To detect Naegleria spp, in particular Naegleria fowleri, the causative agent of human primary amoebic meningoencephalitis, a flagellation test (FT) is routinely used followed by a specific ELISA. A positive FT indicates the presence of Naegleria spp although some false negatives are likely to occur since parameters for enflagellation vary greatly. As negative FTs are not routinely screened any further for the presence of N. fowleri, this could result in an underestimation of the presence of this pathogen. Therefore, amoebae were further analysed using ELISA and standard PCR not only after a positive but also after a negative FT. In this study 39 cultures containing amoebae were tested with FT, ELISA and the two PCR assays with 11 positive for FT. These were submitted to ELISA and four confirmed as N. fowleri. PCR with the common primer-set on these 11 positive FTs revealed all as Naegleria spp. The specific PCR used on these cultures detected four positive for N. fowleri, corresponding totally with the ELISA results. The 28 negative flagellation tests were also submitted to ELISA and PCR. Of these, 11 were identified as Naegleria spp with common PCR and six as N. fowleri as well as with ELISA and the specific PCR. When the detection of Naegleria spp is based on intermediary processes, such as flagellation tests, false negatives are likely to occur leading to severe underestimations. This study has shown that amoebae taken from negative FTs can be identified as Naegleria spp and N. fowleri when using PCR and ELISA. The application of at least one of the specific N. fowleri tests is recommended for routine screening. The heterogeneous distribution of the false negative results between the different power plants suggested the presence of different genotypes.

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