Methodological establishment and diagnostic value of a multiplex fluorescent PCR assay for the detection of three fastidious respiratory pathogens

Abstract

Background: Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are common fastidious bacteria responsible for respiratory tract infections, particularly in children and immunocompromised individuals. Due to their demanding growth requirements, traditional culture methods often yield low sensitivity and delayed results, posing challenges for early and accurate diagnosis.

Objective: To establish a TaqMan probe-based multiplex fluorescent PCR method for the simultaneous rapid detection and identification of three important respiratory fastidious pathogens: Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis.

Methods: By designing and optimizing TaqMan probes and primers targeting Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, the sensitivity, specificity, and reproducibility of the TaqMan probe-based multiplex fluorescent PCR assay were examined. A total of 173 clinical samples which are sputum and alveolar lavage fluid were tested simultaneously using traditional culture methods and multiplex fluorescent PCR assays, and the results were compared for consistency. For samples with inconsistent detection results, targeted high-throughput sequencing (tNGS) was used for confirmation.

Results: The limit of detection of the TaqMan probe-based multiplex fluorescent PCR method was 100 Copies/ml for Streptococcus pneumoniae, 20 Copies/ml for Haemophilus influenzae, and 50 Copies/ml for Moraxella catarrhalis. The detection rate of the multiplex fluorescent PCR method was in high concordance with traditional culture methods and tNGS in the 173 clinical samples (Kappa = 0.819). The multiplex fluorescent PCR method demonstrated high sensitivity and specificity, detecting more cases of mixed infections.

Conclusion: The multiplex fluorescent PCR method established in this study provides a powerful tool for rapid and accurate clinical detection of fastidious respiratory pathogens, with significant clinical application value and promotion prospects. This method offers substantial advantages over traditional culture methods in terms of detection speed and sensitivity, particularly in detecting mixed infections, and has significant potential for clinical application and widespread adoption.

Fig 1. Sensitivity analysis of the multiplex fluorescence PCR method for three fastidious bacteria.

A: 1.5 × 10^6 Copies/ml, B: 1.5 × 10^5 Copies/ml, C: 1.5 × 10^4 Copies/ml, D: 1.5 × 10^3 Copies/ml, E: 500 Copies/ml, F: 200 Copies/ml, G: 100 Copies/ml, H: 50 Copies/ml, I: 20 Copies/ml, J: 10 Copies/ml. Limit of detection for Streptococcus pneumoniae: 100 Copies/ml. Limit of detection for Haemophilus influenzae: 20 Copies/ml. Limit of detection for Moraxella catarrhalis: 50 Copies/ml.

Fig 2. Amplification plots of representative positive and negative clinical specimens using the multiplex fluorescent PCR assay.

Red, green, and blue fluorescence curves represent Streptococcus pneumoniae (SP), Haemophilus influenzae (HI), and Moraxella catarrhalis (MC), respectively. Panel A shows an SP-only positive sample (red curve only), Panel B an HI-only positive sample (green curve only), Panel C an MC-only positive sample (blue curve only), Panel D a negative control (no amplification), and Panel E a triple-positive sample with red, green, and blue curves.

Fig 3. Overview of Multiplex PCR Workflow and Results (by FigDraw).

This figure was created using FigDraw (www.figdraw.com). The images and elements are original and have been authorized for use. This figure was drawn by the first author, Jin Chao Shi.