Numerous toxins and pathogens gain entry into mammalian cells using cell surface glycans. The Iyer group at Georgia State University is working on the development of glycoconjugates for the accurate detection of infectious agents. In this thesis, I have focused on the development of glycans to detect influenza virus and norovirus. In the first section, I have focused on influenza viruses. A panel of synthetic glycans was synthesized as receptor mimics for the specific capture of influenza viruses. The synthetic glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. This glycan printed microarray was evaluated for its ability to capture three strains of influenza viruses. The analytical limit of detection is ~10 pfu/ml, (plaque forming units/milliliter) which is clinical relevant as 102 viral particles are typically required to cause infection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using the microarray and determined the feasibility of this system to determine antiviral resistance for different strains. In addition to optical detection, I developed an electrochemical assay to rapidly detect influenza viruses. Here, we utilized an unique property of influenza viral surface enzyme, Neuraminidase (NA), which cleaves terminal N-Acetyl Neuraminic acid (sialic acid) from cell surfaces and proteins. We designed an electrochemical assay that uses glucose bearing sialic acid substrates. Glucose is released when exposed to viral NA or intact viruses. The released glucose can be detected using repurposed glucose meters. Thus, personal glucose meters that were designed to assist diabetics and prediabetics monitor blood glucose can potentially be used to detect pathogens. Using this approach, we have detected 19 unique strains of influenza viruses. We also demonstrated drug susceptibility using this assay. The limit of detection of this assay is 102 pfu/sample, which is clinically relevant. The results were validated plaque assays and polymerase chain reaction (PCR). In the second part of this thesis, I focused on norovirus detection. I developed a focused glycan microarray that comprised of a library of histo blood group antigens (HBGAs). The HBGAs were attached to a carrier protein and printed onto activated glass slides. A panel of norovirus virus like particles (VLPs) and strains that included different genogroups was exposed to the microarray. We found that different VLPs and strains give rise to unique binding patterns. When the binding pattern of VLPs for a particular strain were compared to the corresponding intact virus, the binding patterns didn't match well, presumably because the virus does not recognize the same antibody as the VLPs. Unfortunately, antibodies for the virus cannot be generated because the virus cannot be grown in a laboratory setting. Indeed, all norovirus samples are obtained from human challenge studies. I also used surface plasmon resonance (SPR) studies in an effort to determine the binding affinities. Divalent biotinylated H type glycans were synthesized and their binding affinities with different VLPs and viral strains were determined. Initial studies suggest that the binding affinities are strain specific. These results demonstrate that glycans can be used to capture and isolate norovirus, although more research is required to develop glycan based norovirus detection kits.

The increasing infection of Cryptococcus species and Tricosporon asahii emerged in clinical patients who were immunocompromised. They usually induce lung, skin, brain and systemic infection. Morbidity and mortality of immunocompromised patients are higher than normal healthy people. Cryptococcus neoformans var. grubii ¡]serotype A¡^ infections were reported in clinical cases predominantly and they were isolated from birds¡¦ droppings in large amount. Cryptococcus neoformans var. gattii ¡]serotype B, C¡^ had a natural life in plants, especially Eucalypticus trees. Isolations from other trees were reported increasingly in the tropical and subtropical areas. Comparing to Cryptococcus species, Tricosporon asahii is the normal mycoses of soil. In this study, we performed an environmental investigation concerning Cryptococcus species and Tricosporon asahii in Southern Taiwan. 120 droppings of racing pigeons and 114 samples from Eucalypticus trees were obtained. The results revealed that 30 Cryptococcus neoformans were isolated from racing pigeons¡¦ droppings ¡]25%¡^, as well as 4 Cryptococcus laurentii ¡]3.3%¡^ and 2 Cryptococcus albidus ¡]1.7%¡^. In addition, 25 Tricosporon asahii ( 20.8% ) were isolated from droppings of racing pigeons. But, none of Cryptococcus species or Tricosporon asahii is isolated from Eucalypticus trees ¡]0%¡^. All of Cryptococcus neoformans isolated from pigeons¡¦ droppings were var. grubii ¡]serotype A¡^ and their drug susceptibility tests showed sensitive to Amphotericin B ¡]minimal inhibitory concentration ¡Ø0.25£gg/ml¡^ and Fluconazole ¡]minimal inhibitory concentration 2£gg/ml¡^ and Flucytosine ¡]minimal inhibitory concentration ¡Ø1£gg/ml¡^. To sum up, both Cryptococcus species and Tricosporon asahii were isolated from droppings of racing pigeons in our study, especially Tricosporon asahii in large amount. Opportunistic infection caused by these species should be given more attention to racing pigeons which have close contact with human . Intensive investigation and surveillance should be carried out in the future to provide an information for the control and prevention of diseases.

BACKGROUND: Multi drug resistant Tuberculosis (MDR-TB) is a serious public health concern in many parts of the world. As per the WHO- 2010 global report on Surveillance and response 3.6% of all incident TB cases globally are multidrug resistant. In this regard, there is an increasing demand for timely, reliable and comprehensive drug susceptibility testing (DST) as MDR-TB surveillance is being geared up. The intent of this analysis is to determine whether there is a need to continue routine confirmatory DST testing at CDC in addition to just sending the isolates for genotyping. Analysis is done by measuring the discordance between the results of laboratory DST at CDC and the local labs drug type, drug testing concentrations, and study sites. METHODS: The data for this analysis was provided by the Tuberculosis Trials Consortium (TBTC), CDC. Data for this analysis was collected over nearly two decades (1993-2011), gathered from 7 clinical trials. Discordance between the local and CDC lab DST results was measured using Kappa statistic. Sensitivity and specificity analysis was done by taking the CDC DST lab results as the gold standard. Discordance levels were calculated by local sites and baseline drug resistance for each antibiotic in each study was measured. RESULTS: Average Kappa values for inter rater agreement for all the studies was 0.6444 whereas the overall level of discordance across all studies is 7.786%. Drug resistance at baseline was highest for Isoniazid and Streptomycin (except Study 23 and 22). CONCLUSION: Though the current results show few DST result discordances between local and CDC labs, it is better to continue to send isolates to the centralized lab (CDC) in view of the worldwide threat of drug resistant TB epidemic, the recommendations of the current literature and the benefits of reliable confirmatory testing services and availability of other molecular diagnostic methods.

Orientadores: Mariangela Ribeiro Resende, Maria Luiza Moretti / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T14:58:52Z (GMT). No. of bitstreams: 1 Ferrari_MichelaDeLuca_M.pdf: 2800534 bytes, checksum: 6c0f0dc17633a5c05f98eaf42e330cb7 (MD5) Previous issue date: 2009 / Resumo: Os objetivos do presente estudo foram estimar a prevalência de resistência do M. tuberculosis aos fármacos de primeira linha entre pacientes com tuberculose diagnosticados no Hospital de Clínicas da Unicamp e avaliar o desempenho do método de microdiluição em placa com [3-(4,5-dimethylthiazol-2-yl)- 2,5diphenyl-tetrazolium bromide] (MTT) comparado ao Bactec MGIT 960®. No período foram notificados 360 casos de tuberculose. Destes 211 casos apresentaram cultura positiva para Mycobacterium sp, sendo incluídos 105 casos, nos quais foi identificado o M. tuberculosis. Dentre estes pacientes a apresentação pulmonar da TB ocorreu em 64,8% e co-infecção pelo HIV em 37,1%. A prevalência de resistência a qualquer fármaco foi de 6,7%; multidrogarresistência (RMP e INH) e resistência a INH e SM foram detectadas em 1%, em 3,8% e em 3,8% dos casos, respectivamente. Todos os isolados foram susceptíveis ao EMB. A resistência primária foi encontrada em quatro casos e resistência adquirida em três. Houve associação entre tratamento prévio e resistência (p=0,0181), as demais variáveis estudadas não foram associadas. Dentre os 119 isolados de M. tuberculosis incluídos para a avaliação do desempenho do MTT, ocorreu concordância de 100% para INH e EMB entre os métodos MTT e Bactec MGIT960®, de 99% para RMP (sensibilidade: 90%), enquanto que para SM houve concordância inferior aquela apresentada pelos outros fármacos (95,2%; sensibilidade: 90,9%). A prevalência de resistência entre os casos novos atendidos no Hospital de Clínicas da Unicamp no período de estudo foi de 3,8% para isoniazida, 1% para rifampicina, 3,8% para etambutol e 1% multidrogarresistente. Resistência a qualquer fármaco foi observada em 6,7% dos casos. O método colorimétrico com MTT apresentou bom desempenho e representa uma alternativa acurada, simples e de reduzidos custos, viável e sustentável para locais e países com limitados recursos. / Abstract: This cross-sectional, hospital-based study conducted between January 2006 and March 2008 sought to estimate the resistance of M. tuberculosis to first-line drugs among patients with tuberculosis confirmed by culture at a Brazilian hospital and evaluate the performance of the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (MTT) microplate assay in comparison to the Bactec MGIT 960. During the study period were reported to the surveillance system 360 tuberculosis cases, 211 out of these were confirmed by positive culture for Mycobacterium sp and 105 out of these had M. tuberculosis isolated on culture and were included to the prevalence study. Pulmonary involvement was present in 64.8% of cases and HIV co-infection was found in 37.1%. The prevalence of M. tuberculosis resistance was 6.7%; multidrug-resistance [rifampin (RMP) and isoniazid (INH)] and resistance to INH and streptomycin (SM) were detected in 1%, in 3.8% and in 3.8% of cases, respectively. All isolates were susceptible to ethambutol (EMB). The resistance was primary in four cases and acquired in three cases. Previous treatment was associated with resistance (p=0.0181). Among 119 isolates of M. tuberculosis included to the MMTT performance evaluation, there was 100% observed agreement for INH and EMB between the MTT and Bactec MGIT 960¿ methods. For RMP, the observed agreement was 99% (sensitivity: 90%), and for SM, the level of agreement was lower (95.2%; sensibility 90.9%) than those of the other drugs. The prevalence of M. tuberculosis resistance among the new cases during the study period was 6.7%; multidrug-resistance and resistance to INH and streptomycin (SM) were detected in 1%, in 3.8% and in 3.8% of cases, respectively. The colorimetric method with tetrazolium (MTT) represents an accurate, simple and low cost alternative that is both viable and sustainable for settings with limited resources. / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

No description available.

Introduction: Les efforts globaux pour contrôler la tuberculose sont présentement restreints par la prévalence croissante du VIH/SIDA. Quoique les éclosions de la tuberculose multi résistante (TB-MDR) soient fréquemment rapportées parmi les populations atteintes du SIDA, le lien entre VIH/SIDA et le développement de résistance n’est pas clair. Objectifs: Cette recherche visait à : (1) développer une base de connaissances concernant les facteurs associés à des éclosions de la TB-MDR parmi les patients atteints du VIH/SIDA; (2) utiliser ce cadre de connaissances pour accroître des mesures préliminaires pour mieux contrôler la tuberculose pulmonaire chez les patients atteints du VIH/SIDA; et (3) afin d’améliorer l’application des ces mesures, affiner les techniques bactériologiques existantes pour Mycobacterium tuberculosis. Méthodologie: Quatre études ont été réalisées : (1) Une étude longitudinale pour identifier les facteurs associés avec une éclosion de la TB-MDR parmi les patients atteints du SIDA qui ont reçu le traitement directement supervisé de courte durée (DOTS) pour la tuberculose pulmonaire au Lima et au Pérou entre 1999 et 2005; (2) Une étude transversale pour décrire différentes étapes de l’histoire naturelle de la tuberculose, la prévalence et les facteurs associés avec la mycobactérie qu’on retrouve dans les selles des patients atteints du SIDA; (3) Un projet pilote pour développer des stratégies de dépistage pour la tuberculose pulmonaire parmi les patients hospitalisés atteints du SIDA, en utilisant l’essaie Microscopic Observation Drug Susceptibility (MODS); et (4) Une étude laboratoire pour identifier les meilleures concentrations critiques pour détecter les souches MDR de M. tuberculosis en utilisant l’essaie MODS. Résultats : Étude 1 démontre qu’une épidémie de TB-MDR parmi les patients atteints du SIDA qui ont reçu DOTS pour la tuberculose pulmonaire ait été causée par la superinfection du clone de M. tuberculosis plutôt que le développement de la résistance secondaire. Bien que ce clone ait été plus commun parmi la cohorte de patients atteints du SIDA, il n’avait aucune différence de risque pour superinfection entre les patients avec ou sans SIDA. Ces résultats suggèrent qu’un autre facteur, possiblement associé à la diarrhée, peu contribuer à la prévalence élevée de ce clone chez les patients atteints du SIDA. Étude 2 suggère que chez la plupart des patients atteints du SIDA il a été retrouvé une mycobactérie dans leurs selles alors qu’ils étaient en phase terminale au niveau de la tuberculose pulmonaire. Or, les patients atteints du SIDA ayant été hospitalisés pendant les deux dernières années pour une autre condition médicale sont moins à risque de se retrouver avec une mycobactérie dans leurs selles. Étude 3 confirme que la tuberculose pulmonaire a été commune à tous les patients hospitalisés atteints du SIDA, mais diagnostiquée incorrectement en utilisant les critères cliniques présentement recommandés pour la tuberculose. Or, l’essaie MODS a détecté pour la plupart de ces cas. De plus, MODS a été également efficace quand la méthode a été dirigée aux patients soupçonnés d’avoir la tuberculose, à cause de leurs symptômes. Étude 4 démontre les difficultés de détecter les souches de M. tuberculosis avec une faible résistance contre ethambutol et streptomycine en utilisant l’essai MODS avec les concentrations de drogue présentement recommandées pour un milieu de culture. Cependant, l’utilité diagnostique de MODS peut être améliorée ; modifier les concentrations critiques et utiliser deux plaques et non une, pour des tests réguliers. Conclusion: Nos études soulèvent la nécessité d’améliorer le diagnostic et le traitement de la tuberculose parmi les patients atteints du SIDA, en particulier ceux qui vivent dans des régions avec moins de ressources. Par ailleurs, nos résultats font ressortir les effets indirects que les soins de santé ont sur les patients infectés par le VIH et qu’ils peuvent avoir sur le développement de la tuberculose. / Background: Global efforts to control tuberculosis are currently being hampered by a continuing rise in the prevalence of HIV/AIDS. Although outbreaks of multidrug resistant tuberculosis (MDR-TB) are commonly reported among AIDS populations, the link between HIV/AIDS and the development of drug-resistance remains unclear. Objectives: This thesis aimed to: (1) build a knowledge foundation regarding underlying factors associated with outbreaks of MDR-TB among HIV/AIDS patients; (2) use this knowledge framework to develop preliminary health measures for controlling pulmonary tuberculosis among HIV/AIDS patients; and (3) in an effort to better implement these health measures, refine existing culture-based diagnostics for Mycobacterium tuberculosis. Methods: Four studies were conducted: (1) a longitudinal study to identify the underlying factors associated with an epidemic of MDR-TB among AIDS patients receiving Directly- Observed Therapy Short-course (DOTS) for pulmonary tuberculosis in Lima, Peru between 1999 and 2005; (2) a cross-sectional study to characterize the prevalence and factors associated with gastrointestinal shedding with mycobacteria among AIDS patients at different stages in the natural history of tuberculosis; (3) a pilot study to develop screening strategies for pulmonary tuberculosis among hospitalized HIV/AIDS patients using the Microscopic Observation Drug Susceptibility (MODS) assay; and (4) a laboratory-based study to define the optimal critical concentrations needed for detecting drug resistance in M. tuberculosis using MODS. Results: Study 1 revealed that an epidemic of MDR-TB among AIDS patients receiving DOTS for pulmonary tuberculosis was due to super-infection with a specific clone of M. tuberculosis rather than the development of secondary drug-resistance. Although this epidemic clone was more common among patients in the AIDS cohort, risk of superinfection did not differ between AIDS and non-AIDS patients after adjusting for baseline risk of exposure, suggesting that another factor possibly associated with diarrhea may be contributing to the strain’s high prevalence among AIDS patients. Study 2 showed that the majority of AIDS patients in the later stages of pulmonary tuberculosis exhibited gastrointestinal shedding with mycobacteria. Stool shedding was rare in the absence of pulmonary tuberculosis. AIDS patients were also less likely to shed mycobacteria if they had been hospitalized during the previous two years for another medical condition. Study 3 confirmed that pulmonary tuberculosis was common among hospitalized AIDS patients but frequently misdiagnosed using currently recommended diagnostic algorithms. The MODS assay detected most cases and was equally effective when targeted to patients clinically suspicious for tuberculosis. Study 4 demonstrated that low grade drug resistance in M. tuberculosis to ethambutol and streptomycin was difficult to detect with MODS using currently recommended drug-concentration standards in broth. Its diagnostic utility could be improved by modifying drug-concentration standards, and including two versus one critical concentration well for standardized testing. Conclusion: Our studies underscore the need to improve the diagnosis and treatment of tuberculosis among AIDS patients living in resource-constrained settings, all in an effort to prevent morbidity, mortality and the transmission of drug-resistant strains. They also highlight the indirect effect that general health care among HIV-infected patients can have on the development of tuberculosis.

Introduction: Les efforts globaux pour contrôler la tuberculose sont présentement restreints par la prévalence croissante du VIH/SIDA. Quoique les éclosions de la tuberculose multi résistante (TB-MDR) soient fréquemment rapportées parmi les populations atteintes du SIDA, le lien entre VIH/SIDA et le développement de résistance n’est pas clair. Objectifs: Cette recherche visait à : (1) développer une base de connaissances concernant les facteurs associés à des éclosions de la TB-MDR parmi les patients atteints du VIH/SIDA; (2) utiliser ce cadre de connaissances pour accroître des mesures préliminaires pour mieux contrôler la tuberculose pulmonaire chez les patients atteints du VIH/SIDA; et (3) afin d’améliorer l’application des ces mesures, affiner les techniques bactériologiques existantes pour Mycobacterium tuberculosis. Méthodologie: Quatre études ont été réalisées : (1) Une étude longitudinale pour identifier les facteurs associés avec une éclosion de la TB-MDR parmi les patients atteints du SIDA qui ont reçu le traitement directement supervisé de courte durée (DOTS) pour la tuberculose pulmonaire au Lima et au Pérou entre 1999 et 2005; (2) Une étude transversale pour décrire différentes étapes de l’histoire naturelle de la tuberculose, la prévalence et les facteurs associés avec la mycobactérie qu’on retrouve dans les selles des patients atteints du SIDA; (3) Un projet pilote pour développer des stratégies de dépistage pour la tuberculose pulmonaire parmi les patients hospitalisés atteints du SIDA, en utilisant l’essaie Microscopic Observation Drug Susceptibility (MODS); et (4) Une étude laboratoire pour identifier les meilleures concentrations critiques pour détecter les souches MDR de M. tuberculosis en utilisant l’essaie MODS. Résultats : Étude 1 démontre qu’une épidémie de TB-MDR parmi les patients atteints du SIDA qui ont reçu DOTS pour la tuberculose pulmonaire ait été causée par la superinfection du clone de M. tuberculosis plutôt que le développement de la résistance secondaire. Bien que ce clone ait été plus commun parmi la cohorte de patients atteints du SIDA, il n’avait aucune différence de risque pour superinfection entre les patients avec ou sans SIDA. Ces résultats suggèrent qu’un autre facteur, possiblement associé à la diarrhée, peu contribuer à la prévalence élevée de ce clone chez les patients atteints du SIDA. Étude 2 suggère que chez la plupart des patients atteints du SIDA il a été retrouvé une mycobactérie dans leurs selles alors qu’ils étaient en phase terminale au niveau de la tuberculose pulmonaire. Or, les patients atteints du SIDA ayant été hospitalisés pendant les deux dernières années pour une autre condition médicale sont moins à risque de se retrouver avec une mycobactérie dans leurs selles. Étude 3 confirme que la tuberculose pulmonaire a été commune à tous les patients hospitalisés atteints du SIDA, mais diagnostiquée incorrectement en utilisant les critères cliniques présentement recommandés pour la tuberculose. Or, l’essaie MODS a détecté pour la plupart de ces cas. De plus, MODS a été également efficace quand la méthode a été dirigée aux patients soupçonnés d’avoir la tuberculose, à cause de leurs symptômes. Étude 4 démontre les difficultés de détecter les souches de M. tuberculosis avec une faible résistance contre ethambutol et streptomycine en utilisant l’essai MODS avec les concentrations de drogue présentement recommandées pour un milieu de culture. Cependant, l’utilité diagnostique de MODS peut être améliorée ; modifier les concentrations critiques et utiliser deux plaques et non une, pour des tests réguliers. Conclusion: Nos études soulèvent la nécessité d’améliorer le diagnostic et le traitement de la tuberculose parmi les patients atteints du SIDA, en particulier ceux qui vivent dans des régions avec moins de ressources. Par ailleurs, nos résultats font ressortir les effets indirects que les soins de santé ont sur les patients infectés par le VIH et qu’ils peuvent avoir sur le développement de la tuberculose. / Background: Global efforts to control tuberculosis are currently being hampered by a continuing rise in the prevalence of HIV/AIDS. Although outbreaks of multidrug resistant tuberculosis (MDR-TB) are commonly reported among AIDS populations, the link between HIV/AIDS and the development of drug-resistance remains unclear. Objectives: This thesis aimed to: (1) build a knowledge foundation regarding underlying factors associated with outbreaks of MDR-TB among HIV/AIDS patients; (2) use this knowledge framework to develop preliminary health measures for controlling pulmonary tuberculosis among HIV/AIDS patients; and (3) in an effort to better implement these health measures, refine existing culture-based diagnostics for Mycobacterium tuberculosis. Methods: Four studies were conducted: (1) a longitudinal study to identify the underlying factors associated with an epidemic of MDR-TB among AIDS patients receiving Directly- Observed Therapy Short-course (DOTS) for pulmonary tuberculosis in Lima, Peru between 1999 and 2005; (2) a cross-sectional study to characterize the prevalence and factors associated with gastrointestinal shedding with mycobacteria among AIDS patients at different stages in the natural history of tuberculosis; (3) a pilot study to develop screening strategies for pulmonary tuberculosis among hospitalized HIV/AIDS patients using the Microscopic Observation Drug Susceptibility (MODS) assay; and (4) a laboratory-based study to define the optimal critical concentrations needed for detecting drug resistance in M. tuberculosis using MODS. Results: Study 1 revealed that an epidemic of MDR-TB among AIDS patients receiving DOTS for pulmonary tuberculosis was due to super-infection with a specific clone of M. tuberculosis rather than the development of secondary drug-resistance. Although this epidemic clone was more common among patients in the AIDS cohort, risk of superinfection did not differ between AIDS and non-AIDS patients after adjusting for baseline risk of exposure, suggesting that another factor possibly associated with diarrhea may be contributing to the strain’s high prevalence among AIDS patients. Study 2 showed that the majority of AIDS patients in the later stages of pulmonary tuberculosis exhibited gastrointestinal shedding with mycobacteria. Stool shedding was rare in the absence of pulmonary tuberculosis. AIDS patients were also less likely to shed mycobacteria if they had been hospitalized during the previous two years for another medical condition. Study 3 confirmed that pulmonary tuberculosis was common among hospitalized AIDS patients but frequently misdiagnosed using currently recommended diagnostic algorithms. The MODS assay detected most cases and was equally effective when targeted to patients clinically suspicious for tuberculosis. Study 4 demonstrated that low grade drug resistance in M. tuberculosis to ethambutol and streptomycin was difficult to detect with MODS using currently recommended drug-concentration standards in broth. Its diagnostic utility could be improved by modifying drug-concentration standards, and including two versus one critical concentration well for standardized testing. Conclusion: Our studies underscore the need to improve the diagnosis and treatment of tuberculosis among AIDS patients living in resource-constrained settings, all in an effort to prevent morbidity, mortality and the transmission of drug-resistant strains. They also highlight the indirect effect that general health care among HIV-infected patients can have on the development of tuberculosis.

<p>Reverse transcriptase (RT) is a crucial enzyme for retrovirus replication, and its presence in the virion is indispensable for infectivity. This thesis illustrates the use of RT activity assays as tools for quantitation and characterization of different retroviruses, particularly HIV. </p><p>A non radioactive assay, using microtiter plates, for the RT of Moloney murine leukemia virus (MMuLV) was developed. Assay conditions for MMuLV and HIV-1 RT, together with isozyme specific RT activity blocking antibodies, were shown useful for discrimination between RTs from different retrovirus genera. RT activity assay for HIV-1 was found to quantitate different subtypes more equally efficient than p24 antigen assays did.</p><p>Viral load (VL), the amount of HIV particles in the blood, is an important marker of the clinical status of an infected person. A method for VL determination based on RT activity (ExaVir Load) was developed. After plasma pretreatment, to inactivate cellular DNA polymerases, virions in patient plasma were immobilized on a gel, which was washed to remove disturbing factors. The virions were lysed with a detergent containing buffer and the lysate eluted. Finally, the RT activity in the lysate was determined and found to correlate strongly to VL by RNA according to a PCR based standard method (Roche Amplicor 1.5). The second version of the method was able to measure VL down to approximately 400 HIV-1 RNA copies/ml. The usefulness of RT from the VL procedure for determination of susceptibility towards anti-HIV drugs was demonstrated, and the results were in agreement with genotypic data. </p><p>Due to its technical simplicity, and ability to detect a broad range of HIV-1 subtypes, ExaVir Load and the drug susceptibility application are interesting for clinical use, particularly but not only in resource limited settings. The concept is also potentially useful for research purposes, e.g. in combination with specific RT assay conditions.</p>

Reverse transcriptase (RT) is a crucial enzyme for retrovirus replication, and its presence in the virion is indispensable for infectivity. This thesis illustrates the use of RT activity assays as tools for quantitation and characterization of different retroviruses, particularly HIV. A non radioactive assay, using microtiter plates, for the RT of Moloney murine leukemia virus (MMuLV) was developed. Assay conditions for MMuLV and HIV-1 RT, together with isozyme specific RT activity blocking antibodies, were shown useful for discrimination between RTs from different retrovirus genera. RT activity assay for HIV-1 was found to quantitate different subtypes more equally efficient than p24 antigen assays did. Viral load (VL), the amount of HIV particles in the blood, is an important marker of the clinical status of an infected person. A method for VL determination based on RT activity (ExaVir Load) was developed. After plasma pretreatment, to inactivate cellular DNA polymerases, virions in patient plasma were immobilized on a gel, which was washed to remove disturbing factors. The virions were lysed with a detergent containing buffer and the lysate eluted. Finally, the RT activity in the lysate was determined and found to correlate strongly to VL by RNA according to a PCR based standard method (Roche Amplicor 1.5). The second version of the method was able to measure VL down to approximately 400 HIV-1 RNA copies/ml. The usefulness of RT from the VL procedure for determination of susceptibility towards anti-HIV drugs was demonstrated, and the results were in agreement with genotypic data. Due to its technical simplicity, and ability to detect a broad range of HIV-1 subtypes, ExaVir Load and the drug susceptibility application are interesting for clinical use, particularly but not only in resource limited settings. The concept is also potentially useful for research purposes, e.g. in combination with specific RT assay conditions.

Efficient control of sea lice is a major challenge for the sustainable production of farmed Atlantic salmon (Salmo salar (Linnaeus, 1758)). These marine ectoparasites feed on mucus, skin and blood of their hosts, thereby reducing the salmon’s growth rate and overall health. In the northern hemisphere, the most prevalent species is Lepeophtheirus salmonis (Krøyer, 1837). In 2006, global costs of sea lice infections are estimated to have exceeded €300 million, with the majority spent on a limited number of chemical delousing agents. Emamectin benzoate (EMB; SLICE®), an avermectin, has been widely used since its introduction in 2000, due to its convenient administration as an in-feed medication and its high efficacy against all parasitic stages of L. salmonis. However, over-reliance on a single or limited range of medicines favours the emergence of drug resistance and, as a result, the efficacy of this compound in treating L. salmonis has decreased in recent years, as reported from e.g. Chile, Norway, Scotland and Canada. Declining efficacy underlines the need for an improved understanding of the molecular mechanisms underlying EMB drug resistance in L. salmonis. Elucidation of these mechanisms would allow for improved monitoring tools, earlier detection of developing resistance, extended usability of current delousing agents and development of new parasiticides. The work described in this thesis sets out to examine the molecular mechanisms underlying EMB resistance in L. salmonis. In earlier studies, research in nematodes and arthropods has linked drug efflux transporters belonging to the family of ATP-binding cassette (ABC) transporters to ivermectin (IVM) resistance, a parasiticide with high chemical similarity to EMB. ABC transporters such as permeability glycoprotein (P-gp), transport a wide range of substrates, including drugs, and have been suggested to provide a potential molecular mechanism through which EMB resistance might be mediated in sea lice. As an example of such mechanisms, increased expression of P-gp is one of the causative factors for drug resistance in human cancer cells and avermectin resistance in nematode parasites such as Caenorhabditis elegans or Haemonchus contortus. Initial research involved screening for novel salmon lice P-gps that might contribute to EMB resistance. A novel P-gp, SL-PGY1, was discovered using a combined bioinformatic and molecular biological approach. The expression was compared in two well-characterised L. salmonis strains differing in their susceptibility to EMB (S = susceptible, R = resistant). Prior to EMB exposure, mRNA levels did not differ from each other, while, after 24 h exposure, a 2.9-fold increase in SL-PGY1 mRNA expression was observed in the R strain. SL-PGY1 appears not to be a major factor contributing to reduced EMB susceptibility, although it could play a role, as expression levels increased upon exposure to EMB. A further four additional drug transporters (ABC C subfamily) were also discovered showing high homology to multidrug-resistance proteins (MRP). The relative expression levels of each MRP was compared in the strains S and R, before and after exposure to EMB. No significant changes were found in their expression patterns. If ABC drug transporters mediate the efflux of EMB and thereby reduce the intracellular concentrations of the drug in exposed animals, the inhibition of those ABC drug transporters was expected to lead to higher intracellular levels of EMB. This could result in an enhanced toxic effect when EMB is co-administered with an inhibitor. Two known inhibitors of human P-gps and MRPs, cyclosporin A (CSA) and verapamil (VER), were co-administered with EMB. CSA increased the toxic effect of EMB in both tested strains, implying that the targets of CSA are expressed at comparable levels and that they may be part of the mechanism conferring EMB resistance. VER increased the toxic effect of EMB in the R strain, but had no significant effects on the S strain. This implies that the expression of factors inhibited by VER differs between the two L. salmonis strains. It is hypothesised that a number of ABC transporters with distinct, yet overlapping patterns of inhibitor specificity are affected by those inhibitors. The search for drug-resistance conferring genes was complemented with a systematic, genome-wide survey of ABC transporters in L. salmonis to find additional members of this important gene family. Next-generation high-throughput RNA sequencing (RNA-seq) was employed to assemble a reference transcriptome from pooled total RNA of salmon lice at different development stages. The transcriptome was assembled against the L. salmonis genome and annotated. Thirty-nine putative ABC transporters were found. Of further interest were transcripts of the subfamily B, C and G, as they contain drug-transporting ABC proteins. For the ABC B subfamily, one full (SL-PGY1) and three half transporter transcripts were found. Only full transporters are known to transport drugs and SL-PGY1 is apparently not a major factor contributing to EMB resistance. Fourteen ABCC sequences were found – 11 MRPs and 3 homologues to sulfonylurea receptors. Of interest are MRPs, as they contribute to drug detoxification in humans and invertebrates. Four MRPs had been identified previously and their expression ratios did not differ between S and R strain parasites. Seven sequences belonging to ABCG subfamily were found. However, none of the L. salmonis ABCG transcripts identified showed sufficient homology to known drug transporters in other species. With the currently limited understanding of the mechanisms conferring EMB resistance, monitoring the susceptibility of L. salmonis subpopulations is essential. Dose-response bioassays are currently widely used. Tests with pre-adult II or adult parasites requires relatively large numbers of parasites (~150) to conduct this type of bioassay, which may not always be available. Addressing this issue, we tested the feasibility of a single-dose bioassay (requiring fewer test animals than dose-response bioassays) to discriminate between L. salmonis strains with differing EMB susceptibility. This alternative approach uses time-course toxicity analysis, where the toxic effect of EMB is monitored over time. After clearly defining the effect criteria, we found that it is possible to discriminate between those L. salmonis strains. However, while requiring fewer test animals, time course toxicity analysis is more labour-intensive, but the alternative design can be suitable under certain circumstances. The work reported here has provided new knowledge concerning the mechanisms of EMB resistance in sea lice. Several novel putative drug transporters have been identified, an important first step toward unravelling the complex interactions of genes involved in EMB resistance in this commercially important parasite.