Recombinase Polymerase Amplification (RPA)-ELISA as an Isothermal Molecular  POCT Method for Bacterial Respiratory Infection Diagnosis

Reza  Azizian; Erfaneh  Jafari; Babak  Pourakabri; Setareh  Mamishi; Reihaneh  Hosseinpour Sadeghi; Maryam Sotoudeh  Anvari

doi:10.18502/ajmb.v17i2.18562

Reza Azizian Pediatric Infectious Diseases Research Center (PIDRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran
Erfaneh Jafari Pediatric Infectious Diseases Research Center (PIDRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran
Babak Pourakabri Pediatric Infectious Diseases Research Center (PIDRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran
Setareh Mamishi Pediatric Infectious Diseases Research Center (PIDRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran
Reihaneh Hosseinpour Sadeghi Pediatric Infectious Diseases Research Center (PIDRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran
Maryam Sotoudeh Anvari Department of Pathology, School of Medicine, Children’s Medical Center, Tehran University of Medical Sciences (TUMS), Tehran, Iran

DOI: https://doi.org/10.18502/ajmb.v17i2.18562

Keywords: Isothermal methods, Point of Care Test (POCT), Respiratory infections, RPA, RPA-ELISA

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 assays. 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 methods. 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.