PCR-based diagnosis of Naegleria sp. infection in formalin-fixed and paraffin-embedded brain sections

Abstract

We developed a real-time PCR which allowed the highly sensitive detection of Naegleria fowleri in histological brain tissue sections from experimentally infected mice. This genus-specific small-subunit (18S) rRNA gene-based PCR can complement conventional (immuno-) histology for the diagnosis of primary amoebic meningoencephalitis in paraffin-embedded brain necropsy specimens that had been fixed in formalin buffered with phosphate-buffered saline.

FIG. 1.

Analyses of trophozoite proliferation and inflammatory response during Naegleria fowleri infections in mice. The results of immunofluorescence analyses that included specific staining for trophozoites (green, trophozoites; blue, 4′,6′diamidino-2-phenylindole counterstain for cell nuclei) in the mouse brain at 24 h p.i. (A) and 96 h p.i. (C) are shown. Corresponding adjacent histological sections taken at 24 h p.i. (B) and 96 h p.i. (D) were stained with HE and demonstrate the degree of the neutrophilic inflammatory response. No trophozoites were visible in the brain at 24 h p. i. (A), but large numbers of trophozoites (green) were detectable at 96 h p. i. (C). Intense inflammation (infiltration of neutrophilic inflammatory cells into the brain parenchyma, as indicated by arrowheads) was detectable at 96 h p.i. (D). Between 72 h p.i. (data not shown) and 96 h p.i. (C) the number of neutrophils was greatly increased, simultaneously including an increase in trophozoite number between 72 h p.i. (data not shown) and 96 h p.i. (C). The insets show typical examples of immunohistologically stained (C) and HE-stained (D) trophic amoebae at higher magnifications. No morphological structures corresponding to encysted amoebae were found.

FIG. 2.

Analysis of diagnostic applicability of the real-time Naegleria spp. PCR for histological specimens. The figure shows the mean logarithmic values (plus standard deviations) from triplicate determinations representing amplification reactions from formalin-fixed, paraffin-embedded histological mouse brain tissue sampled at 0 (experimental time point prior to infection), 24, 48, 72, and 96 h p.i. with Naegleria fowleri. Note that all samples that scored immunohistologically N. fowleri-positive also provided a specific PCR amplification signal.