Analytical performance of an assay for ergosterol, a potential biomarker for Naegleria fowleri in cerebrospinal fluid.

Abstract

Introduction: Naegleria fowleri is a pathogenic free-living amoeba that causes primary amoebic meningoencephalitis, a rare and difficult-to-diagnose form of meningitis. By testing patient cerebrospinal fluid (CSF) for ergosterol, a membrane component unique to fungi and amoeba, cases of primary amoebic meningoencephalitis may be distinguished from cases of bacterial and viral meningitis.

Methods: Pooled CSF was fortified with ergosterol, extracted, filtered, and measured by liquid chromatography-tandem mass spectrometry. Measurement method precision, sensitivity, recovery, and stability were evaluated. In vitro cell cultures of N fowleri and a sample (n = 200) of deidentified residual patient CSF specimens were also tested for ergosterol.

Results: Ergosterol remained stable in CSF at 4 °C and -80 °C. The mean variation and total error of the method were 7.4% and 15.8%, respectively. The method limit of quantification (LOQ) was 1.0 ng/mL, which translated to an in vitro LOQ of 2650 cells/mL or higher. A trace of ergosterol, below the LOQ, was detected in a patient specimen positive for cryptococcal meningitis but not in any other CSF specimens.

Discussion: Ergosterol can be reliably detected in spiked human CSF samples and in the supernatant of N fowleri CSF cultures at concentrations of 1.0 to 500 ng/mL. The clinical utility of this method, however, requires further exploration.