viral não se mostrou inferior a 1000 cópias/μL, enquanto que a técnica permite quantificar até um limiar de 40 copias de DNA/μl. Este trabalho possui relevância para validação de qPCR como diagnóstico da papilomatose bovina e particular importância quando aplicado em estudos da infecção pelo BPV e no monitoramento por veterinários da eficácia das futuras vacinas. / Bovine papillomavirus (BPVs) is the etiologic agent of bovine papilomatose which is characterized by hyper proliferative lesions. Papillomas in cattle are typically benignandoften regress, but occasionally lesions can persist and progress to malignant neoplasia.The majority of current techniques for identification of BPV is unspecific andpossessescross-reactivity with closely related organisms.The Real-time quantitative polymerase chain reaction assay (qPCR) has become an exceptional tool for detection and quantification of oligonucleotides and has been utilized increasingly on viral load evaluation.Aiming to develop a new protocol for fast detection, typification and quantification of BPV in qPCR, we designed five pairs of Oligonucleotides for BPV1, 2, 4, 5 and 6 focusing on L1 gene. The qPCR primers sets were testedin vitroandMadin-Darby Bovine Kidney Cells (MDBK)DNA was also used as negative control.The Real-time qPCR assay provided an accurate detection and quantification for the BPVs 1, 2, 4, 5 and 6. The relative detection limit for the assays was 4fg or 30 to 40genome equivalents. Four primers pairs (qPCRBPV2, 4, 5 and 6) had the same annealing temperature and their products showed differences on meltingpoints analyses. Through the meltingpoint analysis, samples can be identified and discriminated as a screening and then samples can be run for viral load. In our study we tested the viral load in bovine cutaneous skin warts and observed infections with 1000 copies/μl at least. However, this assay could reach levels of 40copies/μL. In conclusion, this methodology has an important impact on the validation of qPCR as a BPV diagnosis. Its relevance is proved when applied to BPV infection studies and the monitoring of the efficacy of future BPV vaccinesby veterinarians.

The rate that HIV-infected individuals progress to AIDS and death varies greatly. Viral load taken during the asymptomatic phase of the disease is one of the best-known predictors of HIV progression rate and transmission risk, and is known to be in uenced by both host and environmental factors. However, the role that the virus itself plays in determining the viral load is less clear. Previous studies have attempted to quantify the amount the viral genome in uences viral load, or the heritability of viral load, using transmission pairs and phylogenetic signal in small sample sizes, but have produced highly disparate estimates. E cient and accurate methods to estimate heritability have been utilised by quantitative geneticists for years, but are rarely applied to non-pedigree data. Here, I present a novel application of a population-scale method based in quantitative genetics to estimate the heritability of viral load in HIV using a viral phylogeny. This new phylogenetic method allows the inclusion of more samples than ever previously used, and avoids confounding e ects associated with transmission pair studies. This new method was applied to the two largest HIV subtypes found in the UK, subtypes B and C, using sequences and clinical data from UK-wide HIV databases. For subtype B (n=8,483) and C (n=1,821), I estimated that 5.7% (CI 2.8{8.6%) and 29.7% (CI 14.8{44.7%) of the variance in viral load is determined by the viral genome, respectively. These estimates suggest that viral in uence on viral load varies greatly between subtypes, with subtype C having much larger viral control over viral load than subtype B. I expanded the phylogenetic method to test whether the component of the viral load determined by the virus has changed over time. In subtype B, I foundevidence of a small but signi cant decrease in the viral component of viral load of -0.05 log10 copies/mL/yr. I built a stochastic, individual-based model capable of simulating a realistic HIV epidemic, with heritable viral loads that in uence transmission and disease progression, capable of generating data sets to assess the accuracy of phylogenetic methods. This was successfully used to generate epidemics approximating those in a small African village and a Western `men who have sex with men' community under a variety of conditions. To test the accuracy of the new phylogenetic heritability estimation method, simulated datasets were generated with the heritability of viral load set at values of 30%, 50%, 70%, and 90%. Unfortunately, complications in the heritability equation used prevented full assessment of the new phylogenetic method on the simulated data. Future development of the model will enable simulation of realistic viral loads under varying heritability values, enabling simulation of data sets that can be used to test this and other heritability estimation methods. This new phylogenetic method allows accurate estimation of heritability in large datasets, and has provided valuable insight into the viral in uence on viral load in HIV.

MSc (Med), Dept of Haematology, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand / BACKGROUND: Human Immunodeficiency Virus (HIV) is a global epidemic with growing numbers of people on highly active anti‐retroviral therapy (HAART) programmes. Effectiveness of treatment needs to be monitored to ensure the uncompromised well being of patients. This is currently done using both Viral Load (VL) and CD4 cell counts for HAART initiation and follow‐up. Although VL is the best predictor of disease progression it is often too expensive for monitoring patients in resource‐limited settings. There is thus a need for a cheaper, more accessible alternative to monitor long term patient response to therapy. METHODS: This study evaluated the use of a recently described flow cytometric assay of CD38 expression (previously developed at the Johannesburg Flow Cytometry Reference Laboratory) in a cohort of HIV+ patients failing 1st line therapy, who were subsequently enrolled onto 2nd line HAART. CD38 and CD8 were “piggy ‐backed” onto the PLG/CD4 protocol and mean fluorescence intensity (MFI) of the CD8/38 expression was monitored longitudinally. Patterns of CD38 expression were compared to 1st line treatment observations to establish equivalence in the predictive power of CD38 expression of fluctuation in viral load on 2nd line treatment patients. In addition, the effect of sample age on assay accuracy was tested before implementation of the CD38 assay at a secondary testing site. RESULTS: The patterns observed in the cohort of 2nd line therapy patients mirrored patterns previously seen in 1st line therapy with 55% of patients showing a continuous decline in CD38 MFI that mimicked changes in VL. The remaining 33% of patients had non‐specific increases in CD38 MFI without concurrent increases in VL and one patient showed irregular VL and CD38 MFI (non‐responder). The CD38 assay showed acceptable accuracy and reproducibility up to 48 hours after venesection (%CV<5%). Implementation at the secondary testing site was successful with 98% similarity (%CV<5%) compared to the reference laboratory. CONCLUSION: CD38 monitoring of 2nd line therapy patients showed comparable patterns to observations in 1st line therapy patients. The assay proved stable over time and easy to implement at another PLG/CD4 testing facility. As such, the CD38 assay offers a cost‐effective, reliable real time supplementary test to long‐term VL monitoring of HIV infected patients on the national ART programme.

L’histoire naturelle et la progression des infections au VPH (virus du papillome humain) sont bien décrites. Cependant, la dynamique de transmission reste faiblement documentée. Une meilleure compréhension de la dynamique de transmission ainsi que de ses facteurs de risque permettrait d’optimiser les stratégies de prévention afin de réduire la prévalence de ces infections dans la population par la vaccination et les méthodes contraceptives. Notre étude vise à déterminer si la charge virale des infections au VPH influence leur transmission entre les partenaires sexuels. Pour ce faire, l’association entre la charge virale au niveau des organes génitaux et la concordance spécifique de type des infections prévalentes au VPH a été évaluée pour 250 couples hétérosexuels récemment formés. Les charges virales de VPH16 (r = 0.30), de VPH18 (r = 0.50) et de VPH51 (r = 0.19) étaient significativement corrélées (p < 0.05) entre les deux partenaires sexuels, contrairement à celles de VPH31 (r = 0.08) et de VPH42 (r = -0.1). Lorsqu’ajusté en fonction de l’âge des participants, une charge virale élevée augmentait significativement le taux de détection du même type chez le partenaire pour les types 16, 31 et 51. Ainsi, dans les couples hétérosexuels récemment formés, des charges virales élevées sont associées à une détection accrue du même type chez le partenaire sexuel. / The natural history and progression of genital HPV infection are well understood. However, less is known about transmission dynamics of HPV between sexual partners. A better knowledge of risk factors and dynamics of HPV transmission is needed to optimize prevention strategies through vaccination and contraceptive measures. Our study aims to determine if the viral load of HPV infection affects transmission between sexual partners. The association between human papillomavirus (HPV) loads in genital swabs and type-specific concordance of prevalent HPV infection was assessed in 250 heterosexual recently-formed couples to further characterize HPV transmission. Viral loads of HPV16 (r=0.30), HPV18 (r=0.50) and HPV51 (r=0.19) were significantly correlated (p<0.05) between partners in opposite to HPV31 (r=0.08) and HPV42 (r=-0.10). A higher HPV load increased significantly the rate of detection of HPV16, 31 and 51 in sexual partners (age-adjusted odds ratios from 1.64 to 7.71). In recently-formed heterosexual couples, higher HPV16, 31 or 51 load was associated with increased detection of the same HPV type in sexual partners.

The South African National Antiretroviral Treatment Guidelines recommend the use of HIV-1 viral load assays for routine monitoring of HIV-1 positive patients receiving highly active antiretroviral therapy (HAART). This thesis describes the innovative approaches to developing more affordable HIV-1 diagnostics and monitoring assays for South Africa, which take into account the tiered laboratory infrastructure of this country. An in-house HIV-1 viral load assay – the LUX assay, was developed and evaluated with a view of implementing this more affordable option in high tier laboratories. The LUX assay represents quantitative real-time RT-PCR that utilizes the LightCycler® technology (Roche) in a novel combination with a LUXÔ primer. The assay showed good analytical sensitivity, specificity and reproducibility of its linear dynamic range of 4x102 to 4x106 RNA copies/ml. Preliminary clinical evaluation (n = 458) of the LUX assay showed good agreement with the COBAS Amplicor assay, and demonstrated its usefulness for long term monitoring of HAART patients. ELISA based viral load testing approaches were investigated as low cost and less technically complex alternatives for medium tier laboratories. The HiSens HIV-1 p24 Ag Ultra (Perkin Elmer) and the ExaVir™ Load Quantitative HIV-RT kits (CAVIDI) were compared with the Roche Amplicor assay. Both assays showed strong association with the Roche Amplicor assay, with R2 = 0.686 and R2 = 0.810, respectively (n = 117). These alternative assays seemed most useful in the serial monitoring of patients on HAART. Major drawbacks included the wide variability of both assays, insufficient sensitivity of the p24 antigen assay and low throughput of the RT assay. Development of a point-of-care HIV-1 RNA assay could address issues related to early and cost effective diagnosis of acute HIV infection. A novel isothermal amplification technique termed the Reverse Transcription Loop Dependant Amplification (RT-LDA) was developed as one component for a potential point-of-care HIV-1 RNA assay. The RT-LDA converted RNA into partially looped ssDNA amplicons, over a wide RNA concentration range (4x103 to 4x108 copies/ml) using a 1 hour incubation at 53ºC. The RT-LDA technology is fully compatible with a lateral flow detection system using dipsticks and highly suitable for point-of-care testing. Overall, this study demonstrates the feasibility of developing novel, more affordable HIV-1 testing options that would be appropriate for the tiered laboratory infrastructure present in South Africa. Evaluation of commercially available, less expensive alternative HIV viral load assays in local settings facilitates their implementation.

Of the 22.5 million individuals infected with the human immunodeficiency virus (HIV) in sub-Saharan Africa, 62% of patients requiring treatment had access to highly active antiretroviral therapy (HAART) in 2011. The delivery of HAART and the appropriate laboratory monitoring of HIV positive individuals in sub-Saharan African countries has become a public health priority, an intervention which has and will continue to dramatically reduce HIV-related morbidity and mortality. Routine laboratory monitoring of HIV infected individuals should ideally include CD4+ T cell testing to assess when to start ART, viral load monitoring to assess virological failure on ART and when indicated, HIVDR genotyping.However, this is often not implemented in resource limited settings due to challenges such as inadequate infrastructure and laboratory capacity, amongst others. Thus the Affordable Resistance Testing for Africa (ART-A) initiative was established to develop an affordable HIV drug resistance testing (HIVDR) algorithm applicable to Africa. The objective of this study was to evaluate the role of in vitro HIVDR phenotyping in the context of HIV-1 subtype C (the most prevalent circulating subtype in sub-Saharan Africa), genotyping and genotypic interpretation tools using existing algorithms, as well as novel virological failure detection tools for clinical patient management. Current gold standard HIVDR phenotyping technologies use an HIV-1 subtype B backbone to create recombinant viruses with patient-derived polymerase (protease and partial reverse transcriptase). This backbone could impact on the in vitro phenotyping results of non-B subtypes, and therefore it was deemed necessary to establish the applicability of HIVDR phenotypic testing of subtype C polymerase when a commercially available subtype B backbone is used. One hundred and fourteen HIV-1 subtype C samples were HIVDR phenotyped against 17 antiretroviral drugs using both subtype B and C backbones and showed a high level of concordance between the two backbone phenotypic resistance profiles (95.8%; 1590 of 1660 fold change comparisons). Natural assay variability was largely responsible for discordant results. Results confirmed that HIV-1 phenotypic reverse transcriptase inhibitor drug resistance test interpretation is independent of the virus backbone subtype. No conclusions could be made for protease inhibitor resistance since limited samples from 2nd line failure were available. Subsequently, the HIVDR genotypic and phenotypic results of the 114 patient samples were compared to determine whether genotyping is a viable alternative to phenotyping. Results showed a 92.3% concordance between genotyping and phenotyping of individual drug comparisons for a number of HIVDR profiles. Discrepancies were attributed to phenotypic assay variability in addition to the role of mutation mixtures, which impacted genotypic interpretations. Overall, HIVDR genotyping is a reliable tool to detect and interpret antiretroviral drug resistance in HIV-1 subtype C infected patients, and can thus be used for clinical patient management. Once the accuracy of HIVDR genotyping was established, the development, validation and evaluation of a potential virological failure assay (ARTA-VFA) and a simplified HIVDR (ARTA-HIVDRultralight) assay was undertaken. A simplified and conceptually novel approach using a qualitative viral load assay with a pre-determined cut-off that gives a threshold above which virological failure (VF) could be confirmed and below which treatment success was likely, was tested. A real-time PCR (ARTA-VFA) assay was developed which involved the amplification of a short sequence of the HIV-1 LTR region from RNA extracted either from plasma and/or dried blood spots (DBS). The ARTA-VFA was tested on 409 patient samples,and successfully amplified samples from all major HIV-1 group M subtypes with equal specificity. The VF was qualitatively classified as a viral load >1000 RNA copies/ml in plasma samples, and >5000 RNA copies/ml in DBS samples. Comparative testing yielded accurate VF determination for therapy-switching in approximately 93% of clinical cases tested, compared to current gold standard quantitative viral load assays. A simplified HIVDR genotyping assay (ARTA-HIVDRultralight) targeting the region of RT harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line regimen failures was developed and assessed. The ARTAHIVDRultralight assay was designed to be practical, faster, and more affordable, show flexibility with respect to equipment (open platform), use DBS or plasma as starting material and amplify and sequence a smaller amplicon (RT). The assay performed well when compared to the in-house assay used in the laboratory at the time for both 212 plasma and 25 DBS samples, yielding identical mutations and subsequent resistant profiles. Furthermore, a theoretical in silico exercise to investigate the consequences of using 125,329 shortened RT genotype (ARTA-HIVDRultralight) as compared to full-length RT sequences showed >95% and >90% concordance when using the Stanford HIVdb algorithm and the virco®TYPE tool, respectively. Differences noted were minor and unlikely to have any impact on clinical decision-making. Overall, this study illustrated that the short RT sequences can be reliably used to generate HIVDR genotypes using the Stanford HIVdb and virco®TYPE algorithms and reduce sequencing costs substantially. A field evaluation using the ARTA-VFA and ARTA-HIVDRultralight on 288 clinical samples was conducted, showing that the accuracy and precision of both assays (using 248 plasma or 40 DBS sampling methods) compared well to the reference methodology, thereby extending access of testing to more remote settings.These assays were designed to either be used as a testing strategy of initially assessing VF,and once confirmed performing an HIVDR assay, or alternatively to be used separately as stand-alone, or within different laboratory tiers in resource limited settings. It is envisaged that the ARTA-VFA could be used in the middle laboratory tier, and if confirmatory, patient samples can be referred to a reference laboratory with the available infrastructure for HIVDR testing using the ARTA-HIVDRultralight. Lastly, an automated sequence analysis and editing software for use in correct base calling of nucleotide/mutation mixtures in HIVDR genotyping was validated on 1624 sequences. Compared to reference software, where interpretation is often operator dependent, this software performed extremely well, with minor discrepancies noted. The automated software can be used to reduce subjectivity, time taken for analysis which is often the rate-limiting step and thus improving the turn-around time and clinical relevance of HIVDR genotyping. Overall, the results obtained describe the validation of using HIVDR genotyping as an alternative tool to phenotyping, and the subsequent development and validation of simple, affordable, "open-platform" alternatives to currently used methods for virological failure monitoring, and accommodate a centralized approach to HIVDR with DBS testing in resource limited settings.

Orientador: Angelo José Magro / Resumo: A Hepatite C é uma doença hepática de etiologia viral cujo agente é o Vírus da Hepatite C (VHC). A infecção pelo vírus, na grande maioria dos casos evolui para a cronicidade, sendo, portanto, um problema de saúde pública devido ao seu grande potencial de evolução para cirrose e hepatocarcinoma. Portanto, atualmente é fundamental a utilização de testes moleculares como diagnóstico complementar e para o acompanhamento da doença no paciente infectado, assim como para a detecção cada vez mais precoce da infecção pelo VHC. Neste sentido, testes para a detecção de ácidos nucléicos podem ser utilizados para a detecção qualitativa e quantitativa do RNA-VHC no sangue dos pacientes, de modo a avaliar a dinâmica da infecção e definir a conduta terapêutica. Desta forma, o Ministério da Saúde adotou no Brasil uma metodologia baseada na Reação em Cadeia da Polimerase em Tempo Real (RT-qPCR) precedida de etapa de transcrição reversa para a detecção do material genético viral em amostras de plasma dos infectados. A presença do VHC está documentada em outros compartimentos biológicos como células mononucleares do sangue periférico (PBMC) e plaquetas, o que sugere que o protocolo atualmente adotado no Brasil pode superestimar o número de partículas virais ativas, visto que o plasma preparado para a realização desta análise contém uma grande quantidade de plaquetas em relação à contagem total dos indivíduos. Desta forma, a finalidade da execução deste projeto foi comparar a carga viral do VHC e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Hepatitis C is a liver disease of viral etiology whose agent is Hepatitis C Virus (HCV). This virus infection, in the vast majority of cases, progresses to chronicity and is therefore a public health problem due to its great potential for progression to cirrhosis and hepatocarcinoma. Therefore, the use of molecular tests as a complementary diagnosis and disease control, as well as for early detection of HCV infection, is currently essential. In this sense, tests for the detection of nucleic acids can be used for the qualitative and quantitative detection of HCV RNA in the blood of the patients, in order to evaluate the dynamics of the infection and define the therapeutics. Thus the Brazilian Ministry of Health adopted a methodology based on the Real-Time Polymerase Chain Reaction (RT-qPCR) preceded by a reverse transcription step for the detection of the viral genetic material in plasma samples of infected individuals. The presence of HCV is well documented in other biological compartments such as peripheral blood mononuclear cells (PBMC) and platelets, suggesting that the protocol currently adopted in Brazil may overestimate the number of active viral particles, since the plasma samples prepared for this analysis contains a large amount of platelets. Thus, the purpose of this project was to compare the HCV viral load in chronically infected patients, taking into account the protocol currently used by the SUS and another one modified, where the plasma samples are practically ... (Complete abstract click electronic access below) / Mestre