Acanthamoeba Isolates Research Articles

Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes from environmental freshwater samples in the Nile Delta region, Egypt

The free-living amoebae of the genus Acanthamoeba include non-pathogenic and pathogenic species and has been recently classified into 15 different genotypes, T1–T15. In this study, a survey was conducted in order to determine the presence and pathogenic potential of free-living amoebae of Acanthamoeba genus in freshwater sources associated with human activities in the Nile Delta region, Egypt. Identification of Acanthamoeba was based on the morphology of cyst and trophozoite forms and PCR amplification with a genus specific primer pair. The pathogenic potential of Acanthamoeba isolates was characterized using temperature and osmotolerance assays and PCR reactions with two primer pairs specific to Acanthamoeba pathogenesis. Isolates genotypes were also determined after ribosomal DNA sequencing. These data revealed that isolates belong to T1, T2, T3, T4 and T7 genotypes. As expected, T4 isolates exhibited the most pathogenic traits and were osmotolerant, temperature tolerant and expressed extracellular serine proteases. This is the first report presenting environmental distribution of Acanthamoeba genotypes in Egypt.

Molecular identification of free-living amoebae of the Vahlkampfiidae and Acanthamoebidae isolated in Arizona (USA)

Sediment samples from rivers, canals and lakes in Arizona (USA) were cultured for free-living amoebae at three different incubation temperatures (22, 37 and 40 °C). Isolates belonging to the Vahlkampfiidae were identified by sequencing the PCR-amplified ITS1, 5.8S and ITS2 rDNA. With this molecular method three Naegleria spp. were identified, N. gruberi sensu stricto, N. australiensis and N. tihangensis. Also a strain each of Willaertia magna and Vahlkampfia avara were identified. Three samples yielded two new Tetramitus spp. of which the closest relative is T. ovis. Many Acanthamoeba strains were also isolated. The genotype of these strains was identified using Acanthamoeba-specific primers (JDP1 and JDP2) amplifying a part of the SSUrDNA and sequencing with an internal primer (892c). Five of the Acanthamoeba isolates belong to genotype T5 ( A. lenticulata), while five are genotype T4.

Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis

BackgroundGranulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined.ResultsUsing an encephalitis isolate belonging to T1 genotype, we observed two major proteases with approximate molecular weights of 150 KD and 130 KD on SDS-PAGE gels using gelatin as substrate. The 130 KD protease was inhibited with phenylmethylsulfonyl fluoride (PMSF) suggesting that it is a serine protease, while the 150 KD protease was inhibited with 1, 10-phenanthroline suggesting that it is a metalloprotease. Both proteases exhibited maximal activity at neutral pH and over a range of temperatures, indicating their physiological relevance. These proteases degrade extracellular matrix (ECM), which provide structural and functional support to the brain tissue, as shown by the degradation of collagen I and III (major components of collagenous ECM), elastin (elastic fibrils of ECM), plasminogen (involved in proteolytic degradation of ECM), as well as casein and haemoglobin. The proteases were purified partially using ion-exchange chromatography and their effects were tested in an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC). Neither the serine nor the metalloprotease exhibited HBMEC cytotoxicity. However, the serine protease exhibited HBMEC monolayer disruptions (trypsin-like) suggesting a role in blood-brain barrier perturbations.ConclusionOverall, these data suggest that Acanthamoeba proteases digest ECM, which may play crucial role(s) in invasion of the brain tissue by amoebae.

In Vitro Pathogenicity ofAcanthamoebaIs Associated with the Expression of the Mannose-Binding Protein

To determine whether the expression of Acanthamoeba mannose-binding protein (MBP) is associated with the pathogenicity of the parasite in vitro. Both active trophozoites and dormant cysts of a pathogenic strain of A. castellanii were analyzed for their ability to bind to corneal epithelium, express MBP, and produce a cytopathic effect (CPE) on host cells. In addition, host cell binding, CPE-inducing ability, and MBP expression pattern of trophozoites of four different isolates of Acanthamoeba with various degrees of in vitro pathogenicity were analyzed. Binding assays were performed with radiolabeled parasites; CPE assays were performed with rabbit corneal epithelial cells as host cells; and the expression of MBP was detected by affinity chromatography of parasite extracts on mannose affinity columns and by immunohistochemical and Western blot analyses. Trophozoites of A. castellanii bound avidly to corneal epithelial cells in a mannose-inhibitable manner, whereas cysts exhibited little binding. The lack of binding of the cysts to host cells was associated with the downregulation of MBP, along with the concomitant loss of CPE. Analysis of trophozoites of five different species of Acanthamoeba exhibiting various degrees of pathogenic potential revealed that the ability of parasites to bind to host cells and produce CPE is directly correlated with the expression of the MBP. Acanthamoeba strains that bound avidly to host cells and produced potent CPE, robustly expressed MBP. In contrast, parasite strains that produced only weak CPE, expressed markedly reduced levels of MBP. The data demonstrating that the pathogenic potential of Acanthamoeba directly correlates with the expression level of the MBP in conjunction with our published studies showing that Acanthamoeba MBP is a major virulence protein suggest that the amoeba lectin has the potential to serve as a marker of pathogenicity.

Genetic diversity of Acanthamoeba isolated from ocean sediments.

Genetic diversity of 18 Acanthamoeba isolates from ocean sediments was evaluated by comparing mitochondrial (mt) DNA RFLP, 18S rDNA sequences and by examining their cytopathic effects on human corneal epithelial cells versus reference strains. All isolates belonged to morphologic group II. Total of 16 restriction phenotypes of mtDNA from 18 isolates demonstrated the genetic diversity of Acanthamoeba in ocean sediments. Phylogenetic analysis using 18s rDNA sequences revealed that the 18 isolates were distinct from morphological groups I and III. Fifteen isolates showed close relatedness with 17 clinical isolates and A. castellanii Castellani and formed a lineage equivalent to T4 genotype of Byers group. Two reference strains from ocean sediment, A. hatchetti BH-2 and A. griffini S-7 clustered unequivocally with these 15 isolates. Diversity among isolates was also evident from their cytopathic effects on human corneal cells. This is the first time describing Acanthamoeba diversity in ocean sediments in Korea.

Genetic Relatedness Among Isolates of Acanthamoeba Based on RAPD Analysis

Acanthamoeba is a pathogen frequently infecting brain, eyes, skin and lung of human and animal. Seven Acanthamoeba local isolates from various sources, two Acanthamoeba type strains and one strain of Hartmannella vermiformis were characterized for their genetic variability using randomly amplified polymorphic DNA (RAPD) technique using four different 10-mer oligonucleotides primers. Electrophoresis of the amplification products generated DNA bands ranging from approximately 0.25 to 7.50 kbp in size. A genetic relatedness among the isolates was examined using Dice similarity coefficient as the genetic distance measured between the strains of Acanthamoeba and H. vermiformis. Three distinct clusters could be separated at genetic distance of approximately 0.330. © 2006 Asian Network for Scientific Information.

PathogenicAcanthamoebastrains from water sources in Jamaica, West Indies

In 2004, samples of tap water and of river and sea water associated with human activities were collected in Jamaica, West Indies, and checked for free-living Acanthamoeba. The morphologies of the cysts and trophozoites observed and the results of PCR-based amplifications with a genus-specific primer pair were used to identify the Acanthamoeba isolates. The potential of each isolate as a human pathogen was then evaluated using thermotolerance and osmotolerance assays and two PCR-based assays for Acanthamoeba pathogenesis. Acanthamoeba were identified in 36.1%, 26.4% and 49.6% of the tap-, river- and sea-water samples collected, respectively. Pathogenic potential was shown by 60.0% of the Acanthamoeba strains isolated from tap water, 68.4% of the strains from river water, and 40.4% of the seawater strains. Sequencing of ribosomal DNA revealed the T1, T2, T4, T5, T7, T9 and T11 genotypes. Isolates of the T4 genotype were collected from tap, rain and sea water and, as expected, exhibited the most pathogenic traits; most were osmotolerant, thermotolerant and expressing extracellular serine protease. This is the first study of the occurrence and distribution of Acanthamoeba in water in the West Indies, and the results confirm the presence of potentially pathogenic strains in Jamaica.

ITS1 sequence variabilities correlate with 18S rDNA sequence types in the genus Acanthamoeba (Protozoa: Amoebozoa)

The subgenus classification of the ubiquitously spread and potentially pathogenic acanthamoebae still poses a great challenge. Fifteen 18S rDNA sequence types (T1-T15) have been established, but the vast majority of isolates fall into sequence type T4, and so far, there is no means to reliably differentiate within T4. In this study, the first internal transcribed spacer (ITS1), a more variable region than the 18S rRNA gene, was sequenced, and the sequences of 15 different Acanthamoeba isolates were compared to reveal if ITS1 sequence variability correlates with 18S rDNA sequence typing and if the ITS1 sequencing allows a differentiation within T4. It was shown that the variability in ITS1 is tenfold higher than in the 18S rDNA, and that ITS1 clusters correlate with the 18S rDNA clusters and thus corroborate the Acanthamoeba sequence type system. Moreover, high sequence dissimilarities and distinctive microsatellite patterns could enable a more detailed differentiation within T4.

Acanthamoeba genotype T4 from the UK and Iran and isolation of the T2 genotype from clinical isolates

The majority of the keratitis-causing Acanthamoeba isolates are genotype T4. In an attempt to determine whether predominance of T4 isolates in Acanthamoeba keratitis is due to greater virulence or greater prevalence of this genotype, Acanthamoeba genotypes were determined for 13 keratitis isolates and 12 environmental isolates from Iran. Among 13 clinical isolates, eight (61.5%) belonged to T4, two (15.3%) belonged to T3 and three (23%) belonged to the T2 genotype. In contrast, the majority of 12 environmental isolates tested in the present study belonged to T2 (7/12, 58.3%), followed by 4/12 T4 isolates (33.3%). In addition, the genotypes of six new Acanthamoeba isolates from UK keratitis cases were determined. Of these, five (83.3%) belonged to T4 and one was T3 (16.6%), supporting the expected high frequency of T4 in Acanthamoeba keratitis. In total, the genotypes of 24 Acanthamoeba keratitis isolates from the UK and Iran were determined. Of these, 17 belonged to T4 (70.8%), three belonged to T2 (12.5%), three belonged to T3 (12.5%) and one belonged to T11 (4.1%), confirming that T4 is the predominant genotype (S2=4.167; P=0.0412) in Acanthamoeba keratitis.

Acanthamoeba isolates belonging to T1, T2, T3, T4 but not T7 encyst in response to increased osmolarity and cysts do not bind to human corneal epithelial cells

Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment and can cause human infections. The life cycle of Acanthamoeba consists of an infective trophozoite form. However under harsh environmental conditions trophozoites differentiate into a double-walled, metabolically inactive and resistant cyst form. Research in Acanthamoeba has mostly focussed on the infective trophozoite form and its pathogenic mechanisms. In this study, we used Acanthamoeba isolates belonging to T1, T2, T3, T4, T7 genotypes and studied their cysts properties. We determined that food deprivation stimulates encystment in Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes in a sodium dodecyl sulfate (SDS)-resistant manner. In addition, increase in osmolarity triggered encystment in T1, T2, T3, T4 isolates (SDS-resistant) but T7 failed to encyst (SDS-labile). Adhesion assays revealed that Acanthamoeba cysts belonging to T1, T2, T3, T4, and T7 genotypes exhibited no and/or minimal binding (<5%) to the host cells. Fluorescein-labelled lectins showed that all Acanthamoeba isolates tested exhibited binding to concanavalin A, indicating the expression of mannosyl- and/or glucosyl-residues. Role of cysts in the transmission of infection is discussed further.

Identification and Distribution of Acanthamoeba Species Genotypes Associated with Nonkeratitis Infections

Acanthamoeba is a free-living protozoan genus found in a wide variety of natural habitats, including water, soil, and air. Pathogenic isolates of Acanthamoeba are medically relevant as the causative agent of sight- threatening Acanthamoeba keratitis (AK), serious infections of other organs, and fatal granulomatous amebic encephalitis. Previous work employing DNA sequences of nuclear and mitochondrial small-subunit rRNA genes (SSU rRNA genes) determined the genotypic diversity of Acanthamoeba and found that many named species of Acanthamoeba are associated with particular genotypes. These studies also concluded that nearly all AK infections result from a single molecular genotype: T4. Here, we asked whether Acanthamoeba clinical isolates from non-AK infections are also associated with particular genotypes. DNA sequence determination of nuclear SSU rRNA genes was employed for genotypic identification of 29 isolates of Acanthamoeba from non-AK infections. Sequence analysis demonstrates that T4 is the predominant genotype in non-AK infections, including those in brain, cerebrospinal fluid, nasal passages, skin, and lung. Rare genotypes (T1, T10, and T12) have been isolated from brain infections. We conclude that genotype T4 is the primary genotype in non-AK Acanthamoeba infections, as was the case in AK infections. However, the genotypes that were isolated from brains have not been observed in environmental isolates of Acanthamoeba, and their natural ecological niche is unknown.

Genotypic identification and species names in Acanthamoeba: correlation and conflict

Many named isolates of Acanthamoeba, e.g. isolates designated Acanthamoeba polyphaga, are used widely in studies of pathogenicity, and other biological characters. However, molecular analyses show that isolates designated A. polyphaga are polyphyletic. Isolates may be very dissimilar at the molecular level, but share a species name, leading investigators to believe that they should share biological characteristics. Here, we present a summary of the correlation in Acanthamoeba between genotypes and the named species of this genus. Analyses of sequences of the nuclear small subunit ribosomal RNA gene (ssu rDNA) have identified at least fifteen genotypes of the free‐living ameba genus Acanthamoeba. Genotype determination has been defined by a sequence dissimilarity of five percent or greater in pairwise sequence comparisons. The ssu rDNA genotypes correlate well with many of the named species of Acanthamoeba. However, in some cases a species name exists as an invalid synonym of other species names within monophyletic genotypic clades. In addition, numerous species names are associated with the large and widely distributed Acanthamoeba genotype T4. Using molecular examination, some of these species designations are shown to be polyphyletic. The genotypes identified by nuclear ssu rDNA are supported by the addition of data from the mitochondrial ssu rDNA, which also provide some resolution of clades within genotype T4. Some lineages within the T4 genotype are associated with named species. Robust identification of Acanthamoeba isolates, including molecular data such as genotypic designation, is required so that data from studies on physiology or pathology may be correctly interpreted.

Isolation and identification of pathogenic Acanthamoeba strains in Tenerife, Canary Islands, Spain from water sources

A comprehensive survey to document the presence of free-living amoebae of the genus Acanthamoeba was conducted in tap water and sea water sources related to human environments in Tenerife, Canary Islands, Spain. Acanthamoeba identification was based on the morphology of cyst and trophozoite forms and PCR amplification with a genus-specific primer pair. The pathogenic potential of Acanthamoeba isolates was characterized by temperature and osmotolerance assays and PCR reactions with two primer pairs related to Acanthamoeba pathogenesis. The results demonstrate the presence of potentially pathogenic strains in both sources. Thus, some of the amoebae in these aquatic habitats can act as opportunistic pathogens, could play a role in the diseases of aquatic organisms, and may present a risk to human health.

Disseminated Acanthamoeba sp. infection in a dog

Several species of free-living amoebae can cause encephalomyelitis in animals and humans. Disseminated acanthamoebiasis was diagnosed in pyogranulomatous lesions in brain, thyroid, pancreas, heart, lymph nodes, and kidney of a one-year-old dog. Acanthamoeba sp. was identified in canine tissues by conventional histology, by immunofluorescence, by cultivation of the parasite from the brain of the dog that had been stored at −70 °C for two months, and by PCR. The sequence obtained from the PCR product from the amoeba from the dog was compared to other sequences in the Acanthamoeba sp. ribosomal DNA database and was determined to be genotype T1, associated with other isolates of Acanthamoeba obtained from granulomatous amebic encephalitis infections in humans.

Ecto-ATPases of clinical and non-clinical isolates of Acanthamoeba

Acanthamoeba are opportunistic protozoan parasites that can cause fatal granulomatous amoebic encephalitis and eye keratitis, however the pathogenic mechanisms of Acanthamoeba remain unclear. In this study, we described the ability of live Acanthamoeba to hydrolyse extracellular ATP. Both clinical and non-clinical isolates belonging to genotypes, T1, T2, T3, T4 and T7 exhibited ecto-ATPase activities in vitro. Using non-denaturing polyacrylamide gel electrophoresis, ecto-ATPases were further characterized. All Acanthamoeba isolates tested, exhibited a single ecto-ATPase band (approximate molecular weight of 272 kDa). However, clinical isolates exhibited additional bands suggesting that ecto-ATPases may play a role in the pathogenesis of Acanthamoeba. This was supported using suramin (ecto-ATPase inhibitor), which inhibited Acanthamoeba-induced host cell cytotoxicity. Previously, we and others have shown that Acanthamoeba binds to host cells using their mannose-binding protein and binding can be blocked using exogenous α-mannose. In this study, we observed that α-mannose significantly increased ecto-ATPase activities of pathogenic Acanthamoeba belonging to T1, T2, T3 and T4 genotypes but had no effect on non-pathogenic Acanthamoeba (belonging to T7 genotype). Overall, we have shown, for the first time, that Acanthamoeba exhibit ecto-ATPase activities, which may play a role in the pathogenesis of Acanthamoeba as well as their potential role in the differentiation of pathogenic Acanthamoeba.

Acanthamoeba induces cell-cycle arrest in host cells.

Acanthamoeba can cause fatal granulomatous amoebic encephalitis (GAE) and eye keratitis. However, the pathogenesis and pathophysiology of these emerging diseases remain unclear. In this study, the effects of Acanthamoeba on the host cell cycle using human brain microvascular endothelial cells (HBMEC) and human corneal epithelial cells (HCEC) were determined. Two isolates of Acanthamoeba belonging to the T1 genotype (GAE isolate) and T4 genotype (keratitis isolate) were used, which showed severe cytotoxicity on HBMEC and HCEC, respectively. No tissue specificity was observed in their ability to exhibit binding to the host cells. To determine the effects of Acanthamoeba on the host cell cycle, a cell-cycle-specific gene array was used. This screened for 96 genes specific for host cell-cycle regulation. It was observed that Acanthamoeba inhibited expression of genes encoding cyclins F and G1 and cyclin-dependent kinase 6, which are proteins important for cell-cycle progression. Moreover, upregulation was observed of the expression of genes such as GADD45A and p130 Rb, associated with cell-cycle arrest, indicating cell-cycle inhibition. Next, the effect of Acanthamoeba on retinoblastoma protein (pRb) phosphorylation was determined. pRb is a potent inhibitor of G1-to-S cell-cycle progression; however, its function is inhibited upon phosphorylation, allowing progression into S phase. Western blotting revealed that Acanthamoeba abolished pRb phosphorylation leading to cell-cycle arrest at the G1-to-S transition. Taken together, these studies demonstrated for the first time that Acanthamoeba inhibits the host cell cycle at the transcriptional level, as well as by modulating pRb phosphorylation using host cell-signalling mechanisms. A complete understanding of Acanthamoeba-host cell interactions may help in developing novel strategies to treat Acanthamoeba infections.

The fate of legionellae within distribution pipe biofilms: measurement of their persistence, inactivation and detachment

Distribution pipe biofilms present a currently unquantified public health risk to consumers receiving water for domestic potable and non-potable use. The aim of this study was to quantify the numbers of legionellae, used here as model bacterial pathogens, that may accumulate, persist within and detach from distribution pipe biofilms. L. pneumophila recovered by standard culture from an 8 week-old biofilm formed within a novel pilot-scale water distribution system represented 1% of those present in the adjacent bulk water. A combined chlorine concentration exceeding 0.2 mg x L(-1) eliminated culturable sessile legionellae altogether, though the reduction in FISH-positive cells represented just 75+/-25% of the original amount, compared to a 5-log reduction in culturable cells during the same period. Where there was < 0.1 mg x L(-1) combined chlorine, an exponential decay/loss of sessile L. pneumophila was observed (k = 0.37 - 0.41) over the course of a 38-day experimental period. The inoculation of the system with 1 microm fluorescent microspheres and legionellae demonstrated that removal of the latter was dominated by chemical disinfection, with erosion and biological grazing playing lesser roles. Under turbulent (Re approximately 5000) conditions, larger clusters of biofilm become detached from substrata, with more than 90% of sessile legionellae mobilised into the bulk water phase. Interaction with both biofilms and a thermophilic Acanthamoeba isolate reduced the susceptibility of legionellae to thermal inactivation by between one and two orders of magnitude, though it increased their sensitivity to chemical (free and combined chlorine) disinfection.

Molecular and physiological evaluation of subtropical environmental isolates of Acanthamoeba spp., causal agent of Acanthamoeba keratitis.

Previous molecular examination of Acanthamoeba spp. has resulted in the determination of distinct genotypes in this genus (designated T1-T12, T14). Genotype T4 has been responsible for the majority of cases of Acanthamoeba keratitis. Here we examine the relative abundance of environmental T4 isolates on beaches and ask whether they have temperature and salinity tolerances that could enhance pathogenicity. Twenty-four Acanthamoeba strains were isolated from beach sand (n = 20), soil (n = 3), and tap water (n = 1) in south Florida. Phylogenetic analysis identified 19 of 24 isolates as T4, the Acanthamoeba keratitis-associated genotype. The remaining isolates were genotype T5 (4) and T11 (1). Nearly all beach isolates were genotype T4, whereas the tap water and soil isolates were mostly T5. All amoebae grew at 0, 1.0, and 2.0% salt and 19 of 20 beach isolates also grew at 3.2%. No soil or tap-water acanthamoebae reproduced at 3.2%. All isolates grew at 37 degrees C and two (T5) at 42 degrees C. Little correlation existed between beach location, salt-tolerance, and genetic relatedness. Overall, the large majority of environmental isolates obtained were genotype T4, suggesting it may be the most common genotype in this environment and could be a potential source of Acanthamoeba keratitis infections.

The identification of free-living environmental isolates of amoebae from Bulgaria.

A survey was carried out in Bulgaria to determine the presence of free-living amoebae (FLA) from environmental sources. In 171 (61.1%) of 280 samples, isolates of Acanthamoeba with group II or III morphology, as well as Hartmannella spp. were recovered. Five isolates named "6" (artificial lake), Ep (lake), G2 (soil), R4* (river) and PK (spring water)--all exhibiting a highly efficient proliferation in axenic cultures--were subsequently cloned and subjected to molecular analyses for identification and genotyping In accordance with morphological findings, PCR-based analyses identified four isolates (6, Ep, G2, R4*) belonging to the genus Acanthamoeba. Confirmation of these findings was obtained by phylogenetic analysis using partial sequencing of the 18S rDNA (ASA.S1) Acanthamoeba-gene. Comparison of these sequences with corresponding regions from other Acanthamoeba strains available from GenBank sorted all four isolates into the sequence type group T4 that contains most of the pathogenic Acanthamoeba strains already identified. The fifth isolate (PK) exhibited morphological characteristics matching those of Hartmannella, and scored negative in the Naegleria fowleri and Acanthamoeba PCRs.

Acanthamoeba keratitis: the role of domestic tap water contamination in the United Kingdom.

The incidence of acanthamoeba keratitis (AK) in the UK is some 15 times that in the United States and seven times that in Holland. To investigate reasons for this higher frequency, a study of the role of domestic tap water as a potential source of AK was undertaken. Tap outlets from the homes of 27 patients with culture-proven AK were sampled and cultured for free-living amoebae (FLA). For all Acanthamoeba isolates, mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) and cytochrome oxidase (cox 1/2) sequence typing was performed to determine the similarity between corneal and tap water isolates. FLA, including Acanthamoeba, were isolated from 24 (89%) of 27 homes, and the presence within the homes varied significantly with tap water temperature and location: 19 (76%) of 25 bathroom sink cold taps sampled compared with 6 (24%) of 25 hot and 9 (47%) of 19 kitchen cold taps compared with 3 (16%) of 19 of hot kitchen taps. Acanthamoeba were isolated from 8 (30%) of 27 homes (five bathroom sink cold taps, one cloakroom cold tap, one bath, and one bedroom sink mixer [hot/cold] taps). In six cases, identical Acanthamoeba mtDNA profiles were found for the clinical and home tap water isolates. In keeping with UK plumbing practice, 24 of 27 homes had internal roof water storage tanks to supply domestic taps, but the mains fed the kitchen cold tap. Water storage tanks promote colonization of domestic water with FLA, including Acanthamoeba, and hence increase the risk of AK. This accounts for the significantly greater incidence of AK in the UK and supports advice to avoid using tap water in contact lens care routines.