Establishment and evaluation of a rapid method for the detection of bacterial pneumonia in hospitalized patients via multiplex PCR-capillary electrophoresis (MPCE)

Cost-effective molecular diagnostic techniques for bacterial pneumonia are limited. We designed primers for 13 bacteria, performed multiplex nucleic acid detection through fragment analysis to obtain pathogen identification results, and established a multiplex PCR-capillary electrophoresis (MPCE) method, which can simultaneously detect 13 pathogens associated with bacterial pneumonia. The sensitivity, specificity, and reproducibility of the MPCE assay were tested, and 420 clinical samples were used to assess the clinical detection ability of MPCE, with the culture method used as a reference. Samples with inconsistent results detected by the two methods were sent for Sanger sequencing. The minimum detection limit of MPCE for 13 bacteria was 6.0 x 10(3) cfu/mL similar to 2.0 x 10(6) cfu/mL. No cross-reactivity was observed with other pathogens. The percentage of agreement for reproducibility analysis reached 100%. For the 420 sputum samples, when the culture method was used as the reference, the sensitivity of MPCE to 13 bacteria ranged from 80% to 100%. The specificity for 13 bacteria ranged from 67.1% to 100%. The percentage of agreement between the MPCE and the culture method ranged from 69.7% to 100%. There was no statistically significant difference (P > 0.05) in the detection of Escherichia coli, Enterobacter cloacae complex, Staphylococcus aureus, methicillin-resistant S. aureus, Streptococcus pyogenes, Moraxella catarrhalis, or Legionella pneumophila between the MPCE and the culture method. Clinical samples with negative cultures but positive MPCE results were validated by Sanger sequencing, and the results were consistent with those of MPCE. The MPCE method has high sensitivity and specificity for bacterial pneumonia, enabling the simultaneous and rapid detection of multiple pathogens. It is cost-effective and has potential for clinical application.