Development of a nested PCR assay to detect the pathogenic free-living amoeba Naegleria fowleri

Abstract

Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis, a fatal disease of the central nervous system that is acquired while swimming or diving in freshwater. A cDNA clone designated Mp2C15 obtained from N. fowleri was used as a probe to distinguish N. fowleri from other free-living amoebae. The Mp2C15 probe hybridized to genomic DNA from pathogenic N. fowleri and antigenically related non-pathogenic N. lovaniensis. Mp2C15 was digested with the restriction enzyme XbaI, resulting in two fragments, Mp2C15.G and Mp2C15.P. Four species of Naegleria and four species of Acanthamoeba were examined for reactivity with Mp2C15.P. Mp2C15.P was specific for N. fowleri and was used in the development of a nested PCR assay which is capable of detecting as little as 5 pg of N. fowleri DNA or five intact N. fowleri amoebae. In summary, a rapid, sensitive, and specific assay for the detection of N. fowleri was developed.

Naegleria fowleriin Well Water

“…To test for viable amebas, we spread aliquots on nonnutrient agar seeded with E. coli at 37°C (3,8). We placed scrapings from the advancing front of subsequent ameba plaques in distilled water to identify enfl agellation (5); however, precise species identification was not possible.…”

“…Live amebae were therefore harvested for PCR analysis to specifi cally identify N. fowleri. We chose PCR over the mouse pathogenicity test because other Naegleria species that are nonpathogenic in humans are lethal in mice (8). The genotype of isolates was not determined because all of the described genotypes found in the United States have been shown to be pathogenic in humans (9).…”

“…Thereafter, serum testing became recommended in Buenos Aires for acute febrile illness, among other dengue surveillance interventions in the area. More recently, epidemiologic surveillance of febrile illness has been strengthened countrywide upon the recent reporting of yellow fever cases in Argentina (8).…”

Multiplex Real-Time PCR Assay for Simultaneous Detection of Acanthamoeba spp., Balamuthia mandrillaris , and Naegleria fowleri

“…Various conventional PCR (which uses gel electrophoresis to determine test results) protocols have been developed for the specific detection of Acanthamoeba (16,17,31,41), N. fowleri (14,24,28,29,35), and B. mandrillaris (3). These protocols have been applied to the detection of Acanthamoeba in corneal scrapings from keratitis cases (18,22,27,34), for confirmatory laboratory diagnosis of N. fowleri PAM (7,13), and to detect B. mandrillaris in clinical specimens (42).…”

Biology and pathogenesis of Naegleria fowleri

“…An indirect immunofluorescence assay (IIF) for the recognition of N. fowleri antigen in paraffin-embedded brain tissue slide is routinely performed at Centers for Disease Control. Additionally, PCR-based assays have been established for the sensitive, rapid, and precise identification of N. fowleri in clinical samples, and cultured amoebae from patients and the environment (14,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) (Table 1).…”