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Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 80-96).

Pneumocystis jiroveci, Mycoplasma pneumoniae and Mycobacterium tuberculosis are respiratory pathogens associated with pneumonia, with increasing prevalence of Pneumocystis pneumonia (PcP) and tuberculosis (TB) in AIDS patients. Increased resistance of M. tuberculosis has emphasized the need for rapid susceptibility testing, such as flow cytometry. Sputum specimens (102) were assessed by PCR employing primers directed at the following genes: P. jiroveci: mitochondrial large subunit ribosomal RNA (mtLSUrRNA), dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR), and for M. pneumoniae: 16S rRNA and P1 adhesin. Positive P. jiroveci samples were genotyped by PCR-SSCP (single-strand conformation polymorphism) targeting the: internal transcribed spacer region (ITS), intron of the nuclear 26S rRNA gene (26S), variable region of the mitochondrial 26S rRNA gene (mt26S) and β-tubulin gene (β-tub). Multi-drug resistant (MDR-TB) cultures grown in the presence and absence of four antibiotics (rifampicin, isoniazid, ethambutol and ofloxacin) were heat killed, stained with SYTO16 and Propidium Iodide and analysed using flow cytometry. Rifampicin resistance gene mutations were screened by PCR and DNA sequencing. Details of patient’s gender, age, HIV and M. tuberculosis status were provided by the hospitals. Women were seen to be at high risk for community-acquired P. jiroveci colonisation. Overall, prevalence of P. jiroveci was 55.1 percent (54/102 patients). P. jiroveci was mainly associated with HIV (25/102 P. jiroveci positive patients for which clinical data was available) and co-colonisation with M. tuberculosis was observed in 11 cases. Sequence analysis of DHPS and DHFR products found no resistance associated mutations. M. pneumoniae was detected in one patient. Four simple SSCP patterns were identified and there were no co-infections with other P. jiroveci strains. Nine M. tuberculosis samples [8 MDR-TB isolates (NHLS) and M. tuberculosis ATCC® 27294TM] were tested. There was a 53 percent (19 out of 36 tests) agreement of flow cytometry with the BACTEC MGIT 960. Mutations (at two specific codons, namely 516 and 531) in the rifampicin resistance-determining region (RRDR) of the rpoB gene were observed in eight M. tuberculosis isolates. Evaluation of methods for genotyping and drug susceptibility testing of PcP and TB are imperative for epidemiology and drug resistance studies, and impact on treatment protocols.

The results from this work are reported into two sections listed below: <br><br> Synthesis: <br><br> Following structural classes of compounds have been designed, synthesized and studied as inhibitors of pjDHFR, RTKs and tubulin: <br> 1. 2,4-Diamino-6-(substituted-arylmethyl)pyrido[2,3-d]pyrimidines <br> 2. 4-((3-Bromophenyl)linked)-6-(substituted-benzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-amines<br> 3. 6-Methyl-5-((substitutedphenyl)thio)-7H-pyrrolo[2,3-d]pyrimidin-2-amines <br> A total of 35 new compounds (excluding intermediates) were synthesized, characterized and submitted for biological evaluation. Results from these studies will be presented in due course. Bulk synthesis of the potent lead compound 170 was carried out to facilitate in vivo evaluation. <br><br> Docking Studies <br><br> Docking studies were performed using LeadIT, MOE, Sybyl or Flexx for target compounds listed above and for other compounds reported by Gangjee et al. against the following targets: <br> 1. Dihydrofolate reductase: human, P. carinii, P. jirovecii (pjDHFR) and T. gondii (tgDHFR)<br> 2. Thymidylate synthase: human (hTS) and T. gondii (tgTS)<br> 3. Receptor tyrosine kinases: VEGFR2, EGFR and PDGFR-β<br> 4. Colchicine binding site of tublulin.<br> Novel homology models were generated and validated for pjDHFR, tgDHFR, tgTS, PDGFR-β and the F36C L65P pjDHFR double mutant. The tgTS homology model generated in this study and employed to design novel inhibitors shows remarkable similarity with the recently published X-ray crystal structures. Docking studies were performed to provide a molecular basis for the observed activity of target compounds against DHFR, RTKs or tubulin. Results from these studies support structure-based and ligand-based medicinal chemistry efforts in order to improve potency and/or selectivity of analogs of the docked compounds against these targets.<br> Novel topomer CoMFA models were developed for tgTS and hTS using a set of 85 bicyclic inhibitors and for RTKs using a set of 60 inhibitors reported by Gangjee et al. The resultant models could be used to explain the potency and/or selectivity differences for selected molecules for tgTS over hTS. Topomer CoMFA maps show differences in steric and/or electronic requirements among the three RTKs, and could be used, in conjuction with other medicinal chemistry approaches, to modulate the selectivity and/or potency of inhibitors with multiple RTK inhibitory potential. Drug design efforts that involve virtual library screening using these topomer CoMFA models in conjunction with traditional medicinal chemistry techniques and docking are currently underway. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Medicinal Chemistry / PhD; / Dissertation;

Infection due to Penicillium marneffei (renamed to Talaromyces marneffei in 2011) has emerged as an important public health problem over the past two decades due to the arrival of the HIV epidemic in Asia. Since 2004, P. marneffei has become the second most common pathogen isolated from routine blood culture, after Cryptococcus neoformans, at the Hospital for Tropical Diseases in Ho Chi Minh City, the largest referral centre for HIV care in southern Vietnam. The clinical epidemiology of P. marneffei infection has not been studied in Vietnam. The fundamental epidemiological questions regarding the pathogen reservoirs and risks of acquisition remain poorly understood. The diagnosis relies on isolation of the pathogen from clinical specimens and can take up to 14 days to identify, resulting in delayed initiation of therapy which is associated with worse treatment outcomes. This thesis aims to increase knowledge and understanding of the clinical and environmental epidemiology of P. marneffei infection and to improve the speed and accuracy of diagnosis of P. marneffei infection. The Précis provides a brief background and rationale for the thesis. Chapter 1 is an introductory chapter and provides an overview of the epidemiology, ecology, mycology, pathology, immunology, clinical features, diagnosis, and treatment of P. marneffei infection. Chapter 2 summarizes the incidence and features of P. marneffei admissions at the Hospital for Tropical Diseases in Ho Chi Minh City over a 13 year period. During this period, 795 patients with P. marneffei infection were identified and hospital charts were obtainable for 513 (65%) patients. The data showed clear seasonality with an increase in incidence of approximately 30% during the rainy season compared to the dry season. The clinical and microbiological features and treatment outcomes of the patients were characterised. Poor outcome, defined as death or worsening disease at hospital discharge, occurred in 28% of patients. History of injection drug use, shorter duration of illness, absence of fever or skin lesions, higher respiratory rates, and lower platelet counts independently predicted poor outcome. Chapter 3 describes an analysis of meteorological factors that determine penicilliosis incidence in Ho Chi Minh City. Humidity, rather than precipitation, was the most important factor that governs the seasonality of penicilliosis. Higher humidity was associated with increased odds of penicilliosis versus cryptococcosis admissions. The infection incubation period was estimated to be between one and three weeks. Chapter 4 describes an analysis of exposure and behavioural risk factors for penicilliosis based on a matched case control study of 205 culture-confirmed HIV-infected penicilliosis cases and 405 HIV-infected controls recruited from two major HIV referral centres in Hanoi and Ho Chi Minh City. Penicilliosis was independently associated with proximity or exposure to tropical plants and exposure to farmed animals. The geographical analysis showed that patients living in or traveling to the highland regions were at increased risk for penicilliosis in southern Vietnam. Chapter 5 describes the development of a Taqman real-time PCR assay based on a novel Mp1 gene target unique to P. marneffei for rapid detection of P. marneffei infection in patient plasma. The assay was tested in 70 plasma samples from HIV-infected patients (50 with culture-confirmed penicilliosis, 20 with other opportunistic infections) and showed a clinical specificity of 100% (20/20) and sensitivity of 70.4% (19/27) and 52.2% (12/23) prior to and within 24-48 hours of antifungal therapy administration, respectively. Chapter 6 is an overview discussion interpreting the implications of the major findings and the future direction of P. marneffei research. The work of this thesis increases knowledge of the clinical epidemiology of P. marneffei infection in Vietnam, providing essential data for the design of prospective studies to improve the diagnosis and treatment of P. marneffei infection in Asia. The data suggest that multiple environmental factors including humidity, tropical plants, farmed animals, and highland location, are important drivers of P. marneffei infection in southern Vietnam. The real-time PCR assay showed potential as a rapid ‘rule-in' test for P. marneffei in this pilot study and should be prospectively evaluated in a large cohort to determine if it can improve diagnostic speed and crucially, impact patient outcomes. Prevention, diagnosis and elimination all require further research to reduce the high mortality following clinical disease caused by P. marneffei in Asia.

Magister Artium - MA / This study falls within the framework of the larger multidisciplinary university health initiative (MUTHI) objectives to investigate and document the use of local medicinal plants for the treatment of HIV and symptoms of related opportunistic infections such as tuberculosis, thrush and shingles in the Western Cape. The study stems from twelve months fieldwork in Strand, Western Cape and the collection of plants from Mpoza, Eastern Cape for a variety of reasons. The study ethnographically documents when, under which circumstances and where plants are collected for use.As far as I am aware this is the first anthropological study which "follows" traditional healers in the Western Cape to a site in the Eastern Cape where they collect plants. Seventeen plants were collected from different genera which traditional healers reported to use as treatment for suspected HIV and related symptoms. For each plant I describe the medicinal uses,preparatory techniques and plant parts used as suggested by traditional healers. I also explore healer's aetiologies concerning plants, treatments and the social-material relations which are prevalent in my research settings.

Human herpesvirus 6 (HHV-6) infections occur in > 95% of humans. Primary infection, which occurs in early childhood as an asymptomatic illness or manifested clinically as roseola infantum, leads to a state of subclinical viral persistence and latency. Reactivation of latent HHV-6 is common after liver transplantation, possibly induced and facilitated by allograft rejection and immunosuppressive therapy. Since the vast majority of humans harbor the virus in a latent state, HHV-6 infections after liver transplantation are believed to be mostly due to endogenous reactivation or superinfection (reactivation in the transplanted organ). In a minority of cases, however, primary HHV-6 infection may occur when an HHV-6 negative individual receives a liver allograft from an HHV-6 positive donor. The vast majority of documented HHV-6 infections after liver transplantation are asymptomatic. In a minority of cases, HHV-6 has been implicated as a cause of febrile illness with rash and myelosuppression, hepatitis, pneumonitis, and encephalitis after liver transplantation. In addition, HHV-6 has been associated with a variety of indirect effects such as allograft rejection, and increased predisposition and severity of other infections including cytomegalovirus (CMV), hepatitis C virus, and opportunistic fungi. Because of the uncommon nature of the clinical illnesses directly attributed to HHV-6, there is currently no recommended HHV-6-specific approach to prevention. However, ganciclovir and valganciclovir, which are primarily intended for the prevention of CMV disease, are also active against HHV-6 and may prevent its reactivation after transplantation. The treatment of established HHV-6 disease is usually with intravenous ganciclovir, cidofovir, or foscarnet, complemented by reduction in the degree of immunosuppression. This article reviews the current advances in the pathogenesis, clinical diagnosis, and therapeutic modalities against HHV6 in the setting of liver transplantation.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

INTRODUÇÃO: Embora a mortalidade geral tenha diminuído na era da terapia antirretroviral de alta potência (TARVAP), o envolvimento do Sistema Nervoso Central (SNC) ainda é elevado nos pacientes com infecção pelo HIV. O estudo sobre possíveis correlações entre neuroinfecções, a presença de déficts neurocognitivos e o uso da TARVAP é importante para se avaliar os efeitos de longo prazo do tratamento com drogas antirretrovirais em uma população. Desta forma, são necessários estudos para se avaliar a neuropatologia da infecção pelo HIV e os efeitos do tratamento bem como a adesão ao mesmo. OBJETIVOS: Analisar a frequência e o tipo de infecções oportunistas e da infecção crônica pelo HIV no SNC na era pós-TARVAP, de pacientes necropsiados em um hospital universitário, no período de 2007 a 2014. Nestes casos, avaliar o substrato neuropatológico de pacientes com Aids e histórico clínico de demência. MATERIAIS E MÉTODOS: Foram analisados os achados dos exames neuropatológicos de 123 encéfalos de pacientes autopsiados com AIDS no Serviço de Patologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCFMRP/USP), seus dados clínicos e respectivos os laudos necroscópicos. RESULTADOS: Dos 123 casos analisados, 73 (59,3%) eram do sexo masculino e a média de idade foi de 40,56 anos. Destes, 69 (56,1%) tiveram neuroinfecções causadas por agentes oportunistas, sendo a neurotoxoplasmose a mais prevalente. Da correlação entre a contagem de células CD4 e o uso da TARVAP houve diferença significativa para uma delas, àquela que foi analisada como sendo a terceira mais próxima da data do óbito (p = 0,03). Apenas 6 pacientes fizeram uso regular da TARVAP no decorrer da doença e nenhum deles apresentaram neuroinfecções durante o tratamento. CONCLUSÕES: Podemos concluir que mesmo na era pós TARVAP as infecções no SNC causadas por agentes infecciosos permanecem presentes e no caso da população estudada podem estar correlacionadas com a baixa adesão ao tratamento. / RATIONALE: Although overall mortality has decreased in the era of highly active antiretroviral therapy (HAART), the involvement of the central nervous system (CNS) is still high in patients with HIV infection. The study of possible correlations between neuroinfections, the presence of neurocognitive payment deficits and the use of HAART is important to evaluate the long-term effects of treatment with antiretroviral drugs in a population. Thus, studies are needed to evaluate the neuropathology of infection by HIV and the treatment effects and it adhesion. OBJECTIVES: To analyze the changes caused by opportunistic infections and chronic HIV infection in the CNS in the post-HAART era of autopsied patients at a university hospital, from 2007 to 2014. In these cases, assess the neuropathologic of AIDS patients and clinical history of dementia. MATERIALS AND METHODS: The findings of neuropathological examinations of 123 brains of patients were analyzed autopsied AIDS in Hospital Pathology Service of the Clinics of Ribeirão Preto Medical School, University of São Paulo (HCFMRP / USP) clinical data and their postmortem reports. RESULTS: The 123 cases analyzed, 73 (59.3%) were male and the average age was 40.56 years. Of these 69 (56,1%) had neuroinfections caused by opportunistic agents, toxoplasmosis being the most prevalent. The correlation between CD4 cell count and use of HAART were no significant differences for one of them to that which was analyzed as being the closest third of the date of death (p = 0.03). Only 6 patients made regular use of HAART during the disease and none of them showed neuroinfections during treatment. CONCLUSIONS: We can conclude that even in the post HAART era CNS infections caused by infectious agents remain present and in the case of the studied population can be correlated with low adherence to the treatment.

As infecções virais são as maiores responsáveis pelas úlceras esofágicas no portador do HIV, sendo o Citomegalovírus (CMV) o agente mais observado, seguido pelo Herpes Vírus Simples (HSV). A abordagem clínica adequada e a utilização de métodos diagnósticos precisos são de grande relevância para o estabelecimento etiológico. Os objetivos deste trabalho foram: avaliar a prevalência de úlceras esofágicas em portadores do HIV; pesquisar os agentes etiológicos associados; verificar a acurácia dos métodos diagnósticos comparando as impressões obtidas pela endoscopia digestiva alta (EDA), histologia utilizando o método de Hematoxilina-Eosina (H&E) e Imuno-histoquímica (IH) para pesquisa de CMV e HSV; avaliar o impacto da coloração para fungos (Gomori metenamina prata- GMS) e bacilos álcool-ácidos resistentes [BAAR- Ziehl-Neelsen (ZN)] e a relevância numérica da amostragem tecidual na avaliação etiológica. Trata-se de um estudo descritivo, retrospectivo, do tipo transversal, baseado em levantamento de dados demográficos, clínico-laboratoriais, endoscópicos obtidos por revisão de prontuários e análise histológica às cegas de biópsias endoscópicas (H&E, IH, GMS e ZN) de úlceras esofágicas em 41 portadores do HIV, no período de agosto de 2002 a setembro de 2006. A IH foi considerada método padrão. No período avaliado, 399 portadores do HIV submeteram-se à EDA, detectando-se úlcera esofágica em 41 pacientes (23 homens, 25 a 56 anos) com uma prevalência de 10,27%. A mediana da contagem de CD4 foi 49 céls/mm3 e da carga viral 58869,5 cópias/ml. A EDA revelou 29/41úlceras esofágicas suspeitas de infecção pelo CMV; 7/41 pelo HSV; 2/41 relacionada ao refluxo gastroesofágico (DRGE); 1/41 por monilíase; 1/41 por paracoccidioidomicose (PCM) e 1/41 inespecífica. O H&E detectou: 25/41 úlceras com aspectos inflamatórios inespecíficos (61%); 6/41 associadas à monilíase (16%); 4/41 por infecção pelo CMV (10%); 2/41 HSV (5%); 1/41CMV e HSV (2%); 1/41 por PCM (2%); 1/41 devido a Histoplasmose (2%) e 1/41 infiltração neoplásica por Linfoma não Hodgkin (2%). A IH para CMV e HSV confirmou os achados do H&E e detectou mais um caso. A EDA revelou sensibilidade alta (100%) para a detecção da úlcera esofágica, especificidade baixa para a caracterização etiológica viral (15%) quando comparada ao H&E e a IH. O H&E mostrou-se um método adequado para avaliação etiológica com sensibilidade de 87% e especificidade de 100% quando comparada a IH. A pesquisa de BAAR pelo ZN foi negativa nas 34 amostras realizadas. O GMS confirmou a presença de fungos detectados ao H&E e foi fundamental na caracterização morfológica do Histoplasma capsulatum e do Paraccocidioides brasiliensis. O número de amostras não influenciou na avaliação etiológica final. Os nossos achados recomendam o H&E como método adequado na avaliação etiológica de úlceras esofágicas no portador do HIV e indicam a IH para pesquisa viral somente nos casos suspeitos, porém, não típicos ao H&E. / Viral infections are the main cause of esophageal ulcers in HIV carriers, cytomegalovirus (CMV) being the most frequently observed, followed by Herpes Simplex Virus (HSV). An appropriate clinical approach and the use of precise diagnostic methods are very important for etiological evaluation. The aim of this work has been: to evaluate the prevalence of esophageal ulcers in HIV carriers; to research the associated etiological agents; to check the accuracy of the diagnostic methods, comparing the impressions obtained by the upper gastrointestinal endoscopy (UGE), histology using the Hematoxiline-Eosin (H&E) method, and immunohistochemistry (IH) to investigate CMV and HSV; to evaluate the impact of the staining for fungus (Gomori methenamine silver GMS) and for resistant alcohol-acid bacillus (BAAR-Ziehl-Neelsen ZN), and the numerical relevance of tissue samples in the etiological characterization. This is a descriptive, retrospective, transversal study, based on demographic, clinic-laboratorial and endoscopic data, obtained by the review of medical charts and blind histological analysis of endoscopic biopsies (H&E, IH, GMS an ZN) of esophageal ulcers of 41 HIV carriers, from August 2002 to September 2006. The IH has been chosen as the standard method. Along the evaluated period, 399 HIV carriers were submitted to UGE, and esophageal ulcer was detected in 41 patients (25 to 56 years old, 23 males), with a prevalence of 10.27%. The median of the CD4 count was 49 cells/mm3, and the viral load 58869.5 copies/ml. UGE has revealed 29/41 esophageal ulcers under suspicion of infection by CMV; 7/41 by HSV; 2/41 related to gastroesophageal reflux (GER)/ 1/41 by moniliasis; 1/41 by paracoccidioidomycosis (PCM) and 1/41 non-specific. H&E has detected 25/41 ulcers with non-specific inflammatory aspects (61%); 6/41 associated with moniliasis (16%); 4/41 caused by CMV infection (10%); 2/41 by HSV (5%); 1/41 by CMV and HSV (2%); 1/41 by PCM (2%); 1/41 due to Hystoplasmosis (2%) and 1/41, to neoplastic infiltration by non-Hodgkin lymphoma (2%). IH for CMV and HSV has confirmed the H&E findings and has detected another case. UGE has revealed high sensitivity (100%) for the detection of esophageal ulcer and low specificity for the viral etiological characterization (15%), as compared to H&E and IH. H&E has shown to be an adequate method for the etiological evaluation, with 87% of sensitivity and 100% of specificity, as compared to IH. BAAR research by ZN was negative in the 34 samples studied. GMS has confirmed the presence of fungus detected by H&E and has been fundamental in the morphological characterization of Histoplasma capsulatum and Paraccocidioides brasiliensis. Our findings support the use of H&E as a suitable method for the etiological evaluation of esophageal ulcers in HIV carriers and indicate IH for viral search only in suspect cases that are non-typical under H&E.