A multiplex PCR for direct detection of Mycoplasma pneumoniae, Chlamydia (Chlamydophila) pneumoniae, and Legionella pneumophila in respiratory specimens

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A multiplex PCR for direct detection of Mycoplasma pneumoniae, Chlamydia (Chlamydophila) pneumoniae, and Legionella pneumophila in respiratory specimens

 

Author: Ma, Kin-chi Marco
Title: A multiplex PCR for direct detection of Mycoplasma pneumoniae, Chlamydia (Chlamydophila) pneumoniae, and Legionella pneumophila in respiratory specimens
Degree: M.Sc.
Year: 2009
Subject: Hong Kong Polytechnic University -- Dissertations.
Polymerase chain reaction.
Pneumonia -- Diagnosis.
Respiratory infections.
Department: Dept. of Health Technology and Informatics
Pages: xi, 86 leaves : ill. ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2305607
URI: http://theses.lib.polyu.edu.hk/handle/200/4428
Abstract: Community acquired pneumonia (CAP) is a worldwide, serious threat to health, and an enormous socio-economic burden for the healthcare system. According to the Hospital Authority statistical report (2005/06), each year three to four thousand patients die of pneumonia. Pneumonia is the third leading cause of death in Hong Kong. A wealth of data accumulated over the past decade has identified atypical pathogens such as M. pneumoniae, C. pneumoniae, and L. pneumophila, as an important cause of acute bronchitis and CAP. The current available methods (direct antigen detection, culture and serology) either lack sensitivity or give only a retrospective diagnosis. Rapid and accurate identification of aetiological agent(s) is an essential procedure for prognostic and therapeutic purposes in the clinical management of patients with CAP. In this study, an in-house real-time multiplex polymerase chain reaction (PCR) using SYBR Green I to detect these three pathogens simultaneously and directly from respiratory specimen was established. Deoxyribonucleic acid (DNA) extracted from respiratory specimens was tested for the presence of Pst-1, PI and rpoB genes which are specific for M pneumoniae, C. pneumoniae and L. pneumophila respectively. In the absence of a reliable gold standard, nucleic acid amplification tests help to overcome the problems in detecting these three pathogens. For cultivable bacteria, the detection limit of the assay was detected by serial dilution of a pure culture of L. pneumophila. For Mycoplasma and Chlamydia, specific DNA targets were quantified by UV spectrometry. The assay was able to reliably detect 10 DNA copies/ul of each pathogen. No significant differences in the sensitivity of each primer set were observed when tested in both multiplex and singleplex PCR assay. A total of 131 patients with symptoms of respiratory tract infection admitted to Princess Margaret Hospital, Yan Chai Hospital and Caritas Medical Centre between June 2007 and February 2008 were retrospectively studied. Of the 10 serologically positive patients, five of them were confirmed by the multiplex real-time PCR to be infected by either one of the three pathogens. 80 serology negative patients were negative with the multiplex real-time PCR. 41 patients were multiplex PCR negative. The serological tests of those patients were not performed as no second convalescence serum was collected. The tum-around time for detection of these three atypical pathogens was reduced from 7 days to about 4 hours. It is concluded that the in-house multiplex PCR assay could be used as a diagnostic and epidemiological tool. The test has the potential to assist clinicians in establishing a specific aetiological diagnosis before initiating therapy, to decrease hospital costs and to prevent inappropriate antimicrobial treatment.

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