Survey and first report of Acanthamoeba T4 genotype in natural spring water resources in the Black Sea, Turkey

Infection with Acanthamoeba spp. may result in granulomatous amoebic encephalitis and Acanthamoeba keratitis. Water is an important habitat where Acanthamoeba species thrive. Therefore, studying the occurrence of this free-living amoeba in water sources will help understand the infection dynamics. The aim of the study was to survey and report on the presence of Acanthamoeba spp. in water resources from the Ordu and Giresun provinces in Black Sea. Acanthamoeba spp. was found in 1/17 natural spring water samples from Ordu and in 2/18 from Giresun. Acanthamoeba species were not detected in any of the investigated tap water samples. Sequencing of the (SSU) rDNA gene resulted in the identification of haplotype I (Acanthamoeba genotype: KJ094684). T4 (8.6%) was the only isolated genotype in both Ordu and Giresun provinces. This is the first report of Acanthamoeba T4 genotype in natural spring water resources in the Black Sea. The occurrence of Acanthamoeba species in natural spring water sources should be considered as a potential risk for human infection, especially to high-risk populations.

Evaluation of Amebicidal and Cysticidal Activities of Antifungal Drug Isavuconazonium Sulfate against Acanthamoeba T4 Strains

Acanthamoeba species of amebae are often associated with Acanthamoeba keratitis, a severe corneal infection. Isavuconazonium sulfate is an FDA-approved drug for the treatment of invasive aspergillosis and mucormycosis. This prodrug is metabolized into the active isavuconazole moiety. Isavuconazole was previously identified to have amebicidal and cysticidal activity against Acanthamoeba T4 strains, but the activity of its prodrug, isavuconazonium sulfate, against trophozoites and cysts remains unknown. Since it is not known if isavuconazonium can be metabolized into isavuconazole in the human eye, we evaluated the activities of isavuconazonium sulfate against trophozoites and cysts of three T4 genotype strains of Acanthamoeba. Isavuconazonium displayed amebicidal activity at nanomolar concentrations as low as 1.4 nM and prevented excystation of cysts at concentrations as low as 136 μM. We also investigated the cysticidal activity of isavuconazonium sulfate in combination with a currently used amebicidal drug polyhexamethylene biguanide (PHMB). Although combination of isavuconazonium with PHMB did not elicit an obvious synergistic cysticidal activity, the combination did not cause an antagonistic effect on the cysts of Acanthamoeba T4 strains. Collectively, these findings suggest isavuconazonium retains potency against Acanthamoeba T4 strains and could be adapted for Acanthamoeba keratitis treatment.

An Evaluation of a Simplified Impression Membrane Sampling Method for the Diagnosis of Microbial Keratitis

The purpose of this study was to compare bacterial isolation rate using a corneal impression membrane (CIM) and a sharp instrument for obtaining corneal samples from patients with suspected microbial keratitis (MK). Data was retrospectively collected for all patients that had corneal samples taken for presumed MK between May 2014 and May 2020. Prior to May 2017 samples were collected by scraping the edges of the ulcer with a blade. From May 2017, samples were collected by placing a CIM (Millicell cell culture insert) against the ulcer. All corneal samples were processed using the same conventional diagnostic culture method. A total of 3099 corneal samples were included, of which 1214 (39.2%) were corneal scrapes and 1885 (60.9%) CIMs. Microorganisms were isolated from 235 (19.4%) and 1229 (65.2%) cases using a corneal scrape and CIM, respectively (p < 0.001). Of routinely described pathogenic microorganisms, there were significant increases in the isolations of S. aureus (2.4% to 11.3%) and Serratia (0.5% to 1.7%) using the CIM and no significant changes in the isolations of S. pneumoniae and P. aeruginosa. No significant differences were seen between the isolation rates of fungi or Acanthamoeba species. There was a significant increase in the isolation rates of other Streptococcal species (0.7% to 6.9%) and CNS species, specifically, S. epidermidis (2.1% to 26.2%), S. capitis (0.4% to 2.6%) and S. warneri (0.3% to 1.6%) using the CIM. The simplified CIM sampling method is an effective method for collecting corneal samples from patients with presumed MK in clinical practice.

Acanthamoeba species isolated from marine water in Malaysia exhibit distinct genotypes and variable physiological properties

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.

Investigating Domestic Shower Settings as a Risk Factor for Acanthamoeba Keratitis

Acanthamoeba keratitis (AK) is a sight-threatening infection of the cornea, which is caused by soil and the waterborne protist Acanthamoeba spp. AK most commonly occurs during contact lens (CL) wear. Risk factors for AK have been linked to non-optimal lens hygiene practices and Acanthamoeba contamination of domestic water. This study investigated the prevalence of Acanthamoeba species in domestic showers in the greater Sydney region, as well as the perception of water contamination of CL as being a risk factor for AK among previous AK patients and their family and friends. Samples from four locations of 13 participants’ shower areas were cultured and Acanthamoeba 18S rRNA was amplified by PCR, followed by sequencing. Twenty-six responses were received to the online questionnaire. Fifteen water samples (29%, 15/52) contained amoeba that were morphologically classified as Acanthamoeba spp. PCR amplification confirmed the presence of Acanthamoeba spp. in four samples (8%, 4/52). Three isolates belonged to the T4, and one isolate to the T3 genotype. On the questionnaire survey, 96% (25/26) of respondents believed that water contamination was likely to be a risk factor for Acanthamoeba keratitis and 58% rated showering with CL as ‘extremely likely’ to be a risk factor for AK. Acanthamoeba pathogenic genotypes (T3 and T4) in the domestic bathroom water suggest that clinicians should remain vigilant in educating CL wearers about avoiding domestic water contamination of CL.

Molecular detection of Acanthamoeba spp. in Seven Crater Lakes of Laguna, Philippines

Acanthamoeba spp. are ubiquitous free-living amoeba with genotypes that cause severe pathology of the eyes, central nervous systems, and rare reports of cutaneous infections. The Seven Crater Lakes are freshwater water resources in Laguna, Philippines primarily used for aquaculture and tourism. A total of 16 surface water samples were collected from different sampling areas per Crater Lake and placed in sterile plastic containers. Samples were filtered using 1.2 μm pore size, glass microfiber filter. Filtered sediments were placed on non-nutrient agar lawned with Escherichia coli and incubated aerobically at 35 °C for 14 days. Six out of 16 water samples exhibited amoebic growth. Cystic stages revealed circular to stellate morphology under light microscopy which were initially classified as Acanthamoeba spp. DNA from positive isolates were made to react with polymerase chain reaction using Acanthamoeba specific primers JDP1 5′-GGCCCAGATCGTTTACCGTGAA-3′ and JDP2 5′-TCTCACAAGCTGCTAGGGAGTCA-3′confirmed the presence of several Acanthamoeba species. Phylogenetic analysis revealed the presence of seven isolates belonging to Acanthamoeba genotypes T4, T5, and T9. The presence of potentially pathogenic Acanthamoeba genotypes in the Seven Crater Lakes of Laguna signifies risk to human health which necessitates the development of programs, policies, and guidelines on the understanding, prevention, and management of potential human infections.

Acanthamoeba Keratitis and Acanthamoeba Conjunctivitis: A Case Report

Acanthamoeba species are vision-threatening agents by causing cornea infections known as Acanthamoeba keratitis. A 5 year-old kid with the complaints of erythema, eyelid edema, inflammation, limitation of eye movements in the right eye, and having no history of wearing contact lenses or trauma, was diagnosed of Acanthamoeba conjunctivitis through laboratory examinations in the Ophthalmology clinic. The visual sharpness of the patient improved after the treatment. A 44 year-old female patient suffering from pain, stinging, irritation, and inability to see in the left eye with the history of wearing contact lenses or trauma was diagnosed of Acanthamoeba keratitis through laboratory examinations. The agent was isolated and identified as "A. castellani" in the Genotype "T2". Examination of the left eye on the 15th day of treatment indicated that all complaints disappeared except for the cataract originated visual loss. However, the first diagnosis of Acanthamoeba keratitis appeared in the literature on a case with no history of wearing contact lenses and trauma it is found to be attention grabbing. We think that Acanthamoeba should not be ignored among microbial agents that cause eye infection with or without trauma and contact lens usage history.