Differentiation of Naegleria fowleri and other naegleriae by polymerase chain reaction and hybridization methods

Abstract

In order to detect and identify Naegleria fowleri strains an assay based on the Polymerase Chain Reaction (PCR) was evaluated. The amplified DNA fragments were detected by gel electrophoresis and ethidium bromide staining, followed by Southern blot hybridization with an internal digoxigenin-labeled probe. A set of primers (B1B2) which flank a 678-bp region within a virulence-associated gene, allowed for the highly specific identification of N. fowleri, since Naegleriae (N. lovaniensis, N. australiensis, N. gruberi, N. andersoni and N. jadini) and other Protozoa did not react. These primers did not detect amplification products from various organisms: Gram-positive bacteria, Gram-negative bacteria, algae, yeasts and human DNA. Whereas a second set of primers (A1A2), which flank a different sequence, detected various Naegleriae and Acanthamoebae strains. After 40 amplification cycles, the limit of detection was a single cell (cyst or trophozoite). Thus, the PCR appears to be a rapid and powerful tool for identification and detection of N. fowleri.

Rapid, Sensitive, and Discriminating Identification ofNaegleriaspp. by Real-Time PCR and Melting-Curve Analysis

“…In other published sequence-based identification methods for N. fowleri, the PCR targets have been an ATPase 6-subunit homologue (16,27) or genes from clone libraries which have unknown functions but have been shown by hybridization to be N. fowleri specific (12,23).…”

“…Finally, it is desirable that the method provide simultaneous identification of as many Naegleria species as possible for the collection of ecologically useful data. The published PCR methods for Naegleria (16,19,23,26,27) meet these criteria only to a limited extent, as all require end-point analysis using agarose gel electrophoresis and none identifies species other than N. fowleri without additional sequencing.…”

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

“…Brief centrifugation of the CSF at 5,000 x g for 5 min is helpful to concentrate amoebae. In addition to microscopy, immunofluorescence assay (IF) (11)(12)(13), enzyme-linked immunosorbent assay (ELISA) (14), flow cytometry (15), and PCR-based assays have been developed. Assays should be employed on both CSF and nasal exudates.…”

“…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).…”