Development of a novel loop-mediated isothermal amplification (LAMP) assay for Balamuthia mandrillaris

Abstract

Balamuthia mandrillaris is a free-living amoeba that can cause cutaneous skin lesions, systemic disease, and a severe life-threatening CNS infection, Balamuthia amoebic encephalitis (BAE), which has a high mortality rate (~92%). The current diagnostic methods for B. mandrillaris have many limitations including the long detection time, low sensitivity, and specific diagnostic tests only available in highly specialized centers. The early stage of diagnosis is important for the treatment because of how quickly BAE infection then develops. Therefore, we developed, optimized, and evaluated a novel loop-mediated isothermal amplification (LAMP) assay for the 18S rRNA gene of B. mandrillaris, which can be observed by naked eyes with phenol red. This novel diagnostic assay can detect ten different B. mandrillaris isolates with no cross-reactivity with the DNA of other free-living amoeba, protozoa, fungi, or bacteria tested. Compared with conventional PCR, our LAMP assay displayed ~10-100-fold higher sensitivity from extracted DNA, heat-treated samples, and non-treated samples, with the limit of detection (LOD) of 10 fg/L DNA, 10 trophozoite/100 L for non-treated cells, and 1 trophozoite/100 L for heat-treated cells, respectively. This method was further validated using two newly developed qPCR and dPCR methods to determine the LOD of approximately 1 copy/L. In addition, there is no tolerance for complicated matrices, that LAMP maintained efficacy in cerebrospinal fluid (CSF), urine, and blood-spiked samples. Due to the simplicity, robustness, and high sensitivity of the developed LAMP method, it will be useful for quick clinical detection and diagnosis of B. mandrillaris, particularly in non-specialized centers and resource-limited areas. Here we report the first LAMP diagnostic method for B. mandrillaris; publicly disclosed on Tuesday, November 4th, 2025 at the 20th (XX) International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM 2025) at Puerto Morelos, Mexico.