Patent Description:
In the elderly population, Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss worldwide<NUM>. It is characterized by confluent soft drusen deposited between retinal pigment epithelium (RPE) and the Bruch's membrane and/or retinal pigmentary changes in the macula at the early stage (intermediate AMD). At later stages, advanced AMD is characterized by two major subtypes, geographic atrophy (dry AMD) or choroidal neovascularization (wet AMD) in the macula<NUM>. While anti-VEGF therapies have been used to control wet AMD, currently there is no approved therapy for dry AMD<NUM>,<NUM>.

The pathogenesis of AMD involves both genetic and environmental factors. Numerous studies have identified variations at the loci of genes that are associated with AMD susceptibility, including complement factor H (CFH), age-related maculopathy susceptibility <NUM> (ARMS2), HtrA serine peptidase <NUM> (HTRA1), indicating that AMD is possible an inflammatory disease<NUM>-<NUM>. Currently, the environmental factors triggering the local inflammation and leading to the early soft drusen in AMD pathology are not clear.

There is a need for improved compositions and methods for assessing, treating or preventing intraocular diseases or disorders in a subject, e.g., a mammal or a human. The present disclosure addresses this and other related needs.

Related technologies are known from the following documents: <NPL>, <NPL>, <NPL>,Rockville, MD, <CIT>, <CIT>, <NPL>, <NPL>, <NPL>, <NPL>; <NPL>, <CIT>, <CIT>, <CIT>, <CIT>; <NPL>, and <NPL>.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunology, and pharmacology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, <NPL>); <NPL>); <NPL>); <NPL>); <NPL>, and periodic updates); <NPL>); and <NPL>).

As used herein, "a" or "an" means "at least one" or "one or more.

The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length, e.g., at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,<NUM> or more amino acids. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

As used herein, the terms "variant" is used in reference to polypeptides that have some degree of amino acid sequence identity to a parent polypeptide sequence. A variant is similar to a parent sequence, but has at least one substitution, deletion or insertion in their amino acid sequence that makes them different in sequence from a parent polypeptide. Additionally, a variant may retain the functional characteristics of the parent polypeptide, e.g., maintaining a biological activity that is at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% of that of the parent polypeptide.

An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule, and can be an immunoglobulin of any class, e.g., IgG, IgM, IgA, IgD and IgE. IgY, which is the major antibody type in avian species such as chicken, is also included within the definition. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')<NUM>, Fv), single chain (ScFv), mutants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.

As used herein, the term "antigen" refers to a target molecule that is specifically bound by an antibody through its antigen recognition site. The antigen may be monovalent or polyvalent, i.e., it may have one or more epitopes recognized by one or more antibodies. Examples of kinds of antigens that can be recognized by antibodies include polypeptides, oligosaccharides, glycoproteins, polynucleotides, lipids, etc..

As used herein, the term "epitope" refers to a portion of an antigen, e.g., a peptide sequence of at least about <NUM> to <NUM>, preferably about <NUM> to <NUM> or <NUM>, and not more than about <NUM>,<NUM> amino acids (or any integer there between), which define a sequence that by itself or as part of a larger sequence, binds to an antibody generated in response to such sequence. There is no critical upper limit to the length of the fragment, which may, for example, comprise nearly the full-length of the antigen sequence, or even a fusion protein comprising two or more epitopes from the target antigen. An epitope for use in the subject invention is not limited to a peptide having the exact sequence of the portion of the parent protein from which it is derived, but also encompasses sequences identical to the native sequence, as well as modifications to the native sequence, such as deletions, additions and substitutions (conservative in nature).

As used herein, the term "specifically binds" refers to the binding specificity of a specific binding pair. Recognition by an antibody of a particular target in the presence of other potential targets is one characteristic of such binding. Specific binding involves two different molecules wherein one of the molecules specifically binds with the second molecule through chemical or physical means. The two molecules are related in the sense that their binding with each other is such that they are capable of distinguishing their binding partner from other assay constituents having similar characteristics. The members of the binding component pair are referred to as ligand and receptor (anti-ligand), specific binding pair (SBP) member and SBP partner, and the like. A molecule may also be an SBP member for an aggregation of molecules; for example an antibody raised against an immune complex of a second antibody and its corresponding antigen may be considered to be an SBP member for the immune complex.

"Polynucleotide," or "nucleic acid," as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The <NUM>' and <NUM>' terminal OH can be phosphorylated or substituted with amines or organic capping groups moieties of from <NUM> to <NUM> carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, <NUM>'-O-methyl-<NUM>'-O- allyl, <NUM>'-fluoro- or <NUM>'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S("thioate"), P(S)S ("dithioate"), "(O)NR <NUM> ("amidate"), P(O)R, P(O)OR', CO or CH <NUM> ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (<NUM>-<NUM> C) optionally containing an ether (--O--) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

"Oligonucleotide," as used herein, generally refers to short, generally single stranded, generally synthetic polynucleotides that are generally, but not necessarily, less than about <NUM> nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.

As used herein, the term "homologue" is used to refer to a nucleic acid which differs from a naturally occurring nucleic acid (e.g., the "prototype" or "wild-type" nucleic acid) by minor modifications to the naturally occurring nucleic acid, but which maintains the basic nucleotide structure of the naturally occurring form. Such changes include, but are not limited to: changes in one or a few nucleotides, including deletions (e.g., a truncated version of the nucleic acid) insertions and/or substitutions. A homologue can have enhanced, decreased, or substantially similar properties as compared to the naturally occurring nucleic acid. A homologue can be complementary or matched to the naturally occurring nucleic acid. Homologues can be produced using techniques known in the art for the production of nucleic acids including, but not limited to, recombinant DNA techniques, chemical synthesis, etc..

As used herein, "substantially complementary or substantially matched" means that two nucleic acid sequences have at least <NUM>% sequence identity. Preferably, the two nucleic acid sequences have at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% of sequence identity. Alternatively, "substantially complementary or substantially matched" means that two nucleic acid sequences can hybridize under high stringency condition(s).

In general, the stability of a hybrid is a function of the ion concentration and temperature. Typically, a hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Moderately stringent hybridization refers to conditions that permit a nucleic acid molecule such as a probe to bind a complementary nucleic acid molecule. The hybridized nucleic acid molecules generally have at least <NUM>% identity, including for example at least any of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% identity. Moderately stringent conditions are conditions equivalent to hybridization in <NUM>% formamide, 5x Denhardt's solution, 5x SSPE, <NUM>% SDS at <NUM>, followed by washing in <NUM>. 2x SSPE, <NUM>% SDS, at <NUM>. High stringency conditions can be provided, for example, by hybridization in <NUM>% formamide, 5x Denhardt's solution, 5x SSPE, <NUM>% SDS at <NUM>, followed by washing in <NUM>. 1x SSPE, and <NUM>% SDS at <NUM>. Low stringency hybridization refers to conditions equivalent to hybridization in <NUM>% formamide, 5x Denhardt's solution, 6x SSPE, <NUM>% SDS at <NUM>, followed by washing in 1x SSPE, <NUM>% SDS, at <NUM>. Denhardt's solution contains <NUM>% Ficoll, <NUM>% polyvinylpyrolidone, and <NUM>% bovine serum albumin (BSA). 20x SSPE (sodium chloride, sodium phosphate, ethylene diamide tetraacetic acid (EDTA)) contains <NUM> sodium chloride, <NUM> sodium phosphate, and <NUM> (EDTA). Other suitable moderate stringency and high stringency hybridization buffers and conditions are well known to those of skill in the art.

As used herein, "vector (or plasmid)" refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well known within the skill of the artisan. An expression vector includes vectors capable of expressing DNA's that are operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

As used herein, "a promoter region or promoter element" refers to a segment of DNA or RNA that controls transcription of the DNA or RNA to which it is operatively linked. The promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter. In addition, the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences may be cis acting or may be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. Exemplary promoters contemplated for use in prokaryotes include the bacteriophage T7 and T3 promoters, and the like.

As used herein, "operatively linked or operationally associated" refers to the functional relationship of DNA with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences. For example, operative linkage of DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA. In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter <NUM>' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation (i.e., start) codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus sites can be inserted immediately <NUM>' of the start codon and may enhance expression. See, e.g., <NPL>. The desirability of (or need for) such modification may be empirically determined.

"Treating" or "treatment" or "alleviation" refers to therapeutic treatment wherein the object is to slow down (lessen) if not cure the targeted pathologic condition or disorder or prevent recurrence of the condition. A subject is successfully "treated" if, after receiving a therapeutic amount of a therapeutic agent or treatment, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of the particular disease. Reduction of the signs or symptoms of a disease may also be felt by the patient. A patient is also considered treated if the patient experiences stable disease. In some embodiments, treatment with a therapeutic agent is effective to result in the patients being disease-free <NUM> months after treatment, preferably <NUM> months, more preferably one year, even more preferably <NUM> or more years post treatment. These parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician of appropriate skill in the art. In some embodiments, "treatment" means any manner in which the symptoms of a condition, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein. In some embodiments, "amelioration" of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.

The term "prediction" or "prognosis" is often used herein to refer to the likelihood that a patient will respond either favorably or unfavorably to a drug or set of drugs, or the likely outcome of a disease. In one embodiment, the prediction relates to the extent of those responses or outcomes. In one embodiment, the prediction relates to whether and/or the probability that a patient will survive or improve following treatment, for example treatment with a particular therapeutic agent, and for a certain period of time without disease recurrence. The predictive methods of the invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient. The predictive methods of the present invention are valuable tools in predicting if a patient is likely to respond favorably to a treatment regimen, such as a given therapeutic regimen, including for example, administration of a given therapeutic agent or combination, surgical intervention, steroid treatment, etc..

As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, e.g., <NPL>). Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.

A "pharmaceutically acceptable salt" is intended to mean a salt of a free acid or base of a compound represented herein that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to the subject. See, generally, <NPL>. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of subjects without undue toxicity, irritation, or allergic response. A compound described herein may possess a sufficiently acidic group, a sufficiently basic group, both types of functional groups, or more than one of each type, and accordingly react with a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt.

Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-<NUM>,<NUM>-dioates, hexyne-<NUM>,<NUM>-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene-<NUM>-sulfonates, naphthalene-<NUM>-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, and mandelates.

As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of a therapeutic agent that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject is effective to prevent or ameliorate an intraocular disease or disorder in a subject. A therapeutically effective dose further refers to that amount of the therapeutic agent sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. In some embodiment, "an effective amount of a compound for treating a particular disease" is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective. The amount may cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. Repeated administration may be required to achieve the desired amelioration of symptoms.

The term "combination" refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where a compound and a combination partner (e.g., another drug as explained below, also referred to as "therapeutic agent" or "co-agent") may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g., synergistic effect. The terms "co-administration" or "combined administration" or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term "pharmaceutical combination" as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

As used herein, "biological sample" refers to any sample obtained from a living or viral source or other source of macromolecules and biomolecules, and includes any cell type or tissue of a subject from which nucleic acid or protein or other macromolecule can be obtained. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. For example, isolated nucleic acids that are amplified constitute a biological sample. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants and processed samples derived therefrom.

The terms "level" or "levels" are used to refer to the presence and/or amount of a target, e.g., a microorganism that is part of the etiology of an intraocular disease or disorder, and can be determined qualitatively or quantitatively. A "qualitative" change in the target, microorganism level refers to the appearance or disappearance of a target, microorganism that is not detectable or is present in samples obtained from normal controls. A "quantitative" change in the levels of one or more targets, microorganisms refers to a measurable increase or decrease in the target, microorganism levels when compared to a healthy control.

A "healthy control" or "normal control" is a biological sample taken from an individual who does not suffer from an intraocular disease or disorder. A "negative control" is a sample that lacks any of the specific analyte the assay is designed to detect and thus provides a reference baseline for the assay.

As used herein, "mammal" refers to any of the mammalian class of species. Frequently, the term "mammal," as used herein, refers to humans, human subjects or human patients. "Mammal" also refers to any of the non-human mammalian class of species, e.g., experimental, companion or economic non-human mammals. Exemplary non-human mammals include mice, rats, rabbits, cats, dogs, pigs, cattle, sheep, goats, horses, monkeys, Gorillas and chimpanzees.

As used herein, "production by recombinant means" refers to production methods that use recombinant nucleic acid methods that rely on well-known methods of molecular biology for expressing proteins encoded by cloned nucleic acids.

As used herein, the term "subject" is not limited to a specific species or sample type. For example, the term "subject" may refer to a patient, and frequently a human patient. However, this term is not limited to humans and thus encompasses a variety of non-human animal or mammalian species.

As used herein, a "prodrug" is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. To produce a prodrug, the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes. The prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design prodrugs of the compound (see, e.g., <NPL>).

It is understood that aspects and embodiments of the invention described herein include "consisting" and/or "consisting essentially of" aspects and embodiments.

Throughout this disclosure, various aspects of this invention are presented in a range format. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from <NUM> to <NUM> should be considered to have specifically disclosed sub-ranges such as from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM> etc., as well as individual numbers within that range, for example, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>.

Other objects, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings.

In another aspect, disclosed herein is a kit or device for assessing an intraocular disease or disorder in a subject, which kit or device comprises reagents for assessing the presence, absence, quantity, the infectious status, and/or the microbiota of a microorganism in an intraocular space, cavity or sample of a subject, wherein the etiology of said intraocular disease or disorder comprises infection of said microorganism in said intraocular space or cavity of said subject.

The present kit or device can comprise any suitable reagents. In some embodiments, the microorganism is a bacterium, a fungus, a virus, or a combination thereof, and the reagents of the present kit or device are configured for assessing the presence, absence, quantity, the infectious status, and/or the microbiota of said microorganism.

The present kit or device can comprise reagents for assessing any suitable number of microorganism(s) in the intraocular space or sample to assess an intraocular disease or disorder in a subject. the present kit or device comprises reagents for assessing the presence, absence, quantity, and/or the infectious status of a single microorganism in the intraocular space, cavity or sample for assessing an intraocular disease or disorder in a subject. The present kit or device comprises reagents for assessing the presence, absence, quantity, and/or the infectious status of multiple microorganisms in the intraocular space, cavity or sample for assessing an intraocular disease or disorder in a subject. The present kit or device comprises reagents for assessing microbiota in the intraocular space, cavity or sample for assessing an intraocular disease or disorder in a subject.

In another example, the present kit or device can comprise reagents to be used in a molecular assay. Any suitable molecular assay can be used. In some embodiments, the present kit or device can comprise reagents for isolating, amplifying, ligating, hybridizing and/or sequencing a polynucleotide of the microorganism. The polynucleotide of the microorganism can be amplified using any suitable technique or procedure. In some embodiments, the present kit or device can comprise reagents for amplifying the polynucleotide using polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA), ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA), primer extension, rolling circle amplification (RCA), self-sustained sequence replication (3SR), or loop-mediated isothermal amplification (LAMP). The polynucleotide of the microorganism can be sequenced using any suitable technique or procedure. In some embodiments, the present kit or device can comprise reagents for polynucleotide sequencing that is conducted with a format selected from the group consisting of Maxam-Gilbert sequencing, a chain-termination method, shotgun sequencing, bridge PCR, single-molecule real-time sequencing, ion semiconductor (ion torrent sequencing), sequencing by synthesis, sequencing by ligation (SOLiD sequencing), chain termination (Sanger sequencing), massively parallel signature sequencing (MPSS), polony sequencing, <NUM> pyrosequencing, Illumina (Solexa) sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, nanopore DNA sequencing, tunnelling currents DNA sequencing, sequencing by hybridization, sequencing with mass spectrometry, microfluidic Sanger sequencing, a microscopy-based technique, RNAP sequencing, and in vitro virus high-throughput sequencing.

In some embodiments, the present kit or device comprises reagents for sequencing analysis and/or PCR analysis. For example, the present kit or device can comprise reagents for metagenomic sequencing analysis, e.g., metagenomic sequencing analysis of the microbiota. In another example, the present kit or device can comprise reagents for real-time PCR analysis, e.g., real-time PCR analysis of the microbiota.

The present kit or device can further comprise additional tools. For example, the present kit or device can further comprise a tool for obtaining a sample from an intraocular space or cavity of a subject. The tool can be configured for obtaining a sample from any suitable intraocular space or cavity of a subject. For example, the tool can be configured for obtaining a sample from aqueous humor in anterior chamber, a suspensory ligament, ciliary body, ciliary body and muscle, vitreous humor in posterior chamber, retina, choroid, optic nerve, lens, or iris of a subject.

The present kit or device can comprise reagents for assessing any suitable intraocular disease or disorder in a subject. The present kit or device can comprise reagents for assessing age-related macular degeneration (AMD) in a subject.

In some embodiments, the present kit or device comprises reagents for assessing the presence, absence, quantity, the infectious status, and/or the microbiota of one or more of the following Bacillus megaterium orBacillus licheniformis in the intraocular space, cavity or sample of a subject for assessing AMD in the subject.

In one embodiment, the present kit or device comprises reagents for assessing the presence, absence, quantity, the infectious status, and/or the microbiota of Bacillus megaterium for assessing AMD in the subject. Bacillus megaterium was assessed using any suitable technique or procedure. The present kit or device comprises reagents for assessing live Bacillus megaterium for assessing AMD in the subject. In another example, the present kit or device comprises reagents for assessing Bacillus megaterium using any suitable immunoassay or molecular assay. In a preferred embodiment, the present kit or device comprises reagents for assessing Bacillus megaterium using sequencing analysis, e.g., metagenomic sequencing analysis of the microbiota, and/or PCR analysis, e.g., real-time PCR analysis of the microbiota. In still another example, Bacillus megaterium can be assessed using technique, procedure or kit in the following Section F. The present kit or device comprises one or more of the following primer pair(s):<NUM>) SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, (<NUM>) SEQ ID NO:<NUM>, SEQ ID NO:<NUM> for assessing Bacillus megaterium.

In some embodiments, the present kit or device comprises a reagent for assessing additional relevant parameters for assessing AMD in the subject. The present kits or devices can be used for any suitable purposes. For example, the present kits or devices can be used for risk assessment, diagnosis, prognosis, stratification and/or treatment monitoring of an intraocular disease or disorder in a subject. For example, the present kits or devices can be used for risk assessment, diagnosis, prognosis, stratification and/or treatment monitoring of AMD of any suitable type or at any suitable stage, e.g., early AMD, intermediate AMD, late AMD, dry AMD, geographic atrophy (also called atrophic AMD), or wet AMD.

In some embodiments, the present disclosure belongs to the technical field of diagnosis of ophthalmic diseases.

It has long been known that the intraocular cavity is absolutely sterile under healthy conditions, because tears and blinks can remove foreign bodies such as microbiota. However, recent studies have also found that even normal healthy eyes have microbiotas. Some of these microbiotas are symbiotic bacteria, while others are pathogenic bacteria.

Age-related macular degeneration (AMD), which can be divided into dry AMD and wet AMD, is an aging change of the macular structure and the leading cause of irreversible vision loss in the elderly.

Dry AMD is due to the long-term chronic progressive atrophy of the RPE-Bruch membrane-choroidal capillary complex. Dry AMD occurs mostly in the elderly over the age of <NUM>. The visual acuity of the bilateral eyes declines symmetrically and extremely slow, and patients often have symptoms such as visual distortion. Fundus examination shows the pigmentation disorder of the macula in the eyes, the disappearance of the foveal light reflex, and sometimes the yellow-white drusen with different sizes and unclear borders in the posterior pole. In the late stage of the disease, due to the atrophy and pigmentation loss of RPE (retinal pigment epithelium (RPE), it can be seen that there is a geographic atrophy zone with relatively clear border in the posterior pole of the retina in some patients. If the choroidal capillaries also get shrink, some thick choroidal blood vessels can be seen in the atrophy zone.

Wet AMD is due to the formation of the choroidal neovascularization (CNV), which is induced by the damage of the Bruch membrane and the growth of choroidal capillaries to the retinal pigment epithelium and retinal nerve epithelium through the lesion of Bruch membrane. Once the CNV is formed, due to the imperfect structure of the new blood vessels, a series of pathological changes such as exudation, hemorrhage, mechanization and scarring appear, and the central vision will lose to be exhausted eventually.

With the increase of age, the phagocytic function of the retinal pigment epithelium declines, therefor, the photoreceptor extracellular disc cannot be completely digested. The residual metabolites continuously discharge from the RPE cells and deposit on the glass membrane to form a drusen.

The drusen is divided into four categories: hard, soft, mixed and degraded drusen. The drusen can be found in the elderly with normal vision. The increase in the number of drusen, the increase in pigment, and the increase in fusion are the characteristics that drusen has risk factors. The accumulation of drusen on the vitreous membrane has an impact on the retina's absorption of nutrients and oxygen through the choroidal vessels, and also destroys the integrity of Bruch's membrane. When the retina is hypoxic or ischemic, it will induce to the up-regulation of vascular endothelial growth factor (VEGF), integrin and protease. When VEGF signal is stronger than pigment epithelium derived factor (PEDF), it will enhance the angiogenesis and stimulate the growth of new blood vessels. Increased expression of VEGF and rupture of Bruch's membrane ultimately lead to the formation of CNV, which in turn causes exudation, hemorrhage and scar formation.

In the past <NUM> years, the diversity and function of microbiota associated with human health and diseases have been extensively studied through high-throughput sequencing technologies and macrobiotic/metagenomic analysis. The local microbiota of the eye under physiological and pathological conditions remains largely uncharacterized. The theory that the intraocular cavity is absolutely sterile under physiological conditions has led many researchers to reason that any types of foreign organisms are exogenous and pathogenic. However, the present disclosure indicates that even normal healthy eyes with no signs of ocular distress or infection have an individualized microbiota with compositional and functional diversity distinct from other body sites and tissues. Interestingly, the fact that P. acnes lives in the majority of human eyes and does not significantly induce intraocular inflammation raises a reasonable hypothesis that the normal intraocular microbiota plays a key role in maintaining the homeostasis of the local ocular environment. Similarly, the dysbiosis of the local microbial community can contribute to the etiology of many infectious, inflammatory, neoplastic, and degenerative ocular diseases. In addition, the idea that culture-positive microbiota such as P. acnes were the major causes of intraocular inflammation warrants reexamination since these microbiota may be part of the intraocular commensal flora while the real pathogens were unexpected and missed. Many ocular procedures such as surgeries and intravitreal injection of anti-VEGF agents may also trigger the exposure of pathogenic intraocular microbiota to host immune system. Therefore, future studies are needed to clearly define the association of intraocular microbiota with integrity of ocular health.

Genetic studies have successfully identified various factors involved in the pathogenesis of AMD. These factors all suggest that the activation of complement cascade and controlling of immune responses are the keys for AMD onset and progress. However, the initiator of AMD pathology (especially how drusen is formed) and the critical link between complement and AMD pathology have been unclear.

The existing technology doesn't clearly clarify the cause of age-related macular degeneration. The diagnosis can only be based on the patient's symptoms. No definitive basis can help the doctor make an accurate judgment, which may lead to misdiagnosis or delay the best time for treatment. In the existing technology, vitrectomy is commonly used to treat age-related macular degeneration, however the improvement of visual acuity after surgery is very limited. Sometimes multiple operations are required to maintain the patient's weak vision and eyeballs. Some patients still have no treatment effect even after multiple operations.

The present disclosure also provides a kit for diagnosing age-related macular degeneration, which is capable of detecting the abundance of microbiota by real-time PCR analysis, so as to diagnose age-related macular degeneration.

In the specific implement way of the present disclosure, a kit for the diagnosis of age-related macular degeneration includes: (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs2) : SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, or, primer pairs3) SEQ ID NO:<NUM>, SEQ ID NO:<NUM>; (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water.

The kit of the present disclosure comprises: (<NUM>) mix; (<NUM>) primer pairs1) : SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, or, primer pairs2) SEQ ID NO:<NUM>, SEQ ID NO:<NUM>; (<NUM>) sterilized water, the mix in the kit contains SYBR GreenI, Taq enzyme, dNTP, buffer.

The present disclosure finds that all human eyes we tested have intraocular microbiota, we next investigated whether a disease-specific intraocular microbiota could characterize ocular manifestations. We carried out metagenomic sequencing analysis on aqueous humor specimens from <NUM> cataract (Cat), <NUM> AMD, <NUM> glaucoma (GLA), <NUM> Betch's disease (BD), <NUM> Vogt-Koyanagi-Harada Syndrome (VKH), and <NUM> endophthalmitis (EOS) patients. Interestingly, the alpha diversity and evenness of the intraocular microbial communities were significantly different among these <NUM> types of patients, despite all patients having bacteria as the major component of their intraocular microbiome. The principal component analysis (PCA) on the composition of the intraocular microbiota (using all microbial species) showed clear differences among cataract, EOS, and some glaucoma patients. However, AMD, VKH, BD, and some glaucoma patients shared indistinguishable features in their intraocular microbiome. Similarly, hierarchical clustering analysis of the abundance of functional microbial genes from all metagenomes indicated that each ocular manifestation had a general signature of microbial function, while there were outliers in every disease group that could be classified to other disease clusters. In spite of the significant individuality presented by the intraocular microbiome, we were able to identify the signature bacterial species for each ocular disease group we tested. Taken together, our results suggest that the composition and function of intraocular microbiota can differentiate ocular diseases such as AMD, cataract, glaucoma, BD, VKH, and EOS.

The present disclosure identified <NUM> bacterial species that were highly enriched in the AH of AMD patients using metagenomic analysis. acnes was the most abundant microorganism in the AH of AMD patients, Bacillus licheniformis (B. licheniformis) and Bacillus megaterium (B. megaterium) were the most enriched species, among the <NUM> AMD-specific ones, in AMD AH specimens. The present disclosure then carried out PCR analysis to investigate whether the <NUM> AMD-specific bacteria could be detected in the hard or soft drusen tissues, as compared to the non-drusen retinal tissues from <NUM> archived ocular slides of AMD patients. Our results showed only <NUM> bacteria could be detected, among which P. acnes was the most abundant species and B. megaterium was the only species enriched in soft drusen. The relative abundance of P. acnes was comparable in hard drusen, soft drusen, and dry AMD lesion tissues as compared to the non-drusen non-lesion retinal tissues. The relative abundance of B. megaterium was elevated by ~<NUM> fold in soft drusen but not the AMD lesions when compared to the non-drusen/non-lesion tissues. These data suggest a possible role of B. megaterium in drusen formation and AMD pathogenesis.

Previous studies demonstrate that drusen contains a variety of complement components and polysaccharides in addition to many other proteins. In addition, the drusen components activate inflammasomes and promote expression of IL-1β and IL-<NUM>. The present disclosure therefore first examined whether B. megaterium, as a component of drusen, was able to induce the activation of complement system and promote the secretion of IL-1β and IL-<NUM>, by acute retinal pigment epitheliitis-<NUM>(ARPE19) cells in vitro. We found B. megaterium but not P. acnes significantly increased the pyroptosis of RPE cells in a time dependent manner. The activation of complement system was confirmed by the production of active form of C5A protein. Both bacteria induced secretion of CFH proteins secreted by ARPE19 cell, while the induction of CFH was more profound by B. megaterium than by P. As the result of pyroptosis, in vitro infection of B. megaterium, but not P. acnes, led to secretion of active IL-1B and IL-<NUM> by RPE cells. These results indicate that infection of B. megaterium can lead to inflammation similarly found in soft drusen.

The present disclosure next tested whether B. megaterium was able to induce inflammation in vivo. We chose the non-human primate macaque (Macaca fascicularis) as our model system considering the ocular anatomy and intraocular environment shared by human and macaque. Infection of live P. acnes bacterium or inoculation of its sonication-inactivated proteins into the eye, as well as live B. licheniformis bacterium or inoculation of its sonication-inactivated proteins into the eye did not induce significant intraocular inflammation. However, infection of live B. megaterium but not its proteins into the eye led to a profound intraocular inflammation. The intraocular inflammation induced by live B. megaterium was characterized by the elevation of TNFA and IL6 but not IFNG and IL17A expression. Importantly, only live B. megaterium was able to activate complement system including C5A and CFH and induce pyroptotic cytokines IL-1β and IL-<NUM> in vivo. The bacteria remained alive in the eyes after inflammation was initiated, suggesting the intraocular inflammation may be long lasting in nature. Taken together, our data demonstrate that infection of B. megaterium can activate complement system and induce pyroptosis of ocular cells in vitro and in vivo.

The present disclosure indicates that bacteria such as B. megaterium located in drusen and activated local complement-mediated immune response can explain the formation of diversified drusen between RPE and Bruch's membrane. The major proteins found in drusen including complement components such as C1Q and immunoglobulin are all first line of anti-infectious agents. Other drusen proteins such as vitronectin and Apolipoprotein E are all recently proved as anti-infectious agents. Therefore, the formation of drusen is very possible the key response of the aging retina in controlling infiltrated bacterial pathogens. Due to the diversity of bacteria, the shape and size of drusen could vary. In the case of hard drusen, where the infection may be cleared, drusen will disappear. However, certain pathogens such as B. megaterium will induce long term activation of immune responses in soft drusen and result in the damage of RPE cells and photoreceptors. Activation of the inflammation of macrophage and pyroptosis of RPE cells are protective responses against local infection, which is consistent with the previous finding that NLRP3 mediated inflammasome activation and IL-<NUM> production protect the retina from neovascularization.

The infectious etiology of AMD is also consistent with the conclusions reached by all genetic studies. For example, a defective CFH, the negative regulator of complement activation induced by B. megaterium infection, will result in uncontrolled complement activation. A defective HTRA1, the protease producing the active form of immunosuppressive cytokine TGF-β, will result in decrease of local TGF-β family proteins. Both of these genetic variations can lead to dysregulation of local anti-infectious responses that damages RPE cells and photoreceptors.

In addition, the potential difference in pathogenic microbiota found in drusen may explain the association of varied genetic risk factors with different ethnic groups (i.e. Caucasian vs Asian). Therefore, based on our data, the infectious etiology of AMD is a plausible mechanism by which early AMD pathology is initiated in the elderly. In summary, the present disclosure provides a novel target for the diagnosis, treatment, and prevention of AMD.

The present disclosure designs and synthesizes universal primers for pathogenic microbiota DNA according to the conserved genomic DNA sequence of common pathogenic microbiota of age-related macular degeneration, and detects pathogenic microbiota by real-time PCR, so as to realize the efficient and accurate detection of pathogenic microbiota of age-related macular degeneration, which help doctors make quick and accurate judgments during the disease diagnosis. And the medicine provided by the present disclosure is prepared based on the cause of age-related macular degeneration and can effectively treat age-related macular degeneration.

A kit for the detection of Propionibacterium acnes contains (<NUM>)SYBR GreenI; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO:<NUM>
GATTGGTTTACTACCCGTGAGCG, SEQ ID NO:<NUM>
ATAGCAGGGATTCCAGCGACA; (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Propionibacterium acnes in the eyes using real-time PCR method.

A kit for the detection of Bacillus megaterium contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO:<NUM> GGTTCAATGAGCCTACT, SEQ ID NO:<NUM> GCCAGCGTCTTTCC; (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Bacillus megaterium in the eyes using real-time PCR method.

A kit for the detection of Bacillus licheniformis contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO:<NUM> TCCCGTCTTCATCTACTGC, SEQ ID NO:<NUM> GGACGCCTACTGGACAA; (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Bacillus licheniformis in the eyes using real-time PCR method.

A kit for the detection of Pseudomonas putida contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO: <NUM> CCGCACAGGTTGTCCCA, SEQ ID NO:<NUM> CTGCTGCGTTGTCGTTCC; (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Pseudomonas putida in the eyes using real-time PCR method.

A kit for the detection of Xanthomonas oryzae contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO:<NUM> TGGTGCGATGGCGATGTT, SEQ ID NO:<NUM> GGTTGCGGCATGTGCTTT; (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Xanthomonas oryzae in the eyes using real-time PCR method.

A kit for the detection of Stenotrophomonas maltophilia contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO:<NUM> GCGTTCGTCCGCTGTCA, SEQ ID NO:<NUM> GGCAACCCGCTAGAATCCC; (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Stenotrophomonas maltophilia in the eyes using real-time PCR method.

A kit for the detection of Lactobacillus reuteri contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO:<NUM> TAGTGGATAATGCCGTTGA, SEQ ID NO:<NUM> CGGTTTGCCAGAAGC; (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Lactobacillus reuteri in the eyes using real-time PCR method.

A kit for the detection of Staphylococcus haemolyticus contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO:<NUM> GTTACACTGCTCCGACAA, SEQ ID NO:<NUM> TTCGCATCAGCAATAA; (<NUM>) dNTP; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Staphylococcus haemolyticus in the eyes using real-time PCR method.

A kit for the detection of Cytophaga hutchinsonii contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO: <NUM> GCTGGCTCCTTTGG, SEQ ID NO:<NUM> GCATTACTGCCTGGTG; (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Cytophaga hutchinsonii in the eyes using real-time PCR method.

A kit for the detection of Gardnerella vaginalis contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO:<NUM> GACTCCGACTTGTTT, SEQ ID NO:<NUM> CATTATCTGGCGTTTTAGC (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Gardnerella vaginalis in the eyes using real-time PCR method.

A kit for the detection of Staphylococcus aureus contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO:<NUM> GAAGCGGAGTTCAAAGG, SEQ ID NO:<NUM> ATGGCAAATCACCAATCA (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Staphylococcus aureus in the eyes using real-time PCR method.

A kit for the detection of Pseudomonas aeruginosa contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO:<NUM> GACCAGGTAGCCGTCGTTCTC, SEQ ID NO:<NUM> TGCTGACCCTGACCGACATTC; (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Pseudomonas aeruginosa in the eyes using real-time PCR method.

A kit for the detection of Bacillus cereus contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO:<NUM> GAAGTGCGTGCGTATAGTGT, SEQ ID NO:<NUM> AAAGAACGACCAAGTGCTG (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Bacillus cereus in the eyes using real-time PCR method.

A kit for the detection of Staphylococcus epidermidis contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO:<NUM> TTGAAGTGAAACGTCCTC, SEQ ID NO:<NUM> TGTCTCATCTAACCACC (<NUM>) dNTP; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Staphylococcus epidermidis in the eyes using real-time PCR method.

A kit for the detection of Enterococcus faecium contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO:<NUM> TGGAGCGATTATACCG, SEQ ID NO:<NUM> GTACCCGCTTGATTGA; (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Enterococcus faecium in the eyes using real-time PCR method.

A kit for the diagnosis of age-related macular degeneration contains (<NUM>)SYBR Green I; (<NUM>) Taq enzyme; (<NUM>) primer pairs SEQ ID NO:<NUM> GGTTCAATGAGCCTACT, SEQ ID NO:<NUM> GCCAGCGTCTTTCC, (<NUM>) dNTP ; (<NUM>) buffer; (<NUM>) sterilized water. The kit is applied on the detection of Bacillus megaterium in the eyes using real-time PCR method.

<NUM> cataract, <NUM> AMD, <NUM> glaucoma, <NUM> BD, <NUM> VKH, and <NUM> EOS patients.

All human eyes we tested have intraocular microbiota, <FIG> is a graph showing the proportion of bacteria, viruses and fungi in the total microbiota in the eyes of patients with different diseases, as shown in <FIG>, all these <NUM> types of patients have bacteria as the major component of their intraocular microbiota. <FIG> is a LefSe analysis graph of bacterial species that were highly enriched in the eyes of patients with different diseases, as shown in <FIG>, patients with different diseases have different kinds of bacteria. <FIG> is a graph showing the relative abundance of P. acnes, Bacillus licheniformis and Bacillus megaterium in the eyes of patients with <NUM> types of diseases. As shown in <FIG>, the eyes of AMD patients contain P. acnes, Bacillus licheniformis and Bacillus megaterium simultaneously, among which the amount of P. acnes is the highest, and the amount of Bacillus licheniformis and Bacillus megaterium is less than that of P. acnes, however it is higher than the amount of Bacillus licheniformis and Bacillus megaterium in the eyes of patients with other <NUM> diseases.

Real-time PCR kit prepared in Examples <NUM>-<NUM>.

Non-drusen, hard drusen, and soft drusen tissues of AMD patients.

As shown in <FIG>, our results showed only <NUM> bacteria could be detected in non-drusen, hard drusen, and soft drusen tissues of AMD using real-time PCR method, P. acnes (a), B. megaterium (b), L. reuteri (c), P. putida (d), P. aeruginosa (e), X. oryzae (f), B. licheniformis (g), and G. vaginalis (h) respectively, among which P. acnes was the most abundant species (<FIG>) and B. megaterium was the only species enriched in soft drusen (<FIG>).

Human ARPE19 cells (American type culture collection, USA).

Bacillus megaterium, Propionibacterium acnes.

Human ARPE19 cells (ATCC, USA) were divided into two groups and cultured in DMEM supplied with <NUM> L-glutamine and <NUM>% FBS. Then, Bacillus megaterium was added to the first group, and P. acnes was added to the second group. After <NUM>, <NUM>, and <NUM> hours of culture in each group, ARPE19 cells were incubated with PI and AnnexcinV-APC (Cat#<NUM>-<NUM>-<NUM>, eBioscience, USA) for <NUM>. The cell death was measured with MACSQuant Analyzer <NUM> flow cytometer (MiltenyiBiotec, Germany).

As shown in <FIG>, we found B. megaterium but not P. acnes significantly increased the pyroptosis of RPE cells in a time dependent manner. As shown in <FIG>, we found the activation of complement system was confirmed by the production of active form of C5A protein. Interestingly, both bacteria induced secretion of CFH proteins by ARPE19 cells, while the induction of CFH was more profound by B. megaterium than by P. As the result of pyroptosis, <FIG> shows that in vitro infection of B. megaterium led to secretion of active IL-1and IL-<NUM> by RPE cells.

These results indicate that infection of B. megaterium can lead to inflammation similarly found in soft drusen.

Bacillus megaterium BNCC190686, Bacillus licheniformis BNCC186069, and Propionibacterium acnes BNCC336649 purchased from BeNa Culture Collection (Beijing, China).

Bacillus megaterium BNCC190686, Bacillus licheniformis BNCC186069, and Propionibacterium acnes BNCC336649 were first cultured overnight at <NUM> on agar plates following the standard protocols provided by the manufacturer. The bacterial cultures were then washed in PBS and resuspended as 1x106CFU/□l solutions and further diluted in PBS as injection solutions.

The macaques were sedated by intramuscular injection of a mixture of Tiletamine Hydrochloride (<NUM>/kg) and Zolazepam Hydrochloride (<NUM>/kg). After topical anesthesia (<NUM>% Proparacaine Hydrochloride), the eyes were immediately visualized in vivo using a light microscope. The pupils were then dilated with <NUM>% tropicamide and <NUM>% phenylephrine to obtain the fundus photographs. Intravitreal injection of bacterial solutions (<NUM> CFU in a volume of 50µl) or sonication-inactivated bacterial proteins (from <NUM> CFU bacteria) was performed with a <NUM> syringe and <NUM>-gauge needle after ocular surface disinfection with <NUM>% PVI solution. In the first group, the left eye of macaques was inoculated with live Propionibacterium acnes bacteria injection, the right eye of macaques was inoculated with live Bacillus licheniformis bacteria injection. In the second group, the left eye of macaques was inoculated with Propionibacterium acnes' sonication-inactivated proteins injection, the right eye of macaques was inoculated with Bacillus licheniformis' sonication-inactivated proteins injection. In the third group, the left eye of macaques was inoculated with live Propionibacterium acnes bacteria injection, the right eye of macaques was inoculated with live Bacillus megaterium bacteria injection. In the fourth group, the left eye of macaques was inoculated with Propionibacterium acnes' sonication-inactivated proteins injection, the right eye of macaques was inoculated with Bacillus megaterium' sonication-inactivated proteins injection.

Slit lamp and fundus examinations were conducted for all macaques within <NUM> days after the injection. The severity of the endophthalmitis was graded according to a previously described standard. The macaques were euthanized <NUM> days post inoculation and both eyeballs were enucleated for histopathological and intraocular cytokine/bacteria analyses. The eyeballs were fixed in <NUM>% paraformaldehyde for <NUM> and then embedded in paraffin. Sections were cut on a microtome at <NUM> and stained with hematoxylin and eosin (H&E).

Results: <FIG> is a fundus photograph of the first and the second group of macaques, and <FIG> is a fundus photograph of the third and the fourth group of macaques, as shown in <FIG>, <NUM> days after the injection of live bacterium or inactivated protein, there was no inflammation in the eyes of the macaques in the first group, the second group, and the fourth group, however, severe inflammation was shown in the right eye of the third group of macaques. Therefore, infection of live P. acnes bacterium or inoculation of its sonication-inactivated proteins into the eye, as well as live B. licheniformis bacterium or inoculation of its sonication-inactivated proteins into the eye did not induce significant intraocular inflammation. However, infection of live B. megaterium but not its proteins into the eye led to a profound intraocular inflammation.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in the elderly worldwide. Despite identification of multiple genetic factors associated with AMD, the environmental factors triggering the damaging local inflammation in AMD remain unclear. Here, using quantitative PCR, negative staining transmission electron microscopy, and high-throughput sequencing technologies, we find that resident microbiota inhabits the intraocular cavities in all eyes. A disease-specific signature of the microbial community differentiates several intraocular diseases including AMD. Importantly, we find that an AMD specific bacterium Bacillus megaterium induces activation of complement and retinal cell death in vitro and in vivo. Our study identifies bacterial infection as the major etiology of early AMD, and provides a novel direction for the diagnosis, treatment, and prevention of the leading blinding disease in late life.

The intraocular environment has long been considered sterile unless invaded by microorganisms due to trauma, surgical procedures, or hematogenous or neurological spread<NUM>. Here, we report that the composition of intraocular microbiota can differentiate ocular diseases. Importantly, we identified an AMD specific bacterium Bacillus megaterium that could promote the development of AMD pathology.

Patients with AMD, cataract (Cat), glaucoma (Gla), Behcet's disease (BD), Vogt-Koyanagi-Harada Syndrome (VKH), and endophthalmitis (EOS) were recruited at Zhongshan Ophthalmic Center (Guangzhou, China) and Tianjin Medical University Eye Hospital (Tianjin, China) between Sep <NUM> and Aug <NUM>. The basic demographic information for these <NUM> cohorts (<NUM> patients) was listed in Table <NUM>. The post mortem eyes were obtained from Guangdong Eye Bank, Guangzhou, China (cohort #<NUM>) (Table <NUM>). This study adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Boards of Zhongshan Ophthalmic Center, Sun Yat-sen University (protocol #2014MEKY024, #2014MEKY032, and #2016KYPJ031), Tianjin Medical University Eye Hospital (protocol #2016KY-<NUM>), and National Eye Institute, National Institutes of Health (protocol #<NUM>-EI-<NUM>). All subjects provided written informed consent before participation.

Fifty µl of fresh aqueous humor (AH) specimens or cultured AH samples were centrifuged at 14000rpm for <NUM>. Supernatant was removed saving <NUM>µl of fluid, which was then loaded onto a copper grid with carbon film. The grid with sample was then stained with <NUM>µl phosphotungstic acid (<NUM>%) for <NUM>. The grid was immediately examined using a JEM2010 electron microscope (JEOL Ltd. The images were acquired on a <NUM> X <NUM> <NUM> CCD camera (Gatan, CA USA). All reagents and grids were sterilized. Water without any AH samples was used as negative controls and no bacteria were found after extensive search in negative controls.

A total of <NUM> ng of DNA from each sample was sonicated into fragments of <NUM>-<NUM> bp using Bioruptor (Diagenode, Belgium) and subjected to sequencing library preparation following the standard protocol provided by the manufacturer using VAHTS Nano DNA Library Prep Kit for Illumina (Vazyme, China). DNA libraries were sequenced to a depth of <NUM>~<NUM> million reads per sample using HiSeq PE Cluster Kit v4 and HiSeq SBS V4 <NUM> cycle kit (Illumina, USA) on the Illumina HiSeq2500 sequencer and subjected to initial processing using CASAVA (v1. <NUM>) (Illumina). All reads were quality controlled and non-human sequences were subjected to analysis of community composition using Kraken<NUM> (with pre-built <NUM> GB database as the reference including complete bacterial, archaeal, and viral genomes in RefSeq as of Dec. <NUM>, <NUM>), as well as functional analysis using HUMAnN2<NUM>. (See also paragraphs [<NUM>]-[<NUM>].

The macaques were sedated by intramuscular injection of a mixture of tiletamine hydrochloride (<NUM>/kg) and xolazepam hydrochloride (<NUM>/kg). After instilling topical anesthesia (<NUM>% proparacaine hydrochloride), the eyes were immediately visualized in vivo using a light microscope. The pupils were then dilated with <NUM>% tropicamide and <NUM>% phenylephrine to obtain the fundus photographs. Then a <NUM> gauge anterior chamber cannula was inserted through a sclerotomy and advanced through the vitreous. Under microscopic monitoring, <NUM> of PBS (with or without bacteria) was injected into the subretinal space between photo receptors and RPE, using a NanoFil Syringe Nanofil-<NUM> for Microinjection (World Precision Instruments, USA). All procedures were done using sterile instruments. The macaque was euthanized <NUM> days post inoculation and the eyeball was enucleated for histological analyses. The eyeballs were fixed in <NUM>% paraformaldehyde for <NUM> and then embedded in paraffin. Sections were cut on a microtome at <NUM> and stained with H&E.

Our preliminary study found Propionibacterium acnes (P. acnes) (both DNA and RNA), a key pathogen inducing endophthalmitis<NUM>, in uninflamed human eyes. This argued against the traditional idea that intraocular environment should be sterile in normal eyes. We therefore collected <NUM> AH specimens from eyes undergoing cataract surgeries that were free of active or history of intraocular inflammation and infection (The summary demographic characteristics of all patients were listed in cohort <NUM> Table <NUM>). Surprisingly, we were able to detect the <NUM> rRNA expression of Propionibacterium spp. in <NUM>% of eyes and RNA expression of a P. acnes specific gene - PPA_RS04200 in <NUM>% of eyes, based on the real-time PCR assays (<FIG>, <FIG>). However, the intraocular load of P. acnes was not age-related (<FIG>). It was unexpected that a commensal bacterium of human skin and opportunistic pathogen associated with post-cataract-surgery endophthalmitis<NUM> could be detected in a majority of eyes from patients experiencing no ocular inflammation before or after the cataract surgery.

Identification of intraocular P. acnes led us to ask the question whether it is possible that other microorganisms also live inside of the normal eyes. Therefore, we carried out single cell analysis of aqueous humors from cataract patients, using Imaging Flow Cytometry technology. By spiking in round beads, we found many microorganisms with a diameter less than <NUM> (the size of beads) in AH (<FIG>). Interestingly, live human cells (DAPI positive) (<FIG>), spherical/rod microorganisms (DAPI positive) (<FIG>), and beads (<FIG>) were all visible on the images of particles. We found similar staining of microbial cells in all <NUM> AHs from <NUM> cataract patients and <NUM> post mortem eyes we tested.

To directly visualize bacteria in intraocular fluid, AH specimens were examined using negative staining transmission electron microscopy. As a positive control, the rod-shaped cultured P. acnes were successfully visualized. In negative controls containing no AH specimens no bacterium could be found using the identical negative staining protocol (<FIG>). Intriguingly, multiple round- and rod-shaped bacteria were found in AH samples (<FIG> and <FIG>). Within some bacteria, endospores were evident. These data demonstrated the existence of multiple types of intraocular bacteria in cataract patients.

It is expected that intraocular bacteria can be cultured if they can be found using a microscope. We therefore made multiple attempts to culture out the bacteria from AH samples. Using <NUM> Agar-based culture mediums with various nutrients we found no positive cultures of AH samples from cataract patients (<FIG>), however cultures using liquid cooked meat medium (<FIG>) were found positive for bacteria and could be visualized using standard light microscopes (<FIG>). On average, we found ~<NUM> CFU (colony forming units) bacteria per <NUM> AH from each eye and <NUM> out of <NUM> (<NUM>%) eyes were found bacteria positive (<FIG>). Cultured bacteria from these AH samples were also examined using negative staining transmission electron microscopy. Both round- and rod-shaped bacteria were found (<FIG>). These data confirmed the existence of multiple types of intraocular bacteria.

Identification and visualization of intraocular bacteria led us to ask the question whether a microbiota lives inside of normal eyes. To identify the composition and function of intraocular microbial communities in AH, we collected specimens of AH, conjunctiva, plasma, and eyelid skin from <NUM> patients undergoing cataract surgery (Table <NUM> presents the demographic characteristics of all patients that are listed in cohort <NUM>). High-throughput sequencing technology was used to profile the metagenomes of these specimens. A large number of human reads were detected among samples from all tissues (<FIG>); however, the relative amount of human reads within the AH samples (~<NUM>%) was significantly lower than the other three tissues (<FIG>). The average numbers of microbial genes detected in AH samples (<FIG>) as well as the alpha diversity (detected by Shannon Index) of the microbial community in AH (<FIG>) were significantly higher than in the other three tissues. Unsupervised hierarchical clustering of metabolic pathways in microbiota from the four tissues found a unique metabolic pattern enriched in AH samples (Table <NUM>) that was distinct from the other three tissues, which were indistinguishable (<FIG>). A principle coordinate analysis (PCoA) of community similarity (including bacteria, fungi, and viruses) indicated that the microbiome of AH was different from the other three tissues (<FIG> and <FIG>-c). The major kingdom of microorganisms was bacteria in all tissues, but the relative abundance of fungal and viral species differed among four tissues (<FIG>). In addition to the detection of.

Importantly, no DNA was detected in multiple negative controls including Blank (no AH), Wash Solution, Anesthesia, Disinfectant, NaCl Solution, and Mydriatic samples and metagenomic sequencing of these negative controls resulted in few quality reads (see supplementary RESULTS and <FIG>-b). These data suggest that the microbiome detected in the AH specimens is not a contamination from conjunctiva, blood, or skin during sampling process. Instead, it represents a unique live community. Importantly, the intraocular microbiome, profiled by metagenomic sequencing analysis, resides in all animals we tested including rat, rabbit, pig, and macaque (<NUM> eyes per species). (See also paragraphs [<NUM>]-[<NUM>].

As all human eyes we tested have intraocular microbiota, we next investigated whether a disease-specific intraocular microbiome could characterize ocular manifestations. We carried out metagenomic sequencing analysis on aqueous humor specimens from <NUM> cataract, <NUM> AMD, <NUM> glaucoma, <NUM> BD, <NUM> VKH, and <NUM> EOS patients (Table <NUM> lists the summary demographic characteristics of all patients in cohort <NUM>-<NUM>). Interestingly, the alpha diversity and evenness of the intraocular microbial communities were significantly different among these <NUM> types of patients (<FIG>), despite all patients having bacteria as the major component of their intraocular microbiome (<FIG>-c). The PCoA on the composition of the intraocular microbiota (using all microbial species) showed clear differences among cataract, EOS, and some glaucoma patients. However, AMD, VKH, BD, and some glaucoma patients shared indistinguishable features in their intraocular microbiome when examining the first two principle coordinates (<FIG> and <FIG>-e). Importantly, the intraocular microbial communities in all patients were significantly different from all sequencing experiments that included specimen-free negative controls (<FIG>). Similarly, hierarchical clustering analysis of the abundance of functional microbial genes from all metagenomes indicated that each ocular manifestation had a general signature of microbial function, while there were outliers in every disease group that could be classified to other disease clusters (<FIG>). In spite of the significant individuality presented by the intraocular microbiome (<FIG>), we were able to identify signature bacterial species for each ocular disease group we tested (<FIG>). Taken together, our results suggest that the composition and function of intraocular microbiota can differentiate ocular diseases such as AMD, cataract, glaucoma, BD, VKH, and EOS.

Our metagenomic analysis identified <NUM> bacterial species that were highly enriched in the AH of AMD patients (<FIG> and Table <NUM>). acnes was the most abundant microorganism in the AH of AMD patients (<FIG>), Bacillus licheniformis (B. licheniformis) and Bacillus megaterium (B. megaterium) (<FIG>) were the most enriched species, among the <NUM> AMD-specific ones, in AMD AH specimens (Table <NUM>). We then carried out PCR analysis to investigate whether the <NUM> AMD-specific bacteria could be detected in the hard or soft drusen tissues, as compared to the non-drusen retinal tissues from <NUM> archived ocular slides of AMD patients. Our results showed only <NUM> bacteria could be detected (<FIG>-h), among which P. acnes was the most abundant species (<FIG>) and B. megaterium was the only species enriched in soft drusen (<FIG>). Intriguingly, the relative abundance of P. acnes was comparable in hard drusen, soft drusen, and dry AMD lesion tissues as compared to the non-drusen non-lesion retinal tissues (<FIG>). The relative abundance of B. megaterium was elevated by ~<NUM> fold in soft drusen when compared to the non-drusen/non-lesion tissues (<FIG>). These data suggest a possible role of B. megaterium in drusen formation and AMD pathogenesis.

Next, we tested whether B. licheniformis and B. megaterium were able to induce inflammation in vivo. We chose the non-human primate macaque (Macaca fascicularis) as our model system considering the ocular anatomy and intraocular environment shared by human and macaque. As shown in <FIG>, intravitreal infection of live P. acnes or B. licheniformis did not induce significant intraocular inflammation. Neither did injection of sonication-inactivated proteins of either P. acnes or B. licheniformis induce significant inflammation. Conversely, infection of live B. megaterium but not its proteins into the eye led to a profound intraocular inflammation (<FIG>). The intraocular inflammation induced by live B. megaterium was characterized by the elevation of TNFA and IL6 but not IFNG or IL17A expression (<FIG>-d). Importantly, only live B. megaterium was able to activate complement system including C5A and CFH (<FIG> and <FIG>-f) and induce pyroptotic cytokines IL-1β and IL-<NUM> in vivo (<FIG>). The bacteria remained alive in the eyes after inflammation was initiated (<FIG>-h), suggesting the intraocular inflammation may be long lasting in nature. These data demonstrate that intravitreal infection of B. megaterium can activate complement system and induce intraocular inflammation in vivo.

megaterium was a cause of AMD, the Kock's postulate has to be satisfied. Our above results demonstrated the existence of B. megaterium in both AH and retinal tissues (<FIG>, <FIG>, and Table <NUM>). We again collected both AH and vitreous humor (VH) specimens from AMD patients and were able to detect B. megaterium DNA in both uncultured and cultured samples (<FIG>-c). We next examined whether subretinal inoculation of AH and VH cultures which had B. megaterium, as well as the cultured single species of B. megaterium led to AMD like pathology in macaque (<FIG>-b). About <NUM> CFU of bacteria (in <NUM> □l PBS) from AH, VH, and B. megaterium cultures were injected subretinally and PBS was used as a control (illustrated in <FIG>). The fundus examination of macaque eye was performed before (Day <NUM>) and after bacterial inoculation on Day <NUM>, Day <NUM>, Day <NUM> (data not shown), as well as Day <NUM> (<FIG>). The PBS injection left only visible scar on the retina, while all bacterial inoculations led to drusenoid lesions on retinal tissues (<FIG>). Drusen-like nodules were also visible under the RPE layer (<FIG>). Fluorescence in situ hybridization results also located B. megaterium in drusenoid but not in the normal tissues post inoculation (<FIG>). An elevation in the expression of C5A, CFH, CASPASE1, and NLRP3 proteins was also detected in the B. megaterium infected drusenoid lesion and para-lesion tissues as compared to the uninfected normal retina in macaque (<FIG>). Taken together, our data demonstrate that infection of B. megaterium can activate complement system and induce drusenoid pathology in vivo.

In the past <NUM> years, the diversity and function of microbiota associated with human health and diseases have been extensively studied through high-throughput sequencing technologies and microbiomic/metagenomic analysis<NUM>. The local microbiota of the eye under physiological and pathological conditions remains largely uncharacterized<NUM>,<NUM>. The theory that the intraocular cavity is absolutely sterile under physiological conditions has led many researchers to reason that any types of foreign organisms are exogenous and pathogenic. However, our data indicate that even normal healthy eyes with no signs of ocular distress or infection have an individualized microbiome with compositional and functional diversity distinct from other body sites and tissues. Interestingly, the fact that P. acnes lives in the majority of human eyes and does not significantly induce intraocular inflammation raises a reasonable hypothesis that the normal intraocular microbiota plays a key role in maintaining the homeostasis of the local ocular environment. Similarly, the dysbiosis of the local microbial community can contribute to the etiology of many infectious, inflammatory, neoplastic, and degenerative ocular diseases. In addition, the idea that culture-positive microorganisms such as P. acnes were the major causes of intraocular inflammation warrants reexamination since these microorganisms may be part of the intraocular commensal microbiome while the real pathogens were uncultivable and missed. Many ocular procedures such as surgeries and intravitreal injection of anti-VEGF agents may also trigger the exposure of pathogenic intraocular microorganisms to host immune system responses. Therefore, future studies are needed to clearly define the symbiotic interactions between host and intraocular microbiota that support ocular health.

Genetic studies have successfully identified various factors involved in the pathogenesis of AMD<NUM>. These factors all suggest that the activation of complement cascade and controlling of immune responses are the keys for AMD onset and progress. However, the initiator of AMD pathology (especially how drusen is formed) and the critical link between complement and AMD pathology have been unclear.

Our findings that intraocular bacteria such as B. megaterium activate local complement-mediated immune responses can explain the formation of diversified drusen between RPE and Bruch's membrane. The major proteins found in drusen including complement components such as C1Q and immunoglobulin are all first-line anti-infectious agents<NUM>. Other drusen proteins such as amyloid Aβ proteins<NUM>,<NUM>, vitronectin<NUM>, and Apolipoprotein E<NUM> exhibited proven roles as anti-infectious agents in recent reports. Therefore, the formation of drusen could represent the key response of the aging retina in controlling infiltrated bacterial pathogens. Due to the diversity of bacteria, the shape and size of drusen could vary<NUM>. In the case of hard drusen, where the infection may be cleared, drusen will disappear. However, certain pathogens such as B. megaterium will induce long term activation of immune responses in soft drusen and result in the damage of RPE cells and photoreceptors. Activation of inflamasome and pyroptosis of RPE or macrophage are protective responses against local infection, which is consistent with the previous finding that NLRP3 mediated inflamasome activation and IL-<NUM> production protect the retina from neovascularization<NUM> (<FIG>).

The infectious etiology of AMD is also consistent with the conclusions reached by all genetic studies. For example, a defective CFH, the negative regulator of complement activation induced by B. megaterium infection, will result in uncontrolled complement activation<NUM>. A defective HTRA1, the protease producing the active form of immunosuppressive cytokine TGF-β, will result in decrease of local TGF-β family proteins<NUM>. Both of these genetic variations can lead to dysregulation of local anti-infectious responses that damages RPE cells and photoreceptors. In addition, the potential difference in pathogenic microorganisms found in drusen may explain the association of varied genetic risk factors with different ethnic groups<NUM> (i.e. Caucasian vs Asian). Additionally, further genetic studies and clinical trials could benefit from metagenomicly informed patient stratification. Therefore, based on our data, the infectious etiology of AMD is a plausible mechanism by which early AMD pathology is initiated in the elderly. In summary, our finding provides a novel target for the diagnosis, treatment, and prevention of AMD.

A topical antimicrobial drug, <NUM>% levofloxacin eye drops (Cravit, Santen Pharmaceutical Co. , Japan), was administered four times a day in both eyes for at least <NUM> days before the cataract surgery. On the day of surgery, patients received conjunctival sac irrigation using <NUM>% sodium chloride solution at least twice and mydriasis using compound tropicamide eye drops. Following disinfection and draping, <NUM>% povidone iodine (PVI) was applied on the eye for <NUM> seconds. The conjunctival sac was then irrigated with tobramycin solution for at least three times. After topical anesthesia with <NUM>% alcaine (at least three times), auxiliary incision was performed using <NUM> stab knife (Alcon, USA) at the <NUM> o'clock position of the limbus. Aqueous humor was sampled via the auxiliary incision using a <NUM> sterile syringe before any other procedures were initiated. Immediately after collection, aqueous humor samples were transferred into sterile eppendorf tubes and stored at -<NUM> prior to DNA extraction. The <NUM>% sodium chloride solution (<NUM>) was also transferred from a fresh <NUM> sterile syringe into a sterile eppendorf tube, which served as the Blank control.

The eye balls from post mortem donors (human donors from unrelated accidental death or laboratory animals free of diseases and genetic manipulation) were sterilized using <NUM>% PVI and tobramycin solution, followed by washing in sterile <NUM>% sodium chloride solution for three times in a cell culture hood. Aqueous humor was sampled using <NUM> sterile syringes.

Around <NUM> of peripheral venous blood was collected in an EDTA-anticoagulated vacutainer tube and then centrifuged for <NUM> at <NUM>,<NUM> rpm. The supernatant was collected and stored at -<NUM> prior to analysis.

The conjunctival impression cytology samples from inferior bulbar conjunctiva were obtained using the following protocol: <NUM>) topically anesthetize the eye with <NUM>-<NUM> drops of Alcaine Eye Drop (Alcon, USA) and keep the eye closed for several minutes; <NUM>) using disposable tweezers, place the MF Membrane filter (Millipore, REF:HAWP01300, <NUM>) on the inferior bulbar conjunctiva with the edge of the membrane clear of the lower tear meniscus and gently press for <NUM>-<NUM> seconds; <NUM>) remove the membrane and store it immediately at -<NUM> in a sterile Eppendorf tube with <NUM>µl Tissue and Cell Lysis Solution containing <NUM>µl of Proteinase K, provided in the MasterPure Complete DNA and RNA Purification Kit (Epicentre, UK); <NUM>) apply <NUM>-<NUM> drops of Neomycin Sulfate eye drops (Alcon, USA) to each examined eye.

Facial skin specimens were collected by scraping the skin of lower eyelid with a sterile MF Membrane filter (Millipore, REF:HAWP01300, <NUM>). The sample was inserted into a sterile eppendorf tube with <NUM>µl lysis solution (Epicentre).

The cooked meat medium (without antibiotics, <NUM>/L Lab-Lemco' powder, <NUM>/L peptone, <NUM>/L yeast extract, <NUM>/L NaH<NUM>PO<NUM>, <NUM>/L glucose, and <NUM>/L soluble starch) was purchased from Huankai Microbial Inc. (Guangzhou, China). Each culture was prepared in a <NUM> glass tube (purchased from Drtech Inc. , Guangzhou China) with <NUM> cooked meat medium, sterilized dry beef granules, and <NUM> liquid paraffin wax (purchased from Huankai Microbial Inc. , Guangzhou, China) on top. All tubes were then sterilized at <NUM> for <NUM> in the HEV-to Autoclave instrument (HIRAYAMA, Japan). Aqueous humor or vitreous humor sample fluid was injected into above tube in sterilized cell culture hood and sealed, followed by culture with shaking (<NUM> rpm) at <NUM> for <NUM> hours in the ZQTY-70F incubator (Zhichu Instrument Co. , Ltd, Shanghai, China). Wax sealed tubes containing the culture medium underwent the incubation protocol but contained no aqueous humor sample to serve as the negative control. All cultures were then gram stained and subjected to microscopic examination.

In addition, the following agar mediums purchased from Huankai Microbial Inc. (Guangzhou, China) were sterilized at <NUM> for <NUM> and used to culture aqueous humor samples at <NUM> for <NUM> days in a HettCube <NUM> incubator (Germany):.

DNA extraction was carried out using MasterPure Complete DNA and RNA Purification Kit (Epicentre, UK) according to the manufacturer's protocol. Briefly, the lysed specimens were vigorously vortexed for <NUM> minutes, followed by incubation at <NUM> for <NUM> minutes. The RNA was removed by <NUM>µg RNase A and DNA was extracted by MPC Protein Precipitation Reagent and isopropanol-ethanol precipitation procedure.

The aqueous humor was centrifuged at <NUM>,<NUM> rpm at <NUM> for <NUM> to collect the precipitate, followed by cytocentrifugation using Cytospin (Thermo Scientific, US). Gram-stain was performed according to the manufacture's instruction. The gram staining procedures were repeated five times.

Pre-processing of sequencing reads: All reads were first evaluated by FastQC for quality control. To maintain the consistency of alignment accuracy among all microbial reads, we first trimmed all reads to <NUM> bp using PrinSeq (v0. <NUM>)<NUM>, which provided a best Q30 in our sample set. Paired-end reads from each sample were combined into one single file and treated as single-end reads. Low quality reads, replicated reads, and potential adapter sequences were removed using Fastx toolkit (v0. The reads containing more than <NUM>% of ambiguous bases were depleted using PrinSeq (v0. Human reads were then removed from the subsequent analysis using HiSAT2 (v2. <NUM>)<NUM>, BMTagger, and DeconSeq<NUM> to obtain clean non-human sequences.

Sequence analysis: The non-human sequences were first analyzed using the Kraken program<NUM>, with the pre-built <NUM> GB database as the reference (including complete bacterial, archaeal, and viral genomes in RefSeq as of Dec. <NUM>, <NUM>) (https://ccb. edu/software/kraken/), followed by mapping of non-human sequences against our custom fungal genomes (containing <NUM> species and <NUM> strains, downloaded from http://fungidb. org/fungidb/, on March 12th <NUM>) using Burrows-Wheeler Aligner (BWA0. 5a) with <NUM> mismatches. The relative abundance of each species was calculated by the ratio of the total mapped reads of each species, normalized by their genome size and the total mapped microbial reads within each sample. Community diversity (Shannon index and evenness) was calculated according to the method described in Mothur program after using a subsampling cutoff of <NUM> microbial sequences per sample. The HMP Unified Metabolic Analysis Network (HUMAnN2)<NUM> was used to analyze the abundance of microbial genes and KEGG pathways. Principal component analysis (PCA) was performed on the relative abundance of bacterial species or microbial genes using Ade4 package in R statistical software (v3. <NUM>) after using a subsampling cutoff of <NUM> microbial sequences per sample or <NUM> read per gene. LDA Effect Size (LefSe)<NUM> was used to identify species, microbial genes, and functional pathways characterizing the differences among sample groups.

The drusen/non-drusen and lesion/non-lesion tissues on AMD slides were microdissected manually from uncovered, hematoxylin and eosin (HE) stained glass slides. Total DNA was isolated using AllPrep DNA/RNA FFPE Kit (Qiagen, USA) following the manufacture's instruction. Real-time PCR was performed to detect the relative amount of all bacteria in retinal tissues using either RT<NUM> SYBR Green qPCR Mastermix (SABiosciences, USA) and ABI <NUM> Real-time PCR System (Life Technologies, USA) or KAPA SYBR FAST Universal qPCR kit (Kapa, USA) and Roche LightCycler <NUM> (Roche, USA). Bacterial relative abundance was normalized to the level of human ACTB.

Multispectral imaging cytometry was used to visualize the size and morphology of microorganisms and human cells. The mixture of <NUM>µl diamidino-phenyl-indole (DAPI) and <NUM>µl microbeads (BD, USA) was added into <NUM>µl aqueous humor and analyzed on the ImageStreamX Mark II Imaging Flow Cytometer (Merck, USA).

All animal experiments were performed following the guidelines for housing and care of laboratory animals. The protocol was approved by the Sun Yat-sen University Animal Care and Use Committee and was consistent with the Association for Research in Vision and Ophthalmology guidelines for studies in animals (protocol #SYXK2014-<NUM> and #SYXK2015-<NUM>). Male New Zealand white rabbits (aged <NUM>-<NUM> weeks and weighted <NUM>-<NUM>) and rats (aged <NUM>-<NUM> weeks) were purchased from Huadong Xinhua Laboratory Animal Center (Guangdong, China). Male adult macaques (Macaca fascicularis, aged <NUM>-<NUM> years) were obtained from the Blooming-Spring Biotechnology Co. Ltd (Guangdong, China).

The macaques were sedated by intramuscular injection of a mixture of Tiletamine Hydrochloride (<NUM>/kg) and Xolazepam Hydrochloride (<NUM>/kg). After topical anesthesia (<NUM>% Proparacaine Hydrochloride), the eyes were immediately visualized in vivo using a light microscope. The pupils were then dilated with <NUM>% tropicamide and <NUM>% phenylephrine to obtain the fundus photographs. Intravitreal injection of bacterial solutions (<NUM> CFU [colony forming units] in a volume of <NUM>µl) or sonication-inactivated bacterial proteins (from <NUM> CFU bacteria) was performed with a <NUM> syringe and <NUM>-gauge needle after ocular surface disinfection with <NUM>% PVI solution. Slit lamp and fundus examinations were conducted for all macaques within <NUM> days after the injection. The severity of the endophthalmitis was graded according to a previously described standard<NUM>. The macaques were euthanized <NUM> days post inoculation and both eyeballs were enucleated for histopathological, intraocular cytokine, and bacteria analyses. The eyeballs were fixed in <NUM>% paraformaldehyde for <NUM> and then embedded in paraffin. Sections were cut on a microtome at <NUM> and stained with hematoxylin and eosin (H&E).

Bacterial strains including Propionibacterium acnes BNCC336649, Bacillus megaterium BNCC190686, and Bacillus licheniformis BNCC186069 were purchased from BeNa Culture Collection (Beijing, China). All strains of bacteria were first cultured overnight at <NUM> on agar plates following the standard protocols provided by the manufacturer. The bacterial cultures were then washed in PBS and resuspended as 1x10<NUM> CFU/µl solutions and further diluted in PBS as injection solutions.

Total RNA was extracted from <NUM>µl aqueous humor using MasterPure Complete DNA & RNA Purification Kit (Epicentre, UK). DNA-free RNA was reverse transcribed into DNA using the All-In-One Master Mix Kit (Kapa, USA). Real-time PCR was performed using KAPA SYBR FAST Universal qPCR kit (Kapa, USA) to quantify bacterial relative abundance and was normalized to the expression of human or macaques ACTB. The primers used were listed in Table <NUM>.

The active forms of cytokines and complement components in cell culture supernatants or aqueous humor specimens from bacteria inoculated macaque eyes were measured using Human IL-1β ELISA kit (Cat#KT98060), Human IL-<NUM> ELISA kit (Cat#KT98065), Human C5a ELISA kit (Cat#KT40003), Monkey IL-1β ELISA kit (Cat#KT49531), Monkey IL-<NUM> ELISA kit (Cat#KT56387), Monkey C5a ELISA kit (Cat#KT40004), and Monkey CFH ELISA kit (Cat#KT45321; MSKBIO, Wuhan, China) according to the manufacture's instruction.

Human ARPE19 cells (ATCC, USA) were cultured in DMEM supplied with <NUM> L-glutamine and <NUM>% FBS. After coculture without or with bacteria, ARPE19 cells were incubated with PI and Annexin V-APC (Cat#<NUM>-<NUM>-<NUM>, eBioscience, USA) for <NUM>. The cell death was measured with MACSQuant Analyzer <NUM> flow cytometer (Miltenyi Biotec, Germany).

Fluorescence in situ hybridization was performed using the FISH kit (Bis-QD355, BersinBio, China). Formalin-fixed paraffin-embedded sections were baked at <NUM> for <NUM>, de-paraffinized in dimethylbenzene (<NUM> per time, three times), and washed in <NUM>% ethanol (<NUM> per time, two times). After air dry on super-clean bench, the protease K was used to digest tissues for <NUM> at <NUM>, followed by washing with sterile PBS solution for <NUM>. Then, the sections were sequentially incubated in <NUM> denatured solution, followed by washing in <NUM>%, <NUM>%, <NUM>% ethanol solutions at -<NUM> (<NUM> per solution). Sections were then hybridized to a general bacteria <NUM> probe labeled by FAM and/or a B. megaterium specific probe labeled by Cy3 overnight at <NUM>. After hybridization, <NUM>% formamide and SSC solution were used to wash all sections. DAPI was used as the control.

All statistical analysis was performed in the SPSS software (v17. Data were represented as mean ± standard error unless otherwise indicated. The parametric (student's) t or F tests were used.

Potential reagent and environmental contamination in high-throughput sequencing experiments were a concern in our study. We thus paid great attention to myriad negative controls for both environments and reagents throughout these studies, including the Blank, Wash Solution, Anesthesia, Disinfectant, NaCl Solution, and Mydriatic samples. Eight independent samples ("Blank") were collected following the exact procedures for aqueous humor (AH) collection, substituting <NUM>µl <NUM>% sodium chloride solution instead of AH into the sample collection tubes in the operation room. Similarly, two each Wash Solution, Anesthesia, Disinfectant, NaCl Solution, and Mydriatic samples were collected as controls for metagenomic sequencing analysis of AH specimens. The <NUM> tissue samples as well as environmental and reagent controls were analyzed using metagenomic sequencing approach, among which we failed to detect any sequencing reads in one unreplaceable conjunctival sample. We found no detectable DNA in all control samples, while an average of <NUM> ng/µl DNA could be detected in AH samples using Qubit (Life, USA) (<FIG>). As a result, over amplified DNA in control samples resulted in almost all low quality and/or repeat reads (<FIG>). These data clearly differentiated the controls from AH samples, and this validation process confirmed that the reads from AH samples were indeed from a true metagenomic community.

To assess whether an intraocular microbiota would be found in both human and animals, we collected postmortem AH specimens from <NUM> fresh human eyes that were free of ocular diseases (individuals deceased due to unrelated accidents, the demographic characteristics of all subjects were listed in Table <NUM>), <NUM> macaque eyes (Macaca fascicularis), <NUM> rabbit eyes (Oryctolagus cuniculus), <NUM> rat eyes (Rattus norvegicus), and <NUM> pig eyes (Sus scrofa) in the sterile Laminar Flow Hood. Metagenomic analysis was performed on these <NUM> AH specimens. Our data suggest that unique live microbiota could be found in human eyes free of any ocular diseases. Animals such as macaque, rabbit, and rat had similar intraocular microbiota as human had, which differed substantially from pigs.

Previous studies demonstrate that drusen contains a variety of complement components and polysaccharides in addition to many other proteins<NUM>. In addition, the drusen components activate inflamasomes and promote expression of IL-<NUM>β and IL-<NUM><NUM>,<NUM>. We therefore first examined whether B. megaterium was able to induce the activation of complement system and promote the secretion of IL-1β and IL-<NUM>, by ARPE19 cells in vitro. As shown in <FIG>, we found B. megaterium but not P. acnes significantly increased the pyroptosis of RPE cells in a time dependent manner. The activation of complement system was confirmed by the production of active form of C5A protein (<FIG>). Interestingly, both bacteria induced secretion of CFH proteins, while the induction of CFH was more profound by B. megaterium than by P. acnes (<FIG>). As the result of pyroptosis, in vitro infection of B. megaterium, but not P. acnes, led to secretion of active IL-1β and IL-<NUM> by RPE cells (<FIG>). These results indicate that infection of B. megaterium can lead to inflammation mediated by RPE.

The test of Antibiotics to treat AMD through inhibiting the growth of microbiota.

The bacterial culture medium (HuanKai Microbial, Guangzhou, China) containing peptone <NUM>, beef extract <NUM>, NaCl <NUM>, agar <NUM>, and MnSO<NUM> <NUM> in <NUM> ddH<NUM>O (pH=<NUM>) was prepared in conical flask (Drtech, Guangzhou, China) and was sterilized in the autoclave (HIRAYAMA, HEV-<NUM>, Japan) at <NUM> for <NUM>. Antibiotics (ampicillin, vancomycin, neomycin, metronidazole, and tetracycline, purchased from Sigma, USA) at various concentrations were added into cooled medium. Bacillus megaterium (total <NUM>*<NUM><NUM> per culture) was cultured in the incubator (HettCube <NUM>, Germany) at <NUM> for <NUM>.

To test whether antibiotics can control the growth of Bacillus megaterium in vitro and in vivo, an antibiotic sensitivity screening test in petri dishes was carried out. The sensitivity of Bacillus megaterium to several major antimicrobial agents including Ampicillin, vancomycin, neomycin, metronidazole, and tetracycline were examined using the minimum inhibitory concentration (MIC) method. As shown in <FIG>, Bacillus megaterium was most sensitive to neomycin, while metronidazole was <NUM>-fold less effective in controlling the growth of Bacillus megaterium.

Claim 1:
A kit or device for use in diagnosing an intraocular disease or disorder in a subject, which kit or device comprises reagents for assessing the presence, absence, quantity, the infectious status, and/or the microbiota of a microorganism in an intraocular space, cavity or sample of a subject, wherein the etiology of the intraocular disease or disorder comprises infection of the microorganism in the intraocular space or cavity of the subject, wherein the intraocular disease or disorder is age-related macular degeneration (AMD); and the microorganism comprises Bacillus megaterium or Bacillus licheniformis, and wherein the reagents comprise a primer pair for Bacillus megaterium, consisting of SEQ ID NO:<NUM> and SEQ ID NO:<NUM>, or a primer pair for Bacillus licheniformis, consisting of SEQ ID NO:<NUM> and SEQ ID NO:<NUM>.