Evaluation of a molecular assay for direct screening of Methicillin-resistant Staphylococcus aureus (MRSA) from nasal swabs

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Evaluation of a molecular assay for direct screening of Methicillin-resistant Staphylococcus aureus (MRSA) from nasal swabs


Author: Hung, Mei-fan Panda
Title: Evaluation of a molecular assay for direct screening of Methicillin-resistant Staphylococcus aureus (MRSA) from nasal swabs
Degree: M.Sc.
Year: 2008
Subject: Hong Kong Polytechnic University -- Dissertations.
Methicillin resistance.
Staphylococcus aureus infections.
Department: Dept. of Health Technology and Informatics
Pages: xi, 59 leaves : ill. ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2305603
URI: http://theses.lib.polyu.edu.hk/handle/200/4606
Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) constitutes a major public health threat, as it is the leading nosocomially acquired pathogen in Hong Kong. Infections caused by MRSA are associated with longer hospital stay, more days of antibiotic administration, higher cost and higher mortality. MRSA is usually introduced into the institution by an infected or colonized patient or by a colonized health care worker. Currently, conventional culture methods are used for its detection. The reporting time of MRSA cultures requires at least 2 days for positive culture and 4 days for negative. Rapid screening followed by accurate identification of MRSA becomes an essential procedure in preventive measures and in infection control. In this study, an in-house real-time polymerase chain reaction (PCR) using SYBR Green I to detect MRSA directly from clinical samples was evaluated. DNA extracted from nasal swabs was tested for the presence of femA and mecA genes which are specific for Staphylococcus aureus and methicillin resistance, respectively. With the presence of both genes and the cycle threshold ratio between 0.9 to 1.1 was reported as positive. Culture-based method was the gold standard. By serial dilution of a pure culture of MRSA, the lowest detection limit of the assay was found to be 10 CFU/ul. The method was specific without cross-reactivity of common respiratory pathogens and commensals (nine species). One hundred and fifty three nasal swabs with a positive rate of 50% from different patients in Princess Margaret Hospital (from Aug-07 to Dec-07) were evaluated. Sixty five were culture-positive and PCR-positive, 74 were culture-negative and PCR-negative, 13 were culture-positive and PCR-negative. One was culture-negative and PCR-positive. Among those 13 false negative, three of which were resolved by enrichment broth real-time PCR (broth-PCR), five had the bacterial count of mecA-positive coagulase negative Staphylococcus species (SCON) more than the bacterial count of MRSA by 1 log, two cases contained MRSA less than the detection limit and inhibition might be present in three cases. Comparing the results of culture, the specificity were 98.7%, sensitivity were 83.3%, positive predictive value were 98.5% and negative predictive value were 85.1%. The sensitivity, positive predictive value and negative predictive value were further improved by using broth-PCR, 87.2%, 98.6% and 88.1%, respectively. The turn-around time for detection of MRSA from clinical samples was shortened from 2 to 4 days to about 1.5 hr. In conclusion, the in-house PCR developed in this assay is a specific test for detection of nasal colonization with MRSA. It provided the same-day result and allowed infection control measures to start early in hospital. However, the sensitivity needed to be improved in the future.

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