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The role of domestic tap water on Acanthamoeba keratitis in non-contact lens wearers and validation of laboratory methods

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Acanthamoeba is increasingly recognized as an important cause of keratitis in non-contact lens wearers while contact lens wear is the leading risk factor for Acanthamoeba keratitis (AK). It is unlikely that the Acanthamoeba colonization is a feature which is effective only in patient's homes with infectious keratitis since the organism has been isolated from domestic tap water. Two hundred and thirty-one (231) corneal scrapings were taken from infectious keratitis cases, and four contact lens solutions and domestic tap waters were taken from 22 out of 44 AK-diagnosed patient's homes. Microscopic examination, culture, PCR, real-time PCR and DNA sequencing analyses were used for AK-diagnosed samples. The real-time PCR was the most sensitive (100 %) one among the methods used in diagnosis of AK. The 44 (19.0 %) out of 231 corneal scrapings, 4/4 (100 %) contact lens solution and 11/22 (50 %) of domestic tap water samples were found to be positive by real-time PCR for Acanthamoeba. A. griffini (T3), A. castellanii (T4) and A. jacobsi (T15) genotypes were obtained from corneal scrapings, contact lens solutions and domestic tap water samples taken from the patient's homes diagnosed with AK. The isolation of Acanthamoeba containing 6/22 (27.3 %) A. griffini (T3), 14/22 (63.6 %) A. castellanii (T4) and 2/22 (9.1 %) A. jacobsi (T15) from the domestic tap water outlets of 22 of 44 (50 %) of patient's homes revealed that is a significant source of these organisms. A. griffini (T3) and A. jacobsi (T15) genotypes have not been determined from AK cases in Turkey previously. Thus, we conclude that Acanthamoeba keratitis is associated with exposition of patients who has ocular trauma or ocular surface disease to domestic tap water in endemic or potentially endemic countries.
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ORIGINAL PAPER
The role of domestic tap water on Acanthamoeba
keratitis in non-contact lens wearers and validation of laboratory
methods
Ismail Soner Koltas
1
&Fadime Eroglu
2
&Elif Erdem
3
&Meltem Yagmur
3
&Ferdi Tanır
4
Received: 9 April 2015 /Accepted: 19 May 2015 /Published online: 29 May 2015
#Springer-Verlag Berlin Heidelberg 2015
Abstract Acanthamoeba is increasingly recognized as an im-
portant cause of keratitis in non-contact lens wearers while con-
tact lens wear is the leading risk factor for Acanthamoeba kera-
titis (AK). It is unlikely that the Acanthamoeba colonization is a
feature which is effective only in patients homes with infectious
keratitis since the organism has been isolated from domestic tap
water. Two hundred and thirty-one (231) corneal scrapings were
taken from infectious keratitis cases, and four contact lens solu-
tions and domestic tap waters were taken from 22 out of 44 AK-
diagnosed patients homes. Microscopic examination, culture,
PCR, real-time PCR and DNA sequencing analyses were used
for AK-diagnosed samples. The real-time PCR was the most
sensitive (100 %) one among the methods used in diagnosis of
AK. The 44 (19.0 %) out of 231 corneal scrapings, 4/4 (100 %)
contact lens solution and 11/22 (50 %) of domestic tap water
samples were found to be positive by real-time PCR for
Acanthamoeba. A. griffini (T3), A. castellanii (T4) and
A. jacobsi (T15) genotypes were obtained from corneal scrap-
ings, contact lens solutions and domestic tap water samples taken
from the patients homes diagnosed with AK. The isolation of
Acanthamoeba containing 6/22 (27.3 %) A. griffini (T3), 14/22
(63.6 %) A. castellanii (T4)and2/22(9.1%)A. jacobsi (T15)
from the domestic tap water outlets of 22 of 44 (50 %) of pa-
tients homes revealed that is a significant source of these organ-
isms. A. griffini (T3) and A. jacobsi (T15) genotypes have not
been determined from AK cases in Turkey previously. Thus, we
conclude that Acanthamoeba keratitis is associated with exposi-
tion of patients who has ocular trauma or ocular surface disease
to domestic tap water in endemic or potentially endemic
countries.
Keywords Acanthamoeba keratitis .Non-contact lens
wearers .Domestic tap water .Acanthamoeba griffini (T3) .
Acanthamoeba castellanii (T4) .Acanthamoeba jacobsi (T15)
Introduction
Acanthamoeba is able to cause a painful sight-threatening
disease of the eyes designated as Acanthamoeba keratitis
(AK) in addition to their role as causative agents of the well-
known Acanthamoeba granulomatous encephalitis (GAE)
(Scheid et al. 2008). AK can be misdiagnosed in clinical ex-
amination since its clinical symptoms are similar to herpetic
and fungal infections (Lehmann et al. 1998). Laboratory
methods are essential for AK diagnosis (Ge et al. 2013;
Claerhout and Kestelyn 1999).Thereareanumberoflabora-
tory techniques to confirm this diagnosis; bacteriological
(smear or cultures) and eventually histopathological
(Claerhout and Kestelyn 1999).
Laboratory methods to diagnose Acanthamoeba are based
on direct microscopic examination using stains coupled with
culture-based methods (Khairnar et al. 2011). However, these
methods are generally not sensitive, convenient or applicable
enough for a precise diagnosis (Ge et al. 2013). Thus, several
investigators have recently demonstrated the usefulness of
*Ismail Soner Koltas
koltas@cu.edu.tr
1
Department of Medical Parasitology, Faculty of Medicine, Cukurova
University, 01330Balcali, Saricam Adana, Turkey
2
Department of Medical Microbiology, Emine-Bahaeddin Nakıboglu
Medical Faculty, Zirve University, 27260, Şahinbey
Gaziantep, Turkey
3
Department of Medical Ophthalmology, Faculty of Medicine,
Cukurova University, 01330Balcali, Saricam Adana, Turkey
4
Department of Public Health, Faculty of Medicine, Cukurova
University, 01330Balcali, Saricam Adana, Turkey
Parasitol Res (2015) 114:32833289
DOI 10.1007/s00436-015-4549-1
molecular methods such as polymerase chain reaction (PCR)
and real-time PCR for AK diagnosis (Khairnar et al. 2011;
Inoue and Ohashi 2013). These methods could be suitable
for both clinical and epidemiological purposes; therefore, they
need to be reliable and sensitive (Pasricha et al. 2003).
AK is also one of the major diseases caused by
Acanthamoeba, which is commonly associated with contact
lens wearers (Alsam et al. 2008). On the other hand, there
have been studies to determine susceptibility of
Acanthamoeba isolates from non-contact lens wearers to var-
ious antimicrobials, and the results of the studies are associat-
ed with use of cationic antiseptics (PHMB and CHx) has re-
sulted in a dramatic improvement in the clinical management
of Acanthamoeba keratitis (Sharma et al. 2000). In addition,
the results of the reports from developing countries reveal that
the main risk factors are corneal trauma and exposure to con-
taminated domestic tap water (Zhao et al. 2010). Water storage
tanks promote colonization of domestic tap water with free-
living amoeba (FLA), including Acanthamoeba, and hence
increase the risk of AK (Kilvington et al. 2004).
This study, therefore, aimed to cross compare the laborato-
ry methods in diagnosis of Acanthamoeba keratitis. The mi-
croscopic examination, culture, PCR and real-time PCR
methods were used for AK diagnosis. We found that genotyp-
ing of Acanthamoeba was causative agents of AK by DNA
sequencing. We also investigated that domestic tap water, con-
tact lens and non-contact lens wearer relationship with AK.
Best of our knowledge, it is the first time that the presence and
distribution of Acanthamoeba genotypes in AK patients and
domestic tap water samples were conducted in Cukurova re-
gion, located in southern Turkey.
Materials and methods
Clinical samples and domestic tap water samples
The 231 corneal scrapings were taken from the patients with
corneal infection and suspected of contacting infectious kera-
titis at Ophthalmology Department, Faculty of Medicine,
Cukurova University, during the period from April 2010 to
December 2013. Corneal scrapings were transferred into the
sterile tube containing phosphate buffer saline (PBS). PBS is
generally utilized to maintain of Acanthamoeba strains for the
short term in a viable condition. The patients responded to the
questions on possible risk factors for the development of AK.
They were related to contact lens wear, disinfection and stor-
age methods, contact lens with domestic tap water and history
of corneal trauma and ocular surface disease. In addition, con-
tact lens solutions were taken from four infectious keratitis
patients, and domestic tap waters were taken from 22 out of
44 AK-diagnosed patients homes. We have not taken tap
water samples from all of the AK-diagnosed patientshomes
because 22 patients did not accept to give tap water samples.
Microscopic examination
The corneal scrapings and contact lens solutions were placed
onto the slides, and the slides were examined for cysts and
trophozoites at ×400 magnification by using a standard light
microscope. We investigated different FLA species such as
Hartmannella using microscopic examination from the corne-
al scrapings and contact lens solutions. Also, FLA parasites
such as Nagleria,Hartmannella,Balamuthia and Sappinia
from the tap water samples were investigated.
Culture
All of the corneal scrapings and contact lens solutions were
inoculated onto plates containing 2 % non-nutrient agar over-
laid with heat-killed Escherichia coli (E. coli) and incubated at
27 °C. The domestic tap water samples taken from AK-
diagnosed patients homes collected in 500-mL sterilized
polypropylene tubes were filtered through 0.45-μMporesize
cellulose filter (Millipore Corporation, Bedford Madison,
USA) under a weak vacuum. The filters were inverted on
heat-inactivated E. coli-treated 2 % non-nutrient agar plates
and incubated at 27 °C. After 34 days, the plates were mon-
itored with an inverted microscope (Leica DM3000, Houston,
Texas, USA) for the outgrowth of Acanthamoeba.
Genus-specif ic PCR for Acanthamoeba
18S rRNA gene amplifications were performed with primers;
forward (5
ı
- GTTTGAGGCAATAACAGGT-3
ı
) and reverse
(5
ı
-GAATTCCTCGTTGAAGAT-3
ı
) (Mathers et al. 2000).
The PCR mix was carried out in 25 μL volume containing
12.5 μl of PCR Master Mix (Promega, USA), 0.5 μlofeach
primer and 5 μl of the DNA samples. The thermal-cycler
programme was composed of an initial denaturation at 95 °C
for10minand35cyclesofdenaturationat9Cfor30s,
annealing at 60 °C for 45 s, extension at 72 °C for 30 s and
final extension at 72 °C for 10 min. The DNA extraction was
performed using the DNA isolation kit (Beckman Coulter,
Beverly, USA). Positive controls with genomic DNA of
Acanthamoeba (ATCC 50370) reference strain and negative
control without DNA template were included in PCR method.
There were no false positive and contamination of clinical
samples and reagents. PCR products were analyzed by
1 % agarose gel stained with ethidium bromide and
visualized under ultraviolet light. The PCR products
were considered to be positive with a fragment of the
correct size approximately 229 bp.
3284 Parasitol Res (2015) 114:32833289
Genus-specif ic real-time PCR for Acanthamoeba
The real-time PCR assay primer AcantF900 (5
ı
-CCCAGATC
GTTTACCGTGAA-3
ı
), AcantR1100 (5
ı
-TAAATATTAATG
CCCCCAACTATCC-3
ı
) and probe (5-FAM-CTGCCACC
GAATACATTAGCATGG-BHQ1-3) were used to amplify
fragments of approximately 180 bp in 18S rRNA gene of
Acanthamoeba (Qvarnstrom et al. 2006). The genus-specific
real-time PCR reaction consisted of a total volume of 20 μl,
containing 10 ng of extracted DNA, 0.2 μM primer mix (for-
ward and reverse), 0.1 μM probe, 1 QuantiFastSYBRGreen
PCR kit (Qiagen, Valencia, CA, USA) and 4 μL distilled
water. Genus-specific real-time PCR assay was carried out in
a Rotor-Gene RG-3000 (Corbett Research, San Francisco,
CA, USA). The thermal cycling conditions were as follows:
initial 50 °C for 2 min, initial denaturation at 95 °C for 2 min,
followed by 40 cycles of denaturation at 95 °C for 15 s, an-
nealing at 60 °C for 60 s, extension at 72 °C for 10 s and final
extension at 72 °C for 10 min.
All of the reactions were analyzed using the software pro-
vided with the instrument. The average cycle threshold (C
T
)
values were determined, and the standard curves were calcu-
lated using the Rotor-Gene 6.1.93 software.
Genotyping of Acanthamoeba with DNA sequencing
The DNA amplification reaction was performed using the
JDP1 (5-GCCCAGATCGTTTACCGGAA3) and JDP2 (5
TCTCACAGCTGCTAGGGGAGTCA-3) primers to ampli-
fy a 423551 bp fragment of 18S rRNA gene in
Acanthamoeba (Schroeder et al. 2001). 50 μLvolumecon-
taining 10 ng DNA template, 1× (50 mM KCl
2
,pH8.3,
10 mM Tris HCl, 2.5 mM MgCl
2
), 0.2 mM dNTP, 0.6 mM
each primer and 0.4 units of Taq polymerase (Fermantas,
Thermo Scientific, Burlington, Canada). The thermal-cycler
programme was an initial denaturation at 95 °C for 10 min and
35 cycles of denaturation at 95 °C for 30 s, annealing at 65 °C
for 45 s, extension at 72 °C for 30 s and final extension at
72 °C for 10 min. Amplification products were analyzed by
2 % agarose gel stained with ethidium bromide and visualized
under ultraviolet light.
PCR products were purified using a SentroPure DNA pu-
rification kit (Sentromer DNA Technologies, Istanbul,
Turkey) and sequenced using the BigDye Terminator V 3.1
cycle sequencing kit (Applied Biosystems, California, USA)
as per the manufacturers protocol on the ABI Prism 310
TM
Genetic Analyzer (Applied Biosystems, California, USA).
Genotype identification was based on the sequence analysis
of 18S rRNA gene as previously described by comparing with
available Acanthamoeba DNA sequences in GenBank data-
base using Basic local alignment search tool (BLAST) analy-
sis software (www.ncbi.nlm.nih.gov/BLAST). Genotype
assignment was performed by sequence comparison, and
multiple alignments were performed using ClustalW2 in
comparison with sequences available in GenBank
TM
for
Acanthamoeba genotypes. Phylogenetic analysis was carried
out using the neighbor-joining (NJ) based on maximum par-
simony, minimum evaluation and maximum likelihood opti-
mality criteria, implemented in Mega software version 6.05.
Bootstrap values were based on 1000 replicates.
Results
The results of microscopic examination, culture, PCR
and real-time PCR methods
Acanthamoeba, 3.9 % (9/231), was found to be positive and
96.1 % (222/231) negative in infectious keratitis cases with
microscopic examination. We did not find other FLA species
in corneal scrapings and contact lens solutions by microscopic
examination. The result of culture was found to be 7.8 % (18/
231) Acanthamoeba positive and 92.2 % (213/231)
Acanthamoeba negative in infectious keratitis cases. In addi-
tion, 17.3 % (40/231) corneal scrapings were Acanthamoeba
positive, and 82.7 % (191/231) corneal scrapings were found
to be Acanthamoeba negative in infectious keratitis cases
when PCR method was used. According to real-time PCR
method, 19.0 % (44/231) were Acanthamoeba positive and
81.0 % (187/231) were Acanthamoeba negative in infectious
keratitis cases (Table 1).
Of the 44 AK patients, four (9.1 %, 4/44) were using contact
lenses while 40 (90.9 %, 40/44) patients were not using contact
lenses. Risk factors in patients who were not contact lens users
were corneal trauma (62.5 %, 25/40) and ocular surface disease
(37.5 %, 15/40). The first symptom noted by the patients was
ocular discomfort and pain, initially stinging or burning, with
very severe pain in later cases. Other symptoms included pho-
tophobia, lacrimation, redness and a decrease in vision. The
common clinical findings were pseudodendritis, radial
keratoneuritis, ring-shaped infiltrate, hypopyon and diffuse
epithelitis. Medical treatment was consisted of the combination
of topical propamidine isethionate (0.1 %), chlorhexidine drop
(0.02 %) and oral ketoconazole. In addition, Acanthamoeba
were determined in 11 (50 %, 11/22) domestic tap water sam-
ples. There were no other detected FLA parasites such as
Tabl e 1 The results of laboratory methods (microscopic examination,
culture, PCR and real-time PCR) for AK diagnosis
AK-suspected cases (n=231) ME Culture PCR Real-time PCR
Positive, % 3.9 7.8 17.3 19.0
Negative, % 96.1 92.2 82.7 81.0
ME microscopic examination
Parasitol Res (2015) 114:32833289 3285
Nagleria,Hartmannella,Balamuthia and Sappinia from the
tap water samples.
The sensitivities and specificities of microscopic
examination, culture, PCR and real-time PCR methods
for Acanthamoeba
The sensitivities and specificities of microscopic examination,
PCR and real-time PCR were determined using culture as a
gold standard. The sensitivity and specificity of microscopic
examination were 50 and 100 %, respectively. The sensitivity
and specificity of PCR and real-time PCR methods were
found to be 100 and 89.6 % and 100 and 87.8 %, respectively.
In addition, the comparison of the sensitivities and specific-
ities of the other methods were seen in Table 2.
The results of genotyping of Acanthamoeba species
The nucleotide sequencing of Acanthamoeba isolates (corneal
scrapings, contact lens solution, domestic tap water) were de-
termined to be showing 100 % similarity with Acanthamoeba
reference strain (ATCC 50370). As for the DNA sequencing,
34.1 % (15/44) A. griffini (T3), 56.8 % (25/44) A. castellanii
(T4) and 9.1 % (4/44) A. jacobsi (T15) were determined in
Acanthamoeba isolates obtained from AK patients. The sim-
ilarity between nucleotide sequencing of Acanthamoeba iso-
lates from contact lens solution and A. castellanii (T4) in
GenBank was 100 %. As compared to the previous records
in GenBank, the similarity between sequences of
Acanthamoeba isolates from domestic tap water samples and
Acanthamoeba strains in GenBank was 100 %. The following
DNA sequencing results 27.3 % (6/22) A. griffini (T3), 63.6 %
(14/22) A. castellanii (T4) and 9.1 % (2/22) A. jacobsi (T15)
were observed in domestic tap water samples (Table 3).
On the other hand, phylogenetic analysis revealed that the
Acanthamoeba isolates were different from nucleotide se-
quencing. Acanthamoeba isolates revealed three different
clusters as A. griffini (T3), A. castellanii (T4) and A. jacobsi
(T15) (Fig. 1).
The nucleotide sequencing of Acanthamoeba isolated from
corneal scrapings, contact lens solution and domestic tap wa-
ter was found to be similar to each other. We observed that
bootstrap values were high and the clustering of strains
remained in the same order if maximum parsimony or other
out-groups were used.
Discussion
The cause of the AK may be difficult to diagnose due to its
variable course and appearance, which is similar to viral ker-
atitis in early stage of disease (Szentmary et al. 2013). The
early suspicion of AK is important since an early diagnosis
might lead to specific early treatment with good response to
the therapy (Lorenzo-Morales et al. 2007). The diagnosis of
AK was made on the basis of clinical and laboratory exami-
nation (Illingworth et al. 1995). Traditional laboratory
methods to diagnose Acanthamoeba are based on microscopic
examination and culture. Microscopic examination has suf-
fered from poor sensitivity, while culture requires several days
to yield a result (Khairnar et al. 2011). Furthermore, 60 % of
AK cases are misdiagnosed using microscopic examination,
and positive cultures are often obtained only after prolonged
incubation (Yera et al. 2007; Kao et al. 2013). Therefore, the
recent trend has focused on the application of PCR and real-
time for the detection of Acanthamoeba with excellent sensi-
tivity when compared to traditional laboratory methods
(Khairnar et al. 2011). Yera et al. have reported the sensitivi-
ties of microscopic examination, culture and PCR as 33 %
(5/15), 6.7 % (1/15) and 94 % (15/16), respectively (Yera
et al. 2007). Kao et al. have suggested that the real-time
PCR is a highly sensitive and specific method, and it is also
simple, rapid, less labor-intensive and less time-consuming
(Kao et al. 2013). We evaluated microscopic examination,
Tabl e 2 The sensitivities and
specificities of laboratory
methods (microscopic
examination, culture, PCR and
real-time PCR) for AK diagnosis
Sensitivity, % Specificity, %
ME Culture PCR Real-time PCR ME Culture PCR Real-time PCR
BL 95.6 86.0 84.2 BL 100 100 100
50 BL 100 100 86.1 BL 89.6 87.8
22.5 45.0 BL 100 100 100 BL 97.9
20.4 40.9 90.9 BL 100 100 100 BL
ME microscopic examination, BL the method was used as baseline
Table 3 The results of genotyping of Acanthamoeba isolates from
corneal scrapings, contact lens solution and domestic tap water
AK cases, % (n=44) Corneal
scrapings
Contact lens
solution
Domestic
tap water
Acanthamoeba griffini (T3) 34.1 0 27.3
Acanthamoeba castellanii (T4) 56.8 100 63.6
Acanthamoeba jacobsi (T15) 9.1 0 9.1
3286 Parasitol Res (2015) 114:32833289
culture, PCR and real-time PCR for the routine diagnosis of
AK directly in corneal scraping obtained from patients with
clinical signs or risk factors suggesting Acanthamoeba corneal
infection. Culture has to be maintained to find other FLA
(Sappinia,Hartmannella) that are known as etiological
agents. Our results supported the usefulness of real-time
PCR additionally to the culture for confirmative, early and
sensitive diagnosis of AK in corneal scraping.
Swimming and showering while wearing contact lenses are
also risk factors for AK (Boggild et al. 2009). Poor hygiene
practices, such as the failure to comply with recommended
lens clearing and disinfection procedures and the rinsing and
storing of lenses in non-sterile saline or domestic tap water, are
recognized risk factors for infect ion (Kilvington et al. 2004).
In recent years, AK has increased, and the main risk factor is
the wear of contact lenses which represent 85 to 90 % of the
AK cases (Dendane et al. 2013). In this study, 100 % (4/4)
Acanthamoeba were determined in contact lens solutions of
AK patients, which confirmed that contact lens solutions play
an important role in contamination of Acanthamoeba.
On the other hand, there are some reports from the USA
and the UK that have described the disease in non-contact lens
wearers (Sharma et al. 2000). In addition, the reported inci-
dence of AK in India varies from 1 to 3 and the cases predom-
inantly in non-contact lens wearers (Pasricha et al. 2003). Our
previously published report of AK in non-contact lens wearers
from Turkey suggested that trauma and exposure to contami-
nated water were major risk factors (Erdem et al. 2014). The
genotyping of Acanthamoeba from AK in non-contact lens
wearers and their domestic tap water were found to be the
same genotyping in this present study. Thus, the results of this
study confirmed our hypothesis in our previous study. These
studies emphasize that ophthalmologists and general physi-
cians in Turkey and other developing countries should be
aware of Acanthamoeba as one of the causes of keratitis in
non-contact lens wearers and hence should take immediate
measures for its early diagnosis and treatment.
Domestic tap water has been commonly cited in literature
as a significant risk factor for the development of AK
(Bonilla-Lemus et al. 2010). Jeong et al. have obtained the
majority of Acanthamoeba isolates from domestic tap water,
and the researchers have reported domestic tap water is poten-
tially keratopathogenic (Jeong et al. 2007). Furthermore,
Lorenzo-Morales et al. reported Acanthamoeba contamina-
tion in 88 out of 148 (59.5 %) tap water samples in Spain
(Lorenzo-Morales et al. 2005).Wefound50%(11/22)
Acanthamoeba and identified as 27.3 % (6/22) A. griffini
(T3), 63.6 % (14/22) A. castellanii (T4) and 9.1 % (2/22)
A. jacobsi (T15) in domestic tap water samples taken from
AK-diagnosed patients homes. Thus, we suggest that domes-
tic tap water plays an important role in contamination of
Acanthamoeba. Domestic tap water is also a significant source
of these organisms, and a reservoir for Acanthamoeba causing
keratitis in non-contact lens wearers. We assume that the dif-
ference in the prevalence of Acanthamoeba contamination in
domestic tap water in different countries might be due to the
difference in the domestic tap water hygiene, climatic condi-
tions and water treatment.
The identification of Acanthamoeba species responsible
for AK and other infections has been an active area of inves-
tigation since the recognition of this amoeba as the etiological
agent in these infections. In addition to bacteria, viruses, fungi
and protozoa have been found intracellulary within FLA, and
a proliferation in the FLA or a distribution through these FLA
at least have been demonstrated. Acanthamoeba are the pre-
dominant FLA discovered as hosts of these endocytobionts.
Especially, if FLA is present in biofilms and there are close
ties with many other microorganisms, the oddsare for some of
these microorganisms to develop human pathogenic proper-
ties (Scheid 2014).
It has been reported that A. palestinensis (T2), A. griffini
(T3), A. castellanii (T4), A. lenticulata (T5), Acanthamoeba
strain KaBo (T13) and A. jacobsi (T15) genotypes have been
associated with AK in worldwide (Jeong and Yu 2005;
Booton et al. 2009;Grünetal.2014). The first AK case was
observed in 1996, and after this, a few AK cases were reported
between years 1999 and 2007 in Turkey (Akyol et al. 1996).
Furthermore, A. castellanii (T4) and A. comandoni (T9) geno-
types were isolated from AK cases, and A. palestinensis (T2),
A. griffini (T3), A. castellanii (T4), A. astronyxis (T7) and
A. comandoni (T9) have been isolated from soil and also from
other environmental samples including domestic tap water,
A.griffini(ref)
A.griffini(domestic tap water)
A.griffini(corneal scrapings)
A.jacobsi(corneal scrapings)
A.jacobsi(ref)
A.jacobsi(domestic tap water)
A.castellanii(ref)
A.castellanii(corneal scrapings)
A.castellanii(contact lens solution)
100
100
100
0.01
Fig. 1 Phylogenetic tree of
Acanthamoeba species from
corneal scrapings, contact lens
solution and domestic tap water.
A reference strain of A. griffini
(T3) (KJ446979), A. castellanii
(T4) (KJ446975) and A. jacobsi
(T15) (KJ446981) was included
in the analysis and phylogenetic
tree construction
Parasitol Res (2015) 114:32833289 3287
hot springs and swimming pools in Turkey (Akyol et al. 1996;
Çoşkun et al. 2013; Ozkoc et al. 2008). Recently,
Acanthamoeba jacobsi (T15) genotype was isolated from wa-
ter samples in Turkey (Evyapan et al. 2015). However,
A. griffini (T3) and A. jacobsi (T15) genotypes were not iso-
lated from AK cases in Turkey before this study.
The causative agent of AK and the risk of domestic tap
water for AK had not been recognized in Cukurova region,
located in southern Turkey. This study showed A. griffini (T3),
A. castellanii (T4) and A. jacobsi (T15) as causative agent of
AK, and domestic tap water is likely to be a significant source
of protozoan in this region. The demonstrated clinical rele-
vance of the genus Acanthamoeba and the relationship
between the genotype and eye infection suggest that
more studies are needed on the identification of the
18S rRNA genotype of Acanthamoeba strains obtained
from corneal samples with keratitis, contact lens solu-
tions and domestic tap water supply.
Acknowledgments The funding was provided by the Cukurova Uni-
versity Research Grant (TF.2012. BAP.21). The study was approved by
the Ethical Committee of the Medicine Faculty of Cukurova University.
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... Those findings were confirmed with other report. Soner Koltas et al. (2015) have confirmed that trauma and exposure to contaminated water were major risk factors to get an AK infection for non-contact lenses users. Moreover, they have demonstrated the presence of the same genotyping of Acanthamoeba from AK cases in non-contact lens wearers and in their corresponding domestic tap water (Koltas et al., 2015). ...
... Soner Koltas et al. (2015) have confirmed that trauma and exposure to contaminated water were major risk factors to get an AK infection for non-contact lenses users. Moreover, they have demonstrated the presence of the same genotyping of Acanthamoeba from AK cases in non-contact lens wearers and in their corresponding domestic tap water (Koltas et al., 2015). Most of those cases were cited in developing countries and thus due to the lack of water treatment process and the presence of water storage tanks (Koltas et al., 2015). ...
... Moreover, they have demonstrated the presence of the same genotyping of Acanthamoeba from AK cases in non-contact lens wearers and in their corresponding domestic tap water (Koltas et al., 2015). Most of those cases were cited in developing countries and thus due to the lack of water treatment process and the presence of water storage tanks (Koltas et al., 2015). ...
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Interest in periocular (eyelid and eyelashes margins) hygiene has attracted attention recently and a growing number of commercials eye cleanser and shampoos have been marketed. In the present study, a particular eye cleanser foam, Belcils® has been tested against trophozoites and cysts on the facultative pathogen Acanthamoeba. Viability was tested by the alamarBlue™ method and the foam was tested for the induction of programmed cell death in order to explore its mode of action. We found that a 1% solution of the foam eliminated both trophozoite and cyst stage of Acanthamoeba spp. After 90 min of incubation, Belcils® induced, DNA condensation, collapse in the mitochondrial membrane potential and reduction of the ATP level production in Acanthamoeba. We conclude that the foam destroys the cells by the induction of an apoptosis-like process. The current eye cleanser could be used as part of AK therapy protocol and as prevention from AK infections for contact lens users and post-ocular trauma patients.
... The prevalence of Acanthamoeba spp. in the domestic water supply is an important public health issue in understanding the potential risk of developing AK in a CL-wearing population. CL-induced AK is associated with the contamination of domestic water with Acanthamoeba, and the use of CL during showering and/or swimming is a major risk factor of AK [20,35,36]. As 90% of AK cases are associated with CL wear [5], Acanthamoeba infection probably begins with the water contamination of CL. ...
... Acanthamoeba spp. are frequently reported from household tap water, particularly in endemic or potentially endemic regions [35,36]. In a study reported from the metropolitan area of Mexico City, 70% (19/27 samples) of domestic tap water was positive for Acanthamoeba spp., and the highest number of amoeba isolates were from roof tanks and water cisterns [28], although most of the amoeba isolates were considered to be non-pathogenic. ...
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Acanthamoeba keratitis (AK) is a sight-threatening infection of the cornea, which is caused by soil and the waterborne protist Acanthamoeba spp. AK most commonly occurs during contact lens (CL) wear. Risk factors for AK have been linked to non-optimal lens hygiene practices and Acanthamoeba contamination of domestic water. This study investigated the prevalence of Acanthamoeba species in domestic showers in the greater Sydney region, as well as the perception of water contamination of CL as being a risk factor for AK among previous AK patients and their family and friends. Samples from four locations of 13 participants’ shower areas were cultured and Acanthamoeba 18S rRNA was amplified by PCR, followed by sequencing. Twenty-six responses were received to the online questionnaire. Fifteen water samples (29%, 15/52) contained amoeba that were morphologically classified as Acanthamoeba spp. PCR amplification confirmed the presence of Acanthamoeba spp. in four samples (8%, 4/52). Three isolates belonged to the T4, and one isolate to the T3 genotype. On the questionnaire survey, 96% (25/26) of respondents believed that water contamination was likely to be a risk factor for Acanthamoeba keratitis and 58% rated showering with CL as ‘extremely likely’ to be a risk factor for AK. Acanthamoeba pathogenic genotypes (T3 and T4) in the domestic bathroom water suggest that clinicians should remain vigilant in educating CL wearers about avoiding domestic water contamination of CL.
... In two separate studies conducted by Kaye and Papaioannou, Acanthamoeba was found in 3.07% and 5.26% of the cases, respectively [22,23]. Acanthamoeba studies carried out in Turkey are limited and mostly case reports or retrospective evaluations of AK cases [24][25][26][27][28][29][30]. In our study, Acanthamoeba was investigated in 60 patients with keratitis and found to be positive in 7 of them (11.66%). ...
... The genotype T4 was reported to be the most common genotype in AK patients in Turkey [25][26][27]. Koltas et al. reported that T3 and T15 genotypes might also be detected as well as Acanthamoeba T4 genotype in corneal scraping samples of AK patients [29]. More recently Alver et al. reported that T2 genotype were isolated from a corneal scraping sample of an AK patient [38]. ...
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Purpose: The primary aim of this study is to investigate Acanthamoeba in clinical samples of keratitis cases (n = 60), in contact lens (CL) and lens care solutions of asymptomatic CL users (n = 41), and to identify the genotypes in positive samples. The secondary aim is to assess the risk factors and clinical features of Acanthamoeba keratitis (AK) patients. Methods: All samples from patients and asymptomatic CL users were examined by microscopy and inoculated in non-nutrient agar plates. PCR was performed using the DNA isolated from corneal scrapings, CL and lens care solution samples. Positive DNA samples were sequenced to determine the genotype of Acanthamoeba. Results: In none of the samples, Acanthamoeba was identified by microscopy, while Acanthamoeba was detected in a patient with keratitis by culture method. However, Acanthamoeba was detected in 11.66% (7/60) of the keratitis patients by PCR. The genotypes of these isolates detected by sequencing were T4 (4), and T5 (3). Acanthamoeba was detected in none of the samples of asymptomatic CL users by any of the three methods. Conclusion: To best of our knowledge, this is the first study to detect T5 genotype in AK patients from Turkey. In addition, the CL use was found to be an important risk factor for AK.
... Although contact lens wear is not commonly associated with AK in India and other developing nations, [45,51] the global surge of myopia and the use of contact lens for preventing myopia, cosmetic purposes and sport activities have increased the risk of AK, especially among youths [1]. As observed in the current study, ocular trauma and contact with contaminated soil or water are the major predisposing risk factors of AK infection not linked with contact lens wear [52][53][54]. Ocular injury with either dust particles or vegetative matters was the most common reported risk factor among AK patients in central China (52.1%) [55] and south India (48.7%) [52]. ...
Article
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Introduction Acanthamoeba is an emerging pathogen, infamous for its resilience against antiprotozoal compounds, disinfectants and harsh environments. It is known to cause keratitis, a sight-threatening, painful and difficult to treat corneal infection which is often reported among contact lens wearers and patients with ocular trauma. Acanthamoeba comprises over 24 species and currently 23 genotypes (T1-T23) have been identified. Aims This retrospective study was designed to examine the Acanthamoeba species and genotypes recovered from patients with Acanthamoeba keratitis (AK), determine the presence of endosymbionts in ocular isolates of Acanthamoeba and review the clinical presentations. Methodology Thirteen culture-confirmed AK patients treated in a tertiary eye care facility in Hyderabad, India from February to October 2020 were included in this study. The clinical manifestations, medications and visual outcomes of all patients were obtained from medical records. The Acanthamoeba isolates were identified by sequencing the ribosomal nuclear subunit ( rns ) gene. Acanthamoeba isolates were assessed for the presence of bacterial or fungal endosymbionts using molecular assays, PCR and fluorescence in situ hybridization (FISH). Results The mean age of the patients was 33 years (SD ± 17.4; 95% CI 22.5 to 43.5 years). Six (46.2%) cases had AK associated risk factors; four patients had ocular trauma and two were contact lens wearers. A. culbertsoni (6/13, 46.2%) was the most common species, followed by A. polyphaga and A. triangularis . Most of the isolates (12/13) belonged to genotype T4 and one was a T12; three sub-clusters T4A, T4B, and T4F were identified within the T4 genotype. There was no significant association between Acanthamoeba types and clinical outcomes. Eight (61.5%) isolates harboured intracellular bacteria and one contained Malassezia restricta . The presence of intracellular microbes was associated with a higher proportion of stromal infiltrates (88.9%, 8/9), epithelial defect (55.6%, 5/9) and hypopyon (55.6%, 5/9) compared to 50% (2/4), 25% (1/4) and 25% (1/4) AK cases without intracellular microbes, respectively. Conclusions Genotype T4 was the predominant isolate in southern India. This is the second report of T12 genotype identified from AK patient in India, which is rarely reported worldwide. The majority of the Acanthamoeba clinical isolates in this study harboured intracellular microbes, which may impact clinical characteristics of AK.
... One of the most important reasons for the high occurrence of Acanthamoeba sp. is its high capability to adapt to harsh environmental conditions (Aksozek et al. 2002). Previous studies have suggested that using that tap water for washing contact lenses is a major risk factor for AK development (Lorenzo-Morales et al. 2005;Shoff et al. 2008;Koltas et al. 2015). The most reported cases of AK are related to people who wear soft contact lenses, which occur due to inappropriate maintenance of their lenses (Lindsay et al. 2007). ...
Article
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Free-living amoebas (FLAs) can cause neurological and ocular complications in humans. Water supplies play a critical role in transmitting FLAs to humans. The aim of the present study was to investigate the presence of FLAs in various aquatic sources including drinking water, stagnant water, and surface water in Alborz province, northern Iran, using morphological and molecular techniques. A total of 70 water samples were collected from 34 drinking waters, 23 surface waters, and 13 stagnant waters. Filtration and cultivation were employed to isolate FLAs. PCR assay was applied by using the genus-specific primers on positive samples. Pathogenicity tests (osmo- and thermo-tolerance properties) were performed for Acanthamoeba spp., positive sample. Considering the morphological criteria, four positive samples of Acanthamoeba sp., three Vermamoeba sp., two mixed Vermamoeba sp. with Vahlkamfiids, and one mixed Acanthamoeba sp. with Vahlkamfiids were isolated. Five Acanthamoeba sp. isolates were amplified using the JDP primer pairs. Among them, two genotypes, T4 (three isolates) and T5 (two isolates) corresponding to A. lenticulata, were identified. Four V. vermiformis samples were confirmed using the sequencing. This study highlighted the occurrence of potentially pathogenic waterborne FLAs in water habitats associated with high human activity. The results of such research on the prevalence of FLAs, as a human hazard, should be communicated to health policymakers. HIGHLIGHTS Water samples were collected from drinking, surface, and stagnant waters.; According to the morphological assessment, Acanthamoeba, Vermamoeba, and Vahlkamfiids were detected among samples.; The genotypes T4 and T5 (corresponding to A. lenticulata) were characterized.;
... The most common genotypes, T4 (93.7%) and T2 (6.25%) were determined in the geothermal river in the southwest of Iran by Niyyati et al. [46]. The most common T4 and T2, T3, T5, T11, T15 genotypes of Acanthamoeba were detected in environmental water samples in Turkey [34,[47][48][49][50]. In the previous study T4 and T5 genotypes were reported in keratitis wild birds in İzmir [51]. ...
Article
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Free-living amoeba (FLA) is widely distributed in the natural environment. Since these amoebae are widely found in various waters, they pose an important public health problem. The aim of this study was to detect the presence of Acanthamoeba, B. mandrillaris, and N. fowleri in various water resources by qPCR in Izmir, Turkey. A total of (n=27) 18.24% Acanthamoeba and (n=4) 2.7% N. fowleri positives were detected in six different water sources using qPCR with ITS regions (ITS1) specific primers. The resulting concentrations varied in various water samples for Acanthamoeba in the range of 3.2x105-1.4x102 plasmid copies/l and for N. fowleri in the range of 8x103-11x102 plasmid copies/l. The highest concentration of Acanthamoeba and N. fowleri was found in seawater and damp samples respectively. All 27 Acanthamoeba isolates were identified in genotype level based on the 18S rRNA gene as T4 (51.85%), T5 (22.22%), T2 (14.81%) and T15 (11.11%). The four positive N. fowleri isolate was confirmed by sequencing the ITS1, ITS2 and 5.8S rRNA regions using specific primers. Four N. fowleri isolates were genotyped (three isolate as type 2 and one isolate as type 5) and detected for the first time from water sources in Turkey. Acanthamoeba and N. fowleri genotypes found in many natural environments are straightly related to human populations to have pathogenic potentials that may pose a risk to human health. Public health professionals should raise awareness on this issue, and public awareness education should be provided by the assistance of civil authorities. To the best of our knowledge, this is the first study on the quantitative detection and distribution of Acanthamoeba and N. fowleri genotypes in various water sources in Turkey.
... It appeared that the niche from which Acanthamoeba had been isolated affected the types of intracellular microbes present, or perhaps affected the ability of particular Acanthamoeba strains to cause infections. This latter hypothesis is presented based on previous investigations that domestic water supplies and contact lenses that are exposed to water are risk factors for Acanthamoeba keratitis [5,[119][120][121]. This suggests that water is the source of the infecting Acanthamoeba [122] and, perhaps, those strains that harbour particular intracellular microbes are more able to instigate corneal (or other) infections [21]. ...
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Acanthamoeba, an opportunistic pathogen is known to cause an infection of the cornea, central nervous system, and skin. Acanthamoeba feeds different microorganisms, including potentially pathogenic prokaryotes; some of microbes have developed ways of surviving intracellularly and this may mean that Acanthamoeba acts as incubator of important pathogens. A systematic review of the literature was performed in order to capture a comprehensive picture of the variety of microbial species identified within Acanthamoeba following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Forty-three studies met the inclusion criteria, 26 studies (60.5%) examined environmental samples, eight (18.6%) studies examined clinical specimens, and another nine (20.9%) studies analysed both types of samples. Polymerase chain reaction (PCR) followed by gene sequencing was the most common technique used to identify the intracellular microorganisms. Important pathogenic bacteria, such as E. coli, Mycobacterium spp. and P. aeruginosa, were observed in clinical isolates of Acanthamoeba, whereas Legionella, adenovirus, mimivirus, and unidentified bacteria (Candidatus) were often identified in environmental Acanthamoeba. Increasing resistance of Acanthamoeba associated intracellular pathogens to antimicrobials is an increased risk to public health. Molecular-based future studies are needed in order to assess the microbiome residing in Acanthamoeba, as a research on the hypotheses that intracellular microbes can affect the pathogenicity of Acanthamoeba infections.
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Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that most frequently affects contact lens wearers. Severe pain out of proportion to the corneal findings, radial keratoneuritis, and ring infiltration are relatively specific clinical manifestations of AK. The diagnosis of AK is challenging and it is often misdiagnosed because of atypical presentations. The definitive diagnosis of AK requires the presence of Acanthamoeba cysts or trophozoites in corneal lesions, and a high index of clinical suspicion is important for early diagnosis. In this review, we describe the clinical manifestations, including atypical findings, and management of AK.
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Purpose: To determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pɛK+) against Acanthamoeba castellanii. Methods: A. castellanii trophozoites and cysts were grown in the presence of pɛK solution (0-2.17 mM), pɛK or pɛK+ hydrogels, or commercial hydrogel contact lens (CL) for 24 hours or 7 days in PBS or Peptone-Yeast-Glucose (PYG) media (nutrient-deplete or nutrient-replete cultures, respectively). Toxicity was determined using propidium iodide and imaged using fluorescence microscopy. Ex vivo porcine corneas were inoculated with A. castellanii trophozoites ± pɛK, pɛK+ hydrogels or commercial hydrogel CL for 7 days. Corneal infection was assessed by periodic acid-Schiff staining and histologic analysis. Regrowth of A. castellanii from hydrogel lenses and corneal discs at 7 days was assessed using microscopy and enumeration. Results: The toxicity of pɛK+ hydrogels resulted in the death of 98.52% or 83.31% of the trophozoites at 24 hours or 7 days, respectively. The toxicity of pɛK+ hydrogels resulted in the death of 70.59% or 82.32% of the cysts in PBS at 24 hours or 7 days, respectively. Cysts exposed to pɛK+ hydrogels in PYG medium resulted in 75.37% and 87.14% death at 24 hours and 7 days. Ex vivo corneas infected with trophozoites and incubated with pɛK+ hydrogels showed the absence of A. castellanii in the stroma, with no regrowth from corneas or pɛK+ hydrogel, compared with infected-only corneas and those incubated in presence of commercial hydrogel CL. Conclusions: pɛK+ hydrogels demonstrated pronounced amoebicidal and cysticidal activity against A. castellanii. pɛK+ hydrogels have the potential for use as CLs that could minimize the risk of CL-associated Acanthamoeba keratitis.
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The aim of the present review is to bring together the main references on the diversity of species, geographic distribution, taxonomy, and phylogeny of naked Amoebozoa from marine and brackish water environments, including those directly connected with the World Ocean, as well as continental saline biotopes. The main body of data is structured in the tables according to the most recent classification of Amoebozoa, which list all published species names with references to the initial descriptions, redescriptions where applicable, data on the biotopes where the species were found, available strains, salinity tolerance ranges, and types of available published data including light and electron microscopical studies, and Genbank accession numbers of published sequences. In addition, we provide a brief overview of the history of investigations of marine amoebae, briefly discuss the current state of their species concept, and current knowledge of their relations to environmental salinity.
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Environmental sources are potential sources for the transmission of Acanthamoeba in humans and other mammals. A total of 50 water samples from hot springs and swimming pools, and 50 soil samples were taken from Adana, Afyon, Kutahya, Mersin and Nigde provinces in Turkey. Samples were analysed using 18S rRNA-DNA sequencing. Acanthamoeba griffini (T3), Acanthamoeba castellanii (T4) and Acanthamoeba jacobsi (T15) were found in water samples. Acanthamoeba griffini (T3) and Acanthamoeba castellanii (T4) were detected in soil samples. In Turkey, this was the first time that Acanthamoeba jacobsi (T15) was detected in water samples. © The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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Several strains of free-living amoebae (FLA) belonging to the genus Acanthamoeba are able to cause a painful sight-threatening disease of the cornea designated as Acanthamoeba keratitis (AK). In this case report, a 22-year-old woman, wearer of soft contact lenses, was treated after the initial examination, and follow-up laboratory results led to the diagnosis of Acanthamoeba keratitis. The patient recovered under the targeted therapy, demonstrating that the acanthamoebae were the etiological agents of the keratitis in this case. The acanthamoebae belonged morphologically to group II. Genotyping of the causative Acanthamoeba strain based on sequences of the PCR amplimer ASA.S1 amplified from 18S ribosomal DNA by using the genus-specific primers JDP1 and JDP2 followed. The phylogenetic comparison of ASA.S1 confirmed that the isolated Acanthamoeba strain is closely related to genotype T13 supported by pairwise sequence identities of 97.1-98.0 % and bootstrap support of 980 replicates with reference sequences of genotype T13. These results regarding the Acanthamoeba keratitis-causing isolate KaBo expands the number of known pathogenic genotypes to 12. To our knowledge, this is the first report of a T13 Acanthamoeba genotype being associated with keratitis in humans.
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The typical clinical appearance of acanthamoeba keratitis includes pseudodendritic epitheliopathy, perineuritis, ring infiltrates or multifocal stromal infiltrates and in some cases limbitis with infiltration of the conjunctiva and/or sterile anterior uveitis. In 83-93 % of cases of acanthamoeba keratitis the patients were contact lens wearers. Acanthamoeba keratitis is diagnosed by polymerase chain reaction (PCR), confocal biomicroscopy, in vitro cultivation and histopathological examination. Information on reliability and efficacy of conservative and surgical therapy of acanthamoeba keratitis has only been published in case series but not yet verified through randomized controlled clinical studies. After early diagnosis acanthamoeba keratitis can often be successfully treated using triple topical therapy with polyhexamide, propamidine isethionate and neomycin. Topical therapy should be continued for up to 1 year. In therapy-resistant cases cryotherapy, amniotic membrane transplantation, crosslinking therapy and therapeutic keratoplasty can be performed. The prognosis of keratoplasty following acanthamoeba keratitis is more favorable if there were no signs of infection at least 3 months before surgery.
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Purpose: To assess the diagnostic methods, risk factors, and clinical features of Acanthamoeba keratitis cases in patients who do not wear contact lenses. Methods: Medical records of 26 consecutive patients with non-contact lens-related Acanthamoeba keratitis, who were followed up at the tertiary eye care center between May 2010 and May 2012, were analyzed. Laboratory, demographic, and clinical findings were evaluated pertaining to the patients. Results: Twenty-six non-contact lens-related Acanthamoeba keratitis cases were included in the study. The main risk factors were trauma (group 1, n = 13 patients) and ocular surface disease (group 2, n = 12 patients). One patient had both of the risk factors mentioned above. Overall test results showed that Acanthamoeba positivity rates were 15.3% for direct microscopy, 46.1% for culture, 92.3% for conventional polymerase chain reaction (PCR), and 100% for real-time PCR. The rates of full-thickness corneal involvement and ring-shaped infiltrations were higher in group 2, whereas superficial keratitis and radial keratoneuritis were higher in group 1. The final visual acuities were significantly better in group 1 than group 2 (p<0.025). Conclusions: This study is the first regional report from Turkey about Acanthamoeba keratitis in non-contact lens users. A majority of cases admitted to a tertiary eye care center were related to trauma or ocular surface disease. Physician suspicion is critically important for the timely diagnosis of these cases. At this point, molecular diagnostic tests (PCR or real-time PCR) seem to support the clinical diagnosis of Acanthamoeba keratitis with the help of fast and reliable results.
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The present work focuses on a local survey of free-living amoebae (FLA) that cause opportunistic and nonopportunistic infections in humans. Determining the prevalence of FLA in water sources can shine a light on the need to prevent FLA related illnesses. A total of 150 samples of tap water were collected from six districts of Sivas province. The samples were filtered and seeded on nonnutrient agar containing Escherichia coli spread. Thirty-three (22%) out of 150 samples were found to be positive for FLA. The FLA were identified by morphology and by PCR using 18S rDNA gene. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of three different species, Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis. Naegleria fowleri, Balamuthia mandrillaris, or Sappinia sp. was not isolated during the study. All A. castellanii and A. polyphaga sequence types were found to be genotype T4 that contains most of the pathogenic Acanthamoeba strains. The results indicated the occurrence and distribution of FLA species in tap water in these localities of Sivas, Turkey. Furthermore, the presence of temperature tolerant Acanthamoeba genotype T4 in tap water in the region must be taken into account for health risks.
Article
Real-time polymerase chain reaction (PCR) is a quantitative method to measure the amount of amplified PCR product in real time with high sensitivity. We have applied this method to detect pathogens in cases of keratitis with an unknown cause. The scraped corneal epithelium for epithelial keratitis or aqueous humor for stromal or endothelial keratitis was obtained and DNA was extracted. The DNA from specific pathogens was amplified using specific primers and TaqMan probe, and assessed quantitatively. Here, we review previously reported noteworthy examples of keratitis diagnosed by our real-time PCR system as follows: cases with Acanthamoeba keratitis whose causative pathogen was only detected by real-time PCR despite not being detected by histological examination and culture; zoster sine herpete with atypical pseudodendrite; acyclovir-resistant herpetic keratitis estimated by changes in viral DNA copy numbers before and after treatment; and corneal endotheliitis positive for cytomegalovirus, human herpes virus-7, or human herpes virus-8. Real-time PCR helps ophthalmologists to make an early diagnosis and provide appropriate treatment for keratitis with complex clinical appearances.
Article
AIM To review the clinical characteristics, diagnosis, and visual outcome in patients with non-contact lens related Acanthamoebakeratitis and compare the findings with reported series of contact lens associated Acanthamoeba keratitis. METHODS Medical and microbiology records of 39 consecutive patients with a diagnosis ofAcanthamoeba keratitis, at a tertiary eyecare centre in India between January 1996 and June 1998, were analysed retrospectively. RESULTS A majority of the patients presented with poor visual acuity and large corneal stromal infiltrates (mean size 38.20 (SD 26.18) mm). A predisposing factor was elicited in 19/39 (48.7%) patients (trauma 15, dirty water splash three, leaf juice one). None of the patients had worn contact lenses. Most patients (26/39 (66.6%)) came from a low socioeconomic background. Complaint of severe pain was not a significant feature and radial keratoneuritis was seen in 1/39 (2.5%) patients. A ring infiltrate was present in 41.1% of cases. A clinical diagnosis of fungal keratitis was made in 45% of the patients before they were seen by us. However, all patients were diagnosed microbiologically at our institute based on demonstration ofAcanthamoeba cysts in corneal scrapings (34/39) and/or culture of Acanthamoeba(34/39). Treatment with biguanides (PHMB, 15/38 (39.4%), PHMB with CHx, 23/38 (60.5%), one patient did not return for treatment) resulted in healing with scar formation in 27 out of 31(87.0%) followed up patients (mean time to healing 106.9 days). Overall visual outcome was poor with no statistical difference between cases diagnosed within 30 days (early) or 30 days after (late) start of symptoms. The visual outcome in cases requiring tissue adhesive (five) and keratoplasty (three) was also poor. CONCLUSIONS This is thought to be the largest series of cases ofAcanthamoeba keratitis in non-contact lens wearers. In such cases, the disease is advanced at presentation in most patients, pathognomonic clinical features are often not seen, disease progression is rapid, and visual outcome is usually poor. Possible existence of Acanthamoeba pathotypes specifically associated with non-contact lens keratitis and unique to certain geographical areas is suggested.
Article
In addition to their role as parasites, free-living amoebae (FLA) can act as hosts of and vehicles for phylogentically diverse microorganisms while some of them replicate intracellularly. These microorganisms are adapted to the intracellular conditions in the amoeba, find suitable conditions and protection from negative environmental influences and take advantage of the dispersal in the environment by their amoebic host. It is expedient to call these organisms “endocytobionts”, at least during the initial steps of any studies. By doing so, it is not necessary to go into potential characteristics of these relationships such as parasitism, phoresy, zoochory, or mutualism at an early stage of study. Among those organisms resisting the lysis within their amoebic host, there are obligate and facultative pathogenic microorganisms affecting the health of humans or animals. FLA-endocytobiont relationships are not only important for the tenacity of the involved microorganisms. Especially if FLA are present in biofilms and there are close ties with many other microorganisms, the odds are for some of these microorganisms to develop human pathogenic properties. Here, the amoebic passage seems to be a prerequisite for the development of virulence factors and it may have an impact on evolutionary processes.
Article
A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of Acanthamoeba. The sensitivity of the LAMP assay was tested using different copies of positive DNA. The specificity of the assay was tested using DNA extracted from Acanthamoeba, Pseudomonas aeruginosa, Candida albicans, herpes simplex virus-1 and human corneal epithelial cells. Its effectiveness was evaluated and compared with culture, corneal smear examination and real-time PCR in corneal samples from mice with Acanthamoeba keratitis. We also tested three corneal samples from patients with suspected Acanthamoeba or fungal infection using LAMP. Loop-mediated isothermal amplification was confirmed to be very sensitive, with the lowest detection limit being ten copies/tube of Acanthamoeba DNA. The LAMP primers only amplified Acanthamoeba DNA. During the development of Acanthamoeba keratitis in mice, almost all of the positive rates of LAMP at each time post-infection were higher than those of culture or corneal smear examination. The total positive rate of LAMP was significantly higher than those of culture and corneal smear examination (p <0.05), whereas the sensitivities of LAMP and real-time PCR were comparable. However, the trends of positive change in these different test methods were generally similar. Of the three clinical corneal specimens, two with suspected Acanthamoeba keratitis tested positive for Acanthamoeba using LAMP along with culture or corneal smear examination, whereas the other suspected fungal keratitis tested negative. The LAMP assay is a simple, rapid, highly specific and sensitive method for the diagnosis of keratitis caused by Acanthamoeba.