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.

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Siamese fighting fish (Betta splendens) are freshwater fish that are commonly found in Thailand and other Southeast Asian countries. In the present study, chrysin-loaded polymeric micelles (CPs) were developed and investigated for the masculinizing effects, survival rate, growth indices, and toxicity on Siamese fighting fish. CPs were prepared using a poloxamer. The micelle system of CPs that were formed at a chrysin-to-polymer ratio of 1:2 was found to be the most suitable monodispersed system and exhibited a nanosized diameter (74.2 ± 1.6 nm) with a narrow size distribution (0.288 ± 0.012). In vivo studies were performed using Siamese fighting fish larvae as animal models. In the in vivo toxicity study, the fish larvae were immersed in aqueous systems containing CPs that had five different chrysin concentrations of 1, 10, 100, 1000, and 10,000 ng/mL for 24, 48, and 72 h. Blank polymeric micelles and water were used as controls. The in vivo masculinization effect of CPs with different chrysin concentrations on the fish larvae was evaluated after 5 weeks of exposure. The results demonstrated that CPs with a chrysin concentration of 1000 ng/mL showed a masculinization effect of 94.59 ± 2.76% with a high fish larvae survival rate of 72.45 ± 5.09% and low toxicity. It was concluded that the developed CPs had a significant effect on the sex reversal of Siamese fighting fish larvae with a high survival rate.