J Med Microbiol 57 (2008), 392-396; DOI: 10.1099/jmm.0.47650-0
© 2008 Society for General Microbiology
ISSN 1473-5644
Identification of Acanthamoeba genotype T4 and Paravahlkampfia sp. from two clinical samples
Soykan Ozkoc1,
Sema Tuncay1,
Songul Bayram Delibas1,
Ciler Akisu1,
Zeynep Ozbek2,
Ismet Durak2 and
Julia Walochnik3
1 Department of Parasitology, Faculty of Medicine, Dokuz Eylul University, Izmir, Turkey
2 Department of Ophthalmology, Faculty of Medicine, Dokuz Eylul University, Izmir, Turkey
3 Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Medical University of Vienna, Vienna, Austria
Correspondence
Soykan Ozkoc
soykan.ozkoc{at}deu.edu.tr
Received 24 September 2007
Accepted 10 November 2007
In this study, two free-living amoebae strains, Acanthamoeba genotype T4 and Paravahlkampfia sp., which were isolated from keratitis cases are presented. While the Acanthamoeba strain was isolated as a single agent, the Paravahlkampfia strain was found together with herpes simplex virus. Neither of the patients were contact lens wearers, but they did have a history of minor corneal trauma. Amoebae were detected on non-nutrient agar covered with Escherichia coli. Based on PCR-amplified 18S rRNA-gene analysis the first isolate was identified as Acanthamoeba genotype T4 and the second as Paravahlkampfia sp. In thermotolerance tests, the maximum temperature at which trophozoites continued to divide was determined as 37 °C for this Acanthamoeba strain and 35 °C for the Paravahlkampfia strain. To the best of our knowledge, the Acanthamoeba strain described herein is the second molecularly identified Acanthamoeba strain in an Acanthamoeba keratitis patient in Turkey. However, the Paravahlkampfia isolate is believed to be the first strain that has been isolated from a keratitis patient and has been molecularly differentiated from Vahlkampfia.
Abbreviations: AK, Acanthamoeba keratitis; FLA, free-living amoebae; HSV, herpes simplex virus.
The GenBank/EMBL/DDBJ accession number for the sequence of Acanthamoeba strain DP/5A is EU266547 and of Acanthamoeba strain SO/1P is EU266548.
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Introduction
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Free-living amoebae (FLA) constitute an important part of the biosphere of soil and water, as well as air, and include amphizoic strains that are pathogenic to humans and animals (Schuster & Visvesvara, 2004). Several species, especially those belonging to the T4 genotype, have been identified as the causative agents of a painful and sight-threatening infection of the cornea, the so called Acanthamoeba keratitis (AK) (Walochnik et al., 2000a; Khan et al., 2001, 2002; Booton et al., 2005; Khan, 2006). These infections can occur in otherwise healthy, non-immunocompromised individuals. While AK infections in developed countries are usually associated with contact lens wear, in developing nations most cases occurred as the result of ocular trauma (Seal et al., 1998; Ibrahim et al., 2007).
In this report we present two keratitis cases and two different amoeba strains isolated from these patients. Both of the patients had a history of minor corneal trauma, but they were not contact lens wearers. Acanthamoeba genotype T4 was detected in the first case while herpes simplex virus (HSV) and Paravahlkampfia sp. were detected in the second. To the best of our knowledge, this is the second study identifying the genotype of an Acanthamoeba strain in an AK patient in Turkey. However, in this current study a non-Acanthamoeba keratitis, from which Paravahlkamphia sp. was isolated, is described for what is believed to be the first time.
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Case 1
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A 38-year-old woman with redness, pain and blurred vision in her right eye was referred to the ophthalmology department in our university hospital for further evaluation in October 2005. She was not a contact lens wearer. Three months before she had sustained corneal abrasion to the right eye from a plant while cleaning a flowerpot. She was seen at that time by several doctors and was treated with a number of different eye drops containing antibiotics, corticosteroids and artificial tears. However, there was no notable improvement in her symptoms. On ophthalmic examination her conjunctiva was hyperaemic, her visual acuity was 4/10, and there was a stromal infiltrate on the central part of the cornea with an overlying epithelial defect and a lesion on the limbus at the 5 o'clock position. Corneal scrapings were obtained for culture and the patient was given empiric treatment with fusidic acid. Cultures were negative for bacteria and fungi. On the 12th day, Acanthamoeba cysts and trophozoites were seen on non-nutrient (NN) agar seeded with Escherichia coli. Treatment was started with polyhexamethylene biguanide, propamidine isethionate and oral itraconazole. A month later her symptoms had relieved and her visual acuity was 7/10, but the patient was lost for further follow up.
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Case 2
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In November 2006, a 61-year-old man was referred to the ophthalmology department in our university hospital for further evaluation. He reported redness, pain and irritation in his left eye. He was not wearing contact lenses. One month previously he had sustained corneal abrasion to the left eye with a piece of straw. He was then seen by an ophthalmologist and was treated with eye drops containing antibiotics and antihistamines. However, there was no notable improvement in his symptoms. On ophthalmic examination his right eye was essentially normal. The acuity of his left eye was at the level of counting fingers from 4 m. A slitlamp examination of his left eye revealed intense corneal oedema, a central epithelial defect and punctate corneal epithelial erosions. Corneal scrapings were obtained for cultures and the patient was given empiric treatment with fusidic acid and acyclovir. On the 13th day, amoebic cysts and trophozoites were seen on NN agar. PCR for HSV was also positive, but the clinical and ophthalmic features of the patient did not look like a typical HSV keratitis. Treatment was started with both antiviral (acyclovir) and antiamoebic drugs (propamidine isethionate and polyhexamethylene biguanide). Three months later his visual acuity was at the level of detecting hand motions from 4 m. On ophthalmic examination the central epithelial defect had become smaller, stromal scarring and neovascularization were also seen.
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Methods
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Isolation and cultivation.
Corneal scraping samples obtained from the two patients, who were presumed to have Acanthamoeba keratitis, were cultured on NN agar plates covered with E. coli. After the samples were directly inoculated on NN agar, plates were sealed, incubated at 30 °C for 14 days and investigated daily by inverted microscopy for amoebal growth. Following isolation, two amoeba isolates (isolate 1, strain DP/5A and isolate 2, strain SO/1P) were transferred into proteose peptone/yeast extract/glucose (PYG) medium and were incubated at 30 °C for 7–21 days as described in the literature (Khan et al., 2001).
Temperature tolerance test.
For temperature tolerance assay of the strain DP/5A, Acanthamoeba axenically grown in culture flasks (25 cm2) were used. As strain SO/1P could not be maintained axenically in PYG medium for a long period, the test was performed on NN agar covered with E. coli. To determine temperature tolerance, both isolates, subcultured as described above, were incubated at various temperatures (34, 37 and 40 °C). After 96 h, the samples were examined for amoebal growth by inverted microscopy and temperature tolerances were recorded.
Isolation of DNA.
For molecular biological investigations actively growing amoebae (
106 cells) were harvested from culture plates with a sterile cotton-tipped applicator and washed three times in sterile 0.9 % NaCl by centrifugation at 500 g for 7 min. Whole-cell DNA was isolated by a modified UNSET (Hugo et al., 1992) procedure as described previously (Walochnik et al., 1999).
PCR and sequence analysis.
For identification of the Acanthamoeba strain a fragment of the 18S rRNA gene was amplified using JPD1 and JPD2 primers, giving a 423–551 bp Acanthamoeba-specific amplicon (Schroeder et al., 2001). We used 1, 3 and 6 µl whole cell DNA in a 50 µl reaction volume and the following amplification program: 45 cycles of 95 °C for 1 min, 60 °C for 1 min, 72 °C for 2 min. For identification of the vahlkampfiid amoeba P2 and P3r primers (Walochnik et al., 2004) were used for amplification. These are universal eukaryotic primers, complementary to strongly conserved regions within the eukaryotic 18S rRNA gene. In vahlkampfiid amoebae the amplicon is approximately 600 bp long and allows an identification down to the genus level. A standard amplification program with 30 cycles of 95 °C for 1 min, 52 °C for 2 min and 72 °C for 3 min was used for PCR.
A genotype T4 Acanthamoeba castellanii strain (strain 1BU, ATCC PRA-105) was used as a positive control for both PCRs. The amplification of the respective fragment of the 18S rRNA gene was visualized by ethidium bromide staining of an agarose electrophoresis gel and compared to a lambda DNA size marker. Amplicons were analysed by direct sequencing of the PCR product using the ABI PRISM BigDye sequencing kit and a 310 ABI PRISM automated sequencer (Applied Biosystems). Sequences were obtained from both strands and data were processed with the GeneDoc sequence editor (Nicholas et al., 1997). Multiple sequence alignment was performed by stepwise pairwise alignment using the CLUSTAL_X application (Thompson et al., 1997). The alignments were assessed by eye and revised manually. Primer sites were excluded from the analysis. The sequences were compared to published sequences from other acanthamoebae and vahlkampfiid amoeba. For the Acanthamoeba strain the genotype was assessed with the model assumption of a <5 % sequence dissimilarity within one genotype as established by Gast et al. (1996). Sequence data were deposited at GenBank under the following accession numbers: EU266547 (strain DP/5A) and EU266548 (strain SO/1P).
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Results
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Acanthamoeba DP/5A strain isolated from patient 1
On the 12th day of inoculation, Acanthamoeba sp.-like trophozoites, and sporadically cysts as well, were clearly visible on the NN agar for the sample from patient 1. Cysts observed had a mean number of seven or less truncate rays, and the ectocyst was not pronounced and wrinkled (Fig. 1
). They were 10–15 µm in diameter. When these amoebae were transferred into PYG medium they exhibited characteristic acanthamoebal growth with an acanthopodial movement, and it could be maintained by serial passages in this medium. The maximum temperature at which trophozoites continued to divide in axenic culture was determined as 37 °C for this Acanthamoeba isolate. Strain DP/5A was identified as morphological group II and genotype T4. When the sequence of our DP/5A strain was compared to published strains, strain DP/5A revealed more than 99 % sequence identity to Acanthamoeba royreba Oak Ridge strain (GenBank accession no. U07417) and Acanthamoeba rhysodes ATCC 30973 strain (GenBank accession no. AY351644).
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Fig. 1. Cysts of Acanthamoeba strain DP/5A genotype T4 viewed by light microscopy (magnification x1000). Bar, 10 µm.
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Paravahlkampfia SO/1P strain isolated from patient 2
Mostly cysts with smooth walls and round to slightly oval shape were observed on the agar plate for the sample from patient 2 on the 13th day of inoculation. Isolated cysts were approximately 5–15 µm in diameter (Fig. 2a). When these cysts were transferred into PYG liquid medium, large numbers of amoebae exhibiting eruptive locomotion different from Acanthamoeba sp. were seen after several hours. Frequently the eruptive bulge appeared to form an advancing front and the posterior end of the locomotory amoebae had uroidal adhesive filaments (collopodia). Trophozoites were 15–35 µm in length in liquid media (Fig. 2b). Strain SO/1P, however, could be maintained in this medium for only 24 h and then the trophozoites turned into cysts, but it could be cultured successfully on NN agar plates coated with E. coli. According to the temperature tolerance test, the maximum temperature at which trophozoites continued to divide on NN agar with E. coli was 35 °C. At 37 °C the cysts did not transform into trophozoites and trophozoites did not continue to divide. Based on the 18S rDNA sequence analysis, strain SO/1P was identified as Paravahlkampfia sp. When the sequences were compared to classified published strains, the closest match was the CDC 453 strain of Paravahlkampfia ustiana (GenBank accession no. AJ550994) isolated from a lizard.
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Fig. 2. (a) Cysts of Paravahlkampfia strain SO/1P viewed by light microscopy (magnification x1000). (b) Trophozoites of Paravahlkampfia strain SO/1P viewed by light microscopy. The collopodia are shown by an arrow (magnification x400). Bars, 10 µm.
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Discussion
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AK is known to be caused by species mostly belonging to the T4 genotype (Khan et al., 2002; Walochnik et al., 2000a). This is an observation valid for Turkey. The indication of the presence of this genotype in soil and tap water through environmental studies, and the isolation of A. castellanii from corneal scrapings of a patient support this idea (Kilic et al., 2004; Ertabaklar et al., 2007). In our study, strain DP/5A was identified as morphological group II and genotype T4. To the best of our knowledge, this is the second study identifying the genotype of an Acanthamoeba strain in an AK patient in Turkey. Previously Ertabaklar et al. (2007) had isolated Acanthamoeba Fa03 strain from an AK patient and identified it as genotype T4. Though there are not so many reports about AK in Turkey, reported keratitis cases are surprisingly not related to the use of contact lenses or corneal trauma (Demirci et al., 2006; Ertabaklar et al., 2007). The keratitis patient from whom we isolated strain DP/5A genotype T4 did not wear contact lenses either. But the patient had a history of minor corneal trauma contrary to published reports. On the other hand, our DP/5A strain did not grow above 37 °C contrary to what has been reported by Ertabaklar et al. (2007). They reported that a T4 strain that was isolated from AK tolerated up to 42 °C (Ertabaklar et al., 2007).
Interestingly, one of the amoebae isolated from keratitis patients in this study was identified as Paravahlkampfia sp. Paravahlkampfia is generally described as a soil amoeba and has been reassigned to a different genus from the genus Vahlkampfia on the basis of 18S rDNA sequence analysis (Brown & De Jonckheere, 1999). Until now two different species of Paravahlkampfia have been defined, P. ustiana and Paravahlkampfia lenta. There is not sufficient information about ecological distribution and pathogenicity of this amoeba. Previously, several reports of human keratitis cases in which vahlkampfiids had been isolated were published. But whether these isolates were Paravahlkampfia or not remains unknown because no molecular identification was done in any of these studies (Aitken et al., 1996; Alexandrakis et al., 1998; Dua et al., 1998). However, P. ustiana (CCAP strain 1588/6 and CDC V453 strain) and P. lenta (6/3AB/1B strain), which have been assigned to the genus Paravahlkampfia, have never been isolated from humans. While P. ustiana CCAP strain 1588/6 was isolated from a swimming pool in Czechoslovakia and P. ustiana CDC V453 strain was isolated from the gut of a lizard, P. lenta 6/3AB/1B strain was isolated from soil (Brown & De Jonckheere, 1999, 2004; Schuster et al., 2003). The strain SO/1P we isolated was detected accidentally. Since the structures of cysts and trophozoites observed in the sample, which was cultured with a prediagnosis as AK, were different than expected, molecular differentiation was carried out and the 18S rDNA sequence analysis identified the isolate as Paravahlkampfia sp. The patient was also positive for HSV and maybe the amoebae were only colonizing the affected eye. To the best of our knowledge, strain SO/1P is the first Paravahlkampfia strain that has been isolated from a keratitis patient and that has been molecularly differentiated from Vahlkampfia.
When compared to other Paravahlkampfia strains, strain SO/1P showed similarities as well as differences with respect to phenotypic features. The ability to grow on NN agar covered with E. coli and the inability to reproduce at temperatures over 35 °C make it similar to P. lenta 6/3Ab/1B strain. But the fast and monopodal eruptive movement of trophozoites in the liquid medium was remarkable. This type of movement was previously identified for P. ustiana, whereas the trophozoite movement of P. lenta was identified as being slower (Brown & De Jonckheere, 2004). However, cysts and trophozoites of strain SO/1P were smaller than the other species morphologically.
Today it is still a controversial issue whether FLA other than Acanthamoeba can produce keratitis or not. As mentioned above there are some reports of isolation of vahlkampfiid amoebae from human cornea that have been published in the last decade (Aitken et al., 1996; Alexandrakis et al., 1998; Dua et al., 1998). In addition to these reports, Kinnear (2003) described that a Vahlkampfia strain and a Hartmannella strain showed similar cytopathogenicity on human keratocytes like an Acanthamoeba strain (Kinnear, 2003). On the other hand, some of the researchers who are specialists in this field propose that there is still insufficient evidence that these strains can actually cause keratitis (De Jonckheere, 2003; De Jonckheere & Brown 2005). In the present case, strain SO/1P was found together with HSV. Although this finding suggests that HSV was the actual causative agent, the patient did not present like a typical HSV keratitis patient. Thus, strain SO/1P cannot be excluded as a pathogenic agent. When tested for thermotolerance, strain SO/1P exhibited a maximum growth temperature of 35 °C, which is similar to the in vivo conditions in the cornea. Walochnik et al. (2000b) have isolated three Vahlkampfia isolates with no clinical relevancy and these did not grow at temperatures higher than 30 °C.
In conclusion we isolated two different FLA strains from the corneal scraping samples of two keratitis patients. To the best of our knowledge the Acanthamoeba strain we described herein is the second molecularly identified Acanthamoeba strain in an AK patient in Turkey. In the other case to the best of our knowledge it is the first time that a Paravahlkampfia strain has been isolated from a keratitis patient and has been molecularly differentiated from Vahlkampfia.
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Introduction
Case 1
