SURVEY OF NAEGLERIA FOWLERI AND PATHOGENIC ACANTHAMOEBA SPP. FROM FRESH WATER AROUND SIRIRAJ HOSPITAL FROM THE CHAO PHRAYA RIVER IN BANGKOK, THAILAND

Transcription

1 SURVEY OF NAEGLERIA FOWLERI AND PATHOGENIC ACANTHAMOEBA SPP. FROM FRESH WATER AROUND SIRIRAJ HOSPITAL FROM THE CHAO PHRAYA RIVER IN BANGKOK, THAILAND Supathra Tiewcharoen, Jundee Rabablert, Kosol Roongruangchai and Virach Junnu 1* Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; 2 Department of Biology, Faculty of Science, Silpakorn University, Nakorn Pathom, Thailand

2 Introduction Freeliving amoeba: the genus Naegleria, Acanthamoeba, Balamuthia and Sappinia are now known to cause brain infections in humans. Naegleria fowleri and pathogenic Acanthamoeba spp. are found to be the causative agent of lethal brain infection (Tiewcharoen et al, 2008; Marciano-Cabral, F., and G. Cabral. 2003). N. fowleri and pathogenic Acanthamoeba spp. in the fresh water system can be sources of infection under natural conditions (Jariya et al, 1997; Nacapunchai et al, 2014). We therefore undertook to investigate the distribution of N.fowleri and Acanthamoeba spp. from Chao Phraya River in Bangkok, Thailand.

3 Materials & Methods A total of 105 water samples from 21 sites in Chao Phraya River around Siriraj Hospital (12 Piers), canals near Siriraj Hospital (9 Piers) was collected at temperature ranges of C, ph ranges of 6.5 to 7.5 between January and April The sediments of samples were culture at 37 C and thermotolerance was tested at 42 C, with subcultured amoebae. The FLA were investigated by morphologically characteristics and molecular method. Results Forty of the 105 samples (38.09%), from Wang Lang, Wat Rakhang, Wat Amarinthararam, District office Bangkoknoi, Wat Taking Chan, Taking Chan floating Market, Wat Khruawan, Wat Phoriang yielded Naegleria spp. at 37 C. and sixteen thermotolerant positive samples (19.04%) were continued to grow at 42 C. By polymerase Chain Reaction, ten thermotolerant Naegleria spp. (9.52%) sample from Wang Lang, Wat Rakhang were identified as Naegleria fowleri. Pathogenic Acanthamoeba spp. was identified by Ac6 gene (23.58 %) from 25 water samples; Wang Lang, Wat Taking Chan, Taking Chan floating Market, Wat Khruawan, Wat Phoriang. They were belonged to Acanthamoeba castellanii % (15/105) and Acanthamoeba tubiashi 9.52% (10/105). Conclusion: Our results suggested that fresh water from four piers: Wang Lang, Wat Rakhang, Wat Khruawan, Wat Phoriang might be the cause of this alarming problem since they can transmit infections to the public.

4 Fig 1. llustrates the map locations of the 16 water sampling sitesmap showing the sampling sites of Chao Phraya River around Siriraj Hospital and cannals near Siriraj Hospital: (a) Rama VIII; (b) Phra Pinkloa; (c) Thonburi Railway; (d) Siriraj; (e) Wang Lang; (f) Wat Rakhang; (g) Wat Arun; (h) Wat Kanlaya; (i) Tha Train; (j) Tha Chang; (k) Tha Prachan; (l) Tha Phra Arthit; (m) Wat Amarinthararam; (n) District office Bangkoknoi; (o) Wat Srisudaram; (p) Wat Takingchan; (q) - Takingchan floating Market; (r) Wat Khruawan; (s) Wat Chinorasaram; (t) Wat Phoriang; (u) Wat Bang Sao Thong. Table 1 Number of isolated amoebae for one week at37 C and 42 C No. Location (n) Naegleria spp Acanthamoeba spp. 37 ๐ C 42 ๐ C 37 ๐ C 42 ๐ C 1 Chao Phraya River around Siriraj Hospital (12) 2 Khlong Bangkoknoi (3) Khlong Chack Prak (2) Khlong Mon (4) Total(21) (38.09%) (19.04%) (23.80%) (0%)

5 A B A B C D C D Fig 2. Unstained or stain morphology of Naegleria a stage under light microscope. ( a), x400 unstained Naegleria trophozoites showing active progressive and directional movement ; (b), x400 cyst showing rounded form with double wall cyst; (c), x1,000 Trichrome s stained trophozoites showing typical semispherical eruptive pseudopodia/ lobopodia and vesicular nucleus ; (d), x1,000 cyst showing prominent vesicular nucleus. Fig 3. Unstained or stain morphology of Acanthamoeba a stage under light microscope.(a), x400 unstained Acanthamoeba trophozoites showing acanthopodia and nucleus; (b), x400 cysts with multiple shapes, round, triangular and square protrusions; (c), stained trophozoites acanthopodia x1,000 Trichrome s and prominent contractile vacuoles; (d), x1,000 cyst with multiple shapes, round, triangular and square protrusion.

6 M Fig 4a. Gel electrophoresis of amplicon from Naegleria spp. PCR conditions showed specific band ITS 450 bp. Lane 1 N. fowleri, positive control showing specific band, Lane2-9 eight major thermophilic Naegleria spp. isolated from Wang Lang, Wat Rakhang, Wat Amarinthararam, District office Bangkoknoi, Wat Taking Chan, Taking Chan floating Market, Wat Khruawan, Wat Phoriangpoativie showing positive band; Lane 10 Pathogenic Acanthamoeba spp. showing negative band. Fig 4b. Gel electrophoresis of amplicon from N. fowleri. PCR conditions showed pathogenic specific band nfa1 360 bp. Lane 1 N. fowleri, positive control showing specific band, Lane2 and Lane 9 N. fowleri isolated from Wang Lang, Wat Rakhang showing positive band; Lane 10 Pathogenic Acanthamoeba spp. showing negative band.

7 Fig 5a. Gel electrophoresis of amplicon from Acanthamoeba spp. PCR conditions showed JDP band at 450 bp. Lane 1 pathogenic Acanthamoeba spp., positive control showing specific band, Lane2-6 five positive samples isolated from Wang Lang, Wat Taking Chan, Taking Chan floating Market, Wat Khruawan, Wat Phoriang, Lane7 N. fowleri, showing negative control. Fig 5b. Gel electrophoresis of amplicon from Acanthamoeba spp. PCR conditions showed Ac6 band at 450 bp. Lane 1pathogenic Acanthamoeba spp., positive control showing specific band 195bp, Lane2 N. fowleri, negative control, 3-4 A.tubishi isolated from Wat Taking Chan, Taking Chan floating Market showing positive band ,460bp, Lane 5-7 A. castellanii Wang Lang, Wat Khruawan, Wat Phoriang showing positive band 195 bp.

8 Conclusion Our results suggested that fresh water from the Wang Lang Pier, Wat Rakhang Pier, Wat Khruawan Pier, and Wat Phoriang Pier may be areas where these pathogens are transmitted to the general public. Public health notices should be developed and advertised to people that frequent and swim in these areas in order to prevent or reduce the number of infections caused by these parasites. Acknowledgements This study was partially supported by the Siriraj Grant for Research Development and Medical Education No. R , Faculty of Medicine Siriraj Hospital, Mahidol University. This study was partially supported by Grant No.SRF-JRG , Department of Biology, Faculty of Science, Silpakorn University. REFERENCES Jariya P, Tiewcharoen S, Junnu V, Lertlaituan P, Suvithayasiri V. Survey of Naegleria fowleri the causative agent of primary amoebic meningoencephalitis (PAM) in stagnant waters around factory area in Thailand. Siriraj Hosp Gaz 1997;49: Marciano-Cabral F and Cabral G. Acanthamoeba spp. as agents of disease in humans. Clin. Microbiol. Rev. 2003; 16: Nacapunchai D, Kino H, Ruangsitticha C, Sriwichai P, Ishih A, Terada M. A brief survey of free-living amebae in Thailand and Hamamatsu District, Japan. Southeast Asian J Trop Med Public Health 2001;32 (suppl 2): Tiewcharoen, S. and Junnu, V. Distribution of pathogenic Naegleria spp in Thailand. Southeast Asian J Trop ,