Isolation and Environmental Detection of <scp> <i>Balamuthia mandrillaris</i> </scp> in Isfahan, Iran

Balamuthia mandrillaris is a free-living opportunistic amoeba known to cause fatal encephalitis. Despite its global environmental presence, data on its distribution in Iran and comparative detection methods remain scarce. This study aimed to investigate the occurrence of B. mandrillaris in various environmental sources in Isfahan, Iran, and to evaluate the performance of culture-based and molecular detection approaches. A total of 96 environmental samples (33 soil, 31 dust, and 32 water) were collected from different locations across Isfahan. All samples were examined in parallel using culture methods and PCR amplification targeting the mitochondrial 16S rDNA gene. Selected PCR-positive samples underwent sequencing to confirm species identity. The overall detection rate of B. mandrillaris was 15.6% using both culture and molecular techniques, with soil samples showing the highest prevalence (24.2% by molecular and 21.2% by culture). Molecular and cultural methods displayed complementary but matrix-dependent detection patterns, with a substantial agreement between methods (κ = 0.72). This study confirms the presence of B. mandrillaris in various environmental sources in Isfahan, highlighting soil as a potential reservoir. The findings emphasise the importance of combined molecular and culture-based approaches and suggest raising public health awareness, even in regions without reported clinical cases.