Analysis of the 5.8S rRNA and internal transcribed spacers regions of the variant Naegleria fowleri Thai strain.

The aim of this study was to analyze the internal transcribed spacers regions (ITS) of the of 5.8S ribosomal RNA (rRNA) gene of four isolants of Naegleria fowleri. Three of four Thai strains were isolated from patients and one from the environment. All four strains were confirmed to be N. fowleri by species-specific PCR using DNA extracted using a QIAamp DNA mini kit. The ITS lengths observed were ITS1, 85 bp; ITS2, 106 bp; and 5.8S, 176 bp. Five discriminating deca-nucleotide primers A1, A15, B10, B12, and B15 were used in this study. Specific prominent bands were observed after PCR with each primer: 600 bp with A1; 615 bp with A15; 1,580 bp with B10; 930 and 510 bp with B12; and 310 bp with B15. All sequences were compared with the Japanese J16-1-42E sequence in the Genbank database. After alignment, our sequences contained only 0.5% variation from the J16-1-42 sequence. The ITS main products of the strain from the environment were similar to those of the three strains from Thai patients. The four Thai strains have essentially the same 5.8S rRNA genes as Cattenom Japanese J16(1) 42E strain.

Distribution of pathogenic Naegleria spp in Thailand.

Research concerning the distribution, isolation, viability, ultrastructure, morphology and immunogenicity of Naegleria fowleri has been increasing in Thailand during 1988-2000. The distribution of the organism was carried out from 1985 to 1987 in Si Sa Ket and Ubon Rachathani Provinces, after the first fatal case was reported in Si Sa Ket. Since then in a 1998 survey of N. fowleri in stagnant water around industrial areas was carried out in Pathum Thani, Samut Prakan and Lopburi provinces. The results showed that 10% of pathogenic Naegleria belonged to species fowleri as characterized by morphology and the occurrence of pathogenesis in mice after nasal inoculation. In the same year, Nacapunchai et al (1999) determined the prevalence of amebae in aquatic habitat of human environments in five parts of Thailand during the summer. Fourteen percent of free living Naegleria spp were found in both soil and water resources. Recent studies of the ultrastructure, factors affecting the viability and SDS-PAGE electrophoretic patterns of 3 Thai strains of pathogenic Naegleria spp indicated their similarities in morphological characteristics of pathogenic reference control, Naegleria fowleri CDC VO 3081. Additional study using a genetic approach to species criteria using allozyme electrophoresis had been conducted.