A

cptPtgge©

K

1

sf

0

Acta Tropica 100 (2006) 63–69

Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7genotypes from environmental freshwater samples

in the Nile Delta region, Egypt

Jacob Lorenzo-Morales a, Antonio Ortega-Rivas a, Enrique Martınez a,Messaoud Khoubbane b, Patricio Artigas b, Marıa Victoria Periago b,

Pilar Foronda a, Nestor Abreu-Acosta a,Basilio Valladares a,∗, Santiago Mas-Coma b

a Instituto Universitario de Enfermedades Tropicales y Salud Publica de Canarias, University of La Laguna,Av. Astrofısico Fco. Sanchez s/n, 38203 La Laguna, Canary Islands, Spain

b Departamento de Parasitologıa, Facultad de Farmacia, Universidad de Valencia,Av. Vicent Andres Estelles s/n, 46100 Burjassot, Valencia, Spain

Received 3 April 2006; received in revised form 14 September 2006; accepted 22 September 2006Available online 31 October 2006

bstract

The free-living amoebae of the genus Acanthamoeba include non-pathogenic and pathogenic species and has been recentlylassified into 15 different genotypes, T1–T15. In this study, a survey was conducted in order to determine the presence andathogenic potential of free-living amoebae of Acanthamoeba genus in freshwater sources associated with human activities inhe Nile Delta region, Egypt. Identification of Acanthamoeba was based on the morphology of cyst and trophozoite forms andCR amplification with a genus specific primer pair. The pathogenic potential of Acanthamoeba isolates was characterized using

emperature and osmotolerance assays and PCR reactions with two primer pairs specific to Acanthamoeba pathogenesis. Isolates

enotypes were also determined after ribosomal DNA sequencing. These data revealed that isolates belong to T1, T2, T3, T4 and T7enotypes. As expected, T4 isolates exhibited the most pathogenic traits and were osmotolerant, temperature tolerant and expressedxtracellular serine proteases. This is the first report presenting environmental distribution of Acanthamoeba genotypes in Egypt.

2006 Elsevier B.V. All rights reserved.

onmen

eywords: Acanthamoeba; Egypt; Pathogen; Nile Delta region; Envir

. Introduction

Acanthamoeba spp. are free-living protozoan para-ites that pervade the entire environment and can beound in many different places such as tap, fresh,

∗ Corresponding author. Tel.: +34 922 318484; fax: +34 922 318514.E-mail address: bvallada@ull.es (B. Valladares).

001-706X/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.actatropica.2006.09.008

tal health; Public health

coastal and bottled mineral water; contact lens solutionsand eyewash stations; soil, dust and air; sewage; heat-ing, ventilation or air-conditioning units and gastroin-testinal washings (Mergeryan, 1991; Armstrong, 2000;Marciano-Cabral et al., 2000; Khan, 2003).

Based on their ecological distribution, there is circu-lation of Acanthamoeba strains between humans and theenvironment. These protozoa are opportunistic causalagents of disseminated infections (mostly cutaneous and

/ Acta T

way were then transferred into axenic cultures by placingthe amoebae into PYG medium (0.75% proteose peptone(wt/vol), 0.75% yeast extract (wt/vol) and 1.5% glucose(wt/vol), with 50 �g/ml gentamicin (Sigma)).

Table 1Acanthamoeba strains used in this study

No. Genotype Strains Source

1 T1 ATCCa 50494 GAE2 T2 CCAPb 1547/1 Soil, Israel3 T3 CCAP 1501/4 Beach, USA

64 J. Lorenzo-Morales et al.

nasopharyngeal infections), a sight-threatening ulcer-ation of the cornea called amoebic keratitis and afatal granulomatous amoebic encephalitis (GAE) (Khan,2003; Marciano-Cabral and Cabral, 2003; Schuster andVisvesvara, 2004; Niederkorn et al., 1999).

The identification of Acanthamoeba at the genuslevel is based on distinctive features of trophozoitesand cysts, especially the double walled cysts. Acan-thamoeba species have been classified into three distinctmorphological groups (I–III) (Pussard and Pons, 1977).However, the taxonomy and classification of these proto-zoa are currently under revision, following the success-ful application of molecular techniques (Stothard et al.,1998; Alves et al., 2000; Vodkin et al., 1992; Szenasi etal., 1998; Khan et al., 2001; Booton et al., 2002; Kong etal., 2002; Walochnik et al., 2000). Evolutionary studieshave led to the identification of 15 different genotypes(T1–T15) based on rRNA gene sequencing (Stothard etal., 1998; Horn et al., 1999; Gast, 2001; Hewett et al.,2003). Attempts to correlate pathogenecity with certaingenotypes are under investigation (Stothard et al., 1998;Gast, 2001; Booton et al., 2004, 2005; Maghsood et al.,2005).

To date studies have shown that 90% of Acan-thamoeba isolates that produce infections belong to theT4 genotype, suggesting that the abundance of T4 iso-lates in human infections is most likely due to theirgreat virulence and/or properties that enhance their trans-missibility as well as their decreased susceptibility tochemotherapeutic agents (Maghsood et al., 2005; Khan,2006).

Acanthamoeba pathogenesis is related to severaldirect and indirect factors. Among these factors, thebinding of Acanthamoeba to host cells is mediated bymannose-binding proteins that lead to secondary eventssuch as phagocytosis, apoptosis and the secretion ofextracellular proteases. Extracellular proteases, mainlyserine and cysteine proteases are major determinantsof the pathogenesis of Acanthamoeba infections, beinginvolved in the degradation of host tissues (Alsam et al.,2003; Khan, 2003; Clarke and Niederkorn, 2006).

The extent to which these organisms are present inwater sources associated with human activity has beenpreviously studied in specific regions in Egypt (Mansouret al., 1991; Hamadto et al., 1993; Sadaka et al., 1994).However, no previous studies on the presence and dis-tribution of Acanthamoeba genotypes have been previ-ously reported in this region. Therefore, the aims of this

study were to determine the presence of Acanthamoeba,using both morphological and molecular tools, in watersources associated with human activities in the NileDelta region, Egypt, and to characterize the pathogenic

ropica 100 (2006) 63–69

potential of the isolated strains using biochemical andPCR assays. Finally, the genotypes of these isolates werealso determined by sequencing the Diagnostic Fragment3 (DF3) of the Acanthamoeba small subunit rRNA gene.

2. Material and methods

Water samples were collected from freshwatersources in the Governorates of Alexandria and Behera,in the Nile Delta region, Egypt (Table 1). This areawas selected because of the importance of water canalsand domestic distribution of water from the Nile river(Curtale et al., 1997, 2003), and the high prevalenceof waterborne parasitic diseases in the human inhabi-tants (Esteban et al., 2003). The area is also included inan international cooperation programme with the WorldHealth Organization, the Egyptian Ministry of Healthand the Italian Cooperation. These studies have allowedthe report of many human activities such as drinking,washing, cooking, fishing and agricultural drainage byEgyptians in the described areas (Table 1).

Once in the laboratory, water samples (approximately500 ml) were filtered through a cellulose nitrate fil-ter, 0.45 �m diameter (Millipore Corporation, Bedford,Madison) with a weak vacuum (flow rate, 1.3 ml/min).The filters were inverted on 2% Neff’s saline non-nutrient agar plates, onto which heat killed bacteria werepoured, and incubated at 25 ◦C. After 3–4 days, the mem-branes were removed and the plates were incubated fora further 1–2 weeks. Swabs were then stroked on 2%Neff’s saline non-nutrient agar plates within an area40 mm × 40 mm in size and incubated at 25 ◦C for upto 2 weeks. These plates were monitored for out-growthof Acanthamoeba microscopically and blocks containingAcanthamoeba were removed and cultures of amoebaewere started by dilution. Acanthamoeba isolated in this

4 T4 ATCC 50238 Fish, France5 T4 ATCC 50492 Keratitis, Ind.6 T7 ATCC 30137 Soil, USA

a American Type Culture Collection.b Culture Collection of Algae and Protozoa.

/ Acta T

lPi

s(tp1asp

ubsApH252D8cpf1w

fctta

a0lgc

trpapdl

b

J. Lorenzo-Morales et al.

Amoeba controls from American Type Culture Col-ection (ATCC) and Culture Collection of Algae androtozoa (CCAP) (Table 1) were grown without shaking

n PYG medium at 25 ◦C.Acanthamoeba trophozoites were harvested at a den-

ity of 2 × 107 parasites/ml. The cells were pelleted500 g) for 10 min at room temperature and washed threeimes with phosphate-buffered saline (PBS), pH 7.2. Cellellets were resuspended in lysis buffer (50 mM NaCl,0 mM EDTA, 50 mM Tris–HCl, pH 8.0) and incubatedt 55 ◦C for 1 h with 0.25 mg/ml Proteinase K. The DNAamples were purified from amoebae isolates by thehenol–chloroform method (Sambrook et al., 1989).

The DNA amplification reactions were performed,sing the genus specific genetic marker that distinguishetween pathogenic and non-pathogenic Acanthamoebatrains, the extracellular serine protease related and the. astronyxis, A. divionensis and A. polyphaga specificrimer pairs (Vodkin et al., 1992; Howe et al., 1997;ong et al., 2000; Ortega-Rivas et al., 2003, 2004,005) in a 30 �l volume containing 10 ng template DNA,0 mM buffer KCl; 10 mM Tris-HCl, 2.5 mM MgCl2,00 mM dNTP, 0.6 mM each primer and 0.4 units of TaqNA polymerase (Applied Biosystems, New Jersey), pH.3 and were performed in a Perkin-Elmer 9600 thermo-ycler. The cycling conditions were: an initial denaturinghase of 94 ◦C for 1 min and 30 repetitions at 94 ◦Cor 1 min, annealing temperature of each primer pair formin and 72 ◦C for 1.5 min. The primer extension phaseas prolonged for 10 min at 72 ◦C in the last cycle.rRNA gene amplifications (DF3 region) were per-

ormed as previously described with minimal modifi-ations (Booton et al., 2002), in a 50 �l volume con-aining 1.25 U Taq DNA polymerase (Applied Biosys-ems), 0.1–1.0 ng DNA, 4 mM MgCl2, 200 �M dNTPnd 0.5 �M each primer.

Amplification products were fractionated by 2%garose electrophoresis stained with a solution of.5 �g/ml of ethidium bromide and visualized under UVight. Known isolates of Acanthamoeba belonging toenotypes T1, T2, T3, T4 and T7 were used as positiveontrols.

To determine the pathogenic potential of Acan-hamoeba isolates, PCR reactions with the primer pairelated to a genetic marker that distinguish betweenathogenic and non-pathogenic Acanthamoeba strainsnd the serine protease primer pair were developed. Tem-erature and osmolarity assays were also developed to

istinguish between pathogenic and non-pathogenic iso-ates (Khan et al., 2001, 2002; Khan and Tareen, 2003).

Acanthamoeba spp. genotype determination wasased on sequence analysis of Diagnostic Fragment 3, a

ropica 100 (2006) 63–69 65

subset of the nuclear small subunit ribosomal RNA geneas previously described (Booton et al., 2002, 2005). Phy-logenetic analyses were carried out using maximum par-simony, minimum evolution and maximum likelihoodoptimality criteria, implemented in Mega 3.0 (Kumaret al., 2004). Transition:transversion ratios were esti-mated by maximum likelihood heuristic searches. Esti-mates of node support were obtained by performing500 bootstrap replicates. Obtained sequences were com-pared to sequences available in GenBank under the fol-lowing accession numbers: Acanthamoeba astronyxisCCAP 1534/1 (Accession # AF239293), A. griffiniCCAP 1501/4 (Accession # AF239295), A. griffini S-7 ATCC 30731 (Accession # U07412), A. palestinen-sis ATCC 30870 (Accession # AF239296), A. palesti-nensis 2802 (Accession # AF019063), A. palestinensis(Accession # L09599), Acanthamoeba polyphaga ATCC50372 (Accession # AY690458) and Acanthamoeba spp.genotype T1 ATCC 50494 (Accession # AF479547).Obtained sequences were also compared with the Acan-thamoeba DNA sequence database (Department ofMolecular Genetics, The Ohio State University, OH,USA). Diagnostic Fragment 3 sequences for the newisolates are deposited in the Genbank database under theaccession numbers: DQ992178–DQ992193.

3. Results

Of 37 samples from freshwater sources, 16 (43.24%)were found to contain Acanthamoeba, after morphol-ogy identification and PCR with the genus specificprimer pair (Table 2). Eleven of these positive sam-ples (69%), demonstrated pathogenic potential as theseisolates showed osmotolerance, thermotolerance andpositive amplification of the genetic marker that dis-tinguish between pathogenic and non-pathogenic Acan-thamoeba strains (Table 2). Furthermore, 7 of the 11 (63.36%) isolates classified as potentially pathogenic, gavepositive amplification with the extracellular serine pro-tease primer pair, showing that these strains were bothpathogenic and virulent (Table 2).

Phylogenetic relationships within the genus wereexamined with maximum parsimony, minimum evolu-tion and maximum likelihood optimality criteria. Thetree topology was identical regardless of the methodused. The potential pathogens, weak potential pathogensand non-pathogens were within separate clusters onthe tree (Fig. 1). The DF3 sequence was also com-

pared with the reference species included in this study.Results revealed that the Acanthamoeba isolated strainsbelong to T1, T2, T3, T4 and T7. Furthermore, assaysfor pathogenecity indicated that all Acanthamoeba iso-

66 J. Lorenzo-Morales et al. / Acta Tropica 100 (2006) 63–69

Table 2Distribution of Acanthamoeba isolates in fresh water sources across Nile Delta Region, Egypt

Isolate Locality Microscopy Pathogenic PCR Protease PCR T◦a/Osmb Genotype Diagnosis

cEFW1 El Kazaa + + + + T4 A. polyphagaEFW2 Ola Gharbea + + + + T4 A. polyphagaEFW3 Abis village + − − ± T2 Acanthamoeba sp.EFW4 Faraum Prince + + + + T4 A. polyphagaEFW5 Tiba + − − − T7 A. astronyxisEFW6 El Kazaa + + − + T4 A. polyphagaEFW7 El Kazaa + + + + T3 Acanthamoeba sp.EFW8 El Kazaa + + + + T4 A. polyphagaEFW9 El Kazaa + + − − T3 Acanthamoeba sp.EFW10 Abis village + + + + T4 A. polyphagaEFW11 Abis village + − − T2 Acanthamoeba sp.EFW12 Abis village + − − ± T2 Acanthamoeba sp.EFW13 Zuhra + + − + T1 Acanthamoeba sp.EFW14 Abis village + + + + T4 A. polyphagaEFW15 Abis village + − − ± T2 Acanthamoeba sp.EFW16 El Kazaa + + − + T3 Acanthamoeba sp.

a T◦, growth of Acanthamoeba isolates at 37 ◦C.

b Osm, growth of Acanthamoeba isolates with 1 M mannitol.c Egypt fresh water (EFW).

lates belonging to T4 were potential pathogens. Isolatesbelonging to T1–T3 genotypes did not exhibit the fullrange of pathogenic traits and were considered as weakpathogens. In contrast, Acanthamoeba isolates belong-ing to T7 genotype did not exhibit any pathogenic traitand were considered as non-pathogenic (Table 2). Inaddition, PCR reactions with the species specific primerpairs identified some of the isolates as A. astronyxis andA. polyphaga (Table 2).

4. Discussion

This is the first study of the occurrence of Acan-thamoeba genotypes in water sources from Egypt,according to our knowledge. The amoebae werewidespread across the water samples surveyed andpotentially pathogenic and virulent strains were isolatedfrom the collected samples. As mentioned before, thesesample sites are included in a surveyed area where activ-ities such as drinking, washing, cooking, fishing andagricultural drainage by Egyptians have been reported.In previous studies on these areas other waterbornepathogens have been identified (Curtale et al., 1997,2003; Esteban et al., 2003).

Such widespread finding of the organisms in waterused for human activity is not surprising since over 80%

of the normal human population may have antibodiesagainst Acanthamoeba (Chappell et al., 2001). Severalstudies have reported Acanthamoeba in tap and springwater systems, rivers and swimming pools worldwide

(Radford et al., 1998; Booton et al., 2002; Ettinger et al.,2003; Seal et al., 2003; Kilvington et al., 2004). Thesewater sources are important in the exposure of humansto Acanthamoeba and may be important in the causationof amoebic keratitis.

Serine proteases play an essential role in the patho-genesis of the amoebae and they are essential for adapta-tion of the organisms to stressful conditions as previouslyreported (Lorenzo-Morales et al., 2005a,b). Therefore,areas with high human activities were likely sites for thedevelopment of pathogenecity in Acanthamoeba.

The molecular and biochemical assays used in thisstudy, have allowed us to identify T1–T4 and T7 geno-types in freshwater sources. Recent studies have estab-lished T4 genotype as the predominant group responsiblefor Acanthamoeba infections in humans (Maghsood etal., 2005; Khan, 2006).

The obtained results showed that the presence ofAcanthamoeba in the Egyptian environment analyzedis not limited to T4 genotype and it is likely that T4isolates have certain properties that make them morevirulent, thus their higher frequency in human infec-tions. It has been recently shown that T4 genotypesexhibit significant higher binding and produced severecytotoxicity on host cells as compared to T2, T3, T7or T11 genotypes (Alsam et al., 2003). In a previ-

ous study, Maghsood et al. proposed the subdivision ofT2 genotype into two separated sequence types, T2Aand T2B. In our study, T2 isolates showed less than a3% dissimilarity in their sequences compared to CCAP

J. Lorenzo-Morales et al. / Acta Tropica 100 (2006) 63–69 67

Fig. 1. 18S rDNA DF3 linearized neighbour-joining tree obtained by using the Kimura two-parameter distance algorithm, produced in MEGA 3.0.The isolates are identified in the tree. The type sequences were taken from GenBank and are presented under the following numbers: CCAP 1534/1(Acanthamoeba astronyxis Accession # AF239293), CCAP 1501/4 (A. griffini Accession # AF239295), ATCC 30731 (A. griffini S-7 Accession# (A. pale# 69045A

1wcii

aptdpt

U07412), ATCC 30870 (A. palestinensis Accession # AF239296),L09599), ATCC 50372 (Acanthamoeba polyphaga Accession # AYF479547).

501/3c and ATCC 30871, and were also classified aseak or non-pathogenic strains. Additionally, cytotoxi-

ity assays (less than 10% cell death) were carried outn order to determine their virulence, confirming thesesolates as weak pathogens.

It is also important to mention that A. polyphagand A. astronyxis species were identified by specificrimer pairs. The latter species is being reported for

he first time from the African region. The widespreadistribution of A. polyphaga across the world and itsrevious association with human disease, points tohe urgent need for awareness among health work-

stinensis 2802 Accession # AF019063), (A. palestinensis Accession8) and ATCC 50494 (Acanthamoeba spp. genotype T1 Accession #

ers and the public of this potentially pathogenicamoeba.

Overall, there is a clear need for more detailed knowl-edge about the distribution of Acanthamoeba genotypesin different environments and their direct and indirectvirulence factors. These findings will be useful in orderto understand the basis of differential distribution ofgenotypes in natural habitats, and to some extent in

clinical cases, which should allow the identification oftheir differential properties contributing to disease andthe development of therapeutic measures against thesepathogens.

cta T

M.A., Bargues, M.D., El Sayed, M., El Wakeel, A., Abdel-Wahab,Y., Montresor, A., Engels, D., Savioli, L., Mas-Coma, S., 2003.Hyperendemic fascioliasis associated with schistosomiasis in vil-lages in the Nile Delta of Egypt. Am. J. Trop. Med. Hyg. 69,429–437.

ropica 100 (2006) 63–69

Ettinger, M.R., Webb, S.R., Harris, S.A., McIninch, S.P., Garman, G.,Brown, B.L., 2003. Distribution of free-living amoebae in JamesRiver, Virginia, USA. Parasitol. Res. 89, 6–15.

Gast, R.J., 2001. Development of an Acanthamoeba specific reversedot-blot and the discovery of a new ribotype. J. Eukaryot. Micro-biol. 48, 609–615.

Hamadto, H.H., Aufy, S.M., el-Hayawan, I.A., Saleh, M.H., Nagaty,I.M., 1993. Study of free living amoebae in Egypt. J. Egypt. Soc.Parasitol. 23, 631–637.

Hewett, M.K., Robinson, B.S., Monis, P.T., Saint, C.P., 2003. Iden-tification of a new Acanthamoeba 18S rRNA gene sequence typecorresponding to the species Acanthamoeba jacobsi Sawyer, Neradand Visvesvara, 1992 (Lobosea: Acanthamoebidae). Acta Proto-zool. 42, 325–329.

Hong, Y.C., Kong, H.H., Ock, M.S., Kim, I.N., Chung, D.I., 2000.Isolation and characterization of a cDNA encoding a subtilisin-like serin proteinase (AhSUB) from Acanthamoeba healyi. Mol.Biol. Parasitol. 111, 441–446.

Horn, M., Fritsche, T.R., Gautom, R.K., Wagner, M., 1999. Novelbacterial endosymbionts of Acanthamoeba spp. related to theParamecium caudatum symbiont Caedibacter caryphilus. Envi-ron. Microbiol. 1, 357–367.

Howe, D.K., Vodkin, M.H., Novak, R.J., Visvesvara, G., McLaughlin,G.L., 1997. Identification of two genetic markers that distinguishpathogenic and nonpathogenic strains of Acanthamoeba spp. Par-asitol. Res. 83, 345–348.

Khan, N.A., Jarroll, E.L., Paget, T.A., 2001. Acanthamoeba can bedifferentiated by the polymerase chain reaction and simple platingassays. Curr. Microbiol. 43, 204–208.

Khan, N.A., Jarroll, E.L., Paget, T.A., 2002. Molecular and physi-ological differentiation between pathogenic and non-pathogenicAcanthamoeba. Curr. Microbiol. 45, 197–202.

Khan, N.A., Tareen, N.K., 2003. Genotypic, phenotypic, biochemi-cal, physiological and pathogenicity-based categorisation of Acan-thamoeba strains. Folia Parasitol. 50, 97–104.

Khan, N.A., 2003. Pathogenesis of Acanthamoeba infections. Microb.Pathog. 34, 277–285.

Khan, N.A., 2006. Acanthamoeba: biology and increasing impor-tance in human health. FEMS Microbiol. Rev. 30, 564–595.

Kilvington, S., Gray, T., Dart, J., Morlet, N., Beeching, J.R., Frazer,D.G., Matheson, M., 2004. Acanthamoeba keratitis: the role ofdomestic tap water contamination in the United Kingdom. Invest.Ophthalmol. Vis. Sci. 45, 165–169.

Kong, H.H., Shin, J.Y., Yu, H.S., Kim, J., Hahn, T.W., Hahn, Y.H.,Chung, D.I., 2002. Mitochondrial dna restriction fragment lengthpolymorphism (RFLP) and 18S small-subunit ribosomal DNAPCR-RFLP analyses of Acanthamoeba isolated from contact lensstorage cases of residents in Southwestern Korea. J. Clin. Micro-biol. 40, 1199–1206.

Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated softwarefor Molecular Evolutionary Genetics Analysis and sequence align-ment. Brief. Bioinform. 5, 150–163.

Lorenzo-Morales, J., Ortega-Rivas, A., Martınez, E., Foronda, P., Val-ladares, B., 2005a. Isolation and identification of pathogenic Acan-thamoeba strains in Tenerife, Canary Islands, Spain from watersources. Parasitol. Res. 95, 273–277.

68 J. Lorenzo-Morales et al. / A

Acknowledgements

Research was supported by a Ph.D. grant fromthe Canary Islands Government and by the ProjectRICET (Project No. C03/04 of the Programme of RedesTematicas de Investigacion Cooperativa, FIS), SpanishMinistry of Health, Madrid, Spain. Research studies inEgypt were supported by Project No. BOS2002-01978of the Spanish Ministry of Science and Technology,Madrid, and PI030545 of FIS.

References

Alsam, S., Kim, K.S., Stins, M., Rivas, A.O., Sissons, J., Khan, N.A.,2003. Acanthamoeba interactions with human brain microvascularendothelial cells. Microb. Pathog. 35, 235–241.

Alves, J.M., Gusmao, C.X., Teixeira, M.M., Freitas, D., Foronda, A.,Affonso, H.T., 2000. Random Amplified Polymorphic DNA pro-files as a tool for the characterization of Brazilian keratitis isolatesof the genus Acanthamoeba. Braz. J. Med. Biol. Res. 33, 19–26.

Armstrong, M., 2000. The pathogenesis of human Acanthamoebainfection. Infect. Dis. Rev. 2, 65–73.

Booton, G.C., Kelly, D.J., Chu, Y.W., Seal, D.V., Houang, E., Lam,D.S., Byers, T.J., Fuerst, P.A., 2002. 18S ribosomal DNA typingand tracking of Acanthamoeba species isolates from corneal scrapespecimens, contact lenses, lens cases, and home water supplies ofAcanthamoeba keratitis patients in Hong Kong. J. Clin. Microbiol.40, 1621–1625.

Booton, G.C., Rogerson, A., Bonilla, T.D., Seal, D.V., Kelly, D.J.,Beattie, T.K., Tomlinson, A., Lares-Villa, F., Fuerst, P.A., Byers,T.J., 2004. Molecular and physiological evaluation of subtropi-cal environmental isolates of Acanthamoeba spp., causal agent ofAcanthamoeba keratitis. J. Eukaryot. Microbiol. 51, 192–200.

Booton, G.C., Visvesvara, G.S., Byers, T.J., Kelly, D.J., Fuerst, P.A.,2005. Identification and distribution of Acanthamoeba speciesgenotypes associated with nonkeratitis infections. J. Clin. Micro-biol. 43, 1689–1693.

Chappell, C.L., Wright, J.A., Coletta, M., Newsome, A.L., 2001. Stan-darized method of measuring Acanthamoeba in sera from healthyhuman subjects. Clin. Diag. Lab. Immunol. 8, 724–730.

Clarke, D.W., Niederkorn, J.Y., 2006. The pathophysiology of Acan-thamoeba keratitis. Trends Parasitol. 22, 175–180.

Curtale, F., Moursy, B.E.M., El-Deen, M.S.S., Nabil, M., El-Wakeel,A., 1997. Operational research on health, nutrition and environ-mental needs in Behera Governorate. Final Report. StrengtheningRural Health Services in Behera, Dakalhia and Qena Governorates.Egyptian–Italian Cooperation, Editing and Printing Center, Exten-sion of the Medical Research Institute of Alexandria University,Alexandria, Egypt, 52 pp. (1–7 Annexes).

Curtale, F., Mas-Coma, S., Hassanein, Y.A.E., Barduagni, P., Pezzotti,P., Savioli, L., 2003. Clinical signs and household characteris-tics associated with human fascioliasis among rural population inEgypt: a case-control study. Parassitologia 45, 5–11.

Esteban, J.G., Gonzalez, C., Curtale, F., Munoz-Antoli, C., Valero,

Lorenzo-Morales, J., Ortega-Rivas, A., Foronda, P., Abreu-Acosta, N.,

Ballart, D., Martınez, E., Valladares, B., 2005b. RNA interference(RNAi) for the silencing of extracellular serine proteases genes inAcanthamoeba: molecular analysis and effect on pathogenecity.Mol. Biochem. Parasitol. 144, 10–15.

/ Acta T

M

M

M

M

M

N

O

O

O

P

J. Lorenzo-Morales et al.

aghsood, A.H., Sissons, J., Rezaian, M., Nolder, D., Warhurst, D.,Khan, N.A., 2005. Acanthamoeba genotype T4 from the UK andIran and isolation of the T2 genotype from clinical isolates. J. Med.Microbiol. 54, 755–759.

ansour, N.S., Saoud, A.F., Nashed, N.N., Youssef, F.G., 1991. Fresh-water amoebae from four aquatic sites in Egypt. J. Egypt. Soc.Parasitol. 21, 15–22.

arciano-Cabral, F., Cabral, G., 2003. Acanthamoeba sp. as agents ofdisease in humans. Clin. Microbiol. Rev. 16, 273–307.

arciano-Cabral, F., Puffenbauer, R., Cabral, G.A., 2000. The increas-ing importance of Acanthamoeba infections. J. Eukaryot. Micro-biol. 47, 29–36.

ergeryan, H., 1991. The prevalence of Acanthamoeba in the humanenvironment. Rev. Infect. Dis. 5, 390–391.

iederkorn, Y., Alizadeh, H., Leher, H., McCulley, J.P., 1999.The pathogenesis of Acanthamoeba keratitis. Microb. Infect. 1,437–443.

rtega-Rivas, A., Lorenzo-Morales, J., Alonso, V., Abreu, N., Foronda,P., Del Castillo, A., Valladares, B., 2003. Random Amplified Poly-morphic DNA as a tool for the identification of Acanthamoebadivionensis. Curr. Microbiol. 47, 84–86.

rtega-Rivas, A., Lorenzo-Morales, J., Villa, M., Martınez, E., Val-ladares, B., Del Castillo, A., 2004. Design and evaluation of a spe-cific primer pair for the identification of Acanthamoeba polyphaga.Curr. Microbiol. 48, 360–363.

rtega-Rivas, A., Lorenzo-Morales, J., Villa, M., Martınez, E., Clavel,

A., Valladares, B., Del Castillo, A., 2005. A Specific Primer Pairfor the diagnosis and identification of Acanthamoeba astronyxis byRAPD-PCR. J. Parasitol. 91, 122–126.

ussard, M., Pons, R., 1977. Morphologie de la paroi kystique et tax-onomie du genre Acanthamoeba. Protistologica 13, 557–598.

ropica 100 (2006) 63–69 69

Radford, C., Lehman, O., Dart, J.K.G., 1998. Acanthamoeba ker-atitis: multicentre survey in England 1992-6. National Acan-thamoeba Keratitis Study Group. Br. J. Ophthalmol. 82, 1387–1392.

Sadaka, H.A., el-Nassery, S.F., abou Samra, L.M., Awadalla, H.N.,1994. Isolation and identification of free-living amoebae from somewater sources in Alexandria. J. Egypt. Soc. Parasitol. 24, 247–257.

Sambrook, J., Fristsch, E.F., Maniatis, T., 1989. Molecular Cloning. ALaboratory Manual, second ed. Cold Spring Harbord LaboratoryPress.

Schuster, F.L., Visvesvara, G.S., 2004. Free-living amoebae as oppor-tunistic and non-opportunistic pathogens of humans and animals.Int. J. Parasitol. 34, 1001–1027.

Seal, D., Beattie, T.K., Tomlinson, A., Fan, D., Wong, E., 2003. Acan-thamoeba keratitis. Br. J. Ophthalmol. 87, 516–517.

Stothard, D.R., Schroeder-Diedrich, J.M., Awwad, M.H., Gast, R.J.,Ledee, D.R., Rodrıguez-Zaragoza, S., Dean, C., Fuerst, P.A.,Byers, T.J., 1998. The evolutionary history of the genus Acan-thamoeba and the identification of eight new 18S rRNA genesequence types. J. Eukaryot. Microbiol. 45, 45–54.

Szenasi, Z., Endo, T., Yagita, K., Nagy, E., 1998. Isolation, identifi-cation and increasing importance of free-living amoebae, causinghuman disease. J. Med. Microbiol. 47, 45–54.

Vodkin, M.H., Howe, D.K., Visvesvara, G., McLaughlin, G.L., 1992.Identification of Acanthamoeba at the generic and specific levels

using the Polymerase Chain Reaction. J. Protozool. 39, 378–385.

Walochnik, J., Obwaller, A., Aspock, H., 2000. Correlations betweenmorphological, molecular, biological and physiological character-istics in clinical and non-clinical isolates of Acanthamoeba spp.Appl. Environ. Microbiol. 66, 4408–4413.