Abstract
Background/Aim: Given the characteristics of Serratia marcescens (S. marcescens), this study aimed at investigating its presence in the hands and contact lens cases of orthokeratology wearers, along with the status of bacterial contamination. Patients and Methods: The 39 patients received the questionnaires about the background of orthokeratology and hygiene habits. A total of 39 contact lens cases and 39 hand samples from the patients were collected at Show Chwan Memorial Hospital from June to August in 2020 and sent to National Chung Cheng University for DNA extraction and PCR identification. Results: The results indicated a detection rate of 5.13% for S. marcescens in the contact lens cases and 12.82% in the hand samples. Additionally, 66.67% of contact lens case samples and 30.77% of hand samples found positive for 16s bacterial amplicons. The relationship between hand contamination and the duration of contact lens usage were revealed for both S. marcescens (p=0.021) and 16s bacterial amplicons (p=0.048). Conclusion: The results indicated that hand hygiene is more critical than focusing on contact lens hygiene when it comes to preventing S. marcescens infections. Nevertheless, both proper hand and contact lens hygiene practices can reduce the detection of bacterial eye pathogens, especially a common intestinal bacterium.
Orthokeratology (Ortho-K) plays a crucial role as an overnight-wear contact lens technique for controlling the progression of myopia (1). However, the overnight wearing of Ortho-K lenses may increase the risk of microbial keratitis and corneal ulcer (2, 3). One of the primary risk factors for ocular infections in Ortho-K is non-compliance with hygiene guidelines (4, 5). The contamination of contact lens and their storage containers have been linked to cases of microbial keratitis (5, 6). The cause of contact lens-associated eye disease includes the introduction of pathogens into the eye and corneal hypoxia resulted from long-term wearing of contact lens, especially those made using low oxygen transmissibility material (low DK/t), and it probably leads to an unhealthy epithelium and is an entry point for microorganisms (7, 8). In contact lens case surveys conducted in various countries, the microbial detection rate in storage cases has been found to exceed 50% (9). The most commonly identified bacteria species included Pseudomonas aeruginosa, Staphylococcus spp., and Serratia marcescens (S. marcescens) (8).
S. marcescens, a member of the Enterobacteriaceae family, demonstrates a diverse spectrum of growth capabilities, flourishing in various ecological niches as well as conventional settings (10). It can also be detected in clinical specimens within hospital settings, such as patients’ saliva, urine, feces, and blood samples, and is recognized as an opportunistic pathogen within the human body (11-13). Additionally, S. marcescens has been isolated from patients with conjunctivitis, corneal ulcers, keratitis and endophthalmitis (14). Wearing contact lens and having an abnormal corneal surface have been recognized as risk factors of S. marcescens ocular-related diseases (15). Over the past two decades, there has been a noticeable increase in its identification, making it a frequently encountered gram-negative bacterium in cases of bacterial keratitis (15-17).
Previous epidemiological studies have identified risk factors associated with contact lens-related microbial keratitis, which include overnight wearing (18), infrequent disinfection of contact lenses (19), and inadequate hand and lens case hygiene (20). The presence of pathogenic microorganisms in lens storage cases could significantly elevate the risk of developing infectious keratitis among lens wearers (21). Furthermore, previous literature has highlighted factors that may influence the contamination of contact lens. These factors encompassed contact lens care disinfection systems, the age of the lens case and the replacement schedule, the wearing schedule and modality of contact lens, the wearer’s experience with contact lens, and the habit of hand washing (22). Nevertheless, further studies are needed to reveal the factors that contribute to lens case contamination in subclinical situation.
As indicated above, given the high prevalence of myopia and high utility rate of Ortho-K in Taiwan (23), the aim of this study was to investigate the association between S. marcescens contamination and hygiene compliance in Ortho-K.
Patients and Methods
Study design, inclusion, and exclusion criteria. This prospective cross-sectional study has been approved by the ethics committee and Institutional Review Board (IRB) of the Show Chwan Memorial Hospital (IRB number-1080507) in accordance with the principles of the Helsinki’s Declaration. All participants had provided written informed consents. The inclusion criteria consisted of cases with myopia who had received Ortho-K treatment during the study period. The exclusion criteria included individuals diagnosed with infectious keratitis, those with concurrent ophthalmic diseases such as allergic or inflamed conjunctivitis or orbital cellulitis, or those with wound infection on the hands.
Study participants. In this study, a total of 39 contact lens samples from 39 patients were collected from June to August in 2020 when they return for myopia following up, whereas the samples of 39 dominant hands were conducted by swab in the hospital. The transportation swabs with Cary Blair Medium (CMP co., New Taipei City, Taiwan, ROC) were used for collecting hand and storage case samples. Before collection, a 12′150 plastic bar with a cotton end was moistened with sterile water and then swabbed back and forth over the palm of the subject’s hand for 5 seconds; and another swab was performed in the storage case for the same time. The swabs were then placed back into the transportation tubes for storage. The blank swab was used as the negative control in following experiments. Hand swab was performed upon the patients’ entering the hospital, where the patients were asked to use disinfectant or alcohol for hand sterilization in pandemic period. The age of the patients ranged from 9 to 50. Subjects included 17 male and 22 female participants. The questionnaires collected information about the duration of Ortho-K and lens cases used, the frequency of lens cases change, whether irrigation with saline solution or rewetting drops before taking on the contact lens, and whether using eyedrops after taking off the contact lens. The contact lens cases and hand samples were collected in sterile containers and cryopreserved at Show Chwan Memorial Hospital in Taiwan. Samples were transported within 24 hours following the biosafety and under controlled temperature to the laboratory at National Chung Cheng University.
DNA extraction. The contact lens cases and hand samples were processed with sterile 2 ml 1′ PBS solution to wash and then collected to a 2 mL tube, for centrifuging by 13,000 g for 10 min, finally removed the supernatant for DNA extraction process. Bacterial gDNA was extracted from a pre-treatment sample using QIAamp DNA Microbiome Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. The purity and concentration of the extracted gDNA were accessed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) at 230-280 nm. The gDNA quality was accessed through gel electrophoresis (1.5% gel in Tris-acetate ethylenediaminetetraacetic acid buffer) at 110 V for 30 min, with DNA bands visualized under ultraviolet light. The purified gDNA was subsequently stored at −20°C for further analysis.
PCR identification of S. marcescens and bacteria-DNA. A reference bacterial genomic DNA (S. marcescens ATCC 8100) extracted by the same kit was used as a positive control. After gDNA extraction, the PCR mixture was prepared using 300 μg of gDNA along with each primer set (5’-GGTGAGCTTAATACGTTCATCAA-3’ and 5’-AATTCCGATTAACGCTTGCAC-3’ for S. marcescens; 27F, 5’-AGAGTTTGATCCTGGCTCAG-3’ and 1492R, 5’-TACGGYTA CCTTGTTACGACTT-3’ for 16s bacteria universal PCR) and a master mix reagent (Fast-RunTM Taq Master Mix with Dye). The total PCR reaction volume was 25 μl, then S. marcescens and 16s bacteria universal PCR reaction conditions are similar to the previous typical study (24). A 16s universal full-length PCR was used to detect the presence of bacteria in the samples. However, 16s sequencing analysis on these samples was not performed; it was conducted solely to investigate the presence of bacteria. Ultimately, all PCR products were examined via electrophoresis (110 V, 30 min in 1.5% agarose gel) to verify the presence of gene-specific amplicons. The S. marcescens PCR products were sequenced by Mission Biotech, Taiwan (Applied Biosystems 3730xl DNA Analyzer). The nucleotide sequences were checked using the Basic Local Alignment Search Tool in the National Center for Biotechnology Information gene database.
Statistical analysis. The chi-square test was performed to analyze the correlation among the presence of S. marcescens, bacterial DNAs and the questionnaire using R software (version 4.0.1; R Foundation for Statistical Computing, Vienna, Austria). We defined the statistical significance threshold as p-value less than 0.05.
Results
Demographics of the participants and parameters in questionnaire. The basic and clinical demographics including sex, age, usage period of contact lens and contact lens cases, frequency of contact lens cases renewal, and cleaning habit, were shown in Table I. All samples were collected from 17 male and 22 female Ortho-K patients, the ages of patients ranged from 9 to 50. Twenty-one patients used the Ortho-K over 12 months and most of them have changed their contact lens cases after using them for 3 to 12 months.
Demographics of the participants.
S. marcescens and 16s bacterial amplicons of Ortho-K cases and hand samples. The appearances of S. marcescens and 16s bacterial DNA isolated from contact lens cases and patients’ hands were shown in Table II. The detection rate of S. marcescens were 5.13% (2/39) and 12.82% (5/39) from contact lens cases and hands samples, respectively. The occurrences of bacterial DNA were 66.67% (26/39) and 30.77% (12/39) from contact lens cases and hands samples, respectively.
Detection of S. marcescens and 16s bacterial amplicons of orthokeratology contact lens cases and hand samples.
Correlations between S. marcescens, 16s bacterial amplicons, and hygiene compliance. The results of chi-square tests examining the association between S. marcescens, 16S bacterial amplicons, and various wearing habit parameters are shown in Table III and Table IV. Among these findings, a significant association was observed between the presence of S. marcescens in hand samples and the duration of Ortho-K lenses usage (p=0.021) (Table III). Besides, another significant association was found between the presence of bacterial 16s in hand samples and the duration of Ortho-K lenses usage (p=0.048) and the use of eye drops after taking off the contact lens (p=0.049) (Table IV).
Stratified analysis of relationship between PCR of S. marcescens and hygiene compliance.
Stratified analysis of culture of 16s bacterial amplicons and hygiene compliance.
Discussion
S. marcescens infection is severe complication for contact lens users, particularly those engaged in overnight Ortho-K. Investigating the correlation between pathogens in the contact lens storage environment and potential risk factors is critical for the prevention of microbial keratitis. In the current study, we reported the detection rate of S. marcescens and other bacterium in contact lens cases and hand cultures. Additionally, we demonstrated the impact of hand sterilization on the detection rate of pathogens.
S. marcescens is frequently detected in corneal samples from patients with bacterial keratitis and in the contact lenses of both symptomatic and asymptomatic individuals (25-28). Consequently, the epidemiological investigations of contamination are imperative to prevent severe complications associated with contact lens use, especially in the cases of over-night Ortho-K. Hand contamination may play an important role in contact lens-related infections. Promoting practices of proper hand and contact lens hygiene is highly recommended to mitigate the risk of bacterial keratitis (29). Given that S. marcescens is a common intestinal bacterium, its presence on hands may be linked to personal hygiene habits. Therefore, adhering to proper hand hygiene when wearing contact lenses can serve as an effective and straightforward means to reduce the risk of being infected.
Even the patients were asked for hand sterilization as soon as their entering the hospital, we observed 5 cases (12.82%) remained positive for S. marcescens and 12 cases (30.77%) tested positive for another bacterium (Table II). Besides, our investigation revealed that the positive samples for S. marcescens and other bacterium in Ortho-K cases were 5.13% and 66.67%, respectively. The S. marcescens detection rates were higher in hands samples than contact lens cases samples, while the detection rates of bacterial DNA were higher in contact lens cases samples than hands samples. This finding suggested it has more risk for patients infected with S. marcescens than other bacterium through hands than contact lens storages. Another possible explanation might be that there are other bacteria presenting in greater quantity than Serratia. Besides, while most contact lens care solutions exhibited excellent antibacterial effects, the detection rate of 16s bacteria in contact lens cases in our study was much higher than that on hands (Table II). This discrepancy may be attributed to infrequent replacement of contact lens care solutions or poor cleaning habits, as the majority of positive samples displayed this trend. Bacterial biofilm may be produced in the contact lens cases without regular disinfection, and S. marcescens is more vulnerable to multipurpose contact lens care solutions (30). Therefore, the frequency and carefulness of replacement of contact lens care solutions emerges as an important factor in preventing bacterial keratitis. Further investigations are needed to validate the correlation between contact lens care solutions and pathogen infection.
The findings from the current study indicated that the detection rate of 16s bacteria on hands was much lower than that in contact lens cases (Table II). This difference may be due to the sample collection process, which took place in a hospital setting where hand disinfection is routinely performed upon entry, especially in COVID-19 pandemic period. The results underscore the significance of hand sterilization in reduction pathogen presence and preventing infections. Previous literature has also highlighted the elevated risk of corneal infections associated with extended contact lens wear and improper usage, particularly among inexperienced wearers. In cases of severe keratitis, failure to regularly replace lenses and inadequate hygiene practices contributed to 63% of the population’s susceptibility to fungal and bacterial infections (31).
In the stratified analysis, we identified statistically significant association between hand contamination and the duration of contact lens usage for both S. marcescens (p=0.021) and 16s groups (p=0.048). Noticeably, the highest contamination rate was observed among individuals with a moderate duration (3-12 months) of contact lens usage (Table III and Table IV). Based on these results, we proposed that the detection rate of pathogens is influenced by a patient’s experience with contact lens use and his/her adherence to recommended practices. The contact lens users with good contact practices, awareness of contact lens-related complications and regularly monitoring might prevent visual damage (32). We arranged clinical following up every month in the beginning three months of Ortho-K prescribing to monitor comprehensive situation, such as corneal infection, topography, refraction, and axial length. Closely monitoring probably detect early lesion, and repeated confirmation and reminding of hygiene habit might prevent serious infection. Contact lenses could potentially introduce behavior-related risks into a patient’s eye, even when the patient had received guidance on proper practices from their eye care professionals (33, 34).
Study limitations. Firstly, the sample size of contact lens cases and hand culturing is limited. Furthermore, the recalled bias of questionnaires decreased the quality of study. Finally, the contact lens care solutions were not unified in every patient due to personal selection, and it had variated bactericidal effect against different pathogens (30, 35). Further studies with control groups based on the validated questionnaire are warranted to confirm our results. Particularly, meticulous questionnaire about lens case hygiene (frequency of lens case cleaning, solution or disinfectant type used, solution replacement in storage case wells) and hand hygiene (hand washing before insertion and removal) could help in further understanding of the association between hand hygiene and bacterial contamination of storage cases.
Conclusion
To the best of our knowledge, this study is the first to investigate S. marcescens and 16s bacterial amplicons simultaneously in both contact lens cases and hands of Ortho-K patients. The detection rates of S. marcescens on hand samples are higher than storage case samples suggesting that hand hygiene is more crucial than contact lens case hygiene for preventing S. marcescens infection and associated risks. Ortho-K patients under repeated guidance of professionals tend to be attentive to the hygiene habits. However, hand hygiene might be overlooked without the regular monitoring, causing the increase of pathogens. Nonetheless, maintaining good hand and contact lens case hygiene practices can decrease bacterial eye infections, particularly those caused by common intestinal bacterium.
Acknowledgements
This study is supported by grant from Show Chwan Memorial Hospital to Dr. Chen (RD108021). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.
Footnotes
Authors’ Contributions
Concept and design: Yang PY, Chao SC; data collection and analysis: Chen HC, Chen JS, Yang PY; statistical analysis: Chen JS, Yang PY; Drafting of the manuscript: Chen HC, Chen JS, Bau DT; manuscript checking and discussing: Chen HC, Yang PY, Chen JS, Bau DT, Chao SC.
Conflicts of Interest
All the Authors declare no conflicts of interest regarding this study.
- Received January 25, 2024.
- Revision received March 3, 2024.
- Accepted March 4, 2024.
- Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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