Multiplex real-time PCR assay for simultaneous detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri

Abstract

Infections caused by Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris occur throughout the world and pose many diagnostic challenges. To date, at least 440 cases of severe central nervous system infections caused by these amebas have been documented worldwide. Rapid and specific identification of these free-living amebas in clinical samples is of crucial importance for efficient case management. We have developed a triplex real-time TaqMan PCR assay that can simultaneously identify Acanthamoeba spp., B. mandrillaris, and N. fowleri in the same PCR vessel. The assay was validated with 22 well-characterized amebic strains harvested from cultures and nine clinical specimens that were previously characterized by in vitro culture and/or immunofluorescence assay. The triplex assay demonstrated high specificity and a rapid test completion time of less than 5 h from the reception of the specimen in the laboratory. This assay was able to detect one single ameba per sample analyzed, as determined with cerebrospinal fluid spiked with diluted cultured amebas. This assay could become useful for fast laboratory diagnostic assessment of amebic infections (caused by free-living amebas) in laboratories with adequate infrastructure to perform real-time PCR testing.

FIG. 1.

Amplification curves from cultured trophozoites, serially diluted in parasite-free CSF. Negative samples were unspiked CSF. Each sample was amplified in parallel in the single-target assay with just one set of primers and probe (dashed lines) and in the triplex assay containing oligonucleotides for detection of all three variants of amebas (solid lines). Only the fluorescence signal from the relevant probe in the triplex reactions is displayed in each figure (the signals from the other two probes remained below threshold for all samples throughout all runs). All experiments were repeated three times; the average results are displayed. dRn, baseline subtracted fluorescence reading normalized to a reference dye.

FIG. 2.

Amplification curves from triplex real-time PCR on brain tissue samples from patient 4 (closed symbols) and 5 (open symbols). Amplification signals from unprocessed tissue are displayed as boxes, those from proteinase-treated tissue as circles, those from DNA extracted from the tissue samples as triangles, those from DNA extracted from cultured N. fowleri (positive control) as multiplication symbols, and those from water (negative control) as a dashed line. Only the fluorescence signal from the N. fowleri-specific probe is displayed (the signals from the other two probes remained below threshold for all samples throughout the run). dRn, baseline subtracted fluorescence reading normalized to a reference dye.