ISSN (Print) 0023-4001 ISSN (Online) 1738-0006

Korean J Parasitol Vol. 57, No. 4: 341-357, August 2019 ▣ MINI-REVIEW https://doi.org/10.3347/kjp.2019.57.4.341

Acanthamoeba in Southeast Asia – Overview and Challenges

Chooseel Bunsuwansakul1, Tooba Mahboob2, Kruawan Hounkong3, Sawanya Laohaprapanon4, Sukhuma Chitapornpan5, Siriuma Jawjit4, Atipat Yasiri6, Sahapat Barusrux1, Kingkan Bunluepuech1, Nongyao Sawangjaroen7, Cristina C. Salibay8, Chalermpon Kaewjai9, Maria de Lourdes Pereira10, 1, Veeranoot Nissapatorn * 1School of Allied Health, Southeast Asia Water Team (SEA Water Team) and World Union for Herbal Drug Discovery (WUHeDD), Walailak University, Nakhon Si Thammarat, Thailand; 2Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 3Department of Microbiology, Princess of Naradhiwas University, Narathiwat, Thailand; 4School of Public Health, Walailak University, Nakhon Si Thammarat, Thailand; 5School of Energy and Environment, University of Phayao, Phayao, Thailand; 6Chulabhorn International College of Medicine, Thammasat University, Pathum Thani, Thailand; 7Department of Microbiology, Prince of Songkla University, Songkhla, Thailand; 8College of Science and Computer Studies, De La Salle University-Dasmarinas, Dasmarinas City, Cavite, Philippines; 9Faculty of Medical Technology, Rangsit University, Pathum Thani, Thailand; 10Department of Medical Sciences & CICECO-Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal

Abstract: Acanthamoeba, one of free-living amoebae (FLA), remains a high risk of direct contact with this protozoan par- asite which is ubiquitous in nature and man-made environment. This pathogenic FLA can cause sight-threatening amoe- bic keratitis (AK) and fatal granulomatous amoebic encephalitis (GAE) though these cases may not commonly be reported in our clinical settings. Acanthamoeba has been detected from different environmental sources namely; soil, water, hot- spring, swimming pool, air-conditioner, or contact lens storage cases. The identification of Acanthamoeba is based on morphological appearance and molecular techniques using PCR and DNA sequencing for clinico-epidemiological pur- poses. Recent treatments have long been ineffective against Acanthamoeba cyst, novel anti-Acanthamoeba agents have therefore been extensively investigated. There are efforts to utilize synthetic chemicals, lead compounds from medicinal plant extracts, and animal products to combat Acanthamoeba infection. Applied nanotechnology, an advanced technolo- gy, has shown to enhance the anti-Acanthamoeba activity in the encapsulated nanoparticles leading to new therapeutic options. This review attempts to provide an overview of the available data and studies on the occurrence of pathogenic Acanthamoeba among the Association of Southeast Asian Nations (ASEAN) members with the aim of identifying some potential contributing factors such as distribution, demographic profile of the patients, possible source of the parasite, mode of transmission and treatment. Further, this review attempts to provide future direction for prevention and control of the Acanthamoeba infection.

Key words: Acanthamoeba, clinico-epidemiology, medicinal plant, molecular, nanotechnology, Southeast Asia

INTRODUCTION mised patients as the main target [1]. Recently, Acanthamoeba spp. are recognized as increasing threat against contact lens Acanthamoeba spp. is one of pathogenic free-living amobae wearers and healthy individuals also take some risks on amoe- (FLA) along with Naegleria fowleri, Balamuthia mandrillaris, and bic keratitis (AK) [2]. Understanding on Acanthamoeba infec- Sappinia sp. which are potential to cause rare infection in cen- tions is therefore crucial but still limited in ASEAN countries tral nervous system. These protozoan parasites are mostly even though studies on anti-Acanthamoeba agent do exist. found in natural soil and water bodies and immunocompro- Herein, an overview of Acanthamoeba was put in a nutshell as well as challenges on recent issues to encounter against this

Received 21 April 2019, revised 18 July 2019, accepted 18 July 2019. amoeba in our regional ASEAN countries including Brunei • Corresponding author ([email protected]) * Darussalam, Cambodia, Indonesia, Lao People’s Democratic © 2019, Korean Society for Parasitology and Tropical Medicine Republic (PDR), Malaysia, Myanmar, the Philippines, Singa- This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) pore, Thailand, and Vietnam. which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

341 342 Korean J Parasitol Vol. 57, No. 4: 341-357, August 2019

ORIGIN OF ACANTHAMOEBA EPIDEMIOLOGY OF ACANTHAMOEBA IN ASEANS

Acanthamoeba spp. is a centrosome-bearing, single-celled, FLA, especially Acanthamoeba spp., occur worldwide and flattened naked amoeba in Order Acanthopodida, Class Cen- have a variety of habitats. Many studies have recorded the wide tramoebia, Phylum Discosea, Amoebozoa clade in Amorphea distribution in soil and water, with differing range of thermal domain of Eukaryotic organisms [3]. Term “Acanth” in Greek tolerance (Table 1). They have been isolated in untreated natu- means spike representing prominent sub-pseudopodia while ral freshwaters, like lakes, ponds, hot springs and waterfalls “amoeba” means alteration like their appearance. The bacteria- [14-17]; and brackish, seawaters, and ocean sediments [18]. phagocytosing protozoa is one of clinical FLA ubiquitous in They were also isolated from treated waters like domestic wa- nature soil and water bodies as well as man-made environ- ter systems, swimming pools, hydrotherapy pools, remedial ment as a secondary decomposer. Ubiquity is implied by pres- spas, tap water and drinking water [14,16,19,20]. Unconven- ence of antibodies in healthy individuals [4]. Acanthamoeba sp. tional water sources like sewage and aquaria were not spared was first recognized as contaminant of Cryptococcus pararoseus with the presence of amoebas [18]. culture by Castellani in 1930 and named as Hartmannella cas- Aside from water, Acanthamoeba spp. were also present in tellanii and then a year later, Acanthamoeba spp. because of its different types of soils such as agricultural, garden and mining double-walled cyst with irregular ectocyst appearance which is [21-23]. Acanthamoeba genotypes of infected cats and dogs different from round and smooth cyst wall of Hartmannella were matched with dry soil and dust. [24]. Acanthamoeba-in- spp. [5]. fected individuals can also be a source of the isolates of organ- ism through sinuses, brain and corneal and skin specimens BRIEF BIOLOGY OF ACANTHAMOEBA [22,25-27] and even in necrotic tissues [18]. The presence of Acanthamoeba spp. has impacted for the last Acanthamoeba spp. appears in 2 forms of life cycle: tropho- decades because of the increasing cases of a rare condition AK, zoite (25-40 µm) and cyst (13-20 µm). Trophozoite is an in- a severe infection of the eye cornea associated with intense fective stage with amoeboid locomotion whilst cyst is a dor- pain. This has been observed in contact lens wearer population mant stage against harsh environment such as temperature [28]. It is believed that the cause of infection is due to the expo- and pH imbalance, malnutrition, or presence of anti-Acan- sure of the eye to the Acanthamoeba-contaminated contact lens thamoeba agents [6]. One third of strength of cyst wall might solutions. Acanthamoeba isolated from contact lens storage cas- come from polymer of glycosidic linkages between saccharides es were confirmed [29]. Further, the usual spread of the con- while another 2/3 are protein and other components, respec- taminant is due to poor hygiene and maintenance of the lens; tively [7]. Furthermore, the protist acts as potential reservoir or and exposure to contaminated water (swimming pool or other vector of human-pathogenic bacteria, fungi, or viruses while recreational waters) while wearing contact lenses. endosymbiont and Acanthamoeba-resistant organisms also are However, the disease has also been reported in non-contact identified [8-10]. Recently, more than 25 species were recorded lens wearers [18,26,27]. This further affirms the possible con- in NCBI taxonomy database and 20 genotypes were published tamination through direct contact to contaminated water and which T4 is a major genotype associated with human infec- soil. The wide dispersal of Acanthamoeba onto the environ- tions [9,11]. For cultivation, xenic culture is obtained by using ment is due to the wind dispersal of its resistant form, the non-nutrient (Page’s amoeba saline) or PYG (peptone 0.05%, cysts. Likely that indoor ventilation system, blowing fan, air yeast extract 0.05%, glucose 0.1%) agar coated with living or diffuser and other furniture contaminated with Acanthamoeba killed bacteria (e.g., Escherichia coli) at 25-28˚C in the dark for can be a cause of spreading indoor [30]. Thus, individuals 2-3 days for trophozoite proliferation and 1-2 weeks for en- who are not contact lens wearers but have been constantly ex- cystment while PYG (peptone 2%, yeast extract 0.5%, glucose posed to dust particles and soil are also at high risk of infec- 0.5%) agar was used for axenic culture [12]. Culture in PYG tion [25]. It is also important to note that exposure to Acan- medium at 4˚C would be convenient method for long-term thamoeba can be as simple as accidental splash of contaminat- preservation at least 1-4 years [13]. ed water to the face or bruised skin [14], making a fast and easy transmission. Bunsuwansakul et al.: Acanthamoeba in Southeast Asia 343 References (Continued to the next page) Nacapunchai et al. (2001) [23] Lekkla et al. (2005) [17] Nuprasert et al. (2010) [59] et al. (2013) [60] Wannasan Buppan et al. (2018) [61] et al. (2009) [62] Wannasan Thammaratana et al. (2016) [15] et al. (2007) [63] Yaicharoen Nacapunchai et al. (2001) [23] et al. (2009) [62] Wannasan Anisah et al. (2003) [64] Init et al. (2010) [32] Onichandran et al. (2013) [16] Gabriel et al. (2019) [65] et al. (2016) [20] Richard Basher et al. (2018) [24] NF ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Group III Group 2.8% (3/105) 2.9% (6/37) 15.5% (16/43) spp. morphology ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Positive Group II Group 16.5% (17/43) 13.7% (28/37) 84.21% (16/19) 73.83% (79/105) Acanthamoeba NF NF ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Group I Group Positive 15.79% (3/19) 23.36% (25/105) spp. NF NF NF NF NF NF 13% (9/69) 35% (105/300) 10% (1/10) 50% (1/2) 50% (2/4) 2.4% (1/42) 100% (2/2) 100% (7/7) 100% (6/6) 100% (4/4) 100% (15/15) 41.7% (43/103) 18.1% (37/64) 24.9% (45/181) 70.2% (40/57) 66.7% (2/3) 18.95% (18/95) 19.05% (16/84) 14.29% (1/7) 16.67% (1/6) 15.87% (10/63) 33.33% (40/120) 16.67% (2/6) 46.19% (388/840) 18.03% (7/11) 73.33% (7/11) Acanthamoeba Positive culture ND ND ND ND ND ND ND ND ND ND FLA 50% (5/10) 50% (3/6) 75% (3/4) 50% (5/10) 75% (3/4) 50% (2/4) 100% (7/7) 100% (2/2) 100% (4/4) 100% (6/6) 100% (2/2) 100% (6/6) 100% (1/1) 66.7% (4/6) 54.4% (457/840) 29.8% (54/181) 66.7% (38/57) 33.3% (1/3) 90.2% (55/61) 51.58% (49/95) 37.68% (28/69) 69.17% (83/120) 7 2 4 6 6 2 4 6 6 7 6 3 4 1 4 95 69 84 10 63 64 10 42 57 61 15 15 spp. in Southeast Asia Southeast in spp. 300 103 120 840 181 No. of sample Acanthamoeba Type of samples Type Water samples Water Hot spring water pond and irrigation canals Freshwater Flood water pond in public parks Freshwater fields Water-logged Ditches Paddy fields Fish farms Large pond Natural water Outdoor air Indoor air Soil swab samples fields Water-logged Ditches Paddy fields Fish farms Large pond Domestic tap water Swimming pools in Kuala Lumpur lake A anthropogenic Recreational lake B anthropogenic Recreational water Tap places Recreational dispenser units Water water Filtered Drain water Paddy fields Drinking water treatment samples Water and stones) Swabs (rocks Water Air Soil Water Distribution of environmental Distribution of environmental Country Thailand Malaysia Table 1. Table 344 Korean J Parasitol Vol. 57, No. 4: 341-357, August 2019 References Chan et al. (2011) [66] Onichandran et al. (2014) [67] Rivera and Adao (2008) [29] Rivera and Adao (2008) [29] Cruz and Rivera (2014) [25] Rivera and Adao (2008) [29] Denet et al. (2017) [21] Majid et al. (2017) [14] ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Group III Group 28.57% (6/21) spp. morphology ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Group II Group 71.43% (15/21) Acanthamoeba NF ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Group I Group spp. NF NF NF NF NF NF NF 23% (20/87) 30% (3/10) 50% (2/4) 50% (1/2) 100% (15/15) 100% (15/15) 100% (20/20) 100% (20/20) 100% (1/1) 100% (3/3) 100% (10/10) 100% (4/4) 100% (4/4) 5.95% 4.76% 4.55% (1/22) 9.68% (3/31) 66.67% (2/3) 33.33% (2/6) 33.33% (1/3) Acanthamoeba Positive culture NF NF ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND FLA 4.55% (1/22) 11.11% (1/9) 18.18% (2/11) 16.13% (5/31) 59 small sub unit rDNA Sequences of Amoebae 3  4 3 6 3 2 2 1 1 1 3 4 4 1 1 9 6 15 15 20 20 87 10 10 22 11 31 15 No. of sample Type of samples Type Wet soil Wet (Dry soil) playgrounds Children Indoors wall surface Outdoor wall surface Air conditioners in KM River Swimming pools Pond Lake water Tap Rain/tap tank dispenser Water Well Spring Mineral Water Soil Soil Contact lens storage cases soil Garden Mining soil Soil Other Water Soil Other Soil water in Lao PDR Treated water in Lao PDR Untreated water in Yangon Treated water in Yangon Untreated water in Singapore Treated water in Singapore Untreated Continued Myanmar, Myanmar, and Country The Philippines Vietnam Others (Lao PDR, Singapore) Table 1. Table ND, Not detected; NF, Not found. ND, Not detected; NF, Bunsuwansakul et al.: Acanthamoeba in Southeast Asia 345

Fig. 1. Epidemiology and clinical cases of Acanthamoeba infection in Southeast Asia. B: Granulomatous amoebic encephalitis; E: Acan- thamoeba keratitis; N: Acanthamoeba sinusitis; and S: Gastric acanthamoebiasis.

Ironically, with the many studies proving the presence of ronment is dry during summer and dust particles can be easily Acanthamoeba in different environmental media (soil, water spread. Likewise, resorts with swimming pools are occupied and air), the dearth of information in Southeast Asian (ASE- the entire summer with local and foreign tourists. Since resorts AN) countries is quite a concern, considering that the varying gain profit only during this time of the year, owners tend to climatic conditions of the region is a favorable habitat for this maximize the use of the swimming pools which may compro- organism which has an unusual geographic distribution [31]. mise the proper cleanup of the swimming facility. This poses The ASEAN countries’ tropical condition, favorite tourist the risk to the swimmers, adding to the fact that Acanthamoeba destinations during summer, consists of beaches, falls, and can also be resistant to disinfectants [26,32]. lakes are among the popular areas where more people involve The detection of Acanthamoeba in soil, water and air in other with these outdoor activities. The congestion can increase risk countries in ASEAN (Fig. 1), confirms that a continual con- of contamination with Acanthamoeba especially when the envi- tamination of the environment persists, and this poses a risk 346 Korean J Parasitol Vol. 57, No. 4: 341-357, August 2019

to people dependent on the soil and water for domestic activi- or aromatic diamidines combination because of resistance of ties, agricultural and farming occupation, and even for recre- cyst form and PHMB is toxic to human corneal cells [40]. ation. The lack of information in some countries (Cambodia For GAE, a very rare condition, is opportunistic and fatal in- and Brunei) does not mean the absence of Acanthamoeba-con- fection with onset of weeks to months mostly in immuno- taminated environment. Albeit, this may result to the inability compromised patients, especially HIV/AIDS patients through of one country to control the spread of possible diseases asso- skin breaks, respiratory tract, and olfactory epithelium. GAE ciated with Acanthamoeba considering that this amoeba may patient will encounter with neurological signs such as confu- also harbor pathogenic bacteria or fungi. sion, headache, and stiff neck as well as psychological change, e.g. irritability generally like other brain infections due to effect CLINICAL SIGNIFICANCE AND DIAGNOSIS of edema, necrosis, and hemorrhages in infected part of brain [43]. To confirm GAE, microscopy and culture from CSF re- Potential pathogenicity of Acanthamoeba was first observed main gold standard methods used after neuroimaging detec- in monkey kidney cell in vitro as well as intracerebral/intraspi- tion of brain lesions while indirect immunofluorescence on nal inoculation in monkeys and intravenous/intranasal inocu- tissue and multiplex real-time PCR assay are available [44]. lation in mice [33,34]. First patient was recognized as GAE in Late/missed diagnosis, blood-brain barrier crossing of antimi- 1972 and a year later, AK [35,36]. Acanthamoeba spp. are there- crobial, drug side effects, drug combination are still an issue fore considered as rare potential pathogen causing cutaneous on GAE treatment and only few patients were cured [45,46]. lesions, sinusitis, AK, GAE, and disseminated form in human There are many reports on Acanthamoeba infection in ASE- and prefer individuals with underlying diseases or immuno- AN countries (Table 2). Most infections are AK with contact compromised host but AK was frequently reported in immu- lens while cases of GAE is rare. Notably, Acanthamoeba can be nocompetent patients especially, contact lens wearers [37]. involved with gastric ulcer and sinusitis and found from nasal For AK, poor sanitation of contact lens wearer is a potential swab from healthy individuals and corneal swab from infected risk and corneal trauma seem required before trophozoite in- animal (Table 2). Undeniably, exposure to soil and contami- fection as well as eye secretion after contact lens wore might be nated water are potential risk but underlying disease might be preferred by Acanthamoeba [38,39]. Onset of AK is days to another one factor for the infections. Misdiagnosis and delay weeks with symptoms of tormenting eye pain, redness, photo- in diagnosis are common among patients leading to perma- phobia, stromal infiltration leading to sight-threatening condi- nent vision blurriness because of injured cornea or deeper lay- tion which are similar and misdiagnosed to Herpes simplex, ers for AK and death for GAE. These problems are still insolv- bacterial or fungal keratitis [39,40]. AK is confirmed by pres- able till date. Rapid and accurate prognosis is therefore an ur- ence of trophozoite with large nucleolus and contractile vacu- gent need for Acanthamoeba infection. oles as well as pseudopodia and transparent protrusions of Ac- anthopodia from corneal scrapings or biopsies under direct mi- CURRENT CHALLENGES AND FUTURE croscopy with several stains. Encystment on non-nutrient agar PERSPECTIVES (NNA) and nucleic acid amplification testing are further inves- tigated for species identification and genotyping, respectively. Contact with Acanthamoeba spp. is common. Immunocom- Taxonomic identification mainly investigated by cyst mor- promised patients should realize this risk and avoid exposure phology under microscope [41] and a hypervariable sequence to, especially, natural soil and water bodies even though it is a part of 18S small subunit rDNA gene called ASA.S1 by Acan- rare disease but GAE is fatal and AK is vision-threatening [6]. thamoeba-specific primers: JDP1 and JDP2 (amplicon size 467 Moreover, no specifically therapeutic course is available for bps for Neff strain of A. castellanii accession number M13435.1) Acanthamoeba spp. infections, in case of GAE. However, com- [42]. Extended or almost complete of 18S rDNA amplicon mercial drugs for AK are highly toxic due to prolonged treat- size provide better solution for genotyping [11,42]. Pathogen ment duration as well as diagnosis and combination of treat- broad-spectrum and most effective anti-Acanthamoeba agents ments depends on medical expertise of physician and avail- against two forms, 0.02% polyhexamethylene biguanide ability of resources [6,47]. The statement diagnostic is chal- (PHMB) or chlorhexidine, still need antibacterial, antifungal, lenge that a new molecular technology can be used in Acan- Bunsuwansakul et al.: Acanthamoeba in Southeast Asia 347 References (2018) [68] (2018) [24] (2018) [19] et al. (2017) [22] (2016) [69] (2014) [27] (2014) [25] et al. (2012) [70] Lim et al. Basher et al. Muslim et al. Wara-Asawapati Gunawan et al. Buerano et al. Cruz and Rivera Wanachiwanawin (Continued to the next page) - - Vision status Loss of patients follow-up Alive with treatment blurriness blurriness Status after up for some Corneal scar loss of follow- blurriness and altered mental altered Improved vision Improved vision Improved ------), ® ® : topical cy - - : Steroid eye drops, eye drops, : Steroid : cefazolin and gen : propamidine isethion propamidine : ) and Polygran Treatment ® After diagnosis  travenous ceftazidime, metronida zole, fluconazole and rifampicin  hlorhexidine, polyhexamethylene bi guanide or propamidine  efore diagnosis  efore diagnosis tamicin; cloplegics; topical 0.1% hexamidine, trans and chlorohexidine, 0.02% plantation natamycin Moxifloxacin eye drops, polymyxin-neomycin- eye drops, (Polygran gramicidin eye drops and voriconazole eye drops ate (Brolene B B A  fter diagnosis and Prednisolone Metronidazole In Chlorhexidine C ------train-matched partial 18S rDNA sequence)  onthly disposable soft con tact lens wearer for 1 year year 1 for wearer lens tact with tap water to rinse con tact lens and case in many occassions it with polyvinylchloride pipe, polyvinylchloride with it topical steroids with lack ontact lens wearer of hygiene ry soil and dust Potential history of patients treet sweeper (4/44), Gar  treet bage collector (2/37), Gar (2/37), collector bage bage sorter (0/16), Land la Bioreactor (1/6), scaper and su (0/4), foremen borer students and (0/3), perviors (1/70) armer with pulmonary tuber culosis history, Raynaud’s Raynaud’s history, culosis phenomenon, mild myositis, and high antinuclear anti body (speckle type) (s  H  D  M F  survivor Drowning Non-contact lens wearer S C  AK AK GAE GAE AK (T4) AK (T4) AK with Condition (Genotype) aeruginosa -(T5, 54, T11) Pseudomonas - method Diagnostic and partial 18S rDNA sequencing and partial 18S rDNA sequencing and partial 18S rDNA sequencing T scan, Microscopy,  T scan, Microscopy, and PCR on partial and PCR on partial 18S rDNA sequenc ing Microscopy and Microscopy culture M  icroscopy, culture, and Microscopy culture C CT scan and microscopy M  icroscopy, culture, M  icroscopy, culture, M  icroscopy and culture infection cases in Southeast Asia Southeast in cases infection fluid Brain swabs Clinical sample Corneal Corneal Corneal Corneal Corneal abscess scraping scraping scraping scraping Nasal swab Cerebrospinal Cerebrospinal - 2 28 32 58 76 (yr) Age

juveniles Acanthamoeba for 8 non- contact lens wearers, 30.6± 15.3 for 12 contact lens wearers Adults and 4  8.3± 14.5 Examples of 8/200 felines and 8/25 canines (56/225 naturally- infected animal) in Malaysia 2 cases in Siriraj hospital, Thailand (1996-2006) Singaporean male Singaporean Ethnicity/Gender 4  Indonesian female Thai female Indonesian male Filipino male 12/180 Filipinos 2  Table 2. Table 348 Korean J Parasitol Vol. 57, No. 4: 341-357, August 2019 References (2009) [71] (2009) [72] (2009) [73] (2008) [74] (2005) [76] (2005) [77] (2004) [78] (2005) [26] Anshu et al. Por et al. Agahan et al. Sirikul et al. Parthasarathy and (2007) [75] Tan Siripanth Sukthana et al. Lim and Wei Kamel et al. -

(Continued to the next page) - up ness Vision Death Cured treatment blurriness blurriness blurriness Status after nearsighted blurriness but loss of follow- blurriness and in 2/3 patients Improved vision Improved vision Improved vision Improved vision Improved Vision blurriness ------

: 0.1% : 0.1% After diagno ; Acantamoebic ; Acantamoebic - - After surgery Treatment : Occulentum Acyclovir, : Occulentum Acyclovir, : 0.1% gutt propamidine isethion propamidine : 0.1% gutt  isdiagnosis Guttae Choramphenicol, and 0.12% Guttae Prednisolone; sis polyhexamethyline gutt 0.02% ate, In Situ ker biguanide, and laser atomileusis (LASIK) for Myopia aldwell-Luc operation, Intravenous  aldwell-Luc operation, Intravenous amphotericin B, oral ketoconazole, amphotericin B, oral ketoconazole, and amoxycillin/clavulanic acid  efore diagnosis agents: 0.02% topical polyhexamide methylene biguanide, 0.02% chloro hexidine, 0.1% hexamidine, and transplantation. phosphate, dexamethasone topical 0.5% levofloxacin, same Acantham oebic agents, and topical preserva steroids. tive-free  olyhexamethylbiguanide, chlorohexi dine, propamidine dexamethasone, dexamethasone, dine, propamidine hexamidine, and transplantation .02% topical polyhexamethylene  .1% topical diclofenac sodium and niguanide, 0.02% chlorohexidine, niguanide, 0.02% chlorohexidine, 0.1% hexamidine, propamidine isethionate, and transplantation. drops. atropine Chlorhexidine 0.02% and fortified fortified and 0.02% Chlorhexidine Gentamycin opical Propamidine isethionate, isethionate,  opical Propamidine P 0  0 B C M T - onstruction worker eye washed with water from open tank after sand and dust strucked in the eye Potential history of patients lens wearer multipurpose disinfectant Suboptimal hygiene practices Non-contact lens wearer Contact lens wearers Rigid gas-permeable contact Swimming in a dam Diving in a natural pond with Contact lens wearer solution C  - - sp. sp.) Acan AK AK AK AK AK AK AK GAE Condition and (Genotype) Naegleria ( thamoeba Amoeba co-in fection sinusitis - method Diagnostic Microscopy and Microscopy culture Microscopy and Microscopy culture and Microscopy culture Microscopy and Microscopy culture and Microscopy culture and Microscopy culture - Nasal Clinical sample Corneal Corneal Corneal Corneal Corneal Corneal autopsy exudate scraping scraping scraping scraping scraping scraping, specimen Biopsy and biopsy, and biopsy, keratoplasy - - - - - 13 36 39 28 (yr) Age  20 years- old= 13, 21-40 years- old= 25, 41-60 years- old= 4 Mean age all 127 mi 40± 22 for < titis patients crobial kera crobial - - - Continued /103 infective kera titis patients with eye surgery 2 Chinese, 8 Malay, 5 Indian, 7 others (2005-2007 in Sin gapore) AK patients/127 keratitis microbial eyes (2001-2004) in Ramathibodi Hos pital, Thailand Ethnicity/Gender 9  2  3 Filipinos 3  Chinese female Thai female Thai male female Singaporean Malaysian male Table 2. Table Bunsuwansakul et al.: Acanthamoeba in Southeast Asia 349 References (2000) [79] (2000) [79] (2000) [80] (2000) [80] (1999) [81] (1990) [81] (1990) [81] (1990) [81] Cheng et al. Cheng et al. Jongwutiwes et al. Jongwutiwes et al. Kosrirukvongs et al. Kosrirukvongs et al. Kosrirukvongs et al. Kosrirukvongs et al.

(Continued to the next page) Vision cataract treatment blurriness blurriness up but no recurrence Status after Recurrence Recurrence Enucleation evisceration Stromal scar Stromal scar Stromal necessitating blurriness with Loss of follow- Improved vision Improved vision Improved ------After After After : 0.006% : 0.006% After diag : ketocon After diagno : miconazole and : miconazole and : spersapolymyxin : spersapolymyxin : tetracycline oint tetracycline : : antimicrobial eye eye : antimicrobial : topical neomycin : topical neomycin : fortified cefazolin, : fortified cefazolin, : 0.006% chlorhexi : antibiotics and plant : gutt spersadexoline; : 0.1% gutt propami : 0.1% gutt Treatment After diagnosis After diagnosis : 0.1% gutt propamidine propamidine gutt 0.1% : : 0.006% chlorhexidine so : 0.006% chlorhexidine solution : propamidine isethionate eye isethionate eye : propamidine  efore diagnosis  efore diagnosis  efore diagnosis  efore diagnosis sis ise dibromopropamidine and drops, thionate eye ointment  efore diagnosis with an chlorhexidine hydrochloride tidlaucoma for recurrence  efore diagnosis dine solution  efore diagnosis After diagnosis dine isethionate, and gutt tobramy cin ment and neosporin eyedrops; diagnosis isethionate and ointment, 1% trifluorothy drops acyclovir eye and eye drops midine ointment; neosporin, poly azole eye drops, myxin, neomycin, gramicidin, pro di drops, eye isethionate pamidine isethionate eye bromopropamidine ointment, and transplantation. neosporin eye drops; gentamicin and cefazolin eyedrops, subconjunctival injection, topical and B, polymyxin neomycin sulfate, gramicidin; sulfate, polymyxin B, and gramicidin; After diagnosis gentamicin, neomycin, topical tobra mycin, topical neomycin sulfate, gramicidin; and B, polymyxin diagnosis lution topical neomycin sulfate, poly root, myxin B, and gramicidin; nosis and 1% topical clotrimazole eye Note: non-compliance drops B B B B B B B B  efore diagnosis - - on-disposable soft contact  on-disposable soft contact contact no and wearer lens lens when swim in lake/pool  on-disposable soft contact lens wearer Potential history of patients plashing fish pond water to  plashing fish pond water to left eye injured by tiny piece piece by tiny eye injured left of bamboo eft eye injured by straw frag straw by injured eye eft ment and dirt cleaned off ment and dirt cleaned off her face using water in from a jar near her home after dig on the ging in the garden outskirts N N L  S Pond water for washing Dust Daily-wear soft contact lenses exposure Plant root AK AK AK AK AK AK AK AK Condition (Genotype) - - - - method Diagnostic indirect immunoflu indirect testing, orescence and isoenzyme analysis immunoflu indirect testing, orescence and isoenzyme analysis immunoflu indirect testing, orescence and isoenzyme analysis immunoflu indirect testing, orescence and isoenzyme analysis and mtDNA-RFLP and mtDNA-RFLP Microscopy and Microscopy culture and Microscopy culture culture Microscopy, culture, Microscopy, M  icroscopy, culture, M  icroscopy, culture, M  icroscopy, culture, M  icroscopy, culture, Clinical sample Corneal Corneal Corneal Corneal Corneal Corneal Corneal Corneal scraping scraping scraping scraping scraping scraping scraping scraping 24 26 58 30 57 36 33 74 (yr) Age Continued Ethnicity/Gender Chinese male Malay male Thai female Thai male Thai female Thai male Thai female Thai male Table 2. Table 350 Korean J Parasitol Vol. 57, No. 4: 341-357, August 2019 References (1990) [81] (1995) [82] (1994) [83] (1994) [83] (1993) [84] Kosrirukvongs et al. Kamel and Norazah Sangruchi et al. Sangruchi et al. Thamprasert et al. - Death Death Death treatment Status after with cataract Vision blurriness - - - - - as After diag Acantham (0.1% Pro (0.1% ® ® P. aeruginosa P. - - : topical neomycin : topical neomycin : Zovirax : cefazolin and genta Treatment : gentamycin and homatropin homatropin and gentamycin : sp.  efore diagnosis for micin eye drops well as chlorhexidine for oeba  efore diagnosis sulfate, polymyxin B, and gramicidin; After diagnosis nosis neosporin, miconazole eye drops, Brolene and eyedrops pamidine isethionate) enesection and rapid fluid replace fluid rapid and  enesection ment, antibiotics, gastrojejunostomy, gastrojejunostomy, antibiotics, ment, ampicillin, gentamicin, and parenteral and metronidazole B B V Potential history of patients Unknown Contact lens wearer Worker Farmer Immunocompetent patients

- -

E. coli AK E. coli K. pneu GAE GAE Condition (Genotype) P. auruginosa P. and gastric ulcer with gastric acanthamoe biasis and sepsis from operative site with and moniae A  K with P  roliferated - - - - - method Diagnostic icroscopy and indi  icroscopy and indi  icroscopy indirect immunoflu indirect testing, orescence and isoenzyme analysis immunofluores rect cence test immunofluores rect cence test icroscopy, M  icroscopy, culture, Microscopy M M R  adiography and microscopy Biopsy Clinical sample Corneal Corneal scraping scraping Brain autopsy Brain autopsy 65 40 26 20 42 (yr) Age keratitis; GAE, Granulomatous amoebic encephalitis; -, Not mentioned in the published paper. Continued Acanthamoeba Ethnicity/Gender Thai female Malaysian female Thai female Thai male Thai female Table 2. Table AK, Bunsuwansakul et al.: Acanthamoeba in Southeast Asia 351 References (2011; Thailand) [91] (2010; Thailand) [92] (2019; Malaysia) [93] (2019; Malaysia) [93] (2018; Malaysia) [94] (2018; Malaysia) [94] (2017; Malaysia) [95] (2017; Malaysia) [95] (2018; Indonesia) [85] (2016; Indonesia) [86] (2016; Indonesia) [86] (2016; Indonesia) [86] (2017; Malaysia) [87] (2016; Malaysia) [88] (2014; Malaysia) [89] (2012; Thailand) [90] (2018; Indonesia) [85] Roongruangchai et al. Roongruangchai et al. Anwar et al. Anwar et al. Mahboob et al. Mahboob et al. Mahboob et al. Mahboob et al. Kusrini et al. Kusrini et al. Kusrini et al. Kusrini et al. Siddiqui et al. Siddiqui et al. Ibrahim et al. Boonman et al. Kusrini et al.

- - - 5 (Continued to the next page)

5

from from

from from from from from from µg/ml µg/ml Acanthamoe A. castellanii A. castellanii Acanthamoeba Acanthamoeba Acanthamoeba Acanthamoeba A. triangularis - - - A. triangularis (ATCC50492) A. triangularis Acanthamoeba A. triangularis Trophozoites (CCAP 1501/3A). (ATCC 50492) at 50 (ATCC 50492) at 50 (ATCC = 25 against = 5 against = 170 against = 1.5 against A. castellanii 50 50 50 50 /IC /IC /IC /IC keratitis isolate and 2 soil isolates, respec =2.39-8.77 µg/ml against = 0.33, 0.66 µM against = 0.7 μg/ml against = 7 μg/ml against = 2.6 μg/ml against = 1.2 μg/ml against = 6.5 μg/ml against 50 50 50 50 50 (CCAP 1501/2A) and 3.74-9.30 µg/ml against A. polyphaga 50 ba tively 50 50 50 50 50 activity against o effect on viability, growth, cellular differentia growth, on viability, o effect tion, and extracellular proteolytic activities activities extracellular proteolytic tion, and against A. castellanii A. castellanii water sample environmental water sample environmental water sample environmental water sample environmental keratitis isolate keratitis isolate keratitis isolate keratitis isolate keratitis isolate 100% abolished amoeba viability of 5× 10 Significanly abolished amoeba viability of 5× 10 CC CC CC CC IC  IC IC IC N  IC  IC IC from from from from from from from from Acanthamoeba Anti- A. triangularis A. triangularis ------A. triangularis A. triangularis (ATCC50492) Cysts keratitis isolate keratitis isolate (ATCC 50492) (ATCC 50492) (ATCC = 100 against = 10 against = 8.5 against = 3.75 against A. castellanii 50 50 50 50 /IC /IC /IC /IC 50 50 50 50 CC= 0.04% dilution after 24 hr against CC= 0.25% dilution after 24 hr against ncystation and excystation inhibition against ncystation induction and excystation inhibition against Acanthamoeba Acanthamoeba A. castellanii A. castellanii environmental water sample environmental water sample environmental water sample environmental water sample environmental M  M  E  Encystation and excystation inhibition against CC CC CC CC E  ------Poly Poly Nanotechnology lized by gum acacia lized by gum tragacanth (DL-lactide-co-glycolide) (DL-lactide-co-glycolide) Gold nanoparticles stabi Silver nanoparticles stabi ](18C6) 3 ) 2 agents and nanoparticles in ASEAN studies (OH 3 ) 3 CN 3 Pericampylus glaucus Pericampylus glaucus Pericampylus glaucus Pericampylus glaucus agents Acanthamoeba O in CH CN 2 3 solution ® .6H 3 sp. sp. solution Anti- ) 3 ® Acanthamoeba Citrus Citrus [Sm(Pic)2(18C6)] (Pic) [Sm(Pic)2(18C6)] (Pic) Virkon flavonoid from Hesperidin, commercial flavonoid, from Naringin, commercial Periglaucine A from Betulinic acid from Periglaucine A from Betulinic acid from Terbium complex [Tb(NO Terbium Tb(NO 18C6 in CH Phosphanegold (I) thiolates 3% DMSO derivatives Carbonyl Thiourea fusaric acid Commercial Betadine Acyclic samarium complexes Cyclic samarium complexes Plant products Anti- Chemicals Table 3. Table 352 Korean J Parasitol Vol. 57, No. 4: 341-357, August 2019 Malaysia) [103] References ; (2012; Thailand) [90] (2019; Malaysia) [100] (2018; Malaysia) [101] (2018; Malaysia) [102] (2012; Thailand) [90] (2018; Malaysia) [96] (2016; Malaysia) [97] (2011; Thailand) [91] (2010; Thailand) [92] (2018; Malaysia) [98] (2016; Thailand) [99] Boonman et al. Anwar et al. Anwar et al. Anwar et al. Siddiqui et al. (2016 Boonman et al. Anwar et al. Mahboob et al. Roongruangchai et al. Roongruangchai et al. Akbar et al. Sampaotong et al. - - - (Continued to the next page) L. A. ac

keratitis keratitis keratitis keratitis A. castellanii effect against against effect trophozoites by by trophozoites effect against effect activity at 10 μM activity at 10 μM (ATCC 50492) (ATCC Acanthamoeba (ATCC 50492) when (ATCC Acanthamoeba Acanthamoeba - - - (ATCC 50492) (ATCC (ATCC 50492) (ATCC activity observed and Trophozoites A. triangularis Acanthamoeba A. castellanii Acanthamoeba Acanthamoeba Acanthamoeba A. castellanii (ATCC 50492) (ATCC ) A. castellanii A. castellanii Acanthamoeba =0.34 µm against =0.31 µm against 50 isolate 50 isolate activity against rowth affected but not viability of affected rowth (ATCC 50492) (ATCC enhanced activity with silver nanoparticles against ignificantly enhanced anti- enhanced  ignificantly  ignificant anti- tivity against with cinnamic acid alone compared environmental and fraction) potent (most acetate ethyl by commer ratio reduction cyst:trophozoites acid (major constituent in cial chlorogenic japonica castellanii (Amphotericin B> Fluconazole> Nystatin) against (Amphotericin B and Nystatin but not Flucon azole) against IC  E  nhanced anti- E  nhanced anti- A  nti- G  S S S  ignificant anti- IC  - - Acanthamoeba A. castellanii castellanii A. Anti- A. castellanii A. castellanii ------Cysts keratitis isolate keratitis isolate CC= 1: 4 dilution after 24 hr against CC= 1: 8 dilution after 24 hr against nti-Encystation activity (Diazepam and Pheno barbitone activity enhanced with silver barbitone activity enhanced with silver nanoparticles) and anti-cyst activity (Pheno barbitone and Phenytoin activity enhanced against nanoparticles) silver with 50492) (ATCC ncystation inhibition against (ATCC 50492) (ATCC (ATCC 50492) (ATCC Acanthamoeba Acanthamoeba in lower than 40% viable cysts A  Encystation inhibition of E  M  M  Undiluted, 1:2, 1:4, 1:6 dilution of EM resulted ------cles Nanotechnology Gold nanopaticles Gold nanoparticles Silver nanoparticles And their silver nanoparti - - sp., and and Thun species species Thunbergia Thunbergia ) TM Citrobacter sp. from Dubia sp. from Fusarium fujikuroi Fusarium fujikuroi spp., Cinnamomum cassia from Madagascar cock from agents Fusarium fujikuroi Serratia marcescens methanolic extract fraction 2 methanolic extract fraction 3 Streptococcus Klebsiella sp., Continued Acanthamoeba iazepam (Valium), Phenobarbitone iazepam (Valium), iclofenac sodium and Indomethacin (NSAIDs) ehydrofuseric acid from acid from  ehydrofuseric species complex Tlau3 isolated from bergia laurifolia upernatants from bacteria isolated from bacteria isolated from  upernatants from cockroach gut: cockroach Escherichia coli two roach; Bacillus cockroach thyl acetate, water, butanol fractions from from fractions butanol water, acetate,  thyl Lonicera japonica usaric acid from  usaric acid from complex Tlau3 isolated from complex Tlau3 isolated from laurifolia (Luminal), and Phenytoin (Dilantin) Nystatin, Fluconazole, and Amphotericin B Nystatin, Fluconazole, and Amphotericin B D  D  Cinnamic acid from Cinnamic acid from E Pouzolzia indica Pouzolzia indica S (EM microorganisms Effective F D Drugs Anti- products Microorganism Table 3. Table Bunsuwansakul et al.: Acanthamoeba in Southeast Asia 353

thamoeba detection and monitoring system to understand these amoebic infections and diagnostic approaches. So far, the gold standard of Acanthamoeba laboratory testing has been cultured on NNA overlaid with E. coli and PYG medium for Malaysia) [103] References ; axenic culture. A modern technique has been applied as far as the laboratory diagnosis is concerned. This can provide a bet- (2016; Malaysia) [104] (2003; Thailand) [105] (2003; Thailand) [105] (2017; Malaysia) [106] (2012; Malaysia) [107] (2016; Malaysia) [104] Siddiqui et al. Nacapunchai et al. Nacapunchai et al. Siddiqui et al. Nakisah et al. Siddiqui et al. Siddiqui et al. (2016 - - -

A. ter routine diagnosis especially using molecular-based inter- -like -like vention such as PCR and MALDI-TOF/MS [5]. It is important (ATCC (ATCC A. cas because mistaken or late diagnosis has been usually reported A. castella A. A. castella due to poor prognosis leading to worsening clinical symptoms polyphaga

and subsequently under postmortem diagnosis [48,49]. More- A. castellanii over, Acanthamoeba spp. are potential Trojan horse of human-

activity against against activity pathogenic viruses, infectious bacteria, and fungi which might Trophozoites

Acanthamoeba be one way of disease spread and gene transfer [10]. Early de-

(ATCC 50492) (ATCC tection technique is needed as well as physician should be aware of Acanthamoeba infection through patient interview and Acanthamoeba (ATCC 50492) (ATCC = 0.615-0.876 µg/ml against clinical (ATCC 50492) (ATCC 50 tellanii activity against o effects on growth and viability of viability and growth on effects o nii o effects on growth of on growth  o effects 50492) ose-dependent growth inhibition (5-700 μg/ (5-700 inhibition growth  ose-dependent against ml) natural water isolated from amoebae were courses nii  nti- tatic effect on growth but not viability of of viability not but growth on effect  tatic castellanii history taking [50,51]. Unfortunately, most patients also come N  D (5-1,000 μg/ml) No effects A IC  N S

- up with lesion in brain for GAE which most of the cases are too late to be cured whilst AK are mainly associated with con-

A. castel tact lens wearer and immunocompetent patients are basically Acanthamoeba A. castellanii A. castellanii

Acanthamoeba affected [52-54]. Anti- Hygiene and proper contact lens usage is a critical point of

- - care which ophthalmologist should pass on knowledge of ap- (ATCC 50492) (ATCC Cysts propriate usage of contact lens [55]. Most disinfectant solu- tions for contact lens are ineffective against Acanthamoeba cyst -like amoebae were isolated from isolated from -like amoebae were which is rich with cellulose structure [56]. Effort on novel anti- A. castellanii

(ATCC 50492) (ATCC Acanthamoeba agents therefore focus on cyst form or other po- o effects on encystation inhibition of o effects lanii resence of cytostatic effect on of cytostatic effect resence polyphaga of concentrations at courses water natural 500-700 μg/ml ncystation inhibition and excystation inhibition  ncystation inhibition and excystation against  ncystation inhibition against (ATCC 50492) (ATCC tential target sites [7]. Biology of Acanthamoeba spp. should be E P  (5-1,000 μg/ml) No effects N  E studied to guide action of desired anti-Acanthamoeba agents which have been identified [57]. In ASEAN nations, Anti- Acanthamoeba activity has been investigated among human------made chemicals, plant extracts, microbial metabolites, anti- Acanthamoeba side effect of drugs, and animal products which Nanotechnology nanoparticles are attractive antimicrobial agent delivery tech-

- nology to enhance the activity of these anti-Acanthamoeba

-lacta agents (Table 3). However, these anti-Acanthamoeba agents Aaptos were tested only in vitro. Blood-brain barrier is another chal- clasto ) serum lenge for anti-Acanthamoeba agents to pass through for the treatment of GAE [58]. There is a long road lying ahead for in vivo experiment and clinical application in ASEAN nations. In agents fact, Southeast Asia (ASEAN) is a gigantic resource of medici- nal plants and bioactive agents. Interestingly, the only PHARM Crocodylus palustris -lactone (proteasome inhibitors) -lactone (proteasome ) from different localities different ) from β Continued database is available at the Faculty of Pharmacy, Mahidol Uni-

Acanthamoeba versity, Thailand in which more than 1,000 collections of Thai  ea sponge crude methanol extracts ( aaptos actacystin and active form as active and  actacystin cystin Artesunate (Antimalaria) Metronidazole ( Crocodile S Bortezomib (proteasome inhibitor) Bortezomib (proteasome L Animal products Anti- Acetaminophen (NSAIDs) IC, Inhibition concentration; CC, Cytotoxicity MCC, Minimal cystcidal concentation; -, Not mentioned in the publ ished paper. Table 3. Table medicinal plants were recorded (http://www.medplant.mahi- 354 Korean J Parasitol Vol. 57, No. 4: 341-357, August 2019

dol.ac.th/pharm/search.asp: March 13, 2019). It is therefore cysts: what are the options? Parasit Vectors 2018; 11: 26. noteworthy to strongly recommend for more research works 8. Scheid P, Schwarzenberger R. Acanthamoeba spp. as vehicle and reservoir of adenoviruses. Parasitol Res 2012; 111: 479-485. that should be further explored on the plants-based medicinal 9. Siddiqui R, Khan NA. Biology and pathogenesis of Acantham- therapy for severe or deadly infections with Acanthamoeba spp. oeba. Parasit Vectors 2012; 5: 6. 10. Guimaraes AJ, Gomes KX, Cortines JR, Peralta JM, Peralta RH. ACKOWLEDGMENTS Acanthamoeba spp. as a universal host for pathogenic microor- ganisms: One bridge from environment to host virulence. Mi- This work is under the project entitled of “Medicinal under- crobiol Res 2016; 193: 30-38. 11. Corsaro D, Walochnik J, Köhsler M, Rott MB. Acanthamoeba exploited Thai native plants against Acanthamoeba, Leishmania misidentification and multiple labels: redefining genotypes donovani, and Plasmodium falciparum – Toward South East Asia T16, T19, and T20 and proposal for Acanthamoeba micheli sp. collaboration initiative (Grant No. 040226) supported by The nov.(genotype T19). Parasitol Res 2015; 114: 2481-2490. Royal Patronage of Her Royal Highness Princess Maha Chakri 12. Schuster FL. Cultivation of pathogenic and opportunistic free- Sirindhorn”. We are also grateful to the Project CICECO-Aveiro living amebae. Clin Microbiol Rev 2002; 15: 342-354. Institute of Materials, FCT Ref. UID/CTM/50011/2019. 13. Axelsson-Olsson D, Olofsson J, Ellström P, Waldenström J, Ol- sen B. A simple method for long-term storage of Acanthamoeba species. Parasitol Res 2009; 104: 935-937. CONFLICT OF INTEREST 14. 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