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

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).

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

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

Thesis (doctoral)--Nijmegen, 1984.

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.

No description available.

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.

Thesis (PhD)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.

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).