Shanmuganathan, Vydeki
(2019)
Epidemiology of Acanthamoeba species in water treatment works in England.
PhD thesis, University of Nottingham.
Abstract
Acanthamoeba spp. are often described as emerging opportunistic protozoan pathogens. Infection is associated with two main clinical diseases: Acanthamoeba keratitis (AK), and infection of the central nervous system in immunocompromised individuals, granulomatous amoebic encephalitis (GAE). The risk factors for AK are contaminated water and wearing contact lenses. Although the occurrence of Acanthamoeba spp. in tap water is well documented, its original source, whether from the public water supply or more locally, within domestic properties, is unknown.
This thesis describes studies of the prevalence of Acanthamoeba spp. in raw water from ground and surface sources, through water treatments works (WTW), to the tap water supplied to customers. The four WTW studied had different processes, depending on their raw water source. Acanthamoeba spp. were isolated from water samples using membrane filtration, cultured on non-nutrient agar (NNA) seeded with E.coli and incubated for 14 days. Light microscopy was used to observe trophozoites and cysts of typical morphology, and isolation was confirmed by PCR using genus-specific primers that target the 18S rDNA and sequencing of the PCR amplicons.
Acanthamoeba spp. were isolated from 100% of the samples taken from raw surface waters (river and lake) and 14% of samples from ground water (aquifers). They were not isolated from any of the samples taken after completion of water treatment, treated water storage tanks, or water from customers’ taps.
All the isolates appeared to be closely related to each other and part of the T4 genotype, which has previously been found to be the most common cause of clinical AK, and also the genotype most widespread in the environment.
Future work should therefore focus on the diversity and distribution of Acanthamoeba spp. within household systems. Whole genome sequence of isolates might identify areas of the genome more useful for studying molecular ecology in water systems. The future development of PCR diagnostics might also improve both our understanding of prevalence and of the molecular ecology of Acanthamoeba spp.
Finally, a previously published protocol was adapted in order to test the susceptibility of an environmental isolate to disinfectants, in this case chlorhexidine (CHX), using a colorimetric sulforhodamine B stain. However, the consistency of the assay, previously used only with laboratory-adapted isolates, was poor, and only non-significant results were obtained.
In conclusion, Acanthamoeba spp. was found to be present in water entering WTW but the processes in place were effective at removing them. In contrast to previous studies, no Acanthamoeba spp. were isolated from tap water, and further investigation is needed to explore the plumbing, tap hygiene and water storage in domestic properties. Finally, it is not clear if the disinfectant assay protocol is suitable for environmental, low passage isolates, so further work is needed to improve the assay.
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