Internal process control

Abstract

This disclosure relates to internal control compositions and methods of use for detecting microbial species.

Description

[0001] INTERNAL PROCESS CONTROL

[0002] RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 605,451, filed December 1, 2023, the contents of which is herein incorporated by reference in its entirety.

[0004] BACKGROUND

[0005] Clinical samples are processed to identify microbes present in subjects (e.g., human subjects) from whom the clinical samples are obtained. Next-generation sequencing (NGS) has become a tool for microbial detection due to its potential for high throughput, high taxonomic resolution, and sensitivity. An NGS workflow typically contains numerous steps, and thus many opportunities for workflow failure.

[0006] SUMMARY

[0007] Rapid and accurate identification of microbes present in clinical samples from subjects is critical for detecting and treating microbial infections in a subject. With the decreasing costs of sequencing, next-generation sequencing (NGS)-based workflows have become a useful tool for microbial detection. Internal controls are essential for validating the performance of such a workflow.

[0008] Currently available internal controls for sequencing-based microbial identification workflows rely on the introduction of naked, synthetic, or artificially packaged nucleic acids, while others simply spike in a microbe after sample processing. Use of naked, synthetic, and artificially packaged nucleic acids does not control for clinical sample processing because these nucleic acids, which are not encompassed in microbial cells, are not affected by clinical sample processing in the manner that microbial cells are affected. Microbes spiked into samples after processing similarly do not control for clinical sample processing because they are not exposed to processing steps. Such solutions do not constitute true internal process controls and cannot account for failures at each step of the sample processing workflow.

[0009] Described herein is an internal process control (IPC) for use in clinical sample processing. Also provided herein are methods of detecting one or more microbial species in a clinical sample containing an IPC. Further provided herein are methods of determining whether a clinical sample comprises one or more microbial species. Additionally provided herein are methods of detecting one or more microbial species in a clinical sample. An IPC of the present disclosure is advantageous over simply spiking in a microbe or other controls (e.g., naked or artificially packaged nucleic acids) introduced before sequencing) because said IPCs are processed alongside the pathogen(s) inherent in the sample and / or the sample matrix and can be used as a control for every step of the workflow.

[0010] Detection of IPC nucleic acids at the end of clinical sample processing ensures that the processing was successful.

[0011] In some aspects, the present disclosure provides a method comprising: (i) obtaining a clinical sample suspected of comprising one or more microbial species from a subject; (ii) introducing an internal process control (IPC) microbe into the clinical sample; (iii) processing the clinical sample with the IPC, including lysing the one or more microbial species and / or the IPC microbe; (iv) amplifying whole genome microbial DNAfrom the lysed microbial species and the lysed IPC microbe; and (v) detecting amplified microbial DNA and / or IPC DNA, wherein the detection of IPC DNA in step (v) indicates that there was not a process failure.

[0012] In some aspects, the present disclosure provides a method comprising: (i) obtaining a clinical sample suspected of comprising one or more microbial species from a subject; (ii) introducing one or more internal process control (IPC) microbes into the clinical sample; (iii) processing the clinical sample with the one or more IPC microbes, including lysing the one or more microbial species and / or the one or more IPC microbes; (iv) amplifying whole genome microbial DNA from the lysed microbial species and the lysed one or more IPC microbes; and (v) detecting amplified microbial DNA and / or IPC DNA, wherein the detection of microbial DNA and / or IPC DNA in step (v) indicates that there was not a process failure.

[0013] In some aspects, the present disclosure provides a method comprising: (i) obtaining a clinical sample suspected of comprising one or more microbial species from a subject; (ii) introducing an internal process control (IPC) microbe into the clinical sample; (iii) processing the clinical sample to release whole genome microbial DNA from one or more microbes present in the sample including the IPC microbe including lysing the one or more microbial species and / or the IPC microbe; (iv) amplifying the released whole genome microbial DNA from the lysed microbial species and the lysed IPC microbe; and (v) detecting amplified microbial DNA and / or IPC DNA, wherein the detection of amplified microbial and / or IPC DNA in step (v) indicates that there was not a process failure, and wherein step (iii) optionally comprises lysing the one or more microbial species and / or the IPC microbes.

[0014] In some aspects, the present disclosure provides a method comprising: (i) introducing an internal process control (IPC) microbe into a clinical sample; (ii) processing the clinical sample with the IPC, including lysing the one or more microbial species and / or the IPC microbe; (iii) amplifying whole genome microbial DNA from the ly: and / or the lysed IPC microbe; and (iv) detecting amplified microbial DNA and / or IPC DNA, wherein the detection of microbial and / or IPC DNA in step (iv) indicates that there was not a process failure.

[0015] In some aspects, the present disclosure provides a method for determining whether a clinical sample comprises one or more microbial species, the method comprising: (i) introducing an internal process control (IPC) microbe into a clinical sample previously obtained from a subject, the sample suspected of comprising one or more microbial species; (ii) processing the clinical sample with the IPC, including lysing the one or more microbial species and / or the IPC microbe; (iii) amplifying whole genome microbial DNA from the lysed microbial species and / or the lysed IPC microbe; and (iv) detecting amplified microbial DNA and / or IPC DNA, wherein the detection of microbial and / or IPC DNA in step (iv) indicates that there was not a process failure.

[0016] In some aspects, the present disclosure provides a method comprising: (i) obtaining a clinical sample suspected of comprising one or more microbial species from a subject; (ii) introducing an internal process control (IPC) microbe into the clinical sample; (iii) processing the clinical sample with the IPC, including lysing the one or more microbial species and / or the IPC microbe; (iv) amplifying microbial DNA from the lysed microbial species and the lysed IPC microbe; and (v) detecting amplified microbial DNA and / or IPC DNA, wherein the detection of microbial and / or IPC DNA in step (v) indicates that there was not a process failure.

[0017] In some aspects, the present disclosure provides a method comprising: (i) obtaining a clinical sample suspected of comprising one or more microbial species from a subject; (ii) introducing one or more internal process control (IPC) microbes into the clinical sample; (iii) processing the clinical sample with the one or more IPC microbes, including lysing the one or more microbial species and / or the one or more IPC microbes; (iv) amplifying microbial DNA from the lysed microbial species and the lysed one or more IPC microbes; and (v) detecting amplified microbial DNA and / or IPC DNA, wherein the detection of microbial and / or IPC DNA in step (v) indicates that there was not a process failure.

[0018] In some aspects, the present disclosure provides a method comprising: (i) obtaining a clinical sample suspected of comprising one or more microbial species from a subject; (ii) introducing an internal process control (IPC) microbe into the clinical sample; (iii) processing the clinical sample to release whole genome microbial DNA from one or more microbes present in the sample including the IPC microbe including lysing the one or more microbial species and / or the IPC microbe; (iv) amplifying the released microbi microbial species and the lysed IPC microbe; and (v) detecting amplified microbial DNA and / or IPC DNA, wherein the detection of amplified microbial and / or IPC DNA in step (v) indicates that there was not a process failure, and wherein step (iii) optionally comprises lysing the one or more microbial species and / or the IPC microbes.

[0019] In some aspects, the present disclosure provides a method comprising: (i) introducing an internal process control (IPC) microbe into a clinical sample; (ii) processing the clinical sample with the IPC, including lysing the one or more microbial species and / or the IPC microbe; (iii) amplifying microbial DNA from the lysed microbial species and / or the lysed IPC microbe; and (iv) detecting amplified microbial DNA and / or IPC DNA, wherein the detection of microbial and / or IPC DNA in step (iv) indicates that there was not a process failure.

[0020] In some aspects, the present disclosure provides a method for determining whether a clinical sample comprises one or more microbial species, the method comprising: (i) introducing an internal process control (IPC) microbe into a clinical sample previously obtained from a subject, the sample suspected of comprising one or more microbial species; (ii) processing the clinical sample with the IPC, including lysing the one or more microbial species and / or the IPC microbe; (iii) amplifying microbial DNA from the lysed microbial species and / or the lysed IPC microbe; and (iv) detecting amplified microbial DNA and / or IPC DNA, wherein the detection of microbial and / or IPC DNA in step (iv) indicates that there was not a process failure.

[0021] In some embodiments, detecting is detecting microbial DNA. In some embodiments detecting is detecting IPC DNA. In some embodiments, detecting is detecting microbial DNA and IPC DNA.

[0022] In some embodiments, the subject is a human. In some embodiments, the one or more microbial species are pathogenic microbial species. In some embodiments, the IPC microbe has 55% - 90% Guanine / Cytosine (G / C) content in its genome. In some embodiments, the IPC microbe is devoid (<50%) of non-integrated self-replicating genetic elements.

[0023] In some embodiments, the IPC microbe is a bacterium. In some embodiments, the IPC microbe is a Gram-positive bacterium. In some embodiments, the IPC microbe is from the genus Leucobacter.

[0024] In some aspects, the present disclosure provides a kit comprising one or more internal process control (IPC) microbes. In some embodiments, the one or more IPC microbes comprises a Gram-positive bacterium. In some embodiments, the Gram-positive bacterium is from the genus Leucobacter . In some embodiments, the one or mon expected to be in a clinical sample from a human subject. In some embodiments, the kit is for detecting one or more microbial species in a clinical sample from a human subject.

[0025] In some aspects, the present disclosure provides a method for making a kit for detection of one or more microbial species in a clinical sample, the method comprising formulating a solution comprising one or more internal process control (IPC) microbes, wherein the IPC microbes are not expected to be in the clinical sample.

[0026] BRIEF DESCRIPTION OF DRAWINGS

[0027] The accompanying drawings are not intended to be drawn to scale. The drawings are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0028] FIG. 1 shows a heatmap of the pairwise genomic relatedness between 53 internal process control (IPC) candidate strains and a selection of 41 bacterial pathogens. Each row and column represent either an IPC candidate (numbers 1-53) or an exemplary bacterial pathogen (numbers 54-94). The percent similarity is represented by the average amino acid identity (AAI) score on the right.

[0029] FIG. 2 shows that an IPC does not interfere with target Pseudomonas aeruginosa (P. aeruginosa) signal in a whole blood sample. P aeruginosa and IPC cells (Leucobacter muris, L. muris) were recovered from blood samples, the bacterial cells were lysed, and bacterial whole genomes were amplified and sequenced. Total sequencing data (megabases) was mapped to P aeruginosa (black) or the IPC (gray). The sequencing reads for P aeruginosa or IPC were normalized to the total amount of sequencing data generated per sample. Concentrations of IPC added to the sample are represented on the x-axis (0-500 CFU / mL) and the ratio of megabases mapped to a target (P aeruginosa or the IPC) to the total number of sequenced megabases on the y-axis.

[0030] FIGs. 3A-3B show that IPC (Leucobacter muris, L. muris) signal recovery in samples spiked with clinically relevant pathogenic microbes is influenced by the guanine-cytosine (GC) content of the target pathogen. FIG. 3A shows pathogen genome recovery (% coverage, y-axis) in the presence (gray; “With IPC”) or absence (black; “No IPC”) of IPC in 10 mL blood samples spiked with 2 CFU / mL of 5 pathogens of increasing GC content (x- axis), with IPC spiked at 100 CFU / ml. No significant difference is observed in pathogen genome recovery between samples with and without the IPC for each of the 5 pathogens. FIG. 3B shows that recovery of the IPC genome (% coverage, y-axi content of target pathogen approaches that of the IPC, compared to low GC pathogens. GC content of species (low to high): Staphylococcus aureus (S. aureus) 32.8%; Candida albicans (C. albicans) 33.4%; Escherichia coli (E. coli) 50.8%; Klebsiella pneumoniae (K. pneumoniae) 57.0%, Pseudomonas aeruginosa (P. aeruginosa) 65.9%; and IPC 70.6%. This result demonstrates that the IPC did not interfere with recovery of pathogens across a wide range of GC content that captures substantially the GC content of all expected pathogen species in typical clinical samples.

[0031] DETAILED DESCRIPTION

[0032] Clinical samples are processed to identify microbes present in subjects (e.g., human subjects) from whom the clinical samples are obtained. Rapid, accurate identification of these microbes is critical for detecting and treating microbial infections in subjects. An internal control that can control the end-to-end processing is required for clinical sample processing to ensure that false negatives are minimized when identifying microbes.

[0033] The present disclosure provides an internal process control (IPC) for clinical sample processing and rapid, accurate identification of microbes in the clinical samples following whole genome amplification and microbial DNA sequencing. An IPC is added into a clinical sample before any processing steps have occurred so that it is processed alongside the one or more microbial species. Processing an IPC as part of a clinical sample allows the IPC to control for the steps of clinical sample processing. An IPC as provided herein is advantageous over simply spiking in a microbe or other controls such as naked or artificially packaged nucleic acids immediately before identification because the IPC is actually processed alongside the microbes endogenous to the sample. This IPC is a control microbe added at the beginning of clinical sample processing, is processed alongside any microbes in the clinical sample, and whose detection at the end of clinical sample processing indicates that the processing was successful. It is beneficial for an IPC to be phylogenetically divergent from microbes in the clinical sample, does not compete with the microbes for signal, and is devoid of small non-integrated self-replicating elements like plasmids.

[0034] The present disclosure therefore advances the field by providing an IPC to control for the steps of clinical sample processing and microbial identification. An IPC as described herein is advantageous because it can be used in clinical sample processing for next generation sequencing (NGS) and act as a control for every step of the workflow. Clinical Sample Processing

[0035] Methods provided herein include, in some embodiments, detecting one or more microbial species in a clinical sample. Such methods include, but are not limited to: (i) obtaining a clinical sample suspected of comprising one or more microbial species from a subject; (ii) introducing an internal process control (IPC) microbe into the clinical sample; (iii) processing the clinical sample with the IPC, including lysing the one or more microbial species and / or the IPC microbe; (iv) amplifying whole genome microbial DNA from the lysed microbial species and / or the lysed IPC microbe; and (v) detecting amplified microbial DNA and / or IPC DNA. The detection of IPC DNA at the end of the method indicates that processing the clinical sample was successful because the IPC was subjected to the same clinical processing steps as the one or more microbial species in a clinical sample. As used herein, suspected means that a clinical sample is from a subject that is thought to have a microbial infection or a subject who has been identified as likely to have a microbial infection. A clinical sample may be directly obtained from a subject as part of a method provided herein. A microbial infection may be with any microbe provided herein.

[0036] In some aspects, the present disclosure provides a method comprising: (i) obtaining a clinical sample suspected of comprising one or more microbial species from a subject; (ii) introducing one or more internal process control (IPC) microbes into the clinical sample; (iii) processing the clinical sample with the one or more IPC microbes, including lysing the one or more microbial species and / or the one or more IPC microbes; (iv) amplifying whole genome microbial DNA from the lysed microbial species and the lysed one or more IPC microbes; and (v) detecting amplified microbial DNA and / or IPC DNA, wherein the detection of IPC DNA in step (v) indicates that there was not a process failure.

[0037] In some aspects, the present disclosure provides a method comprising: (i) obtaining a clinical sample suspected of comprising one or more microbial species from a subject; (ii) introducing an internal process control (IPC) microbe into the clinical sample; (iii) processing the clinical sample to release whole genome microbial DNA from one or more microbes present in the sample including the IPC microbe including lysing the one or more microbial species and / or the IPC microbe; (iv) amplifying the released whole genome microbial DNA from the lysed microbial species and the lysed IPC microbe; and (v) detecting amplified microbial DNA and / or IPC DNA, wherein the detection of amplified IPC DNA in step (v) indicates that there was not a process failure, and wherein step (iii) optionally comprises lysing the one or more microbial species and / or the IPC microbes. In some aspects, the present disclosure provides a method co an internal process control (IPC) microbe into the clinical sample; (ii) processing the clinical sample with the IPC, including lysing the one or more microbial species and / or the IPC microbe; (iii) amplifying whole genome microbial DNA from the lysed microbial species and / or the lysed IPC microbe; and (iv) detecting amplified microbial DNA and / or IPC DNA, wherein the detection of IPC DNA in step (iv) indicates that there was not a process failure.

[0038] Microbial Species

[0039] Methods provided herein include detecting one or more microbial species in a clinical sample. The term “microbial species”, as used herein, is any microorganism that is present in a clinical sample. In some embodiments, the microbial species is a bacterium, a virus, a fungus, a parasite, or any combination thereof. The microbial species present in a sample may be a commensal organism, which is a microorganism (e.g., bacteria, virus, fungus, or parasite) that lives in or on a subject without causing harm to the host or providing any significant benefits, or may be a pathogenic organism, which is a microorganism (e.g., bacteria, virus, fungus, mold, or parasite) that has disease-causing potential. The terms “pathogenic organism” or “pathogenic microbial species”, are used interchangeably herein and refer to a microorganism (e.g., bacteria, virus, fungus, or parasite), that has the ability to cause disease in a subject by invading, colonizing, and / or multiplying within the subject, leading to various illnesses or infections. In some embodiments, a commensal organism can turn into a pathogenic organism in a process referred to as “commensal-to-pathogen transition”. Non-limiting factors contributing to this shift include: i) genetic mutations that may result in the acquisition of new genes or the loss of gene that regulate virulence, increasing the organism’s pathogenic potential; ii) horizontal gene transfer, in which a commensal organism acquires virulence factors or antibiotic resistance genes from another microorganism; iii) changes in environmental conditions, such as antibiotic use or immune system suppression; and, iv) microbiota dysbiosis, in which alterations in a host’s microbiome composition and diversity creates opportunities for commensal organisms to compete with or displace other microorganisms.

[0040] In some embodiments, one or more microbial species in a clinical sample provided herein is a pathogenic microbial species. One microbial species or multiple microbial species in a clinical sample may be pathogenic. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more microbial species are pathogenic microbial species. In some embodiments, the one or more microbial species comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11

[0041] 24, 25, 26, 27, 28, 29, or 30 or more microbial species. In some embodiments, the one or more microbial species comprise commensal microbial species and / or pathogenic microbial species.

[0042] In some embodiments, a pathogenic microbial species is a bacterium. A bacterium is a prokaryote that lacks a true cell nucleus and membrane-bound organelles. Non-limiting examples of pathogenic bacteria include Abiotrophia defective, Acetobacter nitrogenifigens, Achromobacter denitrificans, Achromobacter insolitus, Achromobacter ruhlandii, Achromobacter xylosoxidans, Acidaminococcus intestini, Acidovorax citrulli, Acinetobacter baumannii, Acinetobacter bereziniae, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacter pittii, Acinetobacter radioresistens, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter ursingii, Actinobacillus suis, Actinobacillus ureae, Actinomadura latina, Actinomadura madurae, Actinomyces gerencseriae, Actinomyces graevenitzii, Actinomyces israelii, Actinomyces massiliensis, Actinomyces oris, Actinomyces timonensis, Actinomyces urogenitalis, Actinomyces viscosus, Aerococcus christensenii, Aerococcus sanguinicola, Aerococcus urinae, Aerococcus urinaehominis, Aerococcus viridans, Aeromonas caviae, Aeromonas enteropelogenes, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas schubertii, Aeromonas veronii, Afipia felis, Aggregatibacter actinomycetemcomitans, Aggregatibacter aphrophilus, Aggregatibacter segnis, Agrobacterium tumefaciens, Alcaligenes faecalis, Alloiococcus otitis, Alloscardovia omnicolens, Alysiella crassa, Anaerobiospirillum succiniciproducens, Anaerococcus hydrogenalis, Anaerococcus lactolyticus, Anaerococcus prevotii, Anaerococcus tetradius, Anaeroglobus geminatus, Anaerostipes caccae, Anaplasma phagocytophilum, Arcanobacterium haemolyticum, Aliarcobacter butzleri (Arcobacter butzleri), Aliarcobacter cryaerophilus (Arcobacter cryaerophilus), Aliarcobacter skirrowii (Arcobacter skirrowii), Atlantibacter hermannii (Escherichia hermannii), Lancefieldella parvula (Atopobium parvulum), Lancefieldella rimae (Atopobium rimae), Fannyhessea vaginae (Atopobium vaginae), Aureimonas altamirensis, Azospirillum brasilense (Roseomonas fauriae), Bacillus anthracis, Bacillus cereus, Niallia circulans (Bacillus circulans), Weizmannia coagulans (Bacillus coagulans), Bacillus glycinifermentans, Bacillus licheniformis, Priestia megaterium (Bacillus megaterium), Bacillus mycoides, Bacillus paralicheniformis, Bacillus pumilus, Bacillus safensis, Bacillus subtilis, Bacillus thuringiensis, Bacteroides caccae, Bacteroides eggerthii, Bacteroides faecis, Bacteroides fragilis, Bacteroides ovatus, Bacteroides pyogenes, Bacteroides salyersiae, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Phocaeicola vulgatus (Bacteroides vulgatus), Bartonella ancashensis, Bartonella bacilliformis, Bartonella birtlesii, Bartonella bovis, Bartonella clarridgeiae, Bartonella doshiae, Bartonella elizabethae, Bartonella grahamii, Bartonella henselae, Bartonella koehlerae, Bartonella quintana, Bartonella rattaustraliani, Bartonella rochalimae, Bartonella schoenbuchensis, Bartonella taylorii, Bartonella tribocorum, Bartonella vinsonii, Bergeyella zoohelcum, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium dentium, Bifidobacterium longum, Bifidobacterium psychraerophilum, Bifidobacterium scardovii, Bordetella avium, Bordetella bronchialis, Bordetella bronchiseptica, Bordetella flabilis, Bordetella hinzii, Bordetella holmesii, Bordetella parapertussis, Bordetella pertussis, Bordetella petrii, Bordetella trematum, Borrelia crocidurae, Borrelia duttonii, Borrelia hermsii, Borrelia hispanica, Borreliella mayonii (Borrelia mayonii), Borrelia miyamotoi, Borrelia parkeri, Borrelia persica, Borrelia recurrentis, Borrelia turicatae, Borreliella afzelii (Borrelia afzelii), Borreliella burgdorferi (Borrelia burgdorferi), Borreliella garinii (Borrelia garinii), Brachyspira pilosicoli, Brevibacillus brevis, Brevibacillus laterosporus, Brevibacterium casei, Brevundimonas vesicularis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Budvicia aquatica, Bulleidia extructa, Burkholderia cepacia complex, Burkholderia gladioli, Burkholderia glumae, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia thailandensis, Buttiauxella brennerae, Buttiauxella ferragutiae, Buttiauxella gaviniae, Campylobacter coli, Campylobacter concisus, Campylobacter corcagiensis, Campylobacter cuniculorum, Campylobacter curvus, Campylobacter fetus, Campylobacter gracilis, Campylobacter hominis, Campylobacter hyointestinalis, Campylobacter iguaniorum, Campylobacter jejuni, Campylobacter lari, Campylobacter mucosalis, Campylobacter showae, Campylobacter sputorum, Campylobacter upsaliensis, Campylobacter ureolyticus, Capnocytophaga canimorsus, Capnocytophaga cynodegmi, Capnocytophaga gingivalis, Capnocytophaga granulosa, Capnocytophaga haemolytica, Capnocytophaga ochracea, Capnocytophaga sputigena, Cardiobacterium hominis, Cardiobacterium valvarum, Christensenella hongkongensis (Catabacter hongkongensis), Cedecea davisae, Cedecea neteri, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chromobacterium haemolyticum, Chromobacterium violaceum, Chryseobacterium gleum, Chryseobacterium indologenes, Citrobacter amalonaticus, Citrobacter braakii, Citrobacter farmeri, Citrobacter freundii, Citrobacter koseri, Citrobacter sedlakii, Clostridioides difficile (Clostridium difficile), Clostridium baratii, Clostridium butyricum, Clostridium cadaveris, Clostridium haemolyticum, Clostridium hylemonae, Clostridium innocuum, Clostridium neonatale, Clostridi paraputrificum, Clostridium perfringens, Clostridium tetani, Sarcina ventriculi (Clostridium ventriculi), Comamonas kerstersii, Comamonas terrigena, Corynebacterium accolens, Corynebacterium afermentans, Corynebacterium amycolatum, Corynebacterium argentoratense, Corynebacterium aurimucosum, Corynebacterium diphtheriae, Corynebacterium falsenii, Corynebacterium freiburgense, Corynebacterium freneyi, Corynebacterium glucuronolyticum, Corynebacterium halotolerans, Corynebacterium jeikeium, Corynebacterium kroppenstedtii, Corynebacterium kutscheri, Corynebacterium lipophiloflavum, Corynebacterium massiliense, Corynebacterium matruchotii, Corynebacterium minutissimum, Corynebacterium otitidis (Turicella otitidis), Corynebacterium propinquum, Corynebacterium pseudodiphtheriticum, Corynebacterium pseudotuberculosis, Corynebacterium renale, Corynebacterium riegelii, Corynebacterium simulans, Corynebacterium stationis, Corynebacterium striatum, Corynebacterium timonense, Corynebacterium tuscaniense, Corynebacterium ulcerans, Corynebacterium urealyticum, Corynebacterium ureicelerivorans, Corynebacterium xerosis, Coxiella burnetii, Cronobacter sakazakii (Enterobacter sakazakii), Cupriavidus gilardii, Cupriavidus metallidurans, Cupriavidus pauculus, Cutibacterium granulosum (Propionibacterium granulosum), Delftia acidovorans, Dermabacter hominis, Dermacoccus nishinomiyaensis, Dermatophilus congolensis, Desulfomicrobium orale, Dialister micraerophilus, Dielma fastidiosa, Dolosigranulum pigrum, Dysgonomonas capnocytophagoides, Dysgonomonas gadei, Dysgonomonas hofstadii, Dysgonomonas mossii, Edwardsiella hoshinae, Edwardsiella tarda, Eggerthella lenta, Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia muris, Eikenella corrodens, Elizabethkingia anophelis, Elizabethkingia meningoseptica, Elizabethkingia miricola, Empedobacter brevis, Empedobacter falsenii, Enterobacter cloacae complex, Enterobacter mori, Enterocloster clostridioformis (Clostridium clostridioforme), Enterococcus asini, Enterococcus avium, Enterococcus casselijlavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus gilvus, Enterococcus haemoperoxidus, Enterococcus hirae, Enterococcus italicus, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pallens, Enterococcus phoeniculicola, Enterococcus pseudoavium, Enterococcus raffinosus, Enterococcus saccharolyticus, Enterococcus sulfureus, Enterococcus thailandicus, Erwinia billingiae, Erwinia gerundensis, Erysipelothrix rhusiopathiae, Escherichia albertii, Escherichia coli, Escherichia fergusonii, Eubacterium limosum, Eubacterium nodatum, Facklamia hominis, Facklamia sourekii, Faecalicoccus pleomorphus, Fenollaria massiliensis, Filifactor alocis, Finegoldia j bozemanae (Legionella bozemanae), Fluoribacter dumoffii (Legionella dumoffii), Francisella hispaniensis, Francisella noatunensis, Francisella philomiragia, Francisella tularensis, Franconibacter helveticus, Fusobacterium mortiferum, Fusobacterium necrophorum, Fusobacterium nucleatum, Fusobacterium periodonticum, Fusobacterium russii, Fusobacterium ulcerans, Fusobacterium varium, Gardnerella vaginalis, Gemella bergeri, Gemella haemolysans, Gemella morbillorum, Gemella sanguinis, Gleimia europaea (Actinomyces europaeus), Globicatella sanguinis, Gordonia aichiensis, Gordonia bronchialis, Gordonia otitidis, Gordonia rubripertincta, Gordonia terrae, Gordonibacter pamelaeae, Granulibacter bethesdensis, Granulicatella adiacens, Granulicatella elegans, Grimontia hollisae, Haemophilus aegyptius, Haemophilus ducreyi, Haemophilus haemolyticus, Haemophilus influenzae, Haemophilus parahaemolyticus, Haemophilus parainjluenzae, Haemophilus paraphrohaemolyticus, Haemophilus quentini, Haemophilus sputorum, Hafnia alvei, Hafnia paralvei, Helcococcus kunzii, Helicobacter bills, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter felis, Helicobacter fennelliae, Helicobacter heilmannii, Helicobacter magdeburgensis, Helicobacter pylori, Janibacter indicus, Janibacter melonis, Johnsonella ignava, Jonesia denitrificans, Kerstersia gyiorum, Kingella denitrificans, Kingella kingae, Kingella oralis, Klebsiella aerogenes (Enterobacter aerogenes), Klebsiella michiganensis, Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella variicola, Kluyvera ascorbata, Kluyvera cryocrescens, Kluyvera intermedia, Kocuria rhizophila, Kurthia huakuii, Kurthia massiliensis, Kytococcus sedentarius, Lactobacillus acidophilus, Limosilactobacillus antri (Lactobacillus antri), Lacticaseibacillus casei (Lactobacillus casei), Lactobacillus crispatus, Limosilactobacillus fermentum (Lactobacillus fermentum), Lactobacillus gasseri, Lactobacillus iners, Lactobacillus jensenii, Lactiplantibacillus paraplantarum (Lactobacillus paraplantarum), Lactiplantibacillus plantarum (Lactobacillus plantarum), Limosilactobacillus pontis (Lactobacillus pontis), Lacticaseibacillus rhamnosus (Lactobacillus rhamnosus), Ligilactobacillus saerimneri (Lactobacillus saerimneri), Latilactobacillus sakei (Lactobacillus sakei), Lactobacillus ultunensis, Lactococcus garvieae, Laribacter hongkongensis, Lawsonella clevelandensis, Lawsonia intracellularis, Leclercia adecarboxylata, Legionella adelaidensis, Legionella anisa, Legionella birminghamensis, Legionella brunensis, Legionella cherrii, Legionella cincinnatiensis, Legionella clemsonensis, Legionella drancourtii, Legionella drozanskii, Legionella erythra, Legionella fairfieldensis, Legionella fallonii, Legionella feeleii, Legionella geestiana, Legionella hackeliae, Legionella israelensis, Legionella jamestowniensis, Legic

[0043] Legionella lansingensis, Legionella londiniensis, Legionella longbeachae, Legionella maceachernii, Legionella massiliensis, Legionella moravica, Legionella nautarum, Legionella norrlandica, Legionella oakridgensis, Legionella parisiensis, Legionella pneumophila, Legionella quateirensis, Legionella quinlivanii, Legionella rubrilucens, Legionella sainthelensi, Legionella santicrucis, Legionella shakespearei, Legionella spiritensis, Legionella steelei, Legionella tucsonensis, Legionella tunisiensis, Legionella wadsworthii, Legionella waltersii, Legionella worsleiensis, Leifsonia aquatica, Lelliottia amnigena (Enterobacter amnigenus), Leminorella grimontii, Leptospira alexanderi, Leptospira alstonii, Leptospira biflexa, Leptospira borgpetersenii, Leptospira broomii, Leptospira fainei, Leptospira inadai, Leptospira interrogans, Leptospira kirschneri, Leptospira kmetyi, Leptospira licerasiae, Leptospira mayottensis, Leptospira meyeri, Leptospira noguchii, Leptospira santarosai, Leptospira terpstrae, Leptospira vanthielii, Leptospira weilii, Leptospira wolbachii, Leptospira yanagawae, Leptotrichia buccalis, Pseudoleptotrichia goodfellowii (Leptotrichia goodfellow ii), Leptotrichia shahii, Leptotrichia wadei, Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria monocytogenes, Listeria seeligeri, Listeria welshimeri, Lysinibacillus sphaericus (Bacillus sphaericus), Macrococcus caseolyticus (Staphylococcus caseolyticus), Mannheimia haemolytica, Megasphaera micronuciformis, Microbacterium foliorum, Microbacterium maritypicum, Microbacterium oxydans, Microbacterium paraoxydans, Microbacterium testaceum, Micrococcus luteus, Micrococcus lylae, Mitsuokella multacida, Mobiluncus curtisii, Mobiluncus mulieris, Moellerella wisconsensis, Mogibacterium timidum, Moraxella atlantae, Moraxella catarrhalis, Moraxella lacunata, Moraxella lincolnii, Moraxella nonliquefaciens, Morganella morganii, Morococcus cerebrosus, Mycobacterium asiaticum, Mycobacterium avium complex (MAC), Mycobacterium celatum, Mycobacterium chimaera, Mycobacterium gastri, Mycobacterium genavense, Mycobacterium gordonae, Mycobacterium grossiae, Mycobacterium haemophilum, Mycobacterium heckeshornense, Mycobacterium intermedium, Mycobacterium kansasii, Mycobacterium kyorinense, Mycobacterium leprae, Mycobacterium lepromatosis, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium nebraskense, Mycobacterium paraffmicum, Mycobacterium parascrofulaceum, Mycobacterium scrofulaceum, Mycobacterium sherrisii, Mycobacterium shigaense, Mycobacterium shimoidei, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium talmoniae, Mycobacterium triplex, Mycobacterium tuberculosis complex, Mycobacterium xenopi, Mycobacteroides abs< abscessus), Mycobacteroides chelonae (Mycobacterium chelonae), Mycobacteroides franklinii (Mycobacterium franklinii), Mycobacteroides immunogenum (Mycobacterium immunogenum), Mycobacteroides saopaulense (Mycobacterium saopaulense), Mycolicibacillus koreensis (Mycobacterium koreense), Mycolicibacter arupensis (Mycobacterium arupense), Mycolicibacter heraklionensis (Mycobacterium heraklionense), Mycolicibacter kumamotonensis (Mycobacterium kumamotonense), Mycolicibacterium aurum, Mycolicibacterium brisbanense (Mycobacterium brisbanense), Mycolicibacterium canariasense (Mycobacterium canariasense), Mycolicibacterium chlorophenolicum, Mycolicibacterium chubuense, Mycolicibacterium conceptionense (Mycobacterium conceptionense), Mycolicibacterium cosmeticum (Mycobacterium cosmeticum), Mycolicibacterium diernhoferi (Mycobacterium diernhoferi), Mycolicibacterium elephantis (Mycobacterium elephantis), Mycolicibacterium flavescens (Mycobacterium flavescens), Mycolicibacterium fortuitum (Mycobacterium fortuitum), Mycolicibacterium goodii (Mycobacterium goodii), Mycolicibacterium hassiacum, Mycolicibacterium holsaticum (Mycobacterium holsaticum), Mycolicibacterium iranicum (Mycobacterium iranicum), Mycolicibacterium llatzerense (Mycobacterium llatzerense), Mycolicibacterium mageritense (Mycobacterium mageritense), Mycolicibacterium mucogenicum (Mycobacterium mucogenicum), Mycolicibacterium neoaurum (Mycobacterium neoaurum), Mycolicibacterium novocastrense (Mycobacterium novocastrense), Mycolicibacterium obuense (Mycobacterium obuense), Mycolicibacterium peregrinum (Mycobacterium peregrinum), Mycolicibacterium phlei (Mycobacterium phlei), Mycolicibacterium septicum (Mycobacterium septicum), Mycolicibacterium setense (Mycobacterium setense), Mycolicibacterium smegmatis (Mycobacterium smegmatis), Mycolicibacterium thermoresistibile (Mycobacterium thermoresistibile), Mycolicibacterium tusciae (Mycobacterium tusciae), Mycolicibacterium vaccae (Mycobacterium vaccae), Mycolicibacterium wolinskyi (Mycobacterium wolinskyi), Mycoplasmopsis arginini (Mycoplasma arginini), Mycoplasma capricolum, Mycoplasmopsis cynos (Mycoplasma cynos), Mycoplasmopsis fermentans (Mycoplasma fermentans), Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma hyopneumoniae, Mycoplasma orale, Mycoplasma penetrans, Mycoplasma pirum, Mycoplasma pneumoniae, Mycoplasmopsis pulmonis (Mycoplasma pulmonis), Myroides marinus, Myroides odoratimimus, Myroides odoratus, Neisseria animaloris, Neisseria bacilliformis, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria gonorrhoeae, Neisseria lactamica, Neisseria meningitidis, Neisseria mucosa, Neisseria polysaccharea, Neisseria sicca, Neissei

[0044] Neisseria weaveri, Neorickettsia helminthoeca, Neorickettsia sennetsu, Nocardia abscessus, Nocardia acidivorans, Nocardia africana, Nocardia alba, Nocardia amamiensis, Nocardia anaemiae, Nocardia aobensis, Nocardia araoensis, Nocardia arizonensis, Nocardia arthritidis, Nocardia asiatica, Nocardia beijingensis, Nocardia brasiliensis, Nocardia brevicatena, Nocardia caishijiensis, Nocardia carnea, Nocardia cerradoensis, Nocardia concava, Nocardia coubleae, Nocardia crassostreae, Nocardia cummidelens, Nocardia cyriacigeorgica, Nocardia elegans, Nocardia exalbida, Nocardia farcinica, Nocardia flavorosea, Nocardia fusca, Nocardia gamkensis, Nocardia grenadensis, Nocardia harenae, Nocardia higoensis, Nocardia ignorata, Nocardia inohanensis, Nocardia jejuensis, Nocardia jiangxiensis, Nocardia kruczakiae, Nocardia lijiangensis, Nocardia mexicana, Nocardia mikamii, Nocardia miyunensis, Nocardia niigatensis, Nocardia niwae, Nocardia nova, Nocardia otitidiscaviarum, Nocardia paucivorans, Nocardia pneumoniae, Nocardia pseudobrasiliensis, Nocardia pseudovaccinii, Nocardia puris, Nocardia rhamnosiphila, Nocardia salmonicida, Nocardia seriolae, Nocardia shimofusensis, Nocardia sienata, Nocardia soli, Nocardia speluncae, Nocardia takedensis, Nocardia tenerifensis, Nocardia terpenica, Nocardia testacea, Nocardia thailandica, Nocardia transvalensis, Nocardia uniformis, Nocardia vaccinii, Nocardia vermiculata, Nocardia veterana, Nocardia vinacea, Nocardia violaceofusca, Nocardia vulneris, Nocardia xishanensis, Nocardia yamanashiensis, Nocardiopsis dassonvillei (Nocardia dassonvillei), Obesumbacterium proteus, Brucella anthropi (Ochrobactrum anthropi), Brucella intermedia (Ochrobactrum intermedium), Brucella oryzae (Ochrobactrum oryzae), Odoribacter laneus, Odoribacter splanchnicus, Oerskovia turbata, Oligella ureolytica, Oligella urethralis, Olsenella uli, Oribacterium sinus, Orientia tsutsugamushi, Oscillibacter ruminantium, Paenalcaligenes hominis, Paenibacillus alvei, Paeniclostridium sordellii (Clostridium sordellii), Pandoraea apista, Pandoraea pulmonicola, Pandoraea sputorum, Pannonibacter phragmitetus, Pantoea agglomerans, Pantoea ananatis, Parabacteroides distasonis (Bacteroides distasonis), Parabacteroides goldsteinii, Parabacteroides gordonii, Parabacteroides johnsonii, Parabacteroides merdae (Bacteroides merdae), Paraburkholderia fimgorum, Parachlamydia acanthamoebae, Paraclostridium bifermentans (Clostridium bifermentans), Paracoccus sanguinis, Paracoccus yeei, Parvimonas micra, Pasteurella bettyae, Pasteurella multocida, Pediococcus acidilactici, Pediococcus pentosaceus, Pelobacter propionicus, Peptoniphilus coxii, Peptoniphilus duerdenii, Peptoniphilus harei, Peptoniphilus indolicus, Peptoniphilus lacrimalis, Peptoniphilus rhinitidis, Peptoniphilus senegalensis, Peptostreptococcus anaerobius, Peptostreptococcus stomatis, Photobacterium damselae asymbiotica, Photorhabdus luminescens, Plesiomonas shigelloides, Pluralibacter gergoviae, Porphyromonas asaccharolytica, Porphyromonas gingivalis, Porphyromonas gingivicanis, Prevotella bivia, Prevotella buccae, Prevotella buccalis, Prevotella corporis, Prevotella denticola, Prevotella disiens, Prevotella intermedia, Prevotella loescheii, Prevotella melaninogenica, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Propionibacterium acidifaciens, Cutibacterium namnetense (Propionibacterium namnetense), Propionimicrobium lymphophilum (Corynebacterium lymphophilum), Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Pseudescherichia vulneris (Escherichia vulneris), Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas cannabina, Pseudomonas citronellolis, Pseudomonas fluorescens, Pseudomonas fulva, Pseudomonas luteola, Pseudomonas mendocina, Pseudomonas mosselii, Pseudomonas oryzihabitans, Pseudomonas poae, Pseudomonas protegens, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas veronii, Arachnia propionica (Propionibacterium propionicum), Pseudoramibacter alactolyticus, Psychrobacter cryohalolentis, Psychrobacter phenylpyruvicus (Moraxella phenylpyruvica), Rahnella aquatilis, Ralstonia insidiosa, Ralstonia mannitolilytica, Raoultella ornithinolytica, Raoultella planticola, Rhodococcus erythropolis, Rhodococcus fascians, Rhodococcus hoagii (Rhodococcus equi), Rhodococcus rhodochrous, Rickettsia akari, Rickettsia amblyommatis, Rickettsia australis, Rickettsia canadensis, Rickettsia conorii, Rickettsia felis, Rickettsia helvetica, Rickettsia honei, Rickettsia japonica, Rickettsia massiliae, Rickettsia monacensis, Rickettsia parkeri, Rickettsia prowazekii, Rickettsia raoultii, Rickettsia rickettsii, Rickettsia sibirica, Rickettsia slovaca, Rickettsia typhi, Riemerella anatipestifer, Robinsoniella peoriensis, Rodentibacter pneumotropicus (Pasteurella pneumotropica), Roseobacter denitrificans, Roseomonas cervicalis, Roseomonas gilardii, Roseomonas mucosa, Rothia aeria, Rothia dentocariosa, Rothia kristinae (Kocuria kristinae), Rothia mucilaginosa, Rouxiella chamberiensis, Saccharopolyspora rectivirgula (Micropolyspora faeni), Salmonella bongori, Salmonella enterica, Sanguibacteroides justesenii, Scardovia wiggsiae, Schaalia cardiffensis (Actinomyces cardiffensis), Schaalia georgiae (Actinomyces georgiae), Schaalia meyeri (Actinomyces meyeri), Schaalia odontolytica (Actinomyces odontolyticus), Schaalia turicensis (Actinomyces turicensis), Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Serratia odorifera, Serratia plymuthica, Serratia rubidaea, Serratia ureilytica, Shewanella algae, Shewanella putrefaciens, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Shimwellia blattae (Escherichia blattae), Siccibacter turicensis, Slackia exigua, Sneathia vaginalis (Leptotrichia amnioni. sanguinegens, Solobacterium moorei, Sphingobacterium spiritivorum, Staphylococcus agnetis, Staphylococcus argenteus, Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus chromogenes, Staphylococcus cohnii, Staphylococcus condimenti, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus gallinarum, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus hyicus, Mammaliicoccus lentus (Staphylococcus lentus), Staphylococcus lugdunensis, Staphylococcus pasteuri, Staphylococcus pettenkoferi, Staphylococcus pseudintermedius, Staphylococcus saprophyticus, Staphylococcus schleiferi, Mammaliicoccus sciuri (Staphylococcus sciuri), Staphylococcus simiae, Staphylococcus simulans, Staphylococcus succinus, Mammaliicoccus vitulinus (Staphylococcus vitulinus), Staphylococcus warneri, Staphylococcus xylosus, Stenotrophomonas acidaminiphila, Stenotrophomonas maltophilia, Streptobacillus moniliformis, Streptococcus acidominimus, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus canis, Streptococcus constellatus, Streptococcus criceti, Streptococcus cristatus, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus ferus, Streptococcus gallolyticus, Streptococcus gordonii, Streptococcus hyovaginalis, Streptococcus infantarius, Streptococcus infantis, Streptococcus iniae, Streptococcus intermedins, Streptococcus lutetiensis, Streptococcus macacae, Streptococcus macedonicus, Streptococcus massiliensis, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus oralis subsp. dentisani (Streptococcus dentisani), Streptococcus oralis subsp. tigurinus (Streptococcus tigurinus), Streptococcus parasanguinis, Streptococcus pasteurianus, Streptococcus peroris, Streptococcus pneumoniae, Streptococcus porcinus, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus suis, Streptococcus thermophilus, Streptococcus uberis, Streptococcus vestibularis, Streptomyces cattleya, Streptomyces somaliensis, Sutterella wadsworthensis, Tannerella forsythia (Bacteroides forsythus), Tatlockia micdadei (Legionella micdadei), Tatumella ptyseos, Taylorella asinigenitalis, Taylorella equigenitalis, Terrisporobacter othiniensis, Treponema pallidum, Tropheryma whipplei, Trueperella bernardiae (Arcanobacterium bernardiae), Trueperella pyogenes (Arcanobacterium pyogenes), Tsukamurella paurometabola, Tsukamurella pulmonis, Tsukamurella tyrosinosolvens, Ureaplasma parvum, Ureaplasma urealyticum, Veillonella dispar, Veillonella montpellierensis, Veillonella parvula, Veillonella seminalis, Vibrio albensis, Vibrio alginolyticus, Vibrio cholerae, Vibrio fluvialis, Vibri harveyi, Vibrio metschnikovii, Vibrio mimicus, Vibrio navarrensis, Vibrio parahaemolyticus, Vibrio vulnificus, Waddlia chondrophila, Weeksella virosa, Weissella confusa, Weissella paramesenteroides, Weissella viridescens, Winkia neuii (Actinomyces neuii), Wohlfahrtiimonas chitiniclastica, Wolbachia pipientis, Xanthomonas axonopodis, Xylanimonas cellulosilytica, Yersinia enterocolitica, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia ruckeri, Yokenella regensburgei, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium canettii, Mycobacterium tuberculosis, Burkholderia ambifaria, Burkholderia anthina, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia contaminans, Burkholderia diffusa, Burkholderia dolosa, Burkholderia lata, Burkholderia latens, Burkholderia multivorans, Burkholderia pseudomultivorans, Burkholderia pyrrocinia, Burkholderia seminalis, Burkholderia stabilis, Burkholderia stagnalis, Burkholderia territorii, Burkholderia vietnamiensis, Mycobacterium avium, Mycobacterium colombiense, Mycobacterium indicus pranii, Mycobacterium intr acellular e, Mycobacterium vulneris, Mycobacterium yongonense, Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter cloacae, Enterobacter cloacae complex 'Hoffmann cluster III', Enterobacter cloacae complex 'Hoffmann cluster IV', Enterobacter hormaechei, Enterobacter kobei, Enterobacter lignolyticus, Enterobacter ludwigii, and Enterobacter xiangfangensis .

[0045] In some embodiments, the one or more microbial species are selected from: Acinetobacter baumannii, Acinetobacter ursingii, Aggregatibacter aphrophilus, Bacillus cereus, Bacteroides fragilis, Bacteroides vulgatus, Campylobacter coli, Campylobacter jejuni, Cardiobacterium hominis, Citrobacter freundii, Citrobacter koseri, Clostridium perfringens, Eikenella corrodens, Enterobacter cloacae, Enterococcus avium, Enterococcus casseliflavus, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus raffinosus, Escherichia coli, Haemophilus influenzae, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, Lactobacillus rhamnosus, Listeria monocytogenes, Morganella morganii, Mycobacterium tuberculosis, Pantoea agglomerans, Pasteurella multocida, Propionibacterium acnes, Proteus mirabilis, Protues vulgaris, Pseudomonas aeruginosa, Pseudomonas putida, Raoultella ornithinolytica, Salmonella enterica, Serratia liquefaciens, Serratia marcescens, Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Staphylococcus simulans, Staphylococcus warneri, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus c dysgalactiae, Streptococcus intermedins. Streptococcus mutans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, and Streptococcus sanguinis.

[0046] In some embodiments, a pathogenic microbial species is a virus. A virus is a microscopic infectious agent that contains genetic material (e.g., DNA or RNA) and a protein coat (e.g., capsid). In some embodiments, the virus is an RNA virus or a DNA virus. An RNA virus contains RNA as its genetic material, and a DNA virus contains DNA as its genetic material. In some embodiments where a genetic material is RNA, a method provided herein comprises a reverse transcription step. Reverse transcription is synthesis of a new DNA molecule by the enzyme reverse transcriptase.

[0047] In embodiments of the present disclosure where a pathogenic microbial species is a virus, a method provided herein comprises lysing the virus. Lysing the virus may be by any method known in the art including, but not limited to: chemical lysis (e.g., sodium dodecyl sulfate (SDS) treatment, EDTA treatment, surfactant treatment), temperature lysis (e.g., repeated freeze / thaw cycles), mechanical lysis (e.g., homogenizer, bead beating), or some combination thereof.

[0048] In some embodiments, the RNA virus is a single-stranded RNA virus or a doublestranded RNA virus. In some embodiments, the single-stranded RNA virus is a singlestranded positive-sense RNA virus that can be immediately translated into protein by a host cell (e.g., a cell in a subject). In some embodiments, the single-stranded RNA virus is a single-stranded negative-sense RNA virus that must be converted to positive-sense RNA prior to being translated into protein by a host cell (e.g., a cell in a subject). In some embodiments, the DNA virus is a single-stranded DNA virus, a partially double-stranded DNA virus, or a double-stranded DNA virus. Non-limiting examples of pathogenic viruses include Adeno- associated dependoparvovirus A, Adeno-associated dependoparvovirus B, Alphapapillomavirus 9, BK polyomavirus, Betapapillomavirus 1, Betapapillomavirus 2, Cowpox virus, Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Gammapapillomavirus 1, Gammapapillomavirus 10, Gammapapillomavirus 11, Gammapapillomavirus 13, Gammapapillomavirus 14, Gammapapillomavirus 15, Gammapapillomavirus 16, Gammapapillomavirus 17, Gammapapillomavirus 19, Gammapapillomavirus 2, Gammapapillomavirus 3, Gammapapillomavirus 4, Gammapapillomavirus 5, Gammapapillomavirus 6, Gammapapillomavirus 7, Gammapapillomavirus 8, Gammapapillomavirus 9, Herpes B virus, Herpes simplex virus type 1 (HSV-1), Herpes simplex virus type 2 (HSV-2), Human adenovirus A, Human adenov adenovirus C, Human adenovirus D, Human adenovirus E, Human adenovirus F, Human bocavirus, Human herpesvirus 6A, Human herpesvirus 6B, Human herpesvirus 7, Human papillomavirus, Human papillomavirus 1, Human papillomavirus 10, Human papillomavirus 132, Human papillomavirus 136, Human papillomavirus 140, Human papillomavirus 154, Human papillomavirus 167, Human papillomavirus 18, Human papillomavirus 2, Human papillomavirus 26, Human papillomavirus 32, Human papillomavirus 34, Human papillomavirus 41, Human papillomavirus 49, Human papillomavirus 53, Human papillomavirus 6, Human papillomavirus 61, Human papillomavirus 63, Human papillomavirus 7, Human papillomavirus 90, Human papillomavirus 92, Human papillomavirus 96, Human parvovirus Bl 9, Human polyomavirus 6, Human polyomavirus 7, JC polyomavirus, KI polyomavirus, Kaposi sarcoma-associated herpesvirus, MW polyomavirus, Merkel cell polyomavirus, Molluscum contagiosum virus, Monkeypox virus, Orf virus, Porcine circovirus 1, Porcine circovirus 2, Primate bocaparvovirus 1, Primate bocaparvovirus 2, Primate tetraparvovirus 1 (human PARV-4), Pseudocowpox virus, STL polyomavirus, Tanapox virus, Torque teno virus, Torque teno virus 1, Torque teno virus 10, Torque teno virus 12, Torque teno virus 14, Torque teno virus 15, Torque teno virus 16, Torque teno virus 19, Torque teno virus 2, Torque teno virus 25, Torque teno virus 26, Torque teno virus 27, Torque teno virus 28, Torque teno virus 3, Torque teno virus 4, Torque teno virus 6, Torque teno virus 7, Torque teno virus 8, Trichodysplasia spinulosa-associated polyomavirus, Vaccinia virus, Varicella-zoster virus (VZV), Variola virus, WU Polyomavirus, and Yaba monkey tumor virus.

[0049] In some embodiments, a pathogenic microbial species is a fungus. In some embodiments, the fungus is a mold. A fungus is a eukaryotic microorganism, with a defined nucleus and membrane-bound organelles. Non-limiting examples of pathogenic fungi include Absidia glauca, Absidia repens, Acremonium chrysogenum, Furcasterigmium furcatum (Acremonium furcatum), Actinomucor elegans, Alternaria alternata, Alternaria arborescens, Alternaria brassicicola, Anncaliia algerae, Apiotrichum porosum (Trichosporon porosum), Apophysomyces elegans, Apophysomyces trapeziformis, Apophysomyces variabilis, Aspergillus aculeatus, Aspergillus arachidicola, Aspergillus bombycis, Aspergillus brasiliensis, Aspergillus calidoustus, Aspergillus campestris, Aspergillus candidus, Aspergillus carbonarius, Aspergillus chevalieri, Aspergillus clavatus, Aspergillus cristatus, Aspergillus fischeri, Aspergillus jlavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus hancockii, Aspergillus lentulus, Aspergillus luchuensis, Aspergillus nidulans, Aspergillus niger, Aspergillus nomiae, Aspergillus novofumigatus, A. ochraceoroseus, Aspergillus oryzae, Aspergillus parasiticus, Aspergillus persii, Aspergillus pseudoterreus, Aspergillus rambellii, Aspergillus ruber, Aspergillus sclerotiorum, Aspergillus sojae, Aspergillus steynii, Aspergillus sydowii, Aspergillus taichungensis, Aspergillus terreus, Aspergillus thermomutatus, Aspergillus tubingensis, Aspergillus turcosus, Aspergillus udagawae, Aspergillus ustus, Aspergillus versicolor, Aspergillus wentii, Aspergillus westerdijkiae, Aureobasidium melanogenum, Aureobasidium namibiae, Aureobasidium pullulans, Aureobasidium subglaciale, Basidiobolus meristosporus, Beauveria bassiana, Beauveria rudraprayagi, Blastomyces dermatitidis, Blastomyces percursus, Paecilomyces variotii (Byssochlamys spectabilis), Candida aaseri, Candida albicans, Candida arabinofermentans, Candida auris, Candida boidinii, Candida bracarensis, Candida castellii, Candida dubliniensis, Candida duobushaemulonis, Candida ethanolica, Candida glabrata, Candida haemulonis, Candida intermedia, Candida ipomoeae, Candida nivariensis, Candida orthopsilosis, Candida parapsilosis, Candida pseudohaemulonis, Candida psychrophila, Candida sojae, Candida sorboxylosa, Candida succiphila, Yamadazyma tenuis (Candida tenuis), Candida tropicalis, Ceratocystis adiposa, Ceratocystis albifundus, Ceratocystis eucalypticola, Ceratocystis fimbriata, Ceratocystis manginecans, Ceratocystis platani, Chaetomium globosum, Chaetomium thermophilum, Chrysosporium queenslandicum, Cladophialophora bantiana, Cladophialophora carrionii, Cladophialophora immunda, Cladophialophora psammophila, Cladophialophora yegresii, Cladosporium cladosporioides, Clavispora lusitaniae (Candida lusitaniae), Coccidioides immitis, Coccidioides posadasii, Cokeromyces recurvatus, Colletotrichum acutatum, Colletotrichum falcatum, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Colletotrichum godetiae, Colletotrichum graminicola, Colletotrichum higginsianum, Colletotrichum incanum, Colletotrichum nymphaeae, Colletotrichum orbiculare, Colletotrichum salicis, Colletotrichum simmondsii, Colletotrichum sublineola, Colletotrichum tofieldiae, Conidiobolus coronatus, Conidiobolus incongruus, Coniosporium apollinis, Corynespora cassiicola, Cryptococcus gattii VGI (Cryptococcus gattii), Cryptococcus gattii VGII (Cryptococcus deuterogattii), Cryptococcus gattii VGIII (Cryptococcus bacillisporus), Cryptococcus gattii VGIV (Cryptococcus tetragattii), Cryptococcus neoformans, Cunninghamella, Curvularia lunata, Curvularia papendorfii (Bipolaris papendorfii), Cutaneotrichosporon cutaneum (Trichosporon cutaneum), Cutaneotrichosporon oleaginosum (Trichosporon oleaginosus), Cyberlindnera fabianii (Hansenula fabianii), Cyberlindnera jadinii (Candida utilis), Cyphellophora europaea, Debaryomyces fabryi, Debaryomyces hansenii (Candida famata), D

[0050] Diaporthe aspalathi, Diaporthe longicolla, Emmonsia crescens, Blastomyces silverae (Emmonsia parva), Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis, Encephalitozoon romaleae, Enterocytozoon bieneusi, Exophiala alcalophila, Exophiala aquamarina, Exophiala calicioides, Exophiala dermatitidis, Exophiala mesophila, Exophiala oligosperma, Exophiala sideris, Exophiala spinifera, Exophiala xenobiotica, Filobasidium wieringae, Fonsecaea erecta, Fonsecaea monophora, Fonsecaea multimorphosa, Fonsecaea nubica, Fonsecaea pedrosoi, Fusarium agapanthi, Fusarium asiaticum, Fusarium avenaceum, Fusarium circinatum, Fusarium culmorum, Fusarium euwallaceae, Fusarium fujikuroi, Fusarium graminearum, Fusarium hostae, Fusarium langsethiae, Fusarium mangiferae, Fusarium meridionale, Fusarium nygamai, Fusarium oxysporum, Fusarium pininemorale, Fusarium poae, Fusarium praegraminearum, Fusarium proliferatum, Fusarium pseudograminearum, Fusarium sambucinum, Fusarium solani, Fusarium temperatum, Fusarium udum, Fusarium verticillioides, Geotrichum candidum, Graphilbum fragrans, Hanseniaspora uvarum, Histoplasma capsulatum, Hortaea werneckii, Hyphopichia homilentoma (Candida homilentoma), Kluyveromyces lactis, Kluyveromyces marxianus (Candida kefyr), Kwoniella bestiolae (Cryptococcus bestiolae), Kwoniella dejecticola (Cryptococcus dejecticola), Kwoniella pini (Cryptococcus pinus), Lachancea kluyveri, Lachancea lanzarotensis, Lachancea thermotolerans, Lachancea waltii, Leptosphaeria maculans, Lichtheimia corymbifera, Lichtheimia ramosa, Lodderomyces elongisporus, Lomentospora prolificans, Macrophomina phaseolina, Madurella mycetomatis, Malassezia caprae, Malassezia cuniculi, Malassezia dermatis, Malassezia equina, Malassezia furfur, Malassezia globosa, Malassezia japonica, Malassezia nana, Malassezia obtusa, Malassezia pachydermatis, Malassezia sloofftae, Malassezia sympodialis, Malassezia yamatoensis, Memnoniella echinata, Metarhizium acridum, Metarhizium album, Metarhizium anisopliae, Metarhizium brunneum, Metarhizium guizhouense, Metarhizium majus, Metarhizium rileyi, Metarhizium robertsii, Metschnikowia bicuspidata, Metschnikowia fructicola, Metschnikowia kipukae (Candida kipukae), Meyerozyma carpophila (Candida carpophila), Microsporum canis (Arthroderma otae), Mortierella alpina, Linnemannia elongata (Mortierella elongata), Podila verticillata (Mortierella verticillata), Mucor ambiguus, Mucor circinelloides, Mucor indicus, Mucor irregularis, Mucor velutinosus, Nakaseomyces bacillisporus, Nakaseomyces delphensis, Nakazawaea peltata, Nannizzia gypsea (Microsporum gypseum), Naumovozyma dairenensis, Fusarium vanettenii (Nectria haematococca), Neofusicoccum parvum, Nigrograna mackinnonii (Biatriospora mackinnonii), Nosema apis, Nosema bombycis, Nosema ceranae, Oc

[0051] Ogataea methanolica, Ogataea parapolymorpha, Ogataea polymorpha, Ophiostoma novo- ulmi, Ophiostoma piceae, Samsoniella hepiali (Paecilomyces hepiali), Paracoccidioides brasiliensis, Paracoccidioides lutzii, Pascua guehoae (Trichosporon guehoae), Penicillium antarcticum, Penicillium brasilianum, Penicillium capsulatum, Penicillium carneum, Penicillium coprophilum, Penicillium decumbens, Penicillium digitatum, Penicillium expansum, Penicillium flavigenum, Penicillium freii, Penicillium griseofulvum, Penicillium italicum, Penicillium janthinellum, Penicillium nalgiovense, Penicillium nordicum, Penicillium occitanis, Penicillium oxalicum, Penicillium paneum, Penicillium paxilli, Penicillium roqueforti, Penicillium sclerotiorum, Penicillium steckii, Penicillium subrubescens, Penicillium vulpinum, Phaeoacremonium minimum, Phaeotremella fagi (Cryptococcus fagi), Phaeotremella skinner i (Cryptococcus skinneri), Phanerochaete carnosa, Phanerodontia chrysosporium (Phanerochaete chrysosporium), Pyrrhoderma noxium (Phellinus noxius), Phialophora americana (Capronia semi-immersa), Phialophora attinorum (Phialophora attae), Phoma herbarum, Phycomyces blakesleeanus, Pichia kudriavzevii (Candida krusei), Pneumocystis carinii, Pneumocystis jirovecii, Pneumocystis murina, Pseudocercospora fijiensis (Cercospora fijiensis), Pseudopyrenochaeta lycopersici (Pyrenochaeta lycopersici), Pseudozyma hubeiensis, Purpureocillium lilacinum, Rasamsonia emersonii, Rhinocladiella mackenziei (Ramichloridium mackenziei), Rhizoctonia solani, Rhizomucor miehei, Rhizomucor pusillus, Rhizomucor variabilis, Rhizopus delemar, Rhizopus microsporus, Rhizopus arrhizus (Rhizopus oryzae), Rhizopus stolonifer, Rhodotorula graminis, Rhodotorula mucilaginosa, Rhodotorula toruloides, Rhytidhysteron rufulum, Saccharomyces cerevisiae, Saksenaea oblongispora, Saksenaea vasiformis, Scedosporium apiospermum, Scedosporium aurantiacum, Scedosporium boydii, Scedosporium dehoogii, Schizophyllum commune, Sporopachydermia quercuum, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix insectorum, Sporothrix pallida, Sporothrix schenckii, Stachybotrys chartarum, Stachybotrys chlorohalonata, Starmerella apicola (Candida apicola), Starmerella bacillaris (Candida zemplininia), Stemphylium lycopersici, Suhomyces tanzawaensis (Candida tanzawaensis), Syncephalastrum monosporum, Syncephalastrum racemosum, Talaromyces amestolkiae, Talaromyces atroroseus, Talaromyces cellulolyticus, Talaromyces islandicus (Penicillium islandicum), Evansstolkia leycettana (Talaromyces leycettanus), Talaromyces marneffei (Penicillium marneffei), Talaromyces piceae (Penicillium piceum), Talaromyces pinophilus (Penicillium pinophilum), Talaromyces purpureogenus (Penicillium purpurogenum), Talaromyces stipitatus, Talaromyces verruculosus (Penicillium verruculosum), Talaromyces

[0052] Thermoascus crustaceus, Thermomyces lanuginosus, Thermothelomyces thermophilus (Myceliophthora thermophila), Thermothielavioides terrestris (Thielavia terrestris), Torulaspora delbrueckii, Trachipleistophora hominis, Trichoderma asperellum, Trichoderma atroviride, Trichoderma gamsii, Trichoderma hamatum, Trichoderma harzianum, Trichoderma longibrachiatum, Trichoderma parareesei, Trichoderma reesei, Trichoderma virens, Trichophyton, Trichophyton benhamiae (Arthroderma benhamiae), Trichosporon asahii, Trichosporon coremiiforme, Trichosporon faecale, Trichosporon inkin, Trichosporon ovoides, Ustilago cynodontis, Ustilago esculenta, Ustilago hordei, Ustilago maydis, Ustilago trichophora, Valsa mali, Verruconis gallopava (Ochroconis gallopava), Verticillium alfalfae, Verticillium dahliae, Verticillium longisporum, Verticillium tricorpus, Vittaforma corneae, Volvariella volvacea, Wallemia ichthyophaga, Wallemia mellicola, Wickerhamiella sorbophila (Candida sorbophila), Wickerhamiella versatilis (Candida versatilis), Wickerhamomyces anomalus (Pichia anomala), Wickerhamomyces ciferrii, Yarrowia deformans, Yarrowia keelungensis, Yarrowia lipolytica, Cunninghamella bertholletiae, Cunninghamella, Trichophyton equinum, Trichophyton interdigitale, Trichophyton rubrum, Trichophyton soudanense, Trichophyton tonsurans, Trichophyton verrucosum, and Trichophyton violaceum.

[0053] In some embodiments, a pathogenic microbial species is a fungus selected from Alternaria alternata, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Bipolaris spicifera, Blastomyces dermatitidis, Candida albicans, Candida auris, Candida glabrata, Candida krusei. Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Cladophialphora bantianum, Cladosporium herbarum, Coccidioides immitis / posadasii, Cryptococcus gattii, Cryptococcus neoformans, Curvularia lunata, Dactylaria gallopava, Emmonsia pasteuriana, Exophiala jeanselmei, Exophiala dermatitidis, Fusarium solanum, Fusarium oxysporum, Histoplasma capsulatum, Malassezia furfur, Paracoccidioides brasiliensis, Penicillium chrysogenum, Pneumocystis carinii, Pneumocystis jirovecii, Pseudoalle scher ia boydii, Rhizopus oryzae, Ramichloridium spp., Sporothrix schenckii, Talaromyces marneffei, and Trichosporon asahii.

[0054] In some embodiments, a pathogenic microbial species is a parasite. In some embodiments, a parasite is a protozoan, a helminth, or an arthropod. Protozoa are singlecelled eukaryotic microorganisms belonging to the Kingdom Protist. Helminths are a group of parasitic, multicellular, eukaryotic worms that can be classified into three main groups based on their morphology: 1) trematodes (flukes), which are flatworms that commonly infect the internal organs of their host; 2) cestodes (tapeworms), whi host’s intestine; and, 3) nematodes (roundworms), which can infect various body tissues including the digestive tract, respiratory system, and muscles. Arthropods are invertebrate animals that can transmit disease-causing pathogens (e.g., bacteria, viruses, protozoa, or helminths) to humans and other animals. Non-limiting examples of pathogenic parasites include Acanthamoeba, Ancylostoma ceylanicum, Ancylostoma duodenale, Angiostrongylus cantonensis, Angiostrongylus costaricensis, Anisakis simplex, Ascaris, Babesia divergens, Babesia microti, Balamuthia mandrillaris, Blastocystis hominis, Brugia malayi, Clonorchis sinensis, Cryptosporidium hominis, Cryptosporidium meleagridis, Cryptosporidium muris, Cryptosporidium parvum, Cryptosporidium ubiquitum, Cyclospora cayetanensis, Dictyostelium discoideum, Dirofdaria immitis, Dracunculus medinensis, Echinococcus granulosus, Echinococcus multilocularis, Echinostoma caproni, Entamoeba dispar, Entamoeba histolytica, Enterobius vermicularis, Fasciola hepatica, Giardia intestinalis (Giardia lamblia), Hymenolepis diminuta, Leishmania aethiopica, Leishmania amazonensis, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania major, Leishmania mexicana, Leishmania panamensis, Leishmania tropica, Loa loa, Naegleria fowleri, Necator americanus, Onchocerca volvulus, Opisthorchis viverrini, Plasmodium cynomolgi, Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Pythium insidiosum, Rodentolepis nana (Hymenolepis nana), Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, Strongyloides stercoralis, Taenia asiatica, Taenia saginata, Taenia solium, Toxocara canis, Toxoplasma gondii, Trichinella, Trichomonas vaginalis, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Wuchereria bancrofti, Acanthamoeba astronyxis, Acanthamoeba castellanii, Acanthamoeba comandoni, Acanthamoeba culbertsoni, Acanthamoeba divionensis, Acanthamoeba healyi, Acanthamoeba lenticulata, Acanthamoeba lugdunensis, Acanthamoeba mauritaniensis, Acanthamoeba pearcei, Acanthamoeba polyphaga, Acanthamoeba quina, Acanthamoeba rhysodes, Acanthamoeba royreba, Ascaris lumbricoides, Ascaris suum, Trichinella britovi, Trichinella murrelli, Trichinella nativa, Trichinella nelsoni, Trichinella papuae, Trichinella patagoniensis, Trichinella pseudospiralis, Trichinella spiralis, and Trichinella zimbabwensis.

[0055] In some embodiments, a pathogenic microbial species is a parasite selected from Babesia divergens, Babesia microti, Cryptosporidium hominis, Cryptosporidium meleagridis, Cryptosporidium muris, Cryptosporidium parvum, Cryptosporidium ubiquitum, Cyclospora cayetanensis, Entamoeba dispar, Entamoeba histolytica, Giardia intestinalis, Leishmania aethiopica, Leishmania amazonensis, Leishmania braziliensis, Leisk

[0056] Leishmania infantum, Leishmania major, Leishmania mexicana, Leishmania panamensis, Leishmania tropica, Naegleria fowleri, Plasmodium cynomolgi, Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei, and Trypanosoma cruzi.

[0057] Internal Process Control (IPC)

[0058] In some embodiments, a method provided herein comprises introducing an internal process control (IPC) microbe into a clinical sample suspected of comprising one or more microbial species. An “internal process control (IPC) microbe”, as used herein, is a known control microbe. The IPC microbe may be a microbial species that is not expected to be present in the clinical sample. The IPC microbe may be a well-characterized microbe. IPCs of the present disclosure can be added to a sample at a known concentration prior to clinical sample processing and processed alongside inherent clinical sample microbes in a clinical sample processing workflow.

[0059] As used herein, “end” of a clinical sample processing workflow refers to the last step of a clinical sample processing workflow having been accomplished. In some embodiments, the last step of a clinical sample processing workflow is detection of microbial DNA and / or IPC DNA. In some embodiments, the last step of a clinical sample processing workflow is detection of amplified microbial DNA and / or amplified IPC DNA. In some embodiments, the last step of a clinical sample processing workflow is sequencing of microbial DNA and / or IPC DNA. In some embodiments, the last step of a clinical sample processing workflow is sequencing of amplified microbial DNA and / or amplified IPC DNA. In some embodiments, the last step of a clinical sample processing workflow is quantifying microbial DNA and / or IPC DNA. In some embodiments, the last step of a clinical sample processing workflow is quantifying amplified microbial DNA and / or amplified IPC DNA.

[0060] The detection of the IPC following the clinical sample processing workflow ensures that the processing was successful. In some embodiments, the detection of IPC DNA at the end of the clinical sample processing workflow indicates that the processing was successful. In some embodiments, the detection of IPC DNA at the end of a clinical sample processing workflow validates a negative result (e.g., the detection of zero microbial species in the clinical sample).

[0061] In some embodiments, the detection of IPC DNA in a clinical sample processing indicates that there was not a process failure. A process failure, as used herein, refers to some malfunction of a sample processing workflow, which could occur at processing workflow. A failure in the processing steps might mean that one or more of the processing steps are not successful and the genome of the one or more microbial species suspected of being in the clinical sample is not able to be amplified and thus detected.

[0062] In some embodiments, an IPC microbe is a bacterium. A bacterium is a single-celled microorganism that belongs to the domain Bacteria. They are prokaryotic organisms, meaning that they lack a cell nucleus and other membrane-bound organelles found in eukaryotic cells. In some embodiments, the IPC microbe is a Gram-positive bacterium. A gram-positive bacterium has a thick peptidoglycan cell wall that retains the crystal violet stain used in the Gram staining process and has no outer membrane. Non-limiting examples of Gram-positive bacterial genera include Actinomyces, Aeromicrobium, Agrococcus, Agromyces, Alkalilimnicola, Arenimonas, Auraticoccus, Azoarcus, Azospira, Bacillus, Brachybacterium, Castellaniella, Cellulomonas, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Georgenia, Gephyromycinifex, Ilumatobacter, Isoptericola, Isoptericola, Lactobacillus, Leucobacter, Limnochorda, Listeria, Luteimicrobium, Luteimonas, Lysobacter, Marinicauda, Marmoricola, Melaminivora, Microcella, Miltoncostaea, Mycobacterium, Paraoerskovia, Protaetiibacter, Pulveribacter, Rathayibacter, Sanguibacter, Serinicoccus, Staphylococcus, Starkeya, Streptococcus Tessaracoccus, Thauera, and Thermomonas. In some embodiments, the IPC microbe is from the genus Leucobacter.

[0063] In some embodiments, the IPC microbe is a species selected from Leucobacter muris, Leucobacter triazinivorans, Cellulomonas fimi, Cellulomonas flavigena, Cellulomonas iranensis, Serinicoccus chungangensis, Serinicoccus hydrothermalis, Georgenia wutianyii, Georgenia yuyongxinii, or Agrococcus jejuensis. In some embodiments, the IPC microbe is a species selected from Aeromicrobium choanae, Aeromicrobium marinum, Agromyces archimandritae, Alkalilimnicola ehrlichii, Arenimonas daejeonensis, Auraticoccus monumenti, Azoarcus olearius, Azospira restricta, Brachybacterium faecium, Brachybacterium ginsengisoli, Castellaniella defragrans, Deinococcus maricopensis, Deinococcus radiodurans, Deinococcus radiopugnans, Gephyromycinifex aptenodytis, Ilumatobacter coccineus, Isoptericola dokdonensis, Isoptericola variabilis, Limnochorda pilosa, Luteimicrobium xylanilyticum, Luteimonas chenhongjianii, Luteimonas granuli, Lysobacter caseinilyticus, Lysobacter ciconiae, Lysobacter helvus, Lysobacter maris, Lysobacter soli, Lysobacter solisilvae, Marinicauda algicola, Marmoricola scoriae, Melaminivora jejuensis, Microcella jlavibacter, Paraoerskovia marina, Protaetiibacter larvae, Pulveribacter suum, Rathayibacter iranicus, Rathayibacter T tritici, Sanguibacter keddieii, Starkeya novella, Tessaracoccus lapidicaptus, Thauera chlorobenzoica, or Thermomonas brevis.

[0064] In some embodiments, an IPC microbe is a Gram-negative bacterium. A Gramnegative bacterium has a thin peptidoglycan cell wall, an outer membrane composed of lipopolysaccharides, and does not retain the crystal violet stain used in the Gram staining process. Non-limiting examples of Gram-negative bacterial genera include Acinetobacter, Bdellovibrio, Bordetella, Brucella, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pasturella, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Stenotrophomonas, and Vibrio.

[0065] In some embodiments, an IPC microbe is a Gram-positive bacterium. A Grampositive bacterium has a thick peptidoglycan cell wall, no outer membrane, and retains the crystal violet stain used in the Gram staining process. Non-limiting examples of Grampositive bacterial genera include Staphylococcus, Streptococcus, Bacillus, and Clostridium.

[0066] In some embodiments, an IPC microbe is a virus. A virus is a microscopic infectious agent that lacks the ability to carry out metabolic processes, grow, or reproduce independently. A virus can be a DNA virus (e.g., Adenoviruses, Herpesviruses, Poxviruses, Parvoviruses) or an RNA virus (e.g., Reoviruses, Picornaviruses, Togaviruses, Orthomyxoviruses, Rhabdoviruses, Retroviruses).

[0067] In some embodiments, the IPC microbe is a fungus. A fungus is a eukaryotic organism belonging to the Kingdom Fungi. Non-limiting examples of fungal genera include Agaricus, Amanita, Aspergillus, Candida, Claviceps, Cryptococcus, Fusarium, Morchella, Penicillium, Puccinia, Rhizopus, Saccharomyces, and Trichoderma.

[0068] In some embodiments, the one or more IPC microbes are bacteria (e.g., Gram-positive bacterium and / or Gram-negative bacteria), viruses, fungi, or any combination thereof. In some embodiments, the one or more IPC microbes are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 IPC microbes. In some embodiments, the one or more IPC microbes are 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 5-15, 10-20, 1-20, 2-19, 3-18, 4-17, 5-16, 6-15, 7-14, 8-13, or 9-12 IPC microbes.

[0069] In some embodiments, the IPC microbe has a guanine / cytosine (GC) content that is higher than the one or more microbial species expected to be present in the sample. GC content is a measure of the proportion of guanine and cytosine bases in a DNA or RNA molecule, such as in a genome. In some embodiments, the IPC microbe has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% GC content in its genome. In some emboc has between 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90- 95%, or 95-100% GC content in its genome. In some embodiments, the IPC microbe has between 50-100%, 50-95%, 55-95%, 55-90%, 60-90%, 60-85%, 65-80%, or 70-80% GC content in its genome. In some embodiments, the IPC microbe has 55% - 90% GC content in its genome.

[0070] In some embodiments, the IPC microbe is devoid of non-integrated self-replicating genetic elements (e.g., plasmids). In some embodiments, the IPC microbe is devoid of mobile genetic elements. The term “devoid”, as used herein, refers to having less than 50% mobile genetic element content and / or having less than 50% non-integrated self-replicating genetic elements in a genome. In some embodiments, an IPC microbe has 0%, less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, or less than 50% non-integrated self-replication genetic elements in its genome. In some embodiments, an IPC microbe has 0-5%, 5-10%, 10-15%, 15-20%, 20- 25%, 25-30%, 30-35%, 35-40%, 40-45%, or 45-50% non-integrated self-replication genetic elements in its genome. In some embodiments, an IPC microbe has less 10% non-integrated self-replication genetic elements in its genome. In some embodiments, an IPC microbe has less 5% non-integrated self-replication genetic elements in its genome. In some embodiments, an IPC microbe has between 0-50%, 0-45%, 5-45%, 5-40%, 10-40%, 10-35%, 15-35%, 15-30%, or 20-25% non-integrated self-replication genetic elements in its genome. In some embodiments, an IPC microbe has 0%, less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, or less than 50% mobile genetic element content in its genome. In some embodiments, an IPC microbe has less than 10% mobile genetic element content in its genome. In some embodiments, an IPC microbe has less than 5% mobile genetic element content in its genome. In some embodiments, an IPC microbe has 0-5%, 5-10%, 10-15%, 15-20%, 20- 25%, 25-30%, 30-35%, 35-40%, 40-45%, or 45-50% mobile genetic element content in its genome. In some embodiments, an IPC microbe has between 0-50%, 0-45%, 5-45%, 5-40%, 10-40%, 10-35%, 15-35%, 15-30%, or 20-25% mobile genetic element content in its genome.

[0071] A mobile genetic element, often referred to as a mobile genetic sequence, is a segment of DNAthat has the ability to move or to transpose from one location in a genome to another. Examples of mobile genetic elements include transposons (or transposable elements), which are DNA sequences that can move from one location to another within a genome, disrupting genes, causing mutations, and impacting gene expression; and plasir circular pieces of DNAthat are separate from the chromosomal DNA within a cell.

[0072] In some embodiments, one or more IPC microbes are introduced into a clinical sample. As used herein, “introducing” or “introduced” refers to the process of adding a substance (e.g., an IPC) to a clinical sample (e.g., blood or urine sample). In some embodiments, an IPC microbe (e.g., bacteria, virus, or fungus) is introduced into a clinical sample. In some embodiments, a non-competing amount of an IPC microbe (e.g., bacteria, virus, or fungus) is introduced into a clinical sample. As used herein, a “non-competing amount of an IPC microbe” refers to an amount of an IPC microbe that represents less than 10% of the expected amount of the total microbial species in a clinical sample. In some embodiments, an IPC microbe represents less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% of the total microbial species. In some embodiments, an IPC microbe represents 0.5% - 1%, 0.5% - 2%, 0.5% - 3%, 0.5% - 4%, 0.5% - 5%, 0.5% - 6%, 0.5% - 7%, 0.5% - 8%, 0.5% - 9%, 0.5% - 10%, 1% - 2%, 1% - 3%, 1% - 4%, 1% - 5%, 1% - 6%, 1% - 7%, 1% - 8%, 1% - 9%, 1% - 10%, 2% - 3%, 2%- 4%, 2% - 5%, 2% - 6%, 2% - 7%, 2% - 8%, 2% - 9%, 2% - 10%,

[0073] 3% - 4%, 3% - 5%, 3% - 6%, 3% - 7%, 3% - 8%, 3% - 9%, 3% - 10%, 4% - 5%, 4% - 6%,

[0074] 4% - 7%, 4% - 8%, 4% - 9%, 4% - 10%, 5% - 6%, 5% - 7%, 5% - 8%, 5% - 9%, 5% - 10%,

[0075] 6% - 7%, 6% - 8%, 6% - 9%, 6% - 10%, 7% - 8%, 7% - 9%, 7% - 10%, 8% - 9%, 8% - 10%, or 9% - 10% of the total microbial species.

[0076] In some embodiments, a clinical sample provided herein comprises 1 - 10,000 colony-forming units (CFUs) per milliliter (CFUs / mL) per microbial species before a noncompeting amount of an IPC microbe is added. In such embodiments, a non-competing amount of an IPC microbe is 0.1 - 1,000 CFU / mL. In some embodiments, a clinical sample provided herein comprises 1 - 9,500, 1 - 9,000, 1 - 8,500, 1 - 8,000, 1 - 7,500, 1 - 7,000, 1 - 6,500, 1 - 6,000, 1 - 5,500, 1 - 5,000, 1 - 4,500, 1 - 4,000, 1 - 3,500, 1 - 3,000, 1 - 2,500, 1

[0077] - 2,000, 1 - 1,500, 1 - 1,000, 1 - 500, 1 - 450, 1 - 400, 1 - 350, 1 - 300, 1 - 250, 1 -200, 1 - 150, 1 - 100, 1 - 50, or 1 - 10 CFUs / mL per microbial species before a non-competing among of an IPC is added. In such embodiments, a non-competing amount of an IPC microbe is 0.1 - 950, 0.1 - 900, 0.1 - 850, 0.1 - 800, 0.1 - 750, 0.1 - 700, 0.1 - 650, 0.1 - 600, 0.1 - 550, 0.1 - 500, 0.1 - 450, 0.1 - 400, 0.1 - 350, 0.1 - 300, 0.1 - 250, 0.1 - 200, 0.1

[0078] - 150, 0.1 - 100, 0.1 - 50, 0.1 - 45, 0.1 - 40, 0.1 - 35, 0.1 - 30, 0.1 - 25, 0.1 -20, 0.1 - 15, 0.1 - 10, 0.1 - 5, or 0.1 - 1 CFUs / mL. In some embodiments, where a negative control or clinical s< microbes is used, an IPC microbe represents at least 90% of the detected microbial species. An inherent microbe is a microbe that is already present in a clinical sample prior to the IPC being added to the clinical sample. In some embodiments, where a negative control or clinical sample devoid of inherent microbe is used, an IPC microbe represents at least 95%, at least 97%, at least 99%, or 100% of the detected microbial species. In some embodiments, where a negative control or clinical sample devoid of inherent microbes is used, an IPC microbe represents 90% - 95%, 90% - 97%, 90% - 99%, 90% - 100%, 95% - 97%, 95% - 99%, 95% - 100%, 97% - 99%, 97% - 100%, or 99% - 100% of the detected microbial species.

[0079] In some embodiments, less than 0.01 genome copies, less than 0.05 genome copies, less than 0.1 genome copies, less than 0.5 genome copies, less than 1 genome copies, less than 5 genomes copies, less than 10 genome copies, less than 15 genomes copies, less than 20 genome copies, less than 25 genome copies, less than 50 genome copies, less than 75 genome copies, or less than 100 genome copies of an IPC microbe are introduced into a clinical sample. In some embodiments, at least 0.01 genome copies, at least 0.05 genome copies, at least 0.1 genome copies, at least 0.5 genome copies, at least 1 genome copies, at least 5 genome copies, at least 10 genome copies, at least 15 genome copies, at least 20 genome copies, at least 25 genome copies, at least 50 genome copies, at least 75 genome copies, or at least 100 genome copies of an IPC microbe are introduced into a clinical sample. In some embodiments, 0.01-0.05 genome copies, 0.01-0.1 genome copies, 0.01-0.5 genome copies, 0.01-1 genome copies, 0.01-5 genome copies, 0.01-10 genome copies, 0.01-15 genome copies, 0.01-20 genome copies, 0.01-25 genome copies, 0.01-50 genome copies, 0.01-75 genome copies, 0.01-100 genome copies, 0.05-0.1 genome copies, 0.05-0.5 genome copies, 0.05-1 genome copies, 0.05-5 genome copies, 0.05-10 genome copies, 0.05-15 genome copies, 0.05-20 genome copies, 0.05-25 genome copies, 0.05-50 genome copies, 0.05-75 genome copies, 0.05-100 genome copies, 0.1-0.5 genome copies, 0.1-1 genome copies, 0.1-5 genome copies, 0.1-10 genome copies, 0.1-15 genome copies, 0.1-20 genome copies, 0.1-25 genome copies, 0.1-50 genome copies, 0.1-75 genome copies, 0.1-100 genome copies, 0.5-1 genome copies, 0.5-5 genome copies, 0.5-10 genome copies, 0.5-15 genome copies, 0.5-20 genome copies, 0.5-25 genome copies, 0.5-50 genome copies, 0.5-75 genome copies, 0.5-100 genome copies, 1-5 genome copies, 1-10 genome copies, 1-15 genome copies, 1-20 genome copies, 1-25 genome copies, 1-50 genome copies, 1-75 genome copies, 1-100 genome copies, 5-10 genome copies, 5-15 genome copies, 5-20 genome copies, 5-25 genome copies, 5-50 genome copies, 5-75 genome copies, 5-100 genome copies, 10-15 genome copies, 10-20 genome copies, 10-25 genome copies, 10-50 genome copies, 10-75 j genome copies, 15-20 genome copies, 15-25 genome copies, 15-50 genome copies, 15-75 genome copies, 15-100 genome copies, 20-25 genome copies, 20-50 genome copies, 20-75 genome copies, 20-100 genome copies, 25-50 genome copies, 25-75 genome copies, 25-100 genome copies, 50-75 genome copies, 50-100 genome copies, or 75-100 genome copies of an IPC microbe are introduced into a clinical sample.

[0080] In some embodiments, at least 102genome copies of an IPC microbe are introduced into a clinical sample. In some embodiments, at least 102genome copies, at least 103genome copies, at least 104genome copies, at least 105genome copies, at least 106genome copies, at least 107genome copies, at least 108genome copies, at least 109genome copies, or at least 1010genome copies of an IPC microbe are introduced into a clinical sample. In some embodiments, 102- 103genome copies, 102- 104genome copies, 102- 105genome copies, 102- 106genome copies, 102- 107genome copies, 102- 108genome copies, 102- 109genome copies, 102- 1010genome copies, 103- 104genome copies, 103- 105genome copies, 103- 106genome copies, 103- 107genome copies, 103- 108genome copies, 103- 109genome copies, 103- 1010genome copies, 104- 105genome copies, 104- 106genome copies, 104- 107genome copies, 104- 108genome copies, 104- 109genome copies, 104- 1010genome copies, 105- 106genome copies, 105- 107genome copies, 105- 108genome copies, 105- 109genome copies, 105- 1010genome copies, 106- 107genome copies, 106- 108genome copies, 106- 109genome copies, 106- 1010genome copies, 107- 108genome copies, 107- 109genome copies, 107- 1010genome copies, 108- 109genome copies, 108- 1010genome copies, or 109- 1010genome copies of an IPC microbe are introduced into a clinical sample.

[0081] In some embodiments, one or more IPC microbes (e.g., bacteria, virus, or fungus) are introduced into a clinical sample. In some embodiments, equal amounts of each IPC microbe in the one or more IPC microbes are introduced into a clinical sample. In some embodiments, each IPC microbe in the one or more IPC microbe is introduced into a clinical sample at different amounts.

[0082] In some embodiments, the total amount of one more IPC microbes introduced into a clinical sample is less than 0.01 genome copies, less than 0.05 genome copies, less than 0.1 genome copies, less than 0.5 genome copies, less than 1 genome copies, less than 5 genomes copies, less than 10 genome copies, less than 15 genomes copies, less than 20 genome copies, less than 25 genome copies, less than 50 genome copies, less than 75 genome copies, or less than 100 genome copies. In some embodiments, the total amount of one or more IPC microbes introduced into a clinical sample is at least 0.01 genome cc genome copies, at least 0.1 genome copies, at least 0.5 genome copies, at least 1 genome copies, at least 5 genome copies, at least 10 genome copies, at least 15 genome copies, at least 20 genome copies, at least 25 genome copies, at least 50 genome copies, at least 75 genome copies, or at least 100 genome copies. In some embodiments, the total amount of one or more IPC microbes introduced into a clinical sample is 0.01-0.05 genome copies, 0.01-0.1 genome copies, 0.01-0.5 genome copies, 0.01-1 genome copies, 0.01-5 genome copies, 0.01- 10 genome copies, 0.01-15 genome copies, 0.01-20 genome copies, 0.01-25 genome copies, 0.01-50 genome copies, 0.01-75 genome copies, 0.01-100 genome copies, 0.05-0.1 genome copies, 0.05-0.5 genome copies, 0.05-1 genome copies, 0.05-5 genome copies, 0.05-10 genome copies, 0.05-15 genome copies, 0.05-20 genome copies, 0.05-25 genome copies, 0.05-50 genome copies, 0.05-75 genome copies, 0.05-100 genome copies, 0.1-0.5 genome copies, 0.1-1 genome copies, 0.1-5 genome copies, 0.1-10 genome copies, 0.1-15 genome copies, 0.1-20 genome copies, 0.1-25 genome copies, 0.1-50 genome copies, 0.1-75 genome copies, 0.1-100 genome copies, 0.5-1 genome copies, 0.5-5 genome copies, 0.5-10 genome copies, 0.5-15 genome copies, 0.5-20 genome copies, 0.5-25 genome copies, 0.5-50 genome copies, 0.5-75 genome copies, 0.5-100 genome copies, 1-5 genome copies, 1-10 genome copies, 1-15 genome copies, 1-20 genome copies, 1-25 genome copies, 1-50 genome copies, 1-75 genome copies, 1-100 genome copies, 5-10 genome copies, 5-15 genome copies, 5-20 genome copies, 5-25 genome copies, 5-50 genome copies, 5-75 genome copies, 5-100 genome copies, 10-15 genome copies, 10-20 genome copies, 10-25 genome copies, 10-50 genome copies, 10-75 genome copies, 10-100 genome copies, 15-20 genome copies, 15-25 genome copies, 15-50 genome copies, 15-75 genome copies, 15-100 genome copies, 20-25 genome copies, 20-50 genome copies, 20-75 genome copies, 20-100 genome copies, 25-50 genome copies, 25-75 genome copies, 25-100 genome copies, 50-75 genome copies, 50-100 genome copies, or 75-100 genome copies.

[0083] In some embodiments, the total amount of one or more IPC microbes introduced into a clinical sample is at least 102genome copies. In some embodiments, the total amount of one or more IPC microbes introduced into a clinical sample is at least 102genome copies, at least 103genome copies, at least 104genome copies, at least 105genome copies, at least 106genome copies, at least 107genome copies, at least 108genome copies, at least 109genome copies, or at least 1010genome copies. In some embodiments, the total amount of one or more IPC microbes introduced into a clinical sample is 102- 103genome copies, 102- 104genome copies, 102- 105genome copies, 102- 106genome copies, 102- 107genome copies, 102- 108genome copies, 102- 109genome copies, 102- IO10genome cop copies, 103- 105genome copies, 103- 106genome copies, 103- 107genome copies, 103-

[0084] 108genome copies, 103- 109genome copies, 103- IO10genome copies, 104- 105genome copies, 104- 106genome copies, 104- 107genome copies, 104- 108genome copies, 104-

[0085] 109genome copies, 104- 1010genome copies, 105- 106genome copies, 105- 107genome copies, 105- 108genome copies, 105- 109genome copies, 105- IO10genome copies, 106- 107genome copies, 106- 108genome copies, 106- 109genome copies, 106- IO10genome copies, 107- 108genome copies, 107- 109genome copies, 107- IO10genome copies, 108- 109genome copies, 108- IO10genome copies, or 109- IO10genome copies.

[0086] Detecting

[0087] Methods provided herein include detecting one or more microbial species inherent in a clinical sample. The term “detecting”, as used herein, refers to the process of identifying and / or confirming the presence of one or more microbial species (e.g., bacteria, virus, fungus, or parasite) inherent in a sample (e.g., clinical sample). Those skilled in the art will recognize that such techniques for detecting microbial species from a sample (e.g., a clinical sample) may require one or more processing steps.

[0088] As used herein, “processing” or “processed” refers to preparing (e.g., cell lysis, DNA amplification) a clinical sample for analysis (e.g., sequencing). In some embodiments, processing may include centrifugation (e.g., velocity sedimentation), separating supernatant (e.g., from a pellet) and moving the supernatant to a separate tube (e.g., from the pellet), cell lysis (e.g., host cell lysis, microbial cell lysis, IPC cell lysis), microbial enrichment (e.g., by filtration, centrifugation, or molecular techniques, such as polymerase chain reaction (PCR)), nucleic acid (e.g., DNA or RNA) isolation, or any combination thereof.

[0089] In some embodiments, processing may include one or more centrifugation steps, such as velocity sedimentation. Velocity sedimentation, also known as rate-zonal centrifugation, is a technique used to separate particles in a solution based on their size and shape, where larger molecules accumulate in a pellet at the bottom of the container (e.g., tube) being centrifuged and smaller molecules accumulate in the supernatant above the pellet. This method can be employed to fractionate macromolecules (e.g., proteins, nucleic acids, or subcellular organelles) according to their sedimentation rates in a centrifugal field. In the present disclosure, centrifugation may be used to pellet larger human cells so that smaller microbial cells (and cellular debris) can be separated from the larger human cells. In some embodiments, processing may include separating a s pellet). Separating a supernatant typically involves the separation of a liquid (the supernatant containing one or more microbial species) from solid particles (e.g., the pellet containing human cells and macromolecular aggregates) that have settled at the bottom of a container. The supernatant can be separated by decanting the supernatant into a new container or by pipetting (e.g., using a Pasteur pipette or a micropipette).

[0090] In some embodiments, a supernatant from velocity sedimentation is used for further clinical sample processing.

[0091] In some embodiments, processing may include cell lysis or virus lysis. Cell lysis is the process of breaking open or rupturing cell membranes and / or nuclear membranes to release their contents (e.g., organelles, proteins, DNA, and RNA). Virus lysis is the process of breaking open or rupturing virus particles, virus envelopes, and / or viral capsids to release viral contents (e.g., proteins, DNA, RNA). In some embodiments, the cell lysis is host (e.g., human) cell lysis. In some embodiments, the cell lysis is microbial (e.g., bacteria, fungus, or a parasite) cell lysis. In some embodiments, the cell lysis is internal process control (IPC) lysis. In some embodiments, the host (e.g., human) and microbial (e.g., bacteria, fungus, or a parasite) cells or virus can be differentially lysed and fractionated based on their specific properties, such as size, density, or other chemical properties, including cell membrane, cell wall composition, viral envelope composition, and / or viral capsid. There are several methods for performing cell lysis and virus known in the art, such as mechanical lysis (e.g., homogenization, bead beating), chemical lysis (e.g., detergent lysis, such as with Triton X- 100 or sodium dodecyl sulfate (SDS)), enzymatic lysis (e.g., using lysozyme), freeze-thaw lysis, ultrasonication, high-pressure homogenization, osmotic shock, or electroporation.

[0092] In some embodiments, processing may include microbial enrichment (e.g., by filtration, centrifugation, or molecular techniques, such as polymerase chain reaction (PCR)). Enriching for microbes involves increasing the relative abundance of the target microbial species (e.g., bacteria, virus, fungus, or parasite) so that they can be more easily detected and identified. Relative abundance may be relative to the original clinical sample or relative to non-microbial cells. In some embodiments, microbial enrichment may be performed using filtration, whereby a sample is passed through a filter with a specific pore size that retains the microbial species (e.g., bacteria, virus, fungus, or parasite) while allowing other materials to pass through. In some embodiments, microbial enrichment may be performed using centrifugation, such as ultracentrifugation. In some embodiments, microbial enrichment may be performed using molecular techniques such as polymerase chain reaction (PCR), which can selectively amplify the DNA or RNA of the microbial species in embodiments, microbial enrichment may be performed using a combination of filtration, centrifugation, and / or molecular techniques.

[0093] In some embodiments, processing may include nucleic acid (e.g., DNA or RNA) isolation. Nucleic acid isolation (also known as nucleic acid extraction) is a technique used to obtain DNA or RNA from a biological sample. The DNA or RNA that is isolated can be derived from the host (e.g., human) or from microbial species (e.g., bacteria, virus, fungi, or parasites). Methods of performing nucleic acid isolation are known to those of skill in the art and can include, but are not limited to, phenol-chloroform extraction, silica-based spin columns, magnetic bead-based purification, organic solvent precipitation, solid-phase extraction, or ultracentrifugation.

[0094] In some embodiments, a method for detecting one or more microbial species in a clinical sample comprises processing a clinical sample with an IPC, including lysing the one or more microbial species and / or the IPC microbe.

[0095] In some embodiments, processing may include amplifying genome microbial DNA from lysed microbial species and lysed IPC microbe. In some embodiments, the microbial DNA is whole genome microbial DNA. In some embodiments, the microbial DNA (e.g., whole genome microbial DNA) is from a lysed microbial species and a lysed IPC microbe. Non-limiting examples of methods for amplifying microbial DNA include genome amplification (GA), polymerase chain reaction (PCR), quantitative PCR (qPCR), loop- mediated isothermal amplification (LAMP), whole genome amplification (WGA), multiple displacement amplification (MDA), strand displacement amplification (SDA), random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), and rolling circle amplification (RCA). In some embodiments, whole genome microbial DNA from lysed microbial species and lysed IPC microbe is amplified using MDA.

[0096] Genome amplification may be whole genome amplification (100% of the microbial genome, the IPC genome). Genome amplification may be greater than 1% of the microbial genome, greater than 5% of the microbial genome, greater than 10% of the microbial genome, greater than 15% of the microbial genome, greater than 20% of the microbial genome, greater than 25% of the microbial genome, greater than 30% of the microbial genome, greater than 35% of the microbial genome, greater than 40% of the microbial genome, greater than 45% of the microbial genome, greater than 50% of the microbial genome, greater than 55% of the microbial genome, greater than 60% of the microbial genome, greater than 65% of the microbial genome, greater than 70% of the microbial genome, greater than 75% of the microbial genome, greater than 80° genome, greater than 85% of the microbial genome, greater than 90% of the microbial genome, greater than 95% of the microbial genome, or greater than 99% of the microbial genome. Genome amplification may be greater than 1% of the IPC genome, greater than 5% of the IPC genome, greater than 10% of the IPC genome, greater than 15% of the IPC genome, greater than 20% of the IPC genome, greater than 25% of the IPC genome, greater than 30% of the IPC genome, greater than 35% of the IPC genome, greater than 40% of the IPC genome, greater than 45% of the IPC genome, greater than 50% of the IPC genome, greater than 55% of the IPC genome, greater than 60% of the IPC genome, greater than 65% of the IPC genome, greater than 70% of the IPC genome, greater than 75% of the IPC genome, greater than 80% of the IPC genome, greater than 85% of the IPC genome, greater than 90% of the IPC genome, greater than 95% of the IPC genome, or greater than 99% of the IPC genome. In some embodiments, processing may include amplifying microbial DNA from microbial species and IPC microbe. In some embodiments, processing may include amplifying whole genome microbial DNA from lysed microbial species and lysed IPC microbe.

[0097] In some embodiments, processing may include detecting amplified microbial DNA and IPC DNA. The detected DNA may be amplified (e.g., having undergone DNA amplification) or not amplified. Amplified DNA may be amplified by any process described herein.

[0098] In some embodiments, amplified DNA (e.g., microbial DNA and IPC DNA) is detected. As used herein, detection of amplified DNA (e.g., microbial DNA and IPC DNA) refers to the identification and confirmation of the presence of specific DNA sequences (occurring in a specific microbe or IPC microbe) in a sample following DNA amplification. The detection can be qualitative, to confirm the presence or absence of the target DNA sequence, or quantitative, to provide information about the amount or concentration of the amplified DNA. Methods of detecting amplified DNA (e.g., microbial DNA and IPC DNA) include, but are not limited to, next-generation sequencing (e.g., Illumina sequencing or nanopore sequencing), agarose gel electrophoresis, polyacrylamide gel electrophoresis, ethidium bromide staining, SYBR green staining, fluorescent probes (e.g., TaqMan probes, molecular beacons, dual-labeled probes), DNA intercalating dyes (e.g., EvaGreen or SYBR Safe), DNA fragment analysis (e.g., capillary electrophoresis), real-time PCR (qPCR), digital PCR, nucleic acid hybridization (e.g., microarrays), enzyme-linked immunosorbent assays (ELISA), or microscopy. In some embodiments, one or more microbial species inhere) detected using sequencing (e.g., next-generation sequencing (NGS)). Sequencing data from clinical samples is analyzed to measure the breadth of coverage of a microbial species genome within the sequencing data. This approach is particularly advantageous in sequencing data obtained from clinical samples, which may include human DNA and other confounding DNA sources in addition to DNA from one or more microbial species. By measuring the breadth of coverage of a microbial species genome, contaminating DNA (e.g., human DNA, confounding DNA) may be removed from microbial DNA for processing. In some embodiments, only microbial species with a 0.5x - lOx microbial genome coverage are identified in the present disclosure. In some embodiments, only microbial species with a 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, l.Ox, l.lx, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2. Ox, 2. lx,

[0099] 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3. Ox, 3. lx, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x,

[0100] 3.8x, 3.9x, 4. Ox, 4. lx, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, 4.9x, 5. Ox, 5. lx, 5.2x, 5.3x,

[0101] 5.4x, 5.5x, 5.6x, 5.7x, 5.8x, 5.9x, 6. Ox, 6. lx, 6.2x, 6.3x, 6.4x, 6.5x, 6.6x, 6.7x, 6.8x, 6.9x,

[0102] 7. Ox, 7. lx, 7.2x, 7.3x, 7.4x, 7.5x, 7.6x, 7.7x, 7.8x, 7.9x, 8. Ox, 8. lx, 8.2x, 8.3x, 8.4x, 8.5x,

[0103] 8.6x, 8.7x, 8.8x, 8.9x, 9. Ox, 9. lx, 9.2x, 9.3x, 9.4x, 9.5x, 9.6x, 9.7x, 9.8x, 9.9x, or 10. Ox or more microbial genome coverage are identified in the present disclosure.

[0104] Sequencing reads following processing of a clinical sample are aligned to reference genomes from one or more microbial species. This alignment may be performed manually or by using an open-source software tool (e.g., bwa-mem, rhat, GraphMap, minimap2). The breadth of coverage of a microbial species may be calculated by simply counting sequencing reads for each microbial species or by reconstructing the whole genome of the one or more microbial species from sequencing reads in the clinical sample. Reconstructing the whole genome of one or more microbial species may be performed using an open-source software tool (e.g., / / )'c, Canu, Raven, Shasta, Miniasm) or manually. Once a whole genome of one or more microbial species is reconstructed, the one or more microbial species may be identified using an open-source software tool (e.g., Krakeri) or manually.

[0105] In some embodiments, where amplified microbial DNA and / or the IPC DNA is detected using a sequencing modality (e.g., Illumina sequencing or Nanopore sequencing), the genome coverage of the amplified microbial DNA and / or the amplified IPC DNA is at least 0. lx, at least 0.2x, at least 0.3x, at least 0.4x, at least 0.5x, at least 0.6x, at least 0.7x, at least 0.8x, at least 0.9x, at least l.Ox, at least 1.5x, at least 2. Ox, at least 2.5x, at least 3. Ox, at least 3.5x, at least 4. Ox, at least 4.5x, or at least 5. Ox or more. In some embodiments, where amplified microbial DNA and / or the IPC DNA is detected using a sequencing modality (e.g., Illumina sequencing or Nanopore sequencing), the genome coverage microbial DNA and / or the amplified IPC DNA is 0. lx - 0.2x, 0. lx - 0.3x, 0. lx - 0.4x, 0. lx - 0.5x, O.lx - 0.6x, O.lx - 0.7x, O.lx - 0.8x, O.lx - 0.9x, O.lx - l.Ox, O.lx - 1.5x, O.lx - 2. Ox, O. lx - 2.5x, O. lx - 3. Ox, O. lx - 3.5x, O. lx - 4. Ox, O. lx - 4.5x, O. lx - 5. Ox, 0.2x - 0.3x, 0.2x - 0.4x, 0.2x - 0.5x, 0.2x - 0.6x, 0.2x - 0.7x, 0.2x - 0.8x, 0.2x - 0.9x, 0.2x - l.Ox, 0.2x - 1.5x, 0.2x - 2. Ox, 0.2x - 2.5x, 0.2x - 3. Ox, 0.2x - 3.5x, 0.2x - 4. Ox, 0.2x - 4.5x, 0.2x - 5. Ox, 0.3x - 0.4x, 0.3x - 0.5x, 0.3x - 0.6x, 0.3x - 0.7x, 0.3x - 0.8x, 0.3x - 0.9x, 0.3x - l.Ox, 0.3x - 1.5x, 0.3x - 2. Ox, 0.3x - 2.5x, 0.3x - 3. Ox, 0.3x - 3.5x, 0.3x - 4. Ox, 0.3x - 4.5x, 0.3x - 5. Ox, 0.4x - 0.5x, 0.4x - 0.6x, 0.4x - 0.7x, 0.4x - 0.8x, 0.4x - 0.9x, 0.4x - l.Ox, 0.4x - 1.5x, 0.4x - 2. Ox, 0.4x - 2.5x, 0.4x - 3. Ox, 0.4x - 3.5x, 0.4x - 4. Ox, 0.4x - 4.5x, 0.4x - 5. Ox, 0.5x - 0.6x, 0.5x - 0.7x, 0.5x - 0.8x, 0.5x - 0.9x, 0.5x - l.Ox, 0.5x - 1.5x, 0.5x - 2. Ox, 0.5x - 2.5x, 0.5x - 3. Ox, 0.5x - 3.5x, 0.5x - 4. Ox, 0.5x - 4.5x, 0.5x - 5. Ox, 0.6x - 0.7x, 0.6x - 0.8x, 0.6x - 0.9x, 0.6x - l.Ox, 0.6x - 1.5x, 0.6x - 2. Ox, 0.6x - 2.5x, 0.6x - 3. Ox, 0.6x - 3.5x, 0.6x - 4. Ox, 0.6x - 4.5x, 0.6x - 5. Ox, 0.7x - 0.8x, 0.7x - 0.9x, 0.7x - l.Ox, 0.7x - 1.5x, 0.7x - 2. Ox, 0.7x - 2.5x, 0.7x - 3. Ox, 0.7x - 3.5x, 0.7x - 4. Ox, 0.7x - 4.5x, 0.7x - 5. Ox, 0.8x - 0.9x, 0.8x - l.Ox, 0.8x -1.5x, 0.8x - 2. Ox, 0.8x - 2.5x, 0.8x - 3. Ox, 0.8x - 3.5x, 0.8x - 4. Ox, 0.8x - 4.5x, 0.8x - 5. Ox, 0.9x - l.Ox, 0.9x - 1.5x, 0.9x - 2.0x, 0.9x - 2.5x, 0.9x - 3.0x, 0.9x - 3.5x, 0.9x - 4.0x, 0.9x - 4.5x, 0.9x - 5. Ox, l.Ox - 1.5x, l.Ox - 2. Ox, l.Ox - 2.5x, l.Ox - 3. Ox, l.Ox - 3.5x, l.Ox - 4. Ox, l.Ox - 4.5x, l.Ox - 5. Ox, 1.5x - 2.0x, 1.5x - 2.5x, 1.5x - 3.0x, 1.5x - 3.5x, 1.5x - 4.0x, 1.5x - 4.5x, 1.5x - 5. Ox, 2. Ox - 2.5x, 2. Ox - 3. Ox, 2. Ox - 3.5x, 2. Ox - 4. Ox, 2. Ox - 4.5x, 2. Ox - 5. Ox, 2.5x - 3.0x,2.5x - 3.5x, 2.5x - 4. Ox, 2.5x - 4.5x, 2.5x - 5. Ox, 3. Ox - 3.5x, 3. Ox - 4. Ox, 3. Ox - 4.5x, 3. Ox - 5. Ox, 3.5x - 4. Ox, 3.5x - 4.5x, 3.5x - 5. Ox, 4. Ox - 4.5x, 4. Ox - 5. Ox, or 4.5x - 5. Ox.

[0106] In some embodiments, a method for detecting one or more microbial species in a clinical sample comprises: (i) obtaining a clinical sample (e.g., blood, urine) comprising no microbial species (negative sample) or one or more microbial species (positive sample) from a subject (e.g., a human); (ii) introducing an IPC microbe (e.g., a Gram-negative and / or a Gram-positive bacteria) into the clinical sample; (iii) processing the clinical sample with the IPC, including lysing the one or more microbial species in the clinical sample and the IPC microbe; (iv) amplifying whole genome microbial DNA (e.g., using MDA) from the lysed microbial species and the lysed IPC microbe; and (v) detecting amplified microbial DNA and / or IPC DNA (e.g., using sequencing).

[0107] In some embodiments, a method for detecting one or more microbial species in a clinical sample comprises processing a clinical sample with an IPC. In some embodiments, a method for detecting one or more microbial species in a clinical sample comprises: (i) obtaining a clinical sample (e.g., blood, urine) comprising no micro! sample) or one or more microbial species (positive sample) from a subject (e.g., a human); (ii) introducing an IPC microbe (e.g., a Gram-negative and / or a Gram-positive bacteria) into the clinical sample; (iii) processing the clinical sample with the IPC, including lysing the one or more microbial species inherent in the clinical sample as well as the IPC microbe; (iv) amplifying whole genome microbial DNA (e.g., using MDA) from the lysed microbial species and / or the lysed IPC microbe; and (v) detecting amplified microbial DNA and IPC DNA (e.g., using sequencing), wherein the presence of IPC DNA, microbial species DNA, or IPC DNA and microbial species DNA in step (v) indicates that there was not a process failure and that one or more microbial species would have been detectable if present in the clinical sample. In some embodiments, the presence of IPC DNA in step (v) indicates that there was not a process failure.

[0108] Process failure

[0109] The term “process failure”, as used herein, refers to failure at one or more steps in the sample processing workflow. In some embodiments, failure occurs during a lysis step (e.g., host cell lysis or microbial lysis). Failure during a lysis step can occur for a variety of reasons, including application of improper lysis buffers or buffer conditions, inadequate mechanical disruption methods, or insufficient incubation times. In some embodiments, failure occurs during an amplification step (e.g., microbial genome amplification). Nonlimiting reasons for failure during an amplification step include poor DNA quality, the presence of inhibitors or contaminants that interfere with the amplification process, suboptimal reaction conditions (e.g., improper primer design or reaction conditions, such as temperature and / or time), low template DNA concentrations, or the presence of highly repetitive DNA regions. In some embodiments, failure occurs during the detection step (e.g., sequencing). In some embodiments, failure occurs during sequencing. Sequencing failure may be due to low DNA quality, issues with library preparation, instrumentation issues, insufficient read depth, low signal quality, or poor cluster generation. In some embodiments, a process failure means that a test result is invalid.

[0110] One or more microbial species

[0111] In some embodiments, the one or more different microbial species is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more microbial species. In some embodiments, the one or more different microbial species is greater than 1, greater than 2, greater than 3, greater than 4, greater t greater than 7, greater than 8, greater than 9, greater than 10, greater than 15, greater than 20, greater than 25, greater than 30, greater than 35, greater than 40, greater than 45, or greater than 50, or more different microbial species. In some embodiments, the one or more different microbial species is from 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 5-15, 10-20, 15-25, 20- 30, 25-35, 30-40, 35-45, 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 45-50, 1-30, 2- 29, 3-28, 4-27, 5-26, 6-25, 7-24, 8-23, 9-22, 10-21, 11-20, 12-19, 13-18, or 14-17 different microbial species.

[0112] One or more different microbial species in a clinical sample may all be from the same microbe type (e.g., bacteria, virus, fungus, parasite) or from different microbe types. In some embodiments, one or more different microbial species in a clinical sample are all bacteria, all viruses, all fungi, or all parasites. In some embodiments, one or more different microbial species are bacteria, viruses, fungi, parasites, or some combination thereof.

[0113] Sample

[0114] In some embodiments, a method for detecting one or more microbial species inherent in a clinical sample comprises obtaining a clinical sample comprising one or more microbial species inherent in the clinical sample from a subject. A clinical sample is a biological sample obtained from a subject. In some embodiments, a clinical sample has zero microbial species inherent in the clinical sample. In some embodiments, a clinical sample has one or more microbial species inherent in the clinical sample. Non-limiting examples of clinical samples include a blood sample (e.g., whole blood, plasma, or serum), a urine sample, a saliva sample, a stool sample, a cerebrospinal fluid sample, a synovial fluid sample, a throat swab sample, an oral swab sample, a bronchoalveolar lavage sample, or an intraocular fluid sample. In some embodiments a clinical sample is a blood sample, wherein the blood sample is a whole blood sample, a plasma sample, or a serum sample. In some embodiments, the blood sample is obtained from a subject by venipuncture or finger prick. In some embodiments a clinical sample is a urine sample, wherein the urine sample is obtained by midstream urine collection, catheterization (e.g., using a Foley catheter), or bladder puncture.

[0115] Subjects

[0116] Methods provided herein include obtaining a clinical sample from a subject. A subject may be any organism that can be infected by a microbial species. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human primate (e.g., a laboratory animal such as a rhesus monkey). In some embodiments, such as a rat or mouse. In some embodiments, the subject is a goat, rabbit, sheep, cat, dog, cattle, horse or pig. In some embodiments, the subject is a human.

[0117] Kits

[0118] Also provided by this disclosure is a kit comprising one or more internal process control (IPC) microbes. A kit may also comprise materials for obtaining and processing a clinical sample as described herein.

[0119] IPCs in a kit may be provided in any physical state in which they are stable. As used herein, stable means that the IPCs are not degraded or degenerated by being in the kit. Nonlimiting examples of physical states in which kit can be provided include: lyophilized, dehydrated, sterile spray-dried, aseptic crystallized, dried on a fiber matrix, encapsulated in a lipid matrix or chemically stabilized. In some embodiments, the one or more IPC microbes in a kit are lyophilized. In some embodiments, the one or more IPC microbes in a kit are dehydrated.

[0120] IPC microbes in a kit may be provided in a single mixture or in multiple mixtures. In some embodiments, the one or more IPC microbes in a kit are provided as a single mixture (e.g., in a single tube or container). In some embodiments, the one or more IPC microbes are provided separately (e.g., in separate tubes or containers).

[0121] An IPC in a kit provided herein may include 1 - 20, 2 — 19, 3 — 18, 4 — 17, 5 — 16, 6 —

[0122] 15, 7 - 14, 8 - 13, 9 - 12, or 10 - 11 different microbial species to each serve as IPCs. In some embodiments, an IPC in a kit comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

[0123] 16, 17, 18, 19, or 20 more different microbes to each serve as IPCs. These different microbes may be provided in 1 - 20, 2 - 19, 3 - 18, 4 - 17, 5 - 16, 6 - 15, 7 - 14, 8 - 13, 9 - 12, or 10 -11 different mixtures. In some embodiments, the microbial species are provided in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more different mixtures.

[0124] In some embodiments, a kit comprises one or more internal process (IPC) microbes, wherein the one or more IPC microbes comprises a Gram-positive bacterium. A Grampositive bacterium may be any Gram-positive bacterium provided herein. In some embodiments, a kit provided herein comprises a Gram-positive bacterium from the genus Leucobacter. Non-limiting examples of Gram-positive bacterium from the genus Leucobacter include L. aerolatus, L. albus, L. allii, L. alluvii, L. aridicollis, L. celer, L. chinensis, L. 42axicola42d, L. chromiireducens, L. chromiiresistens, L. chromiisoli, L. coleopterorum, L. 42axicola, L. denitrificans, L. exalbidus , L. holotrichiae, L. humi, L. iarius, L. insecticola, L. japonicus, L. komagatae, L. kyeonggiensis , L. luti, L. manosquensis massiliensis, L. muris, L. musarum, L. populi, L. rhizosphaerae, L. ruminantium, L. salsicius, L. soli, L. tardus, L. tenebrionis, L. triazinivorans, L. viscericola, L. weissii, and A. zeae. In some embodiments, a kit provided herein comprises L. muris.

[0125] In some embodiments, a kit comprises one or more IPC microbes from the genus Deinococcus, Aeromicrobium, Agrococcus, Agromyces, Alkalilimnicola, Arenimonas, Auraticoccus, Azoarcus, Azospira, Brachybacterium, Castellaniella, Cellulomonas, Georgenia, Gephyromycinifex, Ilumatobacter, Isoptericola, Leucobacter, Limnochorda, Luteimicrobium, Luteimonas, Lysobacter, Marinicauda, Marmoricola, Melaminivora, Microcella, Miltoncostaea, Paraoerskovia, Proteatiibacter, Pulver ibacter, Rathayibacter, Sanguibacter, Serinicoccus, Starkeya, Tessaracoccus, Thauera, Thermomonas, or some combination thereof.

[0126] A kit may also comprise one or more buffers for performing a process described herein. Non-limiting examples of buffers include: phosphate buffered saline (PBS), tris buffered saline (TBS), Dulbecco’s phosphate buffered saline (DPBS), Hanks’ balanced salt solution (HBSS), 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), Tris, Tris- HC1, sodium phosphate, potassium phosphate, and potassium chloride.

[0127] Kits may optionally provide additional components such as interpretive information, including, but not limited to a control and / or standard or reference sample. Typically, a kit will comprise a container and a label or package insert(s) providing information on and instructions for using the kit. These instructions may include describing any process provided or described herein.

[0128] Methods of Use

[0129] An internal process control (IPC) may be used in any clinical sample processing protocol that employs molecular techniques to detect one or more microbial species suspected of being in a clinical sample provided herein. Non-limiting examples of molecular techniques that may utilize an IPC include: detecting one or more microbial species, quantifying one or more microbial species, and predicting appropriate treatment for infection with one or more microbial species.

[0130] An IPC may be used in detecting one or more microbial species suspected of being within a clinical sample. Detecting one or more microbial species may be by any process provided herein. In some embodiments, an IPC is introduced into a clinical sample after obtaining the clinical sample from a subject (e.g., a human subject), processed as part of the clinical sample, has its genome amplified alongside one or more mic of being in the clinical sample, after which it is detected.

[0131] An IPC may be used in quantifying one or more microbial species suspected of being in a clinical sample. Quantifying one or more microbial species requires identifying the one or more microbial species that is to be quantified. Identifying the one or more microbial species to be quantified may be by any method provided herein. One or more microbial species to be quantified may be amplified (e.g., by any method provided herein) or may not be amplified. Quantification may be by any method known in the art. Non-limiting examples of quantifying one or more microbial species in a clinical sample include: measuring microbial genome coverage, measuring the number of times a target sequence (e.g., in a microbial genome) appears in a sample, and measuring colony forming units (CFUs). In some embodiments, microbial genome coverage is measured to quantify one or more microbial species suspected of being in a clinical sample. Measuring microbial genome coverage may be by any method provided herein (e.g., manually, using open-source software tools). Quantification of one or more microbial species (e.g., in a clinical sample) may be expressed as a total number (e.g., total number of microbial cells, genomes, total number of CFUs) or as a relative number. If the quantification of one or more microbial species is expressed as a relative number, it may be relative to human or microbe (e.g., human cells, microbial cells, human DNA reads, microbial DNA reads).

[0132] In some embodiments, an IPC may be used to quantify one or more microbial species in a clinical sample. By adding a known concentration of IPC (CFU / mL) added to a clinical sample, it would allow quantification of other microbes by any method provided herein.

[0133] An IPC may be used in clinical sample processing for predicting appropriate treatment for infection with one or more microbial species suspected of being in a clinical sample. A microbe may be susceptible to a potential treatment, resistant to a potential treatment, or neither susceptible nor resistant to a potential treatment. A microbe is susceptible to a potential treatment if contacting the microbe (e.g., in a human subject) with the potential treatment causes reduced survival of the microbe, reduced proliferation of the microbe, or some combination thereof. A microbe is resistant to a potential treatment if contacting the microbe with the potential treatment does not cause reduced survival of the microbe, reduced proliferation of the microbe, or some combination thereof. A microbe may be susceptible to a potential treatment under certain circumstances (e.g., environmental conditions, co-infections with other microbes, etc.) and resistant to the same potential treatment under other circumstances. Predicting appropriate treatment for infection requires identii microbial species, which may be accomplished by any method provided herein. Appropriate treatment may be predicted by any method known in the art. Non-limiting methods of predicting appropriate treatment include: utilizing sequencing data and testing antimicrobial agents that the one or more microbial species are known to be susceptible to. In some embodiments, methods of predicting appropriate antimicrobial treatment include utilizing sequencing data. Sequencing data may be whole genome sequencing data or partial genome sequencing data. When whole genome sequencing data is used to predict appropriate antimicrobial treatment, the genome sequences for a microbe may be compared against a reference database of sequences known to confer antimicrobial agent resistance or susceptibility. Non-limiting examples of reference databases include: Comprehensive Antibiotic Resistance Database (CARD), National Database of Antibiotic Resistant Organisms (NARDO), AMRFinder Plus, ResFinder, ResFinder Plus, and Resistance Map.

[0134] For example, when whole genome sequencing data is used to predict appropriate antimicrobial treatment in Staphylococcus aureus (S. aureus), the whole genome sequencing data may be compared to sequences known to confer resistance to antibiotics such as vancomycin, methicillin, or any other antibiotic used to treat S. aureus infection. Alternatively, whole genome sequencing data may be used to create a de novo reference database that draws on known antimicrobial susceptibility or resistance sequences.

[0135] An antimicrobial agent may be an antibacterial agent, an antifungal agent, an antiviral agent, an anti-parasitic agent, or a combination thereof. Non-limiting examples of antimicrobial agents include: penicillin, methicillin, oxacillin, ampicillin, piperacillin, amoxicillin, cephazolin, cephalexin, cefuroxime, cefoxitin, cefotaxime, ceftriaxone, cefepime, vancomycin, teicoplanin, bleomycin, imipenem, meropenem, gentamicin, tobramycin, amikacin, erythromycin, clarithromycin, azithromycin, tetracycline, doxycycline, chloramphenicol, clindamycin, sulfisoxazole, sulfamethoxazole, metronidazole, trimethoprim, rifampicin, quinolones, clotrimazole, econazole, miconazole, terbinafine, fluconazole, ketoconazole, nystatin, amphotericin, candicidin, fllipin, hamycin, natamycin, rimocidin, bifonazole, butoconazole, econazole, fenticonazole, isoconazole, luliconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, efinaconazole, epoxiconazole, isavuconazole, itraconazole, osaconazole, propi conazole, ravuconazole, terconazole, voriconazole, abafungin, anidulafungin, caspfungin, micafungin, ibrexafungerp, acrisorcin, amorolfme, aurones, benzoic acid, abacavir, acyclovir, adefovir, amantadine, ampligen, amprenavir, umifenovir, atazanavir, tenofovir, baloxavir marboxil, boceprevir, bulevirtide, cidofovir, cobicistat, combivir, daclatasvir, d delaviridine, didanosine, docosanol, dolutegravir, doravirine, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, ensitrelvir, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, foscarnet, ganciclovir, ibacitabine, ibalizumab, idoxuridine, imiquimoid, inosine pranobex, idinavir, lamivudine, letermovir, lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nitazoxanide, norvir, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, remdesivir, ribavirin, rilpivirine, rimantadine, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, taribavirin, telaprevir, telbivudine, tenofovir alafenamide, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, Truvada, umifenovir, valaciclovir, valganciclovir, vicriviroc, vidarabine, zalcitabine, zanamivir, zidovudine, ivermectin, pyrimethamine, dapsone, mefloquine, sulfadiazine, quinine, nitazoxanide, albendazole, piperazine, amodiaquine, furazolidone, mebendazole, sulfametopyrazine, diethylcarbamazine, thiabendazole, pentamidine, levamisole, tinidazole, praziquantel, primaquine, oxamniquine, atovaquone, proguanil, halofantrine, poscaconazole, sulfadoxine, paromomycin, hydroxychloroquine, sinefungin 5-S-methyl-5’-thioadenosine, fumagillin, benzimidazole, diminazene, radicicol, geneticin, halofuginone, oxibendazole, lucanthone, andrographolide, spiramycin, eflornithine, lonidamine, tafenoquine, niclosamide, anisomycin, chlortetracycline, artesunate, pyrantel, hexylresorcinol, dichlorvos, doramectin, moxidectin, phenothiazine, selamectin, salinomycin, artenimol, nifurtimox, triclabendazole, secnidazole, carbendazim, piperaquine, chloroquine, quinacrine, suramin, sodium stibogluconate, artemether, hycanthone, tetrandrine, and lotilaner.

[0136] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the reminder of the disclosure in any way whatsoever.

[0137] EXAMPLES

[0138] Example 1. Internal Process Control Strain Selection

[0139] Molecular diagnostic tests typically employ spiked-in controls that are co-extracted and co-amplified in nucleic acid test-based detection methods. However, in next generation sequencing-based sample-to-answer diagnostic assays, internal process controls (IPCs) are useful to control every step of the diagnostic workflow. In applicatic time is limited, the IPC sequences could have a very high probability to out-compete target sequences at limit of detection concentrations, especially in the presence of other host- derived background nucleic acids. To address this issue and to improve assay specificity, an Internal Process Control (IPC) was developed to differentiate a negative result from a process failure within a sequencing assay without outcompeting target pathogen sequences (Table 1). If a target pathogen is identified in a clinical sample, the target is interpreted as being in the sample. If a target pathogen is not identified in a clinical sample that contains an IPC and the IPC is identified, the target is interpreted as being absent from the sample. If a target pathogen is not identified in a clinical sample and the IPC is not identified, the output is interpreted as a process failure.

[0140] Table 1. Interpreting sequencing assay output using an IPC in a clinical sample.

[0141] A useful IPC possesses particular characteristics. Candidate IPCs were selected in methods of the present disclosure are based on the following criteria:

[0142] • Ability to control for performance (e.g., sensitivity, specificity, accuracy, precision) in an end-to-end process;

[0143] • Having similar behavior to an analyte (e.g., a bacterial or fungal IPC for detecting bacterial or fungal pathogens) during processing;

[0144] • Phylogenetically divergent from analytes (e.g., distinct from pathogens);

[0145] • Having minimal interference or competition with an analyte signal (e.g., does not represent the majority of the sequencing reads);

[0146] • Having process bias (e.g., having high GC content); and

[0147] • Devoid of mobile genetic elements or plasmids that could over-amplify, creating competition with analytes, or be misidentified as belonging to inherent microbes in a clinical sample. A panel of candidate IPCs were analyzed and evaluated for their performance in a clinical sample processing end-to-end process. Candidate IPCs (e.g., Leucobacter muris, Leucobacter triazinivorans, Cellulomans iranesis, and Serinicoccus chugangensis) were spiked-in at a known concentration to a blood sample. This sample was then processed according to a whole blood lysis workflow. Briefly, this workflow includes the following steps: host cell lysis, microbial lysis, microbial genome amplification, sequencing, and bioinformatic analyses.

[0148] The species identified in each sample were evaluated for relatedness using prokaryotic sequence average amino acid identity (AAI) and visualized using hierarchical clustering (FIG. 1). Each of the IPCs scored similarly across all samples, with low AAI values, and demonstrated no significant relatedness between the IPCs and representative bacterial pathogens.

[0149] The results of this study indicate that incorporation of an IPC into an end-to-end process can enable the differentiation of false-negative results due to process failures mediated by the system, reagents, or sample, which are characterized by an absence of an IPC signal, from true negative results, which are characterized by the presence of an IPC signal.

[0150] Example 2. Successful IPC Detection With Target Pathogen in Clinical Samples

[0151] Presence of IPC (Leucobacter muris, L. muris) does not interfere with the target pathogen signal. Increasing concentrations of the IPC (0-500 CFU / ml) were added to 10 ml blood samples spiked in with the pathogen Pseudomonas aeruginosa (P. aeruginosa) at 8 CFU / mL. Pathogen and IPC cells were recovered from blood samples, pathogen and IPC cells were lysed, and pathogen and IPC whole genomes were amplified and sequenced on a nanopore sequencer (Oxford Nanopore PromethlON 2 flow cells) using the RBK114 library preparation kit. Total sequencing data (megabases) mapped to P aeruginosa (black) or the IPC (gray) was normalized to the total amount of sequencing data generated per sample with no significant difference observed for P. aeruginosa sequencing yield between samples with different concentrations of the IPC (FIG. 2). Concentrations of IPC added to the sample are represented on the x-axis (0-500 CFU / mL) with the ratio of megabases mapped to a target (P aeruginosa or the IPC) to the total number of sequenced megabases on the y-axis.

[0152] IPC signal recovery in samples spiked with clinically relevant pathogenic microbes is influenced by the guanine: cytosine (GC) content of the target pathogen. Percentages of total genomes at >lx depth recovered for pathogens (FIG. 3 A) and IPC (.

[0153] (FIG. 3B) from the spiked in blood samples following microbial recovery, whole genome amplification and sequencing is shown. Genome recovery (% coverage, y-axis) in the presence (gray) or absence of IPC (black) in 10 ml blood samples spiked with 2 CFU / mL of 5 pathogens of increasing GC content (x-axis), with IPC spiked at 100 CFU / ml. (FIG. 3A) No significant difference is observed in pathogen genome recovery between samples with and without the IPC for each of the 5 pathogens. Recovery of the IPC genome (% coverage, x- axis) increases as the GC content of target pathogen approaches that of the IPC, compared to low GC pathogens. GC content of species (low to high): Staphylococcus aureus (S. aureus) 32.8%; Candida albicans (C. albicans) 33.4%; Escherichia coli (E. coll) 50.8%; Klebsiella pneumoniae (K. pneumoniae) 57.0%, Pseudomonas aeruginosa (P. aeruginosa) 65.9%; IPC 70.6% (FIG. 3B).

Claims

CLAIMSWhat is claimed is:

1. A method comprising:(i) obtaining a clinical sample suspected of comprising one or more microbial species from a subject;(ii) introducing an internal process control (IPC) microbe into the clinical sample;(iii) processing the clinical sample comprising the IPC microbe, including lysing the one or more microbial species and / or the IPC microbe;(iv) amplifying whole genome microbial DNA from the lysed microbial species and / or the lysed IPC microbe; and(v) detecting amplified microbial DNA and / or IPC DNA, wherein detection of microbial DNA and / or IPC DNA in step (v) indicates that there was not a process failure.

2. A method comprising:(i) introducing an internal process control (IPC) microbe into a clinical sample;(ii) processing the clinical sample comprising the IPC microbe, including lysing the one or more microbial species and / or the IPC microbe;(iii) amplifying whole genome microbial DNA from the lysed microbial species and / or the lysed IPC microbe; and(iv) detecting amplified microbial DNA and / or IPC DNA, wherein detection of microbial DNA and / or IPC DNA in step (iv) indicates that there was not a process failure.

3. The method of claim 1 or claim 2, wherein the subject is a human.

4. The method of any one of claims 1-3, wherein the one or more microbial species are pathogenic microbial species.

5. The method of any one of claims 1-4, wherein the IPC microbe has 55% - 90%Guanine / Cytosine (G / C) content in its genome.

6. The method of any one of claims 1-5, wherein the IPC microbe is devoid of nonintegrated self-replicating genetic elements.

7. The method of any one of claims 1-6, wherein the IPC microbe is a bacterium.

8. The method of claim 7, wherein the IPC microbe is a Gram-positive bacterium.

9. The method of claim 7 or claim 8, wherein the IPC microbe is from genus Leucobacter .

10. A kit comprising one or more internal process control (IPC) microbes.

11. The kit of claim 10, wherein the one or more IPC microbes comprises a Gram-positive bacterium.

12. The kit of claim 11, wherein the Gram-positive bacterium is from genus Leucobacter.

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