Discrimination between clinically relevant and nonrelevant Acanthamoeba strains isolated from contact lens- wearing keratitis patients in Austria.
ABSTRACT Eighteen cases of Acanthamoeba-associated keratitis among contact lens wearers seen at the Department of Ophthalmology, Karl-Franzens-University, Graz, Austria, between 1996 and 1999 are reviewed. The amoebae were proven to be the causative agents in three patients. The aim of our study was to discriminate between clinically relevant and nonrelevant isolates and to assess the relatedness of the isolates to published strains. Altogether, 20 strains of free-living amoebae, including 15 Acanthamoeba strains, 3 Vahlkampfia strains, and 2 Hartmannella strains, were isolated from clinical specimens. The virulent Acanthamoeba strains were identified as A. polyphaga and two strains of A. hatchetti. To our knowledge this is the first determination of keratitis-causing Acanthamoeba strains in Austria. Clinically relevant isolates differed markedly from nonrelevant isolates with respect to their physiological properties. 18S ribosomal DNA sequence types were determined for the three physiologically most-divergent strains including one of the keratitis-causing strains. This highly virulent strain exhibited sequence type T6, a sequence type not previously associated with keratitis. Sequence data indicate that Acanthamoeba strains causing keratitis as well as nonpathogenic strains of Acanthamoeba in Austria are most closely related to published strains from other parts of the world. Moreover, the results of our study support the assumption that pathogenicity in Acanthamoeba is a distinct capability of certain strains and not dependent on appropriate conditions for the establishment of an infection.
- [show abstract] [hide abstract]
ABSTRACT: To support the hypothesis that Acanthamoeba is not a unique cause of amebic keratitis, we report a case of amebic keratitis in which viable Acanthamoeba could not be isolated from corneal tissue. Vahlkampfia and Hartmannella, two other genera of free-living ameba, were isolated, however, using prolonged culture. A 24-year-old wearer of soft contact lenses had keratitis. Extensive histologic and microbiologic investigations were performed on corneal scrape, biopsy, and keratoplasty tissue. Contact lenses, storage case, and the home water supply, where contact lens hygiene was practiced, were examined for the presence of micro-organisms. No viruses, pathogenic bacteria, or fungi were detected from corneal tissue samples. Amebae were observed using light and electron microscopy, but these could not be unequivocally classified using immunocytochemical staining. Viable Vahlkampfia and Hartmannella, but no Acanthamoeba, were isolated from the corneal biopsy sample. Indirect immunofluorescence with a range of polyclonal rabbit antisera raised against axenically cultivated stains of the three amebal genera was unhelpful because of cross-reactivity. A diverse range of micro-organisms was present within the storage case, including the three amebal species. Amebic cysts also were associated with the contact lens. A mixed non-Acanthamoeba amebic keratitis has been identified in a wearer of soft contact lenses where lack of storage case hygiene provided the opportunity for the free-living protozoa Vahlkampfia and Hartmannella to be introduced to the ocular surface. When Acanthamoeba-like keratitis occurs, but where Acanthamoeba cannot be isolated using conventional laboratory culture methods, alternate means should be used to identify other amebae that may be present. Polyclonal immunofluorescent antibody staining was unreliable for generic identification of pathogenic free-living amebae in corneal tissue.Ophthalmology 04/1996; 103(3):485-94. · 5.56 Impact Factor
Article: Basic local alignment search tool.[show abstract] [hide abstract]
ABSTRACT: A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.Journal of Molecular Biology 11/1990; 215(3):403-10. · 3.91 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Acanthamoeba keratitis is a sight-threatening complication of corneal trauma or contact lens wear. Although the majority of corneal isolates of Acanthamoeba belong to Group II in the Pussard-Pons classification based on cyst morphology, they have been placed in at least six species and their genetic relatedness is uncertain. The aim of this study was to determine the virulence of, and the relationship among, strains derived from the cornea, the nasal mucosa, and other environmental sources. To assess virulence, 10(4) trophozoites of each strain were incubated with monolayers of human corneal fibroblasts. By day 7, 12 of 29 strains tested had induced significant cytopathic changes. In addition, inocula of 10(4) cysts or trophozoites with 10(6) Corynebacterium xerosis were injected into the corneas of Porton rats; 11 amoebic strains induced infection within 7 days. The correlation between the virulence of trophozoites in vitro and in vivo was 86%. Using allozyme electrophoresis, 23 Acanthamoeba strains clustered into 5 major phylogenic divisions. Three divisions contained one or more strains that were virulent in the rat cornea. Virulent Pussard-Pons Group II strains clustered tightly to a fixed allelic difference of 13.6%. The eight corneal isolates clustered to 33%, dividing into three lineages. Five avirulent nasal isolates were strongly differentiated from other Group II strains. The results were not in accord with species designations based primarily on morphological criteria. These data suggest that particular subsets of Acanthamoeba strains are virulent in the human cornea.International Journal for Parasitology 03/1995; 25(2):229-39. · 3.64 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY,
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Nov. 2000, p. 3932–3936Vol. 38, No. 11
Discrimination between Clinically Relevant and Nonrelevant
Acanthamoeba Strains Isolated from Contact Lens-
Wearing Keratitis Patients in Austria
J. WALOCHNIK,1E.-M. HALLER-SCHOBER,2H. KO ¨LLI,2O. PICHER,1
A. OBWALLER,1AND H. ASPO ¨CK1*
Department for Medical Parasitology, Clinical Institute of Hygiene, University of Vienna,1
and Department of Ophthalmology, Karl-Franzens-University, Graz,2Austria
Received 24 April 2000/Accepted 18 August 2000
Eighteen cases of Acanthamoeba-associated keratitis among contact lens wearers seen at the Department of
Ophthalmology, Karl-Franzens-University, Graz, Austria, between 1996 and 1999 are reviewed. The amoebae
were proven to be the causative agents in three patients. The aim of our study was to discriminate between
clinically relevant and nonrelevant isolates and to assess the relatedness of the isolates to published strains.
Altogether, 20 strains of free-living amoebae, including 15 Acanthamoeba strains, 3 Vahlkampfia strains, and 2
Hartmannella strains, were isolated from clinical specimens. The virulent Acanthamoeba strains were identified
as A. polyphaga and two strains of A. hatchetti. To our knowledge this is the first determination of keratitis-
causing Acanthamoeba strains in Austria. Clinically relevant isolates differed markedly from nonrelevant iso-
lates with respect to their physiological properties. 18S ribosomal DNA sequence types were determined for the
three physiologically most-divergent strains including one of the keratitis-causing strains. This highly virulent
strain exhibited sequence type T6, a sequence type not previously associated with keratitis. Sequence data
indicate that Acanthamoeba strains causing keratitis as well as nonpathogenic strains of Acanthamoeba in
Austria are most closely related to published strains from other parts of the world. Moreover, the results of our
study support the assumption that pathogenicity in Acanthamoeba is a distinct capability of certain strains and
not dependent on appropriate conditions for the establishment of an infection.
Within the past few years free-living amoebae of the genus
Acanthamoeba have gained increasing clinical relevance main-
ly as causative agents of a very often seriously progressing
keratitis. The first ocular infections with acanthamoebae were
diagnosed in 1974 (21). Since then the occurrence of keratitis
due to Acanthamoeba (Acanthamoeba keratitis) has been es-
calating in correlation with the increasing number of contact
lens wearers. Contaminated contact lens care systems usually
are the first step in Acanthamoeba keratitis pathogenesis. The
most prevalent risk factors are contact lens wear, poor hygiene,
and a compromised corneal barrier. Users of extended-wear
lenses are at special risk. Nevertheless, about 10 to 15% of
cases of Acanthamoeba keratitis occur in persons who do not
wear contact lenses (15).
The prognosis for Acanthamoeba keratitis, when the disease
is diagnosed at an early stage and treated adequately, is rather
good, but fast and reliable diagnosis is of crucial importance.
Clinical signs and symptoms of Acanthamoeba keratitis are
easily confused with fungal or viral keratitis. Initial improve-
ment or stabilization in response to topical antibacterial, anti-
viral, antifungal, or corticosteroid therapy can occur, altering
the clinical picture and thus complicating diagnosis. Clinical
diagnosis should be based on the presence of keratitis with
severe pain and photophobia, stromal infiltrates, radial kera-
toneuritis, and sometimes pseudodendriform epithelial lesions.
Cysts or trophozoites, found in corneal scrapings, on contact
lenses, and inside of lens storage cases, are confirmatory. Agar
culture is the mainstay for laboratory detection of Acantha-
Various Acanthamoeba species have been reported to be
able to cause keratitis: A. castellanii, A. polyphaga, A. hatchetti,
A. culbertsoni, A. rhysodes, A. lugdunensis, A. quina, and A. grif-
fini (26). Although isolates can easily be recognized as belong-
ing to the genus Acanthamoeba by their polygonal cysts, accu-
rate species determination is still problematic. An important
step forward in the differentiation of acanthamoebae was the
division of the genus into three morphological groups by Pus-
sard and Pons (24). Since then a number of attempts to enable
a more precise identification have been made. A very promis-
ing method for identification and phylogenetic studies is the
analysis of the 18S rRNA gene sequence. Recently Stothard et
al. (27) identified 12 Acanthamoeba sequence types, the vast
majority of keratitis-causing strains belonging to sequence type
T4. However, a final system does not yet exist, and, moreover,
representatives from same species differ with respect to their
We report on 18 cases of Acanthamoeba-associated keratitis
seen at the Department of Ophthalmology, Karl-Franzens-
University, Graz, Austria, between 1996 and 1999; in three
cases the amoebae were the disease-causing agents. The aim of
our study was to discriminate between clinically relevant and
nonrelevant isolates and to assess the relatedness of our iso-
lates to published strains by 18S ribosomal DNA (rDNA) se-
MATERIALS AND METHODS
Patients. Clinical specimens of keratitis patients presenting at the Department
of Ophthalmology were on a routine basis investigated for Acanthamoeba spp.
During 1996 to 1999 specimens from 18 keratitis patients, 10 women (55.6%) and
8 men (44.4%), yielded Acanthamoeba. The definitive diagnosis of Acanthamoe-
ba keratitis on the basis of typical clinical signs, no response to antibacterial or
* Corresponding author. Mailing address: Department for Medical
Parasitology, Clinical Institute of Hygiene, University of Vienna, Kin-
derspitalgasse 15, 1095 Vienna, Austria. Phone: 0043-1-4277-79430.
Fax: 0043-1-4277-9794. E-mail: Horst.Aspoeck@univie.ac.at.
antiviral treatment, and detection of acanthamoebae in the corneal epithelium
was verified for three patients (16.7%).
All 18 patients were contact lens wearers, and for all patients cysts were
detectable in contact lens cases by lactophenol cotton blue staining (Table 1).
Thirteen patients (72.2%) wore soft contact lenses, 3 (16.7%) wore rigid gas-
permeable lenses, and 2 (11.1%) wore both types. Patients’ ages ranged from 15
to 54 years (mean, 29 years). The most prevalent clinical signs we observed were
chronic keratitis and keratoconjunctivitis; clinical signs for Acanthamoeba kera-
titis, namely, presence of keratitis with severe pain and photophobia, stromal
infiltrates, radial keratoneuritis, and sometimes pseudodendriform epithelial le-
sions, were seen in seven patients (38.9%). In three of these patients the defin-
itive diagnosis of Acanthamoeba keratitis was verified. Of the 18 patients, only
these 3 showed no response to antibacterial or antiviral treatment. Moreover,
these were the only three cases with acanthamoebae detectable in corneal scrap-
The Acanthamoeba keratitis patients were a 15-year-old female wearer of soft
daily-wear lenses (2HAP), a 41-year-old male with rigid gas-permeable lenses in
the afflicted left eye (11DSP), and a 39-year-old male wearer of rigid gas-
permeable lenses in both eyes (15SOP). In two of these cases the initial diagnosis
had been herpes simplex virus keratitis, which did not improve despite antiviral
therapy. The time between clinical onset and correct diagnosis ranged from 1 to
5 weeks. Antiamoebic treatment consisted of local application of propamidine
isethionate (Brolene eyedrops), hexamidine isethionate (Desomedine eyedrops),
and bacitracin plus neomycin (Nebacetin ointment). No perforating keratoplasty
was necessary. The first patient recovered, with a best corrected visual acuity of
20/20; the visual acuity of the second patient was 20/60 in the affected eye; the
third patient still had corneal erosions, maybe caused by the toxicity of the
Isolation and culture. Contact lenses, corneal scrapings, and swabs from con-
tact lens cases were transferred to nonnutrient agar plates covered with 100 ?l of
a 24-h-old culture of Escherichia coli in brain heart infusion medium. The plates
were sealed and incubated at 30°C for 14 days and examined every 48 h for
amoebal growth. Positive cultures were diluted in order to eliminate coexisting
ciliates, flagellates, bacteria, and fungi by harvesting amoebae at a noncontami-
nated site of the plate with a sterile cotton-tipped applicator and transferring the
amoebae to a fresh plate. All isolates were cloned with the use of a microma-
nipulator and incubated at various temperatures (30, 34, 37, and 42°C). They
were examined daily by phase-contrast microscopy, and amoebal growth and
temperature tolerances were recorded.
Identification and characterization. Amoebae were identified as belonging to
one of the cyst morphological groups (Acanthamoeba sp. groups I to III) estab-
lished by Pussard and Pons (24), and species determination was performed
according to the identification key of Page (23). Differentiation was achieved
mainly on the basis of cyst size, number of opercula, and temperature tolerance.
Moreover, all isolates were examined for their cytopathic effects to a human
cell line (HEp-2). Amoebae were axenized by harvesting cysts from the plate
cultures, incubating them in 3% HCl overnight in order to eliminate the bacteria,
and transferring the amoebae into liquid culture. As a liquid medium we used
proteose peptone-yeast extract-glucose (23). We cultured the amoebae in 150-
cm2tissue culture flasks (Corning Costar, Bodenheim, Germany) at 30°C. Tro-
phozoites were harvested from the axenic cultures by centrifugation (500 ? g for
7 min) and transferred onto a monolayer of HEp-2 cells in an amoeba/cell ratio
of 1/10. The amoebae were designated as highly cytopathic when the monolayer
was completely lysed after 24 to 48 h.
Molecular biology analysis. 18S rDNA sequence analysis was performed for
three isolates most divergent with respect to their physiological properties
(strains 4RE, 9GU, and 11DS) in order to determine the differences of these
strains from and their relatedness to the published strains from other parts of the
world. The 4RE and the 9GU strains were derived from contact lens cases of
non-Acanthamoeba keratitis patients, while the 11DS strain was isolated from the
corneal scraping of Acanthamoeba keratitis patient 11DSP.
For molecular biology investigations amoebae (?106cells) were harvested
from actively growing axenic cultures by centrifugation at 500 ? g for 7 min.
Whole-cell DNA was isolated by a modified UNSET procedure (14). Briefly, the
pellet was resuspended in 500 ?l of UNSET lysis buffer, overlaid with 500 ?l of
phenol-chloroform-isoamylalcohol (PCI), and shaken gently for 5 h. DNA was
extracted by multiple PCI extraction, precipitated in alcohol, air dried and
resuspended in 30 ?l of sterile double-distilled water. The 18S rRNA gene was
amplified using the SSU1 and SSU2 primers (9), complementary to the 5? and 3?
ends of the gene, respectively, and a standard amplification program (30 cycles;
95°C for 1 min, 50°C for 2 min, 72°C for 3 min). Amplification of the 18S rRNA
gene was visualized with ethidium bromide in an agarose gel electrophoresis. The
amplified gene was sequenced stepwise by direct sequencing from the PCR
product using the Thermo Sequenase II sequencing kit (Amersham Pharmacia
Biotech GmbH, Vienna, Austria) and subsequent construction of complemen-
tary internal primers. Sequences were obtained from both strands. Sequencing
was carried out in a 310 ABI PRISM automated sequencer (PE Applied Bio-
systems, Langen, Germany).
Sequence data were processed with the GeneDoc (22) sequence editor, and
sequences were compared to the ones of published strains using a BLAST search
(2). ClustalX (29) was used for pairwise alignment and calculation of the per-
centage of sequence dissimilarity.
Nucleotide sequence accession numbers. Sequence data reported in this
paper were deposited in GenBank and are available under the following refer-
ence numbers: strain 4RE, AF251937; strain 9GU, AF251938; strain 11DS,
TABLE 2. Parasitological data for patients with
Hartmannella sp. 6DOB
aCases in which acanthamoebae were confirmed as causative agents are bold-
face. In all other cases, acanthamoebae were detected only in lens storage cases.
P, positive; N, negative.
bLPCB, lactophenol cotton blue.
cND, no data.
dThe number of plus signs is a measure of the cytopathic effect. ?, no
TABLE 1. Clinical data of patients with Acanthamoeba-associated
RE, soft; LE, rigid
RE, soft; LE, rigid
Rigid gas permeable
Rigid gas permeable
Rigid gas permeable
Keratitis dentritica RE
Corneal infiltration RE
Keratitis superficialis RE
Keratitis superficialis LE
Ulcus corneae LE
Keratitis superficialis RE
Corneal infiltrates LE
aCases in which acanthamoebae were confirmed as causative agents are bold-
face. In all other cases, acanthamoebae were detected only in lens storage cases.
RE, right eye; LE, left eye.
bM, male; F, female.
VOL. 38, 2000DISCRIMINATION AMONG ACANTHAMOEBA STRAINS IN AUSTRIA3933
Identification and characterization of isolates. In all, 20
strains of free-living amoebae including 15 Acanthamoeba
strains, 3 Vahlkampfia strains, and 2 Hartmannella strains were
isolated from clinical specimens (Table 2). Four specimens re-
vealed two variant strains of free-living amoebae each (2HAP,
6DOP, 7TOP, and 8PRP), and in two cases culture was unsuc-
cessful (12JOP and 13PTP). Fourteen of the 15 Acanthamoeba
isolates were identified as belonging to Acanthamoeba sp. group
II. However, several strains exhibited rather varied cyst morphol-
ogies with respect to size and number of opercula although they
were derived from a clone. These strains were classified according
to the average cyst morphology. One strain (18MA) was desig-
nated A. lenticulata (morphological group III), the cysts being
rather small and round. No isolate exhibited a group I morphol-
ogy. In all, A. hatchetti was identified six times, A. rhysodes and A.
polyphaga were each identified three times, A. triangularis was
identified twice, and A. lenticulata was identified once.
Acanthamoeba spp. had been proven to be of clinical rele-
vance in three cases (2HAP, 11DSP, and 15SOP). All of these
isolates showed an Acanthamoeba group II cyst morphology. In
one case (2HAP) two different strains of Acanthamoeba, A. rhy-
sodes and A. hatchetti, were detected. The other keratitis-caus-
ing isolates were identified as A. hatchetti (11DS) and A. poly-
Temperature tolerance tests revealed that all 20 isolates
of free-living amoebae grew at 30°C; 13 of them also grew at
34°C, 11 strains grew at 37°C, and 2 strains grew at 42°C. All
of the thermophilic strains were among the Acanthamoeba
isolates. The two Vahlkampfia strains and the Hartmannella
strain showed no growth above 30°C. Five isolates (2HAB,
9GU, 11DS, 15SO, and 16KV) showed cytopathic effects
against monolayers of HEp-2 cells. Strains 2HAB, 11DS,
and 15SO had been isolated from Acanthamoeba keratitis pa-
tients, yet two strains (9GU and 16KV) isolated from patients
without Acanthamoeba keratitis also exhibited moderate cyto-
pathic effects (monolayer lysis after 3 to 4 days). From the two
different Acanthamoeba strains isolated from patient 2HAP
only one (2HAB) showed pathogenicity-related physiological
characteristics. Eventually only this strain was responsible for
the infection, while the other strain (2HAA), with no patho-
genicity-associated characteristics, was only a concontaminant.
The clinically relevant strains 2HAB, 11DS, and 15SO not
only showed high-temperature tolerance and high cytopathic
effects but also generally exhibited far higher growth rates than
the other isolates.
Molecular biology analysis. All strains investigated revealed
more than 97% sequence identity to classified published
strains (Table 3) and could thus be assigned to one of the 12
published sequence types, as sequence types differ from one
another by at least 5% (27). Nonpathogenic strain 4RE (Gen-
Bank accession no. AF251937), having been identified as A.
hatchetti, displayed sequence type T11, with 99.3% identity to
the BH-2 A. hatchetti strain isolated in brackish water in the
United States (GenBank accession no. AF019068). The 9GU
strain (GenBank accession no. AF251938), which did not cause
disease but which is pathogenic to tissue culture, showed
98.6% identity to the Castellani strain of A. castellanii (Gen-
Bank accession no. U07413), which has sequence type T4. This
strain of A. castellanii has been isolated from a yeast culture in
the United Kingdom and is the type strain for the species
A. castellanii. We thus reclassified our isolate, initially classified
as A. polyphaga, as A. castellanii. Most of the published A. poly-
phaga isolates show sequence type T4, as do most of the A. cas-
tellanii isolates. Stothard et al. (27) proposed to reclassify all
sequence type T4 isolates as A. castellanii. Strain 11DS (Gen-
Bank accession no. AF251939), isolated from the contact lens
case of a patient with serious Acanthamoeba keratitis, showed
an 18S rDNA sequence with highest identity (97.7%) to the 2802
strain of A. palestiniensis with sequence type T6, isolated in a
swimming pool in France (GenBank accession no. AF019063).
However, the 11DS strain exhibits a typical A. hatchetti mor-
phology, with most of the cysts being rectangular (Fig. 1), and
the isolate showed the ability to grow at 42°C, while A. pales-
tiniensis is described as showing no growth even at 37°C (23).
Moreover, because A. hatchetti and A. palestiniensis are described
as polyphyletic (27), we prefer not to reclassify this isolate.
The sequences of our strains clearly diverged from one an-
other, all exhibiting different sequence types, namely, T4, T6,
and T11. The 4RE and the 9GU strains were more closely
related to one another (5.57% dissimilarity) than either was to
the 11DS strain. The 4RE and 9GU strains showed 9.68 and
9.40% sequence dissimilarities, respectively, to the 11DS strain.
TABLE 3. Sequence identity of isolates to published strains
A. hatchetti BH-2
A. castellanii Castellani
A. palestiniensis 2802
Brackish water, United States
Yeast culture, United Kingdom
Swimming pool, France
FIG. 1. Cysts of the 11DS isolate with typical A. hatchetti morphology, as
shown by phase-contrast microscopy. Magnification, ?1,000.
3934 WALOCHNIK ET AL.J. CLIN. MICROBIOL.
Identification and characterization of isolates. Altogether
15 strains of Acanthamoeba, 3 strains of Vahlkampfia, and 2
strains of Hartmannella were isolated. During the last few years
it has become apparent that Vahlkampfia and Naegleria (11) as
well as Hartmannella (1, 16) can cause keratitis or at least be
associated with keratitis. However, neither the Hartmannella
strain nor the Vahlkampfia strains were of clinical relevancy in
In two cases the amoebae could not be grown in vitro. This
may partially be due to the fact that patients had already been
treated for bacteria, and antibacterial and antifungal treatment
is at least partly effective against free-living amoebae. On the
other hand, contact lens disinfectants, even if not used prop-
erly, compromise amoebal viability. Moreover, amoebae pen-
etrate the cornea during the course of infection, protruding up
to Descemet’s membrane. It might therefore in some cases be
impossible to isolate viable amoebae by scraping. Several stud-
ies report on unsuccessful attempts to culture amoebae from
clinical specimens (15, 26).
In general most of the acanthamoebae isolated were iden-
tified as belonging to Acanthamoeba sp. group II, which also is
reported to be the most prevalent group of these microorgan-
isms (23). In a former study we could demonstrate that in the
area of Vienna Naegleria seems to be the predominant genus in
environmental habitats, whereas probing of shower heads and
tap water mainly revealed amoebae of the genera Acanthamoe-
ba and Hartmannella (our unpublished data). This supports the
assumption that, in Acanthamoeba keratitis, infection is pri-
marily acquired via contaminated contact lenses and lens cases,
the amoebae deriving mainly from tap water, rather than by
outdoor swimming. Domestic tap water as source of Acantha-
moeba sp. in Acanthamoeba keratitis was described for the first
time in 1990 (17). Interestingly two of the Acanthamoeba ker-
atitis patients (n ? 3) discussed here wore rigid gas-permeable
contact lenses in the afflicted eyes. Usually wearers of soft
lenses are more likely to acquire Acanthamoeba keratitis, as
the hydrophilic material seems to support attachment and sur-
vival of cysts. However, several cases of Acanthamoeba kera-
titis in wearers of rigid gas-permeable contact lenses have been
reported (4, 25). A case of Acanthamoeba keratitis occurring in
a wearer of daily-disposable contact lenses has also been doc-
umented recently (31). We did not observe any correlation to
age or sex, and in none of the three patients did surgical
treatment become necessary. In a study from the United King-
dom severe visual loss was seen in about 15% of the patients
Morphological determination was rather difficult in some
cases as cysts, although all deriving from one clone, had varied
morphologies, and generally intraspecific polymorphism is ra-
ther common among acanthamoebae (3). The four Acantha-
moeba strains isolated from the contact lens cases of the three
Acanthamoeba keratitis patients were identified by cyst mor-
phology as A. rhysodes, A. polyphaga, and two strains of A.
hatchetti. All of these species are known to cause keratitis
(26). To our knowledge this is the first determination of
keratitis-causing strains in Austria. In 1989, Huber-Spitzy et
al. described a case of Acanthamoeba keratitis in a 37-year-
old woman in Austria (13). They detected cysts of Acantha-
moeba in the corneal epithelium, but the amoeba was neither
isolated nor identified to the species level.
Clinical relevance of strains. Remarkably, in the majority of
keratitis cases we investigated, the acanthamoebae were of no
clinical relevance. Although Acanthamoeba keratitis has be-
come of increasing importance within the last 10 years, corre-
lating to the greater number of contact lens wearers, it is still
a rare disease. The annualized incidence of Acanthamoeba
keratitis is estimated as 0.14 per 100,000 individuals (25).
Also other studies revealed that the majority of people do
not develop a keratitis in spite of coming into contact with
acanthamoebae; the prevalence of acanthamoebae and other
free-living amoebae in asymptomatic contact lens wearers has
been reported frequently (8, 18). Even from the nasal mucosae
of healthy individuals different strains of Acanthamoeba spp.
could be isolated (20). Apart from such widely accepted risk
factors as extended contact lens wear and microlesions in the
cornea, certainly the immune status of the patient may play a
fundamental role in the course of infection and may result in
enhanced susceptibility to developing an Acanthamoeba kera-
titis. It was shown that nearly 50% of healthy individuals carry
antibodies against Acanthamoeba, probably due to the ubiquity
of this microorganism (28).
Moreover, apart from differences in predisposition of pa-
tients, one can assume that amoeba strains vary in pathoge-
nicity. Mazur et al. demonstrated in an animal model a clear
correlation between the occurrence of eye infections and the
degree of virulence of the strains after installation into the
brain (19), which suggests that pathogenicity is not so much
dependent on environmental conditions but rather represents
a distinct characteristic of certain strains. Nevertheless the
initial infective dose is certainly of considerable importance.
The factors which prime amoebae for pathogenicity are poor-
ly understood. Temperature tolerance (10) and cell culture
pathogenicity (3, 5, 6) seem to be related to pathogenicity
potential. Temperature tolerance is widely accepted to be a
prerequisite for pathogenicity in granulomatous amoebic en-
cephalitis caused by Acanthamoeba, as the human body tem-
perature is 37°C. However, the human eye has a mean tem-
perature of only around 34°C. Yet data presented here indicate
that clinically relevant strains not only show higher tempera-
ture tolerances but also generally yield far higher growth rates,
which certainly is of major importance for the establishment of
thamoeba strains is rather conspicuous (3, 7). Moreover, the
keratitis-causing acanthamoebae described here were highly
cytopathic to HEp-2 cells, producing complete destruction of
the monolayer in 1 to 2 days. Altogether, there seems to be a
correlation between clinical relevance and pathogenicity-re-
lated physiological characteristics.
The occurrence of free-living amoebae of nonpathogenic
strains in contact lens cases, however, is still of medical inter-
est, as they can harbor bacteria inside their cysts, protecting
them from disinfectants, and thus function as vectors. In a
recent study we showed that viable Pseudomonas aeruginosa,
one of the major ocular pathogens, can survive in and be
reisolated from cysts of Acanthamoeba (30).
Molecular biology analysis. Interestingly, the three investi-
gated strains showing the most-diverse physiological capacities
also differed with respect to their 18S rDNA sequences, exhib-
iting three different sequence types. The two strains with no
clinical relevancy, both classified as group II acanthamoebae,
showed sequence type T4 (strain 9GU) and sequence type T11
(strain 4RE). Sequence types T3, T4, and T11 are most closely
related, and all represent morphological group II (27). T4 is
reported to be the sequence type isolated most frequently.
Moreover, the majority of keratitis-causing Acanthamoeba
strains are reported to belong to sequence type T4 (27).
The keratitis-causing and highly cytopathic strain 11DS,
identified as A. hatchetti, morphological group II, exhibited
sequence type T6. Sequence type T6 is represented by a single
strain, which had been identified morphologically as A. pales-
VOL. 38, 2000 DISCRIMINATION AMONG ACANTHAMOEBA STRAINS IN AUSTRIA3935
tiniensis belonging to morphological group III. Stothard et al.
also report on a strain presumed to be group II but with a
group III sequence. The authors assume that A. palestiniensis
and A. hatchetti are polyphyletic (27). However, a final system
does not yet exist, and most probably diversification will still be
necessary. T6 isolates have not previously been reported to
cause keratitis. Also no keratitis-causing strain is known to
exhibit sequence type T2, which is the sequence type most
closely related to sequence type T6.
Altogether the results of our study support the assumption
that pathogenicity in Acanthamoeba seems to be a distinct
capability of certain strains and not so much dependent on
appropriate conditions for the establishment of an infection.
Moreover, data presented here indicate that Acanthamoeba
keratitis-causing strains as well as nonpathogenic strains of
Acanthamoeba in Austria are most closely related to published
strains from other parts of the world.
1. Aitken, D., J. Hay, F. B. Kinnear, S. M. Kirkness, W. R. Lee, and D. V. Seal.
1996. Amebic keratitis in a wearer of disposable contact lenses due to a
mixed Vahlkampfia and Hartmannella infection. Ophthalmology 103:485–
2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. Lipman. 1990. Basic
local alignment search tool. J. Mol. Biol. 215:403–410.
3. Badenoch, P. R., M. Adams, and D. J. Coster. 1995. Corneal virulence,
cytopathic effect on human keratocytes and genetic characterisation of Acan-
thamoeba. Int. J. Parasitol. 25:229–239.
4. Cohen, E. J., J. C. Fulton, C. J. Hoffman, C. J. Rapuano, and P. R. Liabson.
1996. Trends in contact lens-associated corneal ulcers. Cornea 15:566–570.
5. Cursons, R. T. M., and T. J. Brown. 1978. Use of cell cultures as an indicator
of pathogenicity of free-living amoebae. J. Clin. Pathol. 31:1–11.
6. De Jonckheere, J. F. 1980. Growth characteristics, cytopathic effect in cell
culture, and virulence in mice of 36 type strains belonging to 19 different
Acanthamoeba spp. Appl. Environ. Microbiol. 39:681–685.
7. Dini, L. A., C. Cockinos, J. A. Frean, I. A. Niszl, and M. B. Markus. 2000.
Unusual case of Acanthamoeba polyphaga and Pseudomonas aeruginosa ker-
atitis in a contact lens wearer from Gauteng, South Africa. J. Clin. Microbiol.
8. Donzis, P. B., B. J. Mondino, B. A. Weissman, and D. A. Bruckner. 1987.
Microbial contamination of contact lens care systems. Am. J. Ophthalmol.
9. Gast, R. J., D. R. Ledee, P. A. Fuerst, and T. Byers. 1996. Subgenus system-
atics of Acanthamoeba: four nuclear 18S rDNA sequence types. J. Eukaryot.
10. Griffin, J. L. 1972. Temperature tolerance of pathogenic and nonpathogenic
free-living amoebae. Science 178:869–870.
11. Harminder, S. D., A. Azurara-Blanco, M. Hossain, and J. Lloyd. 1998.
Non-Acanthamoeba amebic keratitis. Cornea 17:675–677.
12. Hay, J., F. B. Kinnear, C. M. Kirkness, and D. V. Seal. 1995. Acanthamoeba
keratitis: laboratory diagnosis, characterisation of protozoa and treatment.
Scieh Weekly Rep. 29:90–91.
13. Huber-Spitzy, V., G. Grabner, E. Arocker-Mettinger, I. Baumgartner, F.
Skorpik, C. Rappersberger, and R. Haddad. 1989. Acanthamoeba keratitis.
An underdiagnosed entity? Klin. Monatsbl. Augenheilkd. 194:454–457.
14. Hugo, E. R., V. J. Stewart, R. J. Gast, and T. J. Byers. 1992. Purification of
amoeba mtDNA using the UNSET procedure, p. D7.1–7.2. In A. T. Soldo
and J. J. Lee (ed.), Protocols in protozoology. Allen, Lawrence, Kans.
15. Illingworth, C. D., and S. D. Cook. 1998. Acanthamoeba keratitis. Surv.
16. Kennedy, S. M., P. Devine, C. Hurley, Y. S. Ooi, and L. M. T. Collum. 1995.
Corneal infection associated with Hartmannella vermiformis in contact lens
wearer. Lancet 346:637–638.
17. Kilvington, S., D. F. Larkin, D. G. White, and J. R. Beeching. 1990. Labo-
ratory investigation of Acanthamoeba keratitis. J. Clin. Microbiol. 28:2711–
18. Larkin, D. F. P., S. Kilvington, and D. L. Easty. 1990. Contamination of
contact lens storage cases by Acanthamoeba and bacteria. Br. J. Ophthalmol.
19. Mazur, T., E. Hadas, L. Gustowska, J. Winiecka-Krusnell, and E. Linder.
1999. Secondary amoebic eye infections in mice due to Acanthamoeba sp.
Parasitol. Res. 85:776–778.
20. Michel, R., R. Ro ¨hl, and H. Schneider. 1982. Isolation of free-living amoebae
from nasal mucosa of healthy individuals. Zentbl. Bakteriol. Hyg. 176:155–
21. Nagington, F., P. G. Watson, T. J. Playfair, J. McGill, B. R. Hones, and
A. D. M. Steele. 1974. Amoebic infection of the eye. Lancet 2:1537–1540.
22. Nicholas, K. B., H. B. Nicholas, Jr., and D. W. Deerfield II. 1997. GeneDoc:
analysis and visualization of genetic variation. Embnew. News 4:14.
23. Page, F. C. 1991. Nackte Rhizopoda, p. 3–170. In F. C. Page and F. J.
Siemensma (ed.), Nackte Rhizopoda und Heliozoa. G. Fischer, Stuttgart,
24. Pussard, M., and R. Pons. 1977. Morphologie de la paroi kystique et tax-
onomie du genre Acanthamoeba (Protozoa, Amoebida). Protistologica 8:
25. Radford, C. F., O. J. Lehmann, and J. K. G. Dart. 1998. Acanthamoeba
keratitis: multicentre survey in England 1992–6. Br. J. Ophthalmol. 82:1387–
26. Schaumberg, D. A., K. K. Snow, and M. R. Dana. 1998. The epidemic of
Acanthamoeba keratitis: where do we stand? Cornea 17:3–10.
27. Stothard, D. R., J. M. Schroeder-Dietrich, M. H. Awwad, R. J. Gast, D. R.
Ledee, S. Rodriguez-Zaragoza, C. L. Dean, P. A. Fuerst, and T. Byers. 1998.
The evolutionary history of the genus Acanthamoeba and the identification
of eight new 18S rDNA gene sequence types. J. Eukaryot. Microbiol. 45:
28. Tanaka, Y., S. Suguri, M. Harada, T. Hayabara, K. Suzumori, and N. Otha.
1994. Acanthamoeba-specific human T-cell clones isolated from healthy in-
dividuals. Parasitol. Res. 80:549–553.
29. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G.
Higgins. 1997. The ClustalX Windows interface: flexible strategies for mul-
tiple sequence alignment aided by quality analysis tools. Nucleic Acids Res.
30. Walochnik, J., O. Picher, C. Aspo ¨ck, M. Ullmann, R. Sommer, and H.
Aspo ¨ck. 1999. Interactions of “Limax amoebae” and gram-negative bacteria:
experimental studies and review of current problems. Tokai J. Exp. Clin.
31. Woodruff, S. A., and J. K. G. Dart. 1999. Acanthamoeba keratitis occurring
with daily disposable contact lens wear. Br. J. Ophthalmol. 83:1088–1089.
3936WALOCHNIK ET AL.J. CLIN. MICROBIOL.