This study aimed to identify the Acanthamoeba genotypes and their pathogenic potential in five recreational hot springs in Peninsular Malaysia. Fifty water samples were collected between April and September 2018. Physical parameters of water quality were measured in situ while chemical and microbiological analyses were performed in the laboratory. All samples were filtered through the nitrocellulose membrane and tested for Acanthamoeba using both cultivation and polymerase chain reaction (PCR) by targeting the 18S ribosomal RNA gene. The pathogenic potential of all positive isolates was identified using thermo- and osmotolerance tests. Thirty-eight (76.0%) samples were positive for Acanthamoeba. Water temperature (P = 0.035), chemical oxygen demand (P = 0.026), sulphate (P = 0.002) and Escherichia coli (P < 0.001) were found to be significantly correlated with the presence of Acanthamoeba. Phylogenetic analysis revealed that 24 samples belonged to genotype T4, nine (T15), two (T3) and one from each genotype T5, T11 and T17. Thermo- and osmotolerance tests showed that 6 (15.79%) of the Acanthamoeba strains were highly pathogenic. The existence of Acanthamoeba in recreational hot springs should be considered as a health threat among the public especially for high-risk people. Periodic surveillance of hot spring waters and posting warning signs by health authorities is recommended to prevent disease related to pathogenic Acanthamoeba.

The incidence of Acanthamoeba keratitis has been increasing since the previous decades, especially among contact lens users. This infection is majorly caused by the use of ineffective contact lens disinfecting solution. Thus, this study was conducted to evaluate the in vitro effects of multi-purpose disinfecting solutions (MPDS) against Acanthamoeba trophozoites and cysts. Acanthamoeba genotype T4 isolated from contact lens paraphernalia and an environmental strains were propagated for trophozoite or cyst-containing culture and adjusted in final concentration of 1 × 10 5 cells/ml. Amoebicidal and cysticidal assays were conducted by incubating trophozoites and cysts with OPTI-FREE® Express®, ReNu® Fresh™, Complete® Multi-Purpose Solution and AVIZOR Unica® Sensitive according to the manufacturer’s minimum recommended disinfectant time (MMRDT) for up to 12 h at 30 ⁰C. Trypan blue hemocytometer-based microscopic counts determined amoebicidal and cysticidal effects. The viability of Acanthamoeba trophozoites and cysts was confirmed by re-inoculated them in the 1.5% non-nutrient agar plates. It was found that none of the MPDS showed amoebicidal and cysticidal effects during the MMRDT. However, OPTI-FREE® Express® demonstrated a significant differences in average cell reduction for both stages within MMRDT. When subjected to 12 h exposure, both OPTI-FREE® Express® and ReNu® Fresh™ led to significant reduction in the number of trophozoite and cyst cells. Notably, Complete® Multi-Purpose Solution and AVIZOR Unica® Sensitive did appreciably improve the solution effectiveness towards trophozoite cells when incubated for 12 h. All MPDS were largely ineffective, with 100% survival of all isolates at MMRDT, while OPTI-FREE® Express® showed limited amoebicidal activity against the contact lens paraphernalia isolate, however, it was more against the environmental strains after 12 h incubation time. The commercially available MPDS employed in this research offered minimal effectiveness against the protozoa despite the contact time. Improvement or development of new solution should consider the adjustment of the appropriate disinfectant concentration, adequate exposure time or the incorporation of novel chemical elements, which are effective against Acanthamoeba for accelerated disinfecting and more reduction of potential exposure of contact lens users to Acanthamoeba keratitis.

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The present study identifies the Acanthamoeba genotypes and their pathogenic potential in five marine waters in Malaysia. Fifty water samples were collected between January and May 2019. Physical parameters of water quality were measured in situ, whereas chemical and microbiological analyses were conducted in the laboratory. All samples had undergone filtration using nitrocellulose membrane and tested for Acanthamoeba using cultivation and polymerase chain reaction by targeting the 18S ribosomal RNA gene. The pathogenic potential of all positive isolates was identified using physiological tolerance tests. Thirty-six (72.0%) samples were positive for Acanthamoeba. Total coliforms (p = 0.013) and pH level (p = 0.023) displayed significant correlation with Acanthamoeba presence. Phylogenetic analysis showed that 27 samples belonged to genotype T4, four (T11), two (T18) and one from each genotype T5, T15 and T20. Thermo- and osmo-tolerance tests signified that three (8.3%) Acanthamoeba strains displayed highly pathogenic attributes. This study is the first investigation in Malaysia describing Acanthamoeba detection in marine water with molecular techniques and genotyping. The study outcomes revealed that the marine water in Malaysia could be an integral source of acanthamoebic strains potentially pathogenic in humans. Thus, the potential risk of this water should be monitored routinely in each region.

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