Development of a rapid multiplex PCR assay for the detection of common pathogens associated with community-acquired pneumonia.

Abstract

Community-acquired pneumonia (CAP) is one of the most common infectious diseases and is a significant cause of mortality and morbidity globally. A microbial cause was not determined in a sizable percentage of patients with CAP; there are increasing data to suggest regional differences in bacterial aetiology. We devised a multiplex real-time PCR assay for detecting four microorganisms (Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae and Burkholderia pseudomallei) of relevance to CAP infections in Asia. Analytical validation was accomplished using bacterial isolates (n=10-33 of each target organism for analytical sensitivity and n=117 for analytical sensitivity) and clinical validation using 58 culture-positive respiratory tract specimens. The qPCR assay exhibited 100% analytical sensitivity and analytical specificity, and 100% clinical sensitivity and 94-100% clinical specificity. The limit of detection and efficiency for the multiplex PCR assay were 3-33 CFU/mL and 93-110%, respectively. The results showed that the PCR-based method had higher sensitivity than traditional culture-based methods. The assay also demonstrated an ability to semiquantify bacterial loads. We have devised a reliable laboratory-developed multiplex qPCR assay, with a turnaround time of within one working day, for detection of four clinically important CAP-associated microorganisms in Asia. The availability of a test with improved diagnostic capabilities potentially leads to an informed choice of antibiotic usage and appropriate management of the patient to achieve a better treatment outcome and financial savings.