ABSTRACT
Objective: To determine the morphological and molecular characterization of Acanthamoeba isolates from contact lens paraphernalia in Malaysia and to investigate their pathogenic potential based on the physiological tolerance. Methods: One hundred and eighty contact lens wearers donated their contact lens, lens storage cases and lens solutions between 2018 and 2019. The samples were inoculated onto 1.5% non-nutrient agar plates for 14 d. Polymerase chain reaction (PCR) was performed and the amplified PCR products were sequenced and compared with the published sequences in GenBank. The pathogenic potential of positive isolates was further tested using temperature-tolerance and osmo-tolerance assays. Acanthamoeba species were categorized into three distinct morphological groups established by Pussard and Pons. Results: Acanthamoeba was successfully isolated from 14 (7.8%) culture-positive samples in which 11 belong to morphological group II and 3 belong to morphological group III, respectively. The sequencing of 18S ribosomal RNA gene led to the identification of the T4 genotype in all the isolated strains. In vitro assays revealed that 9 (64.3%) Acanthamoeba isolates were able to grow at 42 °C and 1 M mannitol and were thus considered to be highly pathogenic. Conclusions: To the best of our knowledge, this is the first report identifying the Acanthamoeba genotype and their pathogenic potential among contact lens wearers in Malaysia. The potentially pathogenic T4 genotype isolated in this study is the most predominant genotype responsible for human ocular infection worldwide. Hence, increasing attention should be aimed at the prevention of contamination by Acanthamoeba and the disinfection of contact lens paraphernalia.
ABSTRACT
Objective To determine antiacanthamoebic activity of natural and marketed honey samples. Methods Natural honey samples were collected directly from the bee hive and marketed honey samples were purchased from the local market in Karachi, Pakistan. Both honey samples were tested for their flavonoid content (quercetin equivalent per gram of the extract) and phenolic content (gallic acid equivalent per gram). Furthermore, their antioxidant activity was determined by measuring 2,2-diphenyl-1-picrylhydrazyl. Using amoebistatic and amoebicidal assays, the effects of honey samples were tested against growth and viability of Acanthamoeba parasites. Results Natural honey exhibited potent amoebistatic and amoebicidal effects, in a concentration-dependent manner. Honey-treated Acanthamoeba castellanii showed loss of acanthopodia, following which amoebae detached, rounded up, reduced in size, decreased in cytoplasmic mass and they were observed floating in the culture medium. Importantly, honey-treated amoebae did not revive when inoculated in fresh growth medium, however, glycerol-treated amoebae exhibited viable trophozoite and active growth. In contrast, marketed honey samples varied in their efficacy against Acanthamoeba castellanii. The proportion of flavonoid, as determined by quercetin measurements and the proportion of phenolic, as determined by gallic acid measurements was higher in natural honey compared with marketed honey. Similarly, the antioxidant activity, as determined by 2,2-diphenyl-1-picrylhydrazyl scavenging activity was higher in natural honey vs. marketed honey. Conclusions This study shows that natural honey has antiacanthamoebic properties and possesses higher flavonoid, phenolic and antioxidant properties compared with the marketed honey. These findings are of concern to the public, health officials, and to the manufacturers regarding production of honey for medical applications.