Rapid detection of Hemophilus influenzae and Streptococcus pneumoniae simultaneously by using duplex Recombinase Aided Amplification Assay directly from invasive clinical samples

Wenjian Xu¹Yanling Feng²Yuyan Xia²Bing Du²Xianyi Huang¹Lin Zhou¹Jing Li¹Fang Fang¹Chun Meng¹Li Yu¹Lijuan LijuanMa^1*Guanhua Xue^2*Jing Yuan^2*

• ¹Department of Clinical Laboratory，CAPITAL CENTER FOR CHILDREN'S HEALTH，CAPITAL MEDICAL UNIVERSITY, Beijing, China
• ²Capital Institute of Pediatrics, Beijing, China

Community-acquired pneumonia (CAP) is a leading cause of mortality in children under five years of age globally. As Streptococcus pneumoniae and Haemophilus influenzae are the most common pathogens associated with CAP requiring hospitalization, a simple, low-cost, and highly sensitive method is critically needed for rapid and early diagnosis. In this study, we developed an accurate and rapid duplex Recombinase-Aided Amplification (RAA) assay for the simultaneous detection of these pathogens. This assay targets the conserved regions of the omp6 gene for H. influenzae and the lytA gene for S. pneumoniae, which were selected following a comparative genomic analysis across various bacterial strains. The established RAA assay can be completed within 10 minutes at 39°C, with optimal probe concentrations of 0.6uM/0.8uM combination. The limit of detection for this assay was determined to be 72 copies/reaction for S. pneumoniae and 35 copies/reaction for H. influenzae. We validated the clinical performance of the RAA assay using 168 clinical specimens (bronchoalveolar lavage fluid and cerebrospinal fluid) from pediatric patients. For comparison, these specimens were also tested using a real-time PCR assay and traditional bacterial culture. The positive detection rates by RAA for S. pneumoniae (10.71%) and H. influenzae (12.5%) were in 100% concordance with the real-time PCR results, but the RAA assay offered a significantly faster turnaround time. In contrast, the detection rates for bacterial culture were substantially lower, at 5.36% and 4.17%, respectively. In conclusion, the RAA assay described herein offers rapid detection, high sensitivity and specificity, and operational simplicity, making it a highly suitable diagnostic tool for S. pneumoniae and H. influenzae, especially in resource-limited settings.