Drug susceptibility of a clinical isolate of <i>Balamuthia mandrillaris</i> , a pathogenic free-living amoeba

<i>Balamuthia amoebic</i> encephalitis (BAE) is a highly fatal infection caused by <i>Balamuthia mandrillaris</i>, an amoeba that lives in soil and water. In Thailand, three fatal cases of BAE have been documented, but no survivors have been reported, raising questions about current treatment regimens. Previous drug repurposing studies reveal some potent pharmacological compounds, but the drug susceptibility of the clinical isolate of pathogenic amoeba remains variable. Given the success in isolating <i>B. mandrillaris</i> from the human biopsied brain, this study aims to assess the amoebicidal effect of several previously repurposed drugs and suggested therapies for BAE. The trophozoites of a new clinical isolate, the KM-20 strain, were exposed to 12 compounds, including pentamidine, the most widely used antiprotozoal drug, and nitroxoline, the recent radical cure for BAE. The amoebicidal effect was assessed using the ATP level as a cell survival biomarker. The circularity and surface area of the cells were used as recrudescence indicators. Among all drugs tested, nitroxoline is the most potent amoebicidal drug without recrudescence. Topical antiseptic agents caused amoeba lysis at all doses tested, suggesting potential use for cutaneous balamuthiasis. Compared with two laboratory-adapted V039 and PRA-291 strains, the KM-20 isolate had reduced drug susceptibility to all of the tested compounds, suggesting strain dependency of amoebicidal activity. This study provides drug susceptibility data against a novel and geographically diverse clinical isolate of <i>B. mandrillaris</i> to assist in prioritizing anti-<i>Balamuthia</i> agents for further drug development testing, followed by <i>in vivo</i> efficacy testing animal models before clinical trials and drug repurposing.