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Updated: Nov 4, 2025
Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses
Published on: November 10, 2023
Development of a rapid multiplex PCR assay for the detection of common pathogens associated with community-acquired
Seok Hwee Koo1, Boran Jiang2, Pei Qi Lim1
1Clinical Trials and Research Unit, Changi General Hospital, 2 Simei Street 3, Singapore 529889, Singapore.
Background:
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.
Methods:
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.
Results:
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.
Conclusion:
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.
Insights
A new multiplex real-time PCR assay accurately detects four key bacteria causing community-acquired pneumonia (CAP) in Asia. This rapid diagnostic tool improves upon traditional methods, aiding in better patient management and antibiotic selection.
Area of Science:
- Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Community-acquired pneumonia (CAP) is a leading global cause of illness and death.
- The specific microbial cause of CAP remains unidentified in many cases, with regional variations in bacterial pathogens.
- Asia faces unique challenges in CAP diagnosis due to differing etiological agents.
Purpose of the Study:
- To develop and validate a multiplex real-time PCR assay for detecting common CAP pathogens in Asia.
- To provide a rapid and sensitive diagnostic tool for identifying bacterial causes of CAP.
- To improve the management and treatment outcomes of CAP patients in the Asian region.
Main Methods:
- Development of a multiplex real-time PCR assay targeting four key CAP microorganisms: Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Burkholderia pseudomallei.
- Analytical validation using bacterial isolates to determine sensitivity and specificity.
- Clinical validation using 58 culture-positive respiratory tract specimens.
Main Results:
- The qPCR assay demonstrated 100% analytical sensitivity and specificity.
- Clinical sensitivity was 100%, with clinical specificity ranging from 94-100%.
- The assay showed higher sensitivity than conventional culture methods and could semi-quantify bacterial loads.
Conclusions:
- A reliable, laboratory-developed multiplex qPCR assay for four clinically significant CAP microorganisms in Asia has been established.
- This assay offers a rapid turnaround time (within one working day).
- Improved diagnostic capabilities can lead to informed antibiotic choices, better patient management, and improved treatment outcomes.
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