mNGS Analysis of Virus and Colonized Bacteria in Ocular Surface Squamous Neoplasia

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Methods Three patients who were clinically diagnosed with OSSN and underwent ocular surface tumor resection combined with amniotic membrane transplantation from March 1, 2023 to June 1, 2023 were selected as the study group. Specimens were collected during surgery for pathological examination and metagenomic next-generation sequencing (mNGS) of pathogenic microorganisms. Another three patients diagnosed as corneal leukoplakia who were tested negative for viruses by mNGS after penetrating keratoplasty during the same period were selected as the control group. Results Among the 3 OSSN patients, 1 was male and 2 were female, with an average age of 71.3 ± 4.0 (69–76) years. The OSSN in all 3 cases invaded both the cornea and conjunctiva. In this group, mNGS suggested no detection of viral pathogens in the specimens. However, suspected colonized bacteria were detected in the 2 cases of corneal and conjunctival intraepithelial neoplasia (CIN): Propionibacterium acnes, Moraxella osloensis, Pseudomonas fluorescens, Prevotella melaninogenica, and in the 1 case of squamous cell carcinoma of conjunctiva and cornea (SCC): Propionibacterium acnes. Among the 3 cases of corneal leukoplakia in the control group, mNGS indicated no viral pathogens, but detected suspected colonized bacteria of Propionibacterium acnes, Lactobacillus inerta, Staphylococcus hominis, Staphylococcus cohnii, Staphylococcus epidermidis and Escherichia coli. Conclusion The occurrence of OSSN of the patients in this paper was not related to HPV infection. Nevertheless, the increased presence of colonized bacteria of Moraxella osloensis, Pseudomonas fluorescens and Prevotella melaninogenic, as well as the decreased presence of Lactobacillus inerta, Staphylococcus hominis, Staphylococcus cohnii, Staphylococcus epidermidis and Escherichia coli may be the high-risk factors for OSSN. metagenomic next-generation sequencing (mNGS) ocular surface squamous neoplasia (OSSN) colonized bacteria human papillomavirus (HPV) Figures Figure 1 Figure 2 Introduction Ocular surface squamous neoplasia (OSSN), including squamous cell tumor of the conjunctiva and cornea, is the third most prevalent neoplasm affecting the ocular surface, only behind melanoma and lymphoma. Its global incidence is 0.02 to 3.5 cases per 100000 individuals [ 1 ]. In 1995, Lee and Hirst first classified OSSN into two distinct categories: benign and malignant [ 2 ]. Among them, papilloma is the most common benign OSSN and conjunctival intraepithelial neoplasia (CIN) encompasses various degrees of atypical proliferative squamous epithelium and carcinoma in situ, thus making it aptly referred to as a precancerous lesion [ 3 ]. Squamous cell carcinoma (SCC) has been identified as the predominant kind of malignant OSSN [ 4 ]. At present, the etiology of OSSN remains uncertain and its primary risk factors include ultraviolet exposure, human papillomavirus (HPV) or human immunodeficiency virus (HIV) infection [ 5 ]. The comprehension of the correlation between OSSN and viral as well as other pathogenic microbial infections has substantial importance in determining the necessity of suitable anti-pathogen therapy to reduce the risk of recurrence. Human papillomavirus (HPV) is a circular DNA virus that mainly infects epithelial cells of skin and mucous membrane. It is globally recognized as the most prevalent sexually transmitted pathogen [ 6 ]. A comprehensive review of recent years scholarly literature pertaining to the involvement of HPV in OSSN reveals a huge controversy around this topic, which is while some studies have indicated a potential association between HPV infection and the occurrence of OSSN [ 7 , 8 ], some others suggest the opposite [ 9 , 10 ]. Those studies employed conventional HPV detection techniques, such as immunohistochemistry (IHC), polymerase chain reaction (PCR), and in-situ hybridization. The testing methods employed exhibit inherent limitations since they lack the capacity to comprehensively analyze all the pathogens present in OSSN simultaneously. Emerging in recent years, the mNGS technique, however, is a method capable of simultaneously and independently sequencing tens of thousands to millions of DNA fragments [ 11 , 12 ]. Through random high-throughput sequencing techniques of DNA or RNA extracted from the sample, all genomic information in the entire tested specimen can be obtained quickly, efficiently and accurately, so as to analyze pathogenic microorganisms, breaking through the limitation that traditional methods cannot detect multiple pathogenic microorganisms at one time [ 13 ]. Colonized bacteria refer to the bacteria that fall from the external environment to the human body, and settle in certain parts and continue to proliferate and reproduce offspring. When the human body undergoes significant trauma, infection, severe immunodeficiency, and prolonged use of immunosuppressants or glucocorticoids, the colonized bacteria will increase substantially, leading to heightened pathogenicity and the onset of illness in the host [ 14 ]. Studies have shown that ocular colonized bacteria is an important cause of eye infections. Anaerobes, particularly Propionibacterium acnes, have been recognized as one of the important pathogenic bacteria of delayed endophthalmitis after intraocular surgery [ 15 , 16 ]. In Hartikainens et al. study [ 17 ], they analyzed the bacterial culture results of 156 cases of dacryocystitis specimens and found that the positive rate of anaerobic bacteria was 13%. However, whether the distribution characteristics of colonized bacteria will affect the occurrence of OSSN has not been reported in the relevant literature. Therefore, the purpose of this study is to detect the distribution characteristics of pathogenic microorganisms like HPV and ocular colonized bacteria in OSSN and non-OSSN by mNGS, and to explore the relationship between the above pathogens and the occurrence of OSSN. Based on the published literature, this paper is the first report to use mNGS as a detection technique to analyze the distribution characteristics of pathogenic microorganisms like HPV and colonized bacteria in OSSN tissue samples. Methods Patient The was a prospective observational study carried out between March 1, 2023 and June 1, 2023. Under the premise of following the Helsinki Declaration and obtaining informed consent of patients, a total of 3 adult patients (3 eyes) over 18 years old who were diagnosed with OSSN and underwent surgical treatment were included in the study group. In the control group, 3 patients (3 eyes) were diagnosed with corneal leukoplakia and underwent keratoplasty. The patient's medical history information (gender, age, occupation, symptoms, onset time, tumor location, etc.) was systematically collected and evaluated in accordance with the set order of ocular clinical examination. Ophthalmic examination The following examinations were performed: (1) visual acuity and intraocular pressure; (2) slit lamp microscopy and anterior segment photography; (3) anterior segment-optical coherence tomography (AS-OCT); (4) ocular fundus color photography; (5) macular-optical coherence tomography (M-OCT); (6) preoperative blood biochemistry, electrocardiogram, and chest X-ray; (7) pathology examination and mNGS detection for tissues obtained from the surgery. Methods of operation and drug treatment in the study group All patients underwent ocular surface tumor resection combined with amniotic membrane transplantation under general anesthesia. The patient was placed in the supine position and the surgical eye was exposed after standard ophthalmic disinfection and towel laying. Proceeded to open up the eyelid with an eyelid opener and rinse the conjunctival sac with 0.05% iodophor disinfection solution, and then wash with normal saline. Utilized a marker pen to demarcate the resection area about 1mm beside the tumor and then cut the conjunctiva along the marked line while simultaneously cauterizing the scleral nourishing blood vessels beneath the neoplasm to effectively control bleeding. Finally, utilized a cornea lamellar blade to meticulously separate and completely remove the tumor, and scraped clean the corneal wound with a surgery blade. The dimensions of the tumor, specifically its length, breadth, and height, were to be measured in order to determine its size. Subsequently, divided the tumor into two equal halves. A portion of the sample was allocated for pathological examination, while the remaining portion was carefully transferred into a sterile test tube for further mNGS detection. Sutured the conjunctival incision with 10 − 0 nylon surgical suture. Then spread an appropriate size of amniotic membrane out on the surgical wound, and used 10 − 0 nylon surgical suture to continuously suture and fix it on the surface of the cornea at 1mm and 5mm beside the corneal limbus, respectively. One day after the operation, 0.1% tobramycin dexamethasone eye drops (manufactured by Alcon, USA) were applied 4 times a day for one week. The suture and the amniotic membrane were removed a week later, and 0.1% fluorometholone eye drops (manufactured by Alcon, USA) and moxifloxacin hydrochloride eye drops (manufactured by Huarun Zizhu, China) were used 4 times a day respectively. The dosage was halved after 2 weeks, and discontinued after 1 month. Postoperative recurrence criteria in the study group the reappearance and progressive growth of neoplasm in the surgical area. Methods of operation and drug treatment in the control group The control group had penetrating keratoplasty surgery under general anesthesia [ 18 ], when damaged corneal tissues were excised and afterwards tested for mNGS. The results of the mNGS were then compared between the study group and the control group. The recommended treatment starting from the day after the operation involved the administration of 0.1% tobramycin dexamethasone eye drops (manufactured by Alcon, United States) 4 times a day, in addition to 0.1% tacrolimus eye drops (manufactured by Qianshou, Japan) twice daily. Two weeks after that, modified the treatment by substituting the aforementioned eye drops with 0.1% fluorometholone eye drops (manufactured by Alcon, United States) along with moxifloxacin hydrochloride eye drops (manufactured by Huarun Zizhu, China) to be applied 4 times a day, respectively. The dosage was reduced by 50% after 2 weeks. Pathological examination In the study group, the diseased tissues were immediately immersed in 10% neutral formalin buffer solution for 8–12 hours subsequent to the isolation, dehydrated with gradient ethanol, transparentized with xylene, waxed and embedded. The embedded tissue specimens were then cut into 4µm slices, routinely stained with H-E, sealed with neutral gum, and observed under a microscope. The testing indicators utilized in this study were CK5、CK6、P63、P40、P16 and Ki67. mNGS detection Tested the microorganisms in the samples with mNGS, and then compared the results to the nucleic acid sequences of existing microorganisms in the PMDB pathogen database of Huada Biotechnology Co., LTD. (Wuhan) to identify the microorganisms with further analysis. High-throughput sequencing tests is the detection of various nucleic acids in the samples to identify the suspected pathogenic microorganisms, covering 12 types of pathogen detection, including 10989 bacteria (Gram-positive bacteria, Gram-negative bacteria, Mycobacterium), 5050 viruses (single-stranded DNA virus, double-stranded DNA virus, single-stranded RNA virus, double-stranded RNA virus), 1179 fungi, 282 species of parasites and 158 prokaryotes (chlamydia, mycoplasma, rickettsia). The detection process includes sample processing, nucleic acid extraction, genomic library construction, gene sequencing, results analysis, report interpretation, etc. Sample processing and nucleic acid extraction (tissue samples): According to the standard sample collection procedure, the soy-sized tissue blocks were collected and then physically homogenized with 600µL cell lysis buffer and 250µL 0.5mm glass beads. Then 7.2µL Lyticase (RT410-TA, TIANGEN BIOTECH, Beijing, China) was added for enzyme wall-breaking reaction, and 250µL 0.5mm glass beads were added for physical wall-breaking mixed oscillation. Finally 300µL samples were taken to extract nucleic acids according to the instructions of TIANMicrobe magnetic bead pathogenic microbial DNA extraction kit (TIANGEN, NG550-01, China). Genomic library construction and gene sequencing: The extracted nucleic acids were fragmented, end-repaired, adapter-ligated by various enzymes and then amplificated by PCR to build the genomic library. Agilent 2100 Bioanalyzer was used to control the length of the fragments to about 300bp, while Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific Inc.) was used to control the concentration of DNA libraries. Pooled the qualified libraries after detection by equal quality. The libraries after pooling were then to be circularized to form a single-stranded circular structure. Generated by rolling circle amplification (RCA), NDA nanoballs (DNB) were loaded into the sequencing chip and sequenced by BGISEQ-50/MGISEQ-2000 [ 19 ]. Data analysis: After sequencing, low-quality sequences were removed to obtained the high-quality sequences data. With the help of BWA (BWA: http://biobwa.sourceforge.net/ ), the data in the high-quality sequences that were aligned with the human reference genome sequence were removed [ 20 ]. The remaining data were then compared with BGI pathogens metagenomics database (PMDB) after removal of low-complexity reads to obtain the sequence number that could match a certain pathogen, so the possible pathogens could be determined by the sequence number and other clinical examinations. Statistical analysis All summarized data were presented as mean ± standard deviation (SD). No statistical analysis was performed since this was an observational descriptive study. Results Clinical characteristics The three patients, including 1 male and 2 female, at the average age of 71.3 ± 4.0 (69–76) years old, diagnosed with OSSN were all individuals with monocular illness, and the lesions areas were all located in keratoconjunctiva. Please refer to Table 1 for general statistics, family history and other relevant data. The basic conditions of patients with corneal leukoplakia are shown in Table 2 .Ocular symptoms observed in individuals with OSSN included eye redness, foreign body sensation, pain of the eye, and tearing. The occurrence of vision loss could be attributed to the invasion of the central part of the cornea by the tumor. Table 1 Basic information of patients with OSSN Patient Gender Age (years) Course of illness (years) Family History Tumor Basal Diameter (mm) Symptoms Signs 1 Female 76 10 None 13 Foreign-Body Sensation, Tearing, Reduced Vision Pink soft papillary Neoplasm with unclear boundary bulging from the surface of Keratoconjunctiva of the left eye, being unmovable and accompanying with nourishing vessels 2 Female 69 3 None 8 Eye Redness, Photophobia, Pain of the eye, Reduced Vision In the left eye, papillary Neoplasm encircles the corneal limbus by 270° and grows towards the center of it, accompanying with nourishing vessels 3 Male 69 1 None 9 Foreign-Body Sensation, Pain of the eye Photophobia, Tearing Pink soft papillary Neoplasm with clear boundary bulging from the surface of Keratoconjunctiva of the left eye, being unmovable and accompanying with nourishing vessel Table 2 Basic information of patients with Corneal Leukoplakia Patient Gender (years) Age (years) Symptoms Signs 1 Female 58 Vision loss The cornea is grayish white and cloudy with the in-growth of nourishing vessels 2 Female 66 Vision loss The central area of the cornea is white and cloudy, blocking the pupil 3 Female 52 Vision loss The whole layer of the temporal side of the cornea is grayish white and cloudy OSSN showed localized or diffuse pink bulge at the limbus of the cornea, which was papillary or cauliflower-like hyperplasia. The tumor was rich in blood vessels with telangiectasia being visible, which was pine needle-like and invaded the whole superficial layer of the cornea (Fig .1). Postoperative recurrence 2 of the 3 study group patients were followed up during the research period, and there was no recurrence of the tumor. (Fig. 2 ) Pathological Classification Histopathological test results showed that 2 cases of OSSN patients in the study group were CIN and 1 case was squamous cell carcinoma SCC (Fig. 1 ) mNGS examination results Of the 3 cases of OSSN in the study group, mNGS suggested that the specimens were negative for viral pathogens. 2 cases of CIN were detected with suspected colonized organisms: Propionibacterium acnes, Moraxella ossificans, Pseudomonas fluorescens, and Prevotella nigricans, and 1 case of SCC was detected with suspected colonized organisms: Propionibacterium acnes. In the three cases of corneal leukoplakia in the control group, mNGS suggested that no viral pathogens were detected, and suspected colonizing bacteria were detected: Propionibacterium acnes, Lactobacillus inertiae, Staphylococcus mansoni, Staphylococcus kochneri, Staphylococcus epidermidis, and Escherichia coli. Lactobacillus acnes was present in both OSSN and non-OSSN, whereas Moraxella ossificans, Pseudomonas fluorescens, and Prevotella melanogaster were present in OSSN, and Lactobacillus inertiae, Staphylococcus mansoni, Staphylococcus kochneri, Staphylococcus epidermidis, and Escherichia coli were present only in both non-OSSN (Tables 3 and 4 ). Table 3 mNGS detection results of 3 OSSN patients Patient Disease-causing microorganisms Suspected Colonization 1 Not detected Propionibacterium acnes, Moraxella Osloensis, Pseudomonas fluorescens 2 Not detected Propionibacterium acnes, Prevoltella melaninogenica 3 Not detected Lactobacillus acnes Table 4 mNGS detection results for 3 patients with Corneal Leukoplakia Patient Disease-causing microorganisms Suspected Colonization 1 Not detected Propionibacterium acnes, Staphylococcus cohnii 2 Not detected Lactobacillus acnes, Lactobacillus inerta, Staphylococcus hominis 3 Not detected Propionibacterium acnes, Staphylococcus epidermidis, Escherichia coli Discussion OSSN is a relatively rare keratoconjunctival tumor disease. At present, the etiology is still not very clear, but it is very important to clarify the correlation between the occurrence of OSSN and the infection of pathogenic microorganisms. Telling whether the ocular surface of OSSN patients is combined with viral infection has important reference value for deciding whether to choose antiviral drugs to reduce the risk of recurrence and improve the long-term efficacy. Studies have suggested that with the increase of human papillomavirus and human immunodeficiency virus infection rates, the incidence of OSSN may also increase [ 21 ], but there are also other literature suggesting the opposite view. In SCC and CIN, different studies have reported that the HPV infection rates detected in OSSN ranged from 0 to 100%. Tulvatana et al. [ 22 ] used PCR and other methods to perform HPV DNA test on 30 OSSN sample tissues, all of which were negative. Di Girolamo et al. [ 23 ] summarized and compared 34 studies on the correlation between HPV and CIN or squamous cell carcinoma through PubMed, and the results showed that the HPV infection rate was 33.8%. H Carreira et al. [ 24 ] quantified the relationship between HIV and HPV infection and OSSN through systematic review and meta-analysis. The results suggested that HIV was closely related to the increased risk of OSSN (total relative risk = 8.06, 95% confidence interval: 5.29–12.30, 2.25% attributable risk, 56.0% population attributable risk, based on data from 12 studies). The researchers calculated the total relative risk (RR) for mucosal infection with the HPV subtype to be 3.13(95% CI: 1.72–5.71, 1.25–45.6%, based on data from 16 studies). Peter et al. [ 25 ] evaluated the infection rates of human papillomavirus (HPV), Epstein-Barr virus (EBV), Merkel cell polyomavirus (MCPyV), Kaposi 's sarcoma virus (KSV) and adenovirus in 243 OSSN patients by IHC and PCR. Among them, 178 tumor samples were tested positive for EBV, HR-HPV and MCPyV infection by PCR. Notably, EBV exhibited a higher prevalence of 80.3%, while HPV and MCPyV had lower prevalence of 9.0% and 13.5%, respectively. In the retrospective study of Julius et al. [ 26 ], the relationship between HPV infection and the occurrence of OSSN was detected by P16IHC and DNA chip, and HPV infection was detected in 1/3 of tumor samples. In this study, 3 patients with OSSN were all negative for pathogenic microorganisms detected by mNGS. Moreover, the patients in this group were farmers, and the average daily sunlight exposure could reach 6–8 hours. Ultraviolet irradiation may be the main cause of tumor formation. Abnormal ocular microbial flora is considered to be one of the important causes of ophthalmic diseases. More and more cases of infection caused by ocular microbial flora have been reported, including anaerobic bacteria and aerobic bacteria [ 27 ]. After culturing the secretions from 201 patients with meibomian gland dysfunction, Bao Zheng et al. [ 28 ] found that the main strains were Staphylococcus epidermidis (aerobic bacteria) and Propionibacterium acnes (anaerobic bacteria), respectively. In the analysis of 16 cases by Yang et al. [ 29 ], other types of Moraxella played a more important role in keratitis infection, such as Moraxella osloensis. This study compared the mNGS results of both OSSN group and corneal leukoplakia group to explore the differences in the distribution characteristics of ocular surface colonization flora in patients with two diseases, so as to screen the risk factors of OSSN. The results showed that no pathogenic virus was detected in both groups, and among the suspected colonized bacteria, Propionibacterium acnes was positive in both OSSN and corneal leukoplakia specimens, so there was no significant difference between the two groups. Moraxella osloensis, Pseudomonas fluorescens and Prevotella nigrescens only existed in the OSSN group, while Lactobacillus inertis, Staphylococcus hominis, Staphylococcus cohnii, Staphylococcus epidermidis and Escherichia coli only existed in non-OSSN group. It was suggested that the colonization of Moraxella osloensis, Pseudomonas fluorescens and Prevotella nigrescens rather than HPV infection may be the cause of the occurrence and development of OSSN. At present, the standardized treatment for OSSN is surgical resection. In the past 20 years, the treatment of OSSN has changed from simple surgical resection to localized chemotherapy alone or surgical resection combined with localized chemotherapy [ 30 ]. interferonalfa-2b (IFNα-2b) is a glycoprotein produced by recombinant DNA technology, which binds to receptors on the surface of tumor cells to activate effector proteins, resulting in antiviral and anti-tumor effects [ 31 ]. More and more literature have shown that the use of IFNα-2b before and after surgery is more effective in preventing the recurrence of tumor than simple surgical resection, and has fewer side effects than other chemotherapeutic drugs. However, more high-quality clinical studies are still needed to elucidate the efficacy and safety of various drugs under long-term treatment to further improve the postoperative life quality of patients [ 32 ]. In the study conducted by Gu et al. [ 33 ], 43 patients with OSSN underwent simple surgical resection (group A), 16 patients underwent surgical resection and postoperative mitomycin-C treatment (group B), 20 patients underwent surgical resection and postoperative subconjunctival injection of IFNα-2b (group C), postoperative follow-up found that 31 cases (72%) in group A, 5 cases (31%) in group B, and 3 cases (15%) in group C recurred. Nevertheless, the use of eye drops and systemic antiviral drugs may also lead to corresponding drug side effects while increasing the cost of patients. Therefore, if we can accurately determine whether each patient has HPV infection, we can decide whether to give antiviral drugs to avoid the corresponding risk of medication and improve the therapeutic effect. In this study, two patients with CIN and detected negative for virus by mNGS were followed up for 6 months after tumor resection combined with amniotic membrane transplantation, showing good prognosis and no tumor recurrence. conclusion In summary, this article and some published literature suggest that the high incidence of OSSN may not be caused by HPV. The imbalance of ocular surface colonization flora may be a risk factor other than ultraviolet exposure and viral infection. The mNGS detection of ocular viruses and colonized bacteria in OSSN patients is of great significance for rapid diagnosis, guiding perioperative medication, preventing recurrence and improving long-term efficacy. Abbreviations OSSN ocular surface squamous neoplasia HPV human papillomavirus mNGS metagenomic next-generation sequencing CIN corneal and conjunctival intraepithelial neoplasia SCC squamous cell carcinoma of conjunctiva and cornea IHC immunohistochemistry PCR polymerase chain reaction AS-OCT anterior segment-optical coherence tomography M-OCT macular-optical coherence tomography EBV Epstein-Barr virus MCPyV Merkel cell polyomavirus KSV Kaposi 's sarcoma virus IFNα-2b interferonalfa-2b Declarations Acknowledgements The authors thank their department and research team for their help and dedication. Authors’ contributions PW and LG contributed to the study conception and design. Material preparation, data collection and analysis were performed by XY, WW and TZ. The first draft of the manuscript was written by XY and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding This work was supported by the Science and Technology Ministry of China(G2022154028L), National Health Commission of Hubei Province project In 2022WJ2021ZH0005), Subject Construction Foundation of Finance Department of Hubei In 2022(42000022815T000000102), Science and Technology Department of Hubei Province and High level foreign expert project(2022EGD020). Data Availability The data materials were obtained from reasonable request to the corresponding author. All data and materials were unpublished. Competing interests The authors have no relevant financial or non-financial interests to disclose. Ethics approval and consent to participate This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ehics Committee of Affiliated Hospital of Three Gorges University (2023Yj09).Written informed consent was obtained from the patients. Consent for publication Not applicable References Li, Jingwei. 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Bracarda S, Eggermont AMM, Samuelsson J. .Redefining the role of interferon in the treatment of malignant diseases[J]. Eur J Cancer. 2010;46(2):284–97. 10.1016/j.ejca.2009.10.013 . Zhu, Chengfang. Lin Zhirong.Current status of drug therapy for ocular surface squamous cell tumor. Chin J Ocular Trauma Occup Ophthalmopathy. 2021;43(01):72–7. 10.3760/cma.j.cn116022-20200721-00169 . BlasiMA MM. Mitomycin c or interferon as adjuvant therapy to surgery for ocular surface squamous neoplasia: Comparative study[J]. Eur J Ophthalmol. 2018;28(2):204–9. 10.5301/ejo.5001035 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3455936","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":241829647,"identity":"0f8307b7-b394-4903-8065-d6c7c4315e7e","order_by":0,"name":"Xiaoxuan Yang","email":"","orcid":"","institution":"Affiliated Hospital of Three Gorges University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoxuan","middleName":"","lastName":"Yang","suffix":""},{"id":241829648,"identity":"add82b09-2115-43ec-85b4-070184e3ae56","order_by":1,"name":"Wei Wang","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wang","suffix":""},{"id":241829649,"identity":"bc69a1c9-155a-439b-851e-3adb61f6313c","order_by":2,"name":"Tianyu Zhou","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tianyu","middleName":"","lastName":"Zhou","suffix":""},{"id":241829650,"identity":"00aa7e38-c024-44e5-857a-35170d51ba4d","order_by":3,"name":"Ping Wang","email":"","orcid":"","institution":"Affiliated Hospital of Three Gorges University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Wang","suffix":""},{"id":241829651,"identity":"8c74414c-7ef4-451c-acf8-7320063a3cab","order_by":4,"name":"Guigang Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYFACxgYQKcPG3gMVOECkFh42njNAKoEoLRDAwyCRQ6QW+Yjk5hc/d9Ty8Em+PSb58weDHN+NBMbPBXi0GN5IbLPsPXOch006L02aJ4HBWPJGArP0DHxaZiS2GfC2HQNqyTGTBjosccONBDZmHgJaDP+CtEieMZP8kcBQT1CLvERi82PethoeNgkeMwmgwxIMCGkx4HnYxizbdgAYyDnG1jxpEoYzzzxslsZrS3v6449v2+rk5NvPGN78YWMjz3c8+eBnvLYcYGCTYGA4DOMD2dDIxW1LAwPzBwaGOryKRsEoGAWjYIQDAEC+R4DMLiqPAAAAAElFTkSuQmCC","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Guigang","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2023-10-17 06:29:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3455936/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3455936/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":45172074,"identity":"93d92591-28f4-4b82-89df-a9adfe6d8bbd","added_by":"auto","created_at":"2023-10-24 18:05:08","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":257784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA1\u003c/strong\u003e A 76-year-old female patient. The visual acuity was recorded as light perception positive (LP+). \u003cstrong\u003eA2\u003c/strong\u003eThe postoperative condition one day following the surgical removal of the ocular mass , in conjunction with the transplantation of amniotic membrane. \u003cstrong\u003eA3 \u003c/strong\u003eThe amniotic membrane suture was observed to be removed 7 days post-operation. Furthermore, the lens was shown to have turbidity. The individual has a visual acuity of HM/10cm and has been diagnosed with corneal leukomalacia and cataract. \u003cstrong\u003eA4\u003c/strong\u003e HE staining, a microscopic examination revealed the presence of squamous epithelial papilloma with localized moderate-severe atypical hyperplasia. \u003cstrong\u003eA5\u003c/strong\u003e Displaying densely organized epithelial cells. This observation indicated the presence of high-grade CIN of the left eye. \u003cstrong\u003eB1 \u003c/strong\u003eA 69-year-old female, The visual acuity was measured at 0.03. \u003cstrong\u003eB2\u003c/strong\u003e Demonstrated the presence of a blood clot underneath the amniotic membrane subsequent to the surgical removal of a mass , which was followed by an amniotic membrane transplantation. \u003cstrong\u003eB3 \u003c/strong\u003eThe image depicted the removal of the amniotic membrane 7 days post-operation . The measured visual acuity was recorded as 0.03. \u003cstrong\u003eB4\u003c/strong\u003e and \u003cstrong\u003eB5\u003c/strong\u003e Revealed the presence of coated squamous epithelium. Additionally, the local squamous epithelium exhibited significant atypical hyperplasia, indicating the presence of high-grade CIN of the left eye. \u003cstrong\u003eC1\u003c/strong\u003e A 69-year-old male patientin . The patient's visual acuity was measured at 0.3. \u003cstrong\u003eC2\u003c/strong\u003e Transplantation of amniotic membrane in the left eye. \u003cstrong\u003eC3\u003c/strong\u003e Illustrated the extraction of the amniotic sutures one week after the surgical procedure. The visual acuity was measured to be 0.3. \u003cstrong\u003eC4\u003c/strong\u003e and \u003cstrong\u003eC5\u003c/strong\u003e heterogeneous squamous epithelial hyperplasia, characterized by localized basement membrane incompleteness and irregular tumor cell nests infiltrating and proliferating in the mesenchyme, indicated the likelihood of SCC.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3455936/v1/c675b0ee0bee6eb9740983d8.jpeg"},{"id":45172075,"identity":"8d2b5c9e-0c4c-4dee-9d21-5c8a29c5dcc7","added_by":"auto","created_at":"2023-10-24 18:05:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":972193,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA1\u003c/strong\u003e, The 2nd patient, 68 years old, female, CIN of the left corneal conjunctiva. 6 months postoperative ocular appearance images: corneal transparency, no tumor recurrence, temporal conjunctival scar formation. Visual acuity: 0.3. \u003cstrong\u003eB1\u003c/strong\u003e, \u003cstrong\u003eC1\u003c/strong\u003e, \u003cstrong\u003eD1\u003c/strong\u003e, \u003cstrong\u003eE1\u003c/strong\u003e, AS-OCT showed smooth surface of corneal epithelium, well healing, and normal corneal thickness in all quadrants (“衸” was micrometer in the image). \u003cstrong\u003eA2\u003c/strong\u003e, \u003cstrong\u003eB2\u003c/strong\u003e, Patient 3, 69 years old, female, SCC in the left eye. ocular appearance image at 5 months postoperatively: temporal conjunctiva was seen as neoplastic growth and encroachment of the rim of the cornea, cornea was clear, and no tumor recurrence was seen in the preoperative site. No recurrence sign of the tumor. Visual acuity: 0.4 (no improvement in visual acuity after optometry), \u003cstrong\u003eC2\u003c/strong\u003e, \u003cstrong\u003eD2\u003c/strong\u003e, AS-OCT showed smooth corneal epithelial surface with well healing and normal corneal thickness.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3455936/v1/f014c63ea86cfed1053b26f1.png"},{"id":54356905,"identity":"9cdefced-6d1f-4d00-a0e0-2413e8d12492","added_by":"auto","created_at":"2024-04-09 10:05:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1555319,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3455936/v1/dc1b4ddb-64c2-4744-96e0-b5a7dbad041b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"mNGS Analysis of Virus and Colonized Bacteria in Ocular Surface Squamous Neoplasia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOcular surface squamous neoplasia (OSSN), including squamous cell tumor of the conjunctiva and cornea, is the third most prevalent neoplasm affecting the ocular surface, only behind melanoma and lymphoma. Its global incidence is 0.02 to 3.5 cases per 100000 individuals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In 1995, Lee and Hirst first classified OSSN into two distinct categories: benign and malignant [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among them, papilloma is the most common benign OSSN and conjunctival intraepithelial neoplasia (CIN) encompasses various degrees of atypical proliferative squamous epithelium and carcinoma in situ, thus making it aptly referred to as a precancerous lesion [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Squamous cell carcinoma (SCC) has been identified as the predominant kind of malignant OSSN [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. At present, the etiology of OSSN remains uncertain and its primary risk factors include ultraviolet exposure, human papillomavirus (HPV) or human immunodeficiency virus (HIV) infection [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The comprehension of the correlation between OSSN and viral as well as other pathogenic microbial infections has substantial importance in determining the necessity of suitable anti-pathogen therapy to reduce the risk of recurrence. Human papillomavirus (HPV) is a circular DNA virus that mainly infects epithelial cells of skin and mucous membrane. It is globally recognized as the most prevalent sexually transmitted pathogen [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. A comprehensive review of recent years scholarly literature pertaining to the involvement of HPV in OSSN reveals a huge controversy around this topic, which is while some studies have indicated a potential association between HPV infection and the occurrence of OSSN [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], some others suggest the opposite [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Those studies employed conventional HPV detection techniques, such as immunohistochemistry (IHC), polymerase chain reaction (PCR), and in-situ hybridization. The testing methods employed exhibit inherent limitations since they lack the capacity to comprehensively analyze all the pathogens present in OSSN simultaneously. Emerging in recent years, the mNGS technique, however, is a method capable of simultaneously and independently sequencing tens of thousands to millions of DNA fragments [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Through random high-throughput sequencing techniques of DNA or RNA extracted from the sample, all genomic information in the entire tested specimen can be obtained quickly, efficiently and accurately, so as to analyze pathogenic microorganisms, breaking through the limitation that traditional methods cannot detect multiple pathogenic microorganisms at one time [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eColonized bacteria refer to the bacteria that fall from the external environment to the human body, and settle in certain parts and continue to proliferate and reproduce offspring. When the human body undergoes significant trauma, infection, severe immunodeficiency, and prolonged use of immunosuppressants or glucocorticoids, the colonized bacteria will increase substantially, leading to heightened pathogenicity and the onset of illness in the host [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Studies have shown that ocular colonized bacteria is an important cause of eye infections. Anaerobes, particularly Propionibacterium acnes, have been recognized as one of the important pathogenic bacteria of delayed endophthalmitis after intraocular surgery [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In Hartikainens et al. study [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], they analyzed the bacterial culture results of 156 cases of dacryocystitis specimens and found that the positive rate of anaerobic bacteria was 13%. However, whether the distribution characteristics of colonized bacteria will affect the occurrence of OSSN has not been reported in the relevant literature.\u003c/p\u003e \u003cp\u003eTherefore, the purpose of this study is to detect the distribution characteristics of pathogenic microorganisms like HPV and ocular colonized bacteria in OSSN and non-OSSN by mNGS, and to explore the relationship between the above pathogens and the occurrence of OSSN. Based on the published literature, this paper is the first report to use mNGS as a detection technique to analyze the distribution characteristics of pathogenic microorganisms like HPV and colonized bacteria in OSSN tissue samples.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePatient\u003c/h2\u003e\n \u003cp\u003eThe was a prospective observational study carried out between March 1, 2023 and June 1, 2023. Under the premise of following the Helsinki Declaration and obtaining informed consent of patients, a total of 3 adult patients (3 eyes) over 18 years old who were diagnosed with OSSN and underwent surgical treatment were included in the study group. In the control group, 3 patients (3 eyes) were diagnosed with corneal leukoplakia and underwent keratoplasty. The patient\u0026apos;s medical history information (gender, age, occupation, symptoms, onset time, tumor location, etc.) was systematically collected and evaluated in accordance with the set order of ocular clinical examination.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eOphthalmic examination\u003c/h2\u003e\n \u003cp\u003eThe following examinations were performed: (1) visual acuity and intraocular pressure; (2) slit lamp microscopy and anterior segment photography; (3) anterior segment-optical coherence tomography (AS-OCT); (4) ocular fundus color photography; (5) macular-optical coherence tomography (M-OCT); (6) preoperative blood biochemistry, electrocardiogram, and chest X-ray; (7) pathology examination and mNGS detection for tissues obtained from the surgery.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eMethods of operation and drug treatment in the study group\u003c/h2\u003e\n \u003cp\u003eAll patients underwent ocular surface tumor resection combined with amniotic membrane transplantation under general anesthesia. The patient was placed in the supine position and the surgical eye was exposed after standard ophthalmic disinfection and towel laying. Proceeded to open up the eyelid with an eyelid opener and rinse the conjunctival sac with 0.05% iodophor disinfection solution, and then wash with normal saline. Utilized a marker pen to demarcate the resection area about 1mm beside the tumor and then cut the conjunctiva along the marked line while simultaneously cauterizing the scleral nourishing blood vessels beneath the neoplasm to effectively control bleeding. Finally, utilized a cornea lamellar blade to meticulously separate and completely remove the tumor, and scraped clean the corneal wound with a surgery blade. The dimensions of the tumor, specifically its length, breadth, and height, were to be measured in order to determine its size. Subsequently, divided the tumor into two equal halves. A portion of the sample was allocated for pathological examination, while the remaining portion was carefully transferred into a sterile test tube for further mNGS detection. Sutured the conjunctival incision with 10\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon surgical suture. Then spread an appropriate size of amniotic membrane out on the surgical wound, and used 10\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon surgical suture to continuously suture and fix it on the surface of the cornea at 1mm and 5mm beside the corneal limbus, respectively. One day after the operation, 0.1% tobramycin dexamethasone eye drops (manufactured by Alcon, USA) were applied 4 times a day for one week. The suture and the amniotic membrane were removed a week later, and 0.1% fluorometholone eye drops (manufactured by Alcon, USA) and moxifloxacin hydrochloride eye drops (manufactured by Huarun Zizhu, China) were used 4 times a day respectively. The dosage was halved after 2 weeks, and discontinued after 1 month.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003ePostoperative recurrence criteria in the study group\u003c/h2\u003e\n \u003cp\u003ethe reappearance and progressive growth of neoplasm in the surgical area.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eMethods of operation and drug treatment in the control group\u003c/h2\u003e\n \u003cp\u003eThe control group had penetrating keratoplasty surgery under general anesthesia [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e], when damaged corneal tissues were excised and afterwards tested for mNGS. The results of the mNGS were then compared between the study group and the control group. The recommended treatment starting from the day after the operation involved the administration of 0.1% tobramycin dexamethasone eye drops (manufactured by Alcon, United States) 4 times a day, in addition to 0.1% tacrolimus eye drops (manufactured by Qianshou, Japan) twice daily. Two weeks after that, modified the treatment by substituting the aforementioned eye drops with 0.1% fluorometholone eye drops (manufactured by Alcon, United States) along with moxifloxacin hydrochloride eye drops (manufactured by Huarun Zizhu, China) to be applied 4 times a day, respectively. The dosage was reduced by 50% after 2 weeks.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003ePathological examination\u003c/h2\u003e\n \u003cp\u003eIn the study group, the diseased tissues were immediately immersed in 10% neutral formalin buffer solution for 8\u0026ndash;12 hours subsequent to the isolation, dehydrated with gradient ethanol, transparentized with xylene, waxed and embedded. The embedded tissue specimens were then cut into 4\u0026micro;m slices, routinely stained with H-E, sealed with neutral gum, and observed under a microscope. The testing indicators utilized in this study were CK5、CK6、P63、P40、P16 and Ki67.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003emNGS detection\u003c/h2\u003e\n \u003cp\u003eTested the microorganisms in the samples with mNGS, and then compared the results to the nucleic acid sequences of existing microorganisms in the PMDB pathogen database of Huada Biotechnology Co., LTD. (Wuhan) to identify the microorganisms with further analysis. High-throughput sequencing tests is the detection of various nucleic acids in the samples to identify the suspected pathogenic microorganisms, covering 12 types of pathogen detection, including 10989 bacteria (Gram-positive bacteria, Gram-negative bacteria, Mycobacterium), 5050 viruses (single-stranded DNA virus, double-stranded DNA virus, single-stranded RNA virus, double-stranded RNA virus), 1179 fungi, 282 species of parasites and 158 prokaryotes (chlamydia, mycoplasma, rickettsia). The detection process includes sample processing, nucleic acid extraction, genomic library construction, gene sequencing, results analysis, report interpretation, etc.\u003c/p\u003e\n \u003cp\u003eSample processing and nucleic acid extraction (tissue samples): According to the standard sample collection procedure, the soy-sized tissue blocks were collected and then physically homogenized with 600\u0026micro;L cell lysis buffer and 250\u0026micro;L 0.5mm glass beads. Then 7.2\u0026micro;L Lyticase (RT410-TA, TIANGEN BIOTECH, Beijing, China) was added for enzyme wall-breaking reaction, and 250\u0026micro;L 0.5mm glass beads were added for physical wall-breaking mixed oscillation. Finally 300\u0026micro;L samples were taken to extract nucleic acids according to the instructions of TIANMicrobe magnetic bead pathogenic microbial DNA extraction kit (TIANGEN, NG550-01, China).\u003c/p\u003e\n \u003cp\u003eGenomic library construction and gene sequencing: The extracted nucleic acids were fragmented, end-repaired, adapter-ligated by various enzymes and then amplificated by PCR to build the genomic library. Agilent 2100 Bioanalyzer was used to control the length of the fragments to about 300bp, while Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific Inc.) was used to control the concentration of DNA libraries. Pooled the qualified libraries after detection by equal quality. The libraries after pooling were then to be circularized to form a single-stranded circular structure. Generated by rolling circle amplification (RCA), NDA nanoballs (DNB) were loaded into the sequencing chip and sequenced by BGISEQ-50/MGISEQ-2000 [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eData analysis: After sequencing, low-quality sequences were removed to obtained the high-quality sequences data. With the help of BWA (BWA: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://biobwa.sourceforge.net/\u003c/span\u003e\u003c/span\u003e), the data in the high-quality sequences that were aligned with the human reference genome sequence were removed [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. The remaining data were then compared with BGI pathogens metagenomics database (PMDB) after removal of low-complexity reads to obtain the sequence number that could match a certain pathogen, so the possible pathogens could be determined by the sequence number and other clinical examinations.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eAll summarized data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). No statistical analysis was performed since this was an observational descriptive study.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eClinical characteristics\u003c/h2\u003e\n \u003cp\u003eThe three patients, including 1 male and 2 female, at the average age of 71.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0 (69\u0026ndash;76) years old, diagnosed with OSSN were all individuals with monocular illness, and the lesions areas were all located in keratoconjunctiva. Please refer to Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e for general statistics, family history and other relevant data. The basic conditions of patients with corneal leukoplakia are shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.Ocular symptoms observed in individuals with OSSN included eye redness, foreign body sensation, pain of the eye, and tearing. The occurrence of vision loss could be attributed to the invasion of the central part of the cornea by the tumor.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eBasic information of patients with OSSN\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePatient\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003cp\u003e(years)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCourse\u003c/p\u003e\n \u003cp\u003eof illness (years)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFamily History\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTumor Basal Diameter (mm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSymptoms\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSigns\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eForeign-Body Sensation, Tearing, Reduced Vision\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePink soft papillary Neoplasm\u003c/p\u003e\n \u003cp\u003ewith unclear boundary bulging\u003c/p\u003e\n \u003cp\u003efrom the surface of\u003c/p\u003e\n \u003cp\u003eKeratoconjunctiva of the left eye,\u003c/p\u003e\n \u003cp\u003ebeing unmovable and\u003c/p\u003e\n \u003cp\u003eaccompanying with nourishing vessels\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEye Redness, Photophobia, Pain of the eye,\u003c/p\u003e\n \u003cp\u003eReduced Vision\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIn the left eye, papillary Neoplasm\u003c/p\u003e\n \u003cp\u003eencircles the corneal limbus by 270\u0026deg; and grows towards the center of it,\u003c/p\u003e\n \u003cp\u003eaccompanying with nourishing vessels\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eForeign-Body Sensation,\u003c/p\u003e\n \u003cp\u003ePain of the eye Photophobia, Tearing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePink soft papillary Neoplasm\u003c/p\u003e\n \u003cp\u003ewith clear boundary\u003c/p\u003e\n \u003cp\u003ebulging from the surface of Keratoconjunctiva of the left eye,\u003c/p\u003e\n \u003cp\u003ebeing unmovable and\u003c/p\u003e\n \u003cp\u003eaccompanying with nourishing vessel\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eBasic information of patients with Corneal Leukoplakia\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePatient\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003cp\u003e(years)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003cp\u003e(years)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSymptoms\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSigns\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVision loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThe cornea is grayish white and cloudy with the in-growth of nourishing vessels\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVision loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThe central area of the cornea is white and cloudy, blocking the pupil\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVision loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThe whole layer of the temporal side of the cornea is grayish white and cloudy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eOSSN showed localized or diffuse pink bulge at the limbus of the cornea, which was papillary or cauliflower-like hyperplasia. The tumor was rich in blood vessels with telangiectasia being visible, which was pine needle-like and invaded the whole superficial layer of the cornea (Fig .1).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003ePostoperative recurrence\u003c/h2\u003e\n \u003cp\u003e2 of the 3 study group patients were followed up during the research period, and there was no recurrence of the tumor. (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003ePathological Classification\u003c/h2\u003e\n \u003cp\u003eHistopathological test results showed that 2 cases of OSSN patients in the study group were CIN and 1 case was squamous cell carcinoma SCC (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003emNGS examination results\u003c/h2\u003e\n \u003cp\u003eOf the 3 cases of OSSN in the study group, mNGS suggested that the specimens were negative for viral pathogens. 2 cases of CIN were detected with suspected colonized organisms: Propionibacterium acnes, Moraxella ossificans, Pseudomonas fluorescens, and Prevotella nigricans, and 1 case of SCC was detected with suspected colonized organisms: Propionibacterium acnes. In the three cases of corneal leukoplakia in the control group, mNGS suggested that no viral pathogens were detected, and suspected colonizing bacteria were detected: Propionibacterium acnes, Lactobacillus inertiae, Staphylococcus mansoni, Staphylococcus kochneri, Staphylococcus epidermidis, and Escherichia coli. Lactobacillus acnes was present in both OSSN and non-OSSN, whereas Moraxella ossificans, Pseudomonas fluorescens, and Prevotella melanogaster were present in OSSN, and Lactobacillus inertiae, Staphylococcus mansoni, Staphylococcus kochneri, Staphylococcus epidermidis, and Escherichia coli were present only in both non-OSSN (Tables \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003emNGS detection results of 3 OSSN patients\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePatient\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDisease-causing microorganisms\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSuspected Colonization\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNot detected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePropionibacterium acnes,\u003c/p\u003e\n \u003cp\u003eMoraxella Osloensis,\u003c/p\u003e\n \u003cp\u003ePseudomonas fluorescens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNot detected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePropionibacterium acnes,\u003c/p\u003e\n \u003cp\u003ePrevoltella melaninogenica\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNot detected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLactobacillus acnes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003emNGS detection results for 3 patients with Corneal Leukoplakia\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePatient\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDisease-causing microorganisms\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSuspected Colonization\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNot detected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePropionibacterium acnes,\u003c/p\u003e\n \u003cp\u003eStaphylococcus cohnii\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNot detected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLactobacillus acnes,\u003c/p\u003e\n \u003cp\u003eLactobacillus inerta,\u003c/p\u003e\n \u003cp\u003eStaphylococcus hominis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNot detected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePropionibacterium acnes,\u003c/p\u003e\n \u003cp\u003eStaphylococcus epidermidis,\u003c/p\u003e\n \u003cp\u003eEscherichia coli\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOSSN is a relatively rare keratoconjunctival tumor disease. At present, the etiology is still not very clear, but it is very important to clarify the correlation between the occurrence of OSSN and the infection of pathogenic microorganisms. Telling whether the ocular surface of OSSN patients is combined with viral infection has important reference value for deciding whether to choose antiviral drugs to reduce the risk of recurrence and improve the long-term efficacy.\u003c/p\u003e \u003cp\u003eStudies have suggested that with the increase of human papillomavirus and human immunodeficiency virus infection rates, the incidence of OSSN may also increase [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], but there are also other literature suggesting the opposite view. In SCC and CIN, different studies have reported that the HPV infection rates detected in OSSN ranged from 0 to 100%. Tulvatana et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] used PCR and other methods to perform HPV DNA test on 30 OSSN sample tissues, all of which were negative. Di Girolamo et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] summarized and compared 34 studies on the correlation between HPV and CIN or squamous cell carcinoma through PubMed, and the results showed that the HPV infection rate was 33.8%. H Carreira et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] quantified the relationship between HIV and HPV infection and OSSN through systematic review and meta-analysis. The results suggested that HIV was closely related to the increased risk of OSSN (total relative risk\u0026thinsp;=\u0026thinsp;8.06, 95% confidence interval: 5.29\u0026ndash;12.30, 2.25% attributable risk, 56.0% population attributable risk, based on data from 12 studies). The researchers calculated the total relative risk (RR) for mucosal infection with the HPV subtype to be 3.13(95% CI: 1.72\u0026ndash;5.71, 1.25\u0026ndash;45.6%, based on data from 16 studies). Peter et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] evaluated the infection rates of human papillomavirus (HPV), Epstein-Barr virus (EBV), Merkel cell polyomavirus (MCPyV), Kaposi 's sarcoma virus (KSV) and adenovirus in 243 OSSN patients by IHC and PCR. Among them, 178 tumor samples were tested positive for EBV, HR-HPV and MCPyV infection by PCR. Notably, EBV exhibited a higher prevalence of 80.3%, while HPV and MCPyV had lower prevalence of 9.0% and 13.5%, respectively. In the retrospective study of Julius et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], the relationship between HPV infection and the occurrence of OSSN was detected by P16IHC and DNA chip, and HPV infection was detected in 1/3 of tumor samples. In this study, 3 patients with OSSN were all negative for pathogenic microorganisms detected by mNGS. Moreover, the patients in this group were farmers, and the average daily sunlight exposure could reach 6\u0026ndash;8 hours. Ultraviolet irradiation may be the main cause of tumor formation.\u003c/p\u003e \u003cp\u003eAbnormal ocular microbial flora is considered to be one of the important causes of ophthalmic diseases. More and more cases of infection caused by ocular microbial flora have been reported, including anaerobic bacteria and aerobic bacteria [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. After culturing the secretions from 201 patients with meibomian gland dysfunction, Bao Zheng et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] found that the main strains were Staphylococcus epidermidis (aerobic bacteria) and Propionibacterium acnes (anaerobic bacteria), respectively. In the analysis of 16 cases by Yang et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], other types of Moraxella played a more important role in keratitis infection, such as Moraxella osloensis. This study compared the mNGS results of both OSSN group and corneal leukoplakia group to explore the differences in the distribution characteristics of ocular surface colonization flora in patients with two diseases, so as to screen the risk factors of OSSN. The results showed that no pathogenic virus was detected in both groups, and among the suspected colonized bacteria, Propionibacterium acnes was positive in both OSSN and corneal leukoplakia specimens, so there was no significant difference between the two groups. Moraxella osloensis, Pseudomonas fluorescens and Prevotella nigrescens only existed in the OSSN group, while Lactobacillus inertis, Staphylococcus hominis, Staphylococcus cohnii, Staphylococcus epidermidis and Escherichia coli only existed in non-OSSN group. It was suggested that the colonization of Moraxella osloensis, Pseudomonas fluorescens and Prevotella nigrescens rather than HPV infection may be the cause of the occurrence and development of OSSN.\u003c/p\u003e \u003cp\u003eAt present, the standardized treatment for OSSN is surgical resection. In the past 20 years, the treatment of OSSN has changed from simple surgical resection to localized chemotherapy alone or surgical resection combined with localized chemotherapy [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. interferonalfa-2b (IFNα-2b) is a glycoprotein produced by recombinant DNA technology, which binds to receptors on the surface of tumor cells to activate effector proteins, resulting in antiviral and anti-tumor effects [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. More and more literature have shown that the use of IFNα-2b before and after surgery is more effective in preventing the recurrence of tumor than simple surgical resection, and has fewer side effects than other chemotherapeutic drugs. However, more high-quality clinical studies are still needed to elucidate the efficacy and safety of various drugs under long-term treatment to further improve the postoperative life quality of patients [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In the study conducted by Gu et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], 43 patients with OSSN underwent simple surgical resection (group A), 16 patients underwent surgical resection and postoperative mitomycin-C treatment (group B), 20 patients underwent surgical resection and postoperative subconjunctival injection of IFNα-2b (group C), postoperative follow-up found that 31 cases (72%) in group A, 5 cases (31%) in group B, and 3 cases (15%) in group C recurred. Nevertheless, the use of eye drops and systemic antiviral drugs may also lead to corresponding drug side effects while increasing the cost of patients. Therefore, if we can accurately determine whether each patient has HPV infection, we can decide whether to give antiviral drugs to avoid the corresponding risk of medication and improve the therapeutic effect. In this study, two patients with CIN and detected negative for virus by mNGS were followed up for 6 months after tumor resection combined with amniotic membrane transplantation, showing good prognosis and no tumor recurrence.\u003c/p\u003e"},{"header":"conclusion","content":"\u003cp\u003eIn summary, this article and some published literature suggest that the high incidence of OSSN may not be caused by HPV. The imbalance of ocular surface colonization flora may be a risk factor other than ultraviolet exposure and viral infection. The mNGS detection of ocular viruses and colonized bacteria in OSSN patients is of great significance for rapid diagnosis, guiding perioperative medication, preventing recurrence and improving long-term efficacy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eOSSN ocular surface squamous neoplasia\u003c/p\u003e\n\u003cp\u003eHPV human papillomavirus\u003c/p\u003e\n\u003cp\u003emNGS metagenomic next-generation sequencing\u003c/p\u003e\n\u003cp\u003eCIN corneal and conjunctival intraepithelial neoplasia\u003c/p\u003e\n\u003cp\u003eSCC squamous cell carcinoma of conjunctiva and cornea\u003c/p\u003e\n\u003cp\u003eIHC immunohistochemistry\u003c/p\u003e\n\u003cp\u003ePCR polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eAS-OCT anterior segment-optical coherence tomography \u003c/p\u003e\n\u003cp\u003eM-OCT macular-optical coherence tomography \u003c/p\u003e\n\u003cp\u003eEBV Epstein-Barr virus\u003c/p\u003e\n\u003cp\u003eMCPyV Merkel cell polyomavirus \u003c/p\u003e\n\u003cp\u003eKSV Kaposi \u0026apos;s sarcoma virus \u003c/p\u003e\n\u003cp\u003eIFN\u0026alpha;-2b interferonalfa-2b \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe authors thank their department and research team for their help and dedication.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026rsquo; contributions\u003c/p\u003e\n\u003cp\u003ePW and LG contributed to the study conception and design. Material preparation, data collection and analysis were performed by XY, WW and TZ. The first draft of the manuscript was written by XY and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Science and Technology Ministry of China(G2022154028L), National Health Commission of Hubei Province project In 2022WJ2021ZH0005), Subject Construction Foundation of Finance Department of Hubei In 2022(42000022815T000000102), Science and Technology Department of Hubei Province and High level foreign expert project(2022EGD020).\u003c/p\u003e\n\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eThe data materials were obtained from reasonable request to the corresponding author. All data and materials were unpublished.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ehics Committee of Affiliated Hospital of Three Gorges University (2023Yj09).Written informed consent was obtained from the patients.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi, Jingwei. Yang Yanning.Ocular surface squamous epithelial tumor research progress [J]. International journal of ophthalmology, 2018, 18 (6): 4. 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Eur J Ophthalmol. 2018;28(2):204\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.5301/ejo.5001035\u003c/span\u003e\u003cspan address=\"10.5301/ejo.5001035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"metagenomic next-generation sequencing (mNGS), ocular surface squamous neoplasia (OSSN), colonized bacteria, human papillomavirus (HPV)","lastPublishedDoi":"10.21203/rs.3.rs-3455936/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3455936/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eTo analyze the correlation between the onset of ocular surface squamous neoplasia (OSSN) and viral infections such as human papillomavirus (HPV), as well as ocular surface colonized pathogenic microorganisms.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThree patients who were clinically diagnosed with OSSN and underwent ocular surface tumor resection combined with amniotic membrane transplantation from March 1, 2023 to June 1, 2023 were selected as the study group. Specimens were collected during surgery for pathological examination and metagenomic next-generation sequencing (mNGS) of pathogenic microorganisms. Another three patients diagnosed as corneal leukoplakia who were tested negative for viruses by mNGS after penetrating keratoplasty during the same period were selected as the control group.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAmong the 3 OSSN patients, 1 was male and 2 were female, with an average age of 71.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0 (69\u0026ndash;76) years. The OSSN in all 3 cases invaded both the cornea and conjunctiva. In this group, mNGS suggested no detection of viral pathogens in the specimens. However, suspected colonized bacteria were detected in the 2 cases of corneal and conjunctival intraepithelial neoplasia (CIN): Propionibacterium acnes, Moraxella osloensis, Pseudomonas fluorescens, Prevotella melaninogenica, and in the 1 case of squamous cell carcinoma of conjunctiva and cornea (SCC): Propionibacterium acnes. Among the 3 cases of corneal leukoplakia in the control group, mNGS indicated no viral pathogens, but detected suspected colonized bacteria of Propionibacterium acnes, Lactobacillus inerta, Staphylococcus hominis, Staphylococcus cohnii, Staphylococcus epidermidis and Escherichia coli.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe occurrence of OSSN of the patients in this paper was not related to HPV infection. Nevertheless, the increased presence of colonized bacteria of Moraxella osloensis, Pseudomonas fluorescens and Prevotella melaninogenic, as well as the decreased presence of Lactobacillus inerta, Staphylococcus hominis, Staphylococcus cohnii, Staphylococcus epidermidis and Escherichia coli may be the high-risk factors for OSSN.\u003c/p\u003e","manuscriptTitle":"mNGS Analysis of Virus and Colonized Bacteria in Ocular Surface Squamous Neoplasia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-24 18:05:03","doi":"10.21203/rs.3.rs-3455936/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"61d2756b-9364-4bf7-90d0-d9a19a29bca9","owner":[],"postedDate":"October 24th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-09T09:57:14+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-24 18:05:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3455936","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3455936","identity":"rs-3455936","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}