Thesis (doctoral)--Ludwig Maximilians-Universität zu München, 1979.

Pneumocystis jiroveciiis an opportunistic pathogen usually affecting immunocompromised patients. Molecular techniques are increasing used in the diagnosis of Pneumocystis infection, but colonization of Pneumocystis in the respiratory tract is often detected in patients without clinical evidence of Pneumocystis pneumonia. Hence, the epidemiology of colonization in Hong Kong is crucial in the interpretation of test results from these molecular techniques in the diagnosis of Pneumocystis. The purpose of this study wasto find out the prevalence of Pneumocystis colonization by the PCR based analysis of mitochondrial small subunit rRNA (mtSSUrRNA) and to determine fungal load by real time quantitative PCR targeting on mitochondrial large subunit rRNA (mtLSUrRNA).All samples positive for mtSSUrRNA PCR assay were further evaluated to determine the prevalence on the mutations associated with drug resistance in the dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) genes by nested PCR assay and sequencing analysis. In this study, a total of 183 bronchoscopic specimens collected from 155 adult patients were selected. Pneumocystis DNA was detected in 14 patients out of 155 subjects by mtSSUrRNA PCR assay. After the exclusion of three cases of Pneumocystis pneumonia confirmed by microscopy, the overall rate of Pneumocystis colonization was 7.2% (11/152). Among the patients with Pneumocystis colonization, the median age was 72 years in a range of 32 to 84 years and the ratio of male to female was 4.5:1. All patients with Pneumocystis were found in March to August. Apart from one patient with HIV infection and one patient without any chronic illness, the remaining nine non-HIV-infected patients suffered from various underlying diseases including two transplant recipients in kidney and bone marrow, two with lung cancer, two with gastrointestinal cancer, four with hematological malignancies, and two with autoimmune diseases. While fungal load of P. jirovecii were measured in the patients who found positive in mtSSUrRNA PCR assay, one patient showed non-detectable result in real time quantitative PCR (qPCR). The median fungal load among the patients was 82,340 copies per ml. Further amplifications of DHPS and DHFR were successfully performed in eight patients. A synonymous substitution at nucleotide position 312 in the DHFR gene was showed in one patient. Both DHPS and DHFR were found to be wild type in seven patients respectively, corresponding to no amino acid substitution from genetic mutations. In comparison to other studies, the prevalence of Pneumocystis colonization and genotypic mutation on DHPS and DHFR are relatively low. Further studies were suggested for other risk factors such as prophylactic usage, CD4+ T cell count and particular underlying diseases. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Introdução: A Fibrose Cística é uma doença autossômica recessiva associada a infecções respiratórias crônicas e que leva a considerável morbidade e mortalidade. O Pneumocystis jirovecii é um fungo que causa pneumonia em imunossuprimidos e cuja colonização em várias doenças pulmonares mostrou ser um fenômeno clínico importante. Objetivo: Avaliar a taxa de colonização e a distribuição dos genótipos do P. jirovecii entre pacientes com Fibrose Cística. Material e métodos: Foram estudados 34 pacientes com Fibrose Cística que realizaram broncoscopia no Hospital de Clínicas de Porto Alegre entre março de 2004 e agosto de 2007. A detecção do P. jirovecii no lavado broncoalveolar (LBA) foi feita através de nested-PCR com a utilização dos primers pAZ 102-E e pAZ 102-H no primeiro round e pAZ 102-X e pAZ 102-Y no segundo. A caracterização do gene da Diidropteroato sintetase (DIPS) e da Grande Subunidade do RNA ribossômico mitocondrial (mtLSUrRNA) foi realizada através do uso de enzimas de restrição e de seqüenciamento genético, respectivamente. Os dados clínico-demográficos dos pacientes foram obtidos dos prontuários médicos e analisados quanto à presença de colonização. Resultados: A colonização pelo P. jirovecii foi detectada em 38,2% (13/34) dos pacientes com Fibrose Cística. Nenhum dado clínico-demográfico pôde ser associado significativamente (p<0,05) à presença da colonização nos 34 pacientes estudados. Na caracterização genotípica da DIPS, o genótipo selvagem - ausência de mutações nas posições 55 e 57 – foi observado em todos os casos. Na tipagem da mtLSUrRNA, o genótipo 1 (85C/248C) foi observado em 41,6% dos casos, o genótipo 2 (85A/248C) em 16,6% , o genótipo 3 (85T/248C) em 25% e o genótipo misto 1 e 3 em 16,6% dos casos. Conclusão: Foi observada uma alta taxa (38,2%) de colonização pelo P. jirovecii entre os 34 pacientes. Este achado sugere que os portadores de Fibrose Cística podem constituir um reservatório significativo do fungo, uma importante fonte de infecção do microorganismo a outros pacientes. Quanto a uma possível associação entre a colonização pelo P. jirovecii e o curso clínico da doença, faz-se necessário estudar um número maior de pacientes. Os resultados da distribuição genotípica da mtLSUrRNA do P. jirovecii confirmam a predominância dos subtipos 1 e 3 entre os portadores da Fibrose Cística. / Introduction: Cystic Fibrosis is na autosomal recessive disorder associated to chronic respiratory infections, which leads to considerable morbidity and mortality. Pneumocystis jirovecii is a fungus that causes pneumonia in immunosuppressed patients and its colonization rate on several pulmonary diseases is a significant clinical phenomenon. Objective: Assess the rate of P. jirovecii genotypes colonization and distribution in patients with Cystic Fibrosis. Material and methods: 34 patients with Cystic Fibrosis who have undergone bronchoscopy at Hospital de Clínicas de Porto Alegre between March 2004 and August 2007 were evaluated. Detection of P. jirovecii by analysis of bronchoalveolar lavage (BAL) was carried out with nested-PCR, with the use of primers pAZ 102-E and pAZ 102-H in the first round, and pAZ 102-X and pAZ 102-Y in the second round. Characterization of the diihydropteroate synthase (DHPS) gene and the mitochondrial Large Subunit ribosomal RNA (mtLSUrRNA) was performed with the use of restriction enzymes and genetic sequencing, respectively. The clinical and demographic data of patients was obtained from their medical records and analyzed for the presence of colonization. Results: P. jirovecii colonization was detected in 38,2% (13/34) of the patients with Cystic Fibrosis. No clinical-demographic data was significantly associated (p<0,05) to the presence of colonization in the 34 studied patients. Genotypical characterization of DHPS, the wild genotype – absence of mutations in the 55 and 57 positions – was observed in all the cases. In mtLSUrRNA typing genotype 1 (85C/248C) was observed in 41,6% of the cases, genotype 2 (85A/248C) in 16,6%, genotype 3 (85T/248C) in 25% and mixed genotype 1 and 3 in 16,6% of the cases. Conclusion: A high rate (38,2%) of P. jirovecii colonization was found in the 34 patients. This finding suggests that Cystic Fibrosis patients may constitute a major reservoir and source of infection for other patients. As for a possible association between P. jirovecii colonization and the clinical evolution of the disease, further research with a greater number of subjects is needed. The results of P. jirovecii mtLSUrRNA genotypical distribution confirm the predominance of subtypes 1 and 3 among Cystic Fibrosis patients.

Introdução: A Fibrose Cística é uma doença autossômica recessiva associada a infecções respiratórias crônicas e que leva a considerável morbidade e mortalidade. O Pneumocystis jirovecii é um fungo que causa pneumonia em imunossuprimidos e cuja colonização em várias doenças pulmonares mostrou ser um fenômeno clínico importante. Objetivo: Avaliar a taxa de colonização e a distribuição dos genótipos do P. jirovecii entre pacientes com Fibrose Cística. Material e métodos: Foram estudados 34 pacientes com Fibrose Cística que realizaram broncoscopia no Hospital de Clínicas de Porto Alegre entre março de 2004 e agosto de 2007. A detecção do P. jirovecii no lavado broncoalveolar (LBA) foi feita através de nested-PCR com a utilização dos primers pAZ 102-E e pAZ 102-H no primeiro round e pAZ 102-X e pAZ 102-Y no segundo. A caracterização do gene da Diidropteroato sintetase (DIPS) e da Grande Subunidade do RNA ribossômico mitocondrial (mtLSUrRNA) foi realizada através do uso de enzimas de restrição e de seqüenciamento genético, respectivamente. Os dados clínico-demográficos dos pacientes foram obtidos dos prontuários médicos e analisados quanto à presença de colonização. Resultados: A colonização pelo P. jirovecii foi detectada em 38,2% (13/34) dos pacientes com Fibrose Cística. Nenhum dado clínico-demográfico pôde ser associado significativamente (p<0,05) à presença da colonização nos 34 pacientes estudados. Na caracterização genotípica da DIPS, o genótipo selvagem - ausência de mutações nas posições 55 e 57 – foi observado em todos os casos. Na tipagem da mtLSUrRNA, o genótipo 1 (85C/248C) foi observado em 41,6% dos casos, o genótipo 2 (85A/248C) em 16,6% , o genótipo 3 (85T/248C) em 25% e o genótipo misto 1 e 3 em 16,6% dos casos. Conclusão: Foi observada uma alta taxa (38,2%) de colonização pelo P. jirovecii entre os 34 pacientes. Este achado sugere que os portadores de Fibrose Cística podem constituir um reservatório significativo do fungo, uma importante fonte de infecção do microorganismo a outros pacientes. Quanto a uma possível associação entre a colonização pelo P. jirovecii e o curso clínico da doença, faz-se necessário estudar um número maior de pacientes. Os resultados da distribuição genotípica da mtLSUrRNA do P. jirovecii confirmam a predominância dos subtipos 1 e 3 entre os portadores da Fibrose Cística. / Introduction: Cystic Fibrosis is na autosomal recessive disorder associated to chronic respiratory infections, which leads to considerable morbidity and mortality. Pneumocystis jirovecii is a fungus that causes pneumonia in immunosuppressed patients and its colonization rate on several pulmonary diseases is a significant clinical phenomenon. Objective: Assess the rate of P. jirovecii genotypes colonization and distribution in patients with Cystic Fibrosis. Material and methods: 34 patients with Cystic Fibrosis who have undergone bronchoscopy at Hospital de Clínicas de Porto Alegre between March 2004 and August 2007 were evaluated. Detection of P. jirovecii by analysis of bronchoalveolar lavage (BAL) was carried out with nested-PCR, with the use of primers pAZ 102-E and pAZ 102-H in the first round, and pAZ 102-X and pAZ 102-Y in the second round. Characterization of the diihydropteroate synthase (DHPS) gene and the mitochondrial Large Subunit ribosomal RNA (mtLSUrRNA) was performed with the use of restriction enzymes and genetic sequencing, respectively. The clinical and demographic data of patients was obtained from their medical records and analyzed for the presence of colonization. Results: P. jirovecii colonization was detected in 38,2% (13/34) of the patients with Cystic Fibrosis. No clinical-demographic data was significantly associated (p<0,05) to the presence of colonization in the 34 studied patients. Genotypical characterization of DHPS, the wild genotype – absence of mutations in the 55 and 57 positions – was observed in all the cases. In mtLSUrRNA typing genotype 1 (85C/248C) was observed in 41,6% of the cases, genotype 2 (85A/248C) in 16,6%, genotype 3 (85T/248C) in 25% and mixed genotype 1 and 3 in 16,6% of the cases. Conclusion: A high rate (38,2%) of P. jirovecii colonization was found in the 34 patients. This finding suggests that Cystic Fibrosis patients may constitute a major reservoir and source of infection for other patients. As for a possible association between P. jirovecii colonization and the clinical evolution of the disease, further research with a greater number of subjects is needed. The results of P. jirovecii mtLSUrRNA genotypical distribution confirm the predominance of subtypes 1 and 3 among Cystic Fibrosis patients.

Introdução: A Fibrose Cística é uma doença autossômica recessiva associada a infecções respiratórias crônicas e que leva a considerável morbidade e mortalidade. O Pneumocystis jirovecii é um fungo que causa pneumonia em imunossuprimidos e cuja colonização em várias doenças pulmonares mostrou ser um fenômeno clínico importante. Objetivo: Avaliar a taxa de colonização e a distribuição dos genótipos do P. jirovecii entre pacientes com Fibrose Cística. Material e métodos: Foram estudados 34 pacientes com Fibrose Cística que realizaram broncoscopia no Hospital de Clínicas de Porto Alegre entre março de 2004 e agosto de 2007. A detecção do P. jirovecii no lavado broncoalveolar (LBA) foi feita através de nested-PCR com a utilização dos primers pAZ 102-E e pAZ 102-H no primeiro round e pAZ 102-X e pAZ 102-Y no segundo. A caracterização do gene da Diidropteroato sintetase (DIPS) e da Grande Subunidade do RNA ribossômico mitocondrial (mtLSUrRNA) foi realizada através do uso de enzimas de restrição e de seqüenciamento genético, respectivamente. Os dados clínico-demográficos dos pacientes foram obtidos dos prontuários médicos e analisados quanto à presença de colonização. Resultados: A colonização pelo P. jirovecii foi detectada em 38,2% (13/34) dos pacientes com Fibrose Cística. Nenhum dado clínico-demográfico pôde ser associado significativamente (p<0,05) à presença da colonização nos 34 pacientes estudados. Na caracterização genotípica da DIPS, o genótipo selvagem - ausência de mutações nas posições 55 e 57 – foi observado em todos os casos. Na tipagem da mtLSUrRNA, o genótipo 1 (85C/248C) foi observado em 41,6% dos casos, o genótipo 2 (85A/248C) em 16,6% , o genótipo 3 (85T/248C) em 25% e o genótipo misto 1 e 3 em 16,6% dos casos. Conclusão: Foi observada uma alta taxa (38,2%) de colonização pelo P. jirovecii entre os 34 pacientes. Este achado sugere que os portadores de Fibrose Cística podem constituir um reservatório significativo do fungo, uma importante fonte de infecção do microorganismo a outros pacientes. Quanto a uma possível associação entre a colonização pelo P. jirovecii e o curso clínico da doença, faz-se necessário estudar um número maior de pacientes. Os resultados da distribuição genotípica da mtLSUrRNA do P. jirovecii confirmam a predominância dos subtipos 1 e 3 entre os portadores da Fibrose Cística. / Introduction: Cystic Fibrosis is na autosomal recessive disorder associated to chronic respiratory infections, which leads to considerable morbidity and mortality. Pneumocystis jirovecii is a fungus that causes pneumonia in immunosuppressed patients and its colonization rate on several pulmonary diseases is a significant clinical phenomenon. Objective: Assess the rate of P. jirovecii genotypes colonization and distribution in patients with Cystic Fibrosis. Material and methods: 34 patients with Cystic Fibrosis who have undergone bronchoscopy at Hospital de Clínicas de Porto Alegre between March 2004 and August 2007 were evaluated. Detection of P. jirovecii by analysis of bronchoalveolar lavage (BAL) was carried out with nested-PCR, with the use of primers pAZ 102-E and pAZ 102-H in the first round, and pAZ 102-X and pAZ 102-Y in the second round. Characterization of the diihydropteroate synthase (DHPS) gene and the mitochondrial Large Subunit ribosomal RNA (mtLSUrRNA) was performed with the use of restriction enzymes and genetic sequencing, respectively. The clinical and demographic data of patients was obtained from their medical records and analyzed for the presence of colonization. Results: P. jirovecii colonization was detected in 38,2% (13/34) of the patients with Cystic Fibrosis. No clinical-demographic data was significantly associated (p<0,05) to the presence of colonization in the 34 studied patients. Genotypical characterization of DHPS, the wild genotype – absence of mutations in the 55 and 57 positions – was observed in all the cases. In mtLSUrRNA typing genotype 1 (85C/248C) was observed in 41,6% of the cases, genotype 2 (85A/248C) in 16,6%, genotype 3 (85T/248C) in 25% and mixed genotype 1 and 3 in 16,6% of the cases. Conclusion: A high rate (38,2%) of P. jirovecii colonization was found in the 34 patients. This finding suggests that Cystic Fibrosis patients may constitute a major reservoir and source of infection for other patients. As for a possible association between P. jirovecii colonization and the clinical evolution of the disease, further research with a greater number of subjects is needed. The results of P. jirovecii mtLSUrRNA genotypical distribution confirm the predominance of subtypes 1 and 3 among Cystic Fibrosis patients.

Thèse doctorat : Parasitologie : Paris 12 : 2003. / Titre provenant de l'écran-titre.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Abstract not available. / Hui, Mamie. / "Dec 2010." / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 209-236). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.

Tesis presentada a la Universidad de Chile para optar al grado académico de Magíster en Bioquímica área de especialización en Bioquímica Clínica y Memoria para optar al título profesional de Bioquímico / Pneumocystis jirovecii es un microorganismo fúngico capaz de infectar el tracto respiratorio de individuos con algún tipo de inmunocompromiso, y causarles neumonía por Pneumocystis (PCP). Investigaciones más recientes han comprobado que P. jirovecii también se puede hallar en el tracto respiratorio de personas aparentemente inmunocompetentes, sobre todo en lactantes menores de un año de edad, en quienes se denomina “infección primaria”. Aquellos hospederos en quienes se ha detectado el microorganismo en muestras respiratorias, pero que no presentan sintomatología de PCP, se consideran “colonizados” por Pneumocystis. A pesar de los avances en la investigación básica, clínica y epidemiológica de Pneumocystis, no se ha comprobado si el hongo contribuye a la morbilidad de enfermedades respiratorias que a veces presentan pacientes adultos colonizados o lactantes con infección primaria. En vista de lo anterior, y considerando el inconveniente que Pneumocystis no es un hongo cultivable hasta el momento, ha surgido la necesidad de desarrollar nuevos métodos de detección y cuantificación de Pneumocystis que permitan estudiar más a fondo su relevancia clínica y epidemiológica, sobre todo en los grupos de pacientes inmunocompetentes antes mencionados. A este respecto, durante la última década se ha aplicado la técnica de Reacción en Cadena de la Polimerasa en Tiempo Real (q-PCR [quantitative]) para la detección de Pneumocystis, la cual permite cuantificar carga de microorganismo en muestras biológicas, incluso cargas bajísimas que comúnmente no son detectadas por los métodos convencionales. Pero, además de esto, q-PCR permite comparar carga de P. jirovecii entre muestras distintas procedentes del mismo o de distintos pacientes, si se realiza el procedimiento de cuantificación relativa. Esto consiste en normalizar la carga absoluta de microorganismo con la cantidad de DNA humano hallado en la misma muestra, de modo que el cociente resultante es la carga relativa. Teniendo estos antecedentes, se montó y estandarizó el método de q-PCR para cuantificar P. jirovecii en pacientes colonizados, tanto adultos como lactantes de diversas edades, mediante el análisis de muestras invasivas (tejido pulmonar) y no invasivas (aspirado nasofaríngeo, gargarismo e hisopado nasofaríngeo). Se escogió como gen blanco a MSG, un gen multicopia que codifica para la principal glicoproteína de superficie de Pneumocystis. La cuantificación relativa de P. jirovecii mostró que hay cargas significativamente distintas del microorganismo entre ciertos grupos etarios de lactantes colonizados fallecidos por Síndrome de Muerte Súbita (SMS). En otros grupos de pacientes colonizados (adultos y lactantes sanos) no se observaron diferencias significativas de carga relativa de P. jirovecii entre distintas edades. Se evidenció en la mayoría de los grupos de pacientes que la cuantificación relativa presenta un patrón de cargas distinto para cada rango de edad, respecto al patrón obtenido de la cuantificación absoluta. Además, se investigó cuánto afecta la naturaleza de las muestras en la precisión de los resultados. En el caso de las muestras que no son homogéneas, como secreciones respiratorias, se obtuvieron cantidades muy variables de DNA humano, independientemente del tamaño de la muestra, lo cual produjo resultados con mayor variabilidad respecto a los obtenidos para muestras más homogéneas, como tejido pulmonar. En conclusión, este trabajo demuestra la importancia de utilizar resultados expresados como cuantificación relativa, que a diferencia de la absoluta, permite comparar carga de Pneumocystis entre distintas muestras, tras haber eliminado ciertos factores que inducen a obtener conclusiones distorsionadas. Además se evidenció la necesidad de escoger el tipo de muestra más apropiado para obtener resultados más confiables y precisos, al menos para el área de investigación. Esta investigación contribuye a validar una valiosa herramienta de detección que permite comparar carga de Pneumocystis entre distintas muestras. Su alta sensibilidad hace posible su aplicación incluso en muestras de pacientes colonizados por Pneumocystis, cuyas cargas de microorganismo generalmente son demasiado bajas e indetectables por otros métodos. El contar con esta nueva herramienta en nuestro país, nos permitirá investigar más a fondo la trascendencia clínica de P. jirovecii en la salud de pacientes colonizados y lactantes con infección primaria, con o sin enfermedades respiratorias. Así, este trabajo de tesis aporta un respaldo científico a la nueva aplicación de comparar muestras, utilizando el método más avanzado de diagnóstico molecular de Pneumocystis. / Pneumocystis jirovecii is a fungal microorganism that infects the respiratory tract of immunocompromised patients and it may cause Pneumocystis pneumonia (PCP). Recent research has demonstrated that P. jirovecii can also be found in the respiratory tract of apparently immunocompetent patients, especially in infants younger than one year of age; in this case it is called “primary infection”. Those hosts that show this microorganism in their respiratory samples, but who have no PCP symptoms, are known as “Pneumocystis colonized”. Despite of the recent advances in basic, clinical and epidemiologic investigation of Pneumocystis, there is no demonstration whether this fungus affects morbidity of respiratory diseases, which are sometimes present in colonized adult patients or infants with primary infection. In view of this, and considering the inconvenience that, to date, Pneumocystis cannot be cultured, it is necessary to develop new methods to detect and to quantify Pneumocystis in order to study its clinical and epidemiological relevance more deeply, especially in the immunocompetent patients mentioned before. On that score, during the last decade, the technology of Real Time Polymerase Chain Reaction (q-PCR [quantitative]) has been applied for the detection of Pneumocystis. This technology allows to quantify load of microorganism in biological samples, even in very low loads which cannot be commonly detected by conventional methods. In addition to this, q- PCR allows the comparison of P. jirovecii loads among different samples taken from the same or different patients, if relative quantification is calculated. In procedure, the absolute load of microorganisms is normalized with the amount of human DNA found in the same sample, so the result is the relative load. With this information, we established and standarized a q-PCR method in order to quantify P. jirovecii in colonized patients, both adults and infants at several ages, through the analysis of invasive (pulmonary tissue) and non invasive samples (nasopharyngeal aspirate, oral wash and nasopharyngeal swab). MSG was chosen as target gene, this is a multicopy gene which codes for the Major Surface Glycoprotein of Pneumocystis. The relative quantification of P. jirovecii showed significantly different loads of microorganism between several age groups of colonized infants who had died of Sudden Infant Death Syndrome (SIDS). Within others colonized patient groups ―healthy adults and infants― no significantly differences were observed in relative load of P. jirovecii between different ages. In most patient groups, it was apparent that relative quantification shows a different load tendency for each age range, compared with the tendency obtained from absolute quantification. In addition, we investigated the influence of sample composition on the precision of the results. In case of non homogeneous samples, such as respiratory samples, variable quantities of DNA were obtained, independent of the sample size. This produced more variability of results, with regard to those obtained from homogeneous samples, such as pulmonary tissue. In conclusion, this work demonstrates the relevance of using results expressed as relative quantification, which, unlike absolute quantification, allows the comparison of Pneumocystis load between different samples, by eliminating some factors which drive to wrong conclusions. Furthermore, the need of choosing the best sample type in order to obtain results more reliable and precise, at least for research purpose, was demonstrated. This research work contributes to the validation of a useful detection tool, which allows comparing Pneumocystis load between different samples. Its high sensitivity makes it possible to use this technology even in Pneumocystis colonized patients, in which loads of microorganism are generally too low to be detected by other methods. To have this novel tool in our country, will allow us to investigate in depth the clinical relevance of P. jirovecii in health of colonized patients and infants with primary infection ―with or without respiratory diseases. Therefore, this thesis work provides a scientific support to the novel application of comparing samples, using the most advanced, molecular diagnostic method for Pneumocystis.

Thesis (D.P.H.)--University of Michigan.