Development of Colorimetric and Fluorometric Loop-Mediated Isothermal Amplification (LAMP) Assays for the Point-of-Care Molecular Diagnosis of Blastocystis spp

Blastocystis species (spp.) is a genetically diverse protozoan parasite found in the digestive tract of animals and humans. Current clinical diagnosis relies heavily on wet mount microscopy, a method with known limitations, as well as molecular techniques like PCR, nested-PCR, and RT-PCR. However, neither microscopy nor PCR-based approaches are a perfect standard method due to their limitations, such as low accuracy, high equipment cost, and the need for well-trained personnel. Loop-mediated isothermal amplification (LAMP) technique offers the advantage of eliminating these limitations. Therefore, this study aimed to develop rapid and accurate fluorometric LAMP (fLAMP) and colorimetric LAMP (cLAMP) assays. We collected a total of 197 stool samples from both asymptomatic individuals and patients with gastrointestinal conditions. The performance of the LAMP assays was evaluated and compared against a standard RT-PCR assay. Results showed that both LAMP assays provided rapid detection of Blastocystis spp. in 20.0 to 29.2 minutes. The cLAMP assay demonstrated a sensitivity of 95.77%, a specificity of 85.00%, and an accuracy of 90.97%. The fLAMP assay exhibited a sensitivity of 91.43%, a specificity of 93.26%, and an accuracy of 92.27%. The limit-of-detection (LoD) of the fLAMP and cLAMP assays was 5×10 ⁻⁶ ng/µL, which corresponds to 5 fg/µL. The cLAMP assay offers a distinct advantage with its simplified visual result interpretation via a naked-eye color change, eliminating the need for complex instruments. This makes the cLAMP assay suitable for point-of-care testing (PoCT). These characteristics, combined with the femtogram-level detection capability of both assays, make the assays suitable for testing samples containing low DNA copy numbers. This is the first study to develop and elucidate the performance of two LAMP-based assays for the diagnosis of Blastocystis spp. The cLAMP assay exhibits potential to be used for PoCT, particularly in resource-limited settings. Their speed, accuracy, and analytical sensitivity hold promise for improving the diagnosis of Blastocystis spp. infections in patients with gastrointestinal symptoms.