Recombinase Polymerase Amplification (RPA) as an Isothermal Molecular-Point of Care Test (IM-POCT) for Rapid Identification of P. aeruginosa Among Patients with Respiratory Infection
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Published on: April 25, 2025
Reza Azizian1,2, Erfaneh Jafari1,2, Babak Pourakabri3
1Pediatric Infectious Diseases Research Center (PIDRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran.
Insights
A new Recombinase Polymerase Amplification (RPA)-ELISA test rapidly detects Pseudomonas aeruginosa in pediatric respiratory samples. This quick screening tool shows high sensitivity, aiding diagnosis in resource-limited settings.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Pediatric Infectious Diseases
Background:
- Acute Respiratory Infections (ARIs) are a major cause of death in children.
- Pseudomonas aeruginosa is a significant pathogen contributing to ARI mortality.
- Rapid and accurate diagnostic tools are crucial for timely intervention, especially in resource-limited settings.
Purpose of the Study:
- To evaluate the performance of Recombinase Polymerase Amplification (RPA)-ELISA for the rapid detection of P. aeruginosa in pediatric respiratory samples.
- To compare the efficacy of RPA-ELISA with established methods like Real-Time PCR and culture.
- To assess the potential of RPA-ELISA as a point-of-care diagnostic tool.
Main Methods:
- Respiratory samples (sputum, BAL, throat cultures) were collected from 101 pediatric patients.
- P. aeruginosa identification was performed using culture, biochemical tests, and Real-Time PCR.
- RPA-ELISA was developed and optimized for P. aeruginosa detection using labeled primers and probes.
Main Results:
- RPA-ELISA demonstrated high sensitivity (97.6%) and moderate specificity (90%) compared to Real-Time PCR.
- The assay detected P. aeruginosa at a concentration of 41 ng/µL within 30 minutes.
- Out of 101 samples, 83 (82.2%) tested positive for P. aeruginosa.
Conclusions:
- RPA-ELISA is a rapid and sensitive method for detecting P. aeruginosa in pediatric respiratory samples.
- Its speed and simplicity make it suitable for use as an initial screening tool in resource-limited settings.
- Confirmatory testing is recommended due to the moderate specificity of the RPA-ELISA method.
Abstract:
Acute Respiratory Infections (ARIs) are a leading cause of pediatric mortality, with Pseudomonas aeruginosa as a key pathogen. This study evaluated Recombinase Polymerase Amplification (RPA)-ELISA for rapid detection of P. aeruginosa in respiratory samples (n = 101). Compared to Real-Time PCR (98.8% sensitivity, 100% specificity), RPA-ELISA showed 97.6% sensitivity and 90% specificity, detecting pathogens at 41 ng/µL within 30 min. The method's simplicity and speed support its use as a screening tool in resource-limited settings, though confirmatory testing remains advised. Respiratory samples (sputum, BAL, throat cultures) were collected from pediatric patients (August-December 2022). P. aeruginosa was identified via culture, biochemical tests, and Real-Time PCR (targeting gyrB). RPA-ELISA was optimized for P. aeruginosa detection using labeled primers/probes. Among 101 samples, 83 (82.2%) were P. aeruginosa. RPA-ELISA demonstrated 97.6% sensitivity (95% CI: 91.7-99.7%) and 90% specificity (95% CI: 55.5-99.7%) compared to Real-Time PCR (98.8% sensitivity, 95% CI: 93.5-100%; 100% specificity, 95% CI: 69.2-100%). The method detected P. aeruginosa at a concentration of 41 ng/µL with a 30-min turnaround time. Positive results were indicated by a color change in the ELISA wells, contrasting with negative controls. RPA-ELISA is a promising IM-POCT for P. aeruginosa, offering high sensitivity and rapid results. It is suitable for resource-limited settings but requires confirmatory testing due to moderate specificity.
