Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2010 Jul 1.
Published in final edited form as: Cornea. 2009 Jul;28(6):673–676. doi: 10.1097/ICO.0b013e31819342a7

Genotypic Identification of Acanthamoeba sp. Isolates Associated with an Outbreak of Acanthamoeba keratitis (AK)

Gregory C Booton 1,2,3, Charlotte E Joslin 4,5, Megan Shoff 1, Elmer Y Tu 4, Daryl J Kelly 1, Paul A Fuerst 1
PMCID: PMC2701473  NIHMSID: NIHMS87203  PMID: 19512903

Abstract

Purpose

Significant increases in Acanthamoeba keratitis (AK) cases have been observed in the Chicago-Gary-Kenosha area since June, 2003. It was hypothesized that increased rates of AK infections may be due to changes resulting from changes in municipal water treatment, or, alternatively, a more pathogenic strain of Acanthamoeba may be responsible.

Methods

Previous sequence analysis of the 18S rDNA of Acanthamoeba isolates resulted in the identification of 15 different genotypic classes. These analyses indicate AK cases are predominantly associated (~97%) with a single genotype (designated T4) of Acanthamoeba, and rarely with other genotypes (eg., T3 and T11). In this study we test the hypothesis that a new or more pathogenic genotype of Acanthamoeba is the cause of the recent surge in AK.

Results

We determined the genotype of 15 Acanthamoeba sp. isolates from AK cases from this outbreak using sequence analysis of a region of the 18S rDNA. Our results indicate that these isolates are predominantly genotype T4 (87%), with the remaining isolates being genotype T3 (13%). Both genotypes have previously been observed in AK cases.

Conclusion

There is no support for the hypothesis that the current AK outbreak is associated with infection by a new, more pathogenic, Acanthamoeba genotype. In addition, these results offer support for the hypothesis that the increased AK incidence may be due to changes in water treatment protocols leading to increased bacterial colonization of the water supply, and subsequent increases of already present Acanthamoeba sp, ultimately culminating in an increase of AK cases.

Keywords: Acanthamoeba, keratitis, epidemiology, genotype identification

The protistan genus Acanthamoeba consists of approximately twenty-five named species of free-living amoeba that are ubiquitous in nature and have been isolated from a variety of environments including soil, fresh and brackish water, beach sand, air, hot tubs, and recreational swimming pools. 1,2,3,4 Acanthamoeba has a bi-phasic lifestyle consisting of a mobile trophozooite form with characteristic acanthapodia readily visible under light microscopy. A second form, the cyst, is also observed in Acanthamoeba. The ability to encyst enables the amoeba to survive deteriorated or harsh environmental conditions. These cysts can survive for long periods of time and under severe conditions, including in water where chlorine is used as a disinfectant. 5

In addition to its natural distribution, some strains and/or species of Acanthamoeba have been found to be opportunistically pathogenic. The predominant clinical manifestations of Acanthamoeba infection have been cases of Acanthamoeba keratitis (AK) infection, a painful and potentially sight-threatening disease. In North America, AK cases are predominantly associated with contact lens wear. 6,7,8 Other, less common, opportunistic infections caused by Acanthamoeba include those found in the lungs, skin, sinuses, and brains. 2,3,9 Infections of Acanthamoeba in the brain lead to nearly universally fatal cases of granulomatous amebic encephalitis (GAE). 2,3,9 AK infections are found in otherwise healthy individuals, whereas non-keratitis infections are associated with immunocompromised individuals, often in AIDS patients. 2

In the present study we examine the genotypes of Acanthamoeba strains isolated from AK cases associated with an outbreak of infections in the Chicago metropolitan area. From June 1, 2003 until November 30, 2005, 40 cases of AK were studied in previous epidemiological analyses that concluded that the rate of AK cases was significantly higher than historical case numbers predicted. 10,11 The rate was also nearly ten times the expected number of cases based on the number of cases that have been observed in the United States between 1973 and 1988.12 In addition, Joslin et al. found there was a significant difference among the distribution of cases in urban and suburban counties in the outbreak area. 11 Specifically, suburban counties had a higher relative rate of infection compared to an urban county (Cook County, Illinois, USA). 11

It was hypothesized in the Joslin et al. (2006) study that the cause of this increased rate of AK infection was an alteration in the amount and type of water treatment chemicals, following changes in Environmental Protection Agency (EPA) guidelines that mandated a reduction of potentially carcinogenic disinfection byproducts (DBP) in drinking water.11 Alternatively, it is possible that a new, potentially more pathogenic or virulent, strain of Acanthamoeba could be responsible for the increase in cases of AK. Therefore, in the current study we have employed genotypic analysis of the AK-derived Acanthamoeba isolates in order to test the hypothesis that the observed outbreak has resulted from a more pathogenic, or novel, genotype of Acanthamoeba, or alternatively whether the genotypes observed in this outbreak have been seen in previous studies.

Materials and Methods

Corneal scrapes from AK patients were collected by E. Tu of University of Illinois, Chicago (UIC) and sent to Ohio State University (OSU). These were isolates used in previous studies by Joslin et al. 10,11 Upon arrival at OSU, UIC samples were given unique OSU numbers based on arrival date, eg., 05–009, which represents the 9th overall sample received in 2005 at OSU. Next, the samples were briefly vortexed to remove any cells adhering to the sides of the tubes and several drops were plated onto a non-nutrient amoeba saline (NNAS) agar plate seeded with Enterobacter aerogenes (CDC strain #1998-68) as prey. All cultures were maintained at room temperature. To prevent the agar plates from drying out (amoebae migrate in the thin water film on the surface of the agar) all plates were sealed with Parafilm “M” (Pechiney Plastic Packaging, Chicago, IL). Additionally, several drops of the sample were placed in a petri dish containing amoeba saline. The plates were checked for growth at 3d, 7d, and 2 weeks. This process was repeated until positive growth appeared or until no original sample was left. Blocks of agar from plates that showed positive Acanthamoeba growth were then transferred to liquid culture using a Bacto-Casitone/Serum (BCS) media or amoeba saline. 13, 14 DNA was extracted by using the DNeasy kit (Qiagen, Inc., Valencia, CA). Following DNA extraction, PCR was used to amplify the partial nuclear ssu rDNA sequences. DNA sequencing of the partial ssu rDNA sequence was done with an ABI 310 automated fluorescent sequencing system using a set of conserved primers and methods that have been used previously in our phylogenetic studies. 15 The sequences obtained in this study have been deposited in GenBank under the accession numbers EU168067-EU168082.

The sequences were aligned with a standard set of >130 “complete” sequences (over 2000 nucleotides in length) of the Acanthamoeba nuclear small subunit ribosomal RNA gene (ssuDNA) using the sequence alignment application CLUSTALX within the analysis package Mega 3.1. 16 This alignment allowed the identification of those complete Acanthamoeba sequences that most closely matched the sequences from each isolate currently being studied. For most isolates, the overall sequence overlap with the standard set of sequences encompassed approximately 490 of the ~2300 nucleotide long ssuDNA sequence. The phylogenetic relationships of the sequences from the Chicago outbreak were then obtained by using the phylogenetic identification of the closest complete sequence as a surrogate for the shorter sequence obtained in this study.

Results

Sequencing of the nuclear ssu rDNA DF3 diagnostic region in the 15 UIC AK isolates resulted in the genotypic determination of 17 individual DF3 sequences (Table 1). Direct sequence analysis of the PCR product from 06–024 resulted in multiple peaks in the electropherogram, indicitive of a mixed product. Therefore, T/A cloning of the PCR amplimer of 06–024 was performed, and subsequent sequence analysis resulted in the identification of a distinct DF3 sequence, which was genotype T4. Additional sequences were not found following cloning, however the mixed pherogram suggests more products were present. Overall, fourteen of the seventeen AK sequences were determined to be genotype T4 (82%) based on sequence alignment and overall sequence similarity analysis using our Acanthamoeba rDNA database. Similarly, two of the sequences were determined to be genotype T3 (18%), also based on sequence alignment of the DF3 region. Both genotypes have previously been observed in AK cases. In addition, DNA sequences are overwhelmingly similar to previously sequenced isolates of their two respective genotypes. Among the 17 sequences obtained from isolates obtained in this study, 16 could be related to a specific monophyletic subgroup within the phylogenetic tree of the set of complete sequences (Table 1). A single ambiguous isolate sequence represented a generic T4 type sequence, but was equally similar to a number of subgroups within T4 and could not be placed more accurately.

Table 1.

University of Illinois, Chicago Acanthamoeba keratitis (AK) isolates analyzed.

Taxa UIC ID1 OSU ID2 Sample Source3 18S ssu rDNA Genotype GenBank sequence accession #
Acanthamoeba sp. 5731 05-003 AK T4 EU168067
Acanthamoeba sp. 5742 05–009 AK T4 EU168068
Acanthamoeba sp. 1504 05–011 AK T4 EU168069
Acanthamoeba sp. 4736 05–013 AK T4 EU168070
Acanthamoeba sp. 2713 05–014 AK T3 EU168071
Acanthamoeba sp. 1404 05–020 AK T3 EU168072
Acanthamoeba sp. 3403 05–023 AK T4 EU168073
Acanthamoeba sp. 6191 06-001 AK T4 EU168074
Acanthamoeba sp. 2968 06-002 AK T4 EU168075
Acanthamoeba sp. 8599 06-004 AK T4 EU168076
Acanthamoeba sp. 0410 06-005 AK T4 EU168077
Acanthamoeba sp. 4423 06–016 AK T4 EU168078
Acanthamoeba sp. 6050 06–024 AK T4 EU168079
Acanthamoeba sp. 6590 06–025 AK T4 EU168080
Acanthamoeba sp. 1060 06–033 AK-liq T4 EU168081
Acanthamoeba sp. 1060 06–034 AK-plate T4 EU168082
Open in a new tab
1

University of Illinois, Chicago identification number for clinical isolates.

2

Ohio State University identification number, assigned to all samples arriving at OSU for analysis. Format: (year acquired-sequential sample number, eg. 05-003).

3

Sample sources: AK, Acanthamoeba keratitis corneal scrape; AK-liq, AK sample from liquid culture; AK-plate, AK sample from agar plate.

DISCUSSION

Following the recognition of Acanthamoeba’s role in keratitis infections in 1973, there were slightly over 200 cases of AK documented in the US between 1973 and 1988. 12 The rate of infection of contact lens wearers has been estimated in the U.S. to range from 1.65–2.01 per 1,000,000 contact lens users. 10, 17, 18 The identification of particular Acanthamoeba species that are responsible for AK and other infections has been an active area of investigation since the recognition of this amoeba as the etiological agent in these infections. Traditional taxonomy of Acanthamoeba has used morphological markers such as cyst morphology and trophozoite size and shape as classification characters however, this classification is questionable as morphology of cysts and trophozoites changes with culture conditions. 19 Taxa of Acanthamoeba have been categorized into three morphological groups based largely on the cyst morphology of the species. Molecular analyses using nuclear and mitochondrial small subunit ribosomal RNA genes have supported the three morphological groupings, though not species, structure of the genus. 20, 21, 22 In our laboratory we have worked extensively on the analysis of the ssu rDNA of the nucleus and the equivalent gene from the mitochondrial genome and have analyzed more than 200 clinical and environmental isolates using these genes. 20, 21 Work by other laboratories has increased the number of isolates from which sequences have been determined to over 500. 22, 23, 24, 25, 26, 27,28 This database allows us to quickly analyze a clinical or environmental sample using molecular methods to determine and classify the Acanthamoeba sequence genotype. 1, 15

Sequence similarities between isolates using the nuclear and mitochondrial small subunit ribosomal RNA genes have been used to determine phylogenetic relationships between strains and to explore possible correlation with disease phenotypes. The molecular analyses thus far suggest that 15 genotypic type classes exist, designated T1, T2, T3…,T15. 21, 22, 23, 24, 25, 26, 27 Different Acanthamoeba 18S rDNA genotypes are distinguished from one another by a 5% or greater sequence dissimilarity between isolates. The final number of genotypes is an active area of investigation, since it is dependent upon the statistical criteria employed to distinguish genotypes and the expanding number of analyzed isolates. Although the major morphological groups and some of the named species are supported by molecular analyses, a number of the named taxa are not supported as unique monophyletic entities when examined using molecular methods. 21

Nearly all AK cases examined in our lab (as well as by several other investigators) are due to infections involving a closely related group of strains sharing similar ribosomal genotypes. While at least 15 genotypic classes have been identified, nearly all AK-associated strains are classified within a closely related group of genotypes. 21 This group includes genotypes classified as T3, T4, and T11 (and the vast majority of AK strains, >90%, are classified as genotype T4). 21 In addition to the AK isolates, the genotypes T3, T4, and T11 also contain environmental isolates. 21 Further, these three genotypes form a single monophyletic group, including a number of the nominal species of the genus Acanthamoeba. 21 There have been rare examples of other genotypes (T5 and T6) that have been identified in AK infections. 25

As discussed previously, Acanthamoeba is capable of infecting other tissues and organs in addition to the eye, e.g. skin, sinus, liver, and brain. 2,3,9 Our phylogenetic comparisons of the nuclear ssuDNA sequences obtained from these non-AK Acanthamoeba isolates indicated that while the majority of these non-AK infection isolates are genotype T4 (the most common AK and environmental genotype), other rare genotypes were isolated from these non-AK infections (eg. T1, T10, and T12). 29 In addition, two of these non-AK pathogenic genotypes (T10 and T12) have not yet been observed in environmental isolates. 29 More recent reports have identified genotype T5 isolates in disseminated non-AK infections, which as mentioned above, has also rarely been found in AK infections. 25, 30, 31

The results of the current study clearly demonstrate that these isolates are of previously known genotypes: T3 and T4. The sequences determined do not represent novel genotypes, therefore there is no support for the hypothesis that the AK cases in the Chicago area outbreak are the result of infections caused by a new Acanthamoeba genotype(s). In fact, there is very high sequence similarity between the DF3 primary sequences of the Chicago-area isolates and previously sequenced T3 and T4 genotype isolates derived from other AK cases. Specifically, the observation of high sequence similarity in this region with other isolates does not provide support for the hypothesis that these isolates represent more pathogenic Acanthamoeba of previously unknown genotypes. Therefore, the results of this genetic study have conclusively shown that these Chicago-area AK infections are the result of infection by Acanthamoeba of previously known genotypes. Since novel Acanthamoeba genotypes are not the cause of the current outbreak, alternative explanations must be explored to explain the ongoing, increased rate of AK infection observed in the Chicago-area.

One general alternative hypothesis suggests that the increased number of AK cases may be due to an increased abundance of Acanthamoeba in the water supply. 11 The question remains as to the possible reasons for an increased abundance of Acanthamoeba. One possibility is based upon the relatively recent changes that have occurred in the chemical disinfectant treatment of municipal water supplies. EPA guidelines have resulted in the reduction of chemicals whose breakdown products have been shown to be carcinogenic. The decrease in these water treatment disinfectant chemicals may permit increased bacterial colonization of the water, and water supply surfaces. The decreased levels of disinfectant can directly lead to an increased population density of grazing Acanthamoeba that utilize these bacteria as a food source, resulting in an overall increase of Acanthamoeba abundance in the water supply. This scenario may ultimately culminate in an increased number of AK cases due to the general inability of currently available contact lens disinfectant solutions to overcome the increased protozoan load. 32, 33 Regardless of whether this outbreak is determined to be linked to the water supply, the results of this study have unambiguously identified the Chicago-area isolates as genotypically very similar to previously sequenced AK isolates. This lends further support to the alternative hypothesis that the increased AK cases in the Chicago-area may ultimately be linked to mandated changes in the chemical treatment of the household water supply permitting expansion of already-present Acanthamoeba populations of known AK-associated genotypes.

Acknowledgments

GB, MS, DK, and PF acknowledge the support of The United States National Institutes of Health, National Eye Institute (NEI grant EY09073) for this work. E. aerogenes was kindly provided by Department of Microbiology, Ohio State University; OSU#651 deposited by G. Banwart, strain #K-21 derived from 3CDC#1998-68.3

References

  • 1.Booton GC, Rogerson A, Bonilla TD, et al. Molecular and Physiological Evaluation of Subtropical Environmental Isolates of Acanthamoeba spp. Causal Agent of Acanthamoeba Keratitis. J Eukarot Microbiol. 2004;51:192–200. doi: 10.1111/j.1550-7408.2004.tb00545.x. [DOI] [PubMed] [Google Scholar]
  • 2.Marciano-Cabral F, Cabral GA. Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev. 2003;16:273–307. doi: 10.1128/CMR.16.2.273-307.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Marciano-Cabral F, Puffenbarger R, Cabral GA. The increasing importance of Acanthamoeba infections. J Eukaryot Microbiol. 2000;47:29–36. doi: 10.1111/j.1550-7408.2000.tb00007.x. [DOI] [PubMed] [Google Scholar]
  • 4.John DT. Opportunistically pathogenic free-living amebae. In: Kreier JP, Baker JR, editors. Parasitic Protozoa. 2. Vol. 3. San Diego: Academic Press; 1993. pp. 143–246. [Google Scholar]
  • 5.Schuster FL. Cultivation of pathogenic and opportunistic free-living amebas. Clin Micro Rev. 2002;15:342–354. doi: 10.1128/CMR.15.3.342-354.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Beattie TK, Tomlinson A, McFayden AK, et al. Enhanced attachment of Acanthamoeba to extended-wear silicone hydrogel contact lenses: a new risk factor for infection? Ophthalmology. 2003;110:765–771. doi: 10.1016/S0161-6420(02)01971-1. [DOI] [PubMed] [Google Scholar]
  • 7.Schroeder JM, Booton GC, Hay J, et al. Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge. J Clin Microbiol. 2001;39:1903–1911. doi: 10.1128/JCM.39.5.1903-1911.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Tomlinson A, Simmons P, Seal DV, et al. Salicylate inhibition of Acanthamoeba attachment to contact lenses: a way to reduce the risk of infection. Ophthalmology. 2000;107:112–117. doi: 10.1016/s0161-6420(99)00055-x. [DOI] [PubMed] [Google Scholar]
  • 9.Martinez AJ, Visvesvara GS. Free-living amphizoic and opportunistic amebas. Brain Pathol. 1997;7:583–598. doi: 10.1111/j.1750-3639.1997.tb01076.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Joslin CE, Tu EY, Shoff M, Booton G, et al. The Association of Contact Lens Solution Use and Acanthamoeba Keratitis. Am J Ophthalmol. 2007;144:169–180. doi: 10.1016/j.ajo.2007.05.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Joslin CE, Tu EY, Shoff ME, et al. Epidemiological characteristics of a Chicago-area Acanthamoeba keratitis outbreak. Am J Ophthalmol. 2006;142:212–217. doi: 10.1016/j.ajo.2006.04.034. [DOI] [PubMed] [Google Scholar]
  • 12.Stehr-Green JK, Bailey TM, Visvesvara GS. The epidemiology of Acanthamoeba keratitis in the United States. Am J Ophthalmol. 1989 Apr 15;107(4):331–336. doi: 10.1016/0002-9394(89)90654-5. [DOI] [PubMed] [Google Scholar]
  • 13.Page FC. A New Key to Freshwater and Soil Gymnamoebae with instructions for culture. Freshwater Biological Association, The Ferry House; Ambleside, Cumbria: 1988. [Google Scholar]
  • 14.Cerva L. Science. Vol. 209. 1980. Naegleria fowleri: Trimethoprim Sensitivity; p. 1541. [DOI] [PubMed] [Google Scholar]
  • 15.Booton GC, Kelly DJ, Chu Y-W, et al. 18S Ribosomal DNA Typing and Tracking of Acanthamoeba Species Isolates from Corneal Scrape Specimens, Contact Lenses, Lens Cases, and Home Water Supplies of Acanthamoeba Keratitis Patients in Hong Kong. J Clin Microbiol. 2002;40:1621–1625. doi: 10.1128/JCM.40.5.1621-1625.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Kumar S, Tamura K, Nei M. MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Briefings in Bioinformatics. 2004;5:150–163. doi: 10.1093/bib/5.2.150. [DOI] [PubMed] [Google Scholar]
  • 17.Schawnberg DA, Snow KK, Dana MR. The epidemic of Acanthamoeba keratitis: where do we stand? Cornea. 1998;17.1:1–2. doi: 10.1097/00003226-199801000-00001. [DOI] [PubMed] [Google Scholar]
  • 18.Mathers WD, Suphin JE, Folberg R, et al. Outbreak of keratitis presumed to be caused by Acanthamoeba. Am J Ophthalmol. 1996;121:128–42. doi: 10.1016/s0002-9394(14)70577-x. [DOI] [PubMed] [Google Scholar]
  • 19.Pussard M, Pons R. Morphologie de la paroi kystique et taxonomie du genre. Acanthamoeba (Protozoa: Amoebida) Protistologica. 1977;8:557–598. [Google Scholar]
  • 20.Ledee DR, Booton GC, Awwad MH, et al. Advantages of Using Mitochondrial 16S rDNA Sequences to Classify Clinical Isolates of Acanthamoeba. Invest Ophthalmol Vis Sci. 2003;44:1142–1149. doi: 10.1167/iovs.02-0485. [DOI] [PubMed] [Google Scholar]
  • 21.Stothard DR, Schroeder-Diedrich JM, Awwad MH, et al. The evolutionary history of the genus Acanthamoeba and the identification of eight new 18S rRNA gene sequence types. J Eukaryot Microbiol. 1998;45:45–54. doi: 10.1111/j.1550-7408.1998.tb05068.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Gast RJ, Ledee DR, Fuerst PA, et al. Subgenus systematics of Acanthamoeba: Four nuclear 18S rDNA sequence types. J Euk Microbiol. 1996;43:498–504. doi: 10.1111/j.1550-7408.1996.tb04510.x. [DOI] [PubMed] [Google Scholar]
  • 23.Horn M, Fritsche TR, Gautom RK, et al. Novel bacterial endosymbionts of Acanthamoeba spp. related to Paramecium caudatum symbiont. Caedibacter caryophilus Environ Microbiol. 1999;1:357–67. doi: 10.1046/j.1462-2920.1999.00045.x. [DOI] [PubMed] [Google Scholar]
  • 24.Alves JMP, Gusmão CX, Teixeira MMG, et al. Random amplified polymorphic DNA profiles as a tool for the characterization of Brazilian keratitis isolates of the genus Acanthamoeba. Braz J Med Biol Res. 2000;33:19–26. doi: 10.1590/s0100-879x2000000100003. [DOI] [PubMed] [Google Scholar]
  • 25.Walochnik J, Obwaller A, Aspöck H. Correlations between Morphological, Molecular Biological, and Physiological Characteristics in Clinical and Nonclinical Isolates of Acanthamoeba spp. Appl Envron Microbiol. 2000a;66:4408–4413. doi: 10.1128/aem.66.10.4408-4413.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Walochnik J, Haller-Schober E, Kolli H, et al. Discrimination between clinically relevant and non-relevant Acanthamoeba strains isolated from contact lens-wearing keratitis patients in Austria. J Clin Microbiol. 2000b;38:3932–3926. doi: 10.1128/jcm.38.11.3932-3936.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Hewitt MK, Robinson BS, Monis PT, et al. Identification of a New Acanthamoeba 18S rRNA Gene Sequence Type, Corresponding to the Species Acanthamoeba jacobsi Sawyer, Nerad, and Visvesvara, 1992 (Lobosea: Acanthamoebidae) Acta Protozool. 2003;42:325–329. [Google Scholar]
  • 28.NCBI database of available (published and unpublished) Acanthamoeba nuclear 18S ribosomal DNA sequences (ssuDNA, full and partial), available at NCBI website: http://www.ncbi.nlm.nig.gov
  • 29.Booton GC, Visvesvara GS, Byers TJ, et al. Identification and Distribution of Acanthamoeba Species Genotypes Associated with Nonkeratitis Infections. J Clin Micro. 2005;43:1689–1693. doi: 10.1128/JCM.43.4.1689-1693.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Spanakos G, Tzanetou K, Miltsakakis D, et al. Genotyping of pathogenic acanthamoebae isolated from clinical samples in Greece: report of a clinical isolate presenting T5 genotype. Parasitol Int. 2006;55:147–149. doi: 10.1016/j.parint.2005.12.001. [DOI] [PubMed] [Google Scholar]
  • 31.Barete S, Combes A, de Jonckheere JF. Fatal Disseminated Acanthamoeba lenticulata Infection in a Heart Transplant Patient. Emerg Infect Dis. 2007;13:736–738. doi: 10.3201/eid1305.061347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Shoff M, Rogerson A, Schatz S, et al. Variable Responses of Acanthamoeba Strains to Three Multipurpose Lens Cleaning Solutions. Opt Vis Sci. 2007;84:202–207. doi: 10.1097/OPX.0b013e3180339f81. [DOI] [PubMed] [Google Scholar]
  • 33.Shoff M, Joslin CE, Tu EY, et al. Efficacy of Contact Lens Systems Against Recent Clinical and Tap Water Acanthamoeba Isolates. Cornea. 2008;27:713–719. doi: 10.1097/QAI.0b013e31815e7251. [DOI] [PubMed] [Google Scholar]

RESOURCES