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.

The aim of this study is to evaluate cellular interaction between free-living amoebae Naegleria fowleri strains and mammalian target cells in vitro. Two Thai strains of N. fowleri; Khon Kaen strain from the environment and Siriraj strain from the patient's cerebrospinal fluid and the Center of Disease Control VO 3081 strain from Atlanta (US) were studied. Human neuroblastoma (SK-N-MC) and African Green monkey Kidney (Vero) cells were used as target cells. Each cell line was inoculated with each strain of N. fowleri at a ratio of 1:1 and observed for 7 days. The uninoculated target cells and each strain of N. fowleri were used as control. The numbers of the challenged and unchallenged cells as well as the free-living amoebae were counted three times by trypan blue exclusion method. The inoculation began when the amoebae attached to the cell membrane and ingested the target cells. In this study, extensive cytopathogenesis with many floating inoculated cells and abundant number of amoebae were observed. The destruction pattern of both inoculated SK-N-MC and Vero target cells were similar. Interestingly, SK-N-MC was more susceptible to N. fowleri strains than the Vero cell. In addition, N. fowleri Siriraj strain showed the highest destruction pattern for each target cell. Our findings suggest that the SK-N-MC should be used as a base model for studying the neuropathogenesis in primary amoebic meningoencephalitis patients.

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In order to understand the pathogenesis of Naegleria fowleri in primary amoebic meningoencephalitis, the human neuroblastoma (SK-N-MC) and African green monkey kidney (Vero) cells were studied in vitro. Amoeba suspension in cell-culture medium was added to the confluent monolayer of SK-N-MC and Vero cells. The cytopathic activity of N. fowleri trophozoites in co-culture system was elucidated by scanning electron microscope at 3, 6, 9, 12, and 24 h. Two strains of N. fowleri displayed well-organized vigorous pseudopods in Nelson's medium at 37 degrees C. In co-culture, the target monolayer cells were damaged by two mechanisms, phagocytosis by vigorous pseudopods and engulfment by sucker-like apparatus. N. fowleri trophozoites produced amoebostomes only in co-culture with SK-N-MC cells. In contrast, we could not find such apparatus in the co-culture with Vero cells. The complete destruction time (100%) at 1:1 amoeba/cells ratio of SK-N-MC cells (1 day) was shorter than the Vero cells (12 days). In conclusion, SK-N-MC cells were confirmed to be a target model for studying neuropathogenesis of primary amoebic meningoencephalitis.