Methods and systems for detecting a microorganism

Abstract

Described herein methods and systems for detection a fastidious microorganism in a first biological sample and a second biological sample from the subject, wherein the detection of the fastidious microorganism based on the combination of the first and second biological samples yields greater sensitivity or specificity than detection based on either the first biological sample alone or the second biological sample alone.

Description

WSGR Docket No. 68706-703.601METHODS AND SYSTEMS FOR DETECTING A MICROORGANISMCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 743,024, filed January 8, 2025, which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Vector-borne pathogens (e.g., bacteria and protozoa) are transmitted by a variety of vectors, such as ticks and fleas. These pathogens cause or are associated with different diseases and conditions in humans and animals. Surveillance studies of tick populations, for example, have demonstrated the presence of multiple pathogens in individual ticks suggesting a risk of polymicrobial transmission to humans and animals, which can result in the development of coinfections. Co-infections are a relatively poorly understood aspect of vector-borne diseases. A key factor for the deficiency in understanding and / or diagnosing vector-borne diseases is the intrinsic limitations associated with molecular and serologic assays employed for the detection of vector-borne pathogens. For example, limitations in the sensitivity, specificity and the capacity for inclusion of multiple agents within a single assay represent a primary challenge for the accurate detection of polymicrobial vector-borne infections. There is a need in the art for assays and methods that provide enhanced detection and identification of vector-borne pathogens.SUMMARY

[0003] Described herein is a method for detecting a fastidious microorganism in a subject, the method comprising: obtaining a first biological sample and a second biological sample from the subject, wherein the first biological sample and the second biological sample are different sample types; contacting each of the first and second biological samples with an agent to detect a macromolecule derived from the fastidious microorganism; and determining presence or absence of the fastidious microorganism based on the detection from the first biological sample and the second biological sample.

[0004] In certain aspects, this disclosure provides a method for detecting a fastidious microorganism in a subject, the method comprising: obtaining a first biological sample and a second biological sample from the subject, wherein the first biological sample and the second biological sample are different sample types; contacting each of the first and second biological samples with an agent to detect a macromolecule derived from the fastidious microorganism;WSGR Docket No. 68706-703.601and presence or absence of the fastidious microorganism based on the detections, wherein the detection of the fastidious microorganism based on the combination of the first and second biological samples yields greater sensitivity or specificity than detection based on either the first biological sample alone or the second biological sample alone.

[0005] In certain aspects, this disclosure also provides a method for detecting a fastidious microorganism in a subject with increased sensitivity or specificity, the method comprising: obtaining a first biological sample from a subject, wherein the first biological sample is not blood; contacting the first biological sample with an agent to detect a macromolecule derived from the fastidious microorganism; and determining presence or absence of the fastidious microorganism based on the detection, wherein the detection of the fastidious microorganism in the first biological sample comprises greater sensitivity or specificity relative to detection of the fastidious microorganism in a second biological sample from the subject, wherein the first biological sample and the second biological sample are different sample types.

[0006] In some embodiments, the macromolecule comprises a polypeptide associated with the fastidious microorganism. In some embodiments, the polypeptide is expressed on a surface of the fastidious microorganism. In some embodiments, the polypeptide is secreted by the fastidious microorganism. In some embodiments, the macromolecule comprises a polynucleotide associated with the fastidious microorganism. In some embodiments, the polynucleotide comprises DNA of the fastidious microorganism. In some embodiments, the polynucleotide comprises RNA of the fastidious microorganism. In some embodiments, the RNA comprises ribosomal RNA (rRNA). In some embodiments, the rRNA comprises 18S rRNA, 5.8S rRNA, 28 S rRNA, or a combination thereof. In some embodiments, the agent comprises a polypeptide agent. In some embodiments, the polypeptide agent comprises an antibody or an antigen binding fragment thereof. In some embodiments, the polypeptide agent binds to the macromolecule. In some embodiments, the polypeptide agent binding the macromolecule is detected by western blotting, mass spectrometry, immunoassay, or a combination thereof. In some embodiments, the immunoassay comprises ELISA. In some embodiments, the immunoassay comprises detecting the polypeptide agent binding to the at least one polypeptide in suspension.

[0007] In some embodiments, the macromolecule comprises a polypeptide of Borrelia. In some embodiments, the polypeptide of Borrelia comprises outer surface protein A (OspA), OspB, OspC, variable lipoprotein surface-exposed protein (VlsE), oligopeptide ABC transporter I (OppAI), OppAII, OppAIII, OppAIV, OppAV, or a combination thereof. In some embodiments, the macromolecule comprises a polypeptide of Babesia. In some embodiments, theWSGR Docket No. 68706-703.601macromolecule comprises a polypeptide of Bartonella. In some embodiments, the macromolecule comprises a polypeptide of an additional pathogen other than Borrelia, Babesia, and Bartonella.

[0008] In some embodiments, the agent comprises a polynucleotide agent. In some embodiments, the polynucleotide agent comprises a primer. In some embodiments, the primer binds to the macromolecule. In some embodiments, the polynucleotide agent comprises an oligonucleotide probe. In some embodiments, the oligonucleotide probe binds to the macromolecule. In some embodiments, the oligonucleotide probe binds to an amplification product of the macromolecule.

[0009] In some embodiments, the method further comprises performing PCR for amplifying the macromolecule. In some embodiments, the PCR comprises digital PCR (dPCR), quantification PCR (qPCR), conventional PCR (cPCR), next generation sequence (NGS), reverse transcription (RT)-ddPCR, RT-qPCR, RT-cPCR, RNASeq, or a combination thereof.

[0010] In some embodiments, the macromolecule comprises a polynucleotide of Borrelia. In some embodiments, the macromolecule comprises a polynucleotide of Babesia. In some embodiments, the macromolecule comprises a polynucleotide of Bartonella. In some embodiments, the macromolecule comprises a polynucleotide of an additional pathogen other than Borrelia, Babesia, and Bartonella.

[0011] In some embodiments, the sensitivity or selectivity of detecting the fastidious In some embodiments, microorganism based on the combination of the first and second biological samples compared to the sensitivity or specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500% compared to the detection based on either the first biological sample alone or the second biological sample alone.

[0012] In some embodiments, sensitivity or specificity of detecting the fastidious microorganism is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500% compared to the detection of the fastidious microorganism in the second biological sample from the subject.

[0013] In some embodiments, the subject is suspected of being infected by the fastidious microorganism.

[0014] In some embodiments, the method further comprises detecting at least one additional pathogen in the sample. In some embodiments, the at least one additional pathogen comprises a species that is different from the species of the fastidious microorganism. In some embodiments, the at least one additional pathogen comprises a genus that is different from the genus of the fastidious microorganism. In some embodiments, the method further comprises enriching theWSGR Docket No. 68706-703.601fastidious microorganism in a culturing media. In some embodiments, the method further comprises capturing the fastidious microorganism by contacting the fastidious microorganism with a trapping agent. In some embodiments, the trapping agent comprises an antibody or an antigen binding fragment thereof. In some embodiments, the antibody or the antigen binding fragment thereof is conjugated to a solid surface. In some embodiments, the trapping agent comprises a chaotropic agent. In some embodiments, the sample comprises blood, plasma, serum, urine, cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions. In some embodiments, the subject a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is not a mammal. In some embodiments, the subject is not a human. In some embodiments, the subject is a host for the fastidious microorganism. In some embodiments, the subject is not a host for the fastidious microorganism.

[0015] In some embodiments, the method further comprises treating the subject based on the fastidious microorganism in the sample. In some embodiments, the subject is treated based on a life cycle of the fastidious microorganism. In some embodiments, the subject is treated based on a morphology of the fastidious microorganism. In some embodiments, the subject is treated based on species of the fastidious microorganism. In some embodiments, the subject is treated for inflammation associated with the fastidious microorganism. In some embodiments, the subject is treated for a disease or condition associated with the fastidious microorganism. In some embodiments, the disease or condition comprises osteoarthritis (OA), Sjogren’s syndrome (SS), rheumatoid arthritis (RA), fatigue syndrome (CFS), gout, calcium pyrophosphate deposition (CPPD), rotator cuff disease (RC), neuropathy, or a combination thereof. In some embodiments, genus of the fastidious microorganism comprises Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof. In some embodiments, species of the fastidious microorganism comprises Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, Borrelia mayonii, Borrelia miyamotoi, Bartonella vinsonii berkhoffii, Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum, Rickettsia ricketsii, Rickettsia conorii, Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.

[0016] Described herein, in some aspects, is a method of detecting a genus or a species of a fastidious microorganism, comprising: contacting two or more macromolecules in a sample obtained from a subject with two or more agents for detecting a genus or a species of a fastidious microorganism in the sample, wherein the two or more macromolecules compriseWSGR Docket No. 68706-703.601different classes of macromolecule, and wherein the contacting of the two or more macromolecules with the two or more agents increases a sensitivity or a specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to a second sensitivity or a second specificity of detecting the genus or the species of the fastidious microorganism by one agent contacting one class of macromolecule.

[0017] In certain aspects, the present disclosure describes a method of detecting a fastidious microorganism in a subject, comprising: contacting a biological sample from the subject with two or more agents to detect two or more macromolecules derived from the fastidious microorganism; and determining presence or absence of the fastidious microorganism based on the detection, wherein the two or more macromolecules are of different macromolecular classes, and wherein the detection of the fastidious microorganism with two or more macromolecules of the different macromolecular classes provides an increased sensitivity or specificity relative to detection of the fastidious microorganism with one class of macromolecule alone.

[0018] In some embodiments, the two or more macromolecules comprise a polypeptide associated with the fastidious microorganism. In some embodiments, the polypeptide is expressed on a surface of the fastidious microorganism. In some embodiments, the polypeptide is secreted by the fastidious microorganism. In some embodiments, the two or more macromolecules comprise a polynucleotide associated with the fastidious microorganism. In some embodiments, the polynucleotide comprises a DNA of the fastidious microorganism. In some embodiments, the polynucleotide comprises a RNA of the fastidious microorganism. In some embodiments, the RNA comprises ribosomal RNA (rRNA). In some embodiments, the rRNA comprises 18S rRNA, 5.8S rRNA, 28S rRNA, or a combination thereof. In some embodiments, the two or more macromolecules comprise at least one polypeptide and at least one polynucleotide. In some embodiments, the two or more agents comprise a polypeptide agent. In some embodiments, the polypeptide agent comprises an antibody or an antigen binding fragment thereof. In some embodiments, the polypeptide agent binds to the two or more macromolecules. In some embodiments, the polypeptide agent binding the two or more macromolecules is detected by western blotting, mass spectrometry, immunoassay, or a combination thereof. In some embodiments, the immunoassay comprises ELISA. In some embodiments, the immunoassay comprises detecting the polypeptide agent binding to the at least one polypeptide in suspension. In some embodiments, the two or more macromolecules comprise a polypeptide of Borrelia. In some embodiments, the polypeptide of Borrelia comprises outer surface protein A (OspA), OspB, OspC, variable lipoprotein surface-exposed protein (VlsE), oligopeptide ABC transporter I (OppAI), OppAII, OppAIII, OppAIV, OppAV,WSGR Docket No. 68706-703.601or a combination thereof. In some embodiments, the two or more macromolecules comprise a polypeptide of Babesia. In some embodiments, the two or more macromolecules comprise a polypeptide of Bartonella. In some embodiments, the two or more macromolecules comprise a polypeptide of an additional pathogen other than Borrelia, Babesia, and Bartonella. In some embodiments, the two or more agents comprise a polynucleotide agent. In some embodiments, the polynucleotide agent comprises a primer. In some embodiments, the primer binds to the two or more macromolecules. In some embodiments, the polynucleotide agent comprises an oligonucleotide probe. In some embodiments, the oligonucleotide probe binds to the two or more macromolecules. In some embodiments, the oligonucleotide probe binds to an amplification product of the two or more macromolecules. In some embodiments, the method further comprises performing PCR for amplifying the two or more macromolecules. In some embodiments, the PCR comprises digital PCR (dPCR), quantification PCR (qPCR), conventional PCR (cPCR), next generation sequence (NGS), reverse transcription (RT)-ddPCR, RT-qPCR, RT-cPCR, RNASeq, or a combination thereof. In some embodiments, the two or more macromolecules comprise a polynucleotide of Borrelia. In some embodiments, the two or more macromolecules comprise a polynucleotide of Babesia. In some embodiments, the two or more macromolecules comprise a polynucleotide of Bartonella. In some embodiments, the two or more macromolecules comprise a polynucleotide of an additional pathogen other than Borrelia, Babesia, and Bartonella. In some embodiments, the two or more agents comprise at least one polypeptide agent and at least one polynucleotide agent. In some embodiments, the sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, the specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, the subject is suspected of being infected by the fastidious microorganism. In some embodiments, the contacting of the two or more macromolecules with the two or more agents detects the genus or the species of the fastidious microorganism in the subject at an earlier stage of infection by the fastidious microorganism compared to detecting the genus or the species of the fastidious microorganism by the one agent contacting the one class of macromolecule. In some embodiments, the method further comprises detecting at least one additional pathogen in the sample. In some embodiments, the at least one additional pathogen comprises a species that is different from the species of the fastidious microorganism. In some embodiments, the at least one additional pathogen comprises a genus that is different from theWSGR Docket No. 68706-703.601genus of the fastidious microorganism. In some embodiments, the method further comprises enriching the fastidious microorganism in a culturing media. In some embodiments, the method further comprises capturing the fastidious microorganism by contacting the fastidious microorganism with a trapping agent. In some embodiments, the trapping agent comprises an antibody or an antigen binding fragment thereof. In some embodiments, the antibody or the antigen binding fragment thereof is conjugated to a solid surface. In some embodiments, the trapping agent comprises a chaotropic agent. In some embodiments, the sample comprises blood, plasma, serum, urine, cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions. In some embodiments, the subject a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is not a mammal. In some embodiments, the subject is not a human. In some embodiments, the subject is a host for the fastidious microorganism. In some embodiments, the subject is not a host for the fastidious microorganism. In some embodiments, the method further comprises treating the subject based on the genus or the species of the fastidious microorganism in the sample. In some embodiments, the subject is treated based on a life cycle of the fastidious microorganism. In some embodiments, the subject is treated based on a morphology of the fastidious microorganism. In some embodiments, the subject is treated based on the species of the fastidious microorganism. In some embodiments, the subject is treated for inflammation associated with the fastidious microorganism. In some embodiments, the subject is treated for a disease or condition associated with the fastidious microorganism. In some embodiments, the disease or condition comprises osteoarthritis (OA), Sjogren’s syndrome (SS), rheumatoid arthritis (RA), chronic fatigue syndrome (CFS), gout, calcium pyrophosphate deposition (CPPD), rotator cuff disease (RC), neuropathy, or a combination thereof. In some embodiments, the genus of the fastidious microorganism comprises Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof. In some embodiments, the species of the fastidious microorganism comprises Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, Borrelia mayonii, Borrelia miyamotoi, Bartonella vinsonii berkhoffn, Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum, Rickettsia ricketsii, Rickettsia conorii, Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.

[0019] Described herein, in some aspects, is a system comprising: two or more agents for contacting the two or more macromolecules, wherein the two or more macromolecules are different class of macromolecule; wherein the two or more agents contacting the two or moreWSGR Docket No. 68706-703.601macromolecules detects a genus of a species of a fastidious microorganism in a sample obtained from a subject; and wherein contacting the two or more macromolecules with the two or more agents increases a sensitivity or a specificity of detecting the genus or the species of the fastidious microorganism compared to a second sensitivity or specificity of detecting the genus or the species fastidious microorganism one agent contacting one class of macromolecule. In some embodiments, the two or more agents comprise a polypeptide agent. In some embodiments, the polypeptide agent comprises an antibody or an antigen binding fragment thereof. In some embodiments, the polypeptide agent binds to the two or more macromolecules. In some embodiments, the polypeptide agent binding the two or more macromolecules is detected by western blotting, mass spectrometry, immunoassay, or a combination thereof. In some embodiments, the immunoassay comprises ELISA. In some embodiments, the immunoassay comprises detecting the polypeptide agent binding to the at least one polypeptide in suspension. In some embodiments, the two or more macromolecules comprise a polypeptide of Borrelia. In some embodiments, the polypeptide of Borrelia comprises outer surface protein A (OspA), OspB, OspC, variable lipoprotein surface-exposed protein (VlsE), oligopeptide ABC transporter I (OppAI), OppAII, OppAIII, OppAIV, OppAV, or a combination thereof. In some embodiments, the two or more macromolecules comprise a polypeptide of Babesia. In some embodiments, the two or more macromolecules comprise a polypeptide of Bartonella. In some embodiments, the two or more macromolecules comprise a polypeptide of an additional pathogen other than Borrelia, Babesia, and Bartonella. In some embodiments, the two or more agents comprise a polynucleotide agent. In some embodiments, the polynucleotide agent comprises a primer. In some embodiments, the primer binds to the two or more macromolecules. In some embodiments, the polynucleotide agent comprises an oligonucleotide probe. In some embodiments, the oligonucleotide probe binds to the two or more macromolecules. In some embodiments, the oligonucleotide probe binds to an amplification product of the two or more macromolecules. In some embodiments, the two or more macromolecules comprise a polynucleotide of Borrelia. In some embodiments, the two or more macromolecules comprise a polynucleotide of Babesia. In some embodiments, the two or more macromolecules comprise a polynucleotide of Bartonella. In some embodiments, the two or more macromolecules comprise a polynucleotide of an additional pathogen other than Borrelia, Babesia, and Bartonella. In some embodiments, the two or more agents comprise at least one polypeptide agent and at least one polynucleotide agent. In some embodiments, the system further comprises a culturing media for enriching the fastidious microorganism. In some embodiments, the system further comprises a trapping agent for capturing the fastidious microorganism. In some embodiments, the trappingWSGR Docket No. 68706-703.601agent comprises an antibody or an antigen binding fragment thereof. In some embodiments, the antibody or the antigen binding fragment thereof is conjugated to a solid surface. In some embodiments, the trapping agent comprises a chaotropic agent. In some embodiments, the genus of the fastidious microorganism comprises Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof. In some embodiments, the species of the fastidious microorganism comprises Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, Borrelia mayonii, Borrelia miyamotoi, Bartonella vinsonii berkhoffn, Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum, Rickettsia ricketsii, Rickettsia conorii, Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.

[0020] Described herein, in some embodiments, is a kit comprising: two or more agents for contacting the two or more macromolecules, wherein the two or more macromolecules are different class of macromolecule; and wherein the two or more macromolecules are obtained from a fastidious microorganism. In certain embodiments, the present disclosure also provides a kit comprising two or more agents configured to bind to two or more macromolecules derived from the fastidious microorganism and of different macromolecular classes, wherein binding of the two or more agents with the two or more macromolecules is indicative of the presence of the fastidious microorganism in a subject; and wherein the detection of the fastidious microorganism with two or more macromolecules of the different macromolecular classes provides an increased sensitivity or specificity relative to detection of the fastidious microorganism with one class of macromolecule.

[0021] In some embodiments, the fastidious microorganism comprises Borrelia, Babesia, or Bartonella. In some embodiments, the two or more agents comprise a polypeptide agent. In some embodiments, the polypeptide agent comprises an antibody or an antigen binding fragment thereof. In some embodiments, the polypeptide agent binding the two or more macromolecules is detected by western blotting, mass spectrometry, immunoassay, or a combination thereof. In some embodiments, the immunoassay comprises ELISA. In some embodiments, the immunoassay comprises detecting the polypeptide agent binding to at least one polypeptide in suspension. In some embodiments, the two or more agents comprise a polynucleotide agent. In some embodiments, the polynucleotide agent comprises a primer. In some embodiments, the primer binds to the two or more macromolecules. In some embodiments, the polynucleotide agent comprises an oligonucleotide probe. In some embodiments, the oligonucleotide probe binds to the two or more macromolecules. In some embodiments, the oligonucleotide probe binds to an amplification product of the two or more macromolecules. In some embodiments,WSGR Docket No. 68706-703.601the two or more agents comprise at least one polypeptide agent and at least one polynucleotide agent. In some embodiments, the kit further comprises a culturing media for enriching the fastidious microorganism. In some embodiments, the kit further comprises a trapping agent for capturing the fastidious microorganism. In some embodiments, the trapping agent comprises an antibody or an antigen binding fragment thereof. In some embodiments, the antibody or the antigen binding fragment thereof is conjugated to a solid surface. In some embodiments, the trapping agent comprises a chaotropic agent. In some embodiments, the genus of the fastidious microorganism comprises Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof. In some embodiments, the species of the fastidious microorganism comprises Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, Borrelia mayonii, Borrelia miyamotoi, Bartonella vinsonii berkhoffn, Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum, Rickettsia ricketsii, Rickettsia conorii, Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.

[0022] Described herein, in some aspects, is a method for detecting a fastidious microorganism in a transcellular fluid sample obtained from a subject, the method comprising: contacting a macromolecule of a fastidious microorganism with an agent for detecting a genus or a species of the fastidious microorganism in a transcellular fluid sample obtained from a sample, wherein detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample results in an increased sensitivity or specificity of detecting the genus or the species of the fastidious microorganism compared to a second sensitivity or a second specificity of detecting the genus or the species of the fastidious microorganism in an interstitial fluid sample. In some embodiments, the macromolecule comprises a polypeptide associated with the fastidious microorganism. In some embodiments, the polypeptide is expressed on a surface of the fastidious microorganism. In some embodiments, the polypeptide is secreted by the fastidious microorganism. In some embodiments, the macromolecule comprises a polynucleotide associated with the fastidious microorganism. In some embodiments, the polynucleotide comprises a DNA of the fastidious microorganism. In some embodiments, the polynucleotide comprises an RNA of the fastidious microorganism. In some embodiments, the RNA comprises ribosomal RNA (rRNA). In some embodiments, the rRNA comprises 18S rRNA, 5.8S rRNA, 28 S rRNA, or a combination thereof. In some embodiments, the agent comprises a polypeptide agent. In some embodiments, the polypeptide agent comprises an antibody or an antigen binding fragment thereof. In some embodiments, the polypeptide agent binding the macromolecule is detected by western blotting, mass spectrometry, immunoassay, orWSGR Docket No. 68706-703.601a combination thereof. In some embodiments, the immunoassay comprises ELISA. In some embodiments, the immunoassay comprises detecting the polypeptide agent binding to the at least one polypeptide in suspension. In some embodiments, the macromolecule comprises a polypeptide of Borrelia. In some embodiments, the polypeptide of Borrelia comprises outer surface protein A (OspA), OspB, OspC, variable lipoprotein surface-exposed protein (VlsE), oligopeptide ABC transporter I (OppAI), OppAII, OppAIII, OppAIV, OppAV, or a combination thereof. In some embodiments, the macromolecule comprises a polypeptide of Babesia. In some embodiments, the macromolecule comprises a polypeptide of Bartonella. In some embodiments, the agent comprises a polynucleotide agent. In some embodiments, the polynucleotide agent comprises a primer. In some embodiments, the primer binds to the macromolecule. In some embodiments, the polynucleotide agent comprises an oligonucleotide probe. In some embodiments, the oligonucleotide probe binds to the macromolecule. In some embodiments, the oligonucleotide probe binds to an amplification product of the macromolecule. In some embodiments, the method further comprises performing PCR for amplifying the macromolecule. In some embodiments, the PCR comprises digital PCR (dPCR), quantitative PCR (qPCR), conventional PCR (cPCR), next generation sequence (NGS), reverse transcription (RT)-ddPCR, RT-qPCR, RT-cPCR, RNASeq, or a combination thereof. In some embodiments, the macromolecule comprises a polynucleotide of Borrelia. In some embodiments, the macromolecule comprises a polynucleotide of Babesia. In some embodiments, the macromolecule comprises a polynucleotide of Bartonella. In some embodiments, the sensitivity of detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample compared to the second sensitivity obtained from the interstitial fluid sample is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, the specificity of detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample compared to the second specificity obtained from the interstitial fluid sample is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, the subject suspected of being infected by the fastidious microorganism.

[0023] In some embodiments, the contacting of the macromolecule with the agent detects the genus or the species of the fastidious microorganism in the transcellular fluid sample at an earlier stage of infection by the fastidious microorganism compared to detecting the genus or the species of the fastidious microorganism by the agent contacting the macromolecule in the interstitial fluid sample. In some embodiments, the method further comprises detecting at least one additional pathogen in the sample. In some embodiments, the at least one additionalWSGR Docket No. 68706-703.601pathogen comprises a species that is different from the species of the fastidious microorganism. In some embodiments, the at least one additional pathogen comprises a genus that is different from the genus of the fastidious microorganism. In some embodiments, the method further comprises enriching the fastidious microorganism in a culturing media. In some embodiments, the method further comprises capturing the fastidious microorganism by contacting the fastidious microorganism with a trapping agent. In some embodiments, the trapping agent comprises an antibody or an antigen binding fragment thereof. In some embodiments, the antibody or the antigen binding fragment thereof is conjugated to a solid surface. In some embodiments, the trapping agent comprises a chaotropic agent. In some embodiments, the transcellular fluid sample comprises cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions. In some embodiments, the transcellular fluid comprises the synovial fluid. In some embodiments, the interstitial fluid sample comprises blood, serum, plasma, or a combination thereof. In some embodiments, the subject a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is not a mammal. In some embodiments, the subject is not a human. In some embodiments, the subject is a host for the fastidious microorganism. In some embodiments, the subject is not a host for the fastidious microorganism. In some embodiments, the method further comprises treating the subject based on the genus or the species of the fastidious microorganism in the transcellular fluid sample. In some embodiments, the subject is treated based on a life cycle of the fastidious microorganism. In some embodiments, the subject is treated based on a morphology of the fastidious microorganism. In some embodiments, the subject is treated based on the species of the fastidious microorganism. In some embodiments, the subject is treated for inflammation associated with the fastidious microorganism. In some embodiments, the subject is treated for a disease or condition associated with the fastidious microorganism. In some embodiments, the disease or condition comprises osteoarthritis (OA), Sjogren’s syndrome (SS), rheumatoid arthritis (RA), chronic fatigue syndrome (CFS), gout, calcium pyrophosphate deposition (CPPD), rotator cuff disease (RC), neuropathy, or a combination thereof. In some embodiments, the genus of the fastidious microorganism comprises Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof. In some embodiments, the species of the fastidious microorganism comprises Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, Borrelia mayonii, Borrelia miyamotoi, Bartonella vinsonii berkhoffii,WSGR Docket No. 68706-703.601Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum, Rickettsia ricketsii, Rickettsia conorii. Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.INCORPORATION BY REFERENCE

[0024] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” , “FIG.”, and “FIGs” herein), of which:

[0026] FIG. 1 illustrates a flow diagram of an example of an assay workflow for detecting a genus or a species of a fastidious microorganism in a sample.

[0027] FIG. 2 illustrates a non-limiting example of detecting one or more macromolecules for detecting a fastidious microorganism described herein.

[0028] FIGs. 3A and 3B are plots showing the study demographics for age and gender of study participants, and joint sampled for each study participant, respectively.

[0029] FIGs. 4A and 4B are plots showing serology results comparing serum to synovial fluid samples for / / . burgdorferi and Bartonella spp., respectively.

[0030] FIGs. 5A and 5B are bar graphs showing the percentage of participants with each diagnosis of interest and exposure to / / . burgdorferi or Bartonella spp., respectively.

[0031] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments.DETAILED DESCRIPTION

[0032] Described herein, in some aspects, are methods, systems, or kits for detecting a genus or a species of a fastidious microorganism. In some embodiments, the detecting of the genus or theWSGR Docket No. 68706-703.601species of a fastidious microorganism comprises contacting two or more macromolecules in a sample obtained from a subject with two or more agents for detecting a genus or a species of a fastidious microorganism in the sample. In some embodiments, the two or more macromolecules comprise different classes of macromolecule.

[0033] In some embodiments, the present disclosure provides a method, system, or kit for detecting a fastidious microorganism in a subject. In some embodiments, the method comprises obtaining a first biological sample and a second biological sample from the subject, wherein the first biological sample and the second biological sample are different sample types; contacting each of the first and second biological samples with an agent to detect a macromolecule derived from the fastidious microorganism; and determining presence or absence of the fastidious microorganism based on the detection from the first biological sample and the second biological sample.

[0034] In some embodiments, as used herein, a biological sample refers to any material obtained from a subject that comprises or is suspected of comprising a biological analyte comprising a microorganism, nucleic acid, polypeptide, or other macromolecules. The terms “ biological sample” and “sample” may be interchangeably used herein. Biological samples may be obtained from different anatomical sources, physiological compartments, or body fluids of the subject. Non-limiting examples of biological samples include blood-derived samples (e.g., whole blood, plasma, serum, or cellular fraction thereof), tissue-derived samples (e.g., biopsy tissue or tissue homogenates), and non-blood body fluid samples (e.g., urine, saliva, synovial fluid, cerebrospinal fluid, pleural fluid, or interstitial fluid). In some embodiments, the biological sample comprises blood, plasma, serum, urine, cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions. In some embodiments, biological sample may be obtained from different physiological compartments, including intravascular, interstitial, or transcellular compartments. In some embodiments, the biological samples differ in processing state, such as a cell-containing fraction and a cell-free fraction. In some embodiments, a first biological sample and a second biological sample are different types of biological samples. In some embodiments, a first biological sample and a second biological sample are the same types of biological samples. In some embodiments, a first biological sample may be synovial fluid and a second biological sample may be serum. In some embodiments, a first biological sample may be urine and a second biological sample may be whole blood. In some embodiments, a first biological sample may be a cell-containing fraction and a second biological sample may be a cell-free fraction.WSGR Docket No. 68706-703.601

[0035] In some embodiments, the present disclosure describes a method for detecting a fastidious microorganism in a subject, the method comprising: obtaining a first biological sample and a second biological sample from the subject, wherein the first biological sample and the second biological sample are different sample types; contacting each of the first and second biological samples with an agent to detect a macromolecule derived from the fastidious microorganism; and determining presence or absence of the fastidious microorganism based on the detections, wherein the detection of the fastidious microorganism based on the combination of the first and second biological samples yields greater sensitivity or specificity than detection based on either the first biological sample alone or the second biological sample alone. In some embodiments, the biological sample comprises blood, plasma, serum, urine, cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions. In some embodiments, the biological sample may be obtained from different physiological compartments, including intravascular, interstitial, or transcellular compartments. In some embodiments, the biological samples differ in processing state, such as a cell-containing fraction and a cell-free fraction. In some embodiments, the first biological sample may be synovial fluid and the second biological sample may be serum. In some embodiments, the first biological sample may be urine and the second biological sample may be whole blood. In some embodiments, the first biological sample may be a cell-containing fraction and the second biological sample may be a cell-free fraction.

[0036] In some embodiments, the sensitivity or selectivity of detecting the fastidious microorganism based on the combination of the first and second biological samples may be increased by 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500% compared to the detection based on either the first biological sample alone or the second biological sample alone. In some embodiments, the sensitivity or selectivity of detecting the fastidious microorganism based on the combination of the first and second biological samples may be increased by about 3%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500% compared to the detection based on either the first biological sample alone or the second biological sample alone. In some embodiments, the sensitivity or selectivity of detecting the fastidious microorganism based on the combination of the first and second biological samples may be increased by at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%,WSGR Docket No. 68706-703.601at least 400%, or at least 500% compared to the detection based on either the first biological sample alone or the second biological sample alone. In some embodiments, the sensitivity or selectivity of detecting the fastidious microorganism based on the combination of the first and second biological samples may be increased by at most 3%, at most 5%, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 200%, at most 300%, at most 400%, or at most 500% compared to the detection based on either the first biological sample alone or the second biological sample alone.

[0037] In some embodiments, the present disclosure also provides a method for detecting a fastidious microorganism in a subject with increased sensitivity or specificity, the method comprising: obtaining a first biological sample from a subject, wherein the first biological sample is not blood; contacting the first biological sample with an agent to detect a macromolecule derived from the fastidious microorganism; and determining presence or absence of the fastidious microorganism based on the detection, wherein the detection of the fastidious microorganism in the first biological sample comprises greater sensitivity or specificity relative to detection of the fastidious microorganism in a second biological sample from the subject, wherein the first biological sample and the second biological sample are different sample types. In some embodiments, the biological sample comprises blood, plasma, serum, urine, cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions. In some embodiments, biological sample may be obtained from different physiological compartments, including intravascular, interstitial, or transcellular compartments. In some embodiments, the biological samples differ in processing state, such as a cell-containing fraction and a cell-free fraction. In some embodiments, the first biological sample may be synovial fluid. In some embodiments, the first biological sample may be urine. In some embodiments, the first biological sample may be a cell-containing fraction. In some embodiments, the first biological sample may be a cell-free fraction. In some embodiments, the second biological sample may be serum. In some embodiments, the second biological sample may be blood. In some embodiments, the second biological sample may be a cell-containing fraction. In some embodiments, the second biological sample may be a cell-free fraction. In some embodiments, the first biological sample comprises synovial fluid and the second biological sample comprises blood.

[0038] In some embodiments, the sensitivity or selectivity of detecting the fastidious microorganism may be increased by 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,WSGR Docket No. 68706-703.60190%, 100%, 150%, 200%, 300%, 400%, or 500% compared to the detection of the fastidious microorganism in the second biological sample from the subject. In some embodiments, the sensitivity or selectivity of detecting the fastidious microorganism may be increased by about 3%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500% compared to the detection of the fastidious microorganism in the second biological sample from the subject. In some embodiments, the sensitivity or selectivity of detecting the fastidious microorganism may be increased by at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500% compared to the detection of the fastidious microorganism in the second biological sample from the subject. In some embodiments, the sensitivity or selectivity of detecting the fastidious microorganism may be increased by at most 3%, at most 5%, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 200%, at most 300%, at most 400%, or at most 500% compared to the detection of the fastidious microorganism in the second biological sample from the subject.

[0039] FIG. 2 illustrates a non-limiting example of how to detect the macromolecules. In some embodiments, the contacting of the two or more macromolecules with the two or more agents increases a sensitivity or a specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to a second sensitivity or a second specificity of detecting the genus or the species of the fastidious microorganism by one agent contacting one class of macromolecule. In some embodiments, the two or more macromolecules comprise a polypeptide or a polynucleotide associated with the fastidious microorganism. In some embodiments, the two or more macromolecules comprise at least one polypeptide and at least one polynucleotide. In some embodiments, the two or more agents comprise a polypeptide agent or a polynucleotide agent. In some embodiments, the polypeptide agent comprises an antibody or an antigen binding fragment thereof. In some embodiments, the polynucleotide agent comprises a primer or an oligonucleotide probe. In some embodiments, the polypeptide agent or the polynucleotide agent binds to the two or more macromolecules. In some embodiments, the polypeptide agent or the polynucleotide agent binding to the two or more macromolecules can be detected by an immunoassay or a PCR-based assay. In some embodiments, the two or more macromolecules are associated with Borrelia, Babesia, or Bartonella. In some embodiments, by detecting the two or more macromolecules comprising a polypeptide and a polynucleotideWSGR Docket No. 68706-703.601increases a sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity may be increased by 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500%. In some embodiments, by detecting the two or more macromolecules comprising a polypeptide and a polynucleotide increases a sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity may be increased by about 3%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%. In some embodiments, by detecting the two or more macromolecules comprising a polypeptide and a polynucleotide increases a sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity may be increased by at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In some embodiments, by detecting the two or more macromolecules comprising a polypeptide and a polynucleotide increases a sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity may be increased by at most 3%, at most 5%, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 200%, at most 300%, at most 400%, or at most 500%. In some embodiments, by detecting the two or more macromolecules comprising a polypeptide and a polynucleotide increases a specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second specificity may be increased by 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500%. In some embodiments, by detecting the two or more macromolecules comprising a polypeptide and a polynucleotide increases a specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second specificity may be increased by about 3%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%. In some embodiments, by detecting the two or more macromolecules comprising a polypeptide and a polynucleotide increases a specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second specificity may be increased by at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In some embodiments, byWSGR Docket No. 68706-703.601detecting the two or more macromolecules comprising a polypeptide and a polynucleotide increases a specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second specificity may be increased by at most 3%, at most 5%, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 200%, at most 300%, at most 400%, or at most 500%.

[0040] Described herein, in some aspects, are methods, systems, or kits for detecting a fastidious microorganism in a transcellular fluid sample obtained from a subject. In some embodiments, the detecting comprises contacting a macromolecule of a fastidious microorganism with an agent for detecting a genus or a species of the fastidious microorganism in a transcellular fluid sample obtained from a sample. In some embodiments, detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample results in an increased sensitivity or specificity of detecting the genus or the species of the fastidious microorganism compared to a second sensitivity or a second specificity of detecting the genus or the species of the fastidious microorganism in an interstitial fluid sample. In some embodiments, the macromolecule comprises a polypeptide associated with the fastidious microorganism. In some embodiments, the macromolecule comprises a polynucleotide associated with the fastidious microorganism. In some embodiments, the polypeptide or the polynucleotide is associated with the fastidious microorganism. In some embodiments, the agent comprises a polypeptide agent or a polynucleotide agent. In some embodiments, the polypeptide agent or the polynucleotide agent binds to the macromolecule. In some embodiments, the polypeptide agent or the polynucleotide agent binding to the macromolecule can be detected by an immunoassay or a PCR-based assay. In some embodiments, the macromolecule is associated with Borrelia, Babesia, o Bartonella. In some embodiments, the sensitivity of detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample compared to the second sensitivity obtained from the interstitial fluid sample is increased by 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500%. In some embodiments, the sensitivity of detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample compared to the second sensitivity obtained from the interstitial fluid sample is increased by about 3%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%. In some embodiments, the sensitivity of detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample compared to the second sensitivity obtained from the interstitial fluid sample is increasedWSGR Docket No. 68706-703.601by at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In some embodiments, the sensitivity of detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample compared to the second sensitivity obtained from the interstitial fluid sample is increased by at most 3%, at most 5%, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 200%, at most 300%, at most 400%, or at most 500%. In some embodiments, the specificity of detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample compared to the second specificity obtained from the interstitial fluid sample is increased by 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500%. In some embodiments, the specificity of detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample compared to the second specificity obtained from the interstitial fluid sample is increased by about 3%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%. In some embodiments, the specificity of detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample compared to the second specificity obtained from the interstitial fluid sample is increased by at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In some embodiments, the specificity of detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample compared to the second specificity obtained from the interstitial fluid sample is increased by at most 3%, at most 5%, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 200%, at most 300%, at most 400%, or at most 500%.

[0041] In some embodiments, the transcellular fluid sample comprises cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions. In some embodiments, the transcellular fluid comprises the synovial fluid. In some embodiments, the interstitial fluid sample comprises blood, serum, plasma, or a combination thereof. FIGs. 3A-5B illustrate the improvement of detecting the presence of the fastidious microorganism in synovial fluid as opposed to in serum.WSGR Docket No. 68706-703.601

[0042] In some embodiments, the subject is suspected of being infected by the fastidious microorganism. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is not a mammal. In some embodiments, the subject is not a human. In some embodiments, the subject is a host for the fastidious microorganism. In some embodiments, the subject is not a host for the fastidious microorganism. In some embodiments, the sample obtained from the subject can be blood, plasma, serum, urine, cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, effusions, or a combination thereof.

[0043] In some embodiments, the methods, systems, or kits described detect the genus or the species of the fastidious microorganism in the subject at an earlier stage of infection by the fastidious microorganism. In some embodiments, the fastidious microprogram, prior to detection, can be enriched with a culture media described herein. In some embodiments, the fastidious microorganism, prior to detection, can be captured with a trapping agent described herein.

[0044] In some embodiments, the methods, systems, or kits described herein further comprise treating or treatment for a subject infected by the fastidious microorganism. In some embodiments, the subject is treated based on the genus or the species of the fastidious microorganism in the sample. In some embodiments, the subject is treated based on a life cycle of the fastidious microorganism. In some embodiments, the subject is treated based on a morphology of the fastidious microorganism. In some embodiments, the subject is treated based on the species of the fastidious microorganism. In some embodiments, the subject is treated for inflammation associated with the fastidious microorganism. In some embodiments, the subject is treated for a disease or condition associated with the fastidious microorganism. In some embodiments, the disease or condition comprises osteoarthritis (OA), Sjogren’s syndrome (SS), rheumatoid arthritis (RA), chronic fatigue syndrome (CFS), gout, calcium pyrophosphate deposition (CPPD), rotator cuff disease (RC), neuropathy, or a combination thereof. In some embodiments, the genus of the fastidious microorganism comprises Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof. In some embodiments, the species of the fastidious microorganism comprises Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, Borrelia mayonii, Borrelia miyamotoi, Bartonella vinsonii berkhoffti, Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum,WSGR Docket No. 68706-703.601Rickettsia rickelsii. Rickettsia conorii, Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.

[0045] Described herein, in some aspects, are methods and systems for detecting a genus or a species of a microorganism in a sample. At a high level, the methods and systems combine enrichment and capture techniques with multiple detection modalities to detect a microorganism of interest in a subject sample. A microorganism of interest may, for example, be a fastidious microorganism, such as a low abundance pathogen. In various embodiments, a method for detecting a genus or a species of a fastidious microorganism in a subject sample may include detecting a macromolecule associated with the microorganism of interest. In some embodiments, the macromolecule may be a polypeptide associated with the fastidious microorganism. In some embodiments, the macromolecule may be a polynucleotide associated with the fastidious microorganism. In various embodiments, detecting a macromolecule associated with a microorganism of interest may include contacting the macromolecule with an agent for detecting a genus or a species of the microorganism. In some embodiments, a contacting agent may be a polypeptide agent, wherein the polypeptide agent binds to the macromolecule associated with the microorganism. A polypeptide agent may, for example, be an antibody or an antigen binding fragment thereof. In some embodiments, a contacting agent may be a polynucleotide agent, wherein the polynucleotide agent binds to the macromolecule associated with the microorganism. A polynucleotide agent may, for example, be an amplification primer or an oligonucleotide probe. In some embodiments, a method for detecting a genus or a species of a microorganism in a sample may include: (i) obtaining a sample from a subject, such as a subject suspected of having an infection from a fastidious microorganism of interest; (ii) contacting a macromolecule in the sample with an agent for detecting a genus or a species of the microorganism, wherein the macromolecule and the agent are selected to increase a sensitivity or a specificity of detecting the presence of the microorganism in the sample compared to a second sensitivity or a second specificity of detecting the genus or the species of the microorganism; and (iii) based on the detection of the macromolecule, determining the presence of the fastidious microorganism in the sample.

[0046] In one embodiment, a method for detecting a genus or a species of a fastidious microorganism in a subject sample may include detecting two or more macromolecules associated with the microorganism of interest. Accordingly, a method for detecting a genus or a species of a fastidious microorganism in a sample may include: (i) obtaining a sample from a subject, such as a subject suspected of having an infection from a fastidious microorganism of interest; (ii) contacting two or more macromolecules in the sample with two or more agents forWSGR Docket No. 68706-703.601detecting a genus or a species of the microorganism, wherein the two or more macromolecules and the two or more agents are selected to increase a sensitivity or a specificity of detecting the presence of the microorganism in the sample; and (iii) based on the detection of the two or more macromolecules, determining the presence of the fastidious microorganism in the sample. In some embodiments, a sample may be collected from a mammalian subject suspected of having an infection from a fastidious microorganism of interest. In one embodiment, the mammalian subject is a human. In some embodiments, a sample may be collected from a subject that is not a mammal. In some embodiments, a sample may be collected from a subject that is a host reservoir for a fastidious microorganism of interest. In some embodiments, a sample may be collected from a subject that is not a host for a fastidious microorganism of interest. In some embodiments, the two or more macromolecules comprise a polypeptide associated with the fastidious microorganism. In some embodiments, the macromolecule comprises a polypeptide associated with Borrelia, wherein the polypeptide of Borrelia comprises outer surface protein A (OspA), OspB, OspC, variable lipoprotein surface-exposed protein (VlsE), oligopeptide ABC transporter I (OppAI), OppAII, OppAIII, OppAIV, OppAV, or a combination thereof. In some embodiments, the macromolecule comprises a polypeptide associated with Babesia. In some embodiments, the macromolecule comprises a polypeptide associated with Bartonella. In some embodiments, the macromolecule comprises a polypeptide associated with an additional pathogen other than Borrelia, Babesia, and Bartonella. In some embodiments, the two or more macromolecules comprise a polynucleotide associated with the fastidious microorganism. In some embodiments, the macromolecule comprises a polynucleotide associated with Babesia. In some embodiments, the macromolecule comprises a polynucleotide associated with Bartonella. In some embodiments, the macromolecule comprises a polynucleotide associated with an additional pathogen other than Borrelia, Babesia, and Bartonella. In some embodiments, the two or more macromolecules comprise at least one polypeptide and at least one polynucleotide associated with the fastidious microorganism. In some embodiments, the two or more contacting agents comprise a polypeptide agent, wherein the polypeptide agent binds to the two or more macromolecules associated with the microorganism. In some embodiments, the two or more contacting agents comprise a polynucleotide agent, wherein the polynucleotide agent binds to the two or more macromolecules associated with the microorganism. In some embodiments, the two or more contacting agents comprise at least one polypeptide agent and at least one polynucleotide agent that bind to the two or more macromolecules associated with the microorganism.WSGR Docket No. 68706-703.601

[0047] The disclosure provides methods for detecting a genus or a species of a microorganism in a subject sample, wherein the two or more macromolecules and the two or more agents are selected to provide an increased sensitivity or specificity for detecting the presence of the microorganism in the sample compared to a second sensitivity or specificity of one agent contacting one class of macromolecule. In various embodiments, the sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity may be increased by 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500%. In some embodiments, the sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity may be increased by about 3%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%In some embodiments, the sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity may be increased by at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In some embodiments, the sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity may be increased by at most 3%, at most 5%, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 200%, at most 300%, at most 400%, or at most 500%. In various embodiments, the specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second specificity may be increased by 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500%. In some embodiments, the specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second specificity may be increased by about 3%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%. In some embodiments, the specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second specificity may be increased by at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In some embodiments, the specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second specificity may be increased byWSGR Docket No. 68706-703.601at most 3%, at most 5%, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 200%, at most 300%, at most 400%, or at most 500%. In some embodiments, the methods for detecting a genus or a species of a fastidious microorganism of interest described herein may further include culturing the sample in a culture medium selected for enriching the fastidious microorganism in the sample. In some embodiments, the methods for detecting a genus or a species of a fastidious microorganism of interest described herein may further include using a trapping agent to capture the fastidious microorganism in the sample or a cultured sample. In some embodiments, the trapping agent may be an antibody or antigen binding fragment thereof conjugated to a solid surface. In some embodiments, the trapping agent may be a chaotropic agent. The methods for detecting a genus or a species of a microorganism in a subject sample described herein may be used to detect the presence of the fastidious microorganism in the subject at an earlier stage of infection compared to detecting the genus or the species of the fastidious microorganism by one agent contacting one class of macromolecule. In some embodiments, the methods for detecting a genus or a species of a fastidious microorganism in a subject sample described herein may be used to detect the presence of at least one additional fastidious microorganism or other pathogen in the sample. The other pathogen may, for example, comprise a pathogen from a genus that is different from the genus of the fastidious microorganism. The other pathogen may, for example, comprise a species that is different from the species of the fastidious microorganism.

[0048] In some embodiments, a method for detecting the presence of two or more microorganisms in a sample may combine detecting one or more polynucleotide targets associated with one or more microorganisms of interest with detecting one or more polypeptide targets associated with one or more microorganisms of interest. In some embodiments, a method for detecting a genus or a species of a microorganism in a subject sample may further include treating the subject based on the genus or the species of the microorganism(s) (e.g., the fastidious microorganism or additional pathogen) in the sample.

[0049] In some embodiments, the disclosure provides a system for detecting a genus or a species of a fastidious microorganism of interest, the system comprising two or more agents for contacting two or more macromolecules associated with the fastidious microorganism, wherein: (i) the two or more macromolecules are a different class of macromolecule; (ii) the two or more agents contacting the two or more macromolecules detect a genus or a species of a fastidious microorganism in a sample obtained from a subject; and (iii) contacting the two or more macromolecules with the two or more agents increases a sensitivity or a specificity of detectingWSGR Docket No. 68706-703.601the genus or the species of the fastidious microorganism compared to a second sensitivity or specificity of one agent contacting one class of macromolecule. In some embodiments, the two or more agents comprise a polypeptide agent, wherein the polypeptide agent binds to the two or more macromolecules associated with the microorganism. A polypeptide agent may, for example, be an antibody or an antigen binding fragment thereof. Binding of the polypeptide agent to the two or more macromolecules may, for example, be detected by western blotting, mass spectrometry, immunoassay (e.g., ELISA), or a combination thereof. In some embodiments, the immunoassay comprises detecting the polypeptide agent binding to at least one polypeptide in suspension. In some embodiments, a system comprises two or more polypeptide agents for contacting a polypeptide associated with Borrelia, wherein the polypeptide of Borrelia comprises outer surface protein A (OspA), OspB, OspC, variable lipoprotein surface-exposed protein (VlsE), oligopeptide ABC transporter I (OppAI), OppAII, OppAIII, OppAIV, OppAV, or a combination thereof. In some embodiments, a system comprises two or more polypeptide agents for contacting a polypeptide associated with Babesia. In some embodiments, a system comprises two or more polypeptide agents for contacting a polypeptide associated with Bartonella. In some embodiments, a system comprises two or more polypeptide agents for contacting a polypeptide associated with an additional pathogen other than Borrelia, Babesia, and Bartonella. In some embodiments, the two or more agents comprise a polynucleotide agent, wherein the polynucleotide agent binds to two or more polynucleotides associated with the microorganism. A polynucleotide agent may, for example, be an amplification primer, wherein the primer binds to the two or more polynucleotide targets. A polynucleotide agent may, for example, be an oligonucleotide probe, wherein the oligonucleotide probe binds to the two or more polynucleotide targets or an amplification product of the two or more polynucleotide targets. In some embodiments, a system comprises two or more polynucleotide agents for contacting a polynucleotide associated with Borrelia. In some embodiments, a system comprises two or more polynucleotide agents for contacting a polynucleotide associated with Babesia. In some embodiments, a system comprises two or more polynucleotide agents for contacting a polynucleotide associated with Bartonella. In some embodiments, a system comprises two or more polynucleotide agents for contacting a polynucleotide associated with an additional pathogen other than Borrelia, Babesia, and Bartonella. In some embodiments, a system comprises two or more polypeptide agents and two or more polynucleotide agents for contacting a polypeptide or a polynucleotide associated with Borrelia, Babesia, and Bartonella. In one embodiment, a system comprises polynucleotide agents for detection of Bartonella and Babesia, and a polypeptide agent for detection ofWSGR Docket No. 68706-703.601Borrelia. In some embodiments, the system for detecting a genus or a species of a microorganism in a subject sample may be used to detect the presence of at least one additional fastidious microorganism or other pathogen in the sample. The additional pathogen may, for example, comprise a pathogen from a genus that is different from the genus of the fastidious microorganism. The additional pathogen may, for example, comprise a species that is different from the species of the fastidious microorganism. In some embodiments, a system for detecting a genus or a species of a fastidious microorganism of interest may further include a culturing media for enriching the fastidious microorganism.

[0050] In some embodiments, a system for detecting a genus or a species of a fastidious microorganism of interest may further include a trapping agent for capturing the fastidious microorganism. In some embodiments, the trapping agent comprises an antibody or antigen binding fragment thereof conjugated to a solid surface. In some embodiments, the trapping agent comprises a chaotropic agent.

[0051] FIG. 1 is a flow diagram of an example of an assay workflow 100 for detecting a genus or a species of a fastidious microorganism in a sample. Workflow 100 may include, but is not limited to, the following steps. At a step 110, a sample from a subject suspected of having an infection from a fastidious microorganism of interest is collected. The sample may, for example, be collected from a bodily fluid (including tissue fluids) and / or tissue that may correlate with a suspected stage of infection by the microorganism of interest. At a step 115, the subject sample is enriched for the fastidious microorganism of interest to yield an enriched sample for detection the microorganism. In some embodiments, enriching the sample for the fastidious microorganism may include culturing the sample in a culture medium selected to enhance the growth of the fastidious microorganism. In some embodiments, enriching the sample for the fastidious microorganism may include capturing the fastidious microorganism by contacting the microorganism with a trapping agent. The trapping agent may, for example, be an antibody or an antigen binding fragment thereof. In some cases, the trapping agent may, for example, be conjugated to a solid surface. In another example, the trapping agent may be a chaotropic agent. At a step 120, two or more macromolecules of the fastidious microorganism are contacted with two or more agents for detecting a genus or a species of the microorganism in the enriched sample. In some embodiments, the two or more macromolecules comprise a polypeptide associated with the fastidious microorganism. In some embodiments, the two or more macromolecules comprise a polynucleotide associated with the fastidious microorganism. In some embodiments, the two or more macromolecules comprise at least one polypeptide and at least one polynucleotide associated with the fastidious microorganism. In some embodiments,WSGR Docket No. 68706-703.601the two or more agents contacting the two or more macromolecules comprise a polypeptide agent. The polypeptide agent may, for example, be an antibody or an antigen binding fragment thereof that binds to the two or more macromolecules. In some embodiments, the two or more agents contacting the two or more macromolecules comprise a polynucleotide agent. The polynucleotide agent may, for example, be an amplification primer or an oligonucleotide probe for binding to the two or more macromolecules or an amplification product of the two or more macromolecules. At a step 125, the binding of the two or more binding agents to the two or more targeted macromolecules is detected. In one example, binding of the two or more polypeptide agents to the two or more macromolecules may be detected in an immunoassay, (e.g., ELISA). In one example, binding of the two or more polynucleotide agents to the two or more macromolecules may be detected in a PCR amplification reaction (e.g., a digital PCR reaction). At a step 130, the presence of the fastidious microorganism is determined based on the detection of the two or more macromolecules associated with the microorganism of interest.

[0052] In various embodiments, a method for detecting a genus or a species of a fastidious microorganism may include selecting a sample type, wherein the sample type is selected to provide an increased sensitivity or specificity of detecting the microorganism compared to a second sensitivity or a second specificity of detecting the fastidious microorganism in a second sample type. In some embodiments, two or more microorganisms may be detected in a selected sample type. In one embodiment, the sample may be a transcellular fluid sample. A method for detecting a genus or a species of a fastidious microorganism(s) in a transcellular fluid sample may include: (i) obtaining a transcellular fluid sample (e.g., a synovial fluid sample) from a subject, such as a subject suspected of having an infection from a fastidious microorganism of interest; (ii) contacting two or more macromolecules in the transcellular fluid sample with two or more agents for detecting a genus or a species of the microorganism(s), wherein the two or more macromolecules and the two or more agents are selected to increase a sensitivity or a specificity of detecting the presence of the microorganism in the transcellular fluid sample compared to a second sensitivity or a second specificity of detecting the genus or the species of the fastidious microorganism in an interstitial fluid sample; and (iii) based on the detection of the two or more macromolecules, determining the presence of the fastidious microorganism in the transcellular fluid sample. In one embodiment, the transcellular fluid comprises synovial fluid and the interstitial fluid sample comprises blood, serum, plasma, or a combination thereof. In one embodiment, detecting the genus or species of a fastidious microorganism includes detecting a genus or a species of Borrelia and Bartonella in the transcellular fluid sample. For example, Borrelia may be detected in the sample by contacting a polypeptide associated with theWSGR Docket No. 68706-703.601microorganism with a polypeptide agent (e.g., an antibody or antigen binding fragment thereof) and Bartonella may be detected in the sample by contacting a polypeptide associated with the microorganism with a polypeptide agent (e.g., an antibody or antigen binding fragment thereof). The methods and systems for detecting a microorganism disclosed herein provide increased sensitivity in detecting and quantifying fastidious, low-abundance microorganisms (e.g., vector-borne pathogens) in a subject sample. In some embodiments, the increased sensitivity in detecting and quantifying microorganisms in a subject sample may facilitate an earlier diagnosis of a microbial infection in a patient. Early identification of a microbial infection in a patient may be used to improve treatment outcome. The methods and systems for detecting a vector-borne pathogen disclosed herein may be used to inform treatment strategies for a patient diagnosed with a microbial infection. The methods and systems for detecting a microorganism disclosed herein may be used to monitor the efficacy of a therapy (e.g., a drug therapy) for a patient being treated for a microbial infection. The methods for detecting a microorganism of interest (e.g., a vector-borne pathogen) described herein may, for example, be used as a “discovery tool” to develop targeted diagnostic assays with enhanced sensitivity for detection of a pathogen. For example, the methods described herein may be used to identify different biomarkers (target macromolecules) that are associated with early, middle, and late stages of a microbial infection in a subject.Macromolecule Target

[0053] The disclosure provides methods for detecting a genus or a species of a fastidious microorganism(s) in a sample. In various embodiments, the methods generally include detecting two or more macromolecules associated with a microorganism that can be used to detect the presence of the microorganism(s). A macromolecule target may, for example, be a polypeptide or a polynucleotide.Polypeptide target

[0054] In various embodiments, a fastidious microorganism of interest may be detected by detection of a polypeptide associated with the microorganism. In some embodiments, the polypeptide may be a polypeptide expressed on the outer surface of a microorganism of interest. In some embodiments, the polypeptide may be a polypeptide secreted by the fastidious microorganism of interest. In some embodiments, the polypeptide may be a polypeptide associated with a certain stage of a microorganism’s infectious cycle in a host. In some embodiments, the polypeptide may be an unbound polypeptide released after cell lysis. In some embodiments, the polypeptide may be a protein expressed by a microorganism to evade a host’s immune response. In some embodiments, the polypeptide may be a polypeptide expressed by aWSGR Docket No. 68706-703.601microorganism to adapt to a particular environment in a host. In some embodiments, the macromolecule targets comprise a polypeptide of Borrelia, wherein the polypeptide of Borrelia comprises outer surface protein A (OspA), OspB, OspC, variable lipoprotein surface-exposed protein (VlsE), oligopeptide ABC transporter I (OppAI), OppAII, OppAIII, OppAIV, OppAV, or a combination thereof. In some embodiments, the macromolecule targets comprise a polypeptide associated with Babesia. In some embodiments, the macromolecule targets comprise a polypeptide associated with Bartonella. In some embodiments, the macromolecule targets comprise a polypeptide associated with an additional pathogen other than Borrelia, Babesia, and Bartonella.Polynucleotide target

[0055] In various embodiments, polynucleotides associated with a fastidious microorganism of interest may be used to detect the presence of the microorganism in a subject sample. In some embodiments, the polynucleotide comprises a DNA of the fastidious microorganism. In some embodiments, the polynucleotide comprises a RNA of the fastidious microorganism. The RNA may, for example, be a ribosomal RNA (rRNA) or genomic ribosomal DNA (rDNA). The rRNA may, for example, be 18S rRNA, 5.8S rRNA, 28 S rRNA, or a combination thereof.Detection Modalities

[0056] The disclosure provides methods for detecting a genus or a species of a fastidious microorganism in a sample. In various embodiments, the methods generally include contacting two or more macromolecules in a subject sample with two or more agents, wherein the two or more agents contacting the two or more macromolecules detects a genus or a species of a microorganism(s) of interest. A contacting agent may, for example, be a polypeptide or a polynucleotide.Polypeptide Agent

[0057] In various embodiments, a contacting agent may be a polypeptide agent. In some embodiments, the polypeptide agent may be an antibody or an antigen binding fragment thereof that binds to a macromolecule target of interest. A variety of antibody-antigen detection techniques are known in the art. Examples of detection modalities that may be used in the methods disclosed herein include, but are not limited to, immunoassay, western blot assay, mass spectrometry (MS) analysis, Luminex analyte assay, and digital PCR with DNA-tagged targetspecific antibodies (e.g., protein interaction coupling (PICO) technology). In some embodiments, the detection modality comprises an immunoassay. In some embodiments, the immunoassay may be an enzyme-linked immunosorbent assay (ELISA). In some embodiments,WSGR Docket No. 68706-703.601the immunoassay comprises detecting the polypeptide agent binding to at least one polypeptide in suspension. In various embodiments, a method for detecting the presence of a fastidious microorganism in a sample may include: (i) capturing a target polypeptide on a substrate surface, wherein the substrate surface is functionalized with an antibody (or an antigen binding fragment thereof) specific for a macromolecule of interest; and (ii) detecting the captured polypeptide target.Polynucleotide Agent

[0058] In various embodiments, a contacting agent may be a polynucleotide agent, wherein the polynucleotide agent binds to a macromolecule associated with the microorganism. A polynucleotide agent may, for example, be an amplification primer, wherein the primer binds to the macromolecule. A polynucleotide agent may, for example, be an oligonucleotide probe, wherein the oligonucleotide probe binds to a macromolecule or an amplification product of the macromolecule. In some embodiments, a polynucleotide agent may be an amplification primer or an oligonucleotide probe that binds a polynucleotide associated with Borrelia. In some embodiments, a polynucleotide agent may be an amplification primer or an oligonucleotide probe that binds a polynucleotide associated with Babesia. In some embodiments, a polynucleotide agent may be an amplification primer or an oligonucleotide probe that binds a polynucleotide associated with Bartonella. In some embodiments, a polynucleotide agent may be an amplification primer or an oligonucleotide probe that binds a polynucleotide associated with an additional pathogen other than Borrelia, Babesia, and Bartonella.

[0059] A variety of methods for amplifying nucleic acids in a sample and detecting the amplification products are well-known in the art. Examples of nucleic acid amplification techniques that may be used for detection of a microorganism of interest include, but are not limited to, polymerase chain reaction (e.g., conventional PCR (cPCR)), quantitative PCR (qPCR), real-time PCR, digital PCR (dPCR), droplet digital PCR (ddPCR), reverse-transcription (RT) PCR (RT-PCR), RT-dPCR, RT-ddPCR, RT-qPCR or a combination thereof. Examples of techniques that may be used to detect an amplification product include, but are not limited to, hybridization of a fluorescently labeled oligonucleotide probe specific for the amplified sequence and detection of a fluorescence signal produced by the hybridization event; and next generation sequencing (NGS). In one example, a method for detecting the presence of a microorganism in a sample may include: (i) performing a nucleic acid amplification on the sample, or on nucleic acids extracted from the sample to produce an amplification product that is indicative of the microorganism; (ii) and detecting the amplification product. In some embodiments, a method for detecting the presence of a microorganism (e.g., a vector-borneWSGR Docket No. 68706-703.601pathogen) in a sample may include amplifying a target polynucleotide using droplet digital PCR. Methods for detecting and / or identifying a fastidious microorganism in a sample using droplet digital PCR are described in U.S. Patent Application No. 2023 / 0123800, which is incorporated herein by reference in its entirety. For example, a method of detecting and / or identifying a fastidious microorganism in a sample may include: (i) performing a nucleic acid amplification on the sample for a polynucleotide sequence that can identify the presence of the microorganism, wherein performing the nucleic acid amplification includes distributing nucleic acids from the sample into a plurality of partitions; (ii) performing nucleic acid amplification on the plurality of partitions and; (iii) detecting whether a nucleic acid amplification product is produced in the amplification reaction, wherein presence of the amplification product is indicative of the microorganism being present in the sample.EnrichmentCulture Enrichment

[0060] In some embodiments, methods of detecting and / or identifying fastidious microorganisms may also include “enrichment” for the fastidious microorganism prior to performing the methods for detecting and / or identifying the microorganism. For example, a sample derived from a subject that may be suspected of having an infection from a particular fastidious microorganism may be cultured in an enrichment medium as described in in U.S. Pat. No. 7,115,385, which is incorporated herein by reference in its entirety. The cell culture medium may, for example, include free amino acids including glutamine at a concentration of about 300 to about 6000 mg / L; organic acid as a carbon source; hemin; yeast extract; nucleotides; vitamins; acyl homoserine lactone; and sodium bicarbonate, and wherein the culture medium is protein free. In some embodiments, the cell culture medium may be Bartonella a proteobacteria growth medium (BAPGM). In other embodiments, the cell culture media for enrichment of the fastidious microorganism may include, but is not limited to, an insect and / or a mammalian cell culture media. In some embodiments, the cell culture media, for example, BAPGM, may further include any of one, any combination of, and / or all of the following supplements: P-Nicotinamide adenine dinucleotide (NAD); P-Nicotinamide adenine dinucleotide phosphate (NADPH); sodium pyruvate; adenosine 5 '-triphosphate (ATP); essential amino acids; yeast extract; sodium bicarbonate; saponin; sodium polyanethol sulfonate (SPS); and / or serum (such as, but not limited to, fetal, horse, human, etc.), or any other supplement that would be appreciated by one of skill in the art.WSGR Docket No. 68706-703.601Microorganism

[0061] The disclosure provides methods and systems for detecting a genus or a species of one or more microorganisms of interest in a subject sample. In various embodiments, the microorganism may be a fastidious microorganism. In some embodiments, a genus of the fastidious microorganism comprises Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof. In some embodiments, the species of the fastidious microorganism comprises Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, Borrelia mayonii, Borrelia miyamotoi, Bartonella vinsonii berkhoffn, Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum, Rickettsia ricketsii, Rickettsia conorii, Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.Sample Type

[0062] A method for detecting a genus or a species of a fastidious microorganism may include selecting a sample type, wherein the sample type is selected to provide an increased sensitivity or specificity of detecting the microorganism compared to a second sensitivity or a second specificity of detecting the fastidious microorganism in a second sample type. In various embodiments, the sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity may be increased by 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500%. In some embodiments, the sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity may be increased by about 3%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%. In some embodiments, the sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity may be increased by at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In some embodiments, the sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity may be increased by at most 3%, at most 5%, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 200%, at most 300%, at most 400%, or at most 500%. In various embodiments, the specificity of detecting the genus or the species of the fastidiousWSGR Docket No. 68706-703.601microorganism in the sample compared to the second specificity is increased by 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500%. In some embodiments, the specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second specificity is increased by about 3%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%. In some embodiments, the specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second specificity is increased by at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In some embodiments, the specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second specificity is increased by at most 3%, at most 5%, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 150%, at most 200%, at most 300%, at most 400%, or at most 500%.

[0063] In some embodiments, a sample type may be selected to provide for the detection of two or more microorganisms in a single assay. In some embodiments, the sample type may be a transcellular fluid sample. Exemplary sample types include, but are not limited to, blood, plasma, serum, urine, cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions. In some embodiments, the sample type may be an interstitial fluid sample. Exemplary sample types include, but are not limited to, blood, serum, plasma, or a combination thereof. In one embodiment, the transcellular fluid sample may be a synovial fluid sample. In one embodiment, a synovial fluid sample may be selected to provide for the detection of Borrelia and Bartonella in a single assay. In some embodiments, a sample type may be selected to detect a genus or a species of a fastidious microorganism at an earlier stage of infection by the fastidious microorganism.Treatment

[0064] A method for detecting a genus or a species of a microorganism in a subject sample may further include treating the subject based on the genus or the species of the fastidious microorganism detected in the sample. The disclosure provides methods for selecting a therapy for a patient suspected of having an infection from a fastidious microorganism(s) of interest. In one example, a method for selecting a therapy may include: (a) collecting a sample (e.g., aWSGR Docket No. 68706-703.601transcellular fluid sample) from a patient suspected of having an infection from a fastidious microorganism; (b) performing a detection assay to determine a genus or a species of the microorganism(s); and (c) based on the genus or species of the microorganism(s), selecting an effective drug therapy to administer to the patient. In some embodiments, a subject may be treated based on the life cycle of the fastidious microorganism detected in the sample. In some embodiments, a subject may be treated based on the morphology of the fastidious microorganism detected in the sample. In some embodiments, a subject may be treated based on the species of the fastidious microorganism detected in the sample. In some embodiments, a subject may be treated for inflammation associated with the fastidious microorganism detected in the sample. In some embodiments, a subject may be treated for a disease or a condition associated with the fastidious microorganism detected in the sample. Examples of diseases or conditions that may be associated with an infection with a fastidious microorganism include, but are not limited to, osteoarthritis (OA), Sjogren’s syndrome (SS), rheumatoid arthritis (RA), chronic fatigue syndrome (CFS), gout, calcium pyrophosphate deposition (CPPD), rotator cuff disease (RC), neuropathy, or a combination thereof. The disclosure provides methods for monitoring the efficacy of a drug therapy administered to a patient to treat a microbial infection. Monitoring the efficacy of a therapy may, for example, include: (i) determining the quantity (level) of one or more macromolecules associated with the genus or species of the fastidious microorganism over the course of a treatment regimen; and (ii) determining the success or failure of the treatment regimen based on the quantity of the one or more macromolecule targets.Kit

[0065] The disclosure provides a kit for detecting a genus or a species of a fastidious microorganism (e.g., a pathogen). In some embodiments, the kit comprises two or more agents for contacting two or more macromolecules associated with the fastidious microorganism, wherein the two or more macromolecules are different classes of macromolecule. In some embodiments, the present disclosure also provides a kit comprising two or more agents configured to bind to two or more macromolecules derived from the fastidious microorganism and of different macromolecular classes, wherein binding of the two or more agents with the two or more macromolecules is indicative of the presence of the fastidious microorganism in a subject; and wherein the detection of the fastidious microorganism with two or more macromolecules of the different macromolecular classes provides an increased sensitivity or specificity relative to detection of the fastidious microorganism with one class of macromolecule. In some embodiments, the two or more macromolecules are different classes of macromolecule. In some embodiments, the two or more agents comprise an antibody (or anWSGR Docket No. 68706-703.601antigen binding fragment thereof) for binding to the two or more macromolecules. In some embodiments, the two or more agents comprise an antibody (or an antigen binding fragment thereof) for binding to the two or more macromolecules. In some embodiments, the two or more agents comprise an amplification primer, or an oligonucleotide probe for binding to the two or more macromolecules or an amplification product of the two or more macromolecules. In some embodiments, the two or more agents comprise at least on polypeptide agent (i.e., antibody or antigen binding fragment thereof) and at least on polynucleotide agent (i.e., amplification primer or oligonucleotide probe). In some embodiments, the kit comprises a culturing media for enriching the fastidious microorganism. In some embodiments, the kit comprises a trapping agent for capturing the fastidious microorganism. In some embodiments, the trapping agent comprises an antibody or antigen binding fragment thereof conjugated to a solid surface. In some embodiments, the trapping agent comprises a chaotropic agent. In some embodiments, the kit comprises an instruction manual for detecting the binding of the two or more agents to the two or more macromolecules or amplification product thereof. In some embodiments, the kit may be designed to detect a genus of Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof. In some embodiments, the kit may be designed to detect Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, Borrelia mayonii, Borrelia miyamotoi, Bartonella vinsonii berkhoffn, Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum, Rickettsia ricketsii, Rickettsia conorii, Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.

[0066] Use of absolute or sequential terms, for example, “will,” “will not,” “shall,” “shall not,” “must,” “must not,” “first,” “initially,” “next,” “subsequently,” “before,” “after,” “lastly,” and “finally,” are not meant to limit scope of the present embodiments disclosed herein but as exemplary.

[0067] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0068] As used herein, the phrases “at least one”, “one or more”, and “and / or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, andWSGR Docket No. 68706-703.601C”, “one or more of A, B, or C” and “A, B, and / or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0069] As used herein, “or” may refer to “and”, “or,” or “and / or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.

[0070] Any systems, methods, software, and platforms described herein are modular.Accordingly, terms such as “first” and “second” do not necessarily imply priority, order of importance, or order of acts.

[0071] The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and the number or numerical range may vary from, for example, from 1% to 15% of the stated number or numerical range. In examples, the term “about” refers to ±10% of a stated number or value.

[0072] The terms “increased”, “increasing”, or “increase” are used herein to generally mean an increase by a statically significant amount. In some aspects, the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, standard, or control. Other examples of “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.

[0073] The terms “decreased”, “decreasing”, or “decrease” are used herein generally to mean a decrease by a statistically significant amount. In some aspects, “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.WSGR Docket No. 68706-703.601

[0074] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.EXAMPLES

[0075] The following illustrative examples are representative of embodiments of the stimulation, systems, and methods described herein and are not meant to be limiting in any way.Example 1. Detection of two or more macromolecules of a fastidious microorganism

[0076] Detecting two or more macromolecules of a fastidious microorganism can increase the specificity or sensitivity of detecting the presence of the fastidious microorganism in a sample obtained from a subject compared to if the detection of the fastidious microorganism based on detecting only one macromolecule of the fastidious microorganism. The two or more macromolecules can be from different two different classes of macromolecules (e.g., one polypeptide and one polynucleotide).

[0077] A subject is suspected of being infected by Borrelia. A sample (e.g., blood sample) can be obtained from the subject. The sample can be contacted directly with two or more agents for binding to the two or more macromolecules in the sample. The binding of the two or more agents to the two or more macromolecules can be detected by immunoassay or PCR. The sample, prior to contacting with the two or more agents, can be cultured in a culture media for enriching the Borrelia. Alternatively, a trapping agent (e.g., a bead conjugated to an antibody for binding to a polypeptide of the Borrelia or a chaotropic agent for binding to a polynucleotide of the Borrelia) can be contacted with the sample to trap the Borrelia. By detecting at least one polypeptide and at least one polynucleotide of the Borrelia, a specificity or a sensitivity ofWSGR Docket No. 68706-703.601detecting the presence of the Borrelia can be increased compared to detecting only one polypeptide or polynucleotide of the Borrelia. By detecting the Borrelia in the subject with increased specificity or sensitivity, the subject can be treated for the Borrelia infection at an earlier stage of the infection.Example 2. Antibodies to Borrelia burgdorferi and Bartonella species in serum and synovial fluid from people with rheumatic diseases

[0078] A method for detecting a genus or a species of a fastidious microorganism may include selecting a sample type, wherein the sample type is selected to provide an increased sensitivity or specificity of detecting the microorganism compared to a second sensitivity or a second specificity of detecting the fastidious microorganism in a second sample type.

[0079] Vector-borne infections may underlie some rheumatic diseases, particularly in people with joint effusions. We designed a study aimed to compare serum and synovial fluid antibodies toB. burgdorferi and Bartonella spp. in patients with rheumatic diseases as described in Kim, L. et. al, Clinical Microbiology 2024: 12(4), which is incorporated herein by reference in its entirety. This was an observational, cross-sectional study that was performed to determine exposure to B. burgdorferi, B. henselae, and B. quintana in people presenting for rheumatic conditions to the Institute for Specialized Medicine, San Diego, CA.Participants and study size

[0080] Patients who were having blood drawn and arthrocentesis performed as a component of their diagnostic procedures and had ultrasound-confirmed synovial effusions in large-sized or medium-sized joints, with calculated synovial effusion volume equal to or above 2 cubic centimeters per joint were asked to participate in this study. Participants were included if they provided a signed voluntary informed consent and there was an adequate amount of their serum and synovial fluid specimens remaining for research testing (100 pL or more for each sample type) after routine diagnostic tests were performed. This investigation was a clinical study using a convenience sample selected from the first approximately 100 consecutive patients who fit enrollment criteria.

[0081] The study size was determined by the caseload at the study site, with enrollment conducted over a defined time period (10-26-2017 through 01-20-2022). For the 10 most common diagnoses (assuming that approximately half of the participants had the diagnosis and half did not), with 110 participants, there was an 80% power at alpha = 0.05 to detect a difference in pathogen exposure if approximately 10% of participants without the diagnosis had exposure compared to 35% of participants with the diagnosis. For less common diagnoses (assuming that 15% of participants had the diagnosis), with the same parameters, there was 80%WSGR Docket No. 68706-703.601power to detect a statistically significant difference if approximately 45% of participants with the diagnosis had exposure. People were classified by whether they had or did not have each of these specific diagnoses. Each included participant had at least one, if not more, diagnosis.Data sources and variablesSynovial fluid and serum sample collection

[0082] Synovial fluid was obtained via arthrocentesis performed under direct ultrasound guidance. Topical anesthesia was performed using ethyl chloride spray, then local anesthesia was administered by infiltrating soft tissues with 1% lidocaine. Blood samples were collected using a standard venipuncture technique. The collected samples of serum and synovial fluid were processed in accordance with the standard protocol and transported to Galaxy Laboratories, Research Triangle Park, NC, USA for B. burgdorferi and Bartonella spp. testing.B. burgdorferi serology

[0083] B. burgdorferi exposure was determined using Clinical Laboratory Improvement Amendments (CLIA) validated, Commission on Office Laboratory Accreditation (COLA) accredited first-tier IgM and IgG ELISA and second-tier IgM and IgG Western blot testing, interpreted according to CDC criteria, as described in Engstrom SM, et. al, 1995, J Clin Microbiol 33:419-427 and Johnson BJ, et. al, 2011, CAB International, which are incorporated herein by reference in their entirety. Both the first-tier ELISA and second-tier Western blot assays were run against whole cell derivatives of Borrelia burgdorferi B31, as described in Yang L, et. al, 1992, Infect Immun 60:3033-3041; and Dressier F, et. al., 1993, J Infect Dis 167:392-400, which are incorporated herein by reference in their entirety. A serum or synovial fluid sample was considered B. burgdorferi seroreactive if the two-tier test (ELISA and western blot) result was positive for IgM, IgG, or both.Bartonella spp. serology

[0084] CLIA-validated, COLA-accredited B. henselae and B. quintana IgG immunofluorescence antibody assay (IF) serology was performed as previously in Hegarty BC, et.al, 2014, J Vet Intern Med 28:38-41 which are incorporated herein by reference in their entirety. Cell culture-grown B. henselae SA2 and B. quintana were used as antigens. The reported titers for B. henselae and B. quintana IgG IFA ranged from <1 :32 to >1 :256 and are based upon a serial twofold dilution series of each human serum and synovial fluid sample. To further distinguish reactive from non-reactive sera, the laboratory grades fluorescence from 1 + to 4 + reactivity to ensure that fluorescence decreases incrementally as the dilution of patient serum and synovial fluid increases. Samples positive at >1 :256 can be further tested to endpoint titer at the request of the ordering provider. For this study and to limit the potential forWSGR Docket No. 68706-703.601misinterpretation of non-specific binding, especially in a patient population with autoimmune disease, serum and synovial fluid samples were considered B. henselae or B. quintana seroreactive if the indirect fluorescent assay (IF A) resulted in an IgG titer >1 :256. Antibody titers of 1 :64 or 1 : 128 were considered indeterminate and titers of less than 1 :64 were considered non-seroreactive.

[0085] Synovial fluid samples containing visible particulate matter were briefly centrifuged to pellet the material, and the resulting particulate-free material was used in sample analysis. Longterm synovial fluid optimization experiments (conducted between October 2017 and December 2018) were performed by assessment of serial dilutions of synovial fluid samples in assay buffer (1:25, 1:50, 1:100, and 1:200 in PBS-T for ELISA, 1:25 and 1:50 in TBS-T for western blot). Based on the results obtained from the optimization experiments, the 1:25 dilution was selected for IgM assays and 1:50 for IgG assays.Clinical diagnoses

[0086] Clinical diagnoses were determined by the attending physician (AS). The diagnoses of interest used for the secondary aim were determined by evaluating the most common diagnoses in the samples. Some participants were diagnosed with more than one condition. If a participant had multiple common diagnoses, both diagnoses were included in the analysis. Some diagnoses of interest were combined into a more general category as follows: diagnoses of uterine cancer, prostate cancer, and thyroid carcinoma were categorized as “cancer”; any diagnoses that included specific neuropathy such as idiopathic peripheral neuropathy, recurrent median neuropathy, and chronic inflammatory demyelinating polyneuropathy (CIDP) were categorized as “neuropathy”; and diagnoses of both rheumatoid arthritis and juvenile rheumatoid arthritis were categorized as “rheumatoid arthritis.”Laboratory assays

[0087] ELISA and western blot methods detailed in Yang L, et. al, 1992 and Dressier F, et. al., 1993 and references cited therein were used for the development of the laboratory-developed assays utilized in the studies described herein. Galaxy Diagnostics ELISA and western blot assays are CLIA-validated for human serum (accuracy and sensitivity including comparison to standard of care CDC Lyme Reference Panel I samples (as described in Molins CR, et. al., J Clin Microbiol 2014, 52:3755-3762, which is incorporated herein by reference in its entirety.), inter- and intra-operator precision, and analytical specificity including cross-reactivity assessments) and have been commercially available since 2017. Per CLIA guidelines, these assays undergo CAP (College of American Pathologists)-administered proficiency testing twice per year where operators run blinded samples provided by CAP side-by-side with their regularWSGR Docket No. 68706-703.601clinical samples, which are propagated and prepared on slides in-house. The results are graded by CAP and provided to the laboratory. Assay operators also undergo yearly competency assessments, two components of which include running blinded samples and obtaining a >80% grade on a written technical assessment.Bias

[0088] Blood and synovial fluid specimens were drawn from a single physician’s practice in a single geographic location during the course of routine medical diagnosis and treatment, which may limit the generalizability of the results. Confounding effects of gender and age were accounted for in statistical analysis (see below).Statistical methods

[0089] All analysis was done using R v.4.2.2 as described in R Core Team, 2019. “R: a language and environment for statistical computing”, which is incorporated herein by reference in its entirety. Descriptive statistics were calculated for demographic variables, serology results, and diagnoses of interest. Two-by-two tables were created and chi-squared tests were used to compare the proportion of people considered seroreactive using serum compared to synovial fluid. Differences between B. burgdorferi and Bartonella spp. seroreactive and non-seroreactive people for each clinical diagnosis were calculated using chi-squared or Fisher’s exact tests.

[0090] For the two most common clinical diagnoses of interest, multivariate logistic regression was used to determine the association between the diagnosis and pathogen exposure, including age and gender as potentially confounding covariates. We created four models. To investigate associations with osteoarthritis, we created two models with the dependent variable of clinical diagnosis of osteoarthritis (vs no diagnosis of osteoarthritis): one included Bartonella spp. exposure as an explanatory variable and one included B. burgdorferi exposure as an explanatory variable. We also created two models with the dependent variable of clinical diagnosis of Sjogren’s Syndrome (vs no diagnosis of Sjogren’s Syndrome): one included Bartonella spp. exposure as an explanatory variable and one included B. burgdorferi exposure as an explanatory variable. For all models, the Bartonella spp. exposure variable was a binary variable, with a positive defined as IF A seroreactivity against either B. quintana or B. henselae on serum or joint fluid. The B. burgdorferi exposure variable was a binary variable, with a positive defined as a two-tier positive for either IgG or IgM on either joint fluid or serum. For all models, age and gender were included: age as a continuous numerical variable using reported age in years, and gender as a categorical variable using reported gender (male or female). Odds ratios (ORs) and 95% confidence intervals (95% Cis) were calculated. The ORs for associations between OA orWSGR Docket No. 68706-703.601Sjogren’s and pathogen exposure, estimated from the multivariate logistic regression models, are adjusted (aOR). All other ORs are based on univariate models and therefore not adjusted.

[0091] A / < value of <0.05 was used to define statistical significance throughout the study.Though multiple comparisons were made for certain outcomes (e.g., odds of OA diagnosis evaluated with two logistic regression models), this was a retrospective cross-sectional study with small sample size, and the authors felt that using a more conservative / < value would not be useful in this context. The conclusions drawn from this study are hypothesis generating, and in that respect, the authors prioritize associations that may be biologically plausible for future prospective studies.ResultsParticipant demographics, sampling, and diagnoses

[0092] The study included 110 unique participants. Of those, 83 participants were sampled once, 15 participants were sampled twice, 7 participants were sampled three times, and 5 participants were sampled more than three times throughout the study period. For analyses, only the first sample from each unique participant was included (subsequent samples were excluded); the included samples were obtained from 2017-10-26 through 2022-01-20.

[0093] FIG. 3A and 3B are a pair of plots (3 A) and (3B) showing the study demographics for age and gender of study participants, and joint sampled for each study participant, respectively. Referring now to plot (A), of the 110 participants, 79 participants identified as female (72%); 31 participants identified as male (28%); and the age of participants ranged from 10 to 90 years of age, with a median age of 71 years. Referring now to FIG. 3B, joint fluid was collected from 13 different joints, with the right knee being the most frequently sampled, followed by the left knee and then left knee popliteal cysts. The joint sample location was related to the presenting problem of the participants and was included to fully describe the sample population but was not further analyzed in this study.

[0094] The 10 most frequent diagnoses were considered diagnoses of interest: osteoarthritis, Sjogren’s Syndrome, rheumatoid arthritis, chronic fatigue syndrome, gout, calcium pyrophosphate deposition, yersiniosis, cancer, rotator cuff disease, and neuropathy. Some participants were diagnosed with more than one condition and all diagnoses were included in the analysis. However, diagnoses of multiple conditions were not controlled for in the multivariate analysis. In all, 10 participants had no diagnoses of interest; 33 participants had 1 diagnosis of interest; 43 participants had two diagnoses of interest; 19 participants had three diagnoses of interest; and 5 participants had 4 diagnoses of interest. No participants had more than four diagnoses of interest. There were also 59 other diagnoses listed for participants that wereWSGR Docket No. 68706-703.601categorized as “other,” and not listed as diagnoses of interest. Of the diagnoses of interests, osteoarthritis (OA) was the most common clinical diagnosis (67 / 110, 60.9%), followed by Sjogren’s Syndrome (42 / 110, 38.2%), rheumatoid arthritis (RA) (20 / 110, 18.2%), and chronic fatigue syndrome (CFS, 18 / 110, 16.4%); other diagnoses including gout, calcium pyrophosphate deposition, yersiniosis, cancer, neuropathy, and rotator cuff (RC) issues were each diagnosed in less than 16 participants (<15%).B. burgdorferi and Bartonella spp. seroreactivity in serum and synovial fluid

[0095] FIG. 4A and 4B are a pair of plots (4A) and (4B) showing serology results comparing serum to synovial fluid samples for B. burgdorferi and Bartonella spp., respectively. Plot (A), B. burgdorferi two-tier testing results, showing IgM and IgG; and plot (4B) Bartonella spp. IFA results, showing B. henselae and B. quintana. When considering serum or synovial fluid seroreactivity, 30 participants (27%) were B. burgdorferi two-tier seroreactive for IgM, IgG, or both (see plot (A)); 26 participants (24%) were Bartonella spp. seroreactive (see plot (4B)).

[0096] Referring now to plot (A), for B. burgdorferi, based on testing serum samples, there were 17 participants (15%) with evidence of B. burgdorferi exposure: 10 participants had IgM-positive, IgG-negative B. burgdorferi serology results, and 6 participants had IgM-negative, IgG-positive B. burgdorferi serology results. There was one participant with both B. burgdorferi IgM and IgG serum-positive results. Based on testing synovial fluid samples, there were 30 participants (27%) with evidence of B. burgdorferi exposure: 15 participants had IgM-positive, IgG-negative B. burgdorferi serology results, and 8 participants had IgM-negative, IgG-positive B. burgdorferi serology results. There were seven participants with both B. burgdorferi IgM-and IgG-positive synovial fluid results.

[0097] Referring now to plot (4B), for Bartonella spp., 21 participants were seroreactive to both Bartonella spp. (19%); all participants that were / / . quintana seroreactive were also B. henselae seroreactive. Only one participant was B. henselae seroreactive but not B. quintana seroreactive (four participants were B. henselae seroreactive and indeterminate for B. quintana).

[0098] A comparison of results for B. burgdorferi two-tier serology on serum and synovial fluid for B. burgdorferi IgM, B. burgdorferi IgG, and B. burgdorferi IgM and IgG combined is shown in Table 1. Based on the serology results of B. burgdorferi between serum and synovial fluid samples for IgM and IgG, 11 participants were seroreactive for B. burgdorferi IgM in both serum and synovial fluid; 6 participants were seroreactive to B. burgdorferi IgG in both serum and synovial fluid; 17 participants were seroreactive for B. burgdorferi IgM and IgG in both serum and synovial fluid. When comparing B. burgdorferi antibody detection in synovial fluid and serum, IgM and IgG were both detected more frequently in synovial fluid than in serumWSGR Docket No. 68706-703.601samples. For IgM, 11 more participants were positive on synovial fluid than on serum. For IgG, eight more participants were positive on synovial fluid than on serum. One participant tested positive for B. burgdorferi in serum but not in synovial fluid. Combining IgM and IgG results, 27% of participants were antibody positive using synovial fluid, compared to only 15% using serum (P = 0.048).Table 1. Comparison of results for B. burgdorferi two-tier serology on serum and synovial fluid for B. burgdorferi IgM, B. burgdorferi IgG, and B. burgdorferi IgM and IgG combined

[0099] A comparison of results for Bartonella spp. IFA serology between serum and fluid in B. henselae and B. quintana is shown in Table 2.

[0100] A comparison of results for Bartonella spp. IFA serology between B. henselae and B. quintana is shown in Table 3.

[0101] Referring now to Table 2 and Table 3, based on the serology results of Bartonella spp. between serum and synovial fluid samples, two participants were seroreactive for B. henselae in both serum and synovial fluid (see Table 2); five participants were seronegative toB. quintana in synovial fluid but seroreactive to it in serum (seeTable 2). When B. henselae and B. quintana IFA in serum and synovial fluid were compared, 21 participants were positive for both Bartonella spp. (see Table 3). When comparing Bartonella spp. antibody detection in synovial fluid and serum, antibodies to both B. henselae and B. quintana were detected more frequently in serum than in synovial fluid samples (see Table 2). For both B. henselae and B. quintana, there were only two synovial fluid samples that were positive; both participants also were positive on serum samples.WSGR Docket No. 68706-703.601Table 2. Comparison of results for Bartonella spp. IFA serology between serum and synovial fluid in B. henselae and B. quintana0aIndeterminate includes IFA titers 1 :64 or 1 : 128, positive includes IFA titers > 1 :256.Table 3. Comparison of results for Bartonella spp. IFA serology between B. henselae and B. quintancf>>aIndeterminate includes IFA titers 1 :64 or 1 : 128, positive includes IFA titers > 1 :256.

[0102] A comparison of co-exposure between Bartonella spp. IFA serology (serum and synovial fluid) and / f. burgdorferi two-tier serology (serum and synovial fluid) is shown in Table 4. Seroreactivity to both B. burgdorferi and Bartonella spp. together was uncommon: there were only nine participants (8%) seroreactive to both Bartonella spp. and / ?. burgdorferi. In comparison, there were 21 participants with positive B. burgdorferi titers (on serum and / or synovial fluid) but negative for Bartonella spp., and 17 participants were Bartonella spp. IFAWSGR Docket No. 68706-703.601seroreactive, but negative for B. burgdorferi. In all, 63 participants (57%) were seronegative for either pathogen.Table 4. Comparison of co-exposure between Bartonella spp. IFA serology (serum and synovial fluid) and B. burgdorferi two-tier serology (serum and synovial fluid)

[0103] An analysis of associations between age or gender and / ?. burgdorferi exposure (either IgM or IgG) is shown in Table 5. There were no statistically significant associations between age or gender and B. burgdorferi exposure (either IgM or IgG). There was no statistically significant association between age and Bartonella spp. exposure; however, Bartonella spp. exposure was more common in men (13 / 31, 42%) than women (13 / 79, 16%, P = 0.01).Table 5. Associations between age or gender and B. burgdorferi exposure (either IgM or IgG), and Bartonella spp. exposure, with C < 0.05 indicating a statistically significant differenceAssociation of B. burgdorferi and Bartonella spp. on diagnoses of interest

[0104] Multivariate models for two most common diagnoses of interest, osteoarthritis (OA) and Sjogrne’s syndrome are shown in Table 6. OA, the most common diagnosis of interest, was reported in 67 participants (61%). Based on the multivariate logistic regression model, the odds of an OA diagnosis increased with increasing age but were not significantly associated with gender. Controlling for age and gender, OA was not independently significantly associated with B. burgdorferi o Bartonella spp. seroreactivity. Sjogren’s syndrome, the second most commonWSGR Docket No. 68706-703.601diagnosis of interest, was reported in 42 participants (38%). Based on the multivariate logistic regression model, Sjogren’s syndrome was significantly less common in men compared to women but was not significantly associated with age. Controlling for age and gender, Sjogren’s syndrome was not independently significantly associated with B. burgdorferi or Bartonella spp. seroreactivity.Table 6. Multivariate models for two most common diagnoses of interest: osteoarthritis and Sjogren’s syndrome"<<"Bold indicates a statistically significant independent association, with C < 0.05 indicating a statistically significant difference

[0105] FIG. 5A and 5B are a pair of bar graphs (5 A) and (5B) showing the percentage of participants with each diagnosis of interest and exposure to B. burgdorferi or Bartonella spp., respectively. Solid bars indicate participants who did have the diagnosis, striped bars indicate participants without the diagnosis. Abbreviations: OA = osteoarthritis, SS = Sjogren’s syndrome, RA = rheumatoid arthritis, CFS = chronic fatigue syndrome, CPPD = calcium pyrophosphate deposition, RC = rotator cuff disease. A / < value of <0.05 has been used to indicate statistical significance between pathogens and diagnoses of interest. (5 A) B. burgdorferi exposure. Green indicates the proportion of participants with positive two-tier testing for IgM, IgG, or both (on serum and / or synovial fluid). (5B) Bartonella spp. exposure. Orange indicates the proportion of participants with high titer seroreactivity to B. henselae, B. quintana, or both on serum (with or without antibodies detected in synovial fluid).

[0106] FIG. 5A and 5B show the proportion of participants with and without antibodies to each pathogen for each diagnosis of interest. There were no significant univariate associationsWSGR Docket No. 68706-703.601between clinical diagnoses of interest and either B. burgdorferi o Bartonella spp. exposure (as defined by any one or more antibody test positive from either serum or synovial fluid).Study summary

[0107] In this study, we compared serology results on paired serum and synovial fluid, using ELISA and WB (CDC two-tier criteria) for the detection of antibodies against B. burgdorferi, and IFA for the detection of antibodies against B. henselae and B. quintana. For / i. burgdorferi, both IgM and IgG antibodies were significantly more commonly detected in synovial fluid than in serum (P = 0.048) (see Table 1). The opposite was found for Bartonella spp., with only two participants positive on synovial fluid — both of whom had high serum antibody titers. These results suggest that testing synovial fluid for B. burgdorferi antibodies may provide more sensitive detection than testing serum alone. By contrast, testing synovial fluid for antibodies against B. henselae and B. quintana is unlikely to provide diagnostically relevant information for patients.

[0108] Regarding OA and Sjogren’s syndrome, the study was adequately powered based on the power calculation outlined herein above and we were able to evaluate associations for these diagnoses while also controlling for age and gender as confounding demographic factors. In both cases, known associations with demographic factors were found: OA was associated with older age, and Sjogren’s syndrome was associated with the female gender. Even controlling for those factors, there was not a significant association between either of these diagnoses and Bartonella spp. or B. burgdorferi exposure in this study population. Based on our results, we did not find a statistically significant association between any one diagnosis and either pathogen. We were only powered to investigate OA and Sjogren’s syndrome due to the rarity of the other diagnoses of interest.

[0109] Similar proportions of people in this study population were B. burgdorferi and Bartonella spp. seroreactive (27% and 24%, respectively). While the number of named or proposed Bartonella species now approaches 45, with some species, such asB. henselae, having a worldwide geographic distribution (see Alvarez-Fernandez A, et. al, (2018) Parasit Vectors 11 :624; Okaro U, et. al, (2017) Clin Microbiol Rev 30:709-746; and lannino F, et. al, (2018) Vet Ital 54:63-72, which are incorporated herein by reference in their entirety), B. henselae and B. quintana are currently considered the source of a majority of Bartonella infections in North America and Europe (see Agan BK and Dolan MJ. (2002) Clin Lab Med 22:937-962, which is incorporated herein by reference in its entirety). Previous studies across various populations have shown that of patients with diagnosed Cat Scratch Disease (typically caused by B. henselae infection), about 10% developed musculoskeletal manifestations (see Maman E, et. al, (2007)WSGR Docket No. 68706-703.601Clin Infect Dis 45: 1535-1540, which is incorporated herein by reference in its entirety), 5% with atypical manifestations had neuritis or osteomyelitis (Nawrocki CC, et. al, (2020) Emerg Infect Dis 26:1438-1446, which is incorporated herein by reference in its entirety), and about 3% had arthropathies (see Giladi M, et. al, (2005) Arthritis Rheum 52:3611-3617, which is incorporated herein by reference in its entirety). Conversely, of patients presenting for rheumatic conditions, anywhere from 0% to 60% have been reported to have evidence of Bartonella spp. infection or exposure (see Lysakowska ME, et. al, (2019) Rheumatol 38:2691-2698; Dillon B, et. al, (2000) Rheumatol Int 19:219-222; and Maggi RG, et. al, (2012) Emerg Infect Dis 18:783-791, which are incorporated herein by reference in their entirety).

[0110] This study suggests that synovial fluid samples may be important in determining exposure to B. burgdorferi in patients with rheumatic symptoms, though may not provide additional information on exposure toB. henselae or B. quintana compared to serum alone. For B. burgdorferi antibodies, both IgM and IgG were more commonly detected in synovial fluid than in serum and often in synovial fluid only; in contrast, for Bartonella spp. antibodies were rarely detected in synovial fluid and never without their concurrent presence in serum.

[0111] While the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document were individually and separately indicated to be incorporated by reference for all purposes.LIST OF EMBODIMENTS

[0112] The following list of embodiments of the invention are to be considered as disclosing various features of the invention, which features can be considered to be specific to the particular embodiment under which they are discussed, or which are combinable with the various other features as listed in other embodiments. Thus, simply because a feature is discussed under one particular embodiment does not necessarily limit the use of that feature to that embodiment.

[0113] Embodiment 1. A method of detecting a genus or a species of a fastidious microorganism, comprising: contacting two or more macromolecules in a sample obtained from a subject with two or more agents for detecting a genus or a species of a fastidious microorganism in the sample, wherein the two or more macromolecules comprise differentWSGR Docket No. 68706-703.601classes of macromolecule, and wherein the contacting of the two or more macromolecules with the two or more agents increases a sensitivity or a specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to a second sensitivity or a second specificity of detecting the genus or the species of the fastidious microorganism by one agent contacting one class of macromolecule.

[0114] Embodiment 2. The method of embodiment 1, wherein the two or more macromolecules comprise a polypeptide associated with the fastidious microorganism.

[0115] Embodiment 3. The method of embodiment 2, wherein the polypeptide is expressed on a surface of the fastidious microorganism.

[0116] Embodiment 4. The method of embodiment 2, wherein the polypeptide is secreted by the fastidious microorganism.

[0117] Embodiment 5. The method of embodiment 1, wherein the two or more macromolecules comprise a polynucleotide associated with the fastidious microorganism.

[0118] Embodiment 6. The method of embodiment 5, wherein the polynucleotide comprises a DNA of the fastidious microorganism.

[0119] Embodiment 7. The method of embodiment 5, wherein the polynucleotide comprises a RNA of the fastidious microorganism.

[0120] Embodiment 8. The method of embodiment 7, wherein the RNA comprises ribosomal RNA (rRNA).

[0121] Embodiment 9. The method of embodiment 8, wherein the rRNA comprises 18S rRNA, 5.8S rRNA, 28 S rRNA, or a combination thereof.

[0122] Embodiment 10. The method of any one of embodiments 1-9, wherein the two or more macromolecules comprise at least one polypeptide and at least one polynucleotide.

[0123] Embodiment 11. The method of any one of embodiments 1-10, wherein the two or more agents comprise a polypeptide agent.

[0124] Embodiment 12. The method of embodiment 11, wherein the polypeptide agent comprises an antibody or an antigen binding fragment thereof.

[0125] Embodiment 13. The method of embodiment 11 or 12, wherein the polypeptide agent binds to the two or more macromolecules.

[0126] Embodiment 14. The method of embodiment 13, wherein the polypeptide agent binding the two or more macromolecules is detected by western blotting, mass spectrometry, immunoassay, or a combination thereof.

[0127] Embodiment 15. The method of embodiment 14, wherein the immunoassay comprises ELISA.WSGR Docket No. 68706-703.601

[0128] Embodiment 16. The method of embodiment 14, wherein the immunoassay comprises detecting the polypeptide agent binding to the at least one polypeptide in suspension.

[0129] Embodiment 17. The method of any one of embodiments 13-16, wherein the two or more macromolecules comprise a polypeptide of Borrelia.

[0130] Embodiment 18. The method of embodiment 17, wherein the polypeptide of Borrelia comprises outer surface protein A (OspA), OspB, OspC, variable lipoprotein surface-exposed protein (VlsE), oligopeptide ABC transporter I (OppAI), OppAII, OppAIII, OppAIV, OppAV, or a combination thereof.

[0131] Embodiment 19. The method of any one of embodiments 13-16, wherein the two or more macromolecules comprise a polypeptide of Babesia.

[0132] Embodiment 20. The method of any one of embodiments 13-16, wherein the two or more macromolecules comprise a polypeptide of Bartonella.

[0133] Embodiment 21. The method of any one of embodiments 17-20, wherein the two or more macromolecules comprise a polypeptide of an additional pathogen other than Borrelia, Babesia, and Bartonella.

[0134] Embodiment 22. The method of any one of embodiments 1-21, wherein the two or more agents comprise a polynucleotide agent.

[0135] Embodiment 23. The method of embodiment 22, wherein the polynucleotide agent comprises a primer.

[0136] Embodiment 24. The method of embodiment 23, wherein the primer binds to the two or more macromolecules.

[0137] Embodiment 25. The method of embodiment 22, wherein the polynucleotide agent comprises an oligonucleotide probe.

[0138] Embodiment 26. The method of embodiment 25, wherein the oligonucleotide probe binds to the two or more macromolecules.

[0139] Embodiment 27. The method of embodiment 26, wherein the oligonucleotide probe binds to an amplification product of the two or more macromolecules.

[0140] Embodiment 28. The method of any one of embodiments 23-27, further comprising performing PCR for amplifying the two or more macromolecules.

[0141] Embodiment 29. The method of embodiment 28, wherein the PCR comprises digital PCR (dPCR), quantification PCR (qPCR), conventional PCR (cPCR), next generation sequence (NGS), reverse transcription (RT)-ddPCR, RT-qPCR, RT-cPCR, RNASeq, or a combination thereof.WSGR Docket No. 68706-703.601

[0142] Embodiment 30. The method of any one of embodiments 24-29, wherein the two or more macromolecules comprise a polynucleotide of Borrelia.

[0143] Embodiment 31. The method of any one of embodiments 24-29, wherein the two or more macromolecules comprise a polynucleotide of Babesia.

[0144] Embodiment 32. The method of any one of embodiments 24-29, wherein the two or more macromolecules comprise a polynucleotide of Bartonella.

[0145] Embodiment 33. The method of any one of embodiments 30-32, wherein the two or more macromolecules comprise a polynucleotide of an additional pathogen other than Borrelia, Babesia, and Bartonella.

[0146] Embodiment 34. The method of any one of embodiments 1-33, wherein the two or more agents comprise at least one polypeptide agent and at least one polynucleotide agent.

[0147] Embodiment 35. The method of any one of embodiments 1-34, wherein the sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

[0148] Embodiment 36. The method of any one of embodiments 1-35, wherein the specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

[0149] Embodiment 37. The method of any one of embodiments 1-36, wherein the subject is suspected of being infected by the fastidious microorganism.

[0150] Embodiment 38. The method of embodiment 37, wherein the contacting of the two or more macromolecules with the two or more agents detects the genus or the species of the fastidious microorganism in the subject at an earlier stage of infection by the fastidious microorganism compared to detecting the genus or the species of the fastidious microorganism by the one agent contacting the one class of macromolecule.

[0151] Embodiment 39. The method of any one of embodiments 1-38, further comprising detecting at least one additional pathogen in the sample.

[0152] Embodiment 40. The method of embodiment 39, wherein the at least one additional pathogen comprises a species that is different from the species of the fastidious microorganism.

[0153] Embodiment 41. The method of embodiment 39, wherein the at least one additional pathogen comprises a genus that is different from the genus of the fastidious microorganism.

[0154] Embodiment 42. The method of any one of embodiments 1-41, further comprising enriching the fastidious microorganism in a culturing media.WSGR Docket No. 68706-703.601

[0155] Embodiment 43. The method of any one of embodiments 1-42, further comprising capturing the fastidious microorganism by contacting the fastidious microorganism with a trapping agent.

[0156] Embodiment 44. The method of embodiment 43, wherein the trapping agent comprises an antibody or an antigen binding fragment thereof.

[0157] Embodiment 45. The method of embodiment 44, wherein the antibody or the antigen binding fragment thereof is conjugated to a solid surface.

[0158] Embodiment 46. The method of embodiment 43, wherein the trapping agent comprises a chaotropic agent.

[0159] Embodiment 47. The method of any one of embodiments 1-46, wherein the sample comprises blood, plasma, serum, urine, cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions.

[0160] Embodiment 48. The method of embodiment 41, wherein the subject a mammal.

[0161] Embodiment 49. The method of embodiment 48, wherein the subject is a human.

[0162] Embodiment 50. The method of embodiment 1, wherein the subject is not a mammal.

[0163] Embodiment 51. The method of embodiment 1, wherein the subject is not a human.

[0164] Embodiment 52. The method of embodiment 1, wherein the subject is a host for the fastidious microorganism.

[0165] Embodiment 53. The method of embodiment 1, wherein the subject is not a host for the fastidious microorganism.

[0166] Embodiment 54. The method of any one of embodiments 1-53, further comprising treating the subject based on the genus or the species of the fastidious microorganism in the sample.

[0167] Embodiment 55. The method of embodiment 54, wherein the subject is treated based on a life cycle of the fastidious microorganism.

[0168] Embodiment 56. The method of embodiment 54, wherein the subject is treated based on a morphology of the fastidious microorganism.

[0169] Embodiment 57. The method of embodiment 54, wherein the subject is treated based on the species of the fastidious microorganism.

[0170] Embodiment 58. The method of embodiment 54, wherein the subject is treated for inflammation associated with the fastidious microorganism.

[0171] Embodiment 59. The method of embodiment 54, wherein the subject is treated for a disease or condition associated with the fastidious microorganism.WSGR Docket No. 68706-703.601

[0172] Embodiment 60. The method of embodiment 59, wherein the disease or condition comprises osteoarthritis (OA), Sjogren’s syndrome (SS), rheumatoid arthritis (RA), chronic fatigue syndrome (CFS), gout, calcium pyrophosphate deposition (CPPD), rotator cuff disease (RC), neuropathy, or a combination thereof.

[0173] Embodiment 61. The method of any one of embodiments 1-60, wherein the genus of the fastidious microorganism comprises Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof.

[0174] Embodiment 62. The method of any one of embodiments 1-61, wherein the species of the fastidious microorganism comprises Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, Borrelia mayonii, Borrelia miyamotoi, Bartonella vinsonii berkhoffn, Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum, Rickettsia ricketsii, Rickettsia conorii, Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.

[0175] Embodiment 63. A system comprising: two or more agents for contacting the two or more macromolecules, wherein the two or more macromolecules are different class of macromolecule; wherein the two or more agents contacting the two or more macromolecules detects a genus of a species of a fastidious microorganism in a sample obtained from a subject; and wherein contacting the two or more macromolecules with the two or more agents increases a sensitivity or a specificity of detecting the genus or the species of the fastidious microorganism compared to a second sensitivity or specificity of detecting the genus or the species fastidious microorganism one agent contacting one class of macromolecule.

[0176] Embodiment 64. The system of embodiment 63, wherein the two or more agents comprise a polypeptide agent.

[0177] Embodiment 65. The system of embodiment 64, wherein the polypeptide agent comprises an antibody or an antigen binding fragment thereof.

[0178] Embodiment 66. The system of embodiment 64 or 65, wherein the polypeptide agent binds to the two or more macromolecules.

[0179] Embodiment 67. The system of embodiment 66, wherein the polypeptide agent binding the two or more macromolecules is detected by western blotting, mass spectrometry, immunoassay, or a combination thereof.

[0180] Embodiment 68. The system of embodiment 67, wherein the immunoassay comprises ELISA.

[0181] Embodiment 69. The system of embodiment 68, wherein the immunoassay comprises detecting the polypeptide agent binding to the at least one polypeptide in suspension.WSGR Docket No. 68706-703.601

[0182] Embodiment 70. The system of any one of embodiments 63-69, wherein the two or more macromolecules comprise a polypeptide of Borrelia.

[0183] Embodiment 71. The system of embodiments 70, wherein the polypeptide of Borrelia comprises outer surface protein A (OspA), OspB, OspC, variable lipoprotein surface-exposed protein (VlsE), oligopeptide ABC transporter I (OppAI), OppAII, OppAIII, OppAIV, OppAV, or a combination thereof.

[0184] Embodiment 72. The system of any one of embodiments 63-69, wherein the two or more macromolecules comprise a polypeptide of Babesia.

[0185] Embodiment 73. The system of any one of embodiments 63-69, wherein the two or more macromolecules comprise a polypeptide of Bartonella.

[0186] Embodiment 74. The system of any one of embodiments 63-73, wherein the two or more macromolecules comprise a polypeptide of an additional pathogen other than Borrelia, Babesia, and Bartonella.

[0187] Embodiment 75. The system of any one of embodiments 63-74, wherein the two or more agents comprise a polynucleotide agent.

[0188] Embodiment 76. The system of embodiment 75, wherein the polynucleotide agent comprises a primer.

[0189] Embodiment 77. The system of embodiment 76, wherein the primer binds to the two or more macromolecules.

[0190] Embodiment 78. The system of embodiment 75, wherein the polynucleotide agent comprises an oligonucleotide probe.

[0191] Embodiment 79. The system of embodiment 78, wherein the oligonucleotide probe binds to the two or more macromolecules.

[0192] Embodiment 80. The system of embodiment 79, wherein the oligonucleotide probe binds to an amplification product of the two or more macromolecules.

[0193] Embodiment 81. The system of any one of embodiments 63-80, wherein the two or more macromolecules comprise a polynucleotide of Borrelia.

[0194] Embodiment 82. The system of any one of embodiments 63-80, wherein the two or more macromolecules comprise a polynucleotide of Babesia.

[0195] Embodiment 83. The system of any one of embodiments 63-80, wherein the two or more macromolecules comprise a polynucleotide of Bartonella.

[0196] Embodiment 84. The system of any one of embodiments 63-80, wherein the two or more macromolecules comprise a polynucleotide of an additional pathogen other than Borrelia, Babesia, and Bartonella.WSGR Docket No. 68706-703.601

[0197] Embodiment 85. The system of any one of embodiments 63-84, wherein the two or more agents comprise at least one polypeptide agent and at least one polynucleotide agent.

[0198] Embodiment 86. The system of any one of embodiments 63-85, further comprising a culturing media for enriching the fastidious microorganism.

[0199] Embodiment 87. The system of any one of embodiments 63-86, further comprising a trapping agent for capturing the fastidious microorganism.

[0200] Embodiment 88. The system of embodiment 87, wherein the trapping agent comprises an antibody or an antigen binding fragment thereof.

[0201] Embodiment 89. The system of embodiment 88, wherein the antibody or the antigen binding fragment thereof is conjugated to a solid surface.

[0202] Embodiment 90. The system of embodiment 87, wherein the trapping agent comprises a chaotropic agent.

[0203] Embodiment 91. The system of any one of embodiments 63-90, wherein the genus of the fastidious microorganism comprises Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof.

[0204] Embodiment 92. The system of any one of embodiments 63-91, wherein the species of the fastidious microorganism comprises Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, Borrelia mayonii, Borrelia miyamotoi, Bartonella vinsonii berkhoffn, Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum, Rickettsia ricketsii, Rickettsia conorii, Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.

[0205] Embodiment 93. A kit comprising: two or more agents for contacting the two or more macromolecules, wherein the two or more macromolecules are different class of macromolecule; andwherein the two or more macromolecules are obtained from a fastidious microorganism.

[0206] Embodiment 94. The kit of embodiment 93, wherein the fastidious microorganism comprises Borrelia, Babesia, o Bartonella.

[0207] Embodiment 95. The kit of embodiment 93 or 94, the two or more agents comprise a polypeptide agent.

[0208] Embodiment 96. The kit of embodiment 95, wherein the polypeptide agent comprises an antibody or an antigen binding fragment thereof.

[0209] Embodiment 97. The kit of embodiment 96, wherein the polypeptide agent binding the two or more macromolecules is detected by western blotting, mass spectrometry, immunoassay, or a combination thereof.WSGR Docket No. 68706-703.601

[0210] Embodiment 98. The kit of embodiment 97, wherein the immunoassay comprises ELISA.

[0211] Embodiment 99. The kit of embodiment 97, wherein the immunoassay comprises detecting the polypeptide agent binding to at least one polypeptide in suspension.

[0212] Embodiment 100. The kit of embodiment 95, wherein the two or more agents comprise a polynucleotide agent.

[0213] Embodiment 101. The kit of embodiment 100, wherein the polynucleotide agent comprises a primer.

[0214] Embodiment 102. The kit of embodiment 101, wherein the primer binds to the two or more macromolecules.

[0215] Embodiment 103. The kit of embodiment 100, wherein the polynucleotide agent comprises an oligonucleotide probe.

[0216] Embodiment 104. The kit of embodiment 103, wherein the oligonucleotide probe binds to the two or more macromolecules.

[0217] Embodiment 105. The kit of embodiment 104, wherein the oligonucleotide probe binds to an amplification product of the two or more macromolecules.

[0218] Embodiment 106. The kit of embodiment 93, wherein the two or more agents comprise at least one polypeptide agent and at least one polynucleotide agent.

[0219] Embodiment 107. The kit of any one of embodiments 93-105, further comprising a culturing media for enriching the fastidious microorganism.

[0220] Embodiment 108. The kit of any one of embodiments 93-104, further comprising a trapping agent for capturing the fastidious microorganism.

[0221] Embodiment 109. The kit of embodiment 108, wherein the trapping agent comprises an antibody or an antigen binding fragment thereof.

[0222] Embodiment 110. The kit of embodiment 109, wherein the antibody or the antigen binding fragment thereof is conjugated to a solid surface.

[0223] Embodiment 111. The kit of embodiment 104, wherein the trapping agent comprises a chaotropic agent.

[0224] Embodiment 112. The kit of any one of embodiments 93-111, wherein the genus of the fastidious microorganism comprises Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof.

[0225] Embodiment 113. The kit of any one of embodiments 93-112, wherein the species of the fastidious microorganism comprises Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, BorreliaWSGR Docket No. 68706-703.601mayonii, Borrelia miyamotoi, Bartonella vinsonii berkhoffii, Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum, Rickettsia ricketsii, Rickettsia conorii. Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.

[0226] Embodiment 114. A method for detecting a fastidious microorganism in a transcellular fluid sample obtained from a subject, the method comprising: contacting a macromolecule of a fastidious microorganism with an agent for detecting a genus or a species of the fastidious microorganism in a transcellular fluid sample obtained from a sample, wherein detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample results in an increased sensitivity or specificity of detecting the genus or the species of the fastidious microorganism compared to a second sensitivity or a second specificity of detecting the genus or the species of the fastidious microorganism in an interstitial fluid sample.

[0227] Embodiment 115. The method of embodiment 114, wherein the macromolecule comprises a polypeptide associated with the fastidious microorganism.

[0228] Embodiment 116. The method of embodiment 115, wherein the polypeptide is expressed on a surface of the fastidious microorganism.

[0229] Embodiment 117. The method of embodiment 115, wherein the polypeptide is secreted by the fastidious microorganism.

[0230] Embodiment 118. The method of embodiment 114, wherein the macromolecule comprises a polynucleotide associated with the fastidious microorganism.

[0231] Embodiment 119. The method of embodiment 118, wherein the polynucleotide comprises a DNA of the fastidious microorganism.

[0232] Embodiment 120. The method of embodiment 118, wherein the polynucleotide comprises an RNA of the fastidious microorganism.

[0233] Embodiment 121. The method of embodiment 120, wherein the RNA comprises ribosomal RNA (rRNA).

[0234] Embodiment 122. The method of embodiment 121, wherein the rRNA comprises 18S rRNA, 5.8S rRNA, 28 S rRNA, or a combination thereof.

[0235] Embodiment 123. The method of any one of embodiments 114-122, wherein the agent comprises a polypeptide agent.

[0236] Embodiment 124. The method of embodiment 123, wherein the polypeptide agent comprises an antibody or an antigen binding fragment thereof.

[0237] Embodiment 125. The method of embodiment 123, wherein the polypeptide agent binding the macromolecule is detected by western blotting, mass spectrometry, immunoassay, or a combination thereof.WSGR Docket No. 68706-703.601

[0238] Embodiment 126. The method of embodiment 125, wherein the immunoassay comprises ELISA.

[0239] Embodiment 127. The method of embodiment 125, wherein the immunoassay comprises detecting the polypeptide agent binding to the at least one polypeptide in suspension.

[0240] Embodiment 128. The method of any one of embodiments 123-127, wherein the macromolecule comprises a polypeptide of Borrelia.

[0241] Embodiment 129. The method of embodiments 128, wherein the polypeptide of Borrelia comprises outer surface protein A (OspA), OspB, OspC, variable lipoprotein surface-exposed protein (VlsE), oligopeptide ABC transporter I (OppAI), OppAII, OppAIII, OppAIV, OppAV, or a combination thereof.

[0242] Embodiment 130. The method of any one of embodiments 123-127, wherein the macromolecule comprises a polypeptide of Babesia.

[0243] Embodiment 131. The method of any one of embodiments 123-127, wherein the macromolecule comprises a polypeptide of Bartonella.

[0244] Embodiment 132. The method of embodiment 114, wherein the agent comprises a polynucleotide agent.

[0245] Embodiment 133. The method of embodiment 132, wherein the polynucleotide agent comprises a primer.

[0246] Embodiment 134. The method of embodiment 133, wherein the primer binds to the macromolecule.

[0247] Embodiment 135. The method of embodiment 132, wherein the polynucleotide agent comprises an oligonucleotide probe.

[0248] Embodiment 136. The method of embodiment 135, wherein the oligonucleotide probe binds to the macromolecule.

[0249] Embodiment 137. The method of embodiment 136, wherein the oligonucleotide probe binds to an amplification product of the macromolecule.

[0250] Embodiment 138. The method of any one of embodiments 132-137, further comprising performing PCR for amplifying the macromolecule.

[0251] Embodiment 139. The method of embodiment 138, wherein the PCR comprises digital PCR (dPCR), quantitative PCR (qPCR), conventional PCR (cPCR), next generation sequence (NGS), reverse transcription (RT)-ddPCR, RT-qPCR, RT-cPCR, RNASeq, or a combination thereof.

[0252] Embodiment 140. The method of any one of embodiments 134-139, wherein the macromolecule comprises a polynucleotide of Borrelia.WSGR Docket No. 68706-703.601

[0253] Embodiment 141. The method of any one of embodiments 134-139, wherein the macromolecule comprises a polynucleotide of Babesia.

[0254] Embodiment 142. The method of any one of embodiments 134-139, wherein the macromolecule comprises a polynucleotide of Bartonella.

[0255] Embodiment 143. The method of any one of embodiments 114-142, wherein the sensitivity of detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample compared to the second sensitivity obtained from the interstitial fluid sample is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

[0256] Embodiment 144. The method of any one of embodiments 114-142, wherein the specificity of detecting the genus or the species of the fastidious microorganism in the transcellular fluid sample compared to the second specificity obtained from the interstitial fluid sample is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

[0257] Embodiment 145. The method of any one of embodiments 114-144, wherein the subject suspected of being infected by the fastidious microorganism.

[0258] Embodiment 146. The method of any one of embodiments 114-145, wherein the contacting of the macromolecule with the agent detects the genus or the species of the fastidious microorganism in the transcellular fluid sample at an earlier stage of infection by the fastidious microorganism compared to detecting the genus or the species of the fastidious microorganism by the agent contacting the macromolecule in the interstitial fluid sample.

[0259] Embodiment 147. The method of any one of embodiments 114-146, further comprising detecting at least one additional pathogen in the sample.

[0260] Embodiment 148. The method of embodiment 147, wherein the at least one additional pathogen comprises a species that is different from the species of the fastidious microorganism.

[0261] Embodiment 149. The method of embodiment 147, wherein the at least one additional pathogen comprises a genus that is different from the genus of the fastidious microorganism.

[0262] Embodiment 150. The method of any one of embodiments 114-149, further comprising enriching the fastidious microorganism in a culturing media.

[0263] Embodiment 151. The method of any one of embodiments 114-150, further comprising capturing the fastidious microorganism by contacting the fastidious microorganism with a trapping agent.

[0264] Embodiment 152. The method of embodiment 151, wherein the trapping agent comprises an antibody or an antigen binding fragment thereof.WSGR Docket No. 68706-703.601

[0265] Embodiment 153. The method of embodiment 152, wherein the antibody or the antigen binding fragment thereof is conjugated to a solid surface.

[0266] Embodiment 154. The method of embodiment 151, wherein the trapping agent comprises a chaotropic agent.

[0267] Embodiment 155. The method of any one of embodiments 114-154, wherein the transcellular fluid sample comprises cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions.

[0268] Embodiment 156. The method of embodiment 155, wherein the transcellular fluid comprises the synovial fluid.

[0269] Embodiment 157. The method of any one of embodiments 114-156, wherein the interstitial fluid sample comprises blood, serum, plasma, or a combination thereof.

[0270] Embodiment 158. The method of any one of embodiments 114-157, wherein the subject a mammal.

[0271] Embodiment 159. The method of embodiment 158, wherein the subject is a human.

[0272] Embodiment 160. The method of any one of embodiments 114-157, wherein the subject is not a mammal.

[0273] Embodiment 161. The method of any one of embodiments 114-157, wherein the subject is not a human.

[0274] Embodiment 162. The method of any one of embodiments 114-157, wherein the subject is a host for the fastidious microorganism.

[0275] Embodiment 163. The method of any one of embodiments 114-157, wherein the subject is not a host for the fastidious microorganism.

[0276] Embodiment 164. The method of any one of embodiments 114-163, further comprising treating the subject based on the genus or the species of the fastidious microorganism in the transcellular fluid sample.

[0277] Embodiment 165. The method of embodiment 164, wherein the subject is treated based on a life cycle of the fastidious microorganism.

[0278] Embodiment 166. The method of embodiment 164, wherein the subject is treated based on a morphology of the fastidious microorganism.

[0279] Embodiment 167. The method of embodiment 164, wherein the subject is treated based on the species of the fastidious microorganism.

[0280] Embodiment 168. The method of embodiment 164, wherein the subject is treated for inflammation associated with the fastidious microorganism.WSGR Docket No. 68706-703.601

[0281] Embodiment 169. The method of embodiment 164, wherein the subject is treated for a disease or condition associated with the fastidious microorganism.

[0282] Embodiment 170. The method of embodiment 169, wherein the disease or condition comprises osteoarthritis (OA), Sjogren’s syndrome (SS), rheumatoid arthritis (RA), chronic fatigue syndrome (CFS), gout, calcium pyrophosphate deposition (CPPD), rotator cuff disease (RC), neuropathy, or a combination thereof.

[0283] Embodiment 171. The method of any one of embodiments 114-170, wherein the genus of the fastidious microorganism comprises Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof.

[0284] Embodiment 172. The method of any one of embodiments 114-171, wherein the species of the fastidious microorganism comprises Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, Borrelia mayonii, Borrelia miyamotoi, Bartonella vinsonii berkhoffn, Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum, Rickettsia ricketsii, Rickettsia conorii, Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.

Claims

WSGR Docket No. 68706-703.601CLAIMS WHAT IS CLAIMED IS:

1. A method for detecting a fastidious microorganism in a subject, the method comprising:(i) obtaining a first biological sample and a second biological sample from the subject, wherein the first biological sample and the second biological sample are different sample types;(ii) contacting each of the first and second biological samples with an agent to detect a macromolecule derived from the fastidious microorganism; and (iii) determining presence or absence of the fastidious microorganism based on the detection from the first biological sample and the second biological sample.

2. A method for detecting a fastidious microorganism in a subject, the method comprising:(i) obtaining a first biological sample and a second biological sample from the subject, wherein the first biological sample and the second biological sample are different sample types;(ii) contacting each of the first and second biological samples with an agent to detect a macromolecule derived from the fastidious microorganism; and (iii) determining presence or absence of the fastidious microorganism based on the detections,wherein the detection of the fastidious microorganism based on the combination of the first and second biological samples yields greater sensitivity or specificity than detection based on either the first biological sample alone or the second biological sample alone.

3. A method for detecting a fastidious microorganism in a subject with increased sensitivity or specificity, the method comprising:(i) obtaining a first biological sample from a subject, wherein the first biological sample is not blood;(ii) contacting the first biological sample with an agent to detect a macromolecule derived from the fastidious microorganism; and(iii) determining presence or absence of the fastidious microorganism based on the detection,wherein the detection of the fastidious microorganism in the first biological sample comprises greater sensitivity or specificity relative to detection of the fastidious microorganism in a second biological sample from the subject, wherein the first biological sample and the second biological sample are different sample types.WSGR Docket No. 68706-703.6014. The method of any one of claims 1-3, wherein the macromolecule comprises a polypeptide associated with the fastidious microorganism.

5. The method of claim 4, wherein the polypeptide is expressed on a surface of the fastidious microorganism.

6. The method of claim 4, wherein the polypeptide is secreted by the fastidious microorganism.

7. The method of any one of claims 1-3, wherein the macromolecule comprises a polynucleotide associated with the fastidious microorganism.

8. The method of claim 7, wherein the polynucleotide comprises DNA of the fastidious microorganism.

9. The method of claim 7, wherein the polynucleotide comprises RNA of the fastidious microorganism.

10. The method of claim 9, wherein the RNA comprises ribosomal RNA (rRNA).

11. The method of claim 10, wherein the rRNA comprises 18S rRNA, 5.8S rRNA, 28S rRNA, or a combination thereof.

12. The method of any one of claims 1-11, wherein the agent comprises a polypeptide agent.

13. The method of claim 12, wherein the polypeptide agent comprises an antibody or an antigen binding fragment thereof.

14. The method of claim 12 or 13, wherein the polypeptide agent binds to the macromolecule.

15. The method of claim 14, wherein the polypeptide agent binding the macromolecule is detected by western blotting, mass spectrometry, immunoassay, or a combination thereof.

16. The method of claim 15, wherein the immunoassay comprises ELISA.

17. The method of claim 15, wherein the immunoassay comprises detecting the polypeptide agent binding to the at least one polypeptide in suspension.

18. The method of any one of claims 14-16, wherein the macromolecule comprises a polypeptide of Borrelia.

19. The method of claim 18, wherein the polypeptide of Borrelia comprises outer surface protein A (OspA), OspB, OspC, variable lipoprotein surface-exposed protein (VlsE), oligopeptide ABC transporter I (OppAI), OppAII, OppAIII, OppAIV, OppAV, or a combination thereof.

20. The method of any one of claims 14-17, wherein the macromolecule comprises a polypeptide of Babesia.WSGR Docket No. 68706-703.60121. The method of any one of claims 14-17, wherein the macromolecule comprises a polypeptide of Bartonella.

22. The method of any one of claims 18-21, wherein the macromolecule comprises a polypeptide of an additional pathogen other than Borrelia, Babesia, and Bartonella.

23. The method of any one of claims 1-22, wherein the agent comprises a polynucleotide agent.

24. The method of claim 23, wherein the polynucleotide agent comprises a primer.

25. The method of claim 24, wherein the primer binds to the macromolecule.

26. The method of claim 23, wherein the polynucleotide agent comprises an oligonucleotide probe.

27. The method of claim 26, wherein the oligonucleotide probe binds to the macromolecule.

28. The method of claim 27, wherein the oligonucleotide probe binds to an amplification product of the macromolecule.

29. The method of any one of claims 24-28, further comprising performing PCR for amplifying the macromolecule.

30. The method of claim 29, wherein the PCR comprises digital PCR (dPCR), quantification PCR (qPCR), conventional PCR (cPCR), next generation sequence (NGS), reverse transcription (RT)-ddPCR, RT-qPCR, RT-cPCR, RNASeq, or a combination thereof.

31. The method of any one of claims 25-30, wherein the macromolecule comprises a polynucleotide of Borrelia.

32. The method of any one of claims 25-30, wherein the macromolecule comprises a polynucleotide of Babesia.

33. The method of any one of claims 25-30, wherein the macromolecule comprises a polynucleotide of Bartonella.

34. The method of any one of claims 31-33, wherein the macromolecule comprises a polynucleotide of an additional pathogen other than Borrelia, Babesia, and Bartonella.

35. The method of claim 2, wherein the sensitivity or selectivity of detecting the fastidious microorganism based on the combination of the first and second biological samples compared to the sensitivity or specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500% compared to the detection based on either the first biological sample alone or the second biological sample alone.

36. The method of claim 3, wherein the sensitivity or specificity of detecting the fastidious microorganism is increased by at least 5%, at least 10%, at least 20%, at least 50%, at leastWSGR Docket No. 68706-703.601100%, or at least 500% compared to the detection of the fastidious microorganism in the second biological sample from the subject.

37. The method of any one of claims 1-36, wherein the subject is suspected of being infected by the fastidious microorganism.

38. The method of any one of claims 1-37, further comprising detecting at least one additional pathogen in the sample.

39. The method of claim 38, wherein the at least one additional pathogen comprises a species that is different from the species of the fastidious microorganism.

40. The method of claim 38, wherein the at least one additional pathogen comprises a genus that is different from the genus of the fastidious microorganism.

41. The method of any one of claims 1-40, further comprising enriching the fastidious microorganism in a culturing media.

42. The method of any one of claims 1-41, further comprising capturing the fastidious microorganism by contacting the fastidious microorganism with a trapping agent.

43. The method of claim 42, wherein the trapping agent comprises an antibody or an antigen binding fragment thereof.

44. The method of claim 43, wherein the antibody or the antigen binding fragment thereof is conjugated to a solid surface.

45. The method of claim 42, wherein the trapping agent comprises a chaotropic agent.

46. The method of claim 1 or 2, wherein the first biological sample comprises blood, plasma, serum, urine, cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions.

47. The method of claim 1 or 2, wherein the second biological sample comprises blood, plasma, serum, urine, cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions.

48. The method of claim 3, wherein the first biological sample comprises plasma, serum, urine, cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions.

49. The method of claim 3, wherein the second biological sample comprises blood, plasma, serum, urine, cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modifiedWSGR Docket No. 68706-703.601transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions.

50. The method of claim 3, wherein the first biological sample comprises synovial fluid and the second biological sample comprises blood.

51. The method of any one of claims 1-50, wherein the subject a mammal.

52. The method of claim 51, wherein the subject is a human.

53. The method of any one of claims 1-50, wherein the subject is not a mammal.

54. The method of claim 53, wherein the subject is not a human.

55. The method of any one of claims 1-54, wherein the subject is a host for the fastidious microorganism.

56. The method of any one of claims 1-54, wherein the subject is not a host for the fastidious microorganism.

57. The method of any one of claims 1-56, further comprising treating the subject based on the fastidious microorganism in the sample.

58. The method of claim 57, wherein the subject is treated based on a life cycle of the fastidious microorganism.

59. The method of claim 57, wherein the subject is treated based on a morphology of the fastidious microorganism.

60. The method of claim 57, wherein the subject is treated based on species of the fastidious microorganism.

61. The method of claim 57, wherein the subject is treated for inflammation associated with the fastidious microorganism.

62. The method of claim 57, wherein the subject is treated for a disease or condition associated with the fastidious microorganism.

63. The method of claim 62, wherein the disease or condition comprises osteoarthritis (OA), Sjogren’s syndrome (SS), rheumatoid arthritis (RA), chronic fatigue syndrome (CFS), gout, calcium pyrophosphate deposition (CPPD), rotator cuff disease (RC), neuropathy, or a combination thereof.

64. The method of any one of claims 1-63, wherein genus of the fastidious microorganism comprises Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof.

65. The method of any one of claims 1-64, wherein species of the fastidious microorganism comprises Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, Borrelia mayonii, Borrelia miyamotoi,WSGR Docket No. 68706-703.601Bartonella vinsonii berkhoffii, Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum, Rickettsia ricketsii, Rickettsia conorii. Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.

66. A method of detecting a fastidious microorganism in a subject, comprising:(i) contacting a biological sample from the subject with two or more agents to detect two or more macromolecules derived from the fastidious microorganism; and (ii) determining presence or absence of the fastidious microorganism based on the detection,wherein the two or more macromolecules are of different macromolecular classes, and wherein the detection of the fastidious microorganism with two or more macromolecules of the different macromolecular classes provides an increased sensitivity or specificity relative to detection of the fastidious microorganism with one class of macromolecule alone.

67. The method of claim 66, wherein the two or more macromolecules comprise a polypeptide associated with the fastidious microorganism.

68. The method of claim 67, wherein the polypeptide is expressed on a surface of the fastidious microorganism.

69. The method of claim 67, wherein the polypeptide is secreted by the fastidious microorganism.

70. The method of claim 66, wherein the two or more macromolecules comprise a polynucleotide associated with the fastidious microorganism.

71. The method of claim 70, wherein the polynucleotide comprises DNA of the fastidious microorganism.

72. The method of claim 70, wherein the polynucleotide comprises RNA of the fastidious microorganism.

73. The method of claim 72, wherein the RNA comprises ribosomal RNA (rRNA).

74. The method of claim 73, wherein the rRNA comprises 18S rRNA, 5.8S rRNA, 28S rRNA, or a combination thereof.

75. The method of any one of claims 66-74, wherein the two or more macromolecules comprise at least one polypeptide and at least one polynucleotide.

76. The method of any one of claims 67-75, wherein the two or more agents comprise a polypeptide agent.

77. The method of claim 76, wherein the polypeptide agent comprises an antibody or an antigen binding fragment thereof.WSGR Docket No. 68706-703.60178. The method of claim 76 or 77, wherein the polypeptide agent binds to the two or more macromolecules.

79. The method of claim 78, wherein the polypeptide agent binding the two or more macromolecules is detected by western blotting, mass spectrometry, immunoassay, or a combination thereof.

80. The method of claim 79, wherein the immunoassay comprises ELISA.

81. The method of claim 79, wherein the immunoassay comprises detecting the polypeptide agent binding to the at least one polypeptide in suspension.

82. The method of any one of claims 78-81, wherein the two or more macromolecules comprise a polypeptide of Borrelia.

83. The method of claim 82, wherein the polypeptide of Borrelia comprises outer surface protein A (OspA), OspB, OspC, variable lipoprotein surface-exposed protein (VlsE), oligopeptide ABC transporter I (OppAI), OppAII, OppAIII, OppAIV, OppAV, or a combination thereof.

84. The method of any one of claims 78-83, wherein the two or more macromolecules comprise a polypeptide of Babesia.

85. The method of any one of claims 78-81, wherein the two or more macromolecules comprise a polypeptide of Bartonella.

86. The method of any one of claims 80-83, wherein the two or more macromolecules comprise a polypeptide of an additional pathogen other than Borrelia, Babesia, and Bartonella.

87. The method of any one of claims 66-86, wherein the two or more agents comprise a polynucleotide agent.

88. The method of claim 87, wherein the polynucleotide agent comprises a primer.

89. The method of claim 88, wherein the primer binds to the two or more macromolecules.

90. The method of claim 87, wherein the polynucleotide agent comprises an oligonucleotide probe.

91. The method of claim 90, wherein the oligonucleotide probe binds to the two or more macromolecules.

92. The method of claim 91, wherein the oligonucleotide probe binds to an amplification product of the two or more macromolecules.

93. The method of any one of claims 88-92, further comprising performing PCR for amplifying the two or more macromolecules.WSGR Docket No. 68706-703.60194. The method of claim 93, wherein the PCR comprises digital PCR (dPCR), quantification PCR (qPCR), conventional PCR (cPCR), next generation sequence (NGS), reverse transcription (RT)-ddPCR, RT-qPCR, RT-cPCR, RNASeq, or a combination thereof.

95. The method of any one of claims 91-94, wherein the two or more macromolecules comprise a polynucleotide of Borrelia.

96. The method of any one of claims 91-94, wherein the two or more macromolecules comprise a polynucleotide of Babesia.

97. The method of any one of claims 91-94, wherein the two or more macromolecules comprise a polynucleotide of Bartonella.

98. The method of any one of claims 95-97, wherein the two or more macromolecules comprise a polynucleotide of an additional pathogen other than Borrelia, Babesia, and Bartonella.

99. The method of any one of claims 66-98, wherein the two or more agents comprise at least one polypeptide agent and at least one polynucleotide agent.

100. The method of any one of claims 66-99, wherein the sensitivity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

101. The method of any one of claims 66-100, wherein the specificity of detecting the genus or the species of the fastidious microorganism in the sample compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

102. The method of any one of claims 66-101, wherein the subject is suspected of being infected by the fastidious microorganism.

103. The method of claim 102, wherein the contacting of the two or more macromolecules with the two or more agents detects the genus or the species of the fastidious microorganism in the subject at an earlier stage of infection by the fastidious microorganism compared to detecting the genus or the species of the fastidious microorganism by the one agent contacting the one class of macromolecule.

104. The method of any one of claims 66-103, further comprising detecting at least one additional pathogen in the sample.

105. The method of claim 104, wherein the at least one additional pathogen comprises a species that is different from the species of the fastidious microorganism.WSGR Docket No. 68706-703.601106. The method of claim 105, wherein the at least one additional pathogen comprises a genus that is different from the genus of the fastidious microorganism.

107. The method of any one of claims 66-106, further comprising enriching the fastidious microorganism in a culturing media.

108. The method of any one of claims 66-107, further comprising capturing the fastidious microorganism by contacting the fastidious microorganism with a trapping agent.

109. The method of claim 108, wherein the trapping agent comprises an antibody or an antigen binding fragment thereof.

110. The method of claim 109, wherein the antibody or the antigen binding fragment thereof is conjugated to a solid surface.

111. The method of claim 110, wherein the trapping agent comprises a chaotropic agent.

112. The method of any one of claims 66-111, wherein the sample comprises blood, plasma, serum, urine, cerebrospinal fluid, pleural fluid, pulmonary mucus, sputum, transudates, modified transudates, exudates, chest fluid, abdominal fluid, synovial fluid, peritoneal fluid, lymph, saliva, semen, or effusions.

113. The method of claim 66, wherein the subject a mammal.

114. The method of claim 113, wherein the subject is a human.

115. The method of claim 66, wherein the subject is not a mammal.

116. The method of claim 115, wherein the subject is not a human.

117. The method of claim 66, wherein the subject is a host for the fastidious microorganism.

118. The method of claim 66, wherein the subject is not a host for the fastidious microorganism.

119. The method of any one of claims 66-118, further comprising treating the subject based on the genus or the species of the fastidious microorganism in the sample.

120. The method of claim 119, wherein the subject is treated based on a life cycle of the fastidious microorganism.

121. The method of claim 119, wherein the subject is treated based on a morphology of the fastidious microorganism.

122. The method of claim 119, wherein the subject is treated based on the species of the fastidious microorganism.

123. The method of claim 119, wherein the subject is treated for inflammation associated with the fastidious microorganism.

124. The method of claim 119, wherein the subject is treated for a disease or condition associated with the fastidious microorganism.WSGR Docket No. 68706-703.601125. The method of claim 124, wherein the disease or condition comprises osteoarthritis (OA), Sjogren’s syndrome (SS), rheumatoid arthritis (RA), chronic fatigue syndrome (CFS), gout, calcium pyrophosphate deposition (CPPD), rotator cuff disease (RC), neuropathy, or a combination thereof.

126. The method of any one of claims 66-125, wherein the genus of the fastidious microorganism comprises Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof.

127. The method of any one of claims 66-126, wherein the species of the fastidious microorganism comprises Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, Borrelia mayonii, Borrelia miyamotoi, Bartonella vinsonii berkhoffn, Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum, Rickettsia ricketsii, Rickettsia conorii, Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.

128. A kit comprising:two or more agents configured to bind to two or more macromolecules derived from the fastidious microorganism and of different macromolecular classes,wherein binding of the two or more agents with the two or more macromolecules is indicative of the presence of the fastidious microorganism in a subject; andwherein the detection of the fastidious microorganism with two or more macromolecules of the different macromolecular classes provides an increased sensitivity or specificity relative to detection of the fastidious microorganism with one class of macromolecule.

129. The kit of claim 128, wherein the fastidious microorganism comprises Borrelia, Babesia, or Bartonella.

130. The kit of claim 128 or 129, wherein the two or more agents comprise a polypeptide agent.

131. The kit of claim 130, wherein the polypeptide agent comprises an antibody or an antigen binding fragment thereof.

132. The kit of claim 131, wherein the polypeptide agent binding the two or more macromolecules is detected by western blotting, mass spectrometry, immunoassay, or a combination thereof.

133. The kit of claim 132, wherein the immunoassay comprises ELISA.

134. The kit of claim 132, wherein the immunoassay comprises detecting the polypeptide agent binding to at least one polypeptide in suspension.WSGR Docket No. 68706-703.601135. The kit of claim 128 or 129, wherein the two or more agents comprise a polynucleotide agent.

136. The kit of claim 135, wherein the polynucleotide agent comprises a primer.

137. The kit of claim 136, wherein the primer binds to the two or more macromolecules.

138. The kit of claim 137, wherein the polynucleotide agent comprises an oligonucleotide probe.

139. The kit of claim 138, wherein the oligonucleotide probe binds to the two or more macromolecules.

140. The kit of claim 139, wherein the oligonucleotide probe binds to an amplification product of the two or more macromolecules.

141. The kit of claim 128, wherein the two or more agents comprise at least one polypeptide agent and at least one polynucleotide agent.

142. The kit of any one of claims 128-141, further comprising a culturing media for enriching the fastidious microorganism.

143. The kit of any one of claims 128-142, further comprising a trapping agent for capturing the fastidious microorganism.

144. The kit of claim 143, wherein the trapping agent comprises an antibody or an antigen binding fragment thereof.

145. The kit of claim 144, wherein the antibody or the antigen binding fragment thereof is conjugated to a solid surface.

146. The kit of claim 145, wherein the trapping agent comprises a chaotropic agent.

147. The kit of any one of claims 128-146, wherein the genus of the fastidious microorganism comprises Borrelia, Babesia, Bartonella, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, Chlamydia, or a combination thereof.

148. The kit of any one of claims 128-147, wherein the species of the fastidious microorganism comprises Borrelia burgdorferi, Bartonella henselae, Bartonella quintana, Borrelia afzelii, Borrelia garinii, Borrelia hermsi, Borrelia recurrentis, Borrelia mayonii, Borrelia miyamotoi, Bartonella vinsonii berkhoffn, Bartonella koehlerae, Ehrlichia chaffeensis, Anaplasma phagocy tophilum, Rickettsia ricketsii, Rickettsia conorii, Mycoplasma pneumoniae, Chlamydia pneumonia, or a combination thereof.

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