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  • 1 National Public Health Center, Hungary
  • | 2 Semmelweis University, Hungary
  • | 3 Saarland University Medical Center, Germany
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Genus Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment. Within this genus, numerous species are recognized as human pathogens, potentially causing Acanthamoeba keratitis (AK). AK is a corneal disease, associated predominantly with contact lens (CL) wear; its epidemiology is related to the specific Acanthamoeba genotypes. This study reports seven CL wearer, Acanthamoeba PCR-positive patients with AK, diagnosed between January 2015 and 2018. Patients had the diagnosis of AK 1.36 months after first symptoms. Genotyping allowed the identification of six isolates of the T4 and one of the T8 genotypes. At first presentation, pseudendritiformic epithelopathy/dirty epithelium (four eyes, 57.1%), multifocal stromal infiltrates (five eyes, 71.4%), ring infiltrate (three eyes, 42.8%), and perineuritis (one eye, 14.3%) were observed. AK was healed without later recurrence in two eyes (28.5%) using triple-topical therapy, in three eyes (42.8%) following additional penetrating keratoplasty. In one patient (14.3%), AK recurred following successful application of triple-therapy and was treated successfully with repeated triple-topical therapy and in one patient (14.3%), no follow-up data were available after diagnosis. We could not observe correlation of genotype and clinical course or the necessity of corneal transplantation in our case series.

Abstract

Genus Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment. Within this genus, numerous species are recognized as human pathogens, potentially causing Acanthamoeba keratitis (AK). AK is a corneal disease, associated predominantly with contact lens (CL) wear; its epidemiology is related to the specific Acanthamoeba genotypes. This study reports seven CL wearer, Acanthamoeba PCR-positive patients with AK, diagnosed between January 2015 and 2018. Patients had the diagnosis of AK 1.36 months after first symptoms. Genotyping allowed the identification of six isolates of the T4 and one of the T8 genotypes. At first presentation, pseudendritiformic epithelopathy/dirty epithelium (four eyes, 57.1%), multifocal stromal infiltrates (five eyes, 71.4%), ring infiltrate (three eyes, 42.8%), and perineuritis (one eye, 14.3%) were observed. AK was healed without later recurrence in two eyes (28.5%) using triple-topical therapy, in three eyes (42.8%) following additional penetrating keratoplasty. In one patient (14.3%), AK recurred following successful application of triple-therapy and was treated successfully with repeated triple-topical therapy and in one patient (14.3%), no follow-up data were available after diagnosis. We could not observe correlation of genotype and clinical course or the necessity of corneal transplantation in our case series.

Introduction

Acanthamoeba is a genus of free-living amebae widely distributed in various ecological environments. The spectrum ranges from natural biotopes, such as soil, plants, air, dust, freshwater, fishes, sea water, drinking water, swimming pools, and contact lenses (CLs). In addition, they have been isolated from animals such as fish and mammals and also from humans [13].

The first cases, which clearly established Acanthamoeba as a causative agent of disease in humans, were published in the early 1970s [4]. The pathogenesis of AK in humans is currently studied and different Acanthamoeba genotypes have been reported from all over the world [5].

The traditional Acanthamoeba classification has used morphological characteristics, such as morphology, size, and shape [6]. Modern classification uses a molecular biological approach based on 18S ribosomal RNA (rRNA) gene to classify Acanthamoeba isolates as one of the 20 known genotypes (T1–T20). Detection of Acanthamoeba can be rapidly achieved using real-time molecular methods. Each genotype exhibits 5% or more sequence divergences between different genotypes [7]. For diagnostic purposes, the detection of Acanthamoeba at the genus level is sufficient to recognize whether an individual is infected or not. Acanthamoeba keratitis (AK) is mainly present in CL wearers, especially in case of prolonged use of CLs, unappropriate hygienic conditions (contact of the lenses with tap water, swimming pool, dust, etc.) or in case of corneal trauma. AK is mainly caused by isolates with T4 genotype [810]; however, T2, T3, T5, T6, T8, T9, T11, T13, and T15 genotype species have also been identified in patients with AK, as shown in Table I [1118].

Table I.

Non-T4 genotype Acanthamoeba in different countries (literature data)

Acanthamoeba genotypesReport countries
T2Iran [12]
T3Iran [12], England [12], Austria, USA, Spain [1518], and Mexico [17]
T5Austria [16] and USA [14]
T6Austria [16]
T8Hungary [26]
T9Thailand [3]
T10Austria [16]
T11Austria [16], USA [11], and Spain [15]
T15Italy [13]

Following an appropriate clinical and also laboratory diagnosis (confocal microscopy, polymerase chain reaction, histology, and microbiological culture) and having Acanthamoeba in culture, it is still not possible to arrive at a conclusion in which topical treatment could be effectively used.

Our aim was to analyze the effect of Acanthamoeba genotype, isolated from human corneal scrapings and fluid from CL storage, on clinical course of AK.

Materials and Methods

We retrospectively collected diagnostic and clinical data of patients with AK [polymerase chain reaction (PCR) and/or culture positive], with AK diagnosis between January 2015 and 2018 at the Department of Ophthalmology of Semmelweis University.

Sample collection

Acanthamoeba was isolated from corneal scrapings of seven patients [three males (43%) and four females (57%), mean age during diagnosis 30.71 years] between January 2015 and 2018.

Culture-confirmed detection method

The seven samples of corneal scrapings were then transferred to Page’s agar plates overlaid with heat-killed Escherichia coli and cultured at 37 °C for 10 days. The morphology of trophozoites and cysts was studied by light microscopy, according to Page [19]. Plates were monitored for growth of ameba microscopically, from 72 to 96 h for the presence of Acanthamoeba spp. cysts and trophozoites under 320× and 400× magnification.

Molecular methods

Acanthamoeba was isolated from corneal scrapings of the patients and from fluid of CL storage case. The Acanthamoeba species were isolated by dilution method. For this purpose, the samples of corneal scrapings were suspended in 400-μl physiological saline solution (0.85%). After preparation, the DNA extraction was treated with High Pure PCR Template Preparation Kit (Roche, Germany) according to the manufacturer’s instructions. If further processing was delayed, the isolates were stored at 4 °C for 24 h or at −20 °C for a longer period. The DNA amplification was performed using genus-specific primers and genus-specific fluorescence resonance energy transfer (FRET) hybridization probes, previously described by Orosz et al. [20]. Each experiment included one reaction mixture without DNA as a negative control; positive control and each specimen was run in duplicate for real-time PCR assay in parallel.

PCR products were purified with PCR Clean-Up M Kit (Viogene, Sunville, CA). The sequence of each amplicon was determined by cycle sequencing with primers for the 5′-NTR region and with primers with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Germany) according to the manufacturer’s instruction. The electrophoresis was carried out using Applied Biosystems 3500 Genetic Analyzer.

The 5′-NTR and VP1 gene sequences were subject to nucleotide–nucleotide BLAST analysis [21] using the online server at the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast).

The unknown sequences were aligned with known published sequences of the major genotypes using the alignment program MULTALIN (http://multalin.toulouse.inra.fr/multalin) [22]. The genotypes of samples were determined based on this comparison.

The phylogenetic tree was constructed by the neighbor-joining method of genetic distance calculated by the MEGA 6 (http://www.megasoftware.net) [23].

Genotype identification was carried out using a real-time FRET PCR assay based on sequence analysis of the 18S rRNA gene, and sensitivity and specificity were evaluated in comparison with traditional parasitological techniques.

Clinical course

Observing the clinical course, we collected data on (1) time between onset of symptoms and diagnosis of AK, (2) clinical signs of AK, (3) coinfection with bacteria or fungi, (4) conservative treatment, (5) surgical treatment, and (6) outcome. For the outcome, we defined “success” if no sign of active AK was observed at least for 6 months, without the use of topical antiamebic therapy. We defined “failure” if AK persisted or recurred.

Results

Clinical data

Seven patients with AK had the diagnosis 1.36 months after first symptoms, at the Department of Ophthalmology of Semmelweis University as shown in Figure 1.

Figure 1.
Figure 1.

Clinical images of Acanthamoeba keratitis in patients 1–7. Patient 1: multifocalstromal infiltrates. B and C: Patient 2: perineuritis (B) (arrow) and pseudodendritiformic epitheliopathy (“dirty epithelium”) (C). D: Patient 3: multifocal stromal infiltrates and incomplete ring infiltrate (arrow). E and F: Patient 4: multifocal stromal infiltrates (arrows) (E) and ring infiltrate (F) (arrow). G: Patient 5: deep stromal infiltrate (arrow) and fine slightly visible multifocal stromal infiltrates. H: Patient 6: epithelial erosion and broad ring infiltrate (arrow). I: Patient 7: multifocal stromal infiltrates (arrow)

Citation: Acta Microbiologica et Immunologica Hungarica AMicr 66, 3; 10.1556/030.66.2019.008

At the first presentation, pseudendritiformic epithelopathy/dirty epithelium (four eyes, 57.1%), multifocal stromal infiltrates (five eyes, 71.4%), ring infiltrate (three eyes, 42.8%), and perineuritis (one eye, 14.3%) were observed. The clinical data are summarized in Table II.

Table II.

Clinical data during the observation time period in our set of patients

Case no.Age (years), sexTime to diagnosisClinical signsCoinfectionConservative treatmentSurgical treatmentOutcomeAcanthamoeba genotype
124, female3 weeksMSITTTSuccessT4
230, female2 weeksDirty epithelium and perineuritisTTTSuccessT4
316, male2 weeksMSI and ring infiltrateTTTFailureT4
420, female10 daysMSI and ring infiltrateTTTPKP and AMTSuccessT4
537, male2 monthsPersistent epithelial defect and MSIstaph.TTTPKP and AMTSuccessT4
660, male5 monthsPersistent epithelial defect and ring infiltrateTTTPKPSuccessT4
728, female2 weeksMSINo information availableT8

Note: Observing the clinical course, we collected data on (1) time between onset of first symptoms and diagnosis of AK, (2) clinical signs of AK, (3) coinfection with bacteria or fungi, (4) conservative treatment, (5) surgical treatment, and (6) outcome. For the outcome, we defined as “success” if no signs of AK were observed for at least 6 months, without the use of topical antiamebic therapy. We defined as “failure” if AK persisted or recurred. MSI: multifocal stromal infiltrates; staph.: Staphylococcus; TTT: triple-topical therapy; PKP: penetrating keratoplasty; AMT: amniotic membrane transplantation as patch.

AK was healed without later recurrence in two eyes (28.5%) using triple-topical therapy (TTT; polyhexamethylene biguanide, propamidine isethionate, and antibiotics/neomycin) in three eyes (42.8%) following additional penetrating keratoplasty.

In Patient 3, AK recurred following successful application of TTT and was treated successfully with repeated TTT. In Patient 7, no follow-up data were available, following diagnosis. We could not observe similarities in genotype and clinical course or the necessity of corneal transplantation.

Cultivation

All investigated samples revealed Acanthamoeba that were able to grow at 36 °C, the approximate temperature of the human host. Microscopical cultivation was successful in six samples. Probably due to low quantity of corneal scraping in the seventh sample, one sample showed negative result for cultivation. Further examination of the obtained results was carried out by FRET PCR.

Molecular methods

This study reports successful PCR amplification for seven (four females and three males) positive cases. The samples for Acanthamoeba-positive patients, detected by PCR method, were sequenced to identify the species (NCBI Bank: Patient_1-KF873021_T4, Patient_2-KP337296_T4, Patient_3-KU356846_T4, Patient_4-KU356848_T4, Patient_5-KR494236_T4, Patient_6-KJ094693_T4, and Patient_8-MF065931_T8). Sequence analysis using a BLAST search indicated an identity of >98% with Acanthamoeba 18S rRNA gene reference sequences. It was found that the obtained sequences of amebae isolates from the cases belonged to the T4 and T8 genotypes Acanthamoeba spp. neighbor-joining analysis inferred relationships between the PCR products isolated from corneal scrapings and reference strains obtained from NCBI GenBank are shown in Figure 2, respectively.

Figure 2.
Figure 2.

Phylogenetic relations of Acanthamoeba species PCR product Patient_1, Patient_2, Patient_3, Patient_4, Patient_5, Patient_6, Patient_7 and reference strains from NCBI GenBank inferred by neighbor-joining analysis from pairwise comparisons (180-bp fragments)

Citation: Acta Microbiologica et Immunologica Hungarica AMicr 66, 3; 10.1556/030.66.2019.008

Discussion and Conclusions

Detection of Acanthamoeba can be rapidly achieved using real-time FRET molecular methods. For diagnostic purposes, the detection of Acanthamoeba at the genus level is sufficient to recognize whether an individual is infected or not [2425].

Literature describes T4 genotype Acanthamoeba, as the most common in the environment; however, in AK, prevalence of T4 genotype is even more common. The molecular analysis conducted in this study confirmed, in our series of patients, the T4 genotype to be the most frequent cause of AK. In addition, one isolate was T8 genotype and was first associated with AK [26]. These results are consistent with previous findings indicating that T4 is worldwide predominant in AK [2728].

Nevertheless, a heterogeneous virulence of different Acanthamoeba strains has been observed in our case series, for different Acanthamoeba strains. In our opinion, different reference sequences of T4 or other Acanthamoeba genotypes may also be important in prognosting disease progression.

For an appropriate diagnosis, ophthalmological signs of AK must be known. These are “dirty epithelium” (pseudodendritiformic epitheliopathy), non-healing epithelial defects or ulcer, mono- or multifocal stromal infiltrates, perineuritis, ring infiltrate, and in later stages anterior syneciae, iris atrophy, secondary glaucoma, mature cataract, scleritis and chorioretinitis, or even blindness. To date, about 75% of ophthalmologists miss the appropriate clinical diagnosis in AK. Faulty typical diagnosis is herpetic keratitis in most of the cases; however, bacterial or mycotic keratitis may also be suggested [2933]. However, cooperation between clinicians and experts in laboratory diagnostics is indispensable, for adquate treatment in time of these patients.

Nowadays, there is no standardized treatment of AK, as no previous randomized controlled studies have been performed in this rare and often heterogeneous corneal disease. In addition, Acanthamoeba cysts are often resistant or become resistant during treatment to all available topical therapeutic options. The therapeutic regimen used in the recent times includes biguanides (pethylene biguanide 0.02% and chlorhexidine 0.02%), diamidine (propamidine isethionate 0.1% and hexamidine 0.1%), and antibiotics/neomycin as TTT. Antimycotic eye drops, propidium iodide, and miltefosine may also be effective against Acanthamoeba strains. Besides conservative treatment, in advanced cases or in cases without response to topical therapy, penetrating keratoplasty, amniotic membrane transplantation, or crosslinking treatment may be performed [34].

To the best of our knowledge, this is the first study to analyze a correlation between Acanthamoeba genotype and clinical course. However, we could not determine a relationship between both of them in these series of patients. To date, there is no standard treatment of AK; however, we also did not find a study analyzing relationship between genotype and Acanthamoeba susceptibility to different agents. In our opinion, this also needs development in the following years.

There are only case series on safety and effectivity of medical and surgical treatment of AK and to date there are no randomized controlled clinical studies. In Hungary, we suggest topical application of polyhexamethylene biguanide, propamidine isethionate, and antibiotics/neomycin as TTT in case of AK [35].

During the first 2 days, a “surprise attack” or “flash war” is initiated with polyhexamethylene biguanide and propamidine isethionate every quarter to half an hour day and night. Then, until the sixth day, polyhexamethylene biguanide and propamidine isethionate are applied every hour and only over the day. Following 4 weeks, eyedrop use is reduced to every 2 h. In addition, antibiotics/neomycin 5× a day is also applied for some week.

Worldwide incidence of AK increases, presumably due to the increasing use of CLs [3637]. To the best of our actual knowledge, combination therapy using diamidine, biguanide, and antibiotics should be continued in descending dosis until 1 year.

In Hungary, AK has been developed through Acanthamoeba genotypes T4 and T8 in the past 3 years. Analyzing seven patients, we could not determine a relationship between Acanthamoeba genotype and clinical course of the disease. We suggest the development of an international database on AK causative isolates for better understanding of the disease course and better treatment of these patients.

Conflict of Interest

The authors declare no conflict of interest. EO assures that there are no links with a company whose product is mentioned in the article or a company that distributes a competing product. The authors also state that the presentation of the topic is independent and the presentation of the content is product-neutral.

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Senior editors

Editor-in-Chief: Prof. Dóra Szabó (Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary)

Managing Editor: Dr. Béla Kocsis (Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary)

Co-editor: Dr. Andrea Horváth (Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary)

Editorial Board

  • Prof. Éva ÁDÁM (Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary)
  • Prof. Sebastian AMYES (Department of Medical Microbiology, University of Edinburgh, Edinburgh, UK.)
  • Dr. Katalin BURIÁN (Institute of Clinical Microbiology University of Szeged, Szeged, Hungary; Department of Medical Microbiology and Immunobiology, University of Szeged, Szeged, Hungary.)
  • Dr. Orsolya DOBAY (Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary)
  • Prof. Ildikó Rita DUNAY (Institute of Inflammation and Neurodegeneration, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany; Center for Behavioral Brain Sciences (CBBS), Magdeburg, Germany)
  • Prof. Levente EMŐDY(Department of Medical Microbiology and Immunology, University of Pécs, Pécs, Hungary.)
  • Prof. Anna ERDEI (Department of Immunology, Eötvös Loránd University, Budapest, Hungary, MTA-ELTE Immunology Research Group, Eötvös Loránd University, Budapest, Hungary.)
  • Prof. Éva Mária FENYŐ (Division of Medical Microbiology, University of Lund, Lund, Sweden)
  • Prof. László FODOR (Department of Microbiology and Infectious Diseases, University of Veterinary Medicine, Budapest, Hungary)
  • Prof. József KÓNYA (Department of Medical Microbiology, University of Debrecen, Debrecen, Hungary)
  • Prof. Yvette MÁNDI (Department of Medical Microbiology and Immunobiology, University of Szeged, Szeged, Hungary)
  • Prof. Károly MÁRIALIGETI (Department of Microbiology, Eötvös Loránd University, Budapest, Hungary)
  • Prof. János MINÁROVITS (Department of Oral Biology and Experimental Dental Research, University of Szeged, Szeged, Hungary)
  • Prof. Béla NAGY (Centre for Agricultural Research, Institute for Veterinary Medical Research, Budapest, Hungary.)
  • Prof. István NÁSZ (Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary)
  • Prof. Kristóf NÉKÁM (Hospital of the Hospitaller Brothers in Buda, Budapest, Hungary.)
  • Dr. Eszter OSTORHÁZI (Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary)
  • Prof. Rozália PUSZTAI (Department of Medical Microbiology and Immunobiology, University of Szeged, Szeged, Hungary)
  • Prof. Peter L. RÁDY (Department of Dermatology, University of Texas, Houston, Texas, USA)
  • Prof. Éva RAJNAVÖLGYI (Department of Immunology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary)
  • Prof. Ferenc ROZGONYI (Institute of Laboratory Medicine, Semmelweis University, Budapest, Hungary)
  • Prof. Zsuzsanna SCHAFF (2nd Department of Pathology, Semmelweis University, Budapest, Hungary)
  • Prof. Joseph G. SINKOVICS (The Cancer Institute, St. Joseph’s Hospital, Tampa, Florida, USA)
  • Prof. Júlia SZEKERES (Department of Medical Biology, University of Pécs, Pécs, Hungary.)
  • Prof. Mária TAKÁCS (National Reference Laboratory for Viral Zoonoses, National Public Health Center, Budapest, Hungary.)
  • Prof. Edit URBÁN (Department of Medical Microbiology and Immunology University of Pécs, Pécs, Hungary; Institute of Translational Medicine, University of Pécs, Pécs, Hungary.)

 

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2020  
Total Cites 662
WoS
Journal
Impact Factor
2,048
Rank by Immunology 145/162(Q4)
Impact Factor Microbiology 118/137 (Q4)
Impact Factor 1,904
without
Journal Self Cites
5 Year 0,671
Impact Factor
Journal  0,38
Citation Indicator  
Rank by Journal  Immunology 146/174 (Q4)
Citation Indicator  Microbiology 120/142 (Q4)
Citable 42
Items
Total 40
Articles
Total 2
Reviews
Scimago 28
H-index
Scimago 0,439
Journal Rank
Scimago Immunology and Microbiology (miscellaneous) Q4
Quartile Score Medicine (miscellaneous) Q3
Scopus 438/167=2,6
Scite Score  
Scopus General Immunology and Microbiology 31/45 (Q3)
Scite Score Rank  
Scopus 0,760
SNIP
Days from  225
sumbission
to acceptance
Days from  118
acceptance
to publication
Acceptance 19%
Rate

2019  
Total Cites
WoS
485
Impact Factor 1,086
Impact Factor
without
Journal Self Cites
0,864
5 Year
Impact Factor
1,233
Immediacy
Index
0,286
Citable
Items
42
Total
Articles
40
Total
Reviews
2
Cited
Half-Life
5,8
Citing
Half-Life
7,7
Eigenfactor
Score
0,00059
Article Influence
Score
0,246
% Articles
in
Citable Items
95,24
Normalized
Eigenfactor
0,07317
Average
IF
Percentile
7,690
Scimago
H-index
27
Scimago
Journal Rank
0,352
Scopus
Scite Score
320/161=2
Scopus
Scite Score Rank
General Immunology and Microbiology 35/45 (Q4)
Scopus
SNIP
0,492
Acceptance
Rate
16%

 

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Acta Microbiologica et Immunologica Hungarica
Language English
Size A4
Year of
Foundation
1954
Publication
Programme
2021 Volume 68
Volumes
per Year
1
Issues
per Year
4
Founder Magyar Tudományos Akadémia
Founder's
Address
H-1051 Budapest, Hungary, Széchenyi István tér 9.
Publisher Akadémiai Kiadó
Publisher's
Address
H-1117 Budapest, Hungary 1516 Budapest, PO Box 245.
Responsible
Publisher
Chief Executive Officer, Akadémiai Kiadó
ISSN 1217-8950 (Print)
ISSN 1588-2640 (Online)

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