Current Microbiology · 6 authors, 3 centres
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This study developed a recombinase polymerase amplification assay combined with lateral flow strips (RPA-LFS) for rapid detection of Candida parapsilosis. The assay achieved a limit of detection of 5.85 × 10^3 copies per reaction at 95% probability, with 100% specificity against 35 other pathogens. Clinical testing on 281 samples showed 31.7% detection rate, matching qPCR and culture, and results are available within 40 minutes without specialized equipment.
**Background:** Candida parapsilosis is a common cause of candidiasis in low birth weight neonates and critically ill patients, with increasing incidence and a 30-day mortality of 32.4%. Traditional culture methods are slow, and molecular techniques like PCR require expensive equipment. Recombinase polymerase amplification (RPA) combined with lateral flow strips (LFS) offers a rapid, isothermal, and portable alternative. This study aimed to develop and validate an RPA-LFS assay for detecting C. parapsilosis based on the FKS2 gene.
**Methods:** The study used four reference strains (ATCC 22019, 90018, 200954, 7330) and 17 clinical isolates of C. parapsilosis, plus 35 common pathogens for specificity testing. A total of 281 clinical samples (124 blood, 107 sputum, 28 pus, 22 dermal) were collected from patients with suspected Candida infection. Primers and probes targeting the FKS2 gene were designed and screened. The optimal primer/probe set (F6/P/R2B) was selected after testing for amplification efficiency and absence of primer-dimer formation. RPA reactions were performed at 37°C for 20 minutes using the TwistAmp® Liquid DNA Amplification Kit and Twist Amp® DNA amplification nfo kit. Amplification products were detected on lateral flow strips (USTAR Biotechnology). Sensitivity was determined by tenfold serial dilutions from 5.0×10^2 to 5.0×10^7 copies/µl, and the limit of detection (LOD) was calculated by probit regression analysis of eight independent experiments. Specificity was tested against 35 common pathogens. Clinical samples were tested simultaneously by RPA-LFS, qPCR, and conventional culture.
**Key Results:** The optimal RPA-LFS conditions were 37°C for 20 minutes. The LOD was 5.85×10^3 copies per reaction at 95% probability (probit regression). At 5.0×10^2 copies, only 12.5% (1/8) of samples were positive; at 5.0×10^3 copies, 75% (6/8) were positive. The assay showed 100% specificity: all four reference strains and 17 clinical isolates of C. parapsilosis were positive, while all 35 other pathogens (including Candida albicans, Candida tropicalis, Aspergillus fumigatus, Cryptococcus neoformans, and various bacteria) were negative. In clinical samples, 89 out of 281 (31.7%) were positive for C. parapsilosis by RPA-LFS, which was consistent with qPCR and traditional culture results. The assay was not affected by the presence of high concentrations (5.0×10^7 copies/µl) of C. albicans DNA.
**Clinical Implications:** The RPA-LFS assay provides a rapid (within 40 minutes), specific, and sensitive method for detecting C. parapsilosis without the need for expensive thermal cyclers or highly trained personnel. It can be performed at the point of care or even at home, enabling early diagnosis and timely treatment of candidiasis. The assay's performance matches that of qPCR and culture, making it a valuable tool for settings with limited laboratory infrastructure. Potential limitations include the risk of aerosol contamination, though this can be mitigated by home testing. Future studies should evaluate the assay's performance on a larger and more diverse set of clinical samples and assess its utility in guiding antifungal therapy.