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Parasit Host Dis > Volume 57(4):2019 > Article
Bunsuwansakul, Mahboob, Hounkong, Laohaprapanon, Chitapornpan, Jawjit, Yasiri, Barusrux, Bunluepuech, Sawangjaroen, Salibay, Kaewjai, de Lourdes Pereira, and Nissapatorn: Acanthamoeba in Southeast Asia – Overview and Challenges


Acanthamoeba, one of free-living amoebae (FLA), remains a high risk of direct contact with this protozoan parasite which is ubiquitous in nature and man-made environment. This pathogenic FLA can cause sight-threatening amoebic 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 purposes. 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 technology, 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.


Acanthamoeba spp. is one of pathogenic free-living amobae (FLA) along with Naegleria fowleri, Balamuthia mandrillaris, and Sappinia sp. which are potential to cause rare infection in central nervous system. These protozoan parasites are mostly found in natural soil and water bodies and immunocompromised patients as the main target [1]. Recently, Acanthamoeba spp. are recognized as increasing threat against contact lens wearers and healthy individuals also take some risks on amoebic keratitis (AK) [2]. Understanding on Acanthamoeba infections is therefore crucial but still limited in ASEAN countries even though studies on anti-Acanthamoeba agent do exist. Herein, an overview of Acanthamoeba was put in a nutshell as well as challenges on recent issues to encounter against this amoeba in our regional ASEAN countries including Brunei Darussalam, Cambodia, Indonesia, Lao People’s Democratic Republic (PDR), Malaysia, Myanmar, the Philippines, Singapore, Thailand, and Vietnam.


Acanthamoeba spp. is a centrosome-bearing, single-celled, flattened naked amoeba in Order Acanthopodida, Class Centramoebia, Phylum Discosea, Amoebozoa clade in Amorphea domain of Eukaryotic organisms [3]. Term “Acanth” in Greek means spike representing prominent sub-pseudopodia while “amoeba” means alteration like their appearance. The bacteria-phagocytosing protozoa is one of clinical FLA ubiquitous in nature soil and water bodies as well as man-made environment as a secondary decomposer. Ubiquity is implied by presence of antibodies in healthy individuals [4]. Acanthamoeba sp. was first recognized as contaminant of Cryptococcus pararoseus culture by Castellani in 1930 and named as Hartmannella castellanii and then a year later, Acanthamoeba spp. because of its double-walled cyst with irregular ectocyst appearance which is different from round and smooth cyst wall of Hartmannella spp. [5].


Acanthamoeba spp. appears in 2 forms of life cycle: trophozoite (25–40 μm) and cyst (13–20 μm). Trophozoite is an infective stage with amoeboid locomotion whilst cyst is a dormant stage against harsh environment such as temperature and pH imbalance, malnutrition, or presence of anti-Acanthamoeba agents [6]. One third of strength of cyst wall might come from polymer of glycosidic linkages between saccharides while another 2/3 are protein and other components, respectively [7]. Furthermore, the protist acts as potential reservoir or vector of human-pathogenic bacteria, fungi, or viruses while endosymbiont and Acanthamoeba-resistant organisms also are identified [810]. Recently, more than 25 species were recorded in NCBI taxonomy database and 20 genotypes were published which T4 is a major genotype associated with human infections [9,11]. For cultivation, xenic culture is obtained by using non-nutrient (Page’s amoeba saline) or PYG (peptone 0.05%, yeast extract 0.05%, glucose 0.1%) agar coated with living or killed bacteria (e.g., Escherichia coli) at 25–28°C in the dark for 2–3 days for trophozoite proliferation and 1–2 weeks for encystment while PYG (peptone 2%, yeast extract 0.5%, glucose 0.5%) agar was used for axenic culture [12]. Culture in PYG medium at 4°C would be convenient method for long-term preservation at least 1–4 years [13].


FLA, especially Acanthamoeba spp., occur worldwide and have a variety of habitats. Many studies have recorded the wide distribution in soil and water, with differing range of thermal tolerance (Table 1). They have been isolated in untreated natural freshwaters, like lakes, ponds, hot springs and waterfalls [1417]; and brackish, seawaters, and ocean sediments [18]. They were also isolated from treated waters like domestic water systems, swimming pools, hydrotherapy pools, remedial spas, tap water and drinking water [14,16,19,20]. Unconventional water sources like sewage and aquaria were not spared with the presence of amoebas [18].
Aside from water, Acanthamoeba spp. were also present in different types of soils such as agricultural, garden and mining [2123]. Acanthamoeba genotypes of infected cats and dogs were matched with dry soil and dust. [24]. Acanthamoeba-infected individuals can also be a source of the isolates of organism through sinuses, brain and corneal and skin specimens [22,2527] and even in necrotic tissues [18].
The presence of Acanthamoeba spp. has impacted for the last decades because of the increasing cases of a rare condition AK, a severe infection of the eye cornea associated with intense pain. This has been observed in contact lens wearer population [28]. It is believed that the cause of infection is due to the exposure of the eye to the Acanthamoeba-contaminated contact lens solutions. Acanthamoeba isolated from contact lens storage cases were confirmed [29]. Further, the usual spread of the contaminant is due to poor hygiene and maintenance of the lens; and exposure to contaminated water (swimming pool or other recreational waters) while wearing contact lenses.
However, the disease has also been reported in non-contact lens wearers [18,26,27]. This further affirms the possible contamination through direct contact to contaminated water and soil. The wide dispersal of Acanthamoeba onto the environment is due to the wind dispersal of its resistant form, the cysts. Likely that indoor ventilation system, blowing fan, air diffuser and other furniture contaminated with Acanthamoeba can be a cause of spreading indoor [30]. Thus, individuals who are not contact lens wearers but have been constantly exposed to dust particles and soil are also at high risk of infection [25]. It is also important to note that exposure to Acanthamoeba can be as simple as accidental splash of contaminated water to the face or bruised skin [14], making a fast and easy transmission.
Ironically, with the many studies proving the presence of Acanthamoeba in different environmental media (soil, water and air), the dearth of information in Southeast Asian (ASEAN) countries is quite a concern, considering that the varying climatic conditions of the region is a favorable habitat for this organism which has an unusual geographic distribution [31].
The ASEAN countries’ tropical condition, favorite tourist destinations during summer, consists of beaches, falls, and lakes are among the popular areas where more people involve with these outdoor activities. The congestion can increase risk of contamination with Acanthamoeba especially when the environment is dry during summer and dust particles can be easily spread. Likewise, resorts with swimming pools are occupied the entire summer with local and foreign tourists. Since resorts gain profit only during this time of the year, owners tend to maximize the use of the swimming pools which may compromise the proper cleanup of the swimming facility. This poses the risk to the swimmers, adding to the fact that Acanthamoeba can also be resistant to disinfectants [26,32].
The detection of Acanthamoeba in soil, water and air in other countries in ASEAN (Fig. 1), confirms that a continual contamination of the environment persists, and this poses a risk to people dependent on the soil and water for domestic activities, agricultural and farming occupation, and even for recreation. The lack of information in some countries (Cambodia and Brunei) does not mean the absence of Acanthamoeba-contaminated environment. Albeit, this may result to the inability of one country to control the spread of possible diseases associated with Acanthamoeba considering that this amoeba may also harbor pathogenic bacteria or fungi.


Potential pathogenicity of Acanthamoeba was first observed in monkey kidney cell in vitro as well as intracerebral/intraspinal inoculation in monkeys and intravenous/intranasal inoculation in mice [33,34]. First patient was recognized as GAE in 1972 and a year later, AK [35,36]. Acanthamoeba spp. are therefore considered as rare potential pathogen causing cutaneous lesions, sinusitis, AK, GAE, and disseminated form in human and prefer individuals with underlying diseases or immunocompromised host but AK was frequently reported in immunocompetent patients especially, contact lens wearers [37].
For AK, poor sanitation of contact lens wearer is a potential risk and corneal trauma seem required before trophozoite infection as well as eye secretion after contact lens wore might be preferred by Acanthamoeba [38,39]. Onset of AK is days to weeks with symptoms of tormenting eye pain, redness, photophobia, stromal infiltration leading to sight-threatening condition which are similar and misdiagnosed to Herpes simplex, bacterial or fungal keratitis [39,40]. AK is confirmed by presence of trophozoite with large nucleolus and contractile vacuoles as well as pseudopodia and transparent protrusions of Acanthopodia from corneal scrapings or biopsies under direct microscopy with several stains. Encystment on non-nutrient agar (NNA) and nucleic acid amplification testing are further investigated for species identification and genotyping, respectively. Taxonomic identification mainly investigated by cyst morphology under microscope [41] and a hypervariable sequence part of 18S small subunit rDNA gene called ASA.S1 by Acanthamoeba-specific primers: JDP1 and JDP2 (amplicon size 467 bps for Neff strain of A. castellanii accession number M13435.1) [42]. Extended or almost complete of 18S rDNA amplicon size provide better solution for genotyping [11,42]. Pathogen broad-spectrum and most effective anti-Acanthamoeba agents against two forms, 0.02% polyhexamethylene biguanide (PHMB) or chlorhexidine, still need antibacterial, antifungal, or aromatic diamidines combination because of resistance of cyst form and PHMB is toxic to human corneal cells [40].
For GAE, a very rare condition, is opportunistic and fatal infection with onset of weeks to months mostly in immunocompromised patients, especially HIV/AIDS patients through skin breaks, respiratory tract, and olfactory epithelium. GAE patient will encounter with neurological signs such as confusion, headache, and stiff neck as well as psychological change, e.g. irritability generally like other brain infections due to effect of edema, necrosis, and hemorrhages in infected part of brain [43]. To confirm GAE, microscopy and culture from CSF remain gold standard methods used after neuroimaging detection of brain lesions while indirect immunofluorescence on tissue and multiplex real-time PCR assay are available [44]. Late/missed diagnosis, blood-brain barrier crossing of antimicrobial, drug side effects, drug combination are still an issue on GAE treatment and only few patients were cured [45,46].
There are many reports on Acanthamoeba infection in ASEAN countries (Table 2). Most infections are AK with contact lens while cases of GAE is rare. Notably, Acanthamoeba can be involved with gastric ulcer and sinusitis and found from nasal swab from healthy individuals and corneal swab from infected animal (Table 2). Undeniably, exposure to soil and contaminated water are potential risk but underlying disease might be another one factor for the infections. Misdiagnosis and delay in diagnosis are common among patients leading to permanent vision blurriness because of injured cornea or deeper layers for AK and death for GAE. These problems are still insolvable till date. Rapid and accurate prognosis is therefore an urgent need for Acanthamoeba infection.


Contact with Acanthamoeba spp. is common. Immunocompromised patients should realize this risk and avoid exposure to, especially, natural soil and water bodies even though it is a rare disease but GAE is fatal and AK is vision-threatening [6]. Moreover, no specifically therapeutic course is available for Acanthamoeba spp. infections, in case of GAE. However, commercial drugs for AK are highly toxic due to prolonged treatment duration as well as diagnosis and combination of treatments depends on medical expertise of physician and availability of resources [6,47]. The statement diagnostic is challenge that a new molecular technology can be used in Acanthamoeba 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 axenic culture. A modern technique has been applied as far as the laboratory diagnosis is concerned. This can provide a better routine diagnosis especially using molecular-based intervention such as PCR and MALDI-TOF/MS [5]. It is important because mistaken or late diagnosis has been usually reported due to poor prognosis leading to worsening clinical symptoms and subsequently under postmortem diagnosis [48,49]. Moreover, Acanthamoeba spp. are potential Trojan horse of human-pathogenic viruses, infectious bacteria, and fungi which might be one way of disease spread and gene transfer [10]. Early detection technique is needed as well as physician should be aware of Acanthamoeba infection through patient interview and history taking [50,51]. Unfortunately, most patients also come up with lesion in brain for GAE which most of the cases are too late to be cured whilst AK are mainly associated with contact lens wearer and immunocompetent patients are basically affected [5254].
Hygiene and proper contact lens usage is a critical point of care which ophthalmologist should pass on knowledge of appropriate usage of contact lens [55]. Most disinfectant solutions for contact lens are ineffective against Acanthamoeba cyst which is rich with cellulose structure [56]. Effort on novel anti-Acanthamoeba agents therefore focus on cyst form or other potential target sites [7]. Biology of Acanthamoeba spp. should be 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 nanoparticles are attractive antimicrobial agent delivery technology to enhance the activity of these anti-Acanthamoeba agents (Table 3). However, these anti-Acanthamoeba agents were tested only in vitro. Blood-brain barrier is another challenge 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 fact, Southeast Asia (ASEAN) is a gigantic resource of medicinal plants and bioactive agents. Interestingly, the only PHARM database is available at the Faculty of Pharmacy, Mahidol University, Thailand in which more than 1,000 collections of Thai medicinal plants were recorded (http://www.medplant.mahidol.ac.th/pharm/search.asp: March 13, 2019). It is therefore noteworthy to strongly recommend for more research works that should be further explored on the plants-based medicinal therapy for severe or deadly infections with Acanthamoeba spp.


This work is under the project entitled of “Medicinal under-exploited Thai native plants against Acanthamoeba, Leishmania donovani, and Plasmodium falciparum – Toward South East Asia collaboration initiative (Grant No. 040226) supported by The Royal Patronage of Her Royal Highness Princess Maha Chakri Sirindhorn”. We are also grateful to the Project CICECO-Aveiro Institute of Materials, FCT Ref. UID/CTM/50011/2019.

Conflict of interest

The authors declare no conflict of interest related to this study.


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Fig. 1
Epidemiology and clinical cases of Acanthamoeba infection in Southeast Asia. B: Granulomatous amoebic encephalitis; E: Acanthamoeba keratitis; N: Acanthamoeba sinusitis; and S: Gastric acanthamoebiasis.
Table 1
Distribution of environmental Acanthamoeba spp. in Southeast Asia
Country Type of samples No. of sample Positive culture Acanthamoeba spp. morphology References

FLA Acanthamoeba spp. Group I Group II Group III
Thailand Water
 Water samples 95 51.58% (49/95) 18.95% (18/95) ND ND ND Nacapunchai et al. (2001) [23]
 Hot spring water 69 37.68% (28/69) 13% (9/69) ND ND ND Lekkla et al. (2005) [17]
 Freshwater pond and irrigation canals 84 ND 19.05% (16/84) 15.79% (3/19) 84.21% (16/19) NF Nuprasert et al. (2010) [59]
 Flood water 7 100% (7/7) 14.29% (1/7) ND ND ND Wannasan et al. (2013) [60]
 Freshwater pond in public parks 300 ND 35% (105/300) 23.36% (25/105) 73.83% (79/105) 2.8% (3/105) Buppan et al. (2018) [61]
 Water-logged fields 2 100% (2/2) 100% (2/2) ND ND ND Wannasan et al. (2009) [62]
 Ditches 4 100% (4/4) NF ND ND ND
 Paddy fields 6 100% (6/6) 16.67% (1/6) ND ND ND
 Fish farms 10 50% (5/10) 10% (1/10) ND ND ND
 Large pond 6 50% (3/6) NF ND ND ND
 Natural water 63 ND 15.87% (10/63) ND ND ND Thammaratana et al. (2016) [15]
 Outdoor air 103 ND 41.7% (43/103) NF 16.5% (17/43) 15.5% (16/43) Yaicharoen et al. (2007) [63]
 Indoor air 64 ND 18.1% (37/64) NF 13.7% (28/37) 2.9% (6/37)
 Soil swab samples 120 69.17% (83/120) 33.33% (40/120) ND ND ND Nacapunchai et al. (2001) [23]
 Water-logged fields 2 100% (2/2) 50% (1/2) ND ND ND Wannasan et al. (2009) [62]
 Ditches 4 75% (3/4) 50% (2/4) ND ND ND
 Paddy fields 6 100% (6/6) NF ND ND ND
 Fish farms 10 50% (5/10) NF ND ND ND
 Large pond 6 66.7% (4/6) 16.67% (2/6) ND ND ND

Malaysia Water
 Domestic tap water 42 ND 2.4% (1/42) ND ND ND Anisah et al. (2003) [64]
 Swimming pools in Kuala Lumpur 840 54.4% (457/840) 46.19% (388/840) Positive Positive ND Init et al. (2010) [32]
 Recreational anthropogenic lake A 7 ND 100% (7/7) ND ND ND Onichandran et al. (2013) [16]
 Recreational anthropogenic lake B 6 ND 100% (6/6) ND ND ND
 Tap water 181 29.8% (54/181) 24.9% (45/181) ND ND ND Gabriel et al. (2019) [65]
 Recreational places 57 66.7% (38/57) 70.2% (40/57) ND ND ND
 Water dispenser units 3 33.3% (1/3) 66.7% (2/3) ND ND ND
 Filtered water 4 75% (3/4) NF ND ND ND
 Drain water 1 100% (1/1) NF ND ND ND
 Paddy fields 4 50% (2/4) 100% (4/4) ND ND ND
 Drinking water treatment 61 90.2% (55/61) 18.03% (7/11) ND ND ND Richard et al. (2016) [20]
 Water samples 15 ND 100% (15/15) ND ND ND Basher et al. (2018) [24]
 Swabs (rocks and stones) 15 ND 73.33% (7/11) ND ND ND
 Wet soil 15 ND 100% (15/15) ND ND ND
 Children playgrounds (Dry soil) 15 ND 100% (15/15) ND ND ND
 Indoors wall surface 20 ND 100% (20/20) ND ND ND
 Outdoor wall surface 20 ND 100% (20/20) ND ND ND
 Air conditioners in KM 87 ND 23% (20/87) NF 71.43% (15/21) 28.57% (6/21) Chan et al. (2011) [66]

The Philippines Water
 River 10 ND 30% (3/10) ND ND ND Onichandran et al. (2014) [67]
 Swimming pools 4 ND 50% (2/4) ND ND ND
 Pond 3 ND 66.67% (2/3) ND ND ND
 Lake 6 ND 33.33% (2/6) ND ND ND
 Tap water 3 ND 33.33% (1/3) ND ND ND
 Rain/tap tank 2 ND NF ND ND ND
 Water dispenser 2 ND 50% (1/2) ND ND ND
 Well 1 ND 100% (1/1) ND ND ND
 Spring 1 ND NF ND ND ND
 Mineral 1 ND NF ND ND ND
 Water 3 ND 100% (3/3) ND ND ND Rivera and Adao (2008) [29]
 Soil 10 ND 100% (10/10) ND ND ND Rivera and Adao (2008) [29]
 Soil 4 ND 100% (4/4) ND ND ND Cruz and Rivera (2014) [25]
 Contact lens storage cases 4 ND 100% (4/4) ND ND ND Rivera and Adao (2008) [29]

Vietnam Soil
 Garden soil 1 359 small sub unit rDNA Sequences of Amoebae 5.95% ND ND ND Denet et al. (2017) [21]
 Mining soil 1 4.76% ND ND ND

Others (Lao PDR, Myanmar, and Singapore) Treated water in Lao PDR 9 11.11% (1/9) NF ND ND ND Majid et al. (2017) [14]
Untreated water in Lao PDR 22 4.55% (1/22) 4.55% (1/22) ND ND ND
Treated water in Yangon 11 18.18% (2/11) NF ND ND ND
Untreated water in Yangon 31 16.13% (5/31) 9.68% (3/31) ND ND ND
Treated water in Singapore 6 NF NF ND ND ND
Untreated water in Singapore 15 NF NF ND ND ND

ND, Not detected; NF, Not found.

Table 2
Examples of Acanthamoeba infection cases in Southeast Asia
Ethnicity/Gender Age (yr) Clinical sample Diagnostic method Condition (Genotype) Potential history of patients Treatment Status after treatment References
Singaporean male 28 Corneal scraping Microscopy and culture AK with Pseudomonas aeruginosa Hit with polyvinylchloride pipe, topical steroids Before diagnosis: cefazolin and gentamicin; After diagnosis: topical cycloplegics; topical 0.1% hexamidine, 0.02% chlorohexidine, and transplantation Vision blurriness Lim et al. (2018) [68]
48/200 felines and 8/25 canines (56/225 naturally-infected animal) in Malaysia Adults and juveniles Corneal swabs Microscopy, culture, and partial 18S rDNA sequencing AK (T4) Dry soil and dust (strain-matched partial 18S rDNA sequence) - - Basher et al. (2018) [24]
Indonesian female 32 Corneal scraping Microscopy and culture AK Monthly disposable soft contact lens wearer for 1 year with tap water to rinse contact lens and case in many occassions Before diagnosis: Steroid eye drops, Moxifloxacin eye drops, natamycin eye drops, polymyxin-neomycin-gramicidin eye drops (Polygran®), and voriconazole eye drops After diagnosis: propamidine isethionate (Brolene®) and Polygran® Improved vision blurriness Muslim et al. (2018) [19]
Thai female 58 Brain abscess CT scan, Microscopy, and PCR on partial 18S rDNA sequencing GAE Farmer with pulmonary tuberculosis history, Raynaud’s phenomenon, mild myositis, and high antinuclear antibody (speckle type) Metronidazole and Prednisolone Loss of follow-up Wara-Asawapati et al. (2017) [22]
Indonesian male 2 Cerebrospinal fluid CT scan and microscopy GAE Drowning survivor Intravenous ceftazidime, metronidazole, fluconazole and rifampicin Alive with altered mental status Gunawan et al. (2016) [69]
Filipino male 76 Corneal scraping Microscopy, culture, and partial 18S rDNA sequencing AK (T4) Non-contact lens wearer Chlorhexidine Corneal scar Buerano et al. (2014) [27]
12/180 Filipinos - Nasal swab Microscopy, culture, and partial 18S rDNA sequencing −(T5, 54, T11) Street sweeper (4/44), Garbage collector (2/37), Garbage sorter (0/16), Landscaper (1/6), Bioreactor laborer (0/4), foremen and superviors (0/3), and students (1/70) - - Cruz and Rivera (2014) [25]
22 cases in Siriraj hospital, Thailand (1996–2006) 48.3±14.5 for 8 non-contact lens wearers, 30.6±15.3 for 12 contact lens wearers Corneal scraping Microscopy and culture AK Contact lens wearer with lack of hygiene Chlorhexidine, polyhexamethylene biguanide or propamidine Improved vision blurriness and loss of follow-up for some patients Wanachiwanawin et al. (2012) [70]
9/103 infective keratitis patients with eye surgery - - - AK - Polyhexamethylbiguanide, chlorohexidine, propamidine dexamethasone, hexamidine, and transplantation Improved vision blurriness Anshu et al. (2009) [71]
22 Chinese, 8 Malay, 5 Indian, 7 others (2005–2007 in Singapore) <20 years-old=13, 21–40 years-old=25, 41–60 years-old=4 Corneal scraping, biopsy, and keratoplasy specimen Microscopy and culture AK Suboptimal hygiene practices 0.02% topical polyhexamethylene niguanide, 0.02% chlorohexidine, 0.1% hexamidine, 0.1% propamidine isethionate, and transplantation. Vision blurriness Por et al. (2009) [72]
3 Filipinos - Corneal scraping Microscopy AK Non-contact lens wearer 0.1% topical diclofenac sodium and atropine drops. Vision blurriness in 2/3 patients Agahan et al. (2009) [73]
3 AK patients/127 microbial keratitis eyes (2001–2004) in Ramathibodi Hospital, Thailand Mean age 40±22 for all 127 microbial keratitis patients Corneal scraping Microscopy and culture AK Contact lens wearers - - Sirikul et al. (2008) [74]
Chinese female 13 Corneal scraping Microscopy and culture AK Rigid gas-permeable contact lens wearer Before diagnosis; Acantamoebic agents: 0.02% topical polyhexamide methylene biguanide, 0.02% chlorohexidine, 0.1% hexamidine, and transplantation. After surgery: 0.1% topical dexamethasone phosphate, 0.5% levofloxacin, same Acanthamoebic agents, and topical preservative-free steroids. Improved vision blurriness Parthasarathy and Tan (2007) [75]
Thai female - Biopsy and autopsy Microscopy GAE Swimming in a dam - Death Siripanth (2005) [76]
Thai male 36 Nasal exudate Microscopy and culture Amoeba co-infection sinusitis (Naegleria sp. and Acanthamoeba sp.) Diving in a natural pond Caldwell-Luc operation, Intravenous amphotericin B, oral ketoconazole, and amoxycillin/clavulanic acid Cured Sukthana et al. (2005) [77]
Singaporean female 39 Corneal scraping Microscopy and culture AK Contact lens wearer with multipurpose disinfectant solution Misdiagnosis: Occulentum Acyclovir, Guttae Choramphenicol, and 0.12% Guttae Prednisolone; After diagnosis: 0.1% gutt propamidine isethionate, 0.02% gutt polyhexamethyline biguanide, and laser In Situ keratomileusis (LASIK) for Myopia Improved vision blurriness and nearsightedness Lim and Wei (2004) [78]
Malaysian male 28 Corneal scraping Microscopy and culture AK Construction worker eye washed with water from open tank after sand and dust strucked in the eye Topical Propamidine isethionate, Chlorhexidine 0.02% and fortified Gentamycin Improved vision blurriness but loss of follow-up Kamel et al. (2005) [26]
Chinese male 24 Corneal scraping Microscopy and culture AK Non-disposable soft contact lens wearer and no contact lens when swim in lake/pool Before diagnosis: gutt spersadexoline; After diagnosis: 0.1% gutt propamidine isethionate, and gutt tobramycin Stromal scar Cheng et al. (2000) [79]
Malay male 26 Corneal scraping Microscopy and culture AK Non-disposable soft contact lens wearer Before diagnosis: tetracycline ointment and neosporin eyedrops; After diagnosis: 0.1% gutt propamidine isethionate Stromal scar Cheng et al. (2000) [79]
Thai female 58 Corneal scraping Microscopy, culture and mtDNA-RFLP AK Left eye injured by straw fragment and dirt cleaned off from her face using water in a jar near her home after digging in the garden on the outskirts Before diagnosis: antimicrobial eye drops and ointment, 1% trifluorothymidine eye drops and acyclovir eye ointment; After diagnosis: ketoconazole eye drops, neosporin, polymyxin, neomycin, gramicidin, propamidine isethionate eye drops, dibromopropamidine isethionate eye ointment, and transplantation. Recurrence necessitating evisceration Jongwutiwes et al. (2000) [80]
Thai male 30 Corneal scraping Microscopy, culture, and mtDNA-RFLP AK Splashing fish pond water to left eye injured by tiny piece of bamboo Before diagnosis: miconazole and neosporin eye drops; After diagnosis: propamidine isethionate eye drops, and dibromopropamidine isethionate eye ointment Vision blurriness Jongwutiwes et al. (2000) [80]
Thai female 57 Corneal scraping Microscopy, culture, indirect immunofluorescence testing, and isoenzyme analysis AK Pond water for washing Before diagnosis: spersapolymyxin eyedrops, cefazolin and gentamicin subconjunctival injection, topical neomycin sulfate, polymyxin B, and gramicidin; After diagnosis: 0.006% chlorhexidine hydrochloride with antidlaucoma for recurrence Improved vision blurriness with cataract Kosrirukvongs et al. (1999) [81]
Thai male 36 Corneal scraping Microscopy, culture, indirect immunofluorescence testing, and isoenzyme analysis AK Dust Before diagnosis: topical neomycin sulfate, polymyxin B, and gramicidin; After diagnosis: 0.006% chlorhexidine solution Loss of follow-up but no recurrence Kosrirukvongs et al. (1990) [81]
Thai female 33 Corneal scraping Microscopy, culture, indirect immunofluorescence testing, and isoenzyme analysis AK Daily-wear soft contact lenses Before diagnosis: fortified cefazolin, gentamicin, neomycin, topical tobramycin, topical neomycin sulfate, polymyxin B, and gramicidin; After diagnosis: 0.006% chlorhexidine solution Improved vision blurriness Kosrirukvongs et al. (1990) [81]
Thai male 74 Corneal scraping Microscopy, culture, indirect immunofluorescence testing, and isoenzyme analysis AK Plant root exposure Before diagnosis: antibiotics and plant root, topical neomycin sulfate, polymyxin B, and gramicidin; After diagnosis: 0.006% chlorhexidine solution and 1% topical clotrimazole eyedrops Note: non-compliance Enucleation Kosrirukvongs et al. (1990) [81]
Thai female 65 Corneal scraping Microscopy, culture, indirect immunofluorescence testing, and isoenzyme analysis AK Unknown Before diagnosis: topical neomycin sulfate, polymyxin B, and gramicidin; After diagnosis: cefazolin and gentamicin eye drops for P. aeruginosa as well as chlorhexidine for Acanthamoeba sp. Vision blurriness with cataract Kosrirukvongs et al. (1990) [81]
Malaysian female 40 Corneal scraping Microscopy AK with P. auruginosa and E. coli Contact lens wearer Before diagnosis: Zovirax® After diagnosis: gentamycin and homatropin eye drops, neosporin, miconazole eyedrops and Brolene® (0.1% Propamidine isethionate) - Kamel and Norazah (1995) [82]
Thai female 26 Brain autopsy Microscopy and indirect immunofluorescence test GAE Worker - Death Sangruchi et al. (1994) [83]
Thai male 20 Brain autopsy Microscopy and indirect immunofluorescence test GAE Farmer - Death Sangruchi et al. (1994) [83]
Thai female 42 Biopsy Radiography and microscopy Proliferated gastric ulcer with gastric acanthamoebiasis and sepsis from operative site with E. coli and K. pneumoniae Immunocompetent patients Venesection and rapid fluid replacement, antibiotics, gastrojejunostomy, and parenteral ampicillin, gentamicin, and metronidazole Death Thamprasert et al. (1993) [84]

AK, Acanthamoeba keratitis; GAE, Granulomatous amoebic encephalitis; -, Not mentioned in the published paper.

Table 3
Anti-Acanthamoeba agents and nanoparticles in ASEAN studies
Anti-Acanthamoeba agents Nanotechnology Anti-Acanthamoeba activity against References

Cysts Trophozoites
 Cyclic samarium complexes [Sm(Pic)2(18C6)] (Pic) - - IC50=6.5 μg/ml against Acanthamoeba keratitis isolate Kusrini et al. (2018; Indonesia) [85]
 Acyclic samarium complexes [Sm(Pic)2(18C6)] (Pic) - - IC50=0.7 μg/ml against Acanthamoeba keratitis isolate Kusrini et al. (2018; Indonesia) [85]
 Terbium complex [Tb(NO3)3(OH2)3](18C6) - - IC50=7 μg/ml against Acanthamoeba keratitis isolate Kusrini et al. (2016; Indonesia) [86]
 Tb(NO3)3.6H2O in CH3CN - - IC50=2.6 μg/ml against Acanthamoeba keratitis isolate Kusrini et al. (2016; Indonesia) [86]
 18C6 in CH3CN - - IC50=1.2 μg/ml against Acanthamoeba keratitis isolate Kusrini et al. (2016; Indonesia) [86]
 Phosphanegold (I) thiolates - - No effect on viability, growth, cellular differentiation, and extracellular proteolytic activities against A. castellanii (ATCC50492) Siddiqui et al. (2017; Malaysia) [87]
 3% DMSO - Encystation induction and excystation inhibition against A. castellanii (ATCC50492) - Siddiqui et al. (2016; Malaysia) [88]
 Carbonyl Thiourea derivatives - - IC50=2.39–8.77 μg/ml against A. castellanii (CCAP 1501/2A) and 3.74–9.30 μg/ml against A. polyphaga (CCAP 1501/3A). Ibrahim et al. (2014; Malaysia) [89]
 Commercial fusaric acid - - IC50=0.33, 0.33, 0.66 μM against Acanthamoeba keratitis isolate and 2 soil isolates, respectively Boonman et al. (2012; Thailand) [90]
 Betadine® solution - MCC=0.04% dilution after 24 hr against Acanthamoeba keratitis isolate - Roongruangchai et al. (2011; Thailand) [91]
Virkon® solution - MCC=0.25% dilution after 24 hr against Acanthamoeba keratitis isolate - Roongruangchai et al. (2010; Thailand) [92]

Plant products
 Hesperidin, commercial flavonoid from Citrus sp. Silver nanoparticles stabilized by gum acacia Encystation and excystation inhibition against A. castellanii (ATCC 50492) 100% abolished amoeba viability of 5×105 A. castellanii (ATCC 50492) at 50μg/ml Anwar et al. (2019; Malaysia) [93]
 Naringin, commercial flavonoid, from Citrus sp. Gold nanoparticles stabilized by gum tragacanth Encystation and excystation inhibition against A. castellanii (ATCC 50492) Significanly abolished amoeba viability of 5×105 A. castellanii (ATCC 50492) at 50μg/ml Anwar et al. (2019; Malaysia) [93]
 Periglaucine A from Pericampylus glaucus Poly (DL-lactide-co-glycolide) CC50/IC50=100 against A. triangularis from environmental water sample CC50/IC50=25 against A. triangularis from environmental water sample Mahboob et al. (2018; Malaysia) [94]
 Betulinic acid from Pericampylus glaucus Poly (DL-lactide-co-glycolide) CC50/IC50=10 against A. triangularis from environmental water sample CC50/IC50=5 against A. triangularis from environmental water sample Mahboob et al. (2018; Malaysia) [94]
 Periglaucine A from Pericampylus glaucus - CC50/IC50=8.5 against A. triangularis from environmental water sample CC50/IC50=170 against A. triangularis from environmental water sample Mahboob et al. (2017; Malaysia) [95]
 Betulinic acid from Pericampylus glaucus - CC50/IC50=3.75 against A. triangularis from environmental water sample CC50/IC50=1.5 against A. triangularis from environmental water sample Mahboob et al. (2017; Malaysia) [95]
 Cinnamic acid from Cinnamomum cassia Gold nanopaticles Encystation inhibition against A. castellanii (ATCC 50492) Significantly enhanced anti-Acanthamoeba activity against A. castellanii (ATCC 50492) when compared with cinnamic acid alone Anwar et al. (2018; Malaysia) [96]
 Ethyl acetate, water, butanol fractions from Lonicera japonica - - Significant anti-Acanthamoeba effect against environmental A. triangularis trophozoites by ethyl acetate (most potent fraction) and cyst:trophozoites ratio reduction by commercial chlorogenic acid (major constituent in L. japonica) Mahboob et al. (2016; Malaysia) [97]
Pouzolzia indica methanolic extract fraction 2 - MCC=1: 4 dilution after 24 hr against Acanthamoeba keratitis isolate - Roongruangchai et al. (2011; Thailand) [91]
Pouzolzia indica methanolic extract fraction 3 - MCC=1: 8 dilution after 24 hr against Acanthamoeba keratitis isolate - Roongruangchai et al. (2010; Thailand) [92]

Microorganism products
 Supernatants from bacteria isolated from cockroach gut: Serratia marcescens and Escherichia coli from Madagascar cockroach; two Klebsiella spp., Citrobacter sp., Bacillus sp., Streptococcus sp. from Dubia cockroach - - Significant anti-Acanthamoeba effect against A. castellanii (ATCC 50492) Akbar et al. (2018; Malaysia) [98]
 Effective microorganisms (EM™) - Undiluted, 1:2, 1:4, 1:6 dilution of EM resulted in lower than 40% viable cysts - Sampaotong et al. (2016; Thailand) [99]
 Fusaric acid from Fusarium fujikuroi species complex Tlau3 isolated from Thunbergia laurifolia - - IC50=0.31 μm against Acanthamoeba keratitis isolate Boonman et al. (2012; Thailand) [90]
 Dehydrofuseric acid from Fusarium fujikuroi species complex Tlau3 isolated from Thunbergia laurifolia - - IC50=0.34 μm against Acanthamoeba keratitis isolate Boonman et al. (2012; Thailand) [90]

 Nystatin, Fluconazole, and Amphotericin B Gold nanoparticles - Enhanced anti-Acanthamoeba activity at 10 μM (Amphotericin B>Fluconazole>Nystatin) against A. castellanii (ATCC 50492) Anwar et al. (2019; Malaysia) [100]
 Nystatin, Fluconazole, and Amphotericin B Silver nanoparticles - Enhanced anti-Acanthamoeba activity at 10 μM (Amphotericin B and Nystatin but not Fluconazole) against A. castellanii (ATCC 50492) Anwar et al. (2018; Malaysia) [101]
 Diazepam (Valium), Phenobarbitone (Luminal), and Phenytoin (Dilantin) And their silver nanoparticles Anti-Encystation activity (Diazepam and Phenobarbitone activity enhanced with silver nanoparticles) and anti-cyst activity (Phenobarbitone and Phenytoin activity enhanced with silver nanoparticles) against A. castellanii (ATCC 50492) Anti-Acanthamoeba activity observed and enhanced activity with silver nanoparticles against A. castellanii (ATCC 50492) Anwar et al. (2018; Malaysia) [102]
 Diclofenac sodium and Indomethacin (NSAIDs) - Encystation inhibition of A. castellanii (ATCC 50492) Growth affected but not viability of A. castellanii (ATCC 50492) Siddiqui et al. (2016; Malaysia) [103]
Acetaminophen (NSAIDs) - No effects on encystation inhibition of A. castellanii (ATCC 50492) No effects on growth of A. castellanii (ATCC 50492) Siddiqui et al. (2016; Malaysia) [103]
 Bortezomib (proteasome inhibitor) - Encystation inhibition against A. castellanii (ATCC 50492) Static effect on growth but not viability of A. castellanii (ATCC 50492) Siddiqui et al. (2016; Malaysia) [104]
 Lactacystin and active form as clasto-lactacystin β-lactone (proteasome inhibitors) - Encystation inhibition and excystation inhibition against A. castellanii (ATCC 50492) No effects on growth and viability of A. castellanii (ATCC 50492) Siddiqui et al. (2016; Malaysia) [104]
 Artesunate (Antimalaria) - Presence of cytostatic effect on Acanthamoeba polyphaga-like amoebae were isolated from natural water courses at concentrations of 500–700 μg/ml Dose-dependent growth inhibition (5–700 μg/ml) against Acanthamoeba polyphaga-like amoebae were isolated from natural water courses Nacapunchai et al. (2003; Thailand) [105]
 Metronidazole - No effects (5–1,000 μg/ml) No effects (5–1,000 μg/ml) Nacapunchai et al. (2003; Thailand) [105]

Animal products
 Crocodile (Crocodylus palustris) serum - - Anti-Acanthamoeba activity against A. castellanii (ATCC 50492) Siddiqui et al. (2017; Malaysia) [106]
 Sea sponge crude methanol extracts (Aaptos aaptos) from different localities - - IC50=0.615–0.876 μg/ml against clinical A. castellanii Nakisah et al. (2012; Malaysia) [107]

IC, Inhibition concentration; CC, Cytotoxicity concentration; MCC, Minimal cystcidal concentation; -, Not mentioned in the published paper.

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