Clinical Performance of Nanopore Targeted Sequencing for Diagnosing Endophthalmitis

Clinical Performance of Nanopore Targeted Sequencing for Diagnosing Endophthalmitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Clinical Performance of Nanopore Targeted Sequencing for Diagnosing Endophthalmitis Dalan Jing, Xiaodan Jiang, Xiaotong Ren, Ran Hao, Jie Su, Xuemin Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6292219/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jul, 2025 Read the published version in BMC Microbiology → Version 1 posted 13 You are reading this latest preprint version Abstract Purpose Rapid identification of pathogenic bacteria in the vitreous and/or aqueous humor of patients with acute clinical diagnosis of endophthalmitis via nanopore sequencing technology. Methods We recruited a total of 12 patients (12 eyes) who were diagnosed with endophthalmitis at an ophthalmic outpatient clinic from January 2022 to October 2022. Clinical evaluation is conducted in the order of consultation, symptom evaluation, physical sign evaluation, and ophthalmic special examination, all of which are completed by the same experienced clinical physician. Finally, 19 aqueous humor and/or vitreous samples were obtained via anterior chamber wash, vitreous tap and vitrectomy. The samples were separated for cultivation, biochemical drug sensitivity identification, and targeted nanopore sequencing (NTS), and the results of nanopore sequencing were validated via Sanger sequencing. Results In patients with endophthalmitis, NTS can identify infected pathogens within 8–12 hours. Six samples (31.6%) were subjected to culture-based diagnosis, while NTS revealed the presence of pathogenic microorganisms in 19 samples (100%), of which bacteria and fungi were detected in three samples. A total of 19 samples were subjected to Sanger sequencing, of which 16 (84.2%) tested positive, including 6 culture-positive samples and 10 culture-negative samples, of which 5 (26.3%) were positive for two bacterial genera. In culture-positive cases, there is a high-quality match between culture and targeted nanopore sequencing. Conclusions NTS can quickly detect pathogenic bacteria in samples from patients with endophthalmitis. Moreover, the use of vitreous and/or aqueous humor for the NTS has potential. NTS is a promising diagnostic platform for endophthalmitis, especially for mixed infections and culture-negative cases. endophthalmitis microbiome 16S rRNA nanopore sequencing infectious pathogens Figures Figure 1 Figure 2 Figure 3 Introduction Endophthalmitis is one of the most destructive eye infections and may lead to irreversible blindness of infected eyes within hours or days after symptoms appear[ 1 ]. Endophthalmitis is a bacterial or fungal infection of the vitreous and/or aqueous humor[ 2 ]. Most infection routes are exogenous, including eye surgery, penetration of ocular trauma, or the spread of corneal infection, and a few are endogenous and result from hematogenous seeding of the eye by bacteria or fungi[ 3 ]. The incidence of endophthalmitis varies with different causes; cataract surgery is the most common cause, with an incidence ranging from 0.066–0.125% [ 4 ]. Endophthalmitis is a clinical diagnosis supported by vitreous and/or aqueous humor culture or blood culture, although 30% of case culture results are negative [ 2 ]. Multiple factors contribute to the high negative culture rate, including overdiagnosis, a limited sample size, the inability of some bacteria to be cultured in the laboratory, and the use of antibiotic therapy[ 5 ]. Traditional bacterial culture is still the gold standard for the diagnosis of endophthalmitis. The identification of the pathogen takes up to 5 days. Sometimes, 2 to 4 weeks are needed for correct sporulation and pathogen identification[ 6 ]. New methods of molecular microbiology have overcome these difficulties[ 7 ]. DNA sequencing can be broadly classified into two techniques: targeted amplicon sequencing and untargeted whole-genome sequencing (WGS). An example of a sequence of targeted amplifiers is the determination gene of the universal 16S bacterial ribosomal RNA (rRNA) of the amplicon. The first method to sequence DNA was developed by Sanger in 1975[ 8 ]; this method has low throughput and high cost, but it is still the gold standard for sequencing. Pathogen identification through next-generation sequencing (NGS) has been increasingly used in microbial research and clinical diagnosis. NGS can rapidly detect all the different pathogens present in a clinical sample in a single assay, which requires 2 days to 3 days, which is shorter than the turnaround time of routine culture[ 9 ]. Owing to the limitations of the Illumina sequencing platform, which involves short reads (500 base pairs), only part of the 16S rRNA gene can be sequenced, thus limiting the taxonomic resolution to genus-level classification[ 10 ]. Metagenomic NGS techniques provide better taxonomic resolution at the species level, but their sensitivity depends heavily on the background level and is vulnerable to contamination by environmental species [ 11 ]. Simultaneously, it requires greater sequencing depth, leading to higher costs[ 12 ]. Even in cases of severe endophthalmitis caused by virulent pathogens, prompt therapy may save useful vision. The nanopore-targeted sequencing (NTS) method is capable of generating long-read lengths (>5,000 bp) and performing targeted amplification (16S RNA gene for bacteria and ITS for fungi), which do not interfere with the host background DNA[ 13 , 14 ]. Nanopore sequencing enhances our ability to study complex microbial samples by using inexpensive and portable technologies to conduct real-time long-read sequencing[ 12 ]. In addition, it has a short turnaround time of 8–14 h in pleural fluid, ascites fluid, and bronchoalveolar lavage fluid detection[ 15 ]. This method has important advantages, such as the low concentration of bacteria and the small sample size used for endophthalmitis. In recent years, many preliminary studies on amplicon-based nanopore sequencing for endophthalmitis have been reported[ 16 – 18 ]. Through nanopore amplicon sequencing, bacterial and fungal pathogens associated with infectious endophthalmitis can be identified, which is more sensitive and has a faster processing time than conventional culture. In addition, Hao et al. recently reported similar results in patients with endogenous endophthalmitis[ 19 ]. The clinical manifestations of NTS in endophthalmitis patients have not been systematically compared with those of traditional culture analysis, PCR and Sanger sequencing. Therefore, we evaluated the feasibility of NTS in acute endophthalmitis by comparing culture with PCR and Sanger sequencing. Materials and methods Participants This prospective study enrolled patients who presented to the Department of Ophthalmology of Peking University Third Hospital between January 2022 and October 2022. Our study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of the Peking University Third Hospital. (S2021222). The participants provided written informed consent before the intervention. Twelve patients who were clinically presumed to have infectious endophthalmitis (bacterial or fungal) and who were prepared for intravitreal drug injection or pars plana vitrectomy (PPV) were enrolled. Clinical details, including demographic information, predisposing factors, previous treatment, cause and duration of symptoms, and surgical interventions, were also collected. Specimens Aqueous and/or vitreous samples (at least 100 µL) were obtained via three methods: anterior chamber wash, vitreous tap and PPV. All the samples were aseptically divided into two equal amounts without dilution and sent immediately for routine microbiological processing, and the remaining samples for genomic analysis were snap frozen, stored at − 80°C, and transported on dry ice to the laboratories. Conventional bacterial culture The samples were incubated on blood agar and chocolate agar plates at 35°C for 48 hours. When a fungal infection was strongly suspected, additional culture was performed on Sabouraud’s dextrose agar (SDA) plates at 30°C with no CO 2 for 3 weeks. Bacterial or fungal identification was performed from the positive culture plate via standard biochemical tests via a VITEK 2 Compact system (bioMe´rieux, France) . Nanopore-targeted sequencing NTS was conducted according to the method described by Fu et al.[ 15 ]. DNA was extracted via the Sansure DNA Extraction Kit (Changsha, China) following the manufacturer’s instructions. The barcoded amplification products of the 16S rRNA gene, ITS1/2, and rpoB from the same samples were pooled at a mass ratio of 10:3:1. The pooled products from the different samples were equally mixed and used to construct sequencing libraries via a 1D Ligation Kit (SQK-LSK109; Oxford Nanopore). The library was sequenced via Oxford Nanopore GridION X5 with real-time base calling enabled (ont-guppy-for-gridion v. 1.4.3-1 and v. 3.0.3-1; high-accuracy base calling mode)[ 20 ]. TE buffer was used as a negative control in each batch. The criteria for positive results of NTS bacterial or fungal identification were as follows: a map reading of the bacterial species in the sample > 100 or greater than that of any other species and a ratio of the map reading in the sample to the negative control > 10. If the fungal species level was greater than 20 or greater than 50% of the relative abundance, the ratio of the fungal spectrum reading in the sample to the negative control was greater than 10. Important lists of clinically known typical or potentially pathogenic bacteria and fungi are available in the clinical guidelines and literature[ 21 ]. Sanger Sequencing DNA samples were extracted from residual clinical samples. The PCR products were purified and sequenced via an ABI3730 genetic analyser (Beijing Genomics Institute, Beijing, China). After the Bellerophon server was used to evaluate the chimerism formation of the product[ 22 ], the 16S rRNA gene sequences were compared with the GenBank database to search for related sequences via the BLAST program. Statistical analysis All analyses were performed via SPSS version 23.0 software. Only descriptive statistics were used. Treatment and follow-up The treatment plan is selected according to the initially assumed infection type and adjusted according to the NTS and/or culture results. The fixed follow-up time was set at 1 day and 1 week after the operation. Results Patient demographics Nineteen samples from 12 patients were used in the study, 10 of which were vitreous humor samples and 9 were aqueous humor samples. The patients had a median age of 64.3 (range 48–86) years, and 7 were male (58.3%). The predisposing conditions were previous cataract surgery or previous bleb reconstruction surgery (10 patients), trauma (1 patient) and endogenous spread (1 patient). All patients did not undergo prior intraocular antimicrobial therapy before specimen collection. Four patients underwent PPV in the first phase, whereas the remaining 8 patients only underwent intravitreal injection of vancomycin and ceftazidime in the first phase. The majority of the patients presented with visual acuity worse than 20/200 (83.3%) (Table 1). Table 1 Clinical and demographic details of the patients with suspected infectious endophthalmitis ID Sex Age (years) Laterality Clinical diagnosis Predisposing factor Systemic disease Initial VA Treatment Final VA (1 week) 1 M 73 right Postoperative endophthalmitis Cataract surgery: 12 days None CF IOAI + V + C HM 2 M 64 left Postoperative endophthalmitis Bleb reconstruction: 10 days HTN LP PPV + Bleb reconstruction + V + C CF 3 F 86 left Postoperative endophthalmitis Cataract surgery: 2 days HTN, DM HM IOAI + V + C LP 4 F 59 right Postoperative endophthalmitis Cataract surgery: 6 days None 20/200 IOAI + V + C 20/80 5 M 48 left Exogenous endophthalmitis Perforation: steel wire 5days None HM PPV + V + C HM 6 F 57 right Postoperative endophthalmitis Cataract surgery: 10 days DM CF IOAI + V + C CF 7 M 84 left Postoperative endophthalmitis Cataract surgery: 9 days HTN 20/100 IOAI + V + C 20/100 8 M 66 left Postoperative endophthalmitis Cataract surgery: 2 days None HM IOAI + V + C CF 9 F 69 left Postoperative endophthalmitis Cataract surgery: 3 days HTN, hypothyroidism CF IOAI + V + C HM 10 M 52 right Postoperative endophthalmitis Cataract surgery: 4 days None CF IOAI + V + C CF 11 M 68 left Endogenous endophthalmitis Liver abscess (postoperative findings) None NPL PPL + PPV + silicone oil + V + C NPL 12 F 73 left Postoperative endophthalmitis Cataract surgery: 6 days HTN, DM LP PPV + silicone oil + V + C LP Abbreviations: C, ceftazidime; CF, counting fingers; DM:diabetes mellitus; F, female; HM, hand movements; HTP:hypertension; IOAI, intraocular antibiotic injection; LP, light perception; M, male; NPL, no light perception; PPL, pars plana lensectomy; PPV, pars plana vitrectomy; V, vancomycin; VA, visual acuity. Microbiology culture Six samples (31.6%) were positive by microbial culture, of which all 6 samples grew bacteria and none grew fungi. Four samples were positive for S. epidermidis . The remaining three culture-positive samples were positive for Enterococcus faecalis , Sphingomonas oligokinetica , and Klebsiella pneumoniae . Further details are provided in Table 2. Table 2 Identification results of culture-positive samples ID Sample code Sample type Bacterial culture Sanger sequencing NTS 1 2 vitreous body Staphylococcus epidermidis S. epidermidis S. Epidermidis, Corynebacterium Macginleyi, Malassezia restricta, Penicillium citrinum 3 4 vitreous body Enterococcus faecalis E. Faecalis, Oceanobacillus oncorhynchi E. Faecalis 5 7 aqueous humor S. epidermidis S. Epidermidis, Phyllobacterium myrsinacearum S. Epidermidis, Rhodococcus qingshengii 8 vitreous body S. epidermidis S. Epidermidis, P. myrsinacearum S. Epidermidis, Staphylococcus capitis 8 12 aqueous humor Sphingomonas paucimobilis Streptococcus pneumoniae, Lysinibacillus macroides S. Pneumoniae, Streptococcus pseudopneumoniae 11 18 vitreous body Klebsiella pneumoniae K. pneumoniae K. Pneumoniae, Klebsiella variicola Abbreviations: NTS, nanopore targeted sequencing; Nanopore - targeted sequencing NTS detected the presence of microorganisms in 19 samples out of 12 participants (positive rate of 100%), including 6 culture-positive samples and 13 culture-negative samples. In addition to bacteria, fungi, including Restrictive Malassezia , Aspergillus fumigatus , and Penicillium citri , were also detected in 3 samples (Table 2). More bacterial genera and species can be detected in the NTS. Figure 1 displayegd the relative abundances of the bacterial taxa (≥5%) obtained via NTS. Apart from the vitreous sample of code 4, where no microorganisms were detected, and the vitreous sample of code 3, where only Enterococcus faecalis (monomicrobial infection) was detected, two or more bacterial species were found in all 17 samples. The first two bacteria and fungi, on the basis of their abundance, are displayed in Table 2 and 3. By combining the NTS results and generating a heatmap of all the samples, the taxonomic abundance of bacterial species detected in 19 samples (including positive cultures and negative cultures) of presumed infectious endophthalmitis was predominantly gram-positive, as shown in Figure 2. The time range confirmed by the NTS is 8-12 hours. Notably, the type of endophthalmitis in patient 11, who only had a history of retrobulbar injection of triamcinolone acetonide, could not be confirmed. Diagnostic vitrectomy was performed on the patient. The vitreous body was subjected to corresponding testing. NTS first reported results within 9 hours, indicating K. pneumoniae infection. The patient was screened for diabetes and examined by liver and gallbladder ultrasound. The results showed that there were multiple nodules and abscesses in the liver. Table 3 Identification results of culture-negative samples ID Sample code Sample type Bacterial culture Sanger sequencing NTS 1 1 aqueous humor negative negative Staphylococcus epidermidis, Corynebacterium McKinley, Restrictive malassezia, Aspergillus fumigatus 2 3 vitreous body negative negative C. McKinley, Klebsiella aerogenes , R. malassezia 4 5 aqueous humor negative Bacillus.sp Bacillus fumarioli, Halomonas boliviensis 6 vitreous body negative Bacillus.sp, Phyllobacterium.sp Rhodococcus qingshengii , bacillus 6 9 aqueous humor negative Phyllobacterium myrsinacearum Delftia acidovorans, Lawsonella clevelandensis 10 vitreous body negative P.myrsinacearum D. acidovorans 7 11 aqueous humor negative P.myrsinacearum Aquabacterium parvum, Halomonas mongoliensis 8 13 vitreous body negative Streptococcus pneumoniae H.mongoliensis, A. parvum 9 14 aqueous humor negative Staphylococcus aureus, P. myrsinacearum A. parvum, H.mongoliensis 10 15 aqueous humor negative Bacillus.sp Pseudomonas pseudoalcaligenes, H.mongoliensis 16 vitreous body negative negative Sphingomonas oligophenolica, H.mongoliensis 11 17 aqueous humor negative Sphingomonas. sp S. oligophenolica, D. acidovorans 12 19 vitreous body negative Bacillus. sp 绿色芽孢杆菌 Abbreviations: NTS, nanopore targeted sequencing; Comparison of the consistency among the three detection results All 19 samples were subjected to bacterial culture, Sanger sequencing, and NTS. The consistency of the three identification results was compared (Figure 3). Biochemical analysis of bacterial cultures: A total of 4 genera and 4 species of bacteria were isolated. Sanger sequencing microbial community types: A total of 9 genera and 8 species of bacteria were isolated. NTS analysis of bacterial community types: A total of 29 genera and 33 species of bacteria were isolated. The consistency of the three methods at the level of dominant bacterial genera was 5/19 cases (26.3%). A total of 6 positive samples were cultured, of which 5 were consistent with Sanger sequencing (83.3%) and 5 were consistent with NTS (83.3%) (Figure 3). Sixteen samples were positive by Sanger sequencing, and 10 samples were consistent with NTS (62.5%) (Figure 3). Discussion Endophthalmitis is a serious eye infection that may cause permanent loss of useful vision in the affected eye[1]. It can occur following ocular surgery or trauma, as well as through the hematogenous spread of microorganisms from endogenous infection[23]. However, bacteremia or mycosis may be temporary, and patients may experience symptoms of systemic infection [1]. Almost all patients with endophthalmitis exhibit decreased vision, and some patients also experience eye pain. Eye examination usually reveals hypopyon and intraocular inflammation[1]. Traditional cultivation and identification are the gold standards for detecting endophthalmitis, but the positive rate is only 40% and is prone to false negative results[24]. Molecular diagnostic techniques, including culturing negative cases, have been used in the laboratory to identify endophthalmitis pathogens and provide the possibility of rapid diagnosis. These technologies play a key role in the diagnosis of endophthalmitis [25]. Since its first application in ophthalmology in 1993, PCR has shown promising prospects in the treatment of endophthalmitis, increasing the detection rate and shortening the diagnosis time. However, owing to differences in amplification efficiency among different primer groups and limitations in the number of fluorescent markers, the number of fungi and/or bacteria that can be detected simultaneously is limited [7, 26, 27]. Shotgun metagenomics sequences are all derived from the DNA provided by a sample, which theoretically allows for hypothesis-free detection of all types of microorganisms (including rare pathogens) with fast turnover times [28]. It can identify multiple pathogens through a single test and can even be applied to samples after antibiotic treatment [29]. Moreover, amplicon sequencing (based on amplicon metagenomics) amplifies the target DNA of interest by performing PCR before sequencing. Amplicon-based metagenomics enriches target pathogen DNA by reducing the range of detectable pathogens, which is sufficient to classify and analyse pathogens [30, 31]. This method is particularly suitable for detecting low concentrations of bacteria and fungi from clinical samples containing a large amount of host DNA [32]. A previous study revealed good consistency between culture results and NGS results in culture-positive cases; even in culture-negative cases, NGS can identify additional bacteria [33]. Because amplicon sequencing analyses only the target sequence, it is easy to interpret the results. PCR helps to improve sensitivity, ultimately increasing the diagnostic yield [34]. In addition, nanopore sequencing provides a fast library preparation protocol and enables real-time analysis, making it suitable for rapid analysis of clinical samples [35, 36]. In addition, nanopore sequencing can be used to process one sample at a time, while other sequencing platforms, such as Illumina, need to process multiple samples in a batch to achieve economic feasibility, which may have adverse effects on the turnover time of each sample in rare diseases such as endophthalmitis [37, 38]. Through nanopore sequencing, bacterial and fungal pathogens associated with infectious endophthalmitis can be identified, which has higher sensitivity and faster processing time than conventional culture. Previous preliminary studies on targeted nanopore sequencing for endophthalmitis have utilized PCR primers to amplify specific target regions of interest, such as 16S rRNA in bacteria or ITS in fungi. Jun et al. [16] identified pathogens in 5 cases of bacterial endophthalmitis and 3 cases of fungal endophthalmitis via 16S and ITS nanopore amplicon sequencing. Similarly, Huang et al. [17] identified 17 pathogens in 18 cases of endophthalmitis via 16S, ITS, and rpoB gene nanopore amplicon sequencing. In addition, in 8 culture-negative samples and in 1 sample that could not be cultured, nanopore sequencing detected bacteria, fungi, or a mixture of bacteria and fungi in the intraocular fluid. To evaluate the role of nanopore sequencing in identifying potential pathogenic pathogens of endophthalmitis, Low et al. [18] compared the culture results with those of NTS, whole-genome nanopore sequencing, and Illumina sequencing. They reported that nanopore sequencing identified the same bacterial species in all culture-positive cases and identified potential pathogens in two culture-negative cases. Whole-genome nanopores also detected the presence of bacteriophages in three samples. In the past, clinical applications for detecting bacterial and fungal pathogens in clinical samples focused mainly on nanopore metagenomic sequencing, which is characterized by relatively high cost and requires a large amount of bioinformatics processing [39-41]. NTS was used to amplify marker genes and sequence the amplified products via a nanopore sequencing platform, which was developed for the diagnosis of bacterial or fungal infections with lower costs and bioinformatics processing. However, the clinical manifestations of NTS in specimens of infectious endophthalmitis have not been studied compared with traditional culture experiments and Sanger sequencing after PCR in terms of cohorts and systems. Here, we evaluated the feasibility of NTS by comparing culture, PCR, and Sanger sequencing. To achieve greater clinical efficacy in metagenomic sequencing, the following characteristics are necessary: fast turnaround time, low cost, and high sensitivity. From this perspective, NTS is a suitable method for detecting endophthalmitis in samples. This study included a total of 19 samples from 12 patients, of which 10 were vitreous samples and 9 were aqueous humor samples. Among them, 7 were males (58.3%), and 5 were females (41.7%), with more male patients than female patients. Six patients had systemic diseases (50%). The most common triggering factors were previous cataract surgery or follicular reconstruction surgery (10 patients). Understanding the risk factors for endophthalmitis is crucial for its prevention. For postoperative endophthalmitis after cataract surgery, risk factors include advanced age (80 years or older), immune dysfunction secondary to systemic disease, the presence of septic lesions in and around the eye, posterior capsule rupture (PCR), and wound leakage. Lundstrom et al.[42] found that nonuse of anterior chamber antibiotics, contact with the vitreous body, and age over 85 years were the three most common risk factors. In another large-scale epidemiological study by Jabbarvand et al. [43], diabetes, advanced age (over 80 years), conventional large-incision extracapsular cataract extraction and perioperative contact with the vitreous body were significantly associated with an increased risk of endophthalmitis. In addition to these traditional risk factors, the emerging risk factor is cataract surgery in patients who previously received intravitreal injection. In our study, with the exception of surgery-related endophthalmitis, the remaining cases included trauma (1 patient) and endogenous diffusion (1 patient). After penetrating eye injury, 3–10% of patients experience endophthalmitis, although early surgical repair and prophylactic systemic application of antibiotics may reduce the incidence rate to <1%[44]. The risk factors for endophthalmitis include metal foreign body trauma, intraocular foreign body retention, lens rupture, and an initial repair time greater than 24 hours. Endogenous endophthalmitis is most commonly observed in patients with systemic diseases or immune dysfunction, such as diabetes and hepatobiliary diseases [45]. The incidence rate is 0.04%-0.4%, and related risk factors include intravenous medication, diabetes, low immune function, malignant tumors, long hospitalization or intravenous antibiotics. The risk factor for patients with endogenous endophthalmitis in this study was liver abscess. A total of 19 samples were included in the microbial culture, of which 6 samples (31.6%) were culture positive. All 6 samples exhibited bacterial growth, and none of the samples exhibited fungal growth. In contrast, genome sequencing is not limited to a specific species but can detect all different microorganisms present in clinical samples in a single trial. A total of 19 samples were included in the NTS, 19 of which tested positive (100%), including 6 culture-positive samples and 13 culture-negative samples. In addition to bacteria, fungi, including Malassezia restrictive , Aspergillus fumigatus , and Penicillium citri , were also detected in 3 samples. Gene sequencing technology can improve traditional microbial detection and identify microorganisms that were previously unrelated to endophthalmitis [46]. Genomic sequencing has advantages in identifying pathogens of eye infections, potentially improving diagnostic accuracy and selecting appropriate treatment protocols. Biochemical analysis of bacterial cultures: A total of 4 genera and 4 species of bacteria were isolated. Sanger sequencing revealed a total of 9 genera and 8 species of bacteria. NTS analysis of microbial community types: A total of 29 genera and 33 species of bacteria were isolated. The consistency of the three methods in detecting dominant bacterial genera was 5/19 cases (26.3%). A total of 6 positive samples were cultured, of which 5 were consistent with Sanger sequencing (83.3%), 5 were consistent with NTS (83.3%), 16 samples were positive with Sanger sequencing, and 10 samples were consistent with NTS (62.5%). In summary, these results indicate that NTS can provide useful information for identifying pathogens related to endophthalmitis. The positive rate of NTS is significantly greater than that of bacterial culture, as it has high sensitivity even under low microbial loads. Previously, the use of antibiotics had an impact on the diagnostic positivity of traditional culture methods, but nanopore sequencing was not affected because bacterial DNA can be detected even after being killed by antibiotics. In this study, the results of NTS and Sanger sequencing were highly consistent with those of traditional culture. Similar to previous studies [16, 17], using NTS, we detected more than one type of microorganism in some patients, some of whom did not grow in culture. Possible reasons include competition among microorganisms, cultivation conditions, differences in growth rates, and quorum sensing. The multimicrobial infection of endophthalmitis is also a challenging diagnostic and therapeutic situation. The NTS in this study helped to detect a mixture of bacteria and fungi in some patients, which was completely overlooked in the culture. Similar to previous studies [47, 48], we detected multiple microorganisms in some patients. Multi microbial eye infections are not only a challenge in identifying two or more microorganisms, but also in developing appropriate antimicrobial treatments. If fungal infections coexist, consideration should be given to intravitreal antifungal drugs and antibiotics, but personalized decisions should be made based on medical history and clinical examination. There were several limitations to this study. First, the number of patients included was small, and a single center was used. The diagnostic value of the NTS cannot be evaluated via conventional indicators such as sensitivity and specificity. The severity of endophthalmitis may vary among different patients, and the pathogen load may affect the detection results. The etiology, underlying diseases, and treatment methods of different patients may vary, which may affect the analysis of prognostic factors. Therefore, more cases of different severity levels are needed to evaluate the practical performance of NTS in distinguishing the pathogenic agents of endophthalmitis. In addition, owing to its targeted nucleic acid amplification characteristics, NTS still has several drawbacks. Potential contamination may occur during sample preparation and transportation. Therefore, sample quality should be strictly controlled, and in some cases, it may be necessary to repeatedly send samples from the same patient for testing. The limitations of current research on mixed infections of multiple bacteria include retrospective and lack of clear prospective treatment plans. Conclusion Through systematic comparisons with traditional culture assays and PCR followed by Sanger sequencing retrospectively, NTS undoubtedly has unparalleled advantages in accurately detecting the pathogenic bacteria of infectious endophthalmitis, especially for culture-negative and mixed infection cases. NTS is expected to provide an effective pathway for timely and effective identification of endophthalmitis-infected bacteria in clinical practice. Abbreviations NTS, targeted nanopore sequencing; WGS, whole-genome sequencing; rRNA: ribosomal RNA; NGS: next-generation sequencing ; PPV: pars plana vitrectomy; SDA :Sabouraud’s dextrose agar; C, ceftazidime; CF, counting fingers; DM:diabetes mellitus; F, female; HM, hand movements; HTP:hypertension; IOAI, intraocular antibiotic injection; LP, light perception; M, male; NPL, no light perception; PPL, pars plana lensectomy; PPV, pars plana vitrectomy; V, vancomycin; VA, visual acuity; PCR: posterior capsule rupture. Declarations Ethics approval and consent to participate ： Our study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of the Peking University Third Hospital. (S2021222). Written informed consent was obtained from all study participants. Consent for publication ： Not Applicable. Availability of data and materials ： The datasets generated and/or analysed during the current study are available in the NCBI database and are accessible at PRJNA1247725. Competing Interests : All authors declare that they have no conflict of interest. Funding ： The publication of this paper were supported by a grant from Peking University Third Hospital Key Talent Project foundation (Grant no.BYSYZD2021044) and Shandong Provincial Natural Science Foundation (Grant no. ZR2024QH310). The sponsor or funding organisation had no role in the design or conduct of this research. Authors' contributions : All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this article and take responsibility for the integrity of the work as a whole. Dalan Jing: research design, data acquisition, data analysis, and manuscript preparation. Xiaodan Jiang: research design, manuscript modification. Xiaotong Ren: manuscript modification. 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Low L, Nakamichi K, Akileswaran L, Lee CS, Lee AY, Moussa G, Murray PI, Wallace GR, Van Gelder RN, Rauz S: Deep Metagenomic Sequencing for Endophthalmitis Pathogen Detection Using a Nanopore Platform . AM J OPHTHALMOL 2022, 242 :243-251. Hao X, Wang M, Yuan M, Zhang R, Jin W, Yang A: IDENTIFICATION OF PATHOGENS IN THE INTRAOCULAR FLUID SAMPLES OF PATIENTS WITH ENDOGENOUS ENDOPHTHALMITIS USING RAPID NANOPORE TARGETED SEQUENCING . RETINA-J RET VIT DIS 2023, 43 (4):606-615. Bolognini D, Bartalucci N, Mingrino A, Vannucchi AM, Magi A: NanoR: A user-friendly R package to analyze and compare nanopore sequencing data . PLOS ONE 2019, 14 (5):e216471. Miao Q, Ma Y, Wang Q, Pan J, Zhang Y, Jin W, Yao Y, Su Y, Huang Y, Wang M et al : Microbiological Diagnostic Performance of Metagenomic Next-generation Sequencing When Applied to Clinical Practice . CLIN INFECT DIS 2018, 67 (suppl_2):S231-S240. Huber T, Faulkner G, Hugenholtz P: Bellerophon: a program to detect chimeric sequences in multiple sequence alignments . BIOINFORMATICS 2004, 20 (14):2317-2319. Chen KJ, Sun MH, Hou CH, Chen HC, Chen YP, Wang NK, Liu L, Wu WC, Chou HD, Kang EY et al : Susceptibility of bacterial endophthalmitis isolates to vancomycin, ceftazidime, and amikacin . SCI REP-UK 2021, 11 (1):15878. Xu S, Zhou C, Zhang P, Feng C, Zhang T, Sun Z, Zhuang H, Chen H, Chang Q, Jiang R et al : Diagnostic Performance of MALDI-TOF MS Compared to Conventional Microbiological Cultures in Patients with Suspected Endophthalmitis . OCUL IMMUNOL INFLAMM 2020, 28 (3):483-490. Zhu J, Xia H, Tang R, Ng TK, Yao F, Liao X, Zhang Q, Ke X, Shi T, Chen H: METAGENOMIC NEXT-GENERATION SEQUENCING DETECTS PATHOGENS IN ENDOPHTHALMITIS PATIENTS . RETINA-J RET VIT DIS 2022, 42 (5):992-1000. Kosacki J, Boisset S, Maurin M, Cornut PL, Thuret G, Hubanova R, Vandenesch F, Carricajo A, Aptel F, Chiquet C: Specific PCR and Quantitative Real-Time PCR in Ocular Samples from Acute and Delayed-Onset Postoperative Endophthalmitis . AM J OPHTHALMOL 2020, 212 :34-42. Mazoteras P, Bispo PJ, Hofling-Lima AL, Casaroli-Marano RP: DNA extraction methods for panbacterial and panfungal PCR detection in intraocular fluids . CURR EYE RES 2015, 40 (7):697-706. Forbes JD, Knox NC, Ronholm J, Pagotto F, Reimer A: Metagenomics: The Next Culture-Independent Game Changer . FRONT MICROBIOL 2017, 8 :1069. Forbes JD, Knox NC, Peterson CL, Reimer AR: Highlighting Clinical Metagenomics for Enhanced Diagnostic Decision-making: A Step Towards Wider Implementation . COMPUT STRUCT BIOTEC 2018, 16 :108-120. Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glockner FO: Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies . NUCLEIC ACIDS RES 2013, 41 (1):e1. Raja HA, Miller AN, Pearce CJ, Oberlies NH: Fungal Identification Using Molecular Tools: A Primer for the Natural Products Research Community . J NAT PROD 2017, 80 (3):756-770. Dulanto CA, Dekker JP: From the Pipeline to the Bedside: Advances and Challenges in Clinical Metagenomics . J INFECT DIS 2020, 221 (Suppl 3):S331-S340. Deshmukh D, Joseph J, Chakrabarti M, Sharma S, Jayasudha R, Sama KC, Sontam B, Tyagi M, Narayanan R, Shivaji S: New insights into culture negative endophthalmitis by unbiased next generation sequencing . SCI REP-UK 2019, 9 (1):844. Hamad I, Ranque S, Azhar EI, Yasir M, Jiman-Fatani AA, Tissot-Dupont H, Raoult D, Bittar F: Culturomics and Amplicon-based Metagenomic Approaches for the Study of Fungal Population in Human Gut Microbiota . SCI REP-UK 2017, 7 (1):16788. Gardy JL, Loman NJ: Towards a genomics-informed, real-time, global pathogen surveillance system . NAT REV GENET 2018, 19 (1):9-20. Quick J, Grubaugh ND, Pullan ST, Claro IM, Smith AD, Gangavarapu K, Oliveira G, Robles-Sikisaka R, Rogers TF, Beutler NA et al : Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples . NAT PROTOC 2017, 12 (6):1261-1276. Marx V: Nanopores: a sequencer in your backpack . NAT METHODS 2015, 12 (11):1015-1018. Mongan AE, Tuda J, Runtuwene LR: Portable sequencer in the fight against infectious disease . J HUM GENET 2020, 65 (1):35-40. Charalampous T, Kay GL, Richardson H, Aydin A, Baldan R, Jeanes C, Rae D, Grundy S, Turner DJ, Wain J et al : Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection . NAT BIOTECHNOL 2019, 37 (7):783-792. Schmidt K, Mwaigwisya S, Crossman LC, Doumith M, Munroe D, Pires C, Khan AM, Woodford N, Saunders NJ, Wain J et al : Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing . J ANTIMICROB CHEMOTH 2017, 72 (1):104-114. Cheng J, Hu H, Kang Y, Chen W, Fang W, Wang K, Zhang Q, Fu A, Zhou S, Cheng C et al : Identification of pathogens in culture-negative infective endocarditis cases by metagenomic analysis . ANN CLIN MICROB ANTI 2018, 17 (1):43. Garg P, Roy A, Sharma S: Endophthalmitis after cataract surgery: epidemiology, risk factors, and evidence on protection . CURR OPIN OPHTHALMOL 2017, 28 (1):67-72. Jabbarvand M, Hashemian H, Khodaparast M, Jouhari M, Tabatabaei A, Rezaei S: Endophthalmitis Occurring after Cataract Surgery: Outcomes of More Than 480 000 Cataract Surgeries, Epidemiologic Features, and Risk Factors . OPHTHALMOLOGY 2016, 123 (2):295-301. Andreoli CM, Andreoli MT, Kloek CE, Ahuero AE, Vavvas D, Durand ML: Low rate of endophthalmitis in a large series of open globe injuries . AM J OPHTHALMOL 2009, 147 (4):601-608. Silpa-Archa S, Ponwong A, Preble JM, Foster CS: Culture-Positive Endogenous Endophthalmitis: An Eleven-Year Retrospective Study in the Central Region of Thailand . OCUL IMMUNOL INFLAMM 2018, 26 (4):533-542. Doan T, Wilson MR, Crawford ED, Chow ED, Khan LM, Knopp KA, O'Donovan BD, Xia D, Hacker JK, Stewart JM et al : Illuminating uveitis: metagenomic deep sequencing identifies common and rare pathogens . GENOME MED 2016, 8 (1):90. Gandhi J, Jayasudha R, Naik P, Sharma S, Dave VP, Joseph J: Targeted High-Throughput Sequencing Identifies Predominantly Fungal Pathogens in Patients with Clinically Infectious, Culture-Negative Endophthalmitis in South India . MICROORGANISMS 2019, 7 (10). Huang Q, Fu A, Wang Y, Zhang J, Zhao W, Cheng Y: Microbiological diagnosis of endophthalmitis using nanopore targeted sequencing . CLIN EXP OPHTHALMOL 2021, 49 (9):1060-1068. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 02 Jul, 2025 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 06 May, 2025 Reviews received at journal 05 May, 2025 Reviews received at journal 03 May, 2025 Reviewers agreed at journal 01 May, 2025 Reviewers agreed at journal 27 Apr, 2025 Reviewers agreed at journal 26 Apr, 2025 Reviews received at journal 19 Apr, 2025 Reviewers agreed at journal 17 Apr, 2025 Reviewers invited by journal 17 Apr, 2025 Editor assigned by journal 16 Apr, 2025 Editor invited by journal 15 Apr, 2025 Submission checks completed at journal 13 Apr, 2025 First submitted to journal 13 Apr, 2025 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-6292219","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":445206452,"identity":"229e1396-c571-4469-88ec-efab6fe1cf03","order_by":0,"name":"Dalan Jing","email":"","orcid":"","institution":"Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dalan","middleName":"","lastName":"Jing","suffix":""},{"id":445206453,"identity":"fa52bff6-f818-49a7-a42c-4e1642f4d0d1","order_by":1,"name":"Xiaodan Jiang","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaodan","middleName":"","lastName":"Jiang","suffix":""},{"id":445206454,"identity":"52e57faf-b0de-496e-a62d-6380a57717a6","order_by":2,"name":"Xiaotong Ren","email":"","orcid":"","institution":"First Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaotong","middleName":"","lastName":"Ren","suffix":""},{"id":445206455,"identity":"0eeb3d7a-bffb-47b8-9aa7-cd4f1bd54295","order_by":3,"name":"Ran Hao","email":"","orcid":"","institution":"Beijing Tongren Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ran","middleName":"","lastName":"Hao","suffix":""},{"id":445206456,"identity":"a005b7d7-1e60-40da-ac48-36b15daa831b","order_by":4,"name":"Jie Su","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Su","suffix":""},{"id":445206457,"identity":"21035608-c278-451c-a21f-1d4ecb485e05","order_by":5,"name":"Xuemin Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIie3RsWrDMBCA4YMDZTHVKpOSvoIg4KmPkIc44ZKphYweAg24OEMTsmboQ3jseMGgSd01dHCW7t6aJdR7S+RsHfTN+rmTBBBF/5CQJTMVZ5Sj8tBSsQwnN8oabh2P0o3NdetsOJnA4/RwrFhqP8vS4wsOWAycZhKf43QPWWFWAuT6lS4nWC2Ykq+pHK/m3rzfgnIfdWBKUzMpzNM3tt44AVo9hRLqF9P4XHtTLUyFQ5KHPqEGtc8FDEuUJSaeY//IqMjZJHiXu13ZdKfzff+Vu677LpYTud5eTn5JrjseRVEU/ekHHLBSzACnFPgAAAAASUVORK5CYII=","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":true,"prefix":"","firstName":"Xuemin","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-03-24 06:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6292219/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6292219/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-025-04132-y","type":"published","date":"2025-07-02T15:57:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82118441,"identity":"faf44667-8b3e-43c1-b7ce-74068404803b","added_by":"auto","created_at":"2025-05-07 03:07:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2999613,"visible":true,"origin":"","legend":"\u003cp\u003eStacked box plot displaying the relative abundances of the bacterial taxa (≥5%) obtained via NTS. A, Culture-positive endophthalmitis. B, Culture-negative endophthalmitis. C. Stacked box plot displaying the relative abundances of the fungal taxa (≥5%).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6292219/v1/88990739dd3792721028b3ff.png"},{"id":82118442,"identity":"046c4049-949c-4ff1-afc4-a4b4640124c5","added_by":"auto","created_at":"2025-05-07 03:07:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4245421,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of genus-level bacterial abundances in samples from patients with presumed infectious, culture-positive and culture-negative endophthalmitis. The color bar on the right side indicates the average relative abundances of these genera in each patient.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6292219/v1/4a29f4a934901cb707560f06.png"},{"id":82118443,"identity":"df6fdc50-d476-48fd-9027-cbea788091e1","added_by":"auto","created_at":"2025-05-07 03:07:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":337363,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram of consistency between dominant bacterial genera at the level of bacterial culture, Sanger sequencing, and NTS.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6292219/v1/1c4de656764f8cb6803c1ddc.png"},{"id":86179825,"identity":"8c6aff7d-d3bb-4a71-8f0a-14b8704d964e","added_by":"auto","created_at":"2025-07-07 16:19:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10206630,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6292219/v1/8384621d-5808-41af-99ff-a8ae5b43e072.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Clinical Performance of Nanopore Targeted Sequencing for Diagnosing Endophthalmitis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndophthalmitis is one of the most destructive eye infections and may lead to irreversible blindness of infected eyes within hours or days after symptoms appear[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEndophthalmitis is a bacterial or fungal infection of the vitreous and/or aqueous humor[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Most infection routes are exogenous, including eye surgery, penetration of ocular trauma, or the spread of corneal infection, and a few are endogenous and result from hematogenous seeding of the eye by bacteria or fungi[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The incidence of endophthalmitis varies with different causes; cataract surgery is the most common cause, with an incidence ranging from 0.066\u0026ndash;0.125% [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEndophthalmitis is a clinical diagnosis supported by vitreous and/or aqueous humor culture or blood culture, although 30% of case culture results are negative [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Multiple factors contribute to the high negative culture rate, including overdiagnosis, a limited sample size, the inability of some bacteria to be cultured in the laboratory, and the use of antibiotic therapy[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Traditional bacterial culture is still the gold standard for the diagnosis of endophthalmitis. The identification of the pathogen takes up to 5 days. Sometimes, 2 to 4 weeks are needed for correct sporulation and pathogen identification[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. New methods of molecular microbiology have overcome these difficulties[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDNA sequencing can be broadly classified into two techniques: targeted amplicon sequencing and untargeted whole-genome sequencing (WGS). An example of a sequence of targeted amplifiers is the determination gene of the universal 16S bacterial ribosomal RNA (rRNA) of the amplicon. The first method to sequence DNA was developed by Sanger in 1975[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]; this method has low throughput and high cost, but it is still the gold standard for sequencing.\u003c/p\u003e \u003cp\u003ePathogen identification through next-generation sequencing (NGS) has been increasingly used in microbial research and clinical diagnosis. NGS can rapidly detect all the different pathogens present in a clinical sample in a single assay, which requires 2 days to 3 days, which is shorter than the turnaround time of routine culture[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Owing to the limitations of the Illumina sequencing platform, which involves short reads (500 base pairs), only part of the 16S rRNA gene can be sequenced, thus limiting the taxonomic resolution to genus-level classification[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Metagenomic NGS techniques provide better taxonomic resolution at the species level, but their sensitivity depends heavily on the background level and is vulnerable to contamination by environmental species [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Simultaneously, it requires greater sequencing depth, leading to higher costs[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEven in cases of severe endophthalmitis caused by virulent pathogens, prompt therapy may save useful vision. The nanopore-targeted sequencing (NTS) method is capable of generating long-read lengths (\u0026gt;5,000 bp) and performing targeted amplification (16S RNA gene for bacteria and ITS for fungi), which do not interfere with the host background DNA[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Nanopore sequencing enhances our ability to study complex microbial samples by using inexpensive and portable technologies to conduct real-time long-read sequencing[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition, it has a short turnaround time of 8\u0026ndash;14 h in pleural fluid, ascites fluid, and bronchoalveolar lavage fluid detection[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This method has important advantages, such as the low concentration of bacteria and the small sample size used for endophthalmitis. In recent years, many preliminary studies on amplicon-based nanopore sequencing for endophthalmitis have been reported[\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Through nanopore amplicon sequencing, bacterial and fungal pathogens associated with infectious endophthalmitis can be identified, which is more sensitive and has a faster processing time than conventional culture. In addition, Hao et al. recently reported similar results in patients with endogenous endophthalmitis[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe clinical manifestations of NTS in endophthalmitis patients have not been systematically compared with those of traditional culture analysis, PCR and Sanger sequencing. Therefore, we evaluated the feasibility of NTS in acute endophthalmitis by comparing culture with PCR and Sanger sequencing.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticipants\u003c/h2\u003e \u003cp\u003eThis prospective study enrolled patients who presented to the Department of Ophthalmology of Peking University Third Hospital between January 2022 and October 2022. Our study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of the Peking University Third Hospital. (S2021222). The participants provided written informed consent before the intervention. Twelve patients who were clinically presumed to have infectious endophthalmitis (bacterial or fungal) and who were prepared for intravitreal drug injection or pars plana vitrectomy (PPV) were enrolled. Clinical details, including demographic information, predisposing factors, previous treatment, cause and duration of symptoms, and surgical interventions, were also collected.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSpecimens\u003c/h3\u003e\n\u003cp\u003eAqueous and/or vitreous samples (at least 100 \u0026micro;L) were obtained via three methods: anterior chamber wash, vitreous tap and PPV. All the samples were aseptically divided into two equal amounts without dilution and sent immediately for routine microbiological processing, and the remaining samples for genomic analysis were snap frozen, stored at \u0026minus;\u0026thinsp;80\u0026deg;C, and transported on dry ice to the laboratories.\u003c/p\u003e\n\u003ch3\u003eConventional bacterial culture\u003c/h3\u003e\n\u003cp\u003eThe samples were incubated on blood agar and chocolate agar plates at 35\u0026deg;C for 48 hours. When a fungal infection was strongly suspected, additional culture was performed on Sabouraud\u0026rsquo;s dextrose agar (SDA) plates at 30\u0026deg;C with no CO\u003csub\u003e2\u003c/sub\u003e for 3 weeks. Bacterial or fungal identification was performed from the positive culture plate via standard biochemical tests via a VITEK 2 Compact system (bioMe\u0026acute;rieux, France) .\u003c/p\u003e\n\u003ch3\u003eNanopore-targeted sequencing\u003c/h3\u003e\n\u003cp\u003eNTS was conducted according to the method described by Fu et al.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. DNA was extracted via the Sansure DNA Extraction Kit (Changsha, China) following the manufacturer\u0026rsquo;s instructions. The barcoded amplification products of the 16S rRNA gene, ITS1/2, and rpoB from the same samples were pooled at a mass ratio of 10:3:1. The pooled products from the different samples were equally mixed and used to construct sequencing libraries via a 1D Ligation Kit (SQK-LSK109; Oxford Nanopore). The library was sequenced via Oxford Nanopore GridION X5 with real-time base calling enabled (ont-guppy-for-gridion v. 1.4.3-1 and v. 3.0.3-1; high-accuracy base calling mode)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. TE buffer was used as a negative control in each batch. The criteria for positive results of NTS bacterial or fungal identification were as follows: a map reading of the bacterial species in the sample\u0026thinsp;\u0026gt;\u0026thinsp;100 or greater than that of any other species and a ratio of the map reading in the sample to the negative control\u0026thinsp;\u0026gt;\u0026thinsp;10. If the fungal species level was greater than 20 or greater than 50% of the relative abundance, the ratio of the fungal spectrum reading in the sample to the negative control was greater than 10. Important lists of clinically known typical or potentially pathogenic bacteria and fungi are available in the clinical guidelines and literature[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eSanger Sequencing\u003c/h3\u003e\n\u003cp\u003eDNA samples were extracted from residual clinical samples. The PCR products were purified and sequenced via an ABI3730 genetic analyser (Beijing Genomics Institute, Beijing, China). After the Bellerophon server was used to evaluate the chimerism formation of the product[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the 16S rRNA gene sequences were compared with the GenBank database to search for related sequences via the BLAST program.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll analyses were performed via SPSS version 23.0 software. Only descriptive statistics were used.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTreatment and follow-up\u003c/h3\u003e\n\u003cp\u003eThe treatment plan is selected according to the initially assumed infection type and adjusted according to the NTS and/or culture results. The fixed follow-up time was set at 1 day and 1 week after the operation.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003ePatient demographics\u003c/h2\u003e\n \u003cp\u003eNineteen samples from 12 patients were used in the study, 10 of which were vitreous humor samples and 9 were aqueous humor samples. The patients had a median age of 64.3 (range 48–86) years, and 7 were male (58.3%). The predisposing conditions were previous cataract surgery or previous bleb reconstruction surgery (10 patients), trauma (1 patient) and endogenous spread (1 patient). All patients did not undergo prior intraocular antimicrobial therapy before specimen collection. Four patients underwent PPV in the first phase, whereas the remaining 8 patients only underwent intravitreal injection of vancomycin and ceftazidime in the first phase. The majority of the patients presented with visual acuity worse than 20/200 (83.3%) (Table\u0026nbsp;1).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eClinical and demographic details of the patients with suspected infectious endophthalmitis\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"10\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLaterality\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClinical diagnosis\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePredisposing factor\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSystemic disease\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInitial VA\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFinal VA (1 week)\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\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eright\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePostoperative\u003c/p\u003e\n \u003cp\u003eendophthalmitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCataract surgery: 12 days\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=\"left\"\u003e\n \u003cp\u003eCF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIOAI + V + C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM\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\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eleft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePostoperative\u003c/p\u003e\n \u003cp\u003eendophthalmitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBleb reconstruction: 10 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHTN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPV + Bleb reconstruction + V + C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCF\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\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eleft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePostoperative\u003c/p\u003e\n \u003cp\u003eendophthalmitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCataract surgery: 2 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHTN, DM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIOAI + V + C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eright\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePostoperative\u003c/p\u003e\n \u003cp\u003eendophthalmitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCataract surgery: 6 days\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=\"left\"\u003e\n \u003cp\u003e20/200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIOAI + V + C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20/80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eleft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eExogenous endophthalmitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePerforation: steel wire 5days\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=\"left\"\u003e\n \u003cp\u003eHM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPV + V + C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eright\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePostoperative\u003c/p\u003e\n \u003cp\u003eendophthalmitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCataract surgery: 10 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIOAI + V + C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eleft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePostoperative\u003c/p\u003e\n \u003cp\u003eendophthalmitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCataract surgery: 9 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHTN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20/100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIOAI + V + C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20/100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\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\u003eleft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePostoperative\u003c/p\u003e\n \u003cp\u003eendophthalmitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCataract surgery: 2 days\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=\"left\"\u003e\n \u003cp\u003eHM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIOAI + V + C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\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=\"left\"\u003e\n \u003cp\u003eleft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePostoperative\u003c/p\u003e\n \u003cp\u003eendophthalmitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCataract surgery: 3 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHTN, hypothyroidism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIOAI + V + C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\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\u003eright\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePostoperative\u003c/p\u003e\n \u003cp\u003eendophthalmitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCataract surgery: 4 days\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=\"left\"\u003e\n \u003cp\u003eCF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIOAI + V + C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eleft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEndogenous\u003c/p\u003e\n \u003cp\u003eendophthalmitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLiver abscess (postoperative findings)\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=\"left\"\u003e\n \u003cp\u003eNPL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPL + PPV + silicone oil + V + C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNPL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eleft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePostoperative\u003c/p\u003e\n \u003cp\u003eendophthalmitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCataract surgery: 6 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHTN, DM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePPV + silicone oil + V + C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003eAbbreviations: C, ceftazidime; CF, counting fingers; DM:diabetes mellitus; F, female; HM, hand movements; HTP:hypertension; IOAI, intraocular antibiotic injection; LP, light perception; M, male; NPL, no light perception; PPL, pars plana lensectomy; PPV, pars plana vitrectomy; V, vancomycin; VA, visual acuity.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003eMicrobiology culture\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eSix samples (31.6%) were positive by microbial culture, of which all 6 samples grew bacteria and none grew fungi. Four samples were positive for \u003cem\u003eS. epidermidis\u003c/em\u003e. The remaining three culture-positive samples were positive for \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, \u003cem\u003eSphingomonas oligokinetica\u003c/em\u003e, and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. Further details are provided in Table 2.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eIdentification results of culture-positive samples\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample code\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample type\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBacterial culture\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSanger sequencing\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNTS\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=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evitreous body\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. epidermidis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. Epidermidis, Corynebacterium Macginleyi, Malassezia restricta, Penicillium citrinum\u003c/em\u003e\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=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evitreous body\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eEnterococcus faecalis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE. Faecalis, Oceanobacillus oncorhynchi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE. Faecalis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eaqueous humor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. epidermidis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. Epidermidis, Phyllobacterium myrsinacearum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. Epidermidis, Rhodococcus qingshengii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evitreous body\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. epidermidis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. Epidermidis, P. myrsinacearum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. Epidermidis, Staphylococcus capitis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eaqueous humor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eSphingomonas paucimobilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eStreptococcus pneumoniae, Lysinibacillus macroides\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. Pneumoniae, Streptococcus pseudopneumoniae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evitreous body\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eK. Pneumoniae, Klebsiella variicola\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eAbbreviations: NTS, nanopore targeted sequencing;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003eNanopore\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003etargeted sequencing\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNTS detected the presence of microorganisms in 19 samples out of 12 participants (positive rate of 100%), including 6 culture-positive samples and 13 culture-negative samples. In addition to bacteria, fungi, including \u003cem\u003eRestrictive\u0026nbsp;\u003c/em\u003e\u003cem\u003eMalassezia\u003c/em\u003e, \u003cem\u003eAspergillus fumigatus\u003c/em\u003e, and \u003cem\u003ePenicillium citri\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e were also detected in 3 samples (Table 2). More bacterial genera and species can be detected in the NTS. Figure 1 displayegd the relative abundances of the bacterial taxa (≥5%) obtained via NTS. Apart from the vitreous sample of code 4, where no microorganisms were detected, and the vitreous sample of code 3, where only \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (monomicrobial infection) was detected, two or more bacterial species were found in all 17 samples. The first two bacteria and fungi, on the basis of their abundance, are displayed in Table 2 and 3. By combining the NTS results and generating a heatmap of all the samples, the taxonomic abundance of bacterial species detected in 19 samples (including positive cultures and negative cultures) of presumed infectious endophthalmitis was predominantly gram-positive, as shown in Figure 2. The time range confirmed by the NTS is 8-12 hours. Notably, the type of endophthalmitis in patient 11, who only had a history of retrobulbar injection of triamcinolone acetonide, could not be confirmed. Diagnostic vitrectomy was performed on the patient. The vitreous body was subjected to corresponding testing. NTS first reported results within 9 hours, indicating \u003cem\u003eK. pneumoniae\u003c/em\u003e infection. The patient was screened for diabetes and examined by liver and gallbladder ultrasound. The results showed that there were multiple nodules and abscesses in the liver.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eIdentification results of culture-negative samples\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample code\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample type\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBacterial culture\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSanger sequencing\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNTS\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=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eaqueous humor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus epidermidis, Corynebacterium McKinley, Restrictive malassezia, Aspergillus fumigatus\u003c/em\u003e\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=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evitreous body\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eC. McKinley, Klebsiella aerogenes\u003c/em\u003e, \u003cem\u003eR. malassezia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eaqueous humor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus.sp\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus fumarioli, Halomonas boliviensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evitreous body\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus.sp, Phyllobacterium.sp\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eRhodococcus qingshengii\u003c/em\u003e, \u003cem\u003ebacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e6\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\u003eaqueous humor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePhyllobacterium myrsinacearum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eDelftia acidovorans, Lawsonella clevelandensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evitreous body\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP.myrsinacearum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eD. acidovorans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eaqueous humor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP.myrsinacearum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAquabacterium parvum, Halomonas mongoliensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\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\u003evitreous body\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eH.mongoliensis, A. parvum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eaqueous humor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus aureus, P. myrsinacearum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. parvum, H.mongoliensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eaqueous humor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus.sp\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas pseudoalcaligenes, H.mongoliensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evitreous body\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eSphingomonas oligophenolica, H.mongoliensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eaqueous humor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eSphingomonas. sp\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS. \u003cem\u003eoligophenolica, D. acidovorans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evitreous body\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus. sp\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e绿色芽孢杆菌\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eAbbreviations: NTS, nanopore targeted sequencing;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003eComparison of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003econsistency among\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethree detection results\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAll 19 samples were subjected to bacterial culture, Sanger sequencing, and NTS. The consistency of the three identification results was compared (Figure 3). Biochemical analysis of bacterial cultures: A total of 4 genera and 4 species of bacteria were isolated. Sanger sequencing microbial community types: A total of 9 genera and 8 species of bacteria were isolated. NTS analysis of bacterial community types: A total of 29 genera and 33 species of bacteria were isolated. The consistency of the three methods at the level of dominant bacterial genera was 5/19 cases (26.3%). A total of 6 positive samples were cultured, of which 5 were consistent with Sanger sequencing (83.3%) and 5 were consistent with NTS (83.3%) (Figure 3). Sixteen samples were positive by Sanger sequencing, and 10 samples were consistent with NTS (62.5%) (Figure 3).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEndophthalmitis is a serious eye infection that may cause permanent loss of useful vision in the affected eye[1]. It can occur following ocular surgery or trauma, as well as through the hematogenous spread of microorganisms from endogenous infection[23]. However, bacteremia or mycosis may be temporary, and patients may experience symptoms of systemic infection\u0026nbsp;[1]. Almost all patients with endophthalmitis exhibit decreased vision, and some patients also experience eye pain. Eye examination usually\u0026nbsp;reveals\u0026nbsp;hypopyon and intraocular inflammation[1]. Traditional cultivation and identification are the gold\u0026nbsp;standards\u0026nbsp;for detecting endophthalmitis, but the positive rate is only 40% and is prone to false negative results[24]. Molecular diagnostic techniques, including culturing negative cases,\u0026nbsp;have been used in the laboratory to identify endophthalmitis pathogens and provide the possibility of rapid diagnosis. These technologies play a key role in the diagnosis of endophthalmitis\u0026nbsp;[25]. Since its first application in ophthalmology in 1993, PCR has shown promising prospects in the treatment of endophthalmitis, increasing the detection rate and shortening the diagnosis time. However,\u0026nbsp;owing\u0026nbsp;to differences in amplification efficiency among different primer groups and limitations in the number of fluorescent markers, the number of fungi and/or bacteria that can be detected simultaneously is limited\u0026nbsp;[7, 26, 27].\u003c/p\u003e\n\u003cp\u003eShotgun metagenomics sequences are all derived from the DNA provided by a sample, which theoretically allows for hypothesis-free detection of all types of microorganisms (including rare pathogens) with fast turnover times\u0026nbsp;[28]. It can identify multiple pathogens through a single test and can even be applied to samples after antibiotic treatment\u0026nbsp;[29]. Moreover, amplicon sequencing (based on amplicon metagenomics) amplifies the target DNA of interest by performing PCR before sequencing. Amplicon-based metagenomics enriches target pathogen DNA by reducing the range of detectable pathogens, which is sufficient to classify and analyse pathogens\u0026nbsp;[30, 31]. This method is particularly suitable for detecting low concentrations of bacteria and fungi from clinical samples containing a large amount of host DNA\u0026nbsp;[32]. A previous study\u0026nbsp;revealed\u0026nbsp;good consistency between culture results and NGS\u0026nbsp;results\u0026nbsp;in culture-positive cases;\u0026nbsp;even in culture-negative cases, NGS can\u0026nbsp;identify\u0026nbsp;additional bacteria\u0026nbsp;[33].\u003c/p\u003e\n\u003cp\u003eBecause amplicon sequencing analyses only the target sequence, it is easy to interpret the results. PCR helps to improve sensitivity, ultimately increasing the diagnostic yield\u0026nbsp;[34]. In addition, nanopore sequencing provides a fast library preparation protocol and enables real-time analysis, making it suitable for rapid analysis\u0026nbsp;of\u0026nbsp;clinical samples\u0026nbsp;[35, 36].\u0026nbsp;In addition, nanopore sequencing can be used to process one sample at a time, while other sequencing platforms,\u0026nbsp;such as Illumina,\u0026nbsp;need to process multiple samples in a batch to achieve economic feasibility, which may have adverse effects on the turnover time of each sample in rare diseases such as endophthalmitis\u0026nbsp;[37, 38].\u003c/p\u003e\n\u003cp\u003eThrough nanopore sequencing, bacterial and fungal pathogens associated with infectious endophthalmitis can be identified, which has higher sensitivity and faster processing time than conventional culture. Previous preliminary studies on targeted nanopore sequencing for endophthalmitis have utilized PCR primers to amplify specific target regions of interest, such as 16S rRNA in bacteria or ITS in fungi. Jun et al.\u0026nbsp;[16]\u0026nbsp;identified pathogens in 5 cases of bacterial endophthalmitis and 3 cases of fungal endophthalmitis\u0026nbsp;via\u0026nbsp;16S and ITS nanopore amplicon sequencing. Similarly, Huang et al.\u0026nbsp;[17]\u0026nbsp;identified 17 pathogens in 18 cases of endophthalmitis\u0026nbsp;via 16S, ITS, and rpoB gene nanopore amplicon sequencing. In addition, in 8 culture-negative samples and in 1 sample that could not be cultured, nanopore sequencing detected bacteria, fungi, or a mixture of bacteria and fungi in the intraocular fluid. To evaluate the role of nanopore sequencing in identifying potential pathogenic pathogens\u0026nbsp;of endophthalmitis, Low et al.\u0026nbsp;[18]\u0026nbsp;compared the culture results with\u0026nbsp;those of\u0026nbsp;NTS, whole-genome nanopore sequencing, and Illumina sequencing. They\u0026nbsp;reported\u0026nbsp;that nanopore sequencing identified the same bacterial species in all culture-positive cases and identified potential pathogens in two culture-negative cases. Whole-genome nanopores also detected the presence of bacteriophages in three samples.\u003c/p\u003e\n\u003cp\u003eIn the past, clinical applications for detecting bacterial and fungal pathogens in clinical samples focused mainly on nanopore metagenomic sequencing, which is characterized by relatively high cost and requires a large amount of bioinformatics processing\u0026nbsp;[39-41]. NTS\u0026nbsp;was used to amplify\u0026nbsp;marker genes and\u0026nbsp;sequence\u0026nbsp;the amplified products\u0026nbsp;via\u0026nbsp;a nanopore sequencing platform, which\u0026nbsp;was\u0026nbsp;developed for the diagnosis of bacterial or fungal infections with lower costs and bioinformatics processing. However, the clinical manifestations of NTS in specimens of infectious endophthalmitis have not been studied compared with traditional culture experiments and Sanger sequencing after PCR in terms of\u0026nbsp;cohorts\u0026nbsp;and\u0026nbsp;systems. Here, we\u0026nbsp;evaluated\u0026nbsp;the feasibility of NTS by comparing culture, PCR, and Sanger sequencing.\u0026nbsp;To\u0026nbsp;achieve\u0026nbsp;greater\u0026nbsp;clinical efficacy in metagenomic sequencing, the following characteristics\u0026nbsp;are necessary: fast turnaround time, low cost, and high sensitivity. From this perspective, NTS is a suitable method for detecting endophthalmitis\u0026nbsp;in samples.\u003c/p\u003e\n\u003cp\u003eThis study included a total of 19 samples from 12 patients, of which 10 were vitreous samples and 9 were aqueous humor samples. Among them, 7 were males (58.3%), and 5 were females (41.7%), with more male patients than female patients. Six patients had systemic diseases (50%). The most common triggering factors were previous cataract surgery or follicular reconstruction surgery (10 patients). Understanding the risk factors for endophthalmitis is crucial for its prevention. For postoperative endophthalmitis after cataract surgery, risk factors include advanced age (80 years or older), immune dysfunction secondary to systemic disease, the presence of septic lesions in and around the eye, posterior capsule rupture (PCR), and wound leakage. Lundstrom et al.[42]\u0026nbsp;found that\u0026nbsp;nonuse\u0026nbsp;of anterior chamber antibiotics, contact with the vitreous body, and age over 85 years were the three most common risk factors. In another large-scale epidemiological study by Jabbarvand et al.\u0026nbsp;[43], diabetes,\u0026nbsp;advanced age\u0026nbsp;(over 80 years), conventional large-incision extracapsular cataract extraction and\u0026nbsp;perioperative\u0026nbsp;contact with the vitreous body were significantly associated with an increased risk of endophthalmitis. In addition to these traditional risk factors, the emerging risk\u0026nbsp;factor is\u0026nbsp;cataract surgery in patients who previously received intravitreal injection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our study, with the exception of surgery-related endophthalmitis, the remaining cases included trauma (1 patient) and endogenous diffusion (1 patient). After penetrating eye injury, 3\u0026ndash;10% of patients experience endophthalmitis, although early surgical repair and prophylactic systemic application of antibiotics may reduce the incidence rate to \u0026lt;1%[44]. The risk factors for endophthalmitis include metal foreign body trauma, intraocular foreign body retention, lens rupture, and\u0026nbsp;an\u0026nbsp;initial repair time greater than 24 hours.\u0026nbsp;Endogenous\u0026nbsp;endophthalmitis is most commonly\u0026nbsp;observed\u0026nbsp;in patients with systemic diseases or immune dysfunction, such as diabetes and hepatobiliary diseases\u0026nbsp;[45].\u0026nbsp;The\u0026nbsp;incidence rate is 0.04%-0.4%, and related risk factors include intravenous medication, diabetes, low immune function, malignant\u0026nbsp;tumors, long hospitalization or intravenous antibiotics. The risk factor for patients with endogenous endophthalmitis in this study\u0026nbsp;was\u0026nbsp;liver abscess.\u003c/p\u003e\n\u003cp\u003eA total of 19 samples were included in the microbial culture, of which 6 samples (31.6%) were culture positive. All 6 samples exhibited bacterial growth, and none of the samples exhibited fungal growth. In contrast, genome sequencing is not limited to a specific species but can detect all different microorganisms present in clinical samples in a single trial. A total of 19 samples were included in the NTS, 19 of which tested positive (100%), including 6 culture-positive samples and 13 culture-negative samples. In addition to bacteria, fungi, including \u003cem\u003eMalassezia restrictive\u003c/em\u003e, \u003cem\u003eAspergillus fumigatus\u003c/em\u003e, and \u003cem\u003ePenicillium citri\u003c/em\u003e\u003cem\u003e,\u0026nbsp;\u003c/em\u003ewere also detected in 3 samples. Gene sequencing technology can improve traditional microbial detection and identify microorganisms that were previously unrelated to endophthalmitis\u0026nbsp;[46]. Genomic sequencing has advantages in identifying pathogens of eye infections, potentially improving diagnostic accuracy and selecting appropriate treatment protocols. Biochemical analysis of bacterial\u0026nbsp;cultures: A total of 4 genera and 4 species of bacteria were isolated. Sanger sequencing revealed a total of 9 genera and 8 species of bacteria. NTS analysis of microbial community types: A total of 29 genera and 33 species of bacteria were isolated. The consistency of the three methods in detecting dominant bacterial genera was 5/19 cases (26.3%).\u0026nbsp;A total of 6 positive\u0026nbsp;samples\u0026nbsp;were cultured, of which 5 were consistent with Sanger sequencing (83.3%),\u0026nbsp;5 were consistent with NTS (83.3%),\u0026nbsp;16 samples\u0026nbsp;were\u0026nbsp;positive\u0026nbsp;with\u0026nbsp;Sanger sequencing, and 10 samples were consistent with NTS (62.5%). In summary, these results indicate that NTS can provide useful information for\u0026nbsp;identifying\u0026nbsp;pathogens related to endophthalmitis. The positive rate of NTS is significantly\u0026nbsp;greater\u0026nbsp;than that of bacterial culture, as it has high sensitivity even under low microbial loads. Previously, the use of antibiotics had an impact on the diagnostic positivity of traditional culture methods, but nanopore sequencing was not affected because bacterial DNA can be detected even after being killed by antibiotics. In this study, the results of NTS and Sanger\u0026nbsp;sequencing were highly consistent\u0026nbsp;with those of traditional culture. Similar to previous studies\u0026nbsp;[16, 17], using NTS, we detected more than one type of microorganism in some patients, some of\u0026nbsp;whom\u0026nbsp;did not grow in culture. Possible reasons include competition among microorganisms, cultivation conditions, differences in growth rates, and quorum sensing. The multimicrobial infection of endophthalmitis is also a challenging diagnostic and therapeutic situation. The NTS in this study helped to detect a mixture of bacteria and fungi in some patients, which was completely overlooked in the culture.\u0026nbsp;Similar to previous studies\u0026nbsp;[47, 48], we detected multiple microorganisms in some patients. Multi microbial eye infections are not only a challenge in identifying two or more microorganisms, but also in developing appropriate antimicrobial treatments. If fungal infections coexist, consideration should be given to intravitreal antifungal drugs and antibiotics, but personalized decisions should be made based on medical history and clinical examination.\u003c/p\u003e\n\u003cp\u003eThere were several limitations to this study. First, the number of patients included was small, and a single center was used. The diagnostic value of the NTS cannot be evaluated via conventional indicators such as sensitivity and specificity. The severity of endophthalmitis may vary among different patients, and the pathogen load may affect the detection results. The etiology, underlying diseases, and treatment methods of different patients may vary, which may affect the analysis of prognostic factors. Therefore, more cases of different severity levels are needed to evaluate the practical performance of NTS in distinguishing the pathogenic agents of endophthalmitis. In addition, owing to its targeted nucleic acid amplification characteristics, NTS still has several drawbacks. Potential contamination may occur during sample preparation and transportation. Therefore, sample quality should be strictly controlled, and in some cases, it may be necessary to repeatedly send samples from the same patient for testing. The limitations of current research on mixed infections of multiple bacteria include retrospective and lack of clear prospective treatment plans.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThrough systematic comparisons with traditional culture assays and PCR followed by Sanger sequencing retrospectively, NTS undoubtedly has unparalleled advantages in accurately detecting the pathogenic bacteria of infectious endophthalmitis, especially for culture-negative and mixed infection cases. NTS is expected to provide an effective pathway for timely and effective identification of endophthalmitis-infected bacteria in clinical practice.\u003c/p\u003e\n"},{"header":"Abbreviations","content":"\u003cp\u003eNTS, targeted nanopore sequencing; WGS, whole-genome sequencing; rRNA: ribosomal RNA; NGS: next-generation sequencing ; PPV: pars plana vitrectomy; SDA :Sabouraud\u0026rsquo;s dextrose agar; C, ceftazidime; CF, counting fingers; DM:diabetes mellitus; F, female; HM, hand movements; HTP:hypertension; IOAI, intraocular antibiotic injection; LP, light perception; M, male; NPL, no light perception; PPL, pars plana lensectomy; PPV, pars plana vitrectomy; V, vancomycin; VA, visual acuity; PCR: posterior capsule rupture.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eOur study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of the Peking University Third Hospital. (S2021222).\u0026nbsp;Written informed consent was obtained from all study participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eThe datasets generated and/or analysed during the current study are available in the NCBI database and are accessible at \u0026nbsp;PRJNA1247725.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003eAll authors declare that they have no conflict of interest.\u003cbr\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eThe publication of this paper were supported by a grant from Peking University Third Hospital Key\u0026nbsp;Talent Project foundation (Grant no.BYSYZD2021044) and Shandong Provincial Natural Science Foundation (Grant no. ZR2024QH310). The sponsor or funding organisation had no role in the design or conduct of this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003eAll named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this article and take responsibility for the integrity of the work as a whole. Dalan Jing: research design, data acquisition, data analysis, and manuscript preparation. Xiaodan Jiang: research design, manuscript modification. Xiaotong Ren: manuscript modification. Jie Su: data acquisition, manuscript modification. Ran Hao: data analysis. Xuemin Li: research design.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003enone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e: not applicable.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDurand ML: \u003cstrong\u003eBacterial and Fungal Endophthalmitis\u003c/strong\u003e. \u003cem\u003eCLIN MICROBIOL REV\u003c/em\u003e 2017, \u003cstrong\u003e30\u003c/strong\u003e(3):597-613.\u003c/li\u003e\n\u003cli\u003eDurand ML: \u003cstrong\u003eEndophthalmitis\u003c/strong\u003e. \u003cem\u003eCLIN MICROBIOL INFEC\u003c/em\u003e 2013, \u003cstrong\u003e19\u003c/strong\u003e(3):227-234.\u003c/li\u003e\n\u003cli\u003eRelhan N, Forster RK, Flynn HJ: \u003cstrong\u003eEndophthalmitis: Then and Now\u003c/strong\u003e. \u003cem\u003eAM J OPHTHALMOL\u003c/em\u003e 2018, 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\u003cstrong\u003e7\u003c/strong\u003e(10).\u003c/li\u003e\n\u003cli\u003eHuang Q, Fu A, Wang Y, Zhang J, Zhao W, Cheng Y: \u003cstrong\u003eMicrobiological diagnosis of endophthalmitis using nanopore targeted sequencing\u003c/strong\u003e. \u003cem\u003eCLIN EXP OPHTHALMOL\u003c/em\u003e 2021, \u003cstrong\u003e49\u003c/strong\u003e(9):1060-1068.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"endophthalmitis, microbiome, 16S rRNA, nanopore sequencing, infectious pathogens","lastPublishedDoi":"10.21203/rs.3.rs-6292219/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6292219/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eRapid identification of pathogenic bacteria in the vitreous and/or aqueous humor of patients with acute clinical diagnosis of endophthalmitis via nanopore sequencing technology.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe recruited a total of 12 patients (12 eyes) who were diagnosed with endophthalmitis at an ophthalmic outpatient clinic from January 2022 to October 2022. Clinical evaluation is conducted in the order of consultation, symptom evaluation, physical sign evaluation, and ophthalmic special examination, all of which are completed by the same experienced clinical physician. Finally, 19 aqueous humor and/or vitreous samples were obtained via anterior chamber wash, vitreous tap and vitrectomy. The samples were separated for cultivation, biochemical drug sensitivity identification, and targeted nanopore sequencing (NTS), and the results of nanopore sequencing were validated via Sanger sequencing.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn patients with endophthalmitis, NTS can identify infected pathogens within 8\u0026ndash;12 hours. Six samples (31.6%) were subjected to culture-based diagnosis, while NTS revealed the presence of pathogenic microorganisms in 19 samples (100%), of which bacteria and fungi were detected in three samples. A total of 19 samples were subjected to Sanger sequencing, of which 16 (84.2%) tested positive, including 6 culture-positive samples and 10 culture-negative samples, of which 5 (26.3%) were positive for two bacterial genera. In culture-positive cases, there is a high-quality match between culture and targeted nanopore sequencing.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eNTS can quickly detect pathogenic bacteria in samples from patients with endophthalmitis. Moreover, the use of vitreous and/or aqueous humor for the NTS has potential. NTS is a promising diagnostic platform for endophthalmitis, especially for mixed infections and culture-negative cases.\u003c/p\u003e","manuscriptTitle":"Clinical Performance of Nanopore Targeted Sequencing for Diagnosing Endophthalmitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 03:07:10","doi":"10.21203/rs.3.rs-6292219/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-06T11:10:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-05T18:11:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-03T06:30:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3652921009345752089606877551658810761","date":"2025-05-01T08:10:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"227443026145027054414079378880596578929","date":"2025-04-28T03:46:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26556941685217395260432392808380322563","date":"2025-04-26T07:47:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-19T18:41:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50844412535128739965199995361385531241","date":"2025-04-17T06:21:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-17T06:02:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-17T03:44:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-15T21:06:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-13T12:17:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-04-13T12:15:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"92525414-74ba-47f4-bdf8-c316e8d16e2d","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:11:42+00:00","versionOfRecord":{"articleIdentity":"rs-6292219","link":"https://doi.org/10.1186/s12866-025-04132-y","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2025-07-02 15:57:12","publishedOnDateReadable":"July 2nd, 2025"},"versionCreatedAt":"2025-05-07 03:07:10","video":"","vorDoi":"10.1186/s12866-025-04132-y","vorDoiUrl":"https://doi.org/10.1186/s12866-025-04132-y","workflowStages":[]},"version":"v1","identity":"rs-6292219","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6292219","identity":"rs-6292219","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}