Recombinase Polymerase Amplification (Rpa)-Elisa As an Isothermal Molecular Poct Method for Bacterial Respiratory Infection Diagnosis Publisher



Azizian R^{1, 2} ; Jafari E^{1, 2} ; Pourakabri B¹ ; Mamishi S¹ ; Sadeghi RH¹ ; Anvari MS³
Source: Avicenna Journal of Medical Biotechnology Published:2025

Abstract

Background: Acute Respiratory Infections (ARIs) are a leading cause of childhood mortality worldwide, especially in African and Southeast Asian countries. Point of Care Test (POCT) techniques provide faster diagnoses compared to conventional or real-time PCR methods. Recombinase Polymerase Amplification (RPA) offers rapid on-site detection of these infections. Coupling RPA with Enzyme-Linked Immunosorbent Assay (ELISA) (RPA-ELISA) creates a cost-effective alternative, ideal for clinical applications. This study evaluates RPA-ELISA as a rapid diagnostic tool for bacterial respiratory infections. Methods: From 11 August 2022 to 9 February 2023, respiratory samples were collected and processed using culture methods, biochemical tests, real-time PCR, and RPA as-says. The RPA reactions were conducted at 39°C for 30 min, and ELISA was used for detection. Statistical analyses focused on sensitivity, specificity, Positive Predictive Values (PPV), and Negative Predictive Values (NPV). Results: Forty-two respiratory samples, were collected in this period of which 10 samples showed no growth, and 32 tested positive. Among these positive samples, 15 isolates (35.7%) were identified as Klebsiella pneumoniae (K. pneumoniae), 14 isolates (33.3%) as Streptococcus pneumoniae (S. pneumoniae), and 3 isolates (7.1%) as Moraxella catarrhalis (M. catarrhalis). RPA-ELISA demonstrated 100% sensitivity for all pathogens, comparable to or better than RT-PCR, but had slightly lower specificity and PPV. RT-PCR achieved 100% specificity and PPV for all pathogens, indicating higher accuracy; yet, RPA-ELISA's sensitivity points to its effectiveness as a rapid screening tool. Conclusion: RPA-ELISA is significantly faster than real-time PCR and culture meth-ods. Its ease of use makes it suitable for on-site diagnoses in resource-limited environments. Limitations include a small sample size for certain bacteria and the necessity for further validation in varied clinical contexts. © 2025, Avicenna Journal of Medical Biotechnology.