Use of Multiplex PCR and PCR Restriction Enzyme Analysis for Detection and Exploration of the Variability in the Free-Living AmoebaNaegleriain the Environment

Abstract

A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites.The free-living amoebae belonging to the genus Naegleria occur worldwide and inhabit soil and aquatic environments. One member of this genus, Naegleria fowleri, causes primary amoebic meningoencephalitis (PAM) in humans. Although PAM is rare (more than 190 cases reported worldwide [15]), this central nervous system disease is lethal within 1 week in most cases (3). The majority of the fatal infections due to N. fowleri occur in young people exposed to warm water in ponds, swimming pools, and lakes. N. fowleri is thermophilic and generally found in natural and artificially heated water, in particular in the cooling ponds of power plants, in which this species can proliferate intensively (4,11,12,27,28). Two other thermophilic Naegleria species are currently found in these sites: Naegleria lovaniensis, which is harmless, and Naegleria australiensis, which is pathogenic in mice. The thermophilic species Naegleria italica was also reported to be pathogenic in mice but is rarely encountered (7). These two species could be potentially dangerous for humans.In a preventive measure, water monitoring was performed regularly in the nuclear power plants of France in order to check for the proliferation of N. fowleri. The identification of Naegleria species has been carried out by an isoenzymatic procedure which allowed simultaneous detection of the three main thermophilic Naegleria species. Other immunological and molecular techniques are now available to specifically detect N. fowleri in environmental sites (14,25,26). Recently, ribosomal internal transcribed spacers (ITS) were reported to be useful markers for Naegleria (10,20), and species-specific primers were defined for N. fowleri (20). In addition, variations in the sequence and size of the ITS were found within this humanpathogenic species, with five different variants which were mostly detected in France. However, the geographic dispersal and the prevalence of these variants were not well established, since too few samples were examined at different sites.In this study, we applied a simplified ITS PCR procedure to the environmental Naegleria isolates for several reasons: (i) to rapidly and easily analyze a large number of isolates, (ii) to detect the presence of N. fowleri in the potential sites and to further explore the genetic diversity of this species, and (iii) to identify the other thermophilic Naegleria isolates at the sites by using PCR restriction fragment length polymorphism (RFLP) analysis.Amoeba isolation from the environment. Five hundred environmental strains were isolated from water of the cooling ponds and downstream of five different nuclear power plants ...

Human infections caused by free-living amoebae

“…Nested-PCR and PCR can identify the presence of amoeba in microbiological cultures. A PCR assay using primers for the complete ribosomal internal transcribed spacer region (ITS) has been developed that allows for the discrimination of Naegleria species, and a species specific assay allows for the detection of N. fowleri [16,17,18,19].…”

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

“…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.…”

Amoeba-Related Health Risk in Drinking Water Systems: Could Monitoring of Amoebae Be a Complementary Approach to Current Quality Control Strategies?

“…Even if several studies have used qPCR to detect different amoebae species [60][61][62][63], the current knowledge suggest that it will be necessary to use different sets of primers (and probes) to detect all the different species. Recently, the use of qPCR and PMA has been positively evaluated by our group in A. castellani [64].…”