A survey was carried out from August to December 2004 in Pusan, Korea to document the presence of free-living amoeba (FLA), including the genus Acanthamoeba, in both contact lens storage cases and domestic tap water. Acanthamoeba was isolated from 5 (4.2%) in 120 contact lens storage cases. Four house tap water samples from residents, whose contact lens storage cases had been contaminated by Acanthamoeba, were also found to be contaminated with Acanthamoeba. Therefore, the contamination rate of FLA and Acanthamoeba in domestic tap water was investigated in order to examine the role of domestic tap water in Acanthamoeba contamination of contact lens storage cases. FLA and Acanthamoeba were identified in 97 (46.8%) and 16 (7.7%) of the 207 domestic tap water samples, respectively. There were no significant differences between the contamination rates of FLA in tap water according to the filtration plant of origin. No FLA was detected in the tap water directly supplied by the water purification plants. Water storage tanks appear to promote FLA colonization, including Acanthamoeba, in domestic tap water. This increases the risk of Acanthamoeba contamination in contact lens storage cases as well as increasing the risk of Acanthamoeba keratitis.
Acanthamoeba spp. are amphizoic protozoan parasites that are found in the environment as well as in tap water, contact lens cases, soil, dust, and air (Mergeryan, 1991; Armstrong, 2000). These amoebae are the causative agents of amoebic keratitis (AK), pneumonitis, and granulomatous amoebic encephalitis in immunocompromised hosts (Sell et al., 1997; Niederkorn et al., 1999; Rivera and Padya, 2002). AK was firstly reported by Nagington et al. (1974) in Great Britain and by Jones et al. (1975) in the United States. An AK epidemic occurred in the mid-1980s, which was attributed to the increased use of contact lenses and poor lens hygiene (Seal and Hay, 1993, 1994). Recent studies have shown that the rate of Acanthamoeba contamination in contact lens storage cases in Korea was 10.6-15.7% (Lee et al., 1997; Yu et al., 2001; Kong et al., 2002), which is a much higher rate than that reported in the west. Poor hygiene practices, such as the rinsing and storing of lenses in non-sterile saline or tap water are the main risk factors for lens case contamination (Stehr-Green et al., 1987; Seal et al., 1992). Prior to this report, there was no study on the source of Acanthamoeba contamination in contact lens storage cases in Korea. This study investigated the prevalence of Acanthamoeba in contact lens storage cases and domestic tap water in the Pusan province in an attempt to explain the source of Acanthamoeba contamination in contact lens storage cases in Korea.
MATERIALS AND METHODS
Acanthamoeba isolation from contact lens storage cases
Acanthamoeba was isolated from 120 contact lens storage cases from students residing in Pusan according to the method described by Lee et al. (1997).
Tap water sample collection
A total of 207 domestic tap water samples were collected from the resident student houses in the Pusan province from August to December 2004. Six water samples were colleted from 3 water purification plants (Duksan, Hwamyoung, Myoungjang), which supplied tap water to the survey area, at each filtration step.
Acanthamoeba and FLA isolation
One liter of each water sample was filtered through a 5.0 µm pore size cellulose filter (Milipore, Bedford, Madison, USA) under a weak vacuum. The filters were inverted on heat inactivated E. coli treated 1.5% non-nutrient agar plates and incubated at 25℃. After 3-4 days, the plates were monitored microscopically for Acanthamoeba and FLA outgrowth. The genus Acanthamoeba was identified from the FLA based on its distinctive features of trophozoites and cysts, particularly the double-walled cyst shape.
Acanthamoeba in contact lens cases in the Pusan area
In this study, Acanthamoeba was isolated from 5 (4.2%) in 120 contact lens storage cases, and four house tap water samples from residents, whose contact lens storage cases had been contaminated with Acanthamoeba, were also polluted with this organism.
Acanthamoeba and FLA in tap water in the Pusan area
The FLA were isolated from 97 (46.9%) of the 207 domestic tap water samples, of which 12 (5.8%) contained Acanthamoeba, in the Pusan area (Table 1). The Acanthamoeba and FLA contamination rates at each water collection site were similar in this study (Table 1). However, they were more frequently isolated in community dwelling house types than in independent ones (Table 2).
Acanthamoeba and FLA at filtration steps in the purification plants
The level of FLA contamination was examined at each filtration step in the purification plants using a three step system, which supplies domestic tap water to the survey area. Most FLA, including Acanthamoeba, were completely eliminated by the carbon filtration steps (Table 3). No FLA was detected in the water before it was supplied to houses.
AK is an infection that is associated with the wearing of contact lenses. There have been significant improvements in contact lens care systems. This study investigated the possible role of domestic tap water as a reservoir for Acanthamoeba contamination in contact lens storage cases.
In this study, Acanthamoeba was isolated from 5 (4.2%) in 120 contact lens storage cases. The contamination rates were lower than those reported in other provinces in Korea (Lee et al., 1997; Yu et al., 2001; Kong et al., 2002), but were similar to those reported in other countries (Larkin et al., 1990; Watanabe et al., 1994).
The contamination rate of FLA, including Acanthamoeba, in domestic tap water was investigated in order to determine the role of domestic tap water in Acanthamoeba contamination in contact lens storage cases. The FLA were isolated from 46.9% of the domestic tap water samples, of which 5.8% contained Acanthamoeba, in the Pusan area. These results demonstrate that domestic tap water is a significant source of these organisms. These results show a much lower Acanthamoeba contamination rate than in other countries. Kilvington et al. (2004) reported that FLA were cultured from one or more taps from 24 (89%) in 27 households of AK patients in UK. Of these, 8 (26.9%) contained Acanthamoeba. In Spain, Lorenzo-Morales et al. (2005) reported that Acanthamoeba contamination in 88 out of 148 (59.5%) tap water samples. The difference in the prevalence of Acanthamoeba contamination in tap water in different countries might be due to the difference in the tap water hygiene in each country. The Acanthamoeba and FLA were more frequently isolated in community dwelling house types than in independent ones. Community dwelling type houses and most independent ones in this survey area have water storage tanks, which are not often tightly covered. Therefore, environmental organisms can easily contaminate them.
Most FLA, including Acanthamoeba, was completely eliminated by the water purification steps in water purification plants. This shows that FLA contamination, including that from Acanthamoeba, in domestic tap water does not originate from the purification plant, but from the water storage tanks.
Previous studies have shown that many Acanthamoeba isolated from tap water and seawater sources might have some pathogenic ability (Kilvington et al., 2004; Lorenzo-Morales et al., 2005). In addition, some Acanthamoeba isolated in this study have similar molecular characteristics to those of the clinical isolates (data not shown).
In conclusion, domestic tap water, especially when supplied from roof storage tanks, is a source of Acanthamoeba contamination. Contact lens wearers should be aware of the risks associated with Acanthamoeba in tap water supplied from water storage tanks. More education about the hygienic maintenance of water storage tanks is recommended.
1. Armstrong M. The pathogenesis of human Acanthamoeba infection. Infect Dis Rev 2000;2: 65-73.
2. Jones DB, Visvesvara GS, Robinson NM. Acanthamoeba polyphaga keratitis and Acenthamoeba uveitis associated with fatal meningoencephalitis. Trans Ophthalmol Soc UK 1975;95: 221-232. PMID: 1064220.
3. Kilvington S, Gray T, Dart J, Morlet N, Beeching JR, Frazer DG, Matheson M. Acanthamoeba keratitis: The role of domestic tap water contamination in the United Kingdom. Invest Ophthalmol Vis Sci 2004;45: 165-169. PMID: 14691169.
4. Kong HH, Shin JY, Yu HS, Kim J, Hahn TW, Hahn YH, Chung DI. Mitochondrial DNA restriction fragment length polymorphism (RFLP) and 18S small-subunit ribosomal DNA PCR-RFLP analyses of Acanth-amoeba isolated from contact lens storage cases of residents in southwestern Korea. J Clin Microbiol 2002;40: 1199-1206. PMID: 11923331.
5. Larkin DF, Kilvington S, Easty DL. Contamination of contact lens storage cases by Acanthamoeba and bacteria. Br J Ophthalmol 1990;74: 133-135. PMID: 2322508.
6. Lee SM, Choi YJ, Ryu HW, Kong HH, Chung DI. Species identification and molecular characterization of Acanthamoeba isolated from contact lens paraphernalia. Korean J Ophthalmol 1997;11: 39-50. PMID: 9283153.
7. Lorenzo-Morales J, Ortega-Rivas A, Foronda P, Martinez E, Valladares B. Isolation and identification of pathogenic Acanthamoeba strains in Tenerife, Canary Islands, Spain from water sources. Parasitol Res 2005;95: 273-277. PMID: 15678350.
8. Mergeryan H. The prevalence of Acanthamoeba in the human environment. Rev Infect Dis 1991;5: S390-S391. PMID: 2047669.
9. Nagington J, Watson PG, Playfair TJ, McGill J, Jones BR, Steele AD. Amoebic infection of the eye. Lancet 1974;2: 1537-1540. PMID: 4140981.
10. Niederkorn JY, Alizadeh H, Leher H, McCulley JP. The pathogenesis of Acanthamoeba keratitis. Microbes Infect 1999;1: 437-443. PMID: 10602676.
11. Rivera MA, Padhya TA. Acanthamoeba: a rare primary cause of rhinosinusitis. Laryngoscope 2002;112: 1201-1203. PMID: 12169899.
12. Seal D, Stapleton F, Dart J. Possible environmental sources of Acanthamoeba spp in contact lens wearers. Br J Ophthalmol 1992;76: 424-427. PMID: 1627513.
13. Seal DV, Hay J. The microbiologist as a contact lens wearer. J Med Microbiol 1993;39: 1-2. PMID: 8326510.
14. Seal DV, Hay J. Acanthamoeba keratitis. BMJ 1994;309: 1019. PMID: 7848439.
15. Sell JJ, Rupp FW, Orrison WW Jr. Granulomatous amebic encephalitis caused by Acanthamoeba. Neuroradiology 1997;39: 434-436. PMID: 9225325.
16. Stehr-Green JK, Bailey TM, Brandt FH, Carr JH, Bond WW, Visvesvara GS. Acanthamoeba keratitis in soft contact lens wearers: a case-control study. JAMA 1987;258: 57-60. PMID: 3586292.
17. Watanabe R, Ishibashi Y, Hommura S, Ishii K. Acanthamoeba isolation from contact lens solution of contact lens wearers without keratitis. Nippon Ganka Gakkai Zasshi 1994;98: 477-480. PMID: 8197918.
18. Yu HS, Choi KH, Kim HK, Kong HH, Chung DI. Genetic analyses of Acanthamoeba isolates from contact lens storage cases of students in Seoul, Korea. Korean J Parasitol 2001;39: 161-170. PMID: 11441503.