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
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/206492 |
| Date | January 2014 |
| Creators | Wong, Cheuk-yin, Ian, 黃卓賢 |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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