Parasitol Res (2014) 113:2551–2557
DOI 10.1007/s00436-014-3905-x
ORIGINAL PAPER
Incidence and molecular diversity of Acanthamoeba species
isolated from public baths in Hungary
Csaba Kiss & Zsófia Barna & Márta Vargha &
Júlia Katalin Török
Received: 27 November 2013 / Accepted: 9 April 2014 / Published online: 30 April 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract Hungary has a large number of thermal baths and
spa facilities which attract hundreds of thousands of tourists
annually. Until recently, however, the free-living amoebae
were not of public health concern. Genotyping of
Acanthamoeba species, potential agents of keratitis and gran-
ulomatous encephalitis, was carried out in 20 Hungarian pub-
lic baths for the first time to assess the incidence and molec-
ular diversity of the genus in the country. Our results show that
6.7 % of the samples were positive for Acanthamoeba. Of
these positive samples, 6.5 and 7 % was from sterilized and
unsterilized pools, respectively. The 18S rRNA gene investi-
gation of the nine Acanthamoeba strains found reveals that
seven belong to the hazardous T4 genotype. The remaining
two samples were of the T15 type. All the strains kept growing
at 36 °C. Our results underline the need to develop a control
system for free-living amoebae and supervise the disinfection
of Hungarian public baths.
Keywords Hungary . Acanthamoeba . Spa bath
Introduction
Visiting public baths in Hungary may pose a serious public
health threat. Although ubiquitous free-living amoebae inhab-
it wet soil, fresh-water lakes, ponds, and rivers (Page 1988), in
the last decades more and more data indicate that they are
present in man-made environments like swimming-pools,
therapeutic pools, thermal spas, and even the air (Tsvetkova
et al. 2004). They respond to adverse environmental
C. Kiss (*) : Z. Barna : M. Vargha : J. K. Török
Government Office of Győr-Moson-Sopron County Policy
Administration Services of Public Health, Győr, Hungary
e-mail: kiss.csaba@nydr.antsz.hu
conditions by encysting. Their cysts may resist exposure to
chemical or UV light disinfections in baths or drinking water
(Khan 2006). Some genera, including thermotolerant species
such as those of Acanthamoeba and Naegleria, are associated
with human disease.
Recognition of Acanthamoeba pathogenicity in humans
dates back to the early 1970s. The first case of fatal granulo-
matous amebic meningoencephalitis (GAE) in an immuno-
compromised patient was reported in 1972 (Martinez 1982;
Jager and Stamm 1972), and amoebic keratitis (AK) infection
of immunocompetent people wearing contact lenses was doc-
umented in 1974 (Nagington et al. 1974). In Hungary, how-
ever, the examination of Acanthamoeba species, as relevant to
public health agents, initially started in 1985 (Matyi et al.
1985).
Hungarian Acanthamoeba studies have focused on three
different amoeba sources: human samples, environmental
samples from thermal springs, and those from rivers
(Szénási et al. 1998). Thermal baths and spa facilities are
among the major tourist destinations in Hungary, and they
attract large numbers of domestic and foreign visitors. Thus,
there is an urgent need to assess the potential of
Acanthamoeba in Hungarian thermal spas and public baths.
It is known that the virulence of Acanthamoeba genotypes is
diverse. Most manifested human infections (both GAE and
AK) are caused by the genotype designated T4 (Khan et al.
2006; Stothard et al. 1998; Gast et al. 1996; Horn et al. 1999;
Gast 2001). To complete our sparse knowledge of the distri-
bution and incidence of Acanthhamoeba genotypes in
Hungary, we examined the occurrence of Acanthhamoeba
genotypes in selected Hungarian thermal spas and public
baths. This examination established the presence and distribu-
tion of different genotypes, and we correlated the incidence
ratio to the methods used to clean pools in a 1-year study. To
assess the infection potential of the isolated Acanthamoeba
strains, we performed thermotolerance tests.
2552
Materials and methods
Sampling
Sampling was performed anonymously in 20 public baths
including thermal spas and swimming pools in the western
part of Hungary, resulting in a total of 164 water samples. Of
these samples, 107 were from pools that were chemically
disinfected and 57 were from pools that had not been
disinfected.
Three baths with a total of 30 pools were investigated
repeatedly, resulting in 81 samples. From each pool, a
1,000 ml water sample was concentrated to 10 ml using a
cellulose ester membrane filter of 0.45 μm pore size (Merck
Millipore, USA) followed by centrifuging at 600 g for 10 min.
After removing the top 9.9 ml of water, the remaining pellets
were injected into 1.5 % Page’s saline agar plates.
Isolation and culturing
The amoeba plates were incubated at 25 °C and examined
under a light microscope (Zeiss Axioskop) until trophozoites
appeared, generally less than 1 week. The amoebae were then
transferred into new 1.5 % Page’s saline agar plates seeded
with heat-killed Escherichia coli to obtain pure amoeba cul-
tures. Amoeba strains were then subcultured biweekly on
fresh agar plates by transferring one small agar piece with
one amoeba specimen. A total of 11 strains of amoeba were
isolated from these samples.
We identified the amoebae at genus level by microscopic
observation of the throphozoites’ floating and creeping forms,
as well as by the shape of the cysts. The throphozoites ap-
peared within 2–3 days, and the Acanthamoeba specimens
were identified by their characteristic acanthopodia and vacu-
oles. About a week later, the first typical, double-walled cysts
appeared, and within 2 weeks, the majority of the trophozoites
formed cysts. The visually confirmed Acanthamoeba strains
were subjected to subsequent genus-specific PCR analyses.
Amoeba samples without prior genus assignment were proc-
essed with FLA primers.
Molecular biological and sequence analyses
DNA extraction was carried out with the DNeasy Blood &
Tissue Kit (Qiagen, Germany) according to the manufac-
turer’s instructions. The PCR analyses were performed using
genus-specific primers JDP1 and JDP2 (Dykova et al. 1999),
and universal primers designed for free-living amoebae (FLA)
(Tsvetkova et al. 2004). PCR was conducted in a final reaction
volume of 50 μl containing 5 μl template, 0.3 μl (5 U/μl) Taq
DNA Polymerase (Fermentas, Latvia), 1.2 μl dNTP (10 mM),
5 μl MgCl2 (25 mM), and 37 μl of ×1 PCR buffer (Fermentas,
Latvia), with forward and reverse primers 0.75 μl (32.5 nM)
Parasitol Res (2014) 113:2551–2557
each. Table 1 shows the applied PCR protocol and primers
used by us.
The PCR products were detected by 1 % agarose gel
electrophoresis using ethidium bromide staining and purified
using a PCR Advanced™ kit from Viogene (Taiwan). The
purified amplicons were prepared for sequencing using the
following PCR mixture: 1 μl BigDye (Applied Biosystems),
2 μl buffer (Applied Biosystems), 1 μl reverse primer (see
above), 2 μl PCR water, and 4 μl sample. The PCR reaction
conditions were as follows: 96 °C for 10 s, 50 °C for 5 s, and
60 °C for 4 min for 28 cycles, then the DNA was precipitated
and submitted for sequencing (BIOMI, Hungary).
Nucleotide sequences were identified by a BLAST search
optimized for highly similar sequences (NCBI megablast
algorithm). In addition to the newly obtained sequences,
Acanthamoeba sequences from the GenBank database were
included in the phylogenetic analyses (Kong 2009; Corsaro
and Venditti 2010; Chan et al. 2011). The multiple alignments
were performed using the ClustalW algorithm, and the phylo-
genetic tree was constructed using the maximum likelihood
method [GTR, bootstrap test of 1,000 replicates, MEGA5.0.5
(Tamura et al. 2011)].
Thermotolerance analyses
For thermotolerance analyses, all freshly injected
Acanthamoeba plates were incubated for 3–7 days at 25, 30,
36, and 42 °C, respectively, and the tests were repeated five
times for each amoeba strain.
Results
After 1-week incubation of the 164 water samples from pools,
41.4 % tested positive for amoebae (68 samples), and 6.7 % of
this total contained the genus Acanthamoeba. Acanthamoeba
was found in 6.5 % of the samples from disinfected pools and
7 % of the samples from pools that were not disinfected
(Figs. 1 and 2).
Upon higher magnification of the SSU rDNA samples,
nine new Acanthamoeba sequences were found. Identified
Acanthamoeba strains with accession numbers are shown in
Table 2.
Our phylogenetic tree constructed for the Acanthamoeba
species, including the newly found sequences, suggests that
all but two of these sequences belong to the hazardous T4
strain (Fig. 3).
In the present study, we examined whether the obtained
Acanthamoeba strains could survive in a 36–42 °C tempera-
ture range. This temperature range represents clinical mani-
festation (GAE and AK) in a human host. Each isolate was
able to grow and proliferate at 36 °C, but no growth, nor
Parasitol Res (2014) 113:2551–2557 2553

Table 1 Shows applied primers and PCR methods

Genus Forward primer Reverse primer Initial Denaturation/ Final Number

denaturation Anellation/Extension elongation of cycles

Acanthamoeba JDP1 JDP2 95 °C 95 °C 30 s 61 °C 30 s 72 °C 2 min 72 °C 10 min 40

5′-GGCCCAGA 5′-TCTCACAAG
TCGTTTACCG CTGCTAGGG
TGAA-3′ AGTCA-3′
FLA FLAF FLAR 94 °C 94 °C 1 min 63 °C 1 min 74 °C 3,5 min 72 °C 10 min 40
5′-CGCGGTAAT 5′-CAGGTTAAG
TCCAGCTCC GTCTCGTTCGT
AATAGC-3′ TAAC-3′

proliferation, was observed at 42 °C. Table 3 shows the results
of the thermotolerance analyses.
Discussion
The 164 processed samples from 20 public baths demonstrate
that disinfected and non-disinfected pools share an almost
equal proportion of Acanthamoeba colonisation (6.7 and
7 %, respectively). This unexpected finding shows that the
currently applied disinfection methods are inadequate against
Acanthamoeba.
Early classification of Acanthamoeba species was wholly
based on morphology. Based on the shape and size of the
cysts, three major groups of species were established within
the genus Acanthamoeba (Pussard and Pons 1977). However,
this classification had been challenged, because additional
investigation described the dependence of cyst morphology
on the ionic strength of the surrounding environment (Sawyer
1971). Therefore, only molecular methods allow reliable dif-
ferentiation of the Acanthamoeba species (Schuster and
Visvesvara 2004). To date, 18 genotypes have been identified
(Qvarnstrom et al. 2013; Maciver et al. 2013; Trabelsi et al.
2012). Although a number of these methods such as T3, T11,
and even T5 have been noted to result in human clinical
manifestations, as does AK (Maciver et al. 2013; Lorenzo-
Morales et al. 2010; Walochnik et al. 2000; Ledee et al. 2009),
the genotype T4 is particularly important in this regard. The
majority of the human pathogenic Acanthamoeba strains have
proved to belong to the T4 genotype and are believed to be
causative agents of both AK (Acanthamoeba castellani,
Acanthamoeba polyphaga, Acanthamoeba rhysodes, and
Acanthamoeba lugdunensis) (Yu et al. 2004) and GAE
(A. castellani and A. polyphaga) (Castellano-Sanchez et al.
2003; Martinez 1991). Seven of the nine Acanthamoeba
strains identified by sequence analysis in this study belong

Fig. 1 Shows a priori identification of acanthamoebae using light mi- Acanthamoeba (transmitted light), e Trophozoite and cysts together at
croscopy. a trophozoite resting on agar plate (transmitted light), b–c close low magnification (phase contrast micrograph). Scale bars represent 15
up of stretched trophozoites on a microscope slide (Nomarski interference (a), 20 (b–c), 10 (d), and 60 μm (e), respectively
contrast micrograph), d clump of cysts with wall characteristic of
2554 Parasitol Res (2014) 113:2551–2557

pools, the current disinfection methods employed in the inves-

Fig. 2 Shows distribution of water samples for two types of pools,
disinfected and not disinfected. Samples that tested positive for
Acanthamoeba are shown in dark grey
to the T4 group and two strains to the T15 genotype. This
latter genotype appears to be Acanthamoeba jacobsi and
represents the first detection of this species in Hungary.
An amoeba must meet several criteria to be considered
pathogenic in humans. One such requirement is thermotoler-
ance. It has long been known that thermotolerant strains of
Acanthamoeba, which grow at 37 °C, are able to cause AK,
because the average corneal temperature of the human eye is
about 34 °C (Walochnik et al. 2000). Isolates that tolerate
42 °C may cause GAE. In the thermotolerance experiments,
all Acanthamoeba isolates displayed growth at 37 °C, but none
of them showed growth at 42 °C. Thus, the Acanthamoeba
strains isolated from the sampled public pools may have the
potential to cause AK, but presumably not GAE. Since the
potentially hazardous T4 and T15 strains were distributed
roughly equally between the disinfected and non-disinfected
tigated pools should be radically changed so that they are
effective against thermotolerant cyst forming Acanthamoeba
species. Although clinical cases due to Acanthamoeba infec-
tion have not been assigned to public baths in Hungary, the
obtained strains of the T4 and T15 genotypes should be con-
sidered clinically relevant. The Acanthamoeba infections
resulting in GAE are likely to be underdiagnosed in Hungary.
Because autopsies are not legally mandatory any more, direct
causes of death might remain unknown in elderly patients,
especially for those with chronic diseases. Therefore, we can-
not exclude Acanthamoeba infection as a possible causative
agent in unexplained deaths.
Three baths containing 30 pools have been repeatedly
sampled. One of the three baths tested positive for
Acanthamoeba on each sampling, while the other two always
tested negative. In the bath that repeatedly tested positive for
Acanthamoeba, the positive tests were from different pools
during the sampling period. A pool that tested positive was not
found to test positive during subsequent testing. Although a
particular sampling method did not give representative results
for the whole pool, we must emphasize the fact that once the
Acanthamoeba appears in the water treatment system of a
certain bath, its elimination with the current disinfection
methods seems to be insufficient because the Acanthamoeba
trophozoites live mostly within the water’s biofilm and only
occasionally float in the water (Stockman et al. 2011). Our
study did not test the various disinfecting methods employed
in Hungarian baths; therefore, we cannot hypothesize as to the
extent that a well-developed biofilm might protect
Acanthamoeba cells against disinfectants dissolved in the
water. But Hiti et al. (2002) provide data for the difference
in the anti-Acanthamoeba activity of three different contact
lens storage solutions, supporting our assumption that applied
bath-disinfectants might differ in their efficiency in destroying
Acanthamoebae. In addition, Acanthamoeba cysts can be

Table 2 Shows a list of newly found SSU rDNA sequences of Acanthamoeba strains obtained by a BLAST search and the corresponding GenBank
accession numbers

Strain Accession number Published 18S rDNS sequences in GenBank

Closest match Nearest species match

ACA_60/10 HG797013 A. jacobsi AY262364 (99 %) A. jacobsi AY262363 (99 %)

ACA_61/10 HG797015 Acanthamoeba sp. AY703008 (97 %) A. castellanii EF554328 (97 %)
ACA_85/10 HG797014 Acanthamoeba sp. JX423592 (97 %) A. castellanii EF554328 (97 %)
ACA_92/10 HG797016 Acanthamoeba sp. AF005998 (97 %) A. castellanii EF554328 (97 %)
ACA_105/10 HG797017 Acanthamoeba sp. JX423592 (99 %) A. castellanii EF554328 (99 %)
ACA_822/10 HG797018 A. jacobsi AY262364 (100 %) A. jacobsi AY262363 (100 %)
ACA_2636/10 HG797019 Acanthamoeba sp. DQ264391 (99 %) A. mauritaniensis AY351647 (98 %)
ACA_2640/10 HG797020 Acanthamoeba sp. JQ669660 (100 %) A. polyphaga AF019061 (99 %)
ACA_2643/10 HG797021 Acanthamoeba sp. DQ264391 (97 %) A. mauritaniensis AY351647 (95 %)
Parasitol Res (2014) 113:2551–2557 2555

Fig. 3 Shows a maximum likelihood tree of Acanthamoeba partial SSU rDNA sequences. The tree shows the topology of the newly found isolates (in
bold type) and their corresponding genotypes

Table 3 Shows the proportions of surviving strains at different incuba- resistant to certain chemical and UV disinfectants (Khan
tion temperatures
2006).
Strain Incubation temperature and proportion of positive In the present study, we tried to address the incidence and
samples molecular diversity of Acanthamoeba species in Hungarian
public baths. We believe that it is the first systematic regional
25 °C 30 °C 36 °C 42 °C
study pertaining to the presence of Acanthamoebae in
ACA_60/10 5/5 5/5 5/5 0/5 Hungary. During previous pilot studies, concerning incidence
ACA_61/10 5/5 5/5 5/5 0/5 of gymnamoebae species in general, only human pathogenic
ACA_85/10 5/5 5/5 5/5 0/5 amoebae were recorded in the course of occasional sampling
ACA_92/10 5/5 5/5 5/5 0/5 studies. Natural water and soil samples often demonstrate
ACA_105/10 5/5 5/5 5/5 0/5 more than 60 % frequency of Acanthamoeba (e.g.,
ACA_822/10 5/5 5/5 5/5 0/5 Mahmoudi et al. 2012; Reyes-Batlle et al. 2014). Compared
ACA_2636/10 5/5 5/5 5/5 0/5
to similar studies, where the percentage of positive
ACA_2640/10 5/5 5/5 5/5 0/5
Acanthamoeba samples was noted to be 20 (Caumo and
ACA_2643/10 5/5 5/5 5/5 0/5
Rott 2011), 25 (Penas-Ares et al. 1994), 37.5, and 85.7 %
(Tsvetkova et al. 2004), we found that 6.7 % of the total
2556
samples tested positive for Acanthamoeba. However, consid-
ering the high rate (77 %) of the hazardous T4 genotype
recorded in our Acanthamoeba-positive samples, we conclude
that the T4 genotype has a much higher incidence in
Hungarian public baths when compared to previously report-
ed data (23 %) (Caumo and Rott 2011).
Prior to molecular biological genotyping studies, the path-
ogenicity of Acanthamoeba isolates was tested on mice. These
studies suggested high rates of pathogenic isolates [66.7
(Penas-Ares et al. 1994), 70 (De Jonckheere 1979), and
100 % (Rivera et al. 1989; Scaglia et al. 1987)]. These exper-
imental results are well-supported by evidence that shows that
the T4 genotype has the highest rate of incidence in environ-
mental samples, followed by the T3, T2, and T5 genotypes
(Maciver et al. 2013; Reyes-Batlle et al. 2014). Our results
correspond well with the latter experience regarding the pre-
dominance of T4. The public health threat posed by the
presence of the Acanthamoeba species is twofold: (1) active
trophozoites represent possible agents of AK and GAE, and
(2) they can transmit numerous microorganisms harmful to
humans, viruses, bacteria, and microeukaryotes (Scheid and
Schwarzenberger 2011, 2012). Even Acanthamoeba cysts
might host pathogenic prokaryotes (Kilvington and Price
1990). Ultimately, the Hungarian public baths need active
surveillance to identify and control the presence of potentially
hazardous Acanthamoeba species and an urgent revision of
disinfection methods in any affected pools.
Acknowledgments The authors wish to thank Mr. Robert F. Mattick
for his grammatical English language review. Research was financed by
the Hungarian National Scientific Foundation (OTKA) no. T-49632
project.
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