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[10] Tice AK, Shadwick LL, Fiore-Donno AM, Geisen S, Kang S, Schuler GA, et al. Expansion of the molecular and morphological diversity of Acanthamoebidae.

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Acanthamoeba keratitis in noncompliant soft contact lenses users: Genotyping and risk factors, a study from Cairo, Egypt Eman E. Taher a , Eman M.H. Méabed b,∗ , Islam Abdallah c , Wafaa Y. Abdel Wahed d a

Department of Parasitology, Research Institute of Ophthalmology, El-Giza, Egypt Department of Parasitology, Faculty of Medicine, Fayoum University, Egypt Department of Ophthalmology, Research Institute of Ophthalmology, El-Giza, Egypt d Department of Public Health and Community Medicine, Faculty of Medicine, Fayoum University, Egypt b c

a r t i c l e

i n f o

Article history: Received 30 June 2017 Received in revised form 9 August 2017 Accepted 9 September 2017 Keywords: Acanthamoeba keratitis Soft contact lens Genotyping Hygiene Cleaning solutions Tap water

a b s t r a c t Acanthamoeba keratitis (AK) is a severe corneal infection that may occur as a serious outcome of improper use of contact lenses (CL). Objectives: The study aimed to diagnose AK in soft CL users presenting with infectious keratitis, and to identify the prevalent genotypes isolated from different cases. Another aim was to determine the CL hygiene-related risk behaviors, and to explore the risk of water exposure for developing AK. Methods: A cross sectional study was performed. A questionnaire was carried out including 260 clinically diagnosed cases as infectious keratitis (170 females and 90 males); all of them were soft CL users for the suspected risk factors. Corneal scrapes from the affected eyes were cultured to diagnose bacterial and AK. PCR was performed and the amplified products were sequenced and compared with GenBank data. Results: The parasite was positively amplified from 32 samples (12.3%). Acanthamoeba T4 genotype was identified in 27/32 (84.4%) of isolates. Other detected genotypes belonged to T5 and T3 genotypes at rates of 9.4%, and 6.25%, respectively. The most important risk factors associated with development of AK were female sex, sleeping while wearing CL, and exposure to water resources through different practices. These practices included rinsing the CL case in tap water, swimming and/or showering while wearing CL, using multipurpose solution for cleaning the lenses, using water from over-building tanks. Rubbing the eyes due to discomfort when applying CL was an additional important risk factor associated with AK. The protective factor was regular hand washing before using CL. Conclusion: CL users were more exposed to AK and should gain enough health education regarding proper lens hygiene and dangers of tap water exposure. © 2017 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction Acanthamoeba is a genus of free-living amoebae that exists as either active trophozoites or dormant cysts. It is found virtually everywhere, including soil, air, and water [1]. Human infections usually occur through exposure to different water resources, including freshwater, marine water, as well as various domestic water systems such as drinking tap water [2]. Acanthamoeba keratitis is a severe sight-threatening ulceration of the cornea. The early symptoms are typically pain, photophobia and watering. The infection may progress to stromal inflammation,

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (E.M.H. Méabed).

ring ulcers, corneal opacification, hypopyon, corneal perforation, cataract, glaucoma and posterior segment inflammation [3]. Wearing CL is the main risk factor for eye infections, accounting for around 90% of reported cases [3,4]. Soft CL biomaterial acts as vector transferring microorganisms to the ocular surface, producing corneal inflammation, especially if it is not properly disinfected [4]. Wearing CL alters the normal physiological and metabolic activities of corneal cells, causing chronic hypoxic stress on the corneal epithelium leading to premature desquamation of epithelial cells, and significant thinning of the epithelial cell layer [5]. Several unhygienic practices and risk factors interact together, increasing the rates of infectious keratitis in CL users as exposure to corneal trauma and contact with contaminated water [2,4]. However, patient’s compliance and some basic hygienic standards can effectively minimize the risk of AK [5].

http://dx.doi.org/10.1016/j.jiph.2017.09.013 1876-0341/© 2017 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Taher EE, et al. Acanthamoeba keratitis in noncompliant soft contact lenses users: Genotyping and risk factors, a study from Cairo, Egypt. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2017.09.013

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Advanced molecular techniques helped in Taxonomic classification of Acanthamoeba without merely depending on morphological features [2]. Genotyping helped to identify pathogenic isolates [6]. Several species of the genus Acanthamoeba have been known to cause sight-threatening AK. Up till now, around 20 genotypes have been identified; T1–T20 based on the analysis of the diagnostic fragment 3 (DF3) region of the ribosomal DNA (rDNA) gene of Acanthamoeba [1,7]. DF3 encodes the highly variable stem 29-1 of the nuclear small subunit 18S rRNA gene [8,9]. Recently, the genotypes T21 and T22 have been identified [10]. T4 is the most abundant in the environment and includes many pathogenic strains which have been associated with neurological and eye diseases [1,3,6]. The present study aimed to diagnose AK in soft CL users presenting with infectious keratitis, and to identify the prevalent genotypes isolated from patients’ corneal scrapings. Another aim was to identify the CL hygiene-related risk behaviors, and to explore the risk of water exposure for developing AK.

parasite was identified by its characteristic morphology for the cysts (wrinkle double walled) and trophozoites (acanthopodia and pseudopodia). Positive cultures were stored at −20 ◦ C until used for DNA extraction. Bacterial cultures [13] After performing Gram’s staining, each swab was inoculated onto Blood agar, Chocolate agar, MacConkey’s agar and Wilkins media then incubated at 37 ◦ C for 24 h. The cultured plates were checked for growth and finally declared as culture negative after 5 days. Chocolate agar plates were incubated in CO2 incubator and Wilkins media were incubated anaerobically. Culture positive growth was identified by their colony, morphology, Gram staining and motility testing by hanging drop preparation, pigment production and relevant biochemical tests. Molecular analysis

Materials and methods Study population This is a cross sectional study included 260 infectious keratitis cases using soft CL Patients were from the attendee of the Corneal Unit, Research Institute of Ophthalmology, Giza, Egypt (RIO) from 2011 to 2015. Ethical statement Written informed consents were obtained from all participants. The study was approved by the institutional review board and the Ethical Committee of Scientific Research (National Research Centre Ethical Research Committee). Inclusion criteria Patients of any age or sex diagnosed as infectious keratitis and gave a history of the concurrent application of soft CL. Cases who gave a history of using antibiotics in the last previous week were excluded. All patients underwent thorough ocular examination. The clinical history and associated findings were recorded. A structured questionnaire, interview It included the demographic data and the suspected risk factors. The questionnaire was translated to Arabic language and trained personnel met the participants individually to fill it. The socioeconomic standard (SES) of cases was assessed by a scoring system for the occupation, the education level, the crowding index (number of persons per room), the total family income and the sanitation degree [11]. Specimen collection Corneal scraping was performed before giving antibiotic therapy, under complete aseptic precautions, after installing a local anesthetic (4% Xylocaine). The obtained material was inoculated onto the surfaces of different types of agar plates for Acanthamoeba and bacterial identification. The laboratory procedures were performed at the Microbiology and Parasitology laboratories, Research Institute of Ophthalmology (RIO), Giza. Acanthamoeba culture Corneal scrapings were cultivated on non-nutrient agar plates co-seeded with live Escherichia coli [12]. Plates were incubated at 30 ◦ C, observed and examined microscopically daily for 7 days. The

According to the manufacturer’s instructions, cleaned cysts from different culture isolates were subjected to DNA extraction using the QIAamp DNA Mini Kit (Qiagen, Venlo, Netherlands). DNA amplification reactions were performed using genus-specific markers for Acanthamoeba. They amplify a fragment of approximately 500 bp of the ASA.S1 region of the 18S RNA gene. The forward primer is JDP1: 5 -GGCCCAGATCGTTTACCGTGAA and the reverse primer is JDP2: 5 -TCTCACAAGCTGCTAGGGGAGTCA [8]. DNA amplification reaction: The used PCR reaction mixture, per sample, consisted of: 2 ␮L template DNA, 2 ␮L of each primer, 25 ␮L PCR Master Mix (2X), and 19 ␮L sterile de-ionized water. Amplification was done using a thermocycler (PerkinElmer Cestus, Norwalk, CT). The process began with an initial denaturation step at 96 ◦ C for 3 min, followed by 35 cycles of denaturation at 96 ◦ C for 1 min, then primer annealing at 60 ◦ C for 1 min, and extension at 72 ◦ C for 1 min. The final extension step occurred at 72 ◦ C for 7 min. For negative control, distilled water was added instead of DNA. For positive control, Acanthamoeba castellanii Neff ATCC 30010 from the American Type Culture Collection (ATCC) was used. A 250–10,000 base pair (bp) ladder was used as a DNA size marker (Gene RulerTM , Fermentas). Amplified DNA products were then electrophoresed using 1.2% agarose gel stained with ethidium bromide then visualized under UV illumination. Sequencing and genotype identification The amplified PCR products were purified using the AxyPrep PCR Cleanup kit (AXYGEN Biosciences-USA). PCR products were sequenced using an automated fluorescence sequencing system (Applied Biosystems 3730XL Genetic DNA analyzer). Sequence analysis of DF3 region was used to identify specific genotypes through alignment with available Acanthamoeba genotype sequences in the GenBank [9]. Phylogenetic analysis based on the sequences obtained was performed using MAFFT (EMBL-EBI, www.ebi.ac.uk/). Distance-based analyses were conducted using the maximum-parsimony, composite likelihood method, and the phylogenetic tree was constructed using a neighbor-joining algorithm. Statistical analysis The Statistical Package for Social Sciences for Windows (SPSS), version 16 (SPSS Inc., Chicago, IL, USA) was used to perform statistical analysis. The associations of parasite prevalence with the suspected factors were calculated using Pearson’s Chi Square test. Odds ratio (OR) and 95% confidence intervals (CI) were computed. Forward regression analysis (FRA) was performed to identify the

Please cite this article in press as: Taher EE, et al. Acanthamoeba keratitis in noncompliant soft contact lenses users: Genotyping and risk factors, a study from Cairo, Egypt. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2017.09.013

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Fig. 1. A culture shows Acanthamoeba trophozoite and cysts (×1000).

significant predictors of infection. All P values ≤ 0.05 were considered statistically significant. Results Two hundred and sixty cases met the inclusion criteria and were involved in this study. The clinically examined keratitis patients presented with progressive pain, redness, photophobia, and diminution of vision in the affected eye. The females/male ratio was 65.4%/34.6%. Their age ranged from 15 to 35 years. The mean age was 24.4 ± 5.2 years. They were divided into four age groups; the most presenting age group ranged from ≥21 to 25 years (33.8%). Diagnosis of Acanthamoeba and genotyping results Acanthamoeba was identified from 32/260 positive culture plates (12.3%) according to characteristic morphology (Fig. 1), and then were confirmed by PCR test. Through BLAST, all the isolates proved to belong to the genus Acanthamoeba after alignment and comparison with the most common pathogenic isolates from the Genbank. The phylogenetic tree comparing the detected strains with the most common strains was built (Fig. 2). The GenBank accession numbers and the origins of the isolates used for comparison were presented in (Table 1). Results showed that 27/32 isolates (84.4%) belonged to the T4 genotype having 99% similarity rate with Acanthamoeba castellanii Neff — ATCC 50373 (U07416). Three isolates (9.4%) were detected to have 99% similarity rate with T5 Acanthamoeba lenticulata PD2S (U94741) genotype. Another two isolates were detected to have 99% similarity rate with T3 Acanthamoeba griffini S-7 ATCC 30731. The demographic criteria of the examined subjects According to the results of Acanthamoeba culture, cases were classified into two groups; the group positive for Acanthamoeba parasite 32/260 (12.3%), and those negative for Acanthamoeba parasite 228/260 (87.7%). The demographic criteria of cases were presented (Table 2). Acanthamoeba parasite was significantly detected in 18/32 (56.2%) cases their age group ranged from ≥21 to 25 years. Female sex was significantly associated with Acanthamoeba infection more than male sex (P = 0.016). Twenty cases of Acanthamoeba (62.5%) obtained education till the secondary level only. The SES had no significant relation with Acanthamoeba infection. Both summer and spring seasons were significantly associated with this parasitic infection. There was a significant relation of hours of digital straining of the eyes (computer vision syndrome) and Acanthamoeba infection as 13/32 (40.6%) of AK cases reported that they used to spend more than 6 h/day in front of screens. Role of water exposure The distribution of water exposure risk factors in relation to AK, the odd ratio, the upper and the lower 95% confidence interval for odd ratios and the P-values were presented (Table 3).

Totally 188/260 subjects (72.3%) reported they had a daily water supply for their houses from big water tanks placed over buildings’ roofs, exposed to the sun. Significantly, 30/32 (93.8%) of AK cases had their water supply from tanks (P = 0.004). Only, 55/260 (21.2%) of the examined cases reported that they didn’t stick to hand washing before CL handling. However, 20/32 (62.5%) of cases AK ignored hand washing (P = 0.00). CL case was cleaned regularly by 212 (81.5%) of cases, the parasite was isolated from 20 cases of them (62.5%, P = 0.003). Acanthamoeba keratitis was associated also with rinsing the CL or their case with tap water, swimming or showering while wearing CL and using multipurpose solution for cleaning the CL (P ≤ 0.009). Only two diagnosed Acanthamoeba cases reported storing the CL in tap water with insignificant association. Using anti-allergic eye drops or tear substitutions from old nonsealed bottles (≥8 weeks) was reported by 104 participants, of them 7 cases were positive for Acanthamoeba, they represented 21.9% of all diagnosed Acanthamoeba cases (P = 0.025, Table 3).

Other suspected risk factors Bacteria were positively amplified from 194/260 samples (74.6%) (Table 4). The female/male ratio was (76.5%/71.1%). Bacterial Infection was detected in 136/190 patients (70.1%) of age range from 20 to 30 years old (P value = 0.01). Summer and spring were the most common seasons associated with bacterial keratitis (P value = 0.01). Staphylococcus aureus; Staphylococcus epidermidis and Pseudomonas aeruginosa infections were the most encountered bacterial species after cultivation of samples. In this study, we dealt with bacterial infection as a categorical character and cases were divided into either positive or negative for bacterial culture. The detailed association of every bacterial pathogen in relation to AK was previously published in a separate paper. Significantly, 100% of AK cases were positive for bacterial cultures. Acanthamoeba infection was significantly detected in cases gave a history of exposure to dust due to heavy traffic in the crowded Cairo city (p = 0.002), but it was not related to exposure to direct sunlight for prolonged time daily. Infection was significantly detected in those who used the CL daily than occasional users. Surprisingly, 38 (14.6%) of the total examined subjects gave a history of sharing CL with others. All of them were low SES females, from semi-urbanized areas used to share colored cosmetic CL from hairdressers to attend some wedding parties. Acanthamoeba was significantly associated with those who shared CL. Sleeping while wearing the CL was reported by 14/260 of the total examined subjects with significant association with AK (p = 0.003). Using the CL for a prolonged time, despite being expired was reported by 55/260, without significant association with AK. Correction of optical errors was the purpose of CL application in 33.1% of users, their lenses were prescribed by a doctor and they had subjected to ophthalmic examination and received instructions about CL care from their doctors’ prior application. Another 66.9% of

Please cite this article in press as: Taher EE, et al. Acanthamoeba keratitis in noncompliant soft contact lenses users: Genotyping and risk factors, a study from Cairo, Egypt. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2017.09.013

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Fig. 2. Phylogenetic tree representing the multiple sequence alignment of Acanthamoeba samples isolated from patients (PT1–32), with Acanthamoeba stains T1–T10 presented by their GenBank accession numbers and the origins of the isolates. The tree was built by neighbor-joining analysis of the 18S rRNA gene sequence produced in MAFFT — EMBL-EBI (www.ebi.ac.uk/).

Table 1 The 10 species used for comparison of the detected isolates (phylogenetic tree). Species

Genbank accession number

18S rDNA genotype

Acanthamoeba castellanii CDC 0981 V006 Acanthamoeba palestinensis strain Reich Acanthamoeba griffini S7 ATCC 30731 Acanthamoeba castellanii Neff ATCC 50373 Acanthamoeba lenticulata strain PD2S Acanthamoeba palestinensis 2802 Acanthamoeba astronyxis strain CCAP 1534/1 Acanthamoeba tubiashi OC-15C Acanthamoeba comandoni Comandon de Fonbrune Acanthamoeba sp CDC V369

U07400 AY703001 U07412 U07416 U94741 AF019063 AF239293 AF019065 AF019066 AY703001

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

subjects reported that they used CL for cosmetic purposes, and they purchased them from non-specialized person, and applied CL without previous clinical examination of the eyes. Only 100 subjects included in this study reported awareness by hygienic rules about applying and cleaning of CL (P > 0.05). Forty two (16.2%) of the total subjects involved in this study reported that they insisted to apply their CL despite feeling some discomfort in their eyes (P > 0.05). Totally 53.1% of the examined cases reported rubbing of the eyes due to discomfort on applying CL and this practice was significantly associated with AK. Results of the forward regression analysis (FRA) Results of FRA, odds ratio, 95% confidence interval for odd ratio and P values were presented (Table 5). The most important risk

factors were female sex, sleeping while applying CL, rinsing the case in tap water, swimming and/or showering while applying CL, using multipurpose solution for cleaning the lenses, using water from over-building tanks, and rubbing the eyes due to discomfort when applying CL. The protective factor was regular hand washing before using CL. Discussion Contact lenses provide safe and effective vision correction and cheap cosmetic alternatives. However, CL wearers are at risk of AK if they fail to stick to the rules about cleaning, disinfection, and storing their CL [14,15]. In this study, Acanthamoeba isolates belonged to T4, T5 and T3 genotypes at rates of 84.4%, 9.4%, and 6.25%, respectively. Detec-

Please cite this article in press as: Taher EE, et al. Acanthamoeba keratitis in noncompliant soft contact lenses users: Genotyping and risk factors, a study from Cairo, Egypt. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2017.09.013

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Table 2 Demographic features of the examined cases in relation to Acanthamoeba infection. Demographic variables

Acanthamoeba Positive (32) No. (%)

Negative (228) No. (%)

Total (260)

%

P value

Age

15–20 ≥21–25 ≥26–30 ≥31

7 (21.9) 18 (56.2) 6 (18.8) 1 (3.1)

45 (19.7) 70 (30.7) 72 (31.6) 41 (12)

52 88 78 42

20.0 33.8 30.0 16.2

0.013

Sex

Male Female

5 (15.6) 27 (84.4)

85 (37.3) 143 (62.7)

90 170

34.6 65.4

0.016

Educational level

Secondary

7 (21.9) 20 (62.5) 5 (15.6)

57 (25) 122 (53.5) 49 (21.5)

64 142 54

24.6 54.6 20.8

0.061

SES

High Moderate Low

3 (9.4) 17 (53) 12 (38)

19 (8) 133 (58) 76 (33)

22 150 88

8 58 34

0.87

Season

Summer Winter Spring Autumn

14 (43.8) 2 (6.2) 14 (43.8) 2 (6.2)

90 (39.5) 44(19.3) 58 (25.4) 36 (15.8)

104 46 72 38

40.0 17.7 27.7 14.6

0.047

Hours of exposure to computers (digital eye strain)

Not daily 6 h/day

5 (15.6) 6 (18.8) 8 (25.0) 13 (40.6)

35 (15.4) 60 (26.3) 94 (41.2) 39 (17.1)

40 66 102 52

15.4 25.4 39.2 20.0

0.016

Table 3 Water related risk factors in relation to Acanthamoeba infection. Water related variables

Water tanks Hand wash Rinsing CL with tap water Rinsing the case with tap water Storing CL in tap water Regular cleaning case Swimming/showering while wearing CL Multipurpose solution Special saline Using eye drops

Yes Yes No Yes Yes Yes Yes Yes Yes Yes Non sealed

Acanthamoeba Positive (32)

Acanthamoeba Negative (228)

Total

No.

%

No.

%

No.

%

30 12 20 9 11 2 20 4 22 10 7

93.8 37.5 62.5 28.1 34.4 6.2 62.5 12.5 68.8 31.2 21.9

158 193 35 22 28 2 192 4 41 135 97

69.3 84.6 15.4 9.6 12.3 0.9 84.2 1.8 18.0 59.2 42.5

188 205 55 31 39 4 212 8 63 145 104

72.3 78.8 21.2 11.9 15.0 1.5 81.5 3.1 24.2 55.8 40.0

Odd ratio

95% confidence interval

P value

Lower

Upper

6.65 0.11

1.545 0.049

28.58 0.24

0.004 0.00

3.66 3.74 7.53 0.31 8.00 10.0 0.31 0.38

1.509 1.632 1.023 0.14 1.894 4.418 0.14 0.157

8.897 8.579 55.47 0.695 33.78 22.79 0.692 0.91

0.006 0.001 0.075 0.003 0.009 0.00 0.003 0.025

Table 4 Other suspected risk factors in relation to Acanthamoeba infection. CL variables

Bacterial culture Sleeping while wearing CL Using expired CL CL application frequency Exposure to dust Exposure to the Sun Sharing lenses CL purpose CL application after symptoms Rubbing the eye Awareness by CL hygiene

Acanthamoeba Positive (32)

Positive Yes Yes Daily Occasionally Yes Yes Yes Optical Cosmetic Yes Yes Yes

Acanthamoeba Negative (228)

No.

%

No.

%

32 6 11 21 11 28 19 14 9 23 7 27 11

100.0 18.8 34.4 65.6 34.4 87.5 59.4 43.8 28.1 71.9 21.9 84.4 34.4

162 8 44 87 141 134 113 24 77 151 35 111 89

71.1 3.5 19.5 38.2 61.8 58.8 49.6 10.5 33.8 66.2 15.4 48.7 39

tion of genotypes T4 and T3 cleared the relation between AK and the source of infection. They originated from dust and water in patients who used tap, mineral water or the commercially prepared saline for washing and soaking CL [2,16]. T4 is the most separated and virulent genotype from clinical and environmental samples world-

Total (260)

194 14 55 108 152 162 132 38 86 174 42 138 100

%

Odd ratio

95% confidence interval

P value

Lower

Upper

74.6 5.4 21.3 41.5 58.5 62.3 50.8 14.6 33.1 66.9 16.2 53.1 38.5

1.19 6.35 2.17 3.09

1.125 2.04 0.973 1.42

1.275 19.72 4.824 6.729

0.00 0.003 0.054 0.003

4.91 1.49 6.61 0.77

1.667 0.7 2.922 0.339

14.464 3.154 14.958 1.739

0.002 0.298 0.00 0.525

1.54 5.69 0.82

0.62 2.117 0.376

3.844 15.302 1.778

0.35 0.000 0.612

wide [3,17]. It has an increasing importance due to its high range of distribution, resistance of its cysts to antiseptics and its production of more cytotoxic factors than other genotypes [17]. Genotypes T3 and T11 were also commonly associated with AK [18]. This study disagreed with Tawfeek et al. [19] who identified only T7

Please cite this article in press as: Taher EE, et al. Acanthamoeba keratitis in noncompliant soft contact lenses users: Genotyping and risk factors, a study from Cairo, Egypt. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2017.09.013

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Table 5 Risk factors of exposure to Acanthamoeba infection (forward regression analysis). Factors

Sex (female vs male) Sleeping (yes vs no) Rinsing case in tap water (yes vs no) Swimming and or showering (yes vs no) Hand wash (yes vs no) Multipurpose solution (yes vs no) Water tanks (yes vs no) Rubbing the eye (yes vs no) Constant

P value

Odd ratio

0.012 0.008 0.008 0.006 0.000 0.001 0.005 0.006 0.000

6.207 21.154 15.581 128.540 0.028 12.514 24.550 7.854 0.001

from keratitis cases. The current data regarding the distribution of various genotypes in AK cases in Egypt are limited. However, there are some reports on environmentally prevalent genotypes of Acanthamoeba in Egypt. Lorenzo-Morales et al. [20] identified 5 genotypes of genus Acanthamoeba in freshwater sources in the Nile Delta region, Egypt. The isolates belonged to T1, T2, T3, T4 and T7 genotypes. Al-Herrawy et al. morphologically identified six Acanthamoeba species from 10 different swimming pools in Cairo, Egypt. These species were A. polyphaga, A.castellanii, A. rhysodes, A. mauritaniensis, A. royreba and A. triangularis [2]. Tawfeek et al. detected three isolates belong to T4, T3, T5 from the environmental sources [19]. Hassan et al. morphologically identified Acanthamoeba which was isolated from the hydraulic systems of both hemodialysis and dental units in Alexandria, Egypt [21]. The results of this study highlighted the risk factors associated with this disease endangering our community. Currently, the market of soft CL has been markedly extended worldwide including Egypt, due to many factors as the role of fashion and multimedia. This widespread use was not met by the enough medical knowledge about their proper care and hygiene. In the present study, FRA showed that female sex was a significant risk factor for AK as previously reported [14,22]. Young females used to apply CL to avoid wearing glasses and for cosmetic purposes, especially with available cheap purchases. These subjects missed ophthalmic examination. They did not recognize the importance of regular care until they developed infection [5]. Females are more endangered than males, not only because they are more users for CL but also because most of them usually apply cosmetics as eye mascara. Cosmetics’ pigments coat the surface of CLs; allowing the bacteria to adhere to their surface [23]. On the contrary, Ibrahim et al. [24] revealed that the incidence of females was lower than males suffering from AK. Female sex shouldn’t be taken as a separate factor since both Acanthamoeba and microbial keratitis were more prevalent among the age group ≥21–25 years, especially those with moderate to low educational and SES, leading to lack of awareness about the risks associated with CL wear, as previously reported [14,22]. This was manifested as some strange behaviors like sharing or lending CL for cosmetic purposes. This study revealed that AK was more significant during summer and spring seasons (P = 0.047), in agreement with previous studies [25,26]. Also, the results revealed that prolonged exposure to digital screens was associated with AK. Computer vision syndrome causes dry, irritated eyes with eye strain/fatigue, blurred vision, red and burning eyes with excessive tearing, double vision, and headache [27,28]. In this study, bacteria were isolated from 74.6% of the total examined population, and significantly associated with AK, in agreement with previous studies [16,22,29]. The details of bacterial types and their association with AK were previously investigated by the same authors [29].

95.0% CI for odd ratio Lower

Upper

1.500 2.188 2.036 3.984 0.005 2.829 2.656 1.788

25.686 204.526 119.206 414 0.142 55.354 226.906 34.505

According to FRA, exposure to water sources while wearing CL, through swimming and/or showering, and lack of hand washings was an important risk factor for acquiring AK as previously mentioned [14,16,18]. Also the results agreed with Centers for Disease control and prevention (CDC) and the Food and Drug Administration (FDA) as they sent a strong message about the risk of exposure to water sources on development of AK [30]. In the present study, important water-exposure related risk factor was rinsing the CL cases with tap water in agreement with previous reports [3,16,31]. Especially that Lorenzo-Morales et al. reported that CL and their cases should only be cleaned using the appropriate cleaning system, air dried and stored (best: two-step together) [3]. The results of FRA reported using water coming from tanks as a strong risk factor for acquiring AK in agreement with Kilvington et al. [32]. Water tanks fixed over buildings are highly distributed as a source of water supply in crowded Cairo. Unfortunately, these tanks do not receive enough regular cleaning, and even though, the parasite can survive chlorine used for cleaning. Acanthamoeba cysts are ubiquitously present in the environment. They are resistant to extreme temperature, desiccation and disinfection [16]. On the contrary, Chynn et al. [33] suggested that water exposure may be less important in the development of this disease. High percentage of examined subjects in the present study mistakenly thought that the sodium chloride saline solution (multipurpose solution) had the same efficacy as the cleansing and disinfecting solutions. This was a risk factor to acquire AK according to the FRA, as previously reported [15,16]. This necessitates improving CL users’ knowledge about disinfecting solutions. Rubbing the eyes, while wearing CL, was detected as an additional risk factor. This behavior can cause corneal trauma, injury, and facilitate Acanthamoeba invasion of the corneal epithelium [2,3]. The results of FRA reported sleeping while wearing the CL as a risk factor for AK. Sleeping with closed lids, and the lack of eye and lid movements create the suitable conditions for bacterial infection, especially with the presence of CL [34]. This may predispose the cornea to hypoxia, accumulation of surface antigens on lenses, epithelial edema, and occurrence of microtrauma that may lead to superficial punctate keratitis. Contact lenses may compromise the ocular surface by depriving the corneal epithelium of normal tear flushing and from the non-specific humoral immune mechanisms [35]. Another practice that associated with AK was using older non-sealed eye drops as previously reported [3]. Microbial contamination of eye drops occurs after their use, even for antibiotic solutions, especially if the tips were contaminated, and incidence of contamination varies from 0% to 37%. Eye drops can be used without fear of microbiological contamination for at least six weeks after opening the vial [36]. Also, daily application of CL was associated with higher risk of Acanthamoeba infection than occasional users. These data were considered more reasonable due to increased

Please cite this article in press as: Taher EE, et al. Acanthamoeba keratitis in noncompliant soft contact lenses users: Genotyping and risk factors, a study from Cairo, Egypt. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2017.09.013

G Model JIPH-818; No. of Pages 7

ARTICLE IN PRESS E.E. Taher et al. / Journal of Infection and Public Health xxx (2017) xxx–xxx

possibility of contamination with increased frequency of lenses application and removal which was in agreement with Zimmerman et al. [15]. For this reason, Mahittikorn et al. recommended regular replacement of CL and their cases at least every 3 months. This was of utmost importance to minimize chance of contamination [5]. Moreover, Legarreta et al. reported that even the use of daily disposable lenses is not associated with a lower rate of infection than other lens types [30]. Conclusion Acanthamoeba T4 genotype was the most commonly detected isolate. More studies should be conducted in Egypt to identify the prevalent genotypes of Acanthamoeba. CL users were more exposed to AK and should gain enough health education regarding proper CL hygiene and dangers of tap water exposure. Funding No funding sources. Competing interests None declared. Ethical approval Not required. References [1] dos Santos Gomes T, Magnet A, Izquierdo F, Vaccaro L, Redondo F, Bueno S, et al. Acanthamoeba spp. in contact lenses from healthy individuals from Madrid, Spain. PLoS One 2016;11(4):e0154246. [2] Al-Herrawy A, Bahgat M, Mohammed A-E, Ashour A, Hikal W. Acanthamoeba species in swimming pools of Cairo, Egypt. Iran J Parasitol 2014;9(2):194–201. [3] Lorenzo-Morales J, Khan NA, Walochnik J. An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment. Parasite 2015;22:10. [4] Szczotka-Flynn LB, Pearlman E, Ghannoum M. Microbial contamination of contact lenses, lens care solutions, and their accessories: a literature review. Eye Contact Lens 2010;36(2):116–29. [5] Mahittikorn A, Kittichathanakul T, To-I’m J, Nacapunchai D. Knowledge, behavior, and free-living amoebae contamination of cosmetic contact lens among university wearers in Thailand: a cross-sectional study. Eye Contact Lens 2017;43(2):81–8. [6] Maciver SK, Asif M, Simmern MW, Lorenzo-Morales J. A systematic analysis of Acanthamoeba genotype frequency correlated with source and pathogenicity: T4 is confirmed as a pathogen-rich genotype. Eur J Protistol 2013;49(2):217–21. [7] Corsaro D, Walochnik J, Köhsler M, Rott MB. Acanthamoeba misidentification and multiple labels: redefining genotypes T16, T19, and T20 and proposal for Acanthamoeba micheli sp. Nov. (genotype T19). Parasitol Res 2015;114(7):2481–90. [8] Schroeder JM, Booton GC, Hay J, Niszl IA, Seal DV, Markus MB, et al. Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of Acanthamoebae from humans with keratitis and from sewage sludge. J Clin Microbiol 2001;39(5):1903–11. [9] Booton GC, Kelly DJ, Chu YW, Seal DV, Houang E, Lam DS, et al. 18S ribosomal DNA typing and tracking of Acanthamoeba species isolates from corneal scrape specimens, contact lenses, lens cases, and home water supplies of Acanthamoeba keratitis patients in Hong Kong. J Clin Microbiol 2002;40(5):1621–5. [10] Tice AK, Shadwick LL, Fiore-Donno AM, Geisen S, Kang S, Schuler GA, et al. Expansion of the molecular and morphological diversity of Acanthamoebidae (Centramoebida, Amoebozoa) and identification of a novel life cycle type within the group. Biol Direct 2016;11:69.

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Please cite this article in press as: Taher EE, et al. Acanthamoeba keratitis in noncompliant soft contact lenses users: Genotyping and risk factors, a study from Cairo, Egypt. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2017.09.013

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