Molecular detection of Balamuthia Mandrillaris in Soil samples from Kathmandu Valley

Abstract

Abstract Balamuthia mandrillaris is an emerging free-living amoeba (FLA) that inhabits in a wide range of environments including soil, water, air, dust, contact lens storage case, distribution systems, drinking water treatment, and cooling towers. This pathogen is responsible for the amoebic encephalitis caused known as Balamuthia amoebic encephalitis (BAE) with over 90% of cases of this pathogen results in death. The biology and environmental distribution of B. mandrillaris are still insufficiently understood, and the environmental isolation of this amoeba is infrequent. The aim of this study is to detect the presence of B. mandrillaris in soil samples taken from various sites within the Kathmandu Valley. This study assesses the detection of the pathogen in a variety of soil environments that influences its presence, employing molecular approach. Twenty-five (25) soil samples were collected for the investigation and stored in sterilized zip-lock plastic bags at 4°C. B. mandrillaris was identified by isolating DNA from the soil samples, and PCR amplification. The positive samples were separated through electrophoresis and validated the result by sequence analysis and phylogeny. A phylogenetic tree was constructed in MEGA 11 software using the maximum likelihood estimation (MLE) and a substitution Jukes-Cantor model to verify the evolutionary relation and genetic association between predicted B. mandrillaris with previously published GenBank sequences. Out of 25 samples, 5 samples were tested positive for Balamuthia specific DNA in which two were collected from distinct river banks, one from park, one from nursery and one from goat farm premises soil. The NCBI BLAST analysis of the sequences confirmed 100% homology with the corresponding previously reported B. mandrillaris sequence in the GenBank database from Japan. The detection of B. mandrillaris from multiple environmental conditions emphasizes the ubiquitous character of this pathogen. Of the 25 samples, B. mandrillaris was found in five (20%) soil samples through molecular method and sequencing which demonstrate the significantly higher incidence of Balamuthia in environmental habitats. The discovery highlights the importance of public health activities aimed at raising awareness regarding the risks associated with B. mandrillaris.