Methods and kits for identifying and characterizing ocular infections

Abstract

Methods of detection and identification of microbial eye infections by molecular analysis of nucleic acid in tear samples are provided. 16S rDNA is amplified by PCR directly from tear samples without extracting DNA and the amplification products are sequenced by nanopore sequencing so that the microbes can be identified.

Description

[0001] METHODS AND KITS FOR IDENTIFYING AND CHARACTERIZING OCULAR INFECTIONS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 569,532, filed March 25, 2024, the contents of which is incorporated herein by reference in its entirety.

[0004] REFERENCE TO SEQUENCE LISTING

[0005] The Sequence Listing XML submitted as a file named “YU_8845PCT_ST26.xml,” created on March 25, 2025, and having a size of 2,868 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

[0006] FIELD OF THE INVENTION

[0007] The field of invention is generally related to methods and kits for detecting and treating eye infections such as comeal ulcers.

[0008] BACKGROUND OF THE INVENTION

[0009] Comeal ulcers are characterized by a breach in the corneal epithelium, allowing microbial invasion and subsequent inflammation of the stromal layer beneath. Prompt and accurate diagnosis and treatment are important as any delay can lead to scarring and vision loss.

[0010] The impact of corneal ulcers on vision is profound. For instance, a study conducted at a major medical center in Houston, Texas, revealed that only 50% of corneal ulcer patients achieved a post-treatment visual acuity of 20 / 40 or greater. For context, this level of visual acuity is the minimal requirement for obtaining a driver’s license in many states, including Connecticut. Moreover, corneal ulcers often necessitate extensive medical interventions. In the same cohort, about 10.4% of affected individuals experienced comeal perforation, resulting in the need for a comeal transplant. A retrospective study at Queen’s Medical Centre in Nottingham, UK, indicated that 16.3% of patients with bacterial keratitis required surgical intervention to manage the infection or its sequelae, including procedures like corneal gluing, amniotic membrane transplants, and comeal transplantation. These interventions not only signify a considerable expense - with comeal transplants alone costing between $13,000 and $27,000 - but also represent a substantial loss in productivity due to compromised vision, underscoring the significant economic impact of corneal ulcers on both individuals and the healthcare system.

[0011] While the toll of comeal ulcers on vision and its economic burden are considerable in developed nations, their impact is even more detrimental in the developing world. For example, the annual incidence rate of corneal ulcer cases per 100,000 individuals is about 28 in the United States, which pales in comparison to countries like India and Nepal, where the rates increase to 113 and 799 cases per 100,000 individuals, respectively. Corneal ulcers are in fact the primary cause of monocular blindness globally. The higher prevalence of comeal ulcers in these regions, coupled with limited healthcare resources, exacerbates not only the visual impairment but also the economic strain due to medical costs and reduced productivity.

[0012] The visual outcomes for comeal ulcers in less developed countries are indeed more severe due to a variety of factors, including the prevalence of fungal infections which are often more challenging to treat. As opposed to bacterial infections, fungal keratitis often leads to worse visual outcomes due to the fungi's ability to penetrate deeper into the corneal tissue, causing extensive damage and increasing the risk of sight-threatening complications such as comeal perforation. Broad- spectrum antibiotics, commonly administered at the onset of treatment, are ineffective against fungal pathogens, highlighting the important need for accurate diagnostic methods that can swiftly identify the infectious agent and guide the administration of appropriate antifungal therapies early-on. This targeted approach is needed to prevent the progression of the infection to advanced stages.

[0013] While the health and economic impact of comeal ulcers are significant, they are avoidable. Early and accurate diagnosis and treatment can alter the trajectory of the disease, preventing severe complications and the subsequent need for surgical measures. Patients who receive early treatment for corneal ulcers have better visual outcomes.

[0014] However, the traditional diagnostic approach is costly and difficult. Conventional diagnostic and management protocols for corneal ulcers typically start with a clinical evaluation, including a detailed patient history to assess risk factors such as contact lens use or trauma. Slitlamp examination allows visualization of the ulcer and facilitates the collection of samples for culture through comeal scraping. Corneal scraping involves removing cells from the ulcer with a blade. The collected samples are then placed on various culture media to grow and identify the causative organism, including blood agar, chocolate agar for fastidious bacteria that require additional growth factors, Sabouraud dextrose agar for fungi, and thioglycolate broth for anaerobic bacteria. These media help isolate and identify the causative organism, which can range from bacteria and fungi to amoebas and vimses, each requiring specific antimicrobial treatments.

[0015] In the US, the majority of corneal ulcers are caused by bacteria. Therefore, while awaiting culture results, treatment is initiated with broad- spec trum antibiotics to cover a wide range of potential bacterial pathogens. These require frequent dosing and are not readily available in pharmacies. The treatment regimen is refined based on the type of bacteria detected on gram stain or culture. Further refinement of treatment follows antimicrobial sensitivity testing, which informs the choice of antibiotics based on the specific resistance patterns of the bacteria. In cases where there is no improvement with initial treatment, or if cultures fail to grow any organisms despite a high suspicion of infection, a corneal biopsy may be warranted. This more invasive procedure involves obtaining a deeper tissue sample for examination and is typically considered when less invasive measures fail to yield a diagnosis.

[0016] Thus, current diagnostic methods, while the standard-of-care, have many drawbacks.

[0017] Limited access to care: Specialized equipment like slit lamps and board-certified ophthalmologists are necessary to perform corneal scrapings, restricting access. Primary care physicians and optometrists who are often the ones evaluating the patient at the initial encounter face delays in diagnosis when referring patients, and often leading to unnecessary emergency department visits.

[0018] Resource limitations: The need for specialized laboratories for culture and sensitivity tests is a significant barrier in less developed regions, exacerbating health disparities.

[0019] Invasiveness and underutilization: The invasiveness of corneal scraping can lead to patient reluctance as well as underutilization of this crucial diagnostic step by clinicians.

[0020] High false negative rates: Factors such as pre-treatment with antibiotics, improper sample collection, and low microbial loads contribute to high false negative rates (ranging from 33% to 66%), leading to repeated testing and uncertainty in treatment decisions.

[0021] Diagnostic delays: Cultures require significant time to yield results, often several days to weeks, delaying the start of targeted treatment and necessitating the initial use of broad- spectrum antimicrobials.

[0022] Empiric treatment burden: Use of broad-spectrum antibiotics while waiting for culture results places a significant burden on patients, leading to increased costs, increased side effects, and compliance issues. Of note, broad spectrum antibiotics require on-site preparation and are not readily available in most pharmacies.

[0023] Limited window for sample collection: Traditional culture-based diagnostic methods necessitate sample collection prior to starting antibiotic therapy to avoid false-negative results. Once antibiotic treatment begins, the effectiveness of cultures diminishes significantly as the antibiotics can inhibit the growth of the causative organisms, leading to non-detection and thus potential misdiagnosis. Risk of sample contamination: Contamination from normal flora during corneal scraping may result in false positives, complicating the diagnosis and management, especially without the ability to obtain cultures from contralateral unaffected eyes.

[0024] Reduced sensitivity: Traditional culture methods may fail to detect pathogens present in low quantities, certain bacteria, or non-culturable agents such as viruses. Often, this requires the use of additional diagnostic techniques like confocal microscopy or obtaining corneal biopsies for accurate identification.

[0025] Workflow inefficiencies: The complex logistics of corneal scraping, sample transport and processing can introduce significant delays. Moreover, to culture a range of microbes this requires an array of specialized growth media - such as blood agar for aerobic bacteria, chocolate agar for fastidious organisms, thioglycolate for anaerobes, and Sabouraud dextrose for fungi. This not only complicates the diagnostic process but also increases the time and resources needed for a thorough evaluation. This contributes to underutilization of the corneal scraping in management. Reliance on subjective assessment over objective testing, can lead to potential misdiagnosis.

[0026] Each step in the diagnostic process, from scraping to culture to biopsy, incurs costs and can delay definitive treatment. Therefore, there is an important need to streamline this process and obtain faster and more accurate results with the goal of improving patient outcomes.

[0027] Therefore, there is an important need to not only develop accurate diagnostic tools but also expand access to these tools in a timely manner to ensure that patients, irrespective of their geographic location or socioeconomic status, can promptly receive the care they need.

[0028] It is thus an object of the invention to provide improved compositions, devices, and methods of identifying comeal ulcers and other ocular infections.

[0029] It is a further object of the invention to characterize corneal ulcers and other ocular infections to inform subsequent treatment thereof, and to provide the same.

[0030] SUMMARY OF THE INVENTION

[0031] It has been discovered that microbial nucleic acids are present in the tears of microbial infected eyes. As discussed herein, this discovery can be leveraged in various methods of detection, diagnosis, and treatment.

[0032] For example, methods of determining the presence of microbes in the eye of a subject are provided. The methods typically include detecting microbial nucleic acids in a tear sample isolated from the subject. The absence of detection of nucleic acids in the tear sample indicates the eye(s) from which the tear sample was collected is free from microbes. The detection of nucleic acids in the tear sample indicates the eye(s) from which the tear sample was collected is infected with microbes. For purposes of this disclosure, such eyes may still be “infected,” in the sense that the eye is exhibiting a pathology that is consistent with a microbial infection, but microbes are not the cause. For example, “eye infections” of the disclosure include such alternative conditions that present the same or similarly to microbial eye infections, such as some allergic reactions, autoimmune reactions, etc., as discussed in more detail below. Thus, detection of microbes informs the practitioner if a subject presenting a non-specific pathology should be treated with or without an antimicrobial agent, or more specifically which type of antimicrobial agent. Thus, in some embodiments, the subject has a visible or non-visible eye pathology or condition that is prompting the disclosed methods.

[0033] The nucleic acids in the tear sample can be DNA, RNA, or a combination thereof. In some embodiments, when RNA is the target of the molecular analysis, the RNA is converted to DNA by reverse transcription prior to the molecular analysis. Detection can be relative to a control, wherein nucleic acids present in the control are deducted (e.g., physically or in silico) from the molecular analysis before determining if nucleic acids are present in the tear sample. In some embodiments, tears collected from the contralateral eye serve as the control. In this way, for example, human nucleic acids of the subject’s cells are not detected as microbial nucleic acids.

[0034] Isolation of the tears from the subject can include collection of tears using a collection device. Exemplary devices include, but are not limited to, Schirmer strip, capillary action paper, and cotton swab. Isolation of the tears can include drawing the tears out of the collection device, optionally by centrifugation.

[0035] In some embodiments, the nucleic acids are not extracted from the tears prior to detection. The tear sample can be undiluted, or include water or buffer added after the sample is isolated from the subject. Typically, the added water or buffer is nuclease-free.

[0036] The detecting typically includes molecular analysis of the nucleic acids. For example, the detecting can include processing the sample using a machine-based analytical platform. In some embodiments, the molecular analysis includes PCR, sequencing, microarray, or a combination thereof. Exemplary PCR techniques that can be utilized include, for example, Real-Time PCR (quantitative PCR or qPCR), Reverse-Transcriptase (RT-PCR), Multiplex PCR, Nested PCR, High Fidelity PCR, Fast PCR, Hot Start PCR, Long-range PCR, Arbitrary Primed PCR, Digital PCR, Droplet Digital PCR (ddPCR), isothermal amplification PCR, Endpoint PCR (Qualitative PCR), or a combination of any thereof.

[0037] The analysis, e.g., PCR, can be qualitative or quantitative, and can be specific or nonspecific. In some embodiments, the PCR includes one or more sets of primers. In some embodiments, the PCR comprises one or more sets of genus and / or species specific primers specific for a microbe. In some embodiments, the PCR includes non-specific and / or random primers.

[0038] Sequencing can be carried out in alternative to PCR. Thus, in some embodiments the substrate for sequencing is the microbial DNA or cDNA formed from microbial RNA.

[0039] Sequencing can also be carried out in combination with PCR, most typically after the PCR. In such embodiments, the substrate for sequencing can additionally or alternatively be amplicons generated by the PCR. Sequencing techniques include, but are not limited to, Sanger sequencing, single molecule real time (SMRT) sequencing, nanopore DNA sequencing, massively parallel signature sequencing (MPSS), colony sequencing, 454 pyrosequencing, Illumina sequencing, combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, ion torrent semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, and combinations of any thereof.

[0040] The methods can further include the use of bioinformatics tools and techniques, for example to compare the results of the molecular analysis to existing databases such as sequence databases other known sequences, other subject samples, controls, etc. In some embodiments, the methods include determining the type of microbes present in the tear sample. Types of microbes include, for example, bacteria, fungi, viruses, parasites, and combinations thereof. Some embodiments include determining the genus and / or species of the microbes.

[0041] In a particular exemplary embodiment, the method includes PCR amplifying a target portion of the nucleic acids optionally using ribosomal RNA-specific primers, sequencing the resulting amplicons using next generation sequencing optionally nanopore sequencing, and determining the type, genus, species, or a combination thereof of the organism(s) (i.e., microbe(s)) from which the nucleic acids were obtained, optionally using bioinformatics.

[0042] Methods of diagnosing a subject with an eye infection are also provided and including detecting microbes according the disclosed detection methods. The subject can be determined to have a microbial eye infection when nucleic acids are detected in the tear sample, and the subject can be determined not to have a microbial eye infection when nucleic acids are not detected in the tear sample. In some embodiments, the subject is determined to be free from an eye infection.

[0043] Methods of treatment are also provided. The methods can stand alone, or be additional steps of a detection or diagnostic method. For example, a method of treating a subject for an eye infection typically includes administering an antimicrobial agent to a subject diagnosed with a microbial eye infection. The methods can also include abstaining from treating the subject with antimicrobial agents when the subject is determined not to have a microbial eye infection. Where the type, genus, and / or species of the microbes in the sample are determined, this information can further inform treatment. For example, where the microbe(s) is or includes bacteria, and the subject is treated with one or more topical and / or oral antibiotics, optionally wherein antibiotic -resistant bacteria are treated with broad-spectrum antibiotics; where the microbe(s) is or includes a virus and the subject is treated with one or more topical and / or oral antivirals; where the microbe(s) is or includes fungi and the subject is treated with one or more topical and / or oral antifungals; where the microbe(s) is or includes a parasite and the subject is treated with one or more topical and / or oral anti-parasitics.

[0044] Any of the subject can be additionally, or alternatively, treated with an anti-inflammatory, antihistamine, immune suppressant, analgesic, or a combination thereof.

[0045] In some embodiments, the eye infection is keratitis, conjunctivitis, cellulitis, shingles, endophthalmitis, stye, blepharitis, uveitis, dacryocystitis, or canaliculitis.

[0046] In some embodiments, the subject has undergone a comeal transplant. The disclosed compositions and methods can be used to assess the status of the transplant and identify infection, e.g., that occurred during or as a result of the transplant. In some embodiments, the infection is not yet visible. In some embodiments, no microbes are detected and the subject is cleared as being free from infection. In some embodiments, the comeal transplant was about 1-365 days inclusive or any subrange or specific day therebetween, preceding the collection of the tear sample. The subject can be an otherwise healthy subject.

[0047] The subject is typically mammal or bird, and can optionally be selected from humans, goats, sheep, pigs, cattle, zebu, donkeys, water buffaloes, dromedary camel, horse, yak, domestic bactrian camel, llama, alpaca, gayal, bali cattle, domestic rabbit, addax, bison, deer, eland, elk, guinea pig, greater kudu, mule, moose, muskox, and reindeer, chicken, duck, goose, guineafowl, muscovy duck, turkey, emu, egyptian goose, indian peafowl, mute swan, ostrich, partridge, small-billed tinamou, pigeon, quail, edible-nest swiftlet, grey francolin, guineafowl, common pheasant, and golden pheasant.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is an illustration of a pre-curved tear collection strip.

[0050] Figure 2 is a chart summarizing a comparison from Nanopore analysis of tear samples and results from culture.

[0051] Figures 3A-3B are illustrations of results provided by Nanopore for a single run that successfully detected Pseudomonas aeruginosa (A091). Nanopore lists the bacterial species and number of cumulative reads in descending order (Fig. 3A) and a phylogenetic tree, here with a minimum abundance cutoff of 1 % (Fig. 3B)

[0052] Figure 4 is a taxonomic tree of bacterial species identified from the right eye of Subject 1. Subject 1 presented with eye pain and discharge after experiencing a prolonged hospitalization complicated by bacteremia. The bacterial species with the greatest number of reads was Staphylococcus saccharolyticus, accounting for 42.1% of all classified reads.

[0053] Figure 5 is a Taxonomic tree of bacterial species identified from the right eye of Subject 2. Subject 2presented with eye pain, discharge, blurry vision, and pink eye, and had a superficial keratectomy for removal of Salzmann’s nodules 7 days prior to presentation. The bacterial species with the greatest number of reads was Staphylococcus roterodami, accounting for 66.7% of all classified reads.

[0054] Figure 6 is a taxonomic tree of bacterial species identified from the left eye of Subject 3. Subject 3 was a contact lens user who presented with eye pain, discharge, blurry vision, pink eye, and tearing. The bacterial species with the greatest number of reads was Pseudomonas aeruginosa, accounting for 72.0% of all classified reads.

[0055] Figure 7 is a taxonomic tree of bacterial species from the left eye of Subject 4. Subject 4 was a contact lens user who presented with eye pain, discharge, and foreign body sensation. The bacterial species with the greatest number of reads was Pseudomonas aeruginosa, accounting for 62.1% of all classified reads.

[0056] Figure 8 is a taxonomic tree of bacterial species identified from the left eye of Subject 5. Subject 5 had blepharitis and presented with eye pain, discharge, and redness. The bacterial species with the greatest number of reads was Staphylococcus saccharolyticus, accounting for 26.7% of all classified reads.

[0057] Figure 9 is a taxonomic tree of bacterial species identified from the left eye of Subject 6. He was a contact lens user who presented with eye pain, pink eye, blurry vision, tearing, and photosensitivity. The bacterial species with the greatest number of reads was Cutibacterium acnes, accounting for 35.4% of all classified reads.

[0058] Figure 10 is a taxonomic tree of bacterial species identified from the right eye of Subject 9. Subject 9 was a contact lens user who presented with eye pain, pink eye, blurry vision, and photosensitivity. The bacterial species with the greatest number of reads was Staphylococcus saccharolyticus, accounting for 33.3% of all classified reads.

[0059] Figure 11 is a taxonomic tree of bacterial species identified from the right eye of Subject 10. Subject 10 was a contact lens user who presented with eye pain and foreign body sensation. The bacterial species with the greatest number of reads was Kocuria rhizophila, accounting for 78.0% of all classified reads.

[0060] DETAILED DESCRIPTION OF THE INVENTION

[0061] I. Definitions

[0062] The term “conditions sufficient for” refers to any environment that permits the desired activity, for example, that permits specific binding or hybridization between two nucleic acid molecules or that permits reverse transcription and / or amplification of a nucleic acid. Such an environment may include, but is not limited to, particular incubation conditions (such as time and / or temperature) or presence and / or concentration of particular factors, for example in a solution (such as buffer(s), salt(s), metal ion(s), detergent(s), nucleotide(s), enzyme(s), etc.).

[0063] As used herein, the terms “nucleic acid”, “polynucleotide” and “oligonucleotide” refer to primers, probes, oligomer fragments, and oligomer controls and are generic to poly deoxyribonucleotides (containing 2-deoxy-D-ribose), to polyribonucleotides (containing D- ribose), and to any other type of polynucleotide which is an N glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases. There is no intended distinction in length between the term “nucleic acid”, “polynucleotide” and “oligonucleotide”, and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single stranded RNA.

[0064] As used herein, the terms “detect” or “detecting” generally refer to obtaining information. Detecting or determining can utilize any of a variety of techniques available to those skilled in the art, including for example specific techniques explicitly referred to herein. Detecting may involve manipulation of a physical sample, consideration and / or manipulation of data or information, for example utilizing a computer or other processing unit adapted to perform a relevant analysis, and / or receiving relevant information and / or materials from a source. Detecting may also mean comparing an obtained value to a known value, such as a known test value, a known control value, or a threshold value. Detecting may also mean forming a conclusion based on the difference between the obtained value and the known value.

[0065] The terms “contact”, “contacting” or “bringing into contact” describe placement in physical association for example, in solid and / or liquid form. For example, contacting or combining can occur in vitro with one or more primers and / or probes and a biological sample (such as a sample including nucleic acids) in solution. “Amplification” or “amplifying” refers to increasing the number of copies of a nucleic acid molecule, such as a gene, fragment of a gene, or other genomic region. The products of an amplification reaction are called “amplification products” or “amplicons.”

[0066] As used herein, the term “primer” refers to an oligonucleotide, which is capable of acting as a point of initiation of nucleic acid synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a target nucleic acid strand is induced, e.g., in the presence of different nucleotide triphosphates and a polymerase in an appropriate buffer (“buffer” includes pH, ionic strength, cofactors etc.) and at a suitable temperature. In some embodiments, the primer is preferably single-stranded. One or more of the nucleotides of the primer can be modified for instance by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5’ end of the primer, with the remainder of the primer sequence being substantially complementary to the template. Primer includes all forms of primers that may be synthesized including peptide nucleic acid primers, locked nucleic acid primers, phosphorothioate modified primers, labeled primers, and the like. The term “forward primer” as used herein means a primer that anneals to the anti-sense strand of a double-stranded DNA (dsDNA) fragment. A “reverse primer” anneals to the sense-strand of a dsDNA fragment. The terms “primer pair” and “primer set” refers to a forward and reverse primer pair (i.e. , a left and right primer pair) that can be used together to amplify a given region of a nucleic acid of interest.

[0067] The terms “complement”, “complementary” or “complementarity” as used herein with reference to polynucleotides (i.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) refer to the Watson / Crick base-pairing rules. The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5’ end of one sequence is paired with the 3’ end of the other, is in “antiparallel association.” For example, the sequence “5’-A-G-T-3”’ is complementary to the sequence “3’-T-C-A-5’.” Certain bases not commonly found in naturally occurring nucleic acids may be included in the nucleic acids described herein. These include, for example, inosine, 7- deazaguanine, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA). Complementarity need not be perfect (e.g., it can be partial or complete); stable duplexes may contain mismatched base pairs, degenerative, or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs. A complement sequence can also be an RNA sequence complementary to the DNA sequence or its complement sequence, and can also be a cDNA.

[0068] The terms “target nucleic acid” or “target sequence” or “target segment” as used herein refer to a nucleic acid sequence of interest to be detected and / or quantified in the sample to be analyzed. Target nucleic acid may be composed of segments of a genome, a complete gene with or without intergenic sequence, segments or portions of a gene with or without intergenic sequence, or sequence of nucleic acids to which probes or primers are designed to hybridize. Target nucleic acids may include a wild-type sequence(s), a mutation, deletion, insertion or duplication, tandem repeat elements, a gene of interest, a region of a gene of interest or any upstream or downstream region thereof. Target nucleic acids may represent alternative sequences or alleles of a particular gene. Target nucleic acids may be derived from genomic DNA, cDNA, or RNA.

[0069] As used herein, the term “polymorphism” means variations of a nucleotide sequence in a population. For example, polymorphism can be one or more base changes, an insertion, a repeat, or a deletion. Polymorphisms can be single nucleotide polymorphisms (SNP), or simple sequence repeat (SSR). SNPs are variations at a single nucleotide, e.g., when an adenine (A), thymine (T), cytosine (C) or guanine (G) is altered. Generally a variation must generally occur in at least 1 % of the population to be considered a SNP.

[0070] As used herein, the terms “aligning” and “alignment” refer to the comparison of two or more nucleotide sequence based on the presence of short or long stretches of identical or similar nucleotides. Several methods for alignment of nucleotide sequences are known in the art, as will be further explained below.

[0071] The terms “subject,” “individual,” and “patient” refer to any individual who is the target of treatment using the disclosed compositions. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human. The subjects can be symptomatic or asymptomatic. The term does not denote a particular age or sex. A subject can include a control subject or a test subject.

[0072] The term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease state being treated or to otherwise provide a desired pharmacologic and / or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables e.g., age, immune system health, etc.), the disease, and the treatment being administered. The effect of the effective amount can be relative to a control. Such controls are known in the art and discussed herein, and can be, for example the condition of the subject prior to or in the absence of administration of the drug, or drug combination.

[0073] The term “pharmaceutically acceptable” or “biocompatible” refers to compositions, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The phrase “pharmaceutically acceptable carrier” refers to pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, solvent or encapsulating material involved in carrying or transporting any subject composition, from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient.

[0074] The term “treating” or “preventing” a disease, disorder, or condition includes ameliorating at least one symptom of the disease or condition. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating, or palliating the disease state, and remission or improved prognosis. For example, an individual is successfully “treated” if one or more symptoms associated with one or more diseases or disorders are mitigated or eliminated, including, but are not limited to, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and / or prolonging survival of individuals.

[0075] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.

[0076] These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific form or combination of forms of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0077] All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the forms and does not pose a limitation on the scope of the forms unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0078] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0079] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 5%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 2%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 1 %. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

[0080] IL Methods of Detecting Ocular Infections

[0081] The traditional diagnostic approach for comeal ulcers, which is costly and difficult, involves invasive comeal scraping to collect samples for cultures and subsequent antimicrobial susceptibility testing. This process can be lengthy, often requiring several days to produce definitive results. During this waiting period, patients are generally placed on broad-spectrum antimicrobial therapy. This approach can lead to suboptimal treatment, unnecessary side effects, and risks disease progression.

[0082] Thus provided are improved methods of identifying ocular infections, determining the cause of the infection, and treatment of the infection; and compositions and devices for facilitating the same. The provided methodology leverages the groundbreaking discovery that microbial nucleic acids (e.g., DNA and / or RNA) is traceable in tears from eyes affected by ocular infections such as comeal ulcers, and can be amplified without nucleic acid extraction. Utilizing portable nextgeneration sequencing platforms, the causative organism can be identified in less than 3 hours from 1 microliter of tear sample.

[0083] This approach eliminates the need for the invasive comeal scraping procedure and DNA extraction. By offering a non-invasive, expedited, and highly targeted treatment protocol, it presents a significantly better alternative to the standard-of-care, promising increased accuracy and speed in identifying microorganisms associated with comeal ulcers. Additionally, given its non-invasive nature, this approach allows for optometrists, primary care physicians and nurse practitioners to properly diagnose corneal ulcers. This is particularly beneficial for underserved and resourcelimited areas, where it can greatly improve access to care and significantly reduce the necessity for emergency department visits. The approach’s advantages extend beyond addressing the traditional culture-based methods’ limitations, and offer the following general improvements.

[0084] Enhanced accessibility: An important impact of the method is that the simplicity of the tear collection will allow optometrists and practitioners in the primary care setting to diagnose and treat comeal ulcers. Corneal scraping currently used in diagnosing ulcers can only be performed by an ophthalmologist, given its invasive nature and in line with most states’ regulations. This expanded access has the potential to improve the management workflow of comeal ulcers and reduce visits and referrals to emergency departments.

[0085] Rapid diagnostic turnaround time: This methodology reduces the time required to identify the causative organisms, often providing results within hours rather than days or weeks, facilitating quicker initiation of targeted treatments. This has the potential to limit the use of broad-spectrum antibiotics, thereby reducing associated costs and side effects.

[0086] Non-invasiveness: Tear collection is a non-invasive process, and carries no risk to patients, which would increase adoption of this new workflow.

[0087] Low false negative rates: The ability to detect microbial DNA in tears, even in minute quantities, allows for a higher sensitivity compared to culture methods. It can identify a broad range of pathogens, including those that are difficult to culture, like certain fungi, acanthamoeba, and atypical bacteria.

[0088] Increased Specificity: Collecting tears from the unaffected eye as a control can help ensure the specificity of the diagnosis. This is not feasible with the invasive corneal scraping.

[0089] Flexibility in the timing of sample collection: The tear-based diagnostic approach, relying on the detection of microbial DNA, rather than on organism growth, possesses the unique capability to identify the causative organism even after antibiotic treatment has been initiated. Since antibiotics typically take several days to completely eradicate pathogens, their genomic material remains detectable. Data presented in the Examples below indicates that this method can accurately detect and identify pathogens from tear samples collected up to 3 days post-antibiotic administration, providing clinicians with a powerful tool to confirm or adjust treatment strategies based on a reliable diagnosis, without the delay traditionally imposed by the culture-based method. This is particularly beneficial in ambulatory settings where treatment can be started immediately.

[0090] Workflow efficiency: The disclosed tear-based diagnostic workflow is designed for simplicity and ease, requiring minimal technical expertise, and avoiding the need for specialized laboratory facilities. It can be implemented across various clinical settings, including in resource- constrained environments. By streamlining the diagnostic process, it not only improves the efficiency of healthcare delivery but also significantly reduces the potential for errors often associated with the complexity of traditional culture-based methods.

[0091] Broader pathogen identification: The tear-based diagnostic technique has the potential to broaden the spectrum of identifiable pathogens in corneal ulcers, surpassing the limitations of traditional culture methods. The experiment discussed in the Examples below has already identified a pathogen not previously associated with corneal ulcers, highlighting the method's capacity to enhance understanding of ocular infectious diseases, which is important for developing more effective treatment strategies and improving patient outcomes

[0092] Beyond the impact on patients’ vision, implementing the tear-based diagnostic methods has the potential to induce healthcare costs savings, through streamlined diagnostic processes, more informed treatment regimens, and improved patient outcomes.

[0093] Reduced emergency department referrals: By facilitating accurate diagnoses at the point of care (z.e. by optometrists and primary care physicians or nurse practitioners), the tear-based approach can diminish the need for emergency department referrals, which are often significantly more expensive than outpatient visits. This could translate to significant savings to the healthcare system. Decreased culture-related expenses: Traditional cultures require various media and microbiologists for interpretation. The tear-based method without nucleic acid extraction can be performed at a very low cost.

[0094] Minimized empiric treatment costs: Early and accurate pathogen identification allows for targeted therapy, reducing the reliance on broad-spectrum antibiotics, which are expensive. Initiation of targeted treatments early-on can also shorten the duration of therapy.

[0095] Reducing costs from ocular morbidity: Vision loss is associated with significant costs, not only in terms of direct medical expenses but also through indirect costs like lost productivity. Early and precise diagnosis can reduce the progression of disease, thereby decreasing the likelihood of expensive interventions, which can range from several thousand to tens of thousands of dollars.

[0096] A. Methods of Detecting Ocular Infections

[0097] Herein provided are improved methods of identifying and characterizing ocular infections that circumvent many of the challenges faced by the traditional culture-based method. The experiments below illustrate the discovery that microbial DNA is detectable in tears from eyes with corneal infections, and without the need for nucleic extraction. Utilizing molecular analysis such as Polymerase Chain Reaction (PCR), the nature of microbial infections, e.g., bacterial or fungal, can be discerned from just one microliter of a tear sample. Similarly, next-generation sequencing can be applied to DNA for the precise identification of the pathogen's genus and species (taxonomic classification). Tears from infection-free eyes, such as the contralateral eye, can be used as a control.

[0098] Thus, some embodiments utilize molecular analysis such as PCR, sequencing, or a combination thereof. In some embodiments, the method is a two-tiered process, sequentially or in parallel combining PCR and next-generation sequencing.

[0099] The improved methods mark a significant advancement in the swift identification of microorganisms causing corneal ulcers and other eye infections. These methods not only eliminate the need for corneal scraping but facilitate the collection of detailed information about the infectious agent within hours, rather than the days needed to culture samples according to the traditional diagnostic methods. Paired with antimicrobial resistance mapping, this innovative strategy can provide immediate antimicrobial resistance profiling.

[0100] Any of this information detected or determined according to the disclosed methods can be used to inform treatment of the subject. Consequently, this methodology enhances the clinical management of corneal ulcers and other eye infections. Thus, improved methods of treating eye infections are also provided. Any of the disclosed detection methods can further include a treatment step, and likewise methods of treatment can include a step of patient selection. By offering a non-invasive, expedited, and highly targeted treatment protocol, the provided methods present a strong alternative to the conventional standard of care, promising increased accuracy and speed in identifying microorganisms associated with comeal ulcers.

[0101] 1. Tear Collection and Sample Processing

[0102] The disclosed detection methods utilize a lacrimal fluid (also referred to herein as “tears”) as the source of the sample for analysis. It has been discovered that tears collected from infected eyes contain nucleic acids, and such nucleic acids can be used as a substrate for molecular identification of infection and characterization thereof, e.g., using one or more of the molecular techniques discussed below such as PCR, sequencing, or a combination thereof.

[0103] Tear samples can be collected using any suitable means. In some embodiments, tear samples are collected using a tear collection strip. In the non-limiting exemplary experiment below, tear samples were collected using a tear collection strip, typically a Schirmer strip. However, the methodology can be adapted for other tear collection tools such as capillary action paper, cotton swabs, or alternative devices or tools. In some embodiments, the strip is pre-curved to facilitate its insertion into the inferior fornix of the patient’s eye while minimizing contact with the eyelashes. See, e.g., Figure 1.

[0104] The patient can be instructed to look up and away from the strip to reduce the likelihood of contamination. After a sufficient amount of time to collect an effective amount of tear sample, the strip is then carefully removed. Collection time can be, for example, a few seconds to up to 5 minutes. Thus, in some embodiments, the collection time is any integer number of seconds between 1 and 500 inclusive, preferably between 1 and 300 inclusive, or any sub-range of two integers therebetween.

[0105] After collection, the tears are typically placed in a tube for further processing. For example, when a strip is used to collect tears, the strip can be placed into a vial for centrifugation. The vial can be a pre-labeled vial. In some embodiments care is taken not to overtighten the cap, which may lead to strip damage during centrifugation. A custom vial with a smaller size lid can be used to avoid amputation of the strip.

[0106] Tears can be collected from both eyes. In cases of unilateral infection, tears from the contralateral eye can be used as a control.

[0107] To increase the yield of tear fluid, a secondary strip can be used if the first did not reach the desired volume. The samples can be processed immediately or refrigerated or frozen for processing at a later time.

[0108] The tears can be separated from the collection device using any suitable means. For example, vials with a collection strip(s) can be centrifuged at suitable speed and for suitable duration to separate the tear fluid from the collection strip. In the non-limiting exemplary experiment below, tears were separated from test strip by centrifugation at 5000 RPM for 2 minutes. After centrifugation, the strips were carefully removed from the vials to avoid any potential contamination of the tear fluid.

[0109] The tear sample is typically of sufficient or effective such that microbes present in the eye can be detected in the sample, e.g., according to the provided methods. Preferably the volume of liquid tears isolated from the collection device is any integer number of pl between about 1 pl to 100 pl inclusive, preferable about 1 pl to 20 pl inclusive, or any sub-range of two integers therebetween, however, lower and higher volumes are also contemplated.

[0110] The tears can be used undiluted (e.g., “neat”), or diluted, and / or dried tears can be rehydrated. In various embodiments, the suitable solution includes water, preferably nuclease-free water. In some embodiments, the suitable solution includes a buffer (e.g., a neutral or physiological buffer).

[0111] In some embodiments, no fluid is visible on the collection device. In such cases, liquid or buffer such one including a nuclease- free distilled water, can be added to the strip to facilitate elution of the nucleic acids.

[0112] The tear samples can be processed immediately and subjected to one or more molecular techniques such as PCR and / or sequencing, or stored (e.g., refrigeration, or freezing, e.g., at -80 degrees Celsius) for later analysis. Cold storage helps ensure the preservation of microbial nucleic acid integrity for further molecular analysis.

[0113] For the detection and identification of microbial agents using, e.g., PCR and / or sequencing is discussed in more detail below, DNA or RNA can be extracted from the tears using standard nucleic acid extraction kits. Alternatively, the addition of Proteinase K can be used to facilitate the lysis of cells and release of nucleic acids.

[0114] Due to the inherent nature of the chemistry of organic separation, DNA and RNA can be copurified or selectively isolated individually. To selectively isolate DNA, an RNase A treatment may be used to remove RNA present in the aqueous layer (Rogers and Bendich, 1985). For effective DNA isolation, the aqueous layer typically has a basic pH. Acidification using acid guanidinium thiocyanate-phenol-chloroform extraction, forces DNA to be partitioned into the interphase and organic phase, allowing for convenient isolation of RNA directly from the aqueous phase.

[0115] However, the experiments below illustrate that direct PCR amplification could be successfully achieved with as little as 1 microliter of the tear sample, bypassing the need for prior nucleic acid extraction. Thus, in some embodiments, the sample that is subject to, and substrate for, molecular analysis such as PCR and / or sequencing is undiluted or diluted tears that are not subject to further processing. The sample can be used free from nucleic acid extraction. In other embodiments, the nucleic acids of the undiluted or diluted tears can be separated or otherwise processed prior to use.

[0116] 2. Molecular Analysis

[0117] Following collection of tears, the sample is typically subject to one or more forms of molecular analysis to detect the presence or absence of microbes, determine if any present microbes are bacteria, fungi, virus, parasite, or a combination thereof, and / or identify one or more of genus and / or species of the present bacteria, fungi, virus, parasite, or a combination thereof. These various readouts can be obtained using any suitable molecular means, including but not limited to polymerase chain reaction (PCR), arrays, sequencing, and combination thereof. The substrate of the molecular analysis is typically nucleic acids. Depending on the particular technique(s) employed, microbial DNA and / or RNA can be utilized substate. The absence of detected and / or determined nucleic acids can indicate that no microbes are present. Such a determination can indicate that the eye infection is not due to microbial infection, but rather due to an alternative cause. a. Techniques

[0118] Suitable molecular techniques for use in the disclosed methods are known in the art. See, e.g., U.S. Patent No. 11,807,909, and U.S. Publication Nos. 2022 / 0017946, 2021 / 0371940, 2021 / 0308351, and 2021 / 0171898, each of which is specifically incorporated by reference in its entirety.

[0119] Any of the methods can include amplification and / or sequencing of nucleic acids in the collected tears. The sample can include, without limitation, DNA, RNA, cDNA, dsDNA, ssDNA, high Molecular Weight (MW) DNA, chromosomal DNA, genomic DNA, genomic RNA, mitochondrial DNA (mtDNA), and mRNA. In preferred embodiments, the target polynucleotide is selected from genomic DNA, mRNA, and / or genomic RNA (e.g., from a RNA virus). Any of the aforementioned DNA and / or RNA can include or be microbial DNA and / or microbiral RNA, which is typically the target substrate being sought for detection and analysis. The molecular analysis can include the use of primers and / or probes that are target specific for microbes. Such amplification and / or detection can be used to detect and determine the identity of one or more microbes in the sample without the need for sequencing. Additionally, or alternatively, amplification can be with non-specific or random primers. Probes, arrays, sequencing and other detection means can be used to identify the presence and optionally the identity of microbe(s).

[0120] In certain embodiments, RNA is analyzed directly. In other embodiments, RNA is converted to DNA for subsequent molecular analysis. Most typically this is accomplished by a reverse transcription (RT) reaction. Typically, the RT primer is a single- stranded oligonucleotide (e.g., containing DNA) that can act as a point of initiation for nucleic acid synthesis by a RT polymerase under suitable conditions. Typically, the RT primer is able to hybridize to a target RNA (e.g., miRNA, mRNA) to facilitate cDNA synthesis by a reverse transcriptase. The primer can be specific, random, or semi-random.

[0121] As noted, the methods provided herein can include subjecting the tear sample to a polymerase chain reaction (PCR) to amplify the nucleic acid present in the sample. Any polymerase chain reaction known in the art can be used to amplify the nucleic acids. In various aspects, the polymerase chain reaction includes Real-Time PCR (quantitative PCR or qPCR), Reverse-Transcriptase (RT-PCR), Multiplex PCR, Nested PCR, High Fidelity PCR, Fast PCR, Hot Start PCR, Long-range PCR, Arbitrary Primed PCR, Digital PCR, Droplet Digital PCR (ddPCR), isothermal amplification PCR, Endpoint PCR (Qualitative PCR), or a combination of any thereof. In various aspects, the polymerase chain reaction includes qPCR or ddPCR.

[0122] Quantitative PCR (qPCR) can be used to quantify the presence of microbial DNA, providing both the presence and abundance of the target sequence.

[0123] Digital PCR (dPCR) is an alternative approach, providing absolute quantification without the need for external standards.

[0124] Conventional PCR followed by gel electrophoresis can also be used for size-based confirmation of the amplified products. Additionally, loop-mediated isothermal amplification (LAMP) PCR can be employed as a rapid, highly specific, and sensitive method that could be performed without the need for thermal cyclers.

[0125] In some aspects, the analysis can include quantitative detection of the nucleic acid, such as using an oligonucleotide probe or nucleic acid dye. In various aspects, the quantitative detection occurs as part of the polymerase chain reaction performed (e.g., using real time PCR or quantitative PCR (qPCR). In some aspects, the quantitative detection occurs after the polymerase chain reaction is performed. For example, in some aspects, the quantitation can use a Nanopore or similar nanotechnology to detect the nucleic acids. See for example Kang et al., “Ready-to-use nanopore platform for the detection of any DNA / RNA oligo at attomole range using an Osmium tagged complementary probe” Scientific Reports 10, 19790 (2020), which is incorporated herein by reference in its entirety. qPCR is a well-established method for the detection, quantification, and typing of different microbes in clinical samples. Exemplary qPCR methods for use with the present invention include: (1) non-specific fluorescent dye intercalation with any double-stranded DNA and / or (2) sequencespecific oligonucleotides DNA probes that are labelled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary sequence. qPCR has the advantage of being relatively fast and low cost but can only cover limited targets, even with multiplex PCR and multiple-well of amplification.

[0126] Each of the PCR reactions described above can be performed according to standard methods in the art, including those provided in laboratory manuals such as Sambrook, J., et al. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Spector, D. L. et al., Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998; Carruthers, W., and Coldham, I., Modern Methods of Organic Synthesis (4th Edition), Cambridge University Press, Cambridge, U.K., 2004, herein incorporated by reference in its entirety.

[0127] As noted, the methods provided herein include sequencing nucleic acids. The nucleic acids substrate for sequencing can be the nucleic acids from the original tear sample, and / or an amplified nucleic acid product thereof, e.g., produced by a PCR protocol. In various aspects, the analysis can include sequencing the nucleic acids using, but not limited to, Sanger sequencing, single molecule real time (SMRT) sequencing, nanopore DNA sequencing, massively parallel signature sequencing (MPSS), colony sequencing, 454 pyrosequencing, Illumina sequencing, combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, ion torrent semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, or a combination of any thereof, optionally using a microfluidic system. In some embodiments, the sequencing is or includes Illumina, Ion Torrent, PacBio, or Oxford Nanopore technology, or any similar next-generation sequencing platforms.

[0128] Oxford Nanopore sequencing technology used in Nanopore sequencing is the third generation single-molecule sequencing technology which has the advantages of simple sample handling, fast and long sequencing length (>10 kbp) compared with NGS second-generation technology that utilizes amplified signals. Oxford Nanopore sequencing is an emerging third- generation sequencing technology, that can generate ultra-long reads exceeding 800 kb (Jain et al., Nat Biotechnol 36, 338-345, doi:10.1038 / nbt.4060 (2018)) in a portable device called MinlON. These long-reads come without much compromise on reads consensus accuracy since the sequencing errors are mostly random (Loman et al., Nat Methods 12, 733-U751, doi:10.1038 / Nmeth.3444 (2015)). They hold great promise in calling and phasing variants, assembling scaffold, and prospectively detecting epigenetic marks (Cretu et al., Nat Commun 8, 1326, doi: 10.1038 / s41467-017-01343-4 (2017), Simpson et al., Nat Methods 14, 407-410, doi:10.1038 / nmeth.4184 (2017)). These advantages are very suitable for the rapid identification of clinically unknown microbial pathogens.

[0129] In still further aspects, the analysis can include qualitative detection, such as using an agarose gel, polyacrylamide electrophoresis, restriction endonuclease digestion, dot blots, liquid chromatography, electrochemoluminescence, or a combination of any thereof. In general, any method known in the art to sequence, quantify, or detect the nucleic acid may be used in the methods provided herein. In any aspect provided herein, identifying the nucleic acid as indicative of the presence of microbe(s) is contemplated.

[0130] In some embodiments, a PCR, such as qPCR, is used to quantify the populations of microb(s), using described primers and protocols known to persons skilled in the art (Borrel et al, ISME J. 2017 Sep;l l(9):2059-2074. Doi: 10.1038 / ismej.2017.72. Epub 2017 Jun 6. PMID: 28585938; PMCID: PMC5563959.). Additionally, or alternatively, sequencing is used to determine the dominant or various species of the enrichment.

[0131] For example, in the experiments below, PCR amplification was performed using specific primer pairs designed to target the DNA or RNA of potential causative organisms. This method is versatile and can incorporate primers for a wide range of pathogens, including bacteria (e.g., 16S rRNA for broad-range bacterial detection), fungi (e.g., 18S rRNA or ITS regions for broad-range fungal detection), viruses (e.g., specific viral gene targets), and other microorganisms such as mycobacterium or Acanthamoeba (e.g., 18S rRNA).

[0132] For comprehensive microbial identification, raw nucleic acids and / or PCR products can be analyzed using sequence, e.g., next-generation sequencing (NGS). This approach allows for the detailed taxonomic classification of the microbial community within the tear samples. The versatility of this workflow accommodates various sequencing platforms, including but not limited to, Illumina, Ion Torrent, PacBio, and Oxford Nanopore Technologies. In some embodiments, the molecular analysis of a sample includes sample indexing, adaptor ligation and / or library normalization. Sample indexing (“barcoding”) allows multiple targets and / or samples to be run simultaneously taking full advantage of the high-throughput nature of current sequencing platforms. Adapter ligation is sequencing platform specific and standard to manufacturers' protocols. At this step, microbial, or pathogenic, or parasitic DNA or fragments thereof have the platform- specific end sequences needed for sequencing along with index sequences that allow for de-convolution of sequence data by sample. Libraries can be prepared at platform specific concentrations of DNA. Libraries typically require amplification or dilution to achieve the required DNA concentration. The DNA concentration in the library can be determined by, e.g., quantitative real-time PCR using platform specific manufacturer protocols or fluorescence-based measurement using an instrument such as the ThermoFisher Qubit. The sequencing library represents the DNA or fragments or amplicons thereof that make up the genome of the microbes present in the tear sample. These are the molecules whose sequence is determined to generate reads that can be used for k-mer generation and subsequent analyses.

[0133] In the experiment discussed in the Examples below Oxford Nanopore sequencing was used. The use of 16S rRNA gene primers facilitated the identification of bacterial species present within the samples. Additionally, a rapid PCR barcoding kit was also utilized to amplify the entire DNA content of the sample, which included human, bacterial, fungal, viral, and other microbial DNA. To improve the sequencing output for microbial DNA, an adaptive sampling technique was incorporated, which selectively depletes human DNA sequences during the nanopore sequencing run, thereby enriching for non-human DNA and enhancing the detection of pathogenic organisms.

[0134] Molecular analysis of tear samples provides a robust framework for the rapid and precise identification of microbial agents responsible for eye infections including, but not limited to, corneal ulcers, and could be adapted to include other molecular techniques (e.g., emerging sequencing technologies) as they become available or as required by specific research parameters.

[0135] Tears from the contralateral unaffected eye, as well as distilled nuclease-free sterile water, can be used as negative controls. For every primer pair, optimal melting temperature and number of cycles can be determined according to known methods. b. Enzymes and Buffers

[0136] The methods typically utilize, and the provided kits can include, buffers and / or enzymes.

[0137] Typically, the buffers provide appropriate pH and ionic conditions for the one or more enzymes. For example, a buffer can be an aqueous solution that provides optimal pH, ionic strength, cofactors, and the like for optimal enzyme activity. In some embodiments, the buffers are suitable for storage of the enzymes. In preferred embodiments, the buffers are suitable for RT and / or PCR and / or sequenceing.

[0138] Suitable buffer components include, without limitation, one or more salts, reducing agents (e.g., Dithio threitol), buffering agents, deoxynucleoside triphosphates (dNTPs), or combinations thereof. The one or more salts provide monovalent or divalent cations, such as, Mg2+, Mn2+, K+, NH4+, and Na+. Exemplary salts that can be included in the buffers are KC1, MgCh, NaCl, MnCL, NH4CI, MgSO4, (NH4)2SO4, and magnesium acetate. The concentration of the one or more salts can be in the range of from about 1 mM to about 500 mM, about 5 mM to about 250 mM, about 10 mN to about 200 mM, about 25 mM to about 150 mM, or about 50 mM to about 100 mM.

[0139] Suitable buffering agents are known in the art and include, without limitation, tris (e.g., Tris- HC1), tricine, bicine, and HEPES. The buffering agent can have a pH in the range of about 6 to 10 (e.g., a pH of 6.8 to 9, such as about pH 8.5). The concentration of the one or buffering agents can be in the range of from about 10 mM to about 100 mM.

[0140] The compositions can further include one or more nucleotides (e.g., deoxynucleoside triphosphates (dNTPs)). The nucleotide components of the compositions serve as the “building blocks” for newly synthesized nucleic acids, being incorporated therein by the action of the reverse transcriptases or DNA polymerases. Examples of nucleotides suitable for use in the compositions include, but are not limited to, dUTP, dATP, dTTP, dCTP, dGTP, diTP, 7-deaza-dGTP, a-thio- dATP, a-thio-dTTP, a-thio-dGTP, a-thio-dCTP or derivatives thereof, all of which are available commercially from sources including Life Technologies, Inc. (Rockville, Md.), New England BioLabs (Beverly, Mass.) and Sigma Chemical Company (Saint Louis, Mo.). In preferred embodiments, the following dNTPs are included in the compositions: dATP, dTTP, dGTP, and dCTP.

[0141] Suitable reverse transcriptases are known in the art and are commercially available. In some embodiments, the reverse transcriptase is a Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT). Suitable commercial reverse transcriptases include MMLV High Performance Reverse Transcriptase and EpiScript™ RNase H- Reverse Transcriptase (Lucigen), NEB ProtoScript II reverse transcriptase (NEB Cat. No M0368) and Invitrogen SuperScript II, III, and IV reverse transcriptase (Thermo Fisher Scientific Cat. No 18090010).

[0142] Suitable DNA polymerases are known in the art and are commercially available. In some embodiments, the DNA polymerase is a Thermus aquaticus DNA polymerase (Taq Pol). Suitable commercially available DNA polymerases include EconoTaq Polymerase, Phi29 DNA polymerase, Bsu DNA polymerase, OmniAmp™ DNA polymerase (Lucigen), Bst DNA polymerase, Bst 2.0 DNA polymerase, and Bst 2.0 WarmStart™ DNA polymerase (New England Biolabs), Platinum II Taq Polymerase (ThermoFisher Scientific), and KlenTaql from DNA Polymerase Technologies, Inc.

[0143] 3. Bioinformatics

[0144] Any of the disclosed methods can include one or more bioinformatics techniques to facilitate execution of molecular techniques such as those mentioned above and / or analysis and interpretation of information collected by them. Bioinformatics, method of use thereof, and publicly available resources associated therewith are known in the art. See, e.g., U.S. Published Application No. 2020 / 0224248, which is specially incorporated by reference herein in its entirety.

[0145] Molecular methods based on DNA sequence are becoming more and more the reference standard in the post-genomic era, thanks to the availability of an increasing number of DNA sequences of prokaryotic and eukaryotic microorganisms for genotyping and for the identification.

[0146] A key for applying DNA sequences for the characterization, the identification and quantification of any microorganism is the possibility to find in a limited region that is commonly defined “marker” bringing biological information. Nature Magazine defines genetic markers as “DNA sequences with known physical locations on chromosomes. They are points of variation that can be used to identify individuals or species, or may be used to associate an inherited disease with a gene through genetic linkage with nearby but possibly unidentified or uncharacterized genes”. The genetic (DNA) markers applied “to identify individuals or species” share high similarity within a defined “target” group of microorganisms and contemporarily nucleotide variations to differentiate the “target” group from any other.

[0147] In the post-genomic era, the paradigm is shifting from “genetic marker” to “genomic marker”, as well as from “phylogenetics” to “phylogenomics”, due to the availability of an increasing number of whole genome sequences and also to the availability of new bioinformatics tools for data mining. The concept of “genetic marker” and “genomic marker” can be used indeed as synonyms, as they relate on DNA sequences “used to identify individuals or species, or may be used to associate an inherited disease with a gene through genetic linkage”. Either approach can be used in association with, or as steps, in the disclosed methods.

[0148] As is known in the art, the size and the characteristics of the DNA region depends on the characteristics of the diagnostic technological platform which the found DNA sequences (marker) will be applied to. For instance Quantitative Real Time PCR (qPCR) to identify and quantify microorganisms typically amplies short sequences, possibly in the range of 100 bp and 200 bp, while ITS-RFLP to characterize and identify different yeast species is based on DNA sequence sizes usually from 400 bp and 1,050 by. The last two examples introduce the concept of “adaptability” of a certain DNA marker, that can be adapted and used for DNA-based fingerprinting, by implementing different protocols that work on diverse technological platforms such as Real Time qPCR and ITS-RFLP.

[0149] Thus, various DNA-based molecular methods can be used to target the same or different markers to characterize, identify and quantify microorganisms, depending on the characteristics of the microorganisms and also depending on the characteristics of the marker. Examples of marker strategies that can be used in conjunction with the disclosed methods include, but are not limited to, sequence variability generated by events that can be classified in categories such as: a) nucleotide substitutions which generate Single Nucleotide Polymorphisms (SNPs); b) insertions and / or deletions of nucleotides, mobile elements, tandem repeats which generate length polymorphisms. The sequence analysis of the genes coding for the small subunit ribosomal RNA (SSU RNA) and large subunit ribosomal RNA (LSU RNA), namely 16S rRNA and 23S rRNA for prokaryotes and 18S rRNA and 26S rRNA for eukaryotes are generally considered the most effective and robust marker for the identification to the species level. Despite this general rule closely phylogenetically related species are characterized by SSU RNA and LSU RNA close to 100% of sequence similarity. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has been fundamental to addressing questions about bacterial diversity in environmental samples. The 16S rRNA gene is highly conserved, but contains hypervariable regions that can be used to distinguish bacteria at the genus level. Amplicon datasets are often large, and can be rapidly reduced into clusters of operational taxonomic units (OTUs) to quantify the relative abundance of bacteria in a sample.

[0150] Amplicon datasets are useful for surveying bacterial diversity at the genus-level, but provide no information about the function of bacteria in a given environment. Whole genome shotgun (WGS) sequencing offers increased resolution at the species and subspecies level and can be used to infer both taxonomy and function.

[0151] Bioinformatic tools can be used to identify and compare any DNA sequence marker. Numerous software packages, databases, platforms, and score filters involve computational pipelines that assign functions to the genes and facilitate comparative genomic analysis to find any genetic marker candidate for microorganisms identification and quantification. Such tools can be utilized in the disclosed methods to inform the selection of target nucleic acid sequences, design probes and primers, and analyze data collected by molecular techniques such as those mentioned above for the detection of microbes in a tear sample, and optionally for identification thereof. For example, fast k-mer based algorithms have been developed to classify metagenomic reads against known microbial genomes at remarkable throughput and speed. Specifically, Centrifuge (Kim, et al., “Centrifuge: rapid and sensitive classification of metagenomic sequences,” bioRxiv, p. 054965, doi:l 0.1101 / 054965 (2016), CLARK (CLAssifier based on Reduced K-mers) (Ounit, et al., BMC Genomics, 16:236 (2015)), USEARCH (Edgar, et al., BMC Bioinformatics, 26:2460-2461 (2010)), KRAKEN (Wood, et al., Genome Biol, 15:R46 (2014)), and NBC (Naive Bayes Classifier) (Rosen, et al., Adv Bioinformatics, 2008:205969, doi: 10.1155 / 2008 / 205969 (2008)) rapidly identify microbial or parasitic species present in a metagenome using genetic composition-based methods. In each case, frequency profiles of k-mers from microbial genomes are built to rapidly assign reads to genomes in a reference database (Ounit, et al., BMC Genomics, 16:236 (2015); Wood, et al., Genome Biol, 15:R46 (2014); Rosen, et al., BMC Bioinformatics, 27:127-129 (2011); Bazinet, et al., BMC Bioinformatics, 13:92 (2012)). These methods are fast and outperform alignment-based methods, which can also be used.

[0152] B. Organisms and Infections to be Detected and Treated

[0153] Disclosed herein are methods of detecting, identifying, and characterizing eye infections. The methods can, but need not necessarily, include identifying bacteria, fungi, virus and / or parasites that are the causes of the initiation and / or persistence of an eye infection. As discussed in more detail below, these methods can be used to provide diagnostic and prognostic information about suspected infections, pathogens, microbes, or parasites. In some embodiments, the methods include performing molecular analysis of a biological sample such as a patient tear sample, preparing the data obtained from the molecular analysis, developing diagnostic information about the sample and / or prognostic information from the sample. Although the methods described herein typically refer to microbial infections, which include, without limitation, bacterial, viral, fungal, and parasitic infections, or any combination thereof. It is contemplated that the disclosed methods can be utilized to improve clinical outcomes in many types of infections, including, but not limited to, bacterial, viral, fungal, and parasitic infections. Thus, any reference to microbe or microbial should not be viewed as including or excluding any one or more of bacteria, virus, fungi, or parasite (or bacterial, viral, fungal, and parasitic), or any one or more specific example thereof. Any references to bacteria, virus, fungi, or parasite should also be viewed as contemplating one or more of the other microbes.

[0154] 1. Types of Organisms to Be Detected

[0155] The disclosed methods can be used to identify the presence of organisms and infections thereof, such as bacteria, fungal, viral and / or parasitic. In one example, one or more of the following types of organisms can be detected by the present method: Abiotrophia, Acanthamoeba, Acetobacteraceae, Achromobacter, Acidaminococcus, Acidithiobacillus, Acidocella, Acidovorax, Acinetobacter, Acremonium, Actinobacillus, Actinobaculum, Actinomadura, Actinomyces, Adenovirus, Aerococcus, Aeromonas, Aeropyrum, Aggregatibacter, Agrobacterium, Akkermansia, Alcaligenes, Alistipes, Alphacoronavirus, Alternaria, Alteromonas, Anabaena, Anaerobiospirillum, Anaerococcus, Anaeroglobus, Anaerostipes, Anaplasma, Anoxybacillus, Aquabacterium, Arachnia, Aranicola, Arcanobacterium, Arcobacter, Arthrobacter, Arthroderma, Arthrospira, Ascaris, Aspergillus, Astrovirus, Atopobium, Bacillus, Bacteroides, Bacteroidetes, Bartonella, Beauveria, Betacoronavirus, Bifidobacterium, Bilophila, Bipolaris, Blastochloris, Blastococcus, Blastocystis, Blastomyces, Blastoschizomyces, Blautia, Bordetella, Borrelia, Brachymonas, Brachyspira, Bradyrhizobium, Branhamella, Brevibacillus, Brevibacterium, Brevundimonas, Brucella, Buchnera, Bulleidia, Burkholderia, Burkholderiales, Buttiauxella, butyrate-producing organism, Butyrivibrio, Calicivirus, Campylobacter, Candida, Candidatus, Capnocytophaga, Carbolfuchsin, Cardiobacterium, Camobacterium, Catenibacterium, Caulobacter, Cedecea, Cefuroxime, Cellulosimicrobium, Centipeda, Cephalosporins, Cephalosporium, Chaetomium, Chaetothyriales, Chilomastix, Chlamydia, Chlamydophila, Chromobacterium, Chryseobacterium, Chrysosporium, Citrobacter, Cladosporium, Clarithromycin, Clindamycin, Cloacibacterium, Clonorchis, Clostridiales, Clostridium, Coccidioides, Collinsella, Comamonas, Conidiobolus, Coprobacillus, Coprococcus, Corynebacteria, Corynebacterium, Coxiella, Cryptobacterium, Cryptococcus, Cryptosporidium, Cunninghamella, Curvularia, Cyanobacteria, Cyclospora, Cylindrospermopsis, Cytomegalovirus, Dactylaria, Davidiella, Delftia, Deltacoronavirus, Dermabacter, Desmospora, Desulfitobacterium, Desulfomicrobium, Desulfovibrio, Dialister, Didymella, Dientamoeba, Diphyllobothrium, Dolosigranulum, Dorea, Dreschlera, Eboli, Echinococcus, Edwardsiella, Eggerthella, Ehrlichia, Eikenella, Empedobacter, Enhydrobacter, Entamoeba, Enterobacter, Enterobacteriaceae, Enterobius, Enterococci, Enterococcus, Enterovirus, Epicoccum, Epidermophyton, Eremococcus, Erwinia, Erysipelothrix, Erysipelotrichaceae, Erythrobacter, extended spectrum beta-lactamase(ESBL), Escherichia, Eubacterium, Ewingella, Excerohilum, Exiguobacterium, Exoantigen, Exophiala, Facklamia, Faecalibacterium, Filifactor, Finegoldia, Flavobacterium, Flavonifractor, Fonsecaea, Francisella, Frankia, Fusarium, Fusobacterium, Gallicola, Gammacoronavirus, Gardnerella, Gemella, Geobacillus, Geotrichum, Giardia, Giemsa, Gliocladium, Gordonia, Gordonibacter, Granulicatella, Haemophilus, Hafnia, Haloarcula, Halobacterium, Halosimplex, Hansenula, Helcococcus, Helicobacter, Helminthosporium, Hemadsorbing, Herpes, Histoplasma, Holdemania, Hymenolepis, Hyphomicrobium, lodamoeba, Isospora, Janibacter, Janthinobacterium, Jeotgalicoccus, Johnsonella, Kingella, Klebsiella, Kluyvera, Kocuria, Koserella, Lachnospiraceae, Lactobacillus, Lactococcus, Lautropia, Leclercia, Legionella, Leifsonia, Leminorella, Leptospira, Leptotrichia, Leuconostoc, Listeria, Listonella, Lyngbya, Lysinibacillus, Malassezia, Malbranchea, Mannheimia, Megamonas, Megasphaera, Mesorhizobium, Methanobacterium, Methanobrevibacter, Methanosaeta, Methanosarcina, Methanothermobacter, Methylobacterium, Microbacterium, Micrococcus, Microcoleus, Microcystis, Microsporidia, Microsporum, Mobiluncus, Mogibacterium, Mollicutes, Moraxella, Morganella, Mycelia, Mycetocola, Mycobacterium, Mycoplasma, Myroides, Neisseria, Neorickettsia, Nigrospora, Nocardia, Nodularia, Nostoc, Oceanobacillus, Ochrobactrum, Odoribacter, Oenococcus, Oerskovia, Oligella, Olsenella, Oribacterium, Ornithobacterium, Oscillatoria, Oxalobacter, Paecilomyces, Paenibacillus, Pantoea, Parabacteroides, Paracoccus, Paraprevotella, Parascardovia, Parasutterella, Parvimonas, Pasteurella, Pediculus, Pediococcus, Penicillium, Peniophora, Peptococci, Peptococcus, Peptoniphilus, Peptostreptococcus, Petrobacter, Phaeoacremonium, Phaeoannellomyces, Phascolarctobacterium, Phialemonium, Phialophora, Photobacterium, Photorhabdus, Phyllobacterium, Pichia, Picornavirus, Pirellula, Piscirickettsia, Planktothrix, Planomicrobium, Plasmodium, Plesiomonas, Pneumocystis, Poliovirus, Porphyromonas, Prevotella, Propionibacterium, Proteus, Prototheca, Providencia, Pseudallescheria, Pseudomonas, Pseudoramibacter, Pseudoxanthomonas, Rahnella, Ralstonia, Raoultella, Rathayibacter, Rhinocladiella, Rhinosporidium, Rhinovirus, Rhizobium, Rhizomucor, Rhizopus, Rhodanobacter, Rhodococcus, Rhodopirellula, Rhodopseudomonas, Rhodotorula, Riemerella, Roseburia, Roseomonas, Rotavirus, Rothia, Ruminococcaceae, Ruminococcus, Saccharomyces, Salmonella, Sarcoptes, Scardovia, Scedosporium, Schistosoma, Schizophyllum, Schlegelella, Scopulariopsis, Scytalidium, Segniliparus, Selenomonas, Sepedonium, Serratia, Shewanella, Shigella, Simonsiella, Sistotrema, Slackia, Sneathia, Solobacterium, Sphingobacterium, Sphingobium, Sphingomonas, Spirochaeta, Spirochaetaceae, Spirochetes, Spirosoma, Sporobolomyces, Sporothrix, Stachybotrys, Staphylococcus, Stemphylium, Stenotrophomonas, Stenoxybacter, Streptococcus, Streptomyces, Strongyloides, Succinatimonas, Succinivibrio, Sutterella, Syncephalastrum, Synechococcus, Synergistetes, Taenia, Tannerella, Tatumella, Tepidimonas, Tetragenococcus, Tissierella, Treponema, Trichinella, Trichoderma, Trichomonads, Trichomonas, Trichophyton Trichosporon, Trichothecium, Trichuris, Tropheryma, Trypanosoma, Turicibacter, Udeniomyces, Ulocladium, Ureaplasma, Ureibacillus, Ustilago, Vagococcus, Varicella, Variovorax, Veillonella, Verticillium, Vibrio, Virgibacillus, Viridans, Vulcanisaeta, Wangiella, Wautersia, Weeksella, Weissella, Wolbachia, Wolinella, Xanthomonas, Xylohypha, Yersinia, Yokenella, Zoogloea, or Zygomycete.

[0156] In some forms, the organism(s) is one or more of Acanthamoeba, Acinetobacter, Actinobacillus, Actinomyces, Adenovirus, Aspergillus, Bacillus, Bacteroides, Bartonella, Campylobacter, Candida, Clostridium, Coccidioides, Corynebacteria, Corynebacterium, Coxiella, Cryptococcus, Cryptosporidium, Cyclospora, Cytomegalovirus, Flavobacterium, Fusarium, Giardia, Herpes, Histoplasma, Klebsiella, Legionella, Leptospira, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pneumocystis, Propionibacterium, Proteus, Pseudomonas, Rhinovirus, Rhizomucor, Rhizopus, Schistosoma, Serratia, Shigella, Sphingomonas, Spirochaeta, Spirochaetaceae, Staphylococcus, Stenotrophomonas, Streptococcus, Treponema, Trichinella, or Varicella.

[0157] In some examples, one or more pathogenic bacteria are detected with the disclosed method. Examples of pathogenic bacteria which could be detected with the disclosed methods include without limitation any one or more of (or any combination of: Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomyces sp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp. (such as Aeromonas hydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonas caviae), Anaplasma phagocy tophilum, Anaplasma marginale, Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroides sp. (such as Bacteroides fragilis), Bartonella sp. (such as Bartonella bacilliformis and Bartonella henselae, Bifidobacterium sp., Bordetella sp. (such as Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi), Brucella sp. (such as Brucella abortus, Brucella canis, Brucella melintensis and Brucella suis), Burkholderia sp. (such as Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter sp. (such as Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus), Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as, Corynebacterium diphtheriae, Corynebacterium jeikeum and Corynebacterium), Clostridium sp. (such as Clostridium perfringens, Clostridium difficile, Clostridium botulinum and Clostridium tetani), Eikenella corrodens, Enterobacter sp. (such as Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae and Escherichia coli, including opportunistic Escherichia coli, such as enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, ente roagg re gative E. coll and uropatho genic E. coli) Enterococcus sp. (such as Enterococcus faecalis and Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacter pylori, Helicobacter cinaedi and Helicobacter fennelliae), Kingella kingii, Klebsiella sp. (such as Klebsiella pneumoniae, Klebsiella granulomatis and Klebsiella oxytoca), Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Mannheimia hemolytica, Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp., Mycobacterium sp. (such as Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium paratuberculosis, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis, and Mycobacterium marinum), My coplasm sp. (such as Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp. (such as Nocardia asteroides, Nocardia cyriacigeorgica and Nocardia brasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, Plesiomonas shigelloides. Prevotella sp., Porphyromonas sp., Prevotella melaninogenica, Proteus sp. (such as Proteus vulgaris and Proteus mirabilis), Providencia sp. (such as Providencia alcalifaciens, Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (such as Rickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii, Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) and Rickettsia typhi), Rhodococcus sp., Serratia marcescens, Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium), Serratia sp. (such as Serratia marcesans and Serratia liquifaciens), Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei), Staphylococcus sp. (such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus), Streptococcus sp. (such as Streptococcus pneumoniae (for example chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin- resistant serotype 9V Streptococcus pneumoniae, erythromycin-resistant serotype 14 Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, and trimethoprim- resistant serotype 23F Streptococcus pneumoniae, chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, or trimethoprim-resistant serotype 23F Streptococcus pneumoniae), Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, Group A streptococci, Streptococcus pyogenes, Group B streptococci, Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus, Streptococcus equismilis, Group D streptococci, Streptococcus bovis, Group F streptococci, and Streptococcus anginosus Group G streptococci), Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such as Treponema carateum, Treponema petenue, Treponema pallidum and Treponema endemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela and Vibrio fumisii), Yersinia sp. (such as Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis) and Xanthomonas maltophilia among others.

[0158] In some examples, the detected microbes include one or more of one or more Staphylococcus such as Staphylococcus saccharolyticus and / or Staphylococcus roterodami, one or more Pseudomonas such as Pseudomonas aeruginosa, one or more Cutibacterium such as Cutibacterium acnes, one or more Kocuria such as Kocuria rhizophila, or a combination thereof.

[0159] In some examples, one or more pathogenic fungi are detected with the disclosed method. Examples of pathogenic fungi which could be detected with the disclosed methods include without limitation any one or more of (or any combination of) Trichophyton rubrum, T. mentagrophytes, Epidermophytonfloccosum, Microsporum canis, Pityrosporum orbiculare (Malassezia furfur), Candida sp. (such as Candida albicans), Aspergillus sp. (such as Aspergillus fumigatus, Aspergillus flavus and Aspergillus clavatus), Cryptococcus sp. (such as Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus laurentii and Cryptococcus albidus), Histoplasma sp. (such as Histoplasma capsulatum), Pneumocystis sp. (such as Pneumocystis jirovecii), and Stachybotrys (such as Stachybotrys chartarum) among others.

[0160] In some examples, one or more viruses are detected with the disclosed method. Examples of viruses which could be detected with the disclosed methods include without limitation any one or more of (or any combination of) Arenaviruses (such as Guanarito virus, Lassa virus, Junin virus, Machupo virus and Sabia), Arteriviruses, Roniviruses, Astroviruses, Bunyaviruses (such as Crimean-Congo hemorrhagic fever virus and Hantavirus), Barnaviruses, Bimaviruses, Bomaviruses (such as Boma disease virus), Bromoviruses, Caliciviruses, Chrysoviruses, Coronaviruses (such as MERS, SARS-CoV-1, SARS-CoV-2, etc.), Cystoviruses, Clostero viruses, Comoviruses, Dicistroviruses, Flaviruses (such as Yellow fever virus, West Nile virus, Hepatitis C virus, and Dengue fever virus), Filoviruses (such as Ebola virus and Marburg virus), Flexiviruses, Hepeviruses (such as Hepatitis E virus), human adenoviruses (such as human adenovirus A-F), human astroviruses, human BK polyomaviruses, human bocaviruses, human coronavirus (such as a human coronavirus HKU1, NL63, and OC43), human enteroviruses (such as human enterovirus A- D), human erythrovirus V9, human foamy viruses, human herpesviruses (such as human herpesvirus 1 (herpes simplex virus type 1), human herpesvirus 2 (herpes simplex virus type 2), human herpesvirus 3 (Varicella zoster virus), human herpesvirus 4 type 1 (Epstein-Barr virus type 1), human herpesvirus 4 type 2 (Epstein-Barr virus type 2), human herpesvirus 5 strain AD169, human herpesvirus 5 strain Merlin Strain, human herpesvirus 6 A, human herpesvirus 6B, human herpesvirus 7, human herpesvirus 8 type M, human herpesvirus 8 type P and Human Cyotmegalo virus), human immunodeficiency viruses (HIV) (such as HIV 1 and HIV 2), human metapneumoviruses, human papillomaviruses (such as human papillomavirus- 1, human papillomavirus- 18, human papillomavirus-2, human papillomavirus-54, human papillomavirus-61, human papillomavirus-cand90, human papillomavirus RTRX7, human papillomavirus type 10, human papillomavirus type 101, human papillomavirus type 103, human papillomavirus type 107, human papillomavirus type 16, human papillomavirus type 24, human papillomavirus type 26, human papillomavirus type 32, human papillomavirus type 34, human papillomavirus type 4, human papillomavirus type 41, human papillomavirus type 48, human papillomavirus type 49, human papillomavirus type 5, human papillomavirus type 50, human papillomavirus type 53, human papillomavirus type 60, human papillomavirus type 63, human papillomavirus type 6b, human papillomavirus type 7, human papillomavirus type 71, human papillomavirus type 9, human papillomavirus type 92, and human papillomavirus type 96), human parainfluenza viruses (such as human parainfluenza virus 1-3), human parechoviruses, human parvoviruses (such as human parvovirus 4 and human parvovirus B19), human respiratory syncytial viruses, human rhinoviruses (such as human rhinovirus A and human rhinovirus B), human spumaretroviruses, human T- lymphotropic viruses (such as human T-lympho tropic virus 1 and human T-lymphotropic virus 2), Human polyoma viruses, Hypoviruses, Leviviruses, Luteoviruses, Lymphocytic choriomeningitis viruses (LCM), Marnaviruses, Namaviruses, Nidovirales, Nodaviruses, Orthomyxoviruses (such as Influenza viruses), Partitiviruses, Paramyxoviruses (such as Measles virus and Mumps virus), Picornaviruses (such as Poliovirus, the common cold virus, and Hepatitis A virus), Potyviruses, Poxviruses (such as Variola and Cowpox), Sequiviruses, Reoviruses (such as Rotavirus), Rhabdoviruses (such as Rabies virus), Rhabdoviruses (such as Vesicular stomatitis virus, Tetraviruses, Togaviruses (such as Rubella virus and Ross River virus), Tombusviruses, Totiviruses, Tymoviruses, Noroviruses, bovine herpesviruses including Bovine Herpesvirus (BHV) and malignant catarrhal fever virus (MCFV), among others.

[0161] Exemplary parasites that can be identified with the disclosed methods herein include, but are not limited to, Malaria (Plasmodium falciparum, P. vivax, P. malariae), Schistosomes, Trypanosomes, Leishmania, Filarial nematodes, Trichomoniasis, Sarcosporidiasis, Taenia (T. saginata, T. solium), Leishmania, Toxoplasma gondii, Trichinelosis (Trichinella spiralis), Coccidiosis (Eimeria species), and / or amoeba (e.g., Acanthamoeba)

[0162] 2. Subjects

[0163] The disclosed methods are typically used to assist in the diagnosis and treatment of an eye infection in a subject. Although the experiments in the Example below exemplifies ocular infections in humans, the disclosed methods also have important applications in veterinary medicine. Ocular infections are common in domestic animals, with dogs and cats frequently presenting with corneal ulcers and conjunctivitis, conditions that closely parallel those found in humans. In veterinary practice, the challenges of diagnosing ocular infections are compounded by the difficulties in collecting samples from animals. Non-invasive tear collection offers a less stressful alternative to corneal scraping or conjunctival swabs, which are especially challenging in non-compliant or agitated animals.

[0164] Thus, the subject can be human or an animal such as an agricultural animal or a domesticated animals such as pets. Subjects include, but are not limited to, mammals, such as goats, sheep, pigs, cattle, zebu, donkeys, water buffaloes, dromedary camel, horse, yak, domestic bactrian camel, llama, alpaca, gayal, bali cattle, domestic rabbit, addax, bison, deer, eland, elk, guinea pig, greater kudu, mule, moose, muskox, and reindeer, and birds, such as, chicken, duck, goose, guineafowl, muscovy duck, turkey, emu, egyptian goose, indian peafowl, mute swan, ostrich, partridge, small-billed tinamou, pigeon, quail, edible-nest swiftlet, grey francolin, guineafowl, common pheasant, and golden pheasant.

[0165] In some embodiments, the subject is an otherwise healthy subject that has recently undergone a corneal transplant. The disclosed compositions and methods can be used to assess the status of the transplant and identify infection, e.g., that occurred during or as a result of the transplant. In some embodiments, the infection is not yet visible. In some embodiments, no microbes are detected and the subject is cleared as being free from infection. The transplant can be, e.g., days, weeks, or months before tear collection. For example, in some embodiments, the comeal transplant was about 1-365 days inclusive or any subrange or specific day therebetween, preceding the collection of the tear sample. In specific examples, the transplant was 1 month, two weeks, 10 days, or 1 week prior to tear collection. See, e.g., Song, et al., “Post-keratoplasty Infectious Keratitis: Epidemiology, Risk Factors, Management, and Outcomes,” Front Med (Lausanne). 2021 Jul 7;8:707242. doi: 10.3389 / fmed.2021.707242. PMID: 34307431; PMCID: PMC8292647, which is specifically incorporated by references herein in its entirety.

[0166] 3. Infections

[0167] The disclosed methods are typically used to detect and characterized (e.g., identify the cause) or ocular (i.e., eye) infections. In some embodiments, the eye infection is a comeal ulcer. Comeal ulcers are a prominent ocular pathology marked by a disruption in the comeal epithelium. This disruption allows microorganisms to invade, leading to inflammation in the underlying stromal layer. Immediate and accurate identification and treatment are vital, as delays can result in irreversible corneal damage, scarring, and potential loss of vision.

[0168] However, while the experiment in the Example below is directed to identification of the cause of comeal ulcers, this embodiment is exemplary and illustrative that the principles can be applied similarly to other ocular infections, particularly where the infection presents diagnostic and / or treatment challenges. For example, conjunctivitis, commonly referred to as pink eye, can have viral, bacterial, or allergic etiologies. In the US alone, there are approximately 6 million cases of acute conjunctivitis every year. Differentiating between their causes is important, as treatment varies significantly; antibiotics are ineffective against viral and allergic forms. In cases of viral conjunctivitis, which is often self-limiting, identifying specific viruses such as adenovirus or herpes simplex vims can help predict the clinical course and potential complications. Allergic conjunctivitis, on the other hand, lacks microbial signatures; thus, the absence of microbial DNA coupled with clinical presentation can aid in its identification. Another potential application for the disclosed methods includes lacrimal system infections, such as dacryocystitis and canaliculitis. These infections often require microbiological identification to tailor antibiotic therapy.

[0169] By extending the application of the tear-based approach beyond corneal ulcers, a more general, but significant unmet need in ophthalmology is addressed. Access to care is enhanced by expanding microbiology testing that can now be performed by primary care physicians and nurse practitioners. In 2005 alone, there were more than 4 million visits to ambulatory physicians for bacterial conjunctivitis. A third of these were pediatric -related cases and the majority were likely causes by viruses. However, there is a tendency among primary care physicians to diagnose viral conjunctivitis as bacterial. A study conducted in the UK revealed that only 36% of general practitioners felt confident in their ability to distinguish between bacterial and viral conjunctivitis. Despite this uncertainty, an overwhelming majority, about 95%, reported prescribing antibiotics for cases they suspected to be bacterial conjunctivitis.

[0170] This trend indicates that with diagnostic uncertainty there will be overprescription of antibiotics. In the US, the annual cost associated with bacterial conjunctivitis is estimated to range $ 469 million to $705 million. Early and accurate microbial identification can improve patient outcomes across a spectrum of ocular conditions, minimize the use of unnecessary medications, and contribute to more judicious use of antimicrobials. This is particularly beneficial in primary care and urgent care settings, where rapid decision-making is essential and specialized ophthalmic equipment or access to an ophthalmologist may not be readily available.

[0171] Thus, infections applicable to the disclosed methods include, but are not limited to, conjunctivitis (pink eye) (e.g., viral, bacterial, gonococcal, chlamydial, or allergic), keratitis (viral (e.g., Herpes), bacterial, parasitic, or fungal), cellulitis (e.g., preseptal or orbital), shingles, endophthalmitis (e.g., exogenous or endogenous), stye, blepharitis (e.g., anterior or posterior, can be e.g., bacterial, allergic, clogged oil glands, or skin condition), uveitis (e.g., related to or caused by herpes zoster infection, psoriasis, rheumatoid arthritis, syphilis, AIDS, ankylosing spondylitis, multiple sclerosis, tuberculosis, or ulcerative colitis), dacryocystitis, and canaliculitis.

[0172] III. Methods of Use

[0173] A. Diagnostics

[0174] In some embodiments, the disclosed methods are utilized to diagnose a subject with, for example, an infection, such as corneal infection. Patient diagnosis can include utilizing information obtained by comparing information derived from the molecular analysis to known microbial, pathogenic, or parasitic information, e.g., reference sequences, such as those held in a pre-existing database. The analysis can additionally or alternatively be compared to results obtained from patients with similar clinical conditions. Reference sequences for comparison to the sample may be derived from publicly available data, prior samples, etc. Analysis can include, e.g., qualitative analysis such as biomarker presence or absence, and / or analysis of whole or partial genomic sequences along with sequences of specific chromosomes, genes, gene fragments, plasmids, or pathogenicity islands. In the case of prognosis (as discussed in more detail below), sample analysis results can be compared to results derived from other patient samples of known clinical outcome. The samples can be of known or unknown identity and each sample analyzed, along with relevant metadata, may become part of the reference for the next sample analyzed.

[0175] Diagnosing a subject can include summarizing the results of part or all of the molecular analysis into a clinical report. For example, the results can be summarized into a simple table of species found in the sample along with their relative proportion in the sample with additional flags indicating the presence of antibiotic resistance genes.

[0176] Providing diagnostic information on a subject can include providing the results, findings, identifications, relative abundance estimates, predictions and / or treatment recommendations for the subject. For example, the results, findings, identifications, predictions and / or treatment recommendations can be recorded and communicated to technicians, physicians and / or patients or clients. In certain embodiments, computers will be used to communicate such information to interested parties, such as, clients, patients and / or the attending physicians.

[0177] In some embodiments, once a subject’s non-host sequences are identified, an indication of that identity can be displayed and / or conveyed to a clinician, caregiver or a non-clinical provider, including the client / subject. For example, the results of the test are provided to a user (such as a clinician or other health care worker, laboratory personnel, or patient) in a perceivable output that provides information about the results of the method. The output can be, for example, a paper output (for example, a written or printed output), a display on a screen, a graphical output (for example, a graph, chart, or other diagram), or an audible output.

[0178] In some embodiments, the output is a numerical value, such as an amount of a particular set of sequence in the sample as compared to a control. In additional examples, the output is a graphical representation, for example, a graph that indicates the value (such as amount or relative amount) of the particular microbes in the sample from the subject on a standard curve. In a particular example, the output (such as a graphical output) shows or provides a cut-off value or level that indicates the presence of a microbe(s) that could cause an infection. In some examples, the output is communicated to the user, for example by providing an output via physical, audible, or electronic means (for example by mail, telephone, facsimile transmission, email, or communication to an electronic medical record).

[0179] The output can provide quantitative information (for example, an amount of a molecule in a test sample compared to a control sample or value) or can provide qualitative information (moderate to severe microbial infection caused by a particular microbe or parasite indicated). In additional examples, the output can provide qualitative information regarding the relative amount of a particular microbe(s) in the sample, such as identifying presence of an increase relative to a control, a decrease relative to a control, or no change relative to a control.

[0180] In some embodiments, the output is accompanied by guidelines for interpreting the data, for example, numerical or other limits that indicate the presence or absence of a particular microbial or parasitic disorder / condition. The indicia in the output can, for example, include normal or abnormal ranges or a cutoff, which the recipient of the output may then use to interpret the results, for example, to arrive at a diagnosis, prognosis, susceptibility towards or treatment plan. In some examples, the findings are provided in a single page report (e.g., PDF file) for the healthcare provider to use in clinical decision making.

[0181] Based on the findings, the therapy or protocol administered to a subject can be started, modified not started or restarted (in the case of monitoring for a reoccurrence of a particular condition / disorder). Recommendations of what treatment to provide can be provided either in verbal or written communication. The recommendations can be provided to the individual via a computer or in written format and accompany the diagnostic report. For example, a subject may request their report and suggested treatment protocols be provided to them via electronic means, such as by email.

[0182] The report can include determination of other clinical or non-clinical information.

[0183] In some embodiments, the communication containing the diagnostic information and / or treatment recommendations or protocols based on the results, may be generated and delivered automatically to the subject using a combination of computer hardware and software which will be familiar to artisans skilled in telecommunications. One example of a healthcare-oriented communications system is described in U.S. Pat. No. 6,283,761; however, the present disclosure is not limited to methods which utilize this particular communications system. In certain embodiments of the methods of the disclosure, all or some of the method steps, including the assaying of samples, performing the comparisons, and / or communicating of assay results, diagnoses or recommendations, may be carried out together or separately and in the same or in diverse (e.g., foreign) locations.

[0184] In some embodiments, the treatment, dose or dosing regimen is modified based on the information obtained using the disclosed methods.

[0185] A subject can be monitored while undergoing treatment using the methods described herein in order to assess the efficacy of the treatment or protocol. In this manner, the length of time or the amount of treatment given to the subject can be modified based on the results obtained using the methods disclosed herein. The subject can also be monitored after the treatment using the methods described herein to monitor for relapse and thus, the effectiveness of the given treatment. In this manner, whether to resume treatment can be decided based on the results obtained using the methods disclosed herein. In some embodiments, this monitoring is performed by a clinical healthcare provider. This monitoring can be performed by a non-clinical provider and can include self-monitoring or monitoring by a consultant.

[0186] B. Prognosis

[0187] Methods of prognosis are also provided. Clinical prognosis typically includes comparing molecular analysis to, for example, prior samples (e.g., clinical samples) for which outcome is known. In some embodiments, the results of the comparative genomics analysis are summarized into a sample distance matrix.

[0188] Clinical outcome may be determined by analyzing statistically the probabilistic distance a patient sample is from other samples of known outcomes and reporting such as a risk (e.g., risk the patient's wound will be chronic). For example, a deliverable diagnostic report (e.g., PDF file) may be generated for the physician to use in clinical decision making indicating whether a patient sample belongs to a particular prior grouping (healed versus chronic wound). Distances between samples for statistical analyses and visualization can be carried out using clustering methods including, but not limited to, partitioning methods, hierarchical clustering, density-based methods, model-based clustering methods, grid-based methods, and soft-clustering. Typical clustering methods include: k-means clustering, bayesian network analyses, mean shift clustering, densitybased spatial clustering of applications with noise (DBSCAN), expectation maximization using Gaussian Mixture Models (GMM), Agglomerative Hierarchical Clustering. K-mers may also be normalized for specific clinical applications using techniques such as Boolean weighting, logarithmic, and natural log for enhanced visualization of results in clinical reports. Additional formats can be utilized to provide the results including those discussed herein as well as those known to those of ordinary skill in the art.

[0189] The sequence reads that drive prognosis and diagnosis are also extractable from the total data. These reads can be translated in silico into putative protein sequence and analyzed against protein motif databases to identify protein functions that correlate significantly with clinical information (e.g., protease or beta-lactamase activity correlating with tissue invasion or antibiotic resistance). In addition, the sequence reads could provide new biomarkers for the development of rapid diagnostic assays. Thus the disclosed methods can be used to identify protein function as well as new biomarkers. C. Methods of Treating the Subject

[0190] Method of treatment are provided can be used alone or in combination of with any of the methods provided herein. For example, in some embodiments, the treatment methods follow a method of detecting that the subject has a microbial eye infection, and optionally determining the microbe(s) causing the infection. In some embodiments, the methods are directed to a subject that is first selected for treatment according to one or more of the disclose methods or analysis.

[0191] In some embodiments, the methods include providing an appropriate therapy or protocol for the subject after reviewing the diagnostic and / or prognostic results. Additionally or alternatively, the methods can include avoiding an inappropriate therapy or protocol for the subject after reviewing the diagnostic and / or prognostic results. For example, a subject diagnosed with a particular microbial or parasitic infection can be provided a particular therapy selected based on the microbes that are detected. Alternatively, if no microbes are detected, antimicrobial agents can be avoided, and optionally alternative therapies can be selected.

[0192] In some examples, the therapy includes administering an agent to alter one or more signs or symptoms associated with the identified microbial or parasitic disorder / condition. In some examples, the therapy may be altered to adapt to the emergence or change in abundance of new microbes, pathogens, or parasites. The treatment / protocol can be performed multiple times if desired. In one embodiment, the treatment is performed twice a day. In another embodiment, the treatment is performed daily. In other embodiments, the recommendation / treatment is performed weekly.

[0193] The desired treatments or protocols can be administered via any means known to one of skill in the art, including, but not limited to, oral, topical, or systemic administration. In some examples, the treatment is administered to the infected eye(s) in liquid or gel form. In some examples, a composition is administered to the subject orally, such as in a capsule or tablet.

[0194] One or more compositions can be administered via multiple routes at the same or different time period depending upon the disorders / conditions being treated. The percentage of improvement can be, for example, at least about a 5%, such as at least about 10%, at least a 15%, at least a 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% change compared to the baseline score prior to treatment with one or more microbial altering / controlling agents. The improvement can be measured by both subjective and objective methods, and can be quantified using a subjective scoring or a panel scoring, amongst other methods. 1. Treatment - Microbes Detected

[0195] It will be appreciated that a number of eye infections can be caused by different types of microorganisms, and / or by a condition that is not related to a microorganism, such as an allergy, an irritant, a skin condition, an autoimmune disease, etc. Thus, in some embodiments, the disclosed methods lead to determination of the type of microbe(s) and / or the species of microbe(s) causing the infection and thus informs the selection of one or more antimicrobial treatments.

[0196] If microbes are presents, the subject can be treated with one or more topical or oral antimicrobial agents such as naficillin, oxacillin, vancomycin, clindamycin, erythromycin, trimethoprim-sulphamethoxazole, rifampin, ciprofloxacin, broad spectrum penicillin, amoxicillin, gentamicin, ceftriazoxone, cefotaxime, chloramphenicol, clavunate, sulbactam, probenecid, doxycycline, spectinomycin, cefixime, penicillin G, minocycline, P-lactamase inhibitors; meziocillin, piperacillin, aztreonam, norfloxacin, trimethoprim, ceftazidime, ceftriaxone or dapsone.

[0197] For bacterial infections, the subject can be treated with one or more topical or oral antibiotics, or combination thereof.

[0198] In some embodiments, the antibiotic is a penicillin, tetracycline, cephalosporin, quinolone, lincomycin, macrolide, sulfonamide, glycopeptide, aminoglycoside, or a carbapenem.

[0199] Specific exemplary antibiotics drugs include but are not limited to amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem, imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalothin, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, dalbavancin, oritavancin, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, linezolid, posizolid, radezolid, torezolid, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucioxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, temocillin, ticarcillin, calvulanate, ampicillin, subbactam, tazobactam, ticarcillin, clavulanate, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfanamide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole, sulfonamideochrysoidine, demeclocycline, minocycline, oytetracycline, tetracycline, clofazimine, dapsone, dapreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin, dalopristin, thiamphenicol, tigecycyline, tinidazole, trimethoprim, and / or teixobactin.

[0200] For viral infections, the subject can be treated with one or more topical or oral antivirals, or combination thereof. Antivirals include but are not limited to acyclovir, idoxuridine, tromantadine, valacyclovir, valganciclovir, trifluridine, foscamet, ganciclovir, zidovudine, dideoxycytosine, dideoxyinosine, stavudine, famciclovir, didanosine, zalcitabine, rifimantadine, and cytokines.

[0201] For fungal infections, the subject can be treated with one or more topical or oral antifungals (also referred to as antimycotics), or combination thereof.

[0202] There are currently four classes of FDA-approved antifungal agents clinically used in the treatment of invasive fungal infections, namely the polyenes, flucytosine, the azoles, and the echinocandins (Wall G, Lopez-Ribot JL. “Current Antimycotics, New Prospects, and Future Approaches to Antifungal Therapy.” Antibiotics (Basel). 2020 Jul 25 ;9(8):445. doi: 10.3390 / antibiotics9080445. PMID: 32722455; PMCID: PMC7460292, which is specifically incorporated by reference herein in its entirety). Exemplary, non-limiting antimycotics include but are not limited to natamycin, voriconazole, amphotericin B, miconazole, ketoconazole, fluconazole, itraconazole, econazole, clomitrazole, terconazole, griseofulvin, and polyenes such as amphotericin B or nystatin etc.

[0203] For parasitic infections, the subject can be treated with one or more topical or oral antifungals (also referred to as antimycotics), or combination thereof. For example, anti-amoebics include but are not limited to metronidazole, metronidazole benzoate, and tinidazole etc.

[0204] In some embodiments, the subject has a corneal ulcer, and the disclosed methods determine that a microbe is responsible for the causing of the corneal ulcer, optionally, but preferably identifying the type of microbe responsible, and optionally, but preferably determining the genus and / or species of the microbe responsible.

[0205] Determining the cause of the corneal ulcer can inform how the patient is treated for the disease. Compositions and methods for treating corneal ulcers, particularly where the cause has been identified, are known in the art. See, e.g., Lietman, et al., “Update on the Management of Infectious Keratitis,” Ophthalmology, 2017 Nov;124(l 1): 1678-1689. doi:

[0206] 10.1016 / j.ophtha.2017.05.012. Epub 2017 Sep 21. PMID: 28942073; PMCID: PMC5710829, which is specifically incorporated by reference herein in its entirety. a. Bacterial Keratitis

[0207] Topical antibiotics remain the first-line treatment for bacterial keratitis. Clinicians weigh many factors when choosing an antibiotic regimen, including, broad- spectrum coverage, toxicity, availability and cost, and region-specific epidemiology of pathogens and resistance patterns. Indeed, a recent international survey of cornea specialists found that concerns over several of these factors were predictive of antibiotic choice.

[0208] Bacterial ulcers caused by bacteria that are not antibiotic -resistant are usually responsive to treatment with available topical antibiotic drops. The most commonly prescribed antibiotic class for these infections is fluoroquinolones.

[0209] An increase in the rates of antibiotic resistant infections such as methicillin resistant Staphylococcus aureus (MRSA) in North America has caused concern. The United States Center for Disease Control (CDC) estimates that 2 million people are infected with drug resistant microbes each year. Approximately 80% of ocular isolates of MRSA in the US have been reported to be resistant to fluoroquinolones. Thus, if the infection is caused by an antibiotic-resistant strain(s) of bacteria, fortified broad-spectrum antibiotics can be used. Toxicity for broad-spectrum drops can be a factor impacting healing and reducing therapy is often advised, if it is not necessary.

[0210] Additional strategies include treatment with an anticollagenase. Tetracyclines such as doxycycline have been shown to inhibit collagenase and have demonstrated antimetalloproteinase activity in vitro. Adjuvant corticosteroids can be used in an effort to improve outcomes by reducing inflammation, thereby reducing scarring, neovascularization, and stromal melt. b. Fungal Keratitis

[0211] Fungal ulcers often have worse outcomes than bacterial ulcers. Treatment with topical natamycin (e.g., 5%), the only FDA approved treatment, is limited by its poor penetration into the corneal stroma. Topical amphotericin B (e.g., 0.3% to 0.5%) is an alternative. Voriconazole, a newer generation triazole, has gained popularity in the treatment of fungal keratitis due to its excellent ocular penetration. Although topical voriconazole (e.g., 1%) did not show improved outcomes compared with natamycin, there are several reasons that oral voriconazole may have efficacy in the treatment of fungal keratitis. For example,, intermittent dosing of topical medications may result in intervals of sub-therapeutic drug levels and oral medications may provide more steady-state drug levels at the site of infection.

[0212] Other potential adjuvant treatments for fungal keratitis include intracameral injection of amphotericin with or without hypopyon drainage, and intrastromal injection of voriconazole. c. Viral Keratitis

[0213] Herpes simplex virus (HSV) keratitis affects an estimated 500,000 people in the United States and an estimated 1.5 million globally. It is the most common cause of unilateral infectious corneal blindness in much of the developed world. Viral keratitis differs from bacterial and fungal keratitis in that it can become chronic and recurrent. Besides being a painful, sight-threatening infection, HSV keratitis has been shown to significantly impact quality of life even when patients are not experiencing an active infection. Less common forms of viral keratitis include varicellazoster virus (VZV) keratitis, and cytomegalovirus (CMV) keratitis.

[0214] Topical treatments for viral keratitis include antiviral medications and adjuvant topical corticosteroids. The topical antiviral trifluridine is the most commonly prescribed topical antiviral medication for HSV keratitis in the United States. While it is effective in treating HSV keratitis, it has low bioavailability and causes ocular surface toxicity, so its use has become more limited as newer topical antivirals are developed. Topical acyclovir is the first line treatment for HSV keratitis in Europe as it has been shown to be just as effective as trifluridine with less ocular surface toxicity. Ganciclovir is a newer synthetic medication with more broad-spectrum antiviral coverage. In addition to treating HSV and VZV keratitis, topical ganciclovir is also effective in treating keratitis caused by CMV. Ganciclovir has been shown to be just as effective as acyclovir, while causing less ocular toxicity. It may also be less likely to promote drug resistance.

[0215] Topical corticosteroids are also sometimes used as adjuvant therapy to topical antivirals.

[0216] In some embodiments, oral antiviral is prescribed. An example of an oral antiviral is acytclovir, Valacyclovir, a newer antiviral, is well tolerated and there is some evidence that it may have better ocular penetration. Additionally, the treatment dose for valacyclovir is 1 three times daily, as opposed to acyclovir which is 400mg five times daily (800mg five times daily for VZV), which aids in patient compliance. Oral valganciclovir is the preferred treatment for CMV stromal keratitis, but it has significant side effects, including aplastic anemia, which must be closely monitored.

[0217] In some embodiments, subjects likely to develop or experience or recurrence of viral keratitis are treated with an oral antiviral prophylactically or otherwise at a time when not ocular symptoms are apparent.

[0218] 2. Treatment - Adjunct Therapies

[0219] Adjunct and adjuvant therapies are also provided. The adjunct therapy can be used in combination with an anti-microbial when a microbe is detected. In other embodiments, no microbe is detected. In such embodiments, the lack of detection informs that the infection should not be treated with an antimicrobial. In such embodiments, the adjunct therapy can be the primary therapy.

[0220] Adjunct therapies include cleaning and / or flushing with sterile wash, anti-inflammatories, steroids, antihistamines and other allergy medications, anti-collagenases, immunosupressants, and can be selected based on a combination with antimicrobial for infected subjects, or based on the remaining potential underlying causes once microbial infection is eliminated.

[0221] Anti-inflammatories include but are not limited to steroids or NSAID's such as indomethacin, ibuprofen, piroxicam, diclofenac etc.; salicylic acid derivatives (e.g. aspirin), paraminophenol derivative (e.g. acetaminophen), indole and indene acetic acids (indomethacin, sulindac and etodalac), heteroaryl acetic acids (tolmetin diclofenac and ketorolac), aryl propionic acid derivatives (ibuprofen, naproxen, ketoprofen, fenopren, oxaprozine), anthranilic acids (mefenamic acid, meclofenamic acid), and enolic acids (piroxicam, tenoxicam, phenylbutazone and oxyphenthatrazone) .

[0222] Antihistamines are represented by but are not limited to cimetidine, ranitidine, diphenydramine, prylamine, promethazine, chlorpheniramine, chlorcyclizine, terfenadine, carbinoxamine maleate, clemastine fumarate, diphenhydramine hydrochloride, dimenhydrinate, prilamine maleate, tripelennamine hydrochloride, tripelennamine citrate, chlorpheniramine maleate, brompheniramine maleate, hydroxyzine pamoate, hydroxyzine hydrochloride, cyclizine lactate, cyclizine hydrochloride, meclizine hydrochloride, acrivastine, cetirizine hydrochloride, astemizole, levocabastine hydrochloride, and loratadine.

[0223] Anti- allergies include, for example, Disodium cromoglycate etc.

[0224] Immunosuppressive agents include, for example, cyclosporins etc.

[0225] IV. Kits and Devices

[0226] Also disclosed are kits for carrying out the disclosed methods. Compositions, reagents, and other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed methods. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed methods.

[0227] The kits may include a sterile needle, swab, syringe, ampule, tube, container, or other suitable vessels for isolating samples and extracting nucleic acids therefrom, holding assay components and / or performing the assay. For example, in some embodiment, the kit includes a device for collecting tears (e.g., including but not limited to those expressly mentioned above), and optionally tube(s) or vials for freeing the tears from the collection device. Also disclosed are kits with one or more oligonucleotides (e.g., primers, probes, etc.), dNTPs, buffers, and / or enzymes. The kits may include instructions for use.

[0228] The kit can include a sufficient quantity of reverse transcriptase, a DNA polymerase, oligonucleotides, and / or reaction buffer, or any combination thereof, for performing any of the detection assays described above. A kit may further include instructions pertinent for the particular embodiment of the kit, such as providing conditions and steps for operation of the method.

[0229] The kits may contain oligonucleotides (e.g., primers) suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for instance. The container(s) in which the primers are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, multi-well plates, ampoules, or bottles. One or more control probes, primers, and or nucleic acids also may be supplied in the kit. For example, the kit may include one or more positive control samples (such as a sample including a particular nucleic acid) and / or one or more negative control samples (such as a sample known to be negative for a particular nucleic acid).

[0230] In some embodiments, the kit can contain instructions for detecting a target nucleic acid. This can include for example, instructions and / or software for data analysis.

[0231] The methods for detecting ocular infections can be further understood through the following numbered paragraphs.

[0232] Paragraph 1. A method of determining the presence of microbes in the eye of a subject, the method comprising detecting microbial nucleic acids in a tear sample isolated from the subject.

[0233] Paragraph 2. The method of paragraph 1, wherein detecting comprises processing the sample using a machine-based analytical platform.

[0234] Paragraph 3. The method of paragraphs 1 or 2, wherein detecting comprises a molecular analysis comprising PCR, sequencing, microarray, or a combination thereof.

[0235] Paragraph 4. The method of paragraph 3 comprising PCR.

[0236] Paragraph 5. The method of paragraph 4, wherein the PCR is selected from Real-Time PCR (quantitative PCR or qPCR), Reverse-Transcriptase (RT-PCR), Multiplex PCR, Nested PCR, High Fidelity PCR, Fast PCR, Hot Start PCR, Long-range PCR, Arbitrary Primed PCR, Digital PCR, Droplet Digital PCR (ddPCR), isothermal amplification PCR, Endpoint PCR (Qualitative PCR), or a combination of any thereof.

[0237] Paragraph 6. The method of paragraphs 4 or 5, wherein the PCR is qualitative or quantitative. Paragraph 7. The method of any one of paragraphs 4-6, wherein the PCR comprises one or more sets of primers specific for a type of microbe. Paragraph 8. The method of any one of paragraphs 4-7, wherein the PCR comprises one or more sets of genus and / or species specific primers.

[0238] Paragraph 9. The method of any one of paragraphs 4-7, wherein the PCR comprises non-specific and / or random primers.

[0239] Paragraph 10. The method of any one of paragraphs 1-3 comprising sequencing in the absence of PCR, optionally wherein the sequencing is selected from Sanger sequencing, single molecule real time (SMRT) sequencing, nanopore DNA sequencing, massively parallel signature sequencing (MPSS), colony sequencing, 454 pyrosequencing, Illumina sequencing, combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, ion torrent semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, or a combination of any thereof.

[0240] Paragraph 11. The method of paragraph 10, wherein the nucleic acid substrate for sequencing is microbial DNA, or cDNA reverse transcribed from microbial RNA, in the tear sample.

[0241] Paragraph 12. The method of any one of paragraphs 1-9 comprising a combination of PCR and sequencing.

[0242] Paragraph 13. The method of paragraph 12, wherein the sequencing is selected from Sanger sequencing, single molecule real time (SMRT) sequencing, nanopore DNA sequencing, massively parallel signature sequencing (MPSS), colony sequencing, 454 pyrosequencing, Illumina sequencing, combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, ion torrent semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, or a combination of any thereof.

[0243] Paragraph 14. The method of any one of paragraphs 10-13, wherein the nucleic acid substrate for sequencing comprises amplicons generated during the PCR.

[0244] Paragraph 15. The method of any one of paragraphs 1-14, wherein the nucleic acids are not extracted from the tears prior to detection.

[0245] Paragraph 16. The method of any one of paragraphs 1-15, wherein the tear sample further comprises water or buffer added after the sample is isolated from the subject.

[0246] Paragraph 17. The method of any one of paragraphs 1-16, wherein isolation of the tear sample comprises collection of tears using a collection device, optionally wherein the volume of tears isolated from the collection device is any integer number of pl between about 1 pl to 100 pl inclusive, preferable about 1 pl to 20 pl inclusive, or any sub-range of two integers therebetween. Paragraph 18. The method of paragraph 17, wherein the collection device is selected from Schirmer strip, capillary action paper, and cotton swab.

[0247] Paragraph 19. The method of paragraphs 17 or 18, wherein isolation of the tears comprises drawing the tears out of the collection device, optionally by centrifugation.

[0248] Paragraph 20. The method of any one of paragraphs 1-19, wherein the absence of detection of nucleic acids in the tear sample indicates the eye(s) from which the tear sample was collected is free from microbes.

[0249] Paragraph 21. The method of any one of paragraphs 1-20, wherein the detection of nucleic acids in the tear sample indicates the eye(s) from which the tear sample was collected is infected with microbes.

[0250] Paragraph 22. The method of any one of paragraphs 1-21 comprising the use of bioinformatics. Paragraph 23. The method of any one of paragraphs 1-21, further comprising determining the type of microbes present in the tear sample, optionally wherein the microbes are selected from bacteria, fungi, viruses, parasites, and combinations thereof.

[0251] Paragraph 24. The method of any one of paragraphs 1-23 further comprising determining the genus and / or species of the microbes.

[0252] Paragraph 25. The method of any one of paragraphs 1-24, wherein the nucleic acids in the tear sample comprise DNA, RNA, or a combination thereof.

[0253] Paragraph 26. The method of paragraph 25 comprising RNA, wherein the RNA is converted to DNA by reverse transcription prior to molecular analysis.

[0254] Paragraph 27. The method of any one of paragraphs 1-26, wherein detection is relative to a control, wherein nucleic acids present in the control are deducted from the molecular analysis before determining if nucleic acids are present in the tear sample.

[0255] Paragraph 28. The method of paragraph 27, wherein the control is a tear sample collected from the contralateral eye of the same subject.

[0256] Paragraph 29. The method of paragraph 27, wherein the control is a nuclease- free water or buffer. Paragraph 30. The method of any one of paragraphs 1-29, comprising PCR amplifying a target portion of the nucleic acids optionally using ribosomal RNA-specific primers, sequencing the resulting amplicons using next generation sequencing optionally nanopore sequencing, and determining the type, genus, species, or a combination thereof of the organism from which the nucleic acids were obtained, optionally using bioinformatics.

[0257] Paragraph 31. A method of diagnosing a subject with an eye infection comprising detecting microbes according to the method of any one of paragraphs 1-30, wherein the subject is determined to have a microbial eye infection when nucleic acids are detected in the tear sample, and the subject is determined not to have a microbial eye infection when nucleic acids are not detected in the tear sample, optionally wherein the subject is determined to be free from an eye infection. Paragraph 32. The method of paragraph 31, further comprising treating the subject with an antimicrobial agent, when the subject is determined to have a microbial eye infection.

[0258] Paragraph 33. The method of paragraph 31, further comprising abstaining from treating the subject with antimicrobial agents when the subject is determined not to have a microbial eye infection. Paragraph 34. The method of paragraphs 32 or 33 comprising treating the subject with an antiinflammatory, antihistamine, immune suppressant, analgesic, or a combination thereof.

[0259] Paragraph 35. The method of any one of paragraphs 31-34, further comprising determining the type of microbes present in the tear sample.

[0260] Paragraph 36. The method of paragraph 35, wherein the microbes are bacteria, fungi, viruses, parasites, or combinations thereof.

[0261] Paragraph 37. The method of any one of paragraphs 1-36 further comprising determining the genus and / or species of the microbes.

[0262] Paragraph 38. The method of any one of paragraphs 35-37, wherein the subject is treated with an antimicrobial agent effective to treat the microbes that are present.

[0263] Paragraph 39. The method of paragraph 38, wherein the microbe(s) is or comprises a bacteria and the subject is treated with one or more topical and / or oral antibiotics, optionally wherein antibiotic- resistant bacteria are treated with broad- spectrum antibiotics.

[0264] Paragraph 40. The method of paragraphs 38 or 39, wherein the microbe(s) is or comprises a virus and the subject is treated with one or more topical and / or oral antivirals.

[0265] Paragraph 41. The method of any one of paragraphs 38-40, wherein the microbe(s) is or comprises a fungi and the subject is treated with one or more topical and / or oral antifungals.

[0266] Paragraph 42. The method of any one of paragraphs 38-41, wherein the microbe(s) is or comprises a parasite and the subject is treated with one or more topical and / or oral anti-parasitics.

[0267] Paragraph 43. A method of treating a subject for an eye infection comprising administering an antimicrobial agent to a subject diagnosed with a microbial eye infection according to the method of any one of paragraphs 31-37.

[0268] Paragraph 44. The method of paragraph 43, wherein the microbe(s) is or comprises a bacteria and the subject is treated with one or more topical and / or oral antibiotics, optionally wherein antibiotic- resistant bacteria are treated with broad- spectrum antibiotics.

[0269] Paragraph 45. The method of paragraphs 43 or 44, wherein the microbe(s) is or comprises a virus and the subject is treated with one or more topical and / or oral antivirals.

[0270] Paragraph 46. The method of any one of paragraphs 43-45, wherein the microbe(s) is or comprises a fungi and the subject is treated with one or more topical and / or oral antifungals. Paragraph 47. The method of any one of paragraphs 43-46, wherein the microbe(s) is or comprises a parasite and the subject is treated with one or more topical and / or oral anti-parasitics.

[0271] Paragraph 48. A method for treating a subject for an eye infection comprising abstaining from administering antimicrobial agents to a subject diagnosed as not having a microbial eye infection. Paragraph 49. The method of any one of paragraphs 1-48, wherein the eye infection is selected from keratitis, conjunctivitis, cellulitis, shingles, endophthalmitis, stye, blepharitis, uveitis, dacryocystitis, and canaliculitis.

[0272] Paragraph 50. The method of any one of paragraphs 1-49, wherein the subject has undergone a corneal transplant.

[0273] Paragraph 51. The method of paragraph 50, wherein the subject is not showing any symptoms of infections.

[0274] Paragraph 52. The method of paragraphs 50 or 51, wherein the corneal transplant was about 1-365 days inclusive or any subrange or specific day therebetween, preceding the collection of the tear sample.

[0275] Paragraph 53. The method of any one of paragraphs 1-52, wherein the subject is selected from humans, goats, sheep, pigs, cattle, zebu, donkeys, water buffaloes, dromedary camel, horse, yak, domestic bactrian camel, llama, alpaca, gayal, bali cattle, domestic rabbit, addax, bison, deer, eland, elk, guinea pig, greater kudu, mule, moose, muskox, and reindeer, chicken, duck, goose, guineafowl, muscovy duck, turkey, emu, egyptian goose, indian peafowl, mute swan, ostrich, partridge, small-billed tinamou, pigeon, quail, edible-nest swiftlet, grey francolin, guineafowl, common pheasant, and golden pheasant.

[0276] Examples

[0277] Example 1: Detection of Ocular Infections Using Tear Sample Materials and Methods

[0278] Specific parameters for digital PCR and 16S amplification used

[0279] Naica digital PCR system by Stilla was utilized:

[0280] Primer (forward): 16S 341F (20pM)

[0281] Sequence for forward primer: CCTACGGGAGGCAGCAG (SEQ ID NO:1)

[0282] Primer (reverse): 16S 534R (20 pM)

[0283] Sequence for reverse primer: ATTACCGCGGCTGCTGGCA (SEQ ID NO:2) Stilla Buffer A

[0284] Stilla Buffer B

[0285] Evergreen 20X Sapphire Chips

[0286] 40 pM of forward primer and reverse primer were each made from the stock concentration (i.e. I OOpM). Equal amounts of forward and reverse primers were then mixed in a new tube to form a final concentration of 20pM. A master mix of Stilla Buffer A, Buffer B, Evergreen 20x, Primer mix (20pM) and nuclease free water was made for 13 samples. (One extra sample included to account for volume loss). 24 pL of master mix was aliquoted into each PCR tubes (12 total PCR tubes)

[0287] Ratio of reagents were adapted from the Stilla experiment planner as shown below in Table 1.

[0288] Table 1: Ratio of Reagents

[0289] 1 pL of tear sample (undiluted) was placed in each PCR tube . For negative control, 1 pL of nuclease free water was added into PCR tube instead of tear sample. For positive control, 1 pL of oral microbiota was added into PCR tube instead of tear sample. NB: Three positive controls were made for this experiment; two undiluted oral microbiota and one of oral microbiota diluted 1:10

[0290] After vortexing and spinning, the 25 pL of the final mix for each sample was loaded on wells on Sapphire chips.

[0291] PCR Conditions

[0292] Sapphire trays were transferred to the Naica Geode. A PCR protocol was run with the following settings: o Partition at 40°C o Initial denaturation, followed by 33 cycles of 10 min at 95 °C. Denaturation, 30 sec at 95 °C. Annealing, 15 sec at 61 °C. Extension, 1 min at 72 °C. Data analysis for ddPCR

[0293] Image acquisition was performed using the Naica Prism3 reader. Total droplet enumeration and droplet quality control, enabled by the detection of the reference dye in the blue channel, was performed by the Crystal Reader software.

[0294] Extracted fluorescence values for each droplet were then further analyzed using the Crystal Miner software (Stilla Technologies, Villejuif, France).

[0295] Thresholds were set using the automated tool available in the Crystal Miner software. Specific parameters for Nanopore sequencing following 16S amplification 16S rRNA Amplification and Sequencing

[0296] Amplification and sequencing of the 16S rRNA gene were performed using the Oxford Nanopore Technology 16S Barcoding Kit 1-24 (SQK-16S024). The 16S rRNA gene was amplified using the LongAmp Hot Start Taq 2X Master Mix (New England Biolabs, Massachusetts, USA) using the 27F and 1492R primers, which are attached to unique barcode sequences. A mix of 1 uL of the tear sample, 25 uL of the master mix, 10 uL of the barcoded primers, and nuclease free water up to a final reaction mix volume of 50 uL was prepared. Initial denaturation occurred at 95°C for 1 minutes, followed by 50 cycles (instead of the 25 in the original protocol) of 95°C / 20 seconds, 55°C / 30 seconds, and 65°C / 2 minutes. Final extension occurred at 65°C for 5 minutes. The rest of the library preparation was performed using the recommended protocol for ONT kit SQK-16S024 for compatibility with the Flongle flow cell. PCR products were purified using AMPure XP Beads (Beckman Coulter, USA) following the Nanopore protocol. Sequencing runs were performed using the R9.4.1 Flongle flow cell (FLO-FLGOOl, Oxford Nanopore Technologies) on a MinlON MklB Nanopore sequencer. In general, runs were continued until a plateau of reads was achieved, which generally took between 4 and 12 hours. Hardware checks and Flongle flow cell checks were performed before each run to ensure the flow cell had at least 50 available pores.

[0297] Data Analysis for nanopore sequencing

[0298] Basecalling was performed using the built-in Guppy basecaller on MinKnow.

[0299] To identify bacteria, the cloud-based EPI2ME FASTQ16S program (v2023.04.21) provided by Oxford Nanopore Technology was used. The minimum qscore was set at 7. The results from each run were classified according to the NCBI 16S rRNA gene BLAST database.

[0300] Results

[0301] Tear samples were collected from ten patients with a diagnosis of corneal ulcers. Tear samples were collected from each patient’s ulcer eye and the contralateral eye. A total of 19 tear samples were collected as one patient did not have enough tear sample drawn from the contralateral eye due to the patient’s discomfort. A chart review of the 10 patients with a diagnosis of corneal ulcers showed that eight had corneal ulcers that were large enough to allow for corneal scraping and to test for growth on culture. Two patients had corneal ulcers that were too small for corneal scraping and culture.

[0302] Comparison of results by culture and Nanopore sequencing

[0303] A comparison from Nanopore analysis of tear samples and results from culture showed the results, summarized in Figure 2.

[0304] Of the eight samples that were tested for growth on culture, five had bacterial growth. The bacterial species detected via culture on four of these samples were also successfully detected by Nanopore analysis as the bacterial species with the greatest number of reads. Two cultures (Patients A089 and A091) grew Pseudomonas aeruginosa, both of which were successfully detected by Nanopore as the species with the greatest number of reads. One culture (A088) grew Staphylococcus aureus, and Nanopore detected Staphylococcus roterodami as the bacterial species with the greatest number of reads. Staphylococcus roterodami is within the Staphylococcus aureus complex. Another culture grew coagulase-negative Staphylococcus (no species was identified; A051), and Nanopore detected Staphylococcus saccharolyticus as the bacteria with the greatest number of reads. For the fifth sample with bacterial growth (A092), Nanopore successfully detected the bacterial genus on culture. The culture for this sample was positive for Staphylococcus lugdunensis, and Nanopore detected Staphylococcus caprae as the bacterial species with the greatest number of reads.

[0305] Of the three ulcers with no bacterial growth on culture, two ulcerated eyes had bacterial growth detected by Nanopore. For Subject A086, Nanopore detected Staphylococcus saccharolyticus as the bacterial species with the greatest number of reads. For Subject A093, Nanopore detected Cutibacterium acnes as the bacterial species with the greatest number of reads. For Subject A090, whose tear sample had no growth on culture, Nanopore also detected no bacterial species.

[0306] Finally, for the two eyes with ulcers too small to culture, Nanopore detected Staphylococcus saccharolyticus and Kocuria rhizophila, respectively, as the bacterial species with the greatest number of reads.

[0307] Figures 3A-3B gives an example for the results provided by Nanopore for a single run that successfully detected Pseudomonas aeruginosa (A091). Nanopore lists the bacterial species and number of cumulative reads in descending order (Figure 3A). Pseudomonas aeruginosa is the main species detected in this example. Nanopore also provides a phylogenetic tree, shown in Figure 3B, here with a minimum abundance cutoff of 1%.

[0308] The final five patients had the greatest number of reads, respectively, for the following bacterial species: Staphylococcus saccharolyticus (for three samples), Streptococcus sanguinis, and Staphylococcus hominis.

[0309] For the control eyes, four samples had no bacterial results detected by Nanopore. One patient did not have enough tear sample collected to allow for Nanopore analysis.

[0310] Every bacterial agent detected via culture to be the causative agent of a patient’s corneal ulcer was also successfully detected by Nanopore, at least at the genus level. For Subject AO88, the culture grew Staphylococcus aureus, and Nanopore detected Staphylococcus roterodami, which is within the Staphylococcus aureus complex. Therefore, this can be considered a successful result. For Subject A092, the culture grew Staphylococcus lugdunensis, and Nanopore detected Staphylococcus caprae. Both these species are coagulase negative.

[0311] As for antibiotic administration, six of the ten patients were administered antibiotics on the day of tear collection, with the remaining receiving antibiotics either 1, 2, or 3 days prior. This demonstrates that tear collection following antibiotic administration can still be effective in the accurate detection of the causative organism.

[0312] Example 2: An Exemplary Tear-Based Approach for Rapid Identification of Bacterial Pathogens in Corneal Ulcers Using Nanopore Sequencing

[0313] Materials and Methods

[0314] Study Design and Subjects Enrollment

[0315] This was a prospective observational cohort study conducted in accordance with the principles of the Declaration of Helsinki and approved by the Institutional Review Board of Yale University. Informed consent was obtained from all patients. Data on demographics, medical history, and medication use were collected from patients’ medical records at the time of enrollment. Statistical analysis was performed using IBM SPSS Statistics software (version 29).

[0316] Participants included in the study were adults aged 18 years and older who had a clinical diagnosis of microbial keratitis confirmed by an ophthalmologist. Eligible individuals presented with symptoms consistent with corneal ulcers, such as eye redness, pain, and visual impairment, and were able to provide informed consent. Exclusion criteria included individuals with a history of corneal transplantation within the prior 6 months. Individuals diagnosed with viral or fungal keratitis based on preliminary clinical assessments, culture results, or prior medical records were also excluded.

[0317] Tear Collection

[0318] Tear samples were collected from the inferior fornix using a sterile Schirmer strip, avoiding contact with the eyelashes. The strip was subsequently placed in a microcentrifuge tube. Tears were collected following centrifugation for 2 minutes at 5000 RPM and stored at -80°C.

[0319] Comeal Scraping

[0320] Corneal scraping was performed as part of the standard of care for patients presenting with symptoms indicative of microbial keratitis, in accordance with established clinical guidelines. The decision to perform corneal scraping was made at the discretion of the treating physician based on the severity of the ulcer, size, location, and clinical presentation of the infection. The procedure was carried out using a slit lamp for magnification. The patient’s eye was anesthetized with a topical anesthetic to ensure comfort during the procedure. Using a sterile blade, a small sample of the epithelial tissue was carefully removed from the ulcer. The collected samples were immediately placed in appropriate culture media or transport media to preserve microbial viability and transported to the laboratory for prompt culturing and analysis.

[0321] Nanopore 16S rRNA Amplification and Sequencing

[0322] Amplification and sequencing of the 16S rRNA gene, which is universal across bacterial species, were performed using the Oxford Nanopore Technology 16S Barcoding Kit 1-24 (SQK- 16S024). The 16S rRNA gene was amplified using the Long Amp Hot Start Taq 2X Master Mix (New England Biolabs, Massachusetts, USA). One pL of the tear sample was added to the master mix and barcoded primers in a reaction mix volume of 50 pL. Polymerase chain reaction (PCR) was performed at the following settings: an initial denaturation step at 95 °C for 1 minute, followed by 50 cycles at 95 °C for 20 seconds, 55°C for 30 seconds, and 65 °C for 2 minutes, and a final extension step at 65 °C for 5 minutes. The rest of the library preparation was performed using the recommended protocol for ONT kit SQK-16S024 for compatibility with the Flongle flow cell. PCR products were purified using AMPure XP Beads (Beckman Coulter, USA) following the Nanopore protocol. Sequencing runs were performed using the R9.4.1 Flongle flow cell (FLO-FLG001 , Oxford Nanopore Technologies) on a MinlON MklB Nanopore sequencer. Sequencing was allowed to proceed until a plateau of reads was achieved, which generally took between 4 and 12 hours. Analysis and Bacterial Identification

[0323] Basecalling was performed using the built-in Guppy basecaller on MinKnow, which translates the raw signal data from the Nanopore sequencer into nucleotide sequences. For species identification, the cloud-based EPI2ME FASTQ16S pipeline (y2023.04.21) provided by Oxford Nanopore Technology was utilized. The pipeline classifies the results from each sequencing run according to the NCBI 16S rRNA gene BLAST database. A minimum qscore of 7 was set, which indicates the quality threshold for basecalling accuracy; a higher qscore represents a higher confidence in the accuracy of the nucleotide base calls. In cases where multiple reads were identified, the determination of the causative organism was based on the highest read count. Results

[0324] Demographics of study participants and clinical presentation

[0325] There were 10 subjects who were included after meeting the study criteria. The mean age was 50.2 years. There were 6 males and 4 females. The ulcer was present in the left eye of 5 subjects and right eye of 5 subjects. Patient demographics are summarized in Table 2. Table 2: Demographics and Clinical Characteristics of the Study Subjects

[0326] Symptoms at presentation included blurred vision, pain, pink eye, foreign body sensation, discharge, tearing, and sensitivity to light. Time of symptom onset prior to presentation ranged from 1 to 14 days with a mean of 4.7 days. Risk factors for the development of corneal ulcer included use of soft contact lenses (n = 6), history of long hospitalization with associated bacteremia (n = 1), blepharitis (n = 1), status post superficial keratectomy 7 days prior to diagnosis (n = 1), and loose suture in a patient with a penetrating keratoplasty (n = 1). Best corrected or pinhole visual acuity at presentation ranged from 0 to 2.40 LogMAR with a mean of 1.07 LogMAR, and a standard deviation of 0.88 LogMAR.

[0327] On examination, 9 patients presented with a single comeal infiltrate while one patient had 2 infiltrates of comparable size. Comeal ulcers were centrally located in 50% of the cases, with the remainder being peripheral. The average size of infiltrates measured 2.09 mm vertically (range, 1 mm to 5 mm) and 2.20 mm horizontally (range, 1 mm to 5 mm). No ulcers extended to the sclera or limbus. Two patients developed hypopyon.

[0328] Eight subjects underwent comeal scraping for culture, while 2 had ulcers considered too small for scraping. A total of 10 tear samples were collected.

[0329] Summary of culture results

[0330] Of the 8 subjects who underwent corneal scraping and culture, 4 exhibited bacterial growth on culture. The bacteria identified included coagulase-negative Staphylococcus in broth (Subject 1), Staphylococcus aureus (Subject 2), and Pseudomonas aeruginosa (Subjects 3 and 4), all identified one day post-diagnosis. The remaining 4 subjects showed no bacterial growth on culture media including blood agar, chocolate agar, and thioglycolate broth, which were monitored for growth for 5 days. Results from culture are listed in Table 3.

[0331] Table 3: Summary of Culture Results and Nanopore Sequencing Results

[0332] Nanopore sequencing results

[0333] For the 4 subjects whose ulcer scrapings exhibited growth on culture, Nanopore sequencing identified the predominant bacterial pathogens in each tear sample as follows: Staphylococcus saccharolyticus, Staphylococcus roterodami, Pseudomonas aeruginosa, and again Pseudomonas aeruginosa, respectively. Staphylococcus saccharolyticus is coagulase negative11) and Staphylococcus roterodami is within the Staphylococcus aureus complex / 12)

[0334] Among the 4 subjects whose ulcers did not exhibit growth on culture, Nanopore sequencing detected no bacteria in 2 cases, while identifying Staphylococcus saccharolyticus and Cutibacterium acnes in the remaining 2.

[0335] For the 2 subjects with ulcers too small for culture, Nanopore sequencing identified Staphylococcus saccharolyticus in one and Kocuria rhizophila in the other. Results from Nanopore sequencing compared to results from culture are listed in Table 3. Detailed results featuring taxonomic classification for each case along with a synopsis of the clinical presentation are provided in Figures 4-11.

[0336] Discussion

[0337] The findings demonstrate the efficacy of PCR amplification of the 16S gene directly from tears, followed by Nanopore sequencing, in identifying the causative bacterial agents of comeal ulcers. This method demonstrated high sensitivity, successfully detecting bacterial pathogens in all samples that were positive by traditional culture methods. Notably, it also identified bacterial DNA in cases where traditional cultures failed, indicating that tear-based Nanopore sequencing may offer greater sensitivity compared to standard culture-based methods. This non- invasive approach eliminates the need for DNA extraction and delivers results within hours, which is important for the timely management of corneal ulcers.

[0338] Traditional culture-based diagnostics are often limited by high false-negative rates, which range between 32.6-79.4%. <13,14) This limitation stems from several factors, including variability in sample collection techniques and the fastidious nature of certain pathogens that do not grow under standard culture conditions. In contrast, the tear-based sequencing approach, which relies on detecting bacterial DNA, should circumvent these hurdles. In this study, the method identified bacterial reads in 2 of the 4 ulcers that did not exhibit growth on culture, indicating higher sensitivity than traditional methods. Specifically, Nanopore sequencing identified Staphylococcus saccharolyticus and Cutibacterium acnes as the predominant bacterial species in culture-negative ulcers, both of which are associated with corneal ulcers or eye infections / 15,16) Notably, Cutibacterium acnes, formerly known as Propionibacterium acnes, is considered a fastidious organism due to its stringent growth requirements / 17,18)

[0339] Moreover, the tear-based approach could be particularly useful in clinical settings where access to an ophthalmologist is not feasible or laboratory resources are limited, as it requires no specialized equipment or training for sample collection, compared with the traditional culture techniques which require corneal scrapings by an ophthalmologist using a slit-lamp. The sequencing method offers significant benefits because it is non-invasive, eliminating the discomfort and potential complications associated with corneal scrapings.

[0340] Another advantage of this sequencing-based method is its ability to detect low-abundance pathogens and provide insights into the polymicrobial nature of corneal ulcers. In this study, causative organisms identified by Nanopore included pathogens commonly associated with corneal ulcers such as Staphylococcus, Pseudomonas, and Cutibacterium acnes. Notably, Kocuria rhizophila has not been previously reported as a cause of corneal ulcers. It belongs to the Kocuria genus, a group of gram-positive bacteria that, in rare cases, can cause infectious keratitis in immunocompromised patients / 19 2I )Nanopore sequencing was particularly effective at identifying causative bacterial agents in corneal ulcers that were too small for conventional scraping and culture, highlighting its utility in detecting the polymicrobial etiology of these infections. Of the 8 eyes that yielded positive results on Nanopore, 7 showed reads from more than one bacterial genus. This finding aligns with previous reports on the polymicrobial nature of infection-induced corneal ulcers. For instance, in one study, among 81 corneal ulcers analyzed via culture, 43% yielded more than one bacterial organism / 22) Other studies have estimated that between 1.9% and 25% of corneal ulcers are polymicrobial in nature / 23 25)

[0341] These findings indicate that polymicrobial infections may be more common than previously recognized, possibly due to the enhanced detection capabilities of this new method. These findings also indicate that in corneal ulcers, a favorable environment may be conducive to the growth of multiple bacterial species. Ung L, Bispo PJM, Shanbhag SS, Gilmore MS, Chodosh J. The persistent dilemma of microbial keratitis: Global burden, diagnosis, and antimicrobial resistance. Surv Ophthalmol. May-Jun 2019;64(3):255-271.

[0342] Hsu HY, Ernst B, Schmidt EJ, Parihar R, Horwood C, Edelstein SL. Laboratory Results, Epidemiologic Features, and Outcome Analyses of Microbial Keratitis: A 15-Year Review From St. Louis. Am J Ophthalmol. Feb 2019;198:54-62.

[0343] Cabrera- Aguas M, Khoo P, Watson SL. Infectious keratitis: A review. Clin Exp Ophthalmol. Jul 2022;50(5):543-562.

[0344] Low L, Fuentes-Utrilla P, Hodson J, et al. Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis. PeerJ. 2021;9:el0778.

[0345] Khan M, Summers S, Rice SA, Stapleton F, Willcox MDP, Subedi D. Acquired fluoroquinolone resistance genes in corneal isolates of Pseudomonas aeruginosa. Infect Genet Evol. Nov 2020;85: 104574.

[0346] Sauerborn E, Corredor NC, Reska T, et al. Detection of hidden antibiotic resistance through realtime genomics. Nature Communications. 2024 / 06 / 28 2024; 15(1):5494.

[0347] Leggett RM, Alcon-Giner C, Heavens D, et al. Rapid MinlON profiling of preterm microbiota and antimicrobial-resistant pathogens. Nature Microbiology. 2020 / 03 / 01 2020;5(3):430-442.

[0348] Charalampous T, Kay GL, Richardson H, et al. Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection. Nature Biotechnology. 2019 / 07 / 01 2019;37(7):783-792.

[0349] McDermott AM. Antimicrobial compounds in tears. Exp Eye Res. Dec 2013 ; 117:53-61.

[0350] Davidson HJ, Kuonen VJ. The tear film and ocular mucins. Vet Ophthalmol. Mar- Apr 2004;7(2):71-7.

[0351] Ahle CM, Stoddicke K, Afshar M, et al. Staphylococcus saccharolyticus: An Overlooked Human Skin Colonizer. Microorganisms. Jul 23 2020;8(8)

[0352] Schutte AHJ, Strepis N, Zandijk WHA, Bexkens ML, Bode LGM, Klaassen CHW.

[0353] Characterization of Staphylococcus roterodami sp. nov., a new species within the Staphylococcus aureus complex isolated from a human foot infection. Int J Syst Evol Microbiol. Sep 2021 ;71 (9) Ung L, Bispo PJM, Shanbhag SS, Gilmore MS, Chodosh J. The persistent dilemma of microbial keratitis: Global burden, diagnosis, and antimicrobial resistance. Survey of Ophthalmology. 2019 / 05 / 01 / 2019;64(3):255-271. Zemba M, Dumitrescu OM, Dimirache AE, et al. Diagnostic methods for the etiological assessment of infectious corneal pathology (Review). Exp Ther Med. Feb 2022;23(2):137.

[0354] Priya R, Mythili A, Singh YR, et al. Virulence, Speciation and Antibiotic Susceptibility of Ocular Coagulase Negative Staphylococci (CoNS). J Clin Diagn Res. May 2014;8(5):Dc33-7.

[0355] Ashby NS, Johnson TJ, Castillo-Ronquillo Y, et al. Cutibacterium (Formerly Propionibacterium) acnes Keratitis: A Review. Eye & Contact Lens. 2023 ;49(5):212-218.

[0356] Elston MJ, Dupax JP, Opanowo MI, Atkinson RE. Cutibacterium acnes (formerly Propionibacterium acnes) and Shoulder Surgery. Hawaii J Health Soc Welf. Nov 2019;78(11 Suppl 2):3-5.

[0357] Ashby NS, Johnson TJ, Castillo-Ronquillo Y, et al. Cutibacterium (Formerly Propionibacterium) acnes Keratitis: A Review. Eye & Contact Lens. May 1 2023;49(5):212-218.

[0358] Pedro- Aguilar L, Ramirez-Miranda A, Bautista-de Lucio VM, Navas A, Ortiz-Casas M, Graue- Hernandez EO. Epidemiology and Outcomes of Kocuria Keratitis. Eye Contact Lens. Sep 2016;42(5):e20-4.

[0359] Kate A, Bagga B, Joseph J, Mohamed A. Clinical Features and Outcomes of Kocuria Keratitis and Comparison With Coagulase-Negative Staphylococci Keratitis. Cornea. 2020;39(8):957-960.

[0360] Ahmed N, Biswal I, Roy P, Grover R. Kocuria kristinae, an unusual pathogen causing opportunistic infections in patients with malignancy. Indian Journal of Medical Microbiology. 2014 ;32(4) :456. Prahald-Vaezi K, Levasseur SD, Schendel S, et al. The Corneal Ulcer One-Touch Study: A Simplified Microbiological Specimen Collection Method. American Journal of Ophthalmology. 2015 / 01 / 01 / 2015;159(l):37-43.el.

[0361] Chan L, Lopez JB, Saifee M, Padmanabhan S, Chan MF, Yung M. Characterization of Polymicrobial and Antibiotic-Resistant Infectious Keratitis in a County Hospital Setting. Cornea. 2023;42(3):e0016.

[0362] Fernandes M, Vira D, Dey M, Tanzin T, Kumar N, Sharma S. Comparison Between Polymicrobial and Fungal Keratitis: Clinical Features, Risk Factors, and Outcome. American Journal of Ophthalmology. 2015 / 11 / 01 / 2015; 160(5):873-881.e2.

[0363] Lim NCS, Lim DKA, Ray M. Polymicrobial Versus Monomicrobial Keratitis: A Retrospective Comparative Study. Eye & Contact Lens. 2013 ;39(S)

[0364] Peter VG, Morandi SC, Herzog EL, Zinkernagel MS, Zysset-Burri DC. Investigating the Ocular Surface Microbiome: What Can It Tell Us? Clin Ophthalmol. 2023;17:259-271.

[0365] Petrillo F, Pignataro D, Lavano MA, et al. Current Evidence on the Ocular Surface Microbiota and Related Diseases. Microorganisms. Jul 13 2020;8(7) Baudouin C, Labbe A, Liang H, et al. The role of microbial flora on the ocular surface. Curr Opin Allergy Clin Immunol. Oct 2009;9(5):466-70.

[0366] Seven MMI, Williams MR, Shanbhad A, et al. The Ubiquitous Human Skin Commensal Staphylococcus hominis Protects against Opportunistic Pathogens. mBio. Jun 28 2022;13(3):e0093022.

[0367] Garg P, Rao GN. Corneal ulcer: diagnosis and management. Community Eye Health. 1999;12(30):21-3.

[0368] The details of Example 2 are described in A Tear-Based Approach for Rapid Identification of Bacterial Pathogens in Corneal Ulcers Using Nanopore Sequencing, mdRxiV, the contents of which are incorporated by reference in its entirety.

[0369] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A method of determining the presence of microbes in the eye of a subject, the method comprising detecting microbial nucleic acids in a tear sample isolated from the subject.

2. The method of claim 1, wherein detecting comprises processing the sample using a machine-based analytical platform.

3. The method of claims 1 or 2, wherein detecting comprises a molecular analysis comprising PCR, sequencing, microarray, or a combination thereof.

4. The method of claim 3 comprising PCR.

5. The method of claim 4, wherein the PCR is selected from Real-Time PCR (quantitative PCR or qPCR), Reverse-Transcriptase (RT-PCR), Multiplex PCR, Nested PCR, High Fidelity PCR, Fast PCR, Hot Start PCR, Long-range PCR, Arbitrary Primed PCR, Digital PCR, Droplet Digital PCR (ddPCR), isothermal amplification PCR, Endpoint PCR (Qualitative PCR), or a combination of any thereof.

6. The method of claims 4 or 5, wherein the PCR is qualitative or quantitative.

7. The method of any one of claims 4-6, wherein the PCR comprises one or more sets of primers specific for a type of microbe.

8. The method of any one of claims 4-7, wherein the PCR comprises one or more sets of genus and / or species specific primers.

9. The method of any one of claims 4-7, wherein the PCR comprises non-specific and / or random primers.

10. The method of any one of claims 1-3 comprising sequencing in the absence of PCR, optionally wherein the sequencing is selected from Sanger sequencing, single molecule real time (SMRT) sequencing, nanopore DNA sequencing, massively parallel signature sequencing (MPSS), colony sequencing, 454 pyrosequencing, Illumina sequencing, combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, ion torrent semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, or a combination of any thereof.

11. The method of claim 10, wherein the nucleic acid substrate for sequencing is microbial DNA, or cDNA reverse transcribed from microbial RNA, in the tear sample.

12. The method of any one of claims 1-9 comprising a combination of PCR and sequencing.

13. The method of claim 12, wherein the sequencing is selected from Sanger sequencing, single molecule real time (SMRT) sequencing, nanopore DNA sequencing, massively parallel signature sequencing (MPSS), colony sequencing, 454 pyrosequencing, Illumina sequencing, combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, ion torrent semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, or a combination of any thereof.

14. The method of any one of claims 10-13, wherein the nucleic acid substrate for sequencing comprises amplicons generated during the PCR.

15. The method of any one of claims 1-14, wherein the nucleic acids are not extracted from the tears prior to detection.

16. The method of any one of claims 1-15, wherein the tear sample further comprise water or buffer added after the sample is isolated from the subject.

17. The method of any one of claims 1-16, wherein isolation of the tear sample comprises collection of tears using a collection device, optionally wherein the volume of tears isolated from the collection device is any integer number of pl between about 1 pl to 100 pl inclusive, preferable about 1 pl to 20 pl inclusive, or any sub-range of two integers therebetween.

18. The method of claim 17, wherein the collection device is selected from Schirmer strip, capillary action paper, and cotton swab.

19. The method of claims 17 or 18, wherein isolation of the tears comprises drawing the tears out of the collection device, optionally by centrifugation.

20. The method of any one of claims 1-19, wherein the absence of detection of nucleic acids in the tear sample indicates the eye(s) from which the tear sample was collected is free from microbes.

21. The method of any one of claims 1-20, wherein the detection of nucleic acids in the tear sample indicates the eye(s) from which the tear sample was collected is infected with microbes.

22. The method of any one of claims 1-21 comprising the use of bioinformatics.

23. The method of any one of claims 1-21 , further comprising determining the type of microbes present in the tear sample, optionally wherein the microbes are selected from bacteria, fungi, viruses, parasites, and combinations thereof.

24. The method of any one of claims 1-23 further comprising determining the genus and / or species of the microbes.

25. The method of any one of claims 1-24, wherein the nucleic acids in the tear sample comprise DNA, RNA, or a combination thereof.

26. The method of claim 25 comprising RNA, wherein the RNA is converted to DNA by reverse transcription prior to molecular analysis.

27. The method of any one of claims 1-26, wherein detection is relative to a control, wherein nucleic acids present in the control are deducted from the molecular analysis before determining if nucleic acids are present in the tear sample.

28. The method of claim 27, wherein the control is a tear sample collected from the contralateral eye of the same subject.

29. The method of claim 27, wherein the control is a nuclease-free water or buffer.

30. The method of any one of claims 1-29, comprising PCR amplifying a target portion of the nucleic acids optionally using ribosomal RNA-specific primers, sequencing the resulting amplicons using next generation sequencing optionally nanopore sequencing, and determining the type, genus, species, or a combination thereof of the organism from which the nucleic acids were obtained, optionally using bioinformatics.

31. A method of diagnosing a subject with an eye infection comprising detecting microbes according to the method of any one of claims 1-30, wherein the subject is determined to have a microbial eye infection when nucleic acids are detected in the tear sample, and the subject is determined not to have a microbial eye infection when nucleic acids are not detected in the tear sample, optionally wherein the subject is determined to be free from an eye infection.

32. The method of claim 31 , further comprising treating the subject with an antimicrobial agent, when the subject is determined to have a microbial eye infection.

33. The method of claim 31, further comprising abstaining from treating the subject with antimicrobial agents when the subject is determined not to have a microbial eye infection.

34. The method of claims 32 or 33 comprising treating the subject with an antiinflammatory, antihistamine, immune suppressant, analgesic, or a combination thereof.

35. The method of any one of claims 31-34, further comprising determining the type of microbes present in the tear sample.

36. The method of claim 35, wherein the microbes are bacteria, fungi, viruses, parasites, or combinations thereof.

37. The method of any one of claims 1-36 further comprising determining the genus and / or species of the microbes.

38. The method of any one of claims 35-37, wherein the subject is treated with an antimicrobial agent effective to treat the microbes that are present.

39. The method of claim 38, wherein the microbe(s) is or comprises a bacteria and the subject is treated with one or more topical and / or oral antibiotics, optionally wherein antibiotic- resistant bacteria are treated with broad- spectrum antibiotics.

40. The method of claims 38 or 39, wherein the microbe(s) is or comprises a virus and the subject is treated with one or more topical and / or oral antivirals.

41. The method of any one of claims 38-40, wherein the microbe(s) is or comprises a fungi and the subject is treated with one or more topical and / or oral antifungals.

42. The method of any one of claims 38-41, wherein the microbe(s) is or comprises a parasite and the subject is treated with one or more topical and / or oral anti-parasitics.

43. A method of treating a subject for an eye infection comprising administering an antimicrobial agent to a subject diagnosed with a microbial eye infection according to the method of any one of claims 31-37.

44. The method of claim 43, wherein the microbe(s) is or comprises a bacteria and the subject is treated with one or more topical and / or oral antibiotics, optionally wherein antibiotic- resistant bacteria are treated with broad- spectrum antibiotics.

45. The method of claims 43 or 44, wherein the microbe(s) is or comprises a virus and the subject is treated with one or more topical and / or oral antivirals.

46. The method of any one of claims 43-45, wherein the microbe(s) is or comprises a fungi and the subject is treated with one or more topical and / or oral antifungals.

47. The method of any one of claims 43-46, wherein the microbe(s) is or comprises a parasite and the subject is treated with one or more topical and / or oral anti-parasitics.

48. A method for treating a subject for an eye infection comprising abstaining from administering antimicrobial agents to a subject diagnosed as not having a microbial eye infection.

49. The method of any one of claims 1-48, wherein the eye infection is selected from keratitis, conjunctivitis, cellulitis, shingles, endophthalmitis, stye, blepharitis, uveitis, dacryocystitis, and canaliculitis.

50. The method of any one of claims 1-49, wherein the subject undergone a corneal transplant.

51. The method of claim 50, wherein the subject is not showing any symptoms of infections.

52. The method of claims 50 or 51, wherein the corneal transplant was about 1-365 days inclusive or any subrange or specific day therebetween, preceding the collection of the tear sample.

53. The method of any one of claims 1-52, wherein the subject is selected from humans, goats, sheep, pigs, cattle, zebu, donkeys, water buffaloes, dromedary camel, horse, yak, domestic bactrian camel, llama, alpaca, gayal, bali cattle, domestic rabbit, addax, bison, deer, eland, elk, guinea pig, greater kudu, mule, moose, muskox, and reindeer, chicken, duck, goose, guineafowl, muscovy duck, turkey, emu, egyptian goose, indian peafowl, mute swan, ostrich, partridge, smallbilled tinamou, pigeon, quail, edible-nest swiftlet, grey francolin, guineafowl, common pheasant, and golden pheasant.

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