WO2022164968A1 - Methods for enriching microorganisms from low abundance clinical samples for dna sequencing - Google Patents

Methods for enriching microorganisms from low abundance clinical samples for dna sequencing Download PDF

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WO2022164968A1
WO2022164968A1 PCT/US2022/014003 US2022014003W WO2022164968A1 WO 2022164968 A1 WO2022164968 A1 WO 2022164968A1 US 2022014003 W US2022014003 W US 2022014003W WO 2022164968 A1 WO2022164968 A1 WO 2022164968A1
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dna
microbial
amplification
subsamples
clinical sample
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PCT/US2022/014003
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French (fr)
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Chiahao TSUI
Douglas S. KWON
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Day Zero Diagnostics, Inc.
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Publication of WO2022164968A1 publication Critical patent/WO2022164968A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the present disclosure relates generally to the field of diagnostics for microbial infections based on assays for microbial nucleic acids in a clinical sample.
  • the invention relates to improved methods for enriching microbial DNA relative to host DNA in connection with amplification and sequencing to identify microbes present at low levels in a sample.
  • Bacterial infection of the blood known as bacteremia, is one of the most common causes of death worldwide; approximately one in five individuals worldwide will be afflicted by bacteremia. Bacteremia is also the current leading cause of in-hospital deaths in the United States.
  • Blood-borne microbial infections can generally be treated with antibiotics, antifungal, or antiviral medications, but can sometimes result in sepsis, a life-threatening illness in which systemic inflammation can lead to multi-organ collapse and eventually death if left untreated.
  • the early detection of blood infection is paramount as mortality rate and economic cost can dramatically increase when patients are not quickly diagnosed after admission into the hospital.
  • the treatment of microbial infections is becoming even more challenging with the rise of antimicrobial resistance among microbial organisms. (Andersson and Hughes (2010), Nat. Rev. Microbio. 8: 260-71.)
  • NGS next generation sequencing
  • Current approaches utilize enriched cell-free DNA sequencing, amplicon-based sequencing (e.g., 16S targeted amplification), or human DNA depletion methods to isolate causative pathogens for whole-genome amplification (WGA) and sequencing (Blauwkamp, et al., (2019), Nat. Microbiol. 4: 663- 674; Ellis, et al., (2017), J. Microbiol. Methods 138; O’Grady, et al., US Pat. Pub. No. 20190316113 A 1 ).
  • Current enrichment methods for NGS are not effective for rapid identification (e.g., same day) with concomitant high sequencing depth and high genome coverage due to inadequate pathogen enrichment.
  • the present invention depends, in part, upon the development of improved methods for increasing the proportion of microbial cells or DNA relative to other cells or DNA present in a clinical sample obtained from a patient.
  • these improved methods can (i) highly enrich for microbial cells or DNA for detection, (ii) rapidly recover microbial cells or DNA directly from whole blood at low (e.g., ⁇ 5) CFU/mL levels, (iii) amplify microbial DNA in a species specific and/or species non-specific manner, and (iv) sequence the amplified genetic material with quick turn-around times (TATs).
  • the methods can, in some embodiments, produce DNA sequencing data with a significantly enriched proportion of pathogen DNA (e.g., > 25%). This enriched pathogen sequencing data makes it possible to provide fast TATs while providing pathogen whole genome coverage to permit high confidence AMR profiling by a bacteremia diagnostic device.
  • the invention provides methods for determining the presence or absence of a microbial infection in a subject.
  • the methods comprise the following steps: (a) obtaining a clinical sample from the subject; (b) dividing the clinical sample into at least two subsamples; (c) subjecting the clinical sample prior to step (b), and/or the subsamples after step (b), to at least one method of enrichment for microbial DNA; and (d) determining the abundance of microbial DNA in the enriched subsamples.
  • an abundance of microbial DNA greater than zero in at least one subsample indicates the presence of a microbial infection.
  • an abundance of microbial DNA of zero in all subsamples indicates that a microbial infection has not been detected.
  • the invention provides methods for identifying a microbial infection in a subject.
  • the methods comprise the following steps: (a) obtaining a clinical sample from the subject; (b) dividing the clinical sample into at least two subsamples; (c) subjecting the clinical sample prior to step (b), and/or the subsamples after step (b), to at least one method of enrichment for microbial DNA; and (d) analyzing the DNA from the subsamples.
  • the methods comprise the following steps: (a) obtaining a clinical sample from the subject; (b) dividing the clinical sample into at least two subsamples; (c) subjecting the clinical sample prior to step (b), and/or the subsamples after step (b), to at least one method of enrichment for microbial DNA; (d) determining the abundance of microbial DNA in the enriched subsamples; (e) (i) if at least one subsample has an abundance of microbial DNA greater than zero, selecting at least one subsample with a higher abundance of microbial DNA relative to at least one unselected subsample, or (e) (ii) if no subsample has an abundance of microbial DNA greater than zero, determining that the presence of a microbial infection has not been identified; and (f) if at least one subsample has an abundance of microbial DNA greater than zero, analyzing the microbial DNA from the subsamples selected in (e) (i).
  • determining the abundance of microbial DNA comprises species- specific amplification of microbial DNA.
  • the microbial DNA is amplified by a method selected from the group consisting of: polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), reverse-transcriptase PCR (RT-PCR), degenerate oligonucleotide PCR, primer extension pre-amplification, loop-mediated isothermal amplification (LAMP), reverse transcriptase LAMP (RT-LAMP), primer extension pre-amplification (PEP), strand displacement amplification (SDA), helicase dependent amplification (HD A), transcription mediated amplification (TMA), and recombinase polymerase amplification (RPA).
  • PCR polymerase chain reaction
  • qPCR quantitative polymerase chain reaction
  • RT-PCR reverse-transcriptase PCR
  • RT-PCR reverse-transcriptase PCR
  • PCR primer extension pre-amplification
  • LAMP loop-mediated isothermal
  • determining the abundance of microbial DNA comprises whole genome sequencing. In some embodiments, determining the abundance of microbial DNA comprises a polymerase-based or hybridization-based assay. In some embodiments, the polymerase-based assay is selected from the group consisting of a qPCR, LAMP, reverse transcriptase LAMP (RT-LAMP), and RPA-based assay. In some embodiments, the hybridization-based assay is selected from the group consisting of a microarray, southern blot, and northern blot-based assay.
  • Various methods of the present disclosure include a step of enrichment.
  • the clinical sample is subjected to at least one method of enrichment for microbial DNA before the clinical sample is divided into at least 2 subsamples (e.g., before step (b)).
  • the clinical sample is subjected to at least one method of enrichment for microbial DNA after the clinical sample is divided into at least 2 subsamples (e.g., after step (b)).
  • the clinical sample is subjected to at least one method of enrichment for microbial DNA before and after the clinical sample is divided into at least 2 subsamples (e.g., before and after step (b)).
  • the method of enrichment comprises a method of non-amplification enrichment. In some embodiments, the method of non-amplification enrichment comprises reducing the abundance of human cells present in the clinical sample or subsample. In some embodiments, the method of non- amplification enrichment includes at least one method selected from the group consisting of centrifugation, immunoaffinity purification, filtration, and selective lysis to remove human cells. In some embodiments, the method of non-amplification enrichment comprises reducing the abundance of human DNA present in the clinical sample or subsample. In some embodiments, the method of non-amplification enrichment includes at least one method selected from the group consisting of centrifugation, immunoaffinity purification, filtration, and selective lysis to remove human DNA.
  • the method of enrichment comprises a method of global amplification.
  • the method of global amplification comprises a method selected from the group consisting of PCR, qPCR, RT- PCR, degenerate oligonucleotide PCR, primer extension pre-amplification, LAMP, SDA, HDA, TMA, and RPA.
  • the method of global amplification amplifies human and/or microbial DNA present in the clinical sample or subsample.
  • the microbial DNA comprises DNA from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, or more microbial species.
  • the clinical sample comprises an abundance of microbial DNA comprising no more than 10 genomic copies per mL of clinical sample volume prior to enrichment or determining abundance.
  • analyzing the DNA comprises analyzing human and/or microbial DNA present in the subsamples.
  • analyzing the DNA comprises whole genome sequencing.
  • analyzing the DNA comprises generating an Antimicrobial Resistance Sensitivity (AMR/S) profile using a computational algorithm for the microbial species.
  • analyzing the DNA comprises using a molecular- or sequencing-based method to identify the microbial infection.
  • analyzing the DNA comprises using a molecular- or sequencing-based method to identify one or more genus, species, or sub-species strains.
  • the molecular- or sequencing-based method is selected from the group consisting of qPCR, LAMP, RT-LAMP, RPA, microarrays, southern blot, northern blot, next generation sequencing (NGS), and third generation sequencing.
  • NGS or third generation sequencing achieves at least 75% coverage of the microbial genome. In some embodiments, the NGS or third generation sequencing achieves at least 75% coverage of the microbial genome in less than 3 hours.
  • the microbial infection comprises microbes having one or more resistance genes, virulence genes, single nucleotide polymorphisms, accessory genomes, plasmids, and/or recombination regions.
  • one or more detected resistance or virulence genes are amplified.
  • the one or more detected resistance or virulence genes are amplified by PCR, qPCR, LAMP, SDA, HDA, TMA, or RPA.
  • Various methods of the present invention include a step of dividing a clinical sample into subsamples (z.e., a step of subsampling).
  • the clinical sample is obtained from a subject.
  • the clinical sample is a blood sample.
  • the clinical sample is a cerebrospinal fluid (CSF) sample.
  • the clinical sample is a joint fluid, abscess fluid, serum, lymph, urine, stool, or sputum sample.
  • the clinical sample is divided into 4 subsamples.
  • the clinical sample is divided into at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 subsamples.
  • the subsamples comprise substantially equal volumes.
  • the subsamples each comprise at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mL.
  • FIG. 1A shows a direct comparison of replicates from 12 donors in which DNA amplification reactions were conducted using four divided samples and compared to one combined sample.
  • Each replicate represents an individual human donor from which 16 mL of whole blood was processed using the methods as described in Example 1 and amplified as either 4 individual subsamples (4 mL each; dots) or 1 single combined sample (16 mL; squares).
  • the amplified DNA product was processed and sequenced with Oxford Nanopore Technology flow cells.
  • “M:H” is the ratio of microbial (“M”; e.g., bacterial) reads to human (“H”) reads from each individual amplified reaction. The mean is displayed as a horizontal bar when available.
  • FIG. IB shows the CFU / mL plating control range of Klebsiella pneumoniae spiked into each of the 12 donor replicates.
  • FIG. 1C shows the average reads generated for all subsamples compared to the combined samples.
  • FIG. ID shows the average assembly coverage for all subsamples compared to the combined samples.
  • the present invention relates to improved methods that, in some embodiments, can (i) highly enrich for microbial cells for detection, (ii) rapidly recover microbial cells directly from whole blood at low (e.g., ⁇ 5) CFU/mL levels, (iii) agnostically amplify microbial DNA, and (iv) sequence the amplified genetic material with quick turnaround times (TATs).
  • the methods can, in some embodiments, produce DNA sequencing data with a significantly enriched proportion of pathogen DNA (e.g., > 25%). This enriched pathogen sequencing data makes it possible to provide faster TATs while providing whole genome coverage for high confidence AMR profiling using a bacteremia diagnostic device.
  • Bacteremia is currently the leading cause of in-hospital deaths in the United States, yet the standard, culture-based diagnostic methods can require several days to identify microbial species within a sample from a subject, and often include false negatives.
  • Other methods such as polymerase- and hybridization-based assays, provide more rapid identification of microbial species than culture-based methods; however, there are inherent limitations to the breadth of species these methods can identify, and same-day AMR profiling remains difficult to achieve.
  • a critical reason for this is the relative rarity of microbial DNA in a sample (e.g., a blood sample) taken from a human subject with bacteremia or another microbial infection. For example, at 1 CFU/mL, human DNA is up to a billion times more abundant than microbial DNA. This relative rarity makes the use of sequencing-based methods to identify microbial DNA difficult, slow, and cost-inefficient.
  • a possible strategy is to simply increase the sample size.
  • a larger sample would naturally lead to a greater abundance of microbial DNA, which can theoretically enhance the specificity of targeted-amplification methods such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), etc.
  • PCR polymerase chain reaction
  • LAMP loop-mediated isothermal amplification
  • larger sample volumes e.g., > 50 mL
  • require increased handling of the sample which can lead to contamination and added expense, and obtaining larger samples from the subject may be difficult, or not possible. Larger sample volumes may also be harmful to the subject and thus be clinically unacceptable.
  • the present invention provides an alternative way to address both the small quantity and small percentage of microbial DNA present in a clinical sample taken from a subject with bacteremia.
  • the present invention describes a method of increasing the relative abundance of one or more microbial species in a clinical sample from a human subject by dividing the clinical sample into subsamples. Instead of combining samples for a quantitatively larger microbial DNA input, Example 1 surprisingly demonstrates that the relative abundance of microbiakhuman DNA can be increased per sample using smaller subsamples, instead of a combined sample. Unexpectedly, this increase was achieved without negatively impacting overall percent genome coverage.
  • one sample of human blood may contain, for example, 16 “units” of microbial DNA. Dividing that clinical sample into four subsamples, the expected value would be 4 units of microbial DNA per subsample. Indeed, if the size of the clinical sample is sufficiently large and/or the percentage of microbial DNA is sufficiently high, the distribution of the microbial DNA units per subsample might be expected to be 4, 4, 4, and 4 units (z.e., 4 units of microbial DNA per subsample).
  • the random distribution of the microbial DNA units would have greater variance, and might result in 6, 8, 1, and 1 units (z.e., 6 units of microbial DNA in subsample A, 8 units in subsample B, 1 unit in subsample C, and 1 unit in subsample D).
  • the abundance of human DNA in each subsample can be separately determined, or may be assumed to be constant because of its relatively greater abundance and correspondingly lower variance.
  • the relative abundance of microbiakhuman DNA would be increased in subsample A (by 50%) and subsample B (by 100%), and decreased in subsamples C and D (by 75%).
  • the amount of microbial DNA present in each subsample will vary from subsample to subsample, and therefore the relative abundance of microbiakhuman DNA present in at least one subsample is likely to be increased relative to the other subsamples.
  • aspects of the invention provide methods of determining the presence of a microbial infection in a subject.
  • the method comprises the following steps: (a) obtaining a clinical sample from the subject; (b) dividing the clinical sample into at least two subsamples; (c) subjecting the clinical sample prior to step (b), and/or the subsamples after step (b), to at least one method of enrichment for microbial DNA; and (d) determining the abundance of microbial DNA in the enriched subsamples.
  • an abundance of microbial DNA greater than zero in at least one subsample indicates the presence of a microbial infection.
  • an abundance of microbial DNA of zero in all subsamples indicates that a microbial infection has not been detected.
  • aspects of the invention contemplate a method of identifying a microbial infection in a subject.
  • the method comprises the following steps: (a) obtaining a clinical sample from the subject; (b) dividing the clinical sample into at least two subsamples; (c) subjecting the clinical sample prior to step (b), and/or the subsamples after step (b), to at least one method of enrichment for microbial DNA; and (d) analyzing the DNA from the subsamples.
  • the method comprises the following steps: (a) obtaining a clinical sample from the subject; (b) dividing the clinical sample into at least two subsamples; (c) subjecting the clinical sample prior to step (b), and/or the subsamples after step (b), to at least one method of enrichment for microbial DNA; (d) determining the abundance of microbial DNA in the enriched subsamples; (e) (i) if at least one subsample has an abundance of microbial DNA greater than zero, selecting at least one subsample with a higher abundance of microbial DNA relative to at least one unselected subsample, or (e) (ii) if no subsample has an abundance of microbial DNA greater than zero, determining that the presence of a microbial infection has not been identified; and (f) if at least one subsample has an abundance of microbial DNA greater than zero, analyzing the microbial DNA from the subsamples selected in (e) (i).
  • the samples can be combined, and then subsampled again until variance between the subsamples is detected.
  • a can mean one or more than one.
  • a cell can mean a single cell or a multiplicity of cells.
  • variable can be equal to any integer value within the numerical range, including the endpoints of the range.
  • variable can be equal to any real value within the numerical range, including the endpoints of the range.
  • a variable that is described as having values between 0 and 2 can take the values 0, 1, or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values 0 and 2 if the variable is inherently continuous.
  • an “agnostic”, “global”, or “species non-specific” amplification refers to amplification of DNA (e.g., microbial DNA, human DNA, or both) which uses primer sequences which are not limited to a single species but, rather, are characteristic of two or more species.
  • the amplification is agnostic with respect to the multiple species within the DNA being amplified because no particular species is targeted or, in some embodiments, can be targeted.
  • amplification methods using primers directed to 16S rRNA sequences that are conserved across many bacterial species can agnostically or species non- specifically enrich for multiple bacterial 16S rRNA sequences.
  • some agnostic methods can amplify all or most sequences in a population of nucleic acids (e.g., using a mixture of random hexamer primers).
  • amplification refers to the process of increasing the number of copies of a specific nucleotide sequence in a population of nucleic acids by templatedependent and polymerase-dependent chemical synthesis.
  • Methods of amplification include, but are not limited to, polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), reverse-transcriptase PCR (RT-PCR), degenerate oligonucleotide PCR, primer extension pre-amplification, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HD A), transcription mediated amplification (TMA), and recombinase polymerase amplification (RPA).
  • PCR polymerase chain reaction
  • qPCR quantitative polymerase chain reaction
  • RT-PCR reverse-transcriptase PCR
  • PCR primer extension pre-amplification
  • LAMP loop-mediated isothermal amplification
  • SDA strand displacement amplification
  • HD A transcription mediated
  • the nucleic acid amplification is PCR, qPCR, RT-PCR, degenerate oligonucleotide PCR, primer extension pre-amplification, LAMP, RT-LAMP, SDA, HDA, TMA, or RPA.
  • bacteria are single-celled microbes of the kingdom Prokaryota. Of particular interest in the methods of the invention are human pathogenic bacteria species, and more particularly those species associated with bacteremia in humans.
  • the bacteria are Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Enterococcus faecalis , Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, or any other species associated with bacteremia.
  • bacteria refers to the presence of bacteria in the blood.
  • the bacteria present in the blood are infectious bacteria that cause disease in a host.
  • bacteremia can also include non-pathogenic bacteria.
  • a “clinical sample” is a biological sample obtained from a subject.
  • a clinical sample may be directly obtained from the subject (e.g., by collecting the sample from the subject), or may be received indirectly from another person or entity (e.g., a healthcare provider or reference laboratory).
  • a step of “obtaining” can include obtaining directly or indirectly.
  • enrichment refers to the increase in the proportion of one population of DNA (e.g., microbial DNA) relative to a second population of DNA (e.g., human DNA) in a mixed sample comprising at least two populations of DNA. Such an increase in proportion may be achieved by increasing the abundance of the first population of DNA, and/or by decreasing the abundance of the second population of DNA.
  • enrichment comprises a method of non-amplification enrichment. Methods of non-amplification enrichment may include, for example, centrifugation, immunoaffinity purification, filtration, and selective lysis (e.g., to remove human cells and/or human DNA).
  • enrichment comprises a method of amplification. Such amplification may in some embodiments be global or species-specific.
  • host DNA refers to DNA derived from a host (e.g., a human patient or subject), and “non-host DNA” refers to DNA derived from a microbe.
  • mammal refers to a warm-blooded vertebrate that is distinguished by the possession of hair or fur, the secretion of milk by females to nourish the young, and the birth of live young.
  • microbe and “microbial” refer to a microorganism that requires a microscope to be visualized.
  • Non-limiting examples of microbes include: bacteria, archaea, fungi, protists, viruses, and microscopic animals.
  • Pathogenic microbes are capable of causing disease in a host organism.
  • a “mixed sample” or a “mixed clinical sample” is a sample that comprises DNA from at least two sources.
  • the mixed sample comprises a first population and a second population of nucleic acids.
  • the first population of nucleic acids is mammalian DNA (e.g., human DNA) and the second population of nucleic acids is microbial DNA.
  • the first population of nucleic acids is host DNA (e.g., patient DNA) and the second population of nucleic acids is non-host DNA.
  • the first population and the second population of nucleic acids are both microbial DNA.
  • the first population and second population of nucleic acids are both bacterial DNA.
  • the term “relative abundance” refers to the abundance of a nucleotide sequence in a first population of DNA relative to a second population of DNA.
  • the relative abundance can be calculated by dividing (a) the abundance of the nucleotide sequence in the first population by (b) the abundance of the nucleotide sequence in the second population.
  • the abundance of the nucleotide sequence in each population can be estimated by dividing (a) the amount of DNA (e.g., number of bp) in the population corresponding to the nucleotide sequence (including duplicate copies) by (b) the total amount of DNA (e.g., number of bp) in the population.
  • the relative abundance can be approximated based upon knowledge of whole or partial genome sequences or experimentally. If the abundance of the second population of DNA is assumed to be constant (or substantially constant), the relative abundance can be estimated based on the abundance of the first population.
  • sequencing refers to a method for determining the nucleotide sequence of a polynucleotide (e.g.. a genomic DNA sequence).
  • sequencing methods include as non-limiting examples whole genome sequencing (WGS), next generation sequencing (NGS) methods, in which clonally amplified DNA templates or single DNA molecules are sequenced in a massively parallel fashion, or third generation sequencing technology such as nanopore-based methods, in which nucleic acid molecules are carried through a pore and the resulting ionic current change is measured and converted to nucleotide sequences (e.g., as described in Volkerding et al. (2010), Clin. Chem., 55:641-658; Metzker (2010), Nature Rev.
  • the NGS or third generation sequencing achieves at least 75% coverage of the microbial genome. Such coverage may, in some embodiments, be achieved in less than 3 hours.
  • kits non-specific microbial DNA refers to microbial DNA having a sequence found in two or more microbial species, but not found in human DNA.
  • samples are two or more samples that have been derived from a single, original sample.
  • “Clinical subsamples” are subsamples that have been divided from a single, original clinical sample obtained from a subject (e.g., a blood, CSF, joint fluid, or abscess sample).
  • substantially equal refers to a first measurement (e.g., a volume) that differs from a second measurement by a value less than about 1 percent.
  • the first measurement differs from the second measurement by less than 0.9 percent, 0.8 percent, less than 0.7 percent, less than 0.6 percent, less than 0.5 percent, less than 0.4 percent, less than 0.3 percent, less than 0.2 percent, or less than 0.1 percent.
  • WGA Whole Genome Amplification
  • WGA generally refers to a method for amplification of a limited DNA sample in a non-specific manner in order to generate a new sample that has a higher DNA concentration.
  • DOP degenerate oligonucleotide-primed PCR
  • PEP primer extension PCR
  • MDA multiple displacement amplification
  • WGA is a method of global amplification.
  • WGS Whole Genome Sequencing
  • the WGS methods of the invention are those sequencing methods that, when applied to a sample of genomic DNA, are capable of obtaining the sequence of substantially the entire genome.
  • WGS can be performed using any next-generation or third generation sequencing technology known in the art. In some embodiments, WGS is a method of global sequencing.
  • the present invention provides methods for determining the presence of and/or identifying one or more microbial species present in a clinical sample obtained from a mammalian subject, typically a human subject.
  • clinical samples are obtained directly or indirectly from human subjects.
  • the clinical samples are obtained from non-human mammalian subjects.
  • the non-human mammalian subjects are companion animals such as dogs or cats; agricultural animals such as cows, pigs, sheep, goats or horses; or common laboratory animals such as rodents, rabbits or non-human primates.
  • the clinical sample is obtained or derived from blood, joint fluid, abscess fluid, serum, sputum, mucus, saliva, wound drainage, urine, stool, lymph, lavage, cerebral- spinal fluid (CSF), or any fluid aspirate or tissue extraction of human and/or other mammalian origin.
  • the clinical sample is obtained or derived from blood.
  • the clinical sample is obtained or derived from CSF.
  • the clinical sample is obtained or derived from joint fluid (e.g., a prosthetic joint).
  • the clinical sample is obtained or derived from tissue abscess fluid.
  • the clinical sample comprises a quantity of DNA including from between 1 and 10 8 genomes (e.g., human, microbial, and any other genomes) per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1 and 10 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1 and 10 2 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1 and 10 3 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1 and 10 4 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1 and 10 5 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1 and 10 6 genomes per milliliter.
  • the clinical sample comprises DNA including from between 1 and 10 8 genomes (e.g., human, microbial, and any other genomes) per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1 and 10 genomes per milliliter. In some embodiments, the clinical sample
  • the clinical sample comprises DNA including from between 1 and 10 7 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 10 3 and 10 5 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 10 5 and 10 6 genomes per milliliter.
  • the clinical sample comprises DNA including from between 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000 , 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 50,000,000, 60,000,000, 70,000,000, 80,000,000, 90,000,000, or 100,000,000 genomes per milliliter, or any value contained therein.
  • the clinical sample comprises a quantity of DNA including from between 0.1 and 10 4 microbial genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 0.1 and 10 microbial genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 0.1 and 10 2 microbial genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 0.1 and 10 3 microbial genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 10 2 and 10 3 microbial genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 10 3 and 10 4 microbial genomes per milliliter.
  • the clinical sample comprises DNA including from between 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 microbial genomes per milliliter, or any value contained therein.
  • the clinical sample comprises an abundance of microbial DNA comprising no more than 10 genomic copies per milliliter of clinical sample volume prior to enrichment or determining abundance.
  • the clinical sample comprises a quantity of DNA including from between 200 and 40,000,000 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 200 and 400 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 200 and 4,000 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 200 and 40,000 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 200 and 10 5 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including between 200 and 10 6 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 200 and 10 7 human genomes per milliliter.
  • the clinical sample comprises DNA including from between 200 and 20,000,000 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including between 200 and 30,000,000 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, or 40,000,000 human genomes per milliliter, or any value contained therein.
  • the clinical sample comprises DNA including from between 8,000,000 to 22,000,000 human genomes per milliliter (e.g., 8,000,000, 9,000,000, 10,000,000, 11,000,000, 12,000,000, 13,000,000, 14,000,000, 15,000,000, 16,000,000, 17,000,000, 18,000,000, 19,000,000, 20,000,000, 21,000,000, or 22,000,000 human genomes per milliliter, or any value contained therein).
  • the subject has, or is suspected of having, a microbial infection (e.g., bacteremia).
  • a microbial infection e.g., bacteremia
  • the clinical sample comprises DNA from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, or more microbial species.
  • the clinical sample is directly obtained by a person practicing the methods of the present invention.
  • the clinical sample is obtained indirectly by a person practicing the methods of the present invention.
  • the clinical sample may be directly obtained from a subject, by either the subject or by a physician, physician’s assistant, nurse, laboratory technician or other healthcare personnel, and then may be indirectly obtained by a person practicing the methods of the present invention.
  • “obtaining” a clinical sample encompasses both directly and indirectly obtaining the sample according to the description herein.
  • Methods for obtaining clinical samples from a subject are known in the art, and may include, for example, identifying the patient prior to collecting a sample (e.g., by checking identification, armbands, etc.), labelling collection containers with appropriate patient identifiers in the presence of the patient, using at least two patient identifiers to label each container, sterilizing the collection site, drawing the samples into collection tubes in the proper sequence (e.g., blood culture tubes; coagulation tubes; serum tubes with or without clot activator, and with or without gel; heparin tubes, with or without gel plasma separator; EDTA tubes; oxalate and fluoride tubes; etc.), inverting the collection tubes end-to-end (/'. ⁇ ?., gentle inversion) multiple times (e.g., 10 times) after collection, using proper collection containers, not transferring samples into secondary containers, delivering samples to the laboratory promptly after collection and/or processing the samples promptly after collection, avoiding hemolysis, drawing a first “flush” syringe prior to
  • a clinical sample comprises anywhere between 0.1 mL and 50 mL of sample material.
  • a clinical sample comprises 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 45, 40, 45, or 50 mL, or any value included therein, such as 1.1, 1.2, 1.3, etc., mL of sample material.
  • a clinical sample comprises 16 mL of sample material.
  • clinical samples are stored at room temperature (e.g., -20-25 °C).
  • clinical samples are refrigerated (e.g., -2-8 °C, including 4 °C).
  • clinical samples are frozen (e.g., less than or equal to 0 °C).
  • clinical samples are stored in sample containers.
  • sample containers A variety of different sample containers are used in the art, and may be adapted to different types of clinical samples.
  • commonly used blood collection containers, blood culture bottles, plasma tubes, and blood culture media may include heparin, including lithium heparin and/or sodium heparin (e.g., Cat. Nos. 364960, 366667, 367871, 367878, 367884, 367886, 367960, 367961, 367962, and 367964 Vacutainer® collection tubes, BD Biosciences, Franklin Lakes, NJ), SPS (e.g., Cat. No. 364960 Vacutainer® collection tubes, Cat. Nos.
  • sample containers are pre-loaded with preservatives and/or anticoagulants (e.g., sodium polyanetholesulfonate (SPS), heparin, lithium heparin, sodium heparin, citrate, sodium citrate, acid citrate dextrose (ACD), hyaluronate, dermatan sulfate polyanion, EDTA, potassium EDTA (K2EDTA), and chondroitin D-glucuronate anion) that may have the unintended effect of acting as nucleic acid amplification inhibitors during genetic identification or analysis of the various nucleic acids present in a sample (see, e.g., Fredericks and Reiman (1998), J.
  • preservatives and/or anticoagulants e.g., sodium polyanetholesulfonate (SPS), heparin, lithium heparin, sodium heparin, citrate, sodium citrate, acid citrate dextrose (ACD), hyaluronate, dermat
  • the methods of the invention comprise dividing a clinical sample into two or more subsamples.
  • the clinical sample is approximately evenly divided such that each subsample contains an equal volume of clinical sample material.
  • the subsamples comprise substantially equal volumes.
  • the clinical sample is unevenly divided, either intentionally or unintentionally, such that at least one subsample contains an unequal volume of clinical sample material relative to the other subsamples.
  • Methods for dividing a clinical sample would be readily apparent to those skilled in the art. Briefly, a clinical sample may be divided into subsamples by aliquoting certain amounts of sample material from a first container into two or more secondary containers (e.g.. test tubes, vials, etc.). Methods for aliquoting may be performed by a human or by a machine, and include, for example, pouring, dispensing from a syringe, or dispensing from a pipette.
  • subsamples are handled and stored using the same methods that would be practiced in the handling and storage of similar clinical samples, as known by those of skill in the art and described herein.
  • a clinical sample is divided into two or more subsamples. In some embodiments, a clinical sample is divided into between 2 and 1,000 subsamples. In some embodiments, a clinical sample is divided into between 2 and 10 subsamples. In some embodiments, a clinical sample is divided into between 2 and 20 subsamples. In some embodiments, a clinical sample is divided into between 2 and 50 subsamples. In some embodiments, a clinical sample is divided into between 2 and 100 subsamples. In some embodiments, a clinical sample is divided into between 2 and 500 subsamples.
  • a clinical sample is divided into 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 subsamples. In some embodiments, a clinical sample is divided into 4 subsamples. In some embodiments, a clinical sample is divided into at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 subsamples.
  • a 16 mL clinical sample is divided into 4 subsamples, each with a volume of approximately 4 mL.
  • the subsamples each comprise at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mL.
  • the abundance of microbial DNA present in each of the two or more divided subsamples is determined, and one or more of the divided subsamples is selected for further analysis.
  • subsamples are further divided into two or more subsamples.
  • a subsample is divided into between 2 and 1,000 subsamples.
  • a subsample is divided into between 2 and 10 subsamples.
  • a subsample is divided into between 2 and 20 subsamples.
  • a subsample is divided into between 2 and 50 subsamples.
  • a subsample is divided into between 2 and 100 subsamples.
  • a subsample is divided into between 2 and 500 subsamples.
  • a subsample is divided into 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 subsamples.
  • the abundance of microbial DNA present in each of the two or more further divided subsamples is determined, and one or more of the further divided subsamples is selected for further analysis.
  • At least two selected subsamples are recombined (e.g., pooled) to create one or more combined selected subsamples.
  • the step of pooling may be performed before or after various steps of the methods of the present invention.
  • the step of pooling is performed prior to detecting and/or identifying one or more specific microbial species based upon the presence of species- specific microbial DNA present in at least one selected subsample.
  • the step of pooling is performed prior to subjecting the selected subsample to a qualitative second analysis to identify one or more specific microbial species within the sub sample.
  • Subsamples may be pooled in order to create combined selected subsamples which contain a higher relative abundance of microbial DNA than was present in one or more nonpooled subsamples, or than was present in the original clinical sample.
  • the selected subsamples chosen for the step of pooling are those with a higher relative abundance of microbial DNA than any of the non-selected subsamples.
  • the selected subsamples have a 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%,
  • one or more secondary clinical samples are further divided into two or more tertiary subsamples (e.g., subsamples created upon dividing the pooled secondary clinical samples) according to the subsampling methods described herein.
  • two or more secondary subsamples are re-combined (e.g..
  • tertiary clinical samples e.g., clinical samples created from the pooling of selected secondary subsamples.
  • the steps of dividing and pooling can be repeated one time, two times, three times, or more than three times, according to the methods described herein.
  • the subsamples e.g., subsamples, secondary subsamples, tertiary subsamples, etc.
  • clinical samples e.g., clinical samples, secondary clinical samples, tertiary clinical samples, etc.
  • the abundance of microbial DNA present in each of the two or more divided subsamples may be determined, and one or more of the divided subsamples may be selected for further analysis.
  • Certain methods of the invention include a step of enriching for microbial DNA. Such enrichment may occur before and/or after the step of subsampling (e.g., before and/or after step (b)).
  • the clinical sample is subjected to at least one method of enrichment for microbial DNA before the step of subsampling.
  • the clinical sample is subjected to at least one method of enrichment for microbial DNA after the step of subsampling.
  • the clinical sample is subjected to at least one method of enrichment for microbial DNA before and after the step of subsampling.
  • the methods of the invention therefore involve processing (e.g., enriching) the clinical sample to alter the abundance of certain cells and/or DNA present in the sample prior to determining the abundance of microbial DNA in the enriched subsamples.
  • enriching for microbial DNA may be achieved by increasing the abundance of microbial cells and/or DNA present in the clinical sample or subsample (e.g., by a method of species-specific enrichment, such as by species-specific amplification), and/or by decreasing the abundance of human cells and/or DNA present in the clinical sample or subsample (e.g., by a method of non-amplification enrichment, such as selective lysis).
  • the method of enrichment comprises a method of nonamplification enrichment.
  • the method of non-amplification enrichment comprises reducing of the abundance of human cells present in the clinical sample or subsample.
  • the method of non-amplification enrichment comprises reducing of the abundance of human DNA present in the clinical sample or subsample.
  • the method of non-amplification enrichment includes at least one method selected from the group consisting of centrifugation, immunoaffinity purification, filtration, and selective lysis to remove human cells.
  • Centrifugation may in some embodiments include differential centrifugation to separate one population of cells and/or DNA from another population based on size (see, e.g., Amasia and Madou (2010), Bioanalysis, 2(10):1701-10; Pitt et al.
  • Selective lysis may in some embodiments be applied to one population of cells and/or DNA using detergents such as Triton X-100, Tween 20, Tween 80, or saponin (see, e.g., Hasan et al. (2016), J. Clin. Microbio. 54(4): 919-27; Shehadul Islam et al. (2017), Micromachines 8(3): 83), or using proteins targeting human cells and/or human DNA (see, e.g., O’Grady et al., US Pat. Pub. No. 20190316113A1).
  • the method of non-amplification enrichment comprises a combination of selective lysis and DNase I (see, e.g., Charalampous et al. (2019), Nat. Biotech. 'NK 'y.'l 83-92), or any other combination of the foregoing.
  • mtDNA human mitochondrial DNA
  • sample processing methods and blocking oligonucleotides are described in International Patent Application No. PCT/US2018/056598, published on April 25, 2019 as WO2019/079656, the entire disclosure of which is incorporated herein in its entirety.
  • these sample processing methods result in at least a 50% increase in relative abundance of a targeted population of nucleic acids (e.g., microbial, bacterial, or non-host) in clinical samples and/or subsamples, relative to clinical samples and/or subsamples which have not undergone the sample processing methods.
  • nucleic acids e.g., microbial, bacterial, or non-host
  • nucleic acids e.g., microbial, bacterial, or non-host
  • nucleic acids e.g., microbial, bacterial, or non-host
  • a non-targeted population of nucleic acids e.g., mammalian, human, or host
  • nucleic acids e.g., mammalian, human, or host
  • nucleic acids e.g., mammalian, human, or host
  • At least one subsample will have a higher relative abundance of microbial DNA (e.g., an increased ratio of M:H) than other subsamples.
  • at least one subsample with a higher relative abundance of microbial DNA, relative to the other subsamples is selected for further analysis, such as, for example, amplification, sequencing, and/or identification of microbial species present in the sample, etc.
  • at least one selected subsample has a higher relative abundance of microbial DNA relative to at least one non-selected subsample.
  • the method of enrichment comprises a method of global amplification. It will be understood that methods of global amplification would encompass the amplification of microbial DNA, if such DNA is present in the clinical sample or subsample. In some embodiments, the method of global amplification amplifies human and microbial DNA present in the clinical sample or subsample.
  • the method of global amplification comprises a method selected from the group consisting of polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), reversetranscriptase PCR (RT-PCR), degenerate oligonucleotide PCR, primer extension preamplification, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HD A), transcription mediated amplification (TMA), and recombinase polymerase amplification (RPA).
  • PCR polymerase chain reaction
  • qPCR quantitative polymerase chain reaction
  • RT-PCR reversetranscriptase PCR
  • RT-PCR reversetranscriptase PCR
  • PCR primer extension preamplification
  • LAMP loop-mediated isothermal amplification
  • SDA strand displacement amplification
  • HD A helicase dependent amplification
  • TMA transcription mediated amplification
  • RPA recombinase polymerase amplification
  • Certain methods of the invention include a step in which the abundance of microbial DNA present in the subsamples is determined.
  • this step may include species-specific amplification (e.g., species-specific enrichment) of microbial DNA in each subsample, quantification of the amount of microbial DNA in each subsample, and/or calculation of the abundance of microbial DNA, or relative abundance of microbial DNA : host DNA, in each subsample.
  • the abundance of microbial DNA present in the subsamples can be determined using whole genome sequencing (WGS) or targeted 16S sequencing (see, e.g., Xia et al. (2011), PLoS ONE 6(12), p.e27992), hybridization assays (e.g., sandwich hybridization assays, competitive hybridization assays, hybridization-ligation assays, dual ligation hybridization assays, nuclease hybridization assays, etc.) (see, e.g., Wu et al. (2006), Ana/. Biochem.
  • WGS whole genome sequencing
  • targeted 16S sequencing see, e.g., Xia et al. (2011), PLoS ONE 6(12), p.e27992
  • hybridization assays e.g., sandwich hybridization assays, competitive hybridization assays, hybridization-ligation assays, dual ligation hybridization assays, nuclease hybridization assay
  • polymerase-based amplification assays e.g., quantitative polymerase chain reaction (qPCR), loop mediated isothermal amplification (LAMP), reverse transcriptase LAMP (RT-LAMP), shotgun sequencing
  • qPCR quantitative polymerase chain reaction
  • LAMP loop mediated isothermal amplification
  • RT-LAMP reverse transcriptase LAMP
  • shotgun sequencing e.g., Ivy, et al. (2016), J. Clin. Microbiol. 56(9): e00402-18
  • RPA recombinase polymerase amplification
  • the polymerase-based assay is selected from the group consisting of quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP), reverse transcriptase LAMP (RT-LAMP), helicase dependent amplification (HD A), and recombinase polymerase amplification (RPA) based assays.
  • the hybridization-based assay is selected from the group consisting of microarray, southern blot, and northern blot-based assays.
  • determining the abundance of microbial DNA comprises species-specific amplification of microbial DNA. It will be understood that methods of species-specific amplification would encompass the amplification of microbial DNA, if such DNA is present in the clinical sample or subsample. In some embodiments, the method of species-specific amplification amplifies microbial DNA present in the clinical sample or subsample.
  • the microbial DNA is amplified by a method selected from the group consisting of: polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), reverse-transcriptase PCR (RT-PCR), degenerate oligonucleotide PCR, primer extension pre-amplification, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HD A), transcription mediated amplification (TMA), and recombinase polymerase amplification (RPA).
  • PCR polymerase chain reaction
  • qPCR quantitative polymerase chain reaction
  • RT-PCR reverse-transcriptase PCR
  • PCR reverse-transcriptase PCR
  • RT-PCR reverse-transcriptase PCR
  • HD A helicase dependent amplification
  • TMA transcription mediated amplification
  • RPA recombinase polymerase amplification
  • Certain methods of the invention include a step of analyzing the DNA from the subsamples.
  • the analyzed DNA may in some embodiments be microbial DNA, human DNA, or both.
  • analyzing the DNA comprises analyzing human and/or microbial DNA, if such DNA is present in the subsamples.
  • analyzing the DNA comprises whole genome sequencing (WGS).
  • WGS employs the DNA sequencing technology of Helicos True Single Molecule Sequencing (tSMS) (e.g., as described in Harris et al. (2008), Science 320:106-09).
  • tSMS Helicos True Single Molecule Sequencing
  • WGS employs the DNA sequencing technology of 454 sequencing (Roche) (e.g., as described in Margulies et al. (2005), Nature 437:376-380).
  • WGS employs the DNA sequencing technology of nanopore sequencing (e.g., as described in Soni and Meller (2007), Clin. Chem. 53:1996-2001).
  • WGS employs the DNA sequencing technology of chemical-sensitive field effect transistor (chemFET) array (e.g., as described in U.S. Patent Application Publication No. 2009/0026082).
  • chemFET chemical-sensitive field effect transistor
  • WGS employs the DNA sequencing technology of Halcyon Molecular's method that uses transmission electron microscopy (TEM) (e.g., as described in PCT Patent Publication No. W02009/046445).
  • TEM transmission electron microscopy
  • WGS employs parallel sequencing of millions of DNA fragments using Illumina’s sequencing-by- synthesis and reversible terminator-based sequencing chemistry (e.g., as described in Bentley et al.
  • WGS employs the DNA sequencing technology of sequencing-by-ligation (see, e.g., Ho et al. (2011), BMC Genom. 12:598), which is available commercially as SOLiDTM technology (Applied Biosystems).
  • WGS employs single molecule, real-time (SMRT) DNA sequencing (see, e.g., Ardui et al. (2016), Nucl. Acids Res. 46(5):2159-68), which is available commercially as SMRTTM sequencing technology of Pacific Biosciences.
  • SMRT real-time
  • WGS employs the DNA sequencing technology of Ion Torrent single molecule sequencing (see, e.g., Buermans et al.
  • analyzing the DNA identifies the DNA of one or more specific microbial species which may be present in a subsample.
  • one or more specific microbial species are identified by analyzing differences in polymorphic sequences that are present in the mixed clinical sample or subsample. Differences in polymorphic microbial sequences present in samples comprising a mixture of DNA from two or more different microbial genomes may be identified by any of a variety of methods known in the art.
  • WGS that employs next generation sequencing technologies (NGS) in which clonally amplified DNA templates or single DNA molecules are sequenced in a massively parallel fashion
  • NGS next generation sequencing technologies
  • WGS can provide the most definitive identification of species-specific microbial sequences.
  • NGS provides digital quantitative information, in that each sequence read is a countable “sequence tag” representing an individual clonal DNA template or a single DNA molecule.
  • the sequencing technologies of NGS include pyro sequencing, sequencing-by- synthesis with reversible dye terminators, sequencing by oligonucleotide probe ligation, real time sequencing, and biological or solid state nanopore sequencing.
  • NGS technologies are known in the art, and some are commercially available, such as the sequencing-by-hybridization platform from Affymetrix, Inc. (Sunnyvale, Calif.), the sequencing-by-synthesis platforms from 454 Life Sciences (Bradford, Conn.), Illumina/Solexa (Hayward, Calif.), Helicos Biosciences (Cambridge, Mass.), and the sequencing-by-ligation platform from Applied Biosystems (Foster City, Calif.), as described below.
  • single molecule sequencing technologies are encompassed by the method of the invention and include the SMRTTM technology of Pacific Biosciences, the Ion TorrentTM technology, Hyb & SeqTM NGS Technology (Nanostring Technologies, Inc.), and nanopore sequencing being developed by, for example, Oxford Nanopore Technologies.
  • Sanger sequencing including automated Sanger sequencing, can be employed in the methods of the invention, despite being considered a ‘first generation’ technology.
  • Additional sequencing methods that comprise the use of nucleic acid imaging technologies, such as atomic force microscopy (AFM) or transmission electron microscopy (TEM), may also be used.
  • AFM atomic force microscopy
  • TEM transmission electron microscopy
  • hybridization assays e.g., sandwich hybridization assays, competitive hybridization assays, hybridization-ligation assays, dual ligation hybridization assays, nuclease hybridization assays, etc.
  • hybridization assays e.g., sandwich hybridization assays, competitive hybridization assays, hybridization-ligation assays, dual ligation hybridization assays, nuclease hybridization assays, etc.
  • polymerase-based amplification assays e.g., quantitative polymerase chain reaction (qPCR), loop mediated isothermal amplification (LAMP), reverse transcriptase LAMP (RT-LAMP), helicase dependent amplification (HDA) or recombinase polymerase amplification (RPA)
  • qPCR quantitative polymerase chain reaction
  • LAMP loop mediated isothermal amplification
  • RTA helicase dependent amplification
  • RPA recombinase polymerase amplification
  • microarrays see, e.g., Rajilic-Stojanovic et al. (2009), Environ. Microbio.
  • analyzing the DNA comprises using a molecular- or sequencing-based method to identify the microbial infection.
  • analyzing the DNA comprises using a molecular- or sequencing-based method to identify one or more genus, species, or sub-species strains.
  • the molecular- or sequencing-based method is selected from the group consisting of quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP), reverse transcriptase LAMP (RT-LAMP), helicase dependent amplification (HDA), recombinase polymerase amplification (RPA), next generation sequencing (NGS), third generation sequencing, microarrays, southern blot, and northern blot.
  • third generation sequencing comprises nanopore -based methods, in which nucleic acid molecules are carried through a protein pore and the resulting ionic current change is measured and converted to nucleotide sequences (e.g., as described in Volkerding et al. (2010), Clin. Chem. 55:641-658; Metzker (2010), Nature Rev. 11:31-46; Minervini et al. (2020), Frontiers in Genetics 11; Petersen et al. (2019), J. Clin. Microbio. 58(1)). Examples of third generation sequencing methods may be found, for example, in Schadt et al. (2010), Hum. Mol. Gen. 19(R2): R227-240.
  • the NGS or third generation sequencing achieves at least 75% coverage of the microbial genome. In some embodiments, the NGS or third generation sequencing achieves at least 75% coverage of the microbial genome in less than 3 hours.
  • k-mer matching-based approaches see, e.g., Ye et al. (2019), Cell 178(4):779-94
  • alignment-based approaches and commercially-available tools.
  • K-mer matching-based approaches rely on the unique subsequences of a sequence of length “k”.
  • K-mer matching-based approaches are known in the art, and include, for example, Kraken (see, e.g., Wood and Salzberg (2014), Genome Bio. 15(3):R46), Kraken2 (see, e.g., Wood et al. (2019), Genome Bio. 20(1)), Bracken (see, e.g., Lu et al. (2017), Peer J Comp. Sci. 3:el04), KrakenUniq (see, e.g., Breitwieser et al. (2016), Genome Bio. 19(1)), and Centrifuge (see, e.g., Kim et al. (2016), Genome Res. 26(12): 1721-29).
  • Alignment-based approaches involve aligning a sequence of interest (e.g., one belonging to the amplified and detected microbial species) to a reference genome (e.g., a bacterial genome).
  • Alignment-based approaches are known in the art, and may include, for example SURPI (see, e.g., Naccache et al. (2014), Genome Res. 24(7): 1180-92), MEGAN- LR (see, e.g., Huson et al. (2016), Bio. Direct 13(1)); Segata et al. (2012), Nat. Meth. 8: 811— 14), and LORCAN (see, e.g., Neuenschwander et al. (2020), J. Clin. Microbio. 58(6)).
  • identifying microbial species are commercially available, and may include, for example, identification services such as those offered by CosmosID® (Rockville, MD), One Codex (San Francisco, CA), and IDbyDNA, Inc. (Salt Lake City, UT).
  • Identifying the specific species of microbes present in a clinical sample or subsample which has been subjected to the methods of the present disclosure allows for the appropriate diagnosis and treatment of microbial infections, including bacteremia. While broad- spectrum antibiotics may be used to treat infections upon the detection of microbes in the clinical sample, species- specific treatment is preferred not only for enhanced treatment efficacy, but also to mitigate the risk of developing antibiotic-resistant bacteria in the patient (see, e.g., Ventola (2015), Pharma. Therap. 40(4): 277-83).
  • analyzing the DNA comprises generating an Antimicrobial Resistance Sensitivity (AMR/S) profile using a computational algorithm for the microbial species.
  • AMR/S profile is generated using a polymerase- or hybridization-based assay, and/or a computational algorithm. Polymerase- and hybridizationbased assays are described elsewhere herein.
  • the generation of an AMR/S profile may in some embodiments be useful for the treatment of bacteremia, for example by allowing for strain-specific treatment.
  • the methods of the present invention are used to determine the antibiotic susceptibility of a microbial infection (e.g., bacteremia).
  • the microbial DNA sequence data resulting from the methods of the present invention can be used to determine antibiotic susceptibility through a “rulebased” computational algorithm by cross referencing against known genetic antibiotic determinants as described in the art (see, e.g., Su et al. (2016), J. Clin. Microbio. 57(3)).
  • the microbial DNA sequence data resulting from the methods of the present invention can be used to determine antibiotic susceptibility through a “model-based” computational algorithm (e.g., a machine learning or statistical modeling approach) as described in the art (see, e.g., Su et al. (2018), supra). Any number of databases may be used to construct either rule-based or model-based computational algorithms, including CARD, ResFinder, ARG-ANNOT, ARDB, MEGARes, Resfams, RAST, BARRGD, etc.
  • the methods of the present invention include one or more steps suitable for amplifying a nucleic acid population.
  • nucleic acid amplification techniques known in the art include: polymerase chain reaction (PCR), quantitative PCR (qPCR), reverse-transcriptase PCR (RT-PCR), degenerate oligonucleotide PCR, primer extension pre-amplification (PEP), strand displacement amplification (SDA), helicase dependent amplification (HD A), recombinase polymerase amplification (RPA), loop- mediated isothermal amplification (LAMP), reverse transcriptase LAMP (RT-LAMP), and transcription mediated amplification (TMA).
  • PCR polymerase chain reaction
  • qPCR quantitative PCR
  • RT-PCR reverse-transcriptase PCR
  • RT-PCR degenerate oligonucleotide PCR
  • PEP primer extension pre-amplification
  • SDA strand displacement amplification
  • HD A helicase dependent amplification
  • a “primer” is an oligonucleotide that is complementary to a sequence in the population of nucleic acids to be amplified.
  • “complementary” refers to the ability of a nucleotide sequence to base-pair with another nucleotide sequence. Base-pairing may be by Watson-Crick base pairing, Hoogsteen base pairing, or any other method of base-pairing known in the art.
  • a “polymerase” is an enzyme that synthesizes relatively long stretches of nucleic acids, including DNA and RNA, by extending a primer.
  • Polymerases utilize an existing nucleic acid strand as a template for nucleic acid synthesis and typically employ Watson- Crick base pairing to select the correct nucleotide to add to the growing nucleic acid strands, wherein adenine (A) pairs with thymine (T), A pairs with uracil (U), and cytosine (C) pairs with guanosine (G).
  • A adenine
  • T thymine
  • U A pairs with uracil
  • C cytosine
  • Non-limiting examples of polymerases include eukaryotic polymerases such as polymerase alpha, polymerase delta, and polymerase epsilon; bacterial polymerases such as Thermits aquaticus (Taq), Deep Vent, and Therminator; RNA polymerases such as RNA polymerase I and RNA polymerase II; and strand-displacing polymerases.
  • eukaryotic polymerases such as polymerase alpha, polymerase delta, and polymerase epsilon
  • bacterial polymerases such as Thermits aquaticus (Taq), Deep Vent, and Therminator
  • RNA polymerases such as RNA polymerase I and RNA polymerase II
  • strand-displacing polymerases include strand-displacing polymerases.
  • a “strand-displacing polymerase” refers to an enzyme which separates the strands of a DNA double helix as it extends a primer template.
  • the strand displacement polymerase is selected from the group consisting of: phi29 polymerase; Bst DNA polymerase, large fragmentTM; Bsu DNA polymerase, large fragmentTM; Deep Vent DNA polymerase®; Deep Vent (exo) DNA polymerase®; Klenow fragment; DNA polymerase I, large fragment; M-MuLV reverse transcriptase; Therminator DNA polymerase®; Vent DNA polymerase®; Vent (exo) DNA polymerase®; and SD polymerase.
  • phi29 refers to the replicative polymerase from the Bacillus subtilis phage phi29.
  • the phi29 polymerase has exceptional strand displacement and processive synthesis properties, as well as an inherent 3’— >5’ exonuclease proofreading ability.
  • nucleic acid amplification is whole genome amplification (WGA).
  • WGA whole genome amplification
  • DOP-PCR degenerate oligonucleotide primed-polymerase chain reaction
  • MDA multiple displacement amplification
  • MALBAC multiple annealing and looping-based amplification cycles
  • a key aspect of WGA by phi29 is usage of random hexamer oligonucleotides to amplify all possible genomic sequences. While this is crucial for an unbiased DNA amplification approach, the use of random hexamers becomes a liability when dealing with samples of pathogenic microbial DNA isolated from a human biological sample due to undesired human nucleic acid contaminants.
  • the human genome is approximately 1,000 times larger than the average microbial genome, which presents a daunting challenge when sequencing pathogenic microbes that are vastly outnumbered by human cells.
  • microbial nucleic acids present in the clinical sample obtained from the subject are species non-specific ally amplified (e.g., nucleic acids from two or more microbial species present in the clinical sample are amplified).
  • the step of amplification occurs before the step of subsampling.
  • the sample is processed before subsampling to species non- specifically amplify microbial DNA in the sample.
  • the step of amplification occurs after the step of subsampling.
  • the subsamples are processed after subsampling to species non- specifically amplify microbial DNA in each subsample.
  • the microbial nucleic acids present in the clinical sample obtained from the subject are species-specifically amplified (e.g., nucleic acids from a single microbial species present in the clinical sample are amplified).
  • the step of amplification occurs before the step of subsampling.
  • the sample is processed before subsampling to species-specifically amplify microbial DNA in the sample.
  • the step of amplification occurs after the step of subsampling.
  • the subsamples are processed after subsampling to species-specifically amplify microbial DNA in each sub sample.
  • the present invention provides improved methods for determining the presence of and/or identifying a microbial infection in a subject.
  • the microbial infection is determined and/or identified by the detection of one or more microbial species present in a clinical sample or subsamples using the methods described herein.
  • the one or more microbial species comprise one or more of a bacterium, a virus, a fungus, a protist, or a yeast. In some embodiments, the one or more microbial species comprise one or more bacterial species. Although some bacteria are normally present in healthy mammals, disruption of the normal balance between the bacteria and the human host, or the presence of abnormal or pathogenic bacteria within the host, can lead to infection.
  • Staphylococcus aureus is a bacterium that is normally present in the human body and is frequently found in the nose, respiratory tract, and on the skin. Although S. aureus is not always pathogenic, it is a common cause of skin infections including abscesses, respiratory infections, and food poisoning. The common method of treating
  • S. aureus infections is using antibiotics, although the emergence of antibiotic -resistant strains of S. aureus such as Methicillin-Resistant S. aureus (MRS A) and Vancomycin-Resistant S. aureus (VRSA) have become worldwide clinical health challenges.
  • MRS A Methicillin-Resistant S. aureus
  • VRSA Vancomycin-Resistant S. aureus
  • Staphylococcus epidermidis is a bacterium that is normally present in the human body, where it is frequently found on the skin. Although S. epidermidis is not generally pathogenic, subjects with compromised immune systems are at risk of developing S. epidermidis infections, and S. epidermidis poses a particular threat to subjects with surgical implants because it can grow on plastic surfaces and spread to the human body.
  • S. epidermidis strains are often resistant to antibiotics, including rifamycin, fluoroquinolones, gentamicin, tetracycline, clindamycin, and sulfonamides.
  • Streptococcus agalactiae ( S. agalactiae ) is a bacterium that is generally not pathogenic and can be found in the gastrointestinal and genitourinary tract in up to 30% of humans. Pathogenic infections due to S. agalactiae are of concern for neonates and immunocompromised individuals. S. agalactiae infections in adults can be life-threatening and include bacteremia, soft-tissue infections, osteomyelitis, endocarditis, and meningitis. S. agalactiae is increasingly resistant to clindamycin and erythromycin.
  • Enterococcus faecalis (E. faecalis) is a bacterium that inhabits the gastrointestinal tracts of humans and other mammals. However, E. faecalis can cause endocarditis, septicemia, urinary tract infections, and meningitis. E. faecalis infections can be lifethreatening, particularly when the E. faecalis is resistant to treatment with gentamicin and vancomycin.
  • Enterococcus f aecium is a bacterium that inhabits the gastrointestinal tracts of humans and other mammals, but it may also be pathogenic, resulting in diseases such as meningitis and endocarditis. E. faecium infections can be life-threatening, particularly when the E. faecium is resistant to treatment with vancomycin.
  • Escherichia coli is a bacterium that inhabits the gastrointestinal tracts of humans and other mammals, but it may also be pathogenic, resulting in conditions such as gastroenteritis, urinary tract infections, neonatal meningitis, hemorrhagic colitis, and bacteremia. E. coli is increasingly resistant to multiple antibiotics, including fluoroquinolones, cephalosporins, and carbapenems.
  • Klebsiella pneumoniae ( K. pneumoniae ) is a bacterium that is normally found in the mouth, skin, and intestines of humans and other mammals. However, it can cause destructive changes to mammalian (e.g., human) lungs if inhaled, particularly to alveoli.
  • K. pneumoniae infections are generally seen in subjects with a compromised immune system, including subjects with diabetes, alcoholism, cancer, liver disease, chronic obstructive pulmonary diseases, glucocorticoid therapy, and renal failure.
  • K. pneumoniae is increasingly resistant to multiple antibiotics, including fluoroquinolones, cephalosporins, tetracycline, chloramphenicol, carbapenems, and trimethoprim/sulfamethoxazole.
  • the microbe is a pathogenic microbe. In some embodiments, the microbe is a bacterium. In some embodiments, the bacterium is associated with bacteremia. In some embodiments, the bacterium is S. aureus, S. epidermidis, S. agalactiae, E. faecalis, E. faecium, E. coli, K. pneumoniae, or any other bacterial species associated with clinical infection.
  • methods of the present invention are used to identify a pathogenic microbe selected from: Achromobacter spp., Acinetobacter calcoaceticus/baumannii complex, Acinetobacter haemolyticus, Acinetobacter junii, Acinetobacter radioresistens, Acinetobacter ursingii, Acinetobacter Iwoffii, Actinomyces israelii, Actinomyces meyeri, Actinomyces naeslundii, Actinomyces neuii, Actinomyces odontolyticus, Actinomyces pyogenes, Actinomyces viscosus, Aerococcus urinae, Aerococcus viridans, Aeromonas spp., Alcaligenes faecalis, Alcaligenes xylosoxidans, Alpha hemolytic streptococcus, Arcanobacterium haemolyticum, Aspergillus s
  • the microbial infection comprises microbes having one or more resistance genes, virulence genes, single nucleotide polymorphisms, accessory genomes, plasmids, and/or recombination regions.
  • one or more detected resistance or virulence genes are amplified.
  • the one or more detected resistance or virulence genes are amplified by polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP), helicase dependent amplification (HD A), or recombinase polymerase amplification (RPA).
  • PCR polymerase chain reaction
  • qPCR quantitative polymerase chain reaction
  • LAMP loop-mediated isothermal amplification
  • HD A helicase dependent amplification
  • RPA recombinase polymerase amplification
  • the methods of the present invention are used to select an appropriate treatment for a pathogenic microbe infection.
  • a clinical sample is obtained from a subject having or suspected of having a pathogenic microbial infection.
  • the infection is a bacteremia.
  • the appropriate treatment is treatment with an antibiotic.
  • Non-limiting examples of antibiotics include: vancomycin, bacterium, methicillin, ceftobiprole, ceftaroline, dalbacancin, daptomycin, fusidic acid, linezolid, mupirocin, oritavancin, tedzolid, telavancin, tetracycline, amoxicillin, penicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole/trimethoprim, and levofloxacin.
  • the infection is a viral infection.
  • the appropriate treatment is an anti-viral.
  • Non-limiting examples of anti-virals include: abacavir, acyclovir, balavir, cidofovir, darunavir, entecavir, famciclovir, ganciclovir, ostellamivir, penciclovir, and zalcitabine.
  • the infection is a fungal infection.
  • the appropriate treatment is an anti-fungal.
  • Non-limiting examples of anti-fungals include: amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, rimocidin, bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconzaole, and anidulafungin.
  • Example 1 Subsampling results in a quicker and more effective microbial enrichment process.
  • Blood from two vacutainers (approximately 20 mL total of blood) was combined and 4 x 4 mL of blood was aliquoted into 4 x 15 mL conical tubes containing 8 to 16 mL of 1 x TBS (20 mM Tris-Cl, pH 7.0, 150 mM NaCl). To mix, the tubes were then inverted. To simulate a bacteremic blood sample, bacteria Klebsiella pneumoniae') was added or “spiked” into the blood samples at a final concentration of 2-3 colony forming units (CFU) / mL of blood.
  • CFU colony forming units
  • the bacteremic blood samples were centrifuged at low speeds (for example, 500g to 700g for 3 to 7 minutes) in order to compact the red blood cells (RBC) at the bottom of the tube and maximize bacterial retention in the supernatant.
  • the maximal amount of the first supernatant (SI) from each tube was aspirated and the process was repeated after diluting the RBC with the same amount of 1 x TBS used previously. All supernatant-containing subsamples were then treated with Proteinase K (NEB P8107), as described in US Patent No. 10,544,446, to remove SPS carry over and subsequent molecular amplification inhibition.
  • Detergents commonly used for selective lysis such as Tween-20 (Sigma Aldrich P9416) and Saponin (Sigma Aldrich Cat. #47036) were used at a final concentration between 0.1 and 2% along with DNase I (see, e.g., Charalampous et al. (2019), Nat. Biotechnol. 37 , 783-92; Street et al. (2019), J. Clin. Microbio. 58(3); Hasan et al. (2016), J. Clin. Microbio. 54(4); Shehadul Islam et al. (2017), Micromachines 8(3): 83) to efficiently deplete human DNA.
  • the detergent concentration used was informed by the specific bacteria’s viability in the detergent used.
  • samples were spun down (for example, 2000g to 3000g for 10 to 20 minutes) to remove most of the supernatant. Subsequent washes were performed with TBST (1 x TBS + 0.1% Tween 20) and spun down at 10,000g for 3 minutes to collect the bacterial pellet.
  • the concentrated sample was then subjected to a modified REPLI-g Single Cell protocol (Qiagen Cat. #150345) to lyse the collected cells.
  • Samples were resuspended in 4.8 pL of 1 x PBS and 3.6 pL of D2 buffer was added. The samples were then incubated at 65°C for 10 minutes. 3.6 pL of STOP buffer was added post-incubation.
  • each 12 pL lysate was used for a 25 pL REPLI-g Single Cell MDA reaction and another 5pL was combined with the rest of the donor subsamples (4 x 5 pL) for a lOOpL REPLI-g Single Cell MDA reaction.
  • Each REPLI-g SC reaction was supplemented with 2% BSA (Sigma Aldrich Cat # A7638) + 2% DMSO (Sigma Aldrich Cat # D8418) and amplified for 4 hours.
  • Amplified DNA samples were quantified with the Qubit dsDNA HS kit (Thermo Fisher Cat #Q32854) and Qubit Fluorometer 3.0 (Thermo Fisher). 1 pg of each amplified DNA sample was used for library preparation with the SQK-LSK109 library preparation kit from Oxford Nanopore Technology (ONT) and sequenced with nanopore flow cells. The resulting sequencing data was base-called / demultiplexed by ONT’s Guppy Basecaller (v3.3.O).
  • Sequencing files for each barcode were combined into a single file and processed through the following pipeline: each read was classified using Kraken, sequences were aligned to both a human reference genome and to the internal DZD reference genome assembly for that strain using minimap 2 (v2.9; see, e.g., Li (2016), Minimap2: Pairwise alignment for nucleotide sequences, Bioinformatics 34(18): 3094-3100), and alignment statistics were computed using minimap2 (v2.9) and SAMtools (vl.3.1; see, e.g., Li et al. (2009), 1000 Genome Project Data Processing Subgroup, The Sequence Alignment/Map format and SAMtools, Bioinformatics 25(16): 2078-79). M:H ratios were measured by dividing the megabases classified to the pathogen of interest to the megabases classified to the human genome.
  • analysis of the sequencing results shows the ratio of microbial (bacterial) to human (M:H ratio) reads in the subsamples.
  • M:H ratio ratio of microbial (bacterial) to human
  • the results show that each replicate had at least 1 and up to 3 subsample(s) with a higher M:H ratio as compared to the combined sample.
  • a higher M:H ratio means a larger fraction of microbial sequences per given sample or subsample, which effectively decreases the necessary sequencing time for a bacteremia diagnostics device and results in a shorter sample-to-result time.
  • a diagnostic device has one hour to generate data for roughly > 25 x K.
  • pneumoniae genome coverage (e.g., > 140 Mb), a data output rate of 500 Mb per hour, and is working with low bacterial load samples (FIG. IB), 100% of the subsamples met those criteria, versus only 66.7% of the combined sample replicates.
  • This example shows that subsampling results in a microbial enrichment process to generate a higher ratio of useful microbial data when working with low microbial load blood samples.
  • a comparison of the genome coverage between the subsamples (4 mL) and the combined samples (16 mL) also shows that there is no significant difference in resulting genome coverage with similar number of total sequencing reads generated, despite using a fraction of the total microbial input (FIGs. 1C and ID).
  • Example 2 Subsampling methods increase speed and accuracy of determining presence of and/or identifying microbial infection.
  • the human genome comprises approximately 3.3 x 10 9 bp of DNA and, therefore, a diploid human cell comprises approximately 6.6 x 10 9 bp.
  • a standard clinical sample of blood drawn directly from a patient will include approximately 4 million white blood cells per milliliter (mL) and, therefore, 1 mL of blood can be expected to contain roughly 2.64 x 10 16 bp of human DNA.
  • the pathogenic bacteria present in human blood have much smaller genomes, and are likely to be present in much lower numbers.
  • the genomes of the bacteria commonly isolated in sepsis or bacteremia typically range between 2 x 10 6 to 6 x 10 6 bp (e.g., S. pyogenes'. 1.8 mbp, S. aureus'. 2.8 mbp, E. coli: 4.6 mbp, Klebsiella spp.'. 5.5 mbp, P. aeruginosa'. 6.3 mbp).
  • the number of bacteria present in a clinical sample from a sepsis patient is usually less than 50 cells/mL, commonly less than 10 cells/mL, and often less than 1 cell/mL.
  • the bacterial DNA present in a clinical sample from a sepsis patient may typically range from 2-300 x 10 6 bp/mL.
  • the initial ratio of bacterial DNA (2-300 x 10 6 bp/mL) to human DNA (2.64 x 10 16 bp/mL) may be expected to range from about 7.6 x 10“ 11 to 1.1 x 10’ 8 , which makes detection of bacterial DNA exceedingly difficult.
  • the ratio of bacterial DNA (2-300 x 10 6 bp/mL) to human DNA (2.64 x 10 7 bp/mL) may be expected to range from about 7.6 x IO’ 2 to 11.
  • a single 16 mL clinical sample of blood is obtained from a patient.
  • the sample is divided in 4 subsamples.
  • Each subsample is subjected to standard techniques (e.g., lysis, centrifugation, etc.) for reducing human cells and human DNA by a factor of 10 9 .
  • sample 1 contains 4.0 x 10 6 bp/ml
  • sample 2 contains 1.0 x 10 6 bp/ml
  • sample 3 contains 3.0 x 10 6 bp/ml
  • sample 4 contains 8.0 x 10 6 bp/ml of bacterial DNA.
  • the average abundance of bacterial DNA across the four samples is 4 x 10 6 , but the abundance in sample 4 is twice that, at 8.0 x 10 6 .
  • the abundance of human DNA is 2.64 x 10 7
  • the ratio of bacteriakhuman DNA in sample 4 is 0.30.
  • the amount of DNA to be sequenced is reduced 2-fold by choosing to sequence only one of four samples instead of all four, and the relative abundance of bacteriakhuman DNA is increased 2-fold by choosing subsample 4, which has twice the bacteriakhuman DNA ratio of the original clinical sample.

Abstract

The present disclosure describes methods for determining the presence of and/or identifying a microbial infection in a subject. Such methods are facilitated by increasing the proportion of microbial cells and/or DNA present in a clinical sample obtained from a patient. The invention depends, in part, upon the development of improved methods that (i) highly enrich for microbial cells for detection, (ii) rapidly recover microbial cells directly from whole blood at low (< 5) CFU/mL levels, (iii) agnostically amplify microbial DNA, and (iv) sequence the amplified genetic material with quick turnaround times (TATs). Moreover, the methods described herein can produce DNA sequencing data with a significantly enriched proportion of pathogen DNA (> 25%). This enriched pathogen sequencing data makes it possible to provide the fastest tum-around-time (TAT) while providing whole genome coverage for high confidence AMR profiling using a bacteremia diagnostic device.

Description

METHODS FOR ENRICHING MICROORGANISMS FROM LOW ABUNDANCE CLINICAL SAMPLES FOR DNA SEQUENCING
FIELD OF THE INVENTION
The present disclosure relates generally to the field of diagnostics for microbial infections based on assays for microbial nucleic acids in a clinical sample. In particular, the invention relates to improved methods for enriching microbial DNA relative to host DNA in connection with amplification and sequencing to identify microbes present at low levels in a sample.
BACKGROUND
Infections with pathogenic microbial cells in mammalian tissues are a major healthcare and economic concern. (Deresinski (2007), Clin. Infect. Dis. 15(45 Supp. 3 :S): 177-83.) Bacterial infection of the blood, known as bacteremia, is one of the most common causes of death worldwide; approximately one in five individuals worldwide will be afflicted by bacteremia. Bacteremia is also the current leading cause of in-hospital deaths in the United States. (Rudd, et al., (2020), Lancet 395(10219): 200-211.) Blood-borne microbial infections can generally be treated with antibiotics, antifungal, or antiviral medications, but can sometimes result in sepsis, a life-threatening illness in which systemic inflammation can lead to multi-organ collapse and eventually death if left untreated. (Fleischmann, et al., (2016), Am. J. Respir. Crit. Care Med. 193(3): 259-272.) The early detection of blood infection is paramount as mortality rate and economic cost can dramatically increase when patients are not quickly diagnosed after admission into the hospital. (Paoli, et al., (2018), Crit. Care Med. 46(12): 1889-1897.) The treatment of microbial infections is becoming even more challenging with the rise of antimicrobial resistance among microbial organisms. (Andersson and Hughes (2010), Nat. Rev. Microbio. 8: 260-71.)
The World Health Organization (WHO) has listed the spread of resistance to antimicrobials as one of the most pressing concerns the world’s population will face over the upcoming years. The number of deaths attributed to antimicrobial-resistant bacteria globally could reach as high as 10 million in 2050 if current practices are not altered. (O'Neill (2016), supra.) In the United States alone, approximately 2,000,000 patients per year are infected with antimicrobial-resistant organisms and roughly 100,000 of those patients will die as a result of these infections. The rapid and correct identification of the pathogen(s) involved in an infection and its antimicrobial susceptibility pattern can be critical in reducing economic costs, as well avoiding permanent injury or death. (Deresinski (2007), Clin. Infect. Dis. 15(45 Supp. 3:S): 177-83.)
The current standard of care for bacteremia is culture-based diagnostics. (Dellinger, et al., (2013), Crit. Care Med. 41(2): 580-637.) However, blood culture approaches have significant disadvantages, such as an extended period of time until positive identification and false negatives due to fastidious pathogens. (Opota, et al., (2015), Clin. Microbiol. Infect. 21(4): 323-331.) Polymerase chain reaction (PCR)-based diagnostics have provided a faster alternative to culture-based diagnostics with positive identification in hours instead of days. However, inherent limitations of PCR-based technology constrain the breadth of species coverage and antimicrobial resistance (AMR) profiling to select targets. Recently, next generation sequencing (NGS) approaches have been developed as an agnostic identification method for causative pathogens in bacteremia cases. Current approaches utilize enriched cell-free DNA sequencing, amplicon-based sequencing (e.g., 16S targeted amplification), or human DNA depletion methods to isolate causative pathogens for whole-genome amplification (WGA) and sequencing (Blauwkamp, et al., (2019), Nat. Microbiol. 4: 663- 674; Ellis, et al., (2017), J. Microbiol. Methods 138; O’Grady, et al., US Pat. Pub. No. 20190316113 A 1 ). Current enrichment methods for NGS are not effective for rapid identification (e.g., same day) with concomitant high sequencing depth and high genome coverage due to inadequate pathogen enrichment.
Therefore, there is a clear and present need for rapid and accurate diagnostic methods and products capable of producing sequencing data with a significantly enriched proportion of pathogenic DNA for confident species identification and AMR profiling.
SUMMARY
The present invention depends, in part, upon the development of improved methods for increasing the proportion of microbial cells or DNA relative to other cells or DNA present in a clinical sample obtained from a patient. In some embodiments, these improved methods can (i) highly enrich for microbial cells or DNA for detection, (ii) rapidly recover microbial cells or DNA directly from whole blood at low (e.g., < 5) CFU/mL levels, (iii) amplify microbial DNA in a species specific and/or species non-specific manner, and (iv) sequence the amplified genetic material with quick turn-around times (TATs). Moreover, the methods can, in some embodiments, produce DNA sequencing data with a significantly enriched proportion of pathogen DNA (e.g., > 25%). This enriched pathogen sequencing data makes it possible to provide fast TATs while providing pathogen whole genome coverage to permit high confidence AMR profiling by a bacteremia diagnostic device.
Accordingly, in some aspects, the invention provides methods for determining the presence or absence of a microbial infection in a subject. In some embodiments, the methods comprise the following steps: (a) obtaining a clinical sample from the subject; (b) dividing the clinical sample into at least two subsamples; (c) subjecting the clinical sample prior to step (b), and/or the subsamples after step (b), to at least one method of enrichment for microbial DNA; and (d) determining the abundance of microbial DNA in the enriched subsamples. In some embodiments, an abundance of microbial DNA greater than zero in at least one subsample indicates the presence of a microbial infection. In some embodiments, an abundance of microbial DNA of zero in all subsamples indicates that a microbial infection has not been detected.
In other aspects, the invention provides methods for identifying a microbial infection in a subject. In some embodiments, the methods comprise the following steps: (a) obtaining a clinical sample from the subject; (b) dividing the clinical sample into at least two subsamples; (c) subjecting the clinical sample prior to step (b), and/or the subsamples after step (b), to at least one method of enrichment for microbial DNA; and (d) analyzing the DNA from the subsamples. In some embodiments, the methods comprise the following steps: (a) obtaining a clinical sample from the subject; (b) dividing the clinical sample into at least two subsamples; (c) subjecting the clinical sample prior to step (b), and/or the subsamples after step (b), to at least one method of enrichment for microbial DNA; (d) determining the abundance of microbial DNA in the enriched subsamples; (e) (i) if at least one subsample has an abundance of microbial DNA greater than zero, selecting at least one subsample with a higher abundance of microbial DNA relative to at least one unselected subsample, or (e) (ii) if no subsample has an abundance of microbial DNA greater than zero, determining that the presence of a microbial infection has not been identified; and (f) if at least one subsample has an abundance of microbial DNA greater than zero, analyzing the microbial DNA from the subsamples selected in (e) (i). In some embodiments of any of the foregoing methods, the microbial infection is identified based upon the analyzed DNA.
Various methods of the present disclosure include a step of determining abundance. In some embodiments, determining the abundance of microbial DNA comprises species- specific amplification of microbial DNA. In some embodiments, the microbial DNA is amplified by a method selected from the group consisting of: polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), reverse-transcriptase PCR (RT-PCR), degenerate oligonucleotide PCR, primer extension pre-amplification, loop-mediated isothermal amplification (LAMP), reverse transcriptase LAMP (RT-LAMP), primer extension pre-amplification (PEP), strand displacement amplification (SDA), helicase dependent amplification (HD A), transcription mediated amplification (TMA), and recombinase polymerase amplification (RPA). In some embodiments, determining the abundance of microbial DNA comprises whole genome sequencing. In some embodiments, determining the abundance of microbial DNA comprises a polymerase-based or hybridization-based assay. In some embodiments, the polymerase-based assay is selected from the group consisting of a qPCR, LAMP, reverse transcriptase LAMP (RT-LAMP), and RPA-based assay. In some embodiments, the hybridization-based assay is selected from the group consisting of a microarray, southern blot, and northern blot-based assay.
Various methods of the present disclosure include a step of enrichment. In some embodiments, the clinical sample is subjected to at least one method of enrichment for microbial DNA before the clinical sample is divided into at least 2 subsamples (e.g., before step (b)). In some embodiments, the clinical sample is subjected to at least one method of enrichment for microbial DNA after the clinical sample is divided into at least 2 subsamples (e.g., after step (b)). In some embodiments, the clinical sample is subjected to at least one method of enrichment for microbial DNA before and after the clinical sample is divided into at least 2 subsamples (e.g., before and after step (b)). In some embodiments, the method of enrichment comprises a method of non-amplification enrichment. In some embodiments, the method of non-amplification enrichment comprises reducing the abundance of human cells present in the clinical sample or subsample. In some embodiments, the method of non- amplification enrichment includes at least one method selected from the group consisting of centrifugation, immunoaffinity purification, filtration, and selective lysis to remove human cells. In some embodiments, the method of non-amplification enrichment comprises reducing the abundance of human DNA present in the clinical sample or subsample. In some embodiments, the method of non-amplification enrichment includes at least one method selected from the group consisting of centrifugation, immunoaffinity purification, filtration, and selective lysis to remove human DNA. In some embodiments, the method of enrichment comprises a method of global amplification. In some embodiments, the method of global amplification comprises a method selected from the group consisting of PCR, qPCR, RT- PCR, degenerate oligonucleotide PCR, primer extension pre-amplification, LAMP, SDA, HDA, TMA, and RPA. In some embodiments, the method of global amplification amplifies human and/or microbial DNA present in the clinical sample or subsample.
In some embodiments, the microbial DNA comprises DNA from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, or more microbial species. In some embodiments, the clinical sample comprises an abundance of microbial DNA comprising no more than 10 genomic copies per mL of clinical sample volume prior to enrichment or determining abundance.
Various methods of the present disclosure include a step of analyzing DNA (e.g., microbial DNA, human DNA, etc.) from the subsamples. In some embodiments, analyzing the DNA comprises analyzing human and/or microbial DNA present in the subsamples. In some embodiments, analyzing the DNA comprises whole genome sequencing. In some embodiments, analyzing the DNA comprises generating an Antimicrobial Resistance Sensitivity (AMR/S) profile using a computational algorithm for the microbial species. In some embodiments, analyzing the DNA comprises using a molecular- or sequencing-based method to identify the microbial infection. In some embodiments, analyzing the DNA comprises using a molecular- or sequencing-based method to identify one or more genus, species, or sub-species strains. In some embodiments, the molecular- or sequencing-based method is selected from the group consisting of qPCR, LAMP, RT-LAMP, RPA, microarrays, southern blot, northern blot, next generation sequencing (NGS), and third generation sequencing. In some embodiments, the NGS or third generation sequencing achieves at least 75% coverage of the microbial genome. In some embodiments, the NGS or third generation sequencing achieves at least 75% coverage of the microbial genome in less than 3 hours. In some embodiments, the microbial infection comprises microbes having one or more resistance genes, virulence genes, single nucleotide polymorphisms, accessory genomes, plasmids, and/or recombination regions. In some embodiments, one or more detected resistance or virulence genes are amplified. In some embodiments, the one or more detected resistance or virulence genes are amplified by PCR, qPCR, LAMP, SDA, HDA, TMA, or RPA.
Various methods of the present invention include a step of dividing a clinical sample into subsamples (z.e., a step of subsampling). In some embodiments, the clinical sample is obtained from a subject. In some embodiments, the clinical sample is a blood sample. In some embodiments, the clinical sample is a cerebrospinal fluid (CSF) sample. In some embodiments, the clinical sample is a joint fluid, abscess fluid, serum, lymph, urine, stool, or sputum sample. In some embodiments, the clinical sample is divided into 4 subsamples. In some embodiments, the clinical sample is divided into at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 subsamples. In some embodiments, the subsamples comprise substantially equal volumes. In some embodiments, the subsamples each comprise at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mL.
These and other aspects and embodiments of the invention are illustrated and described below. Other compositions, methods and features will be apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional compositions and methods and features are within the scope of the present invention.
BRIEF DESCRIPTION OF THE FIGURES
The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
FIG. 1A shows a direct comparison of replicates from 12 donors in which DNA amplification reactions were conducted using four divided samples and compared to one combined sample. Each replicate represents an individual human donor from which 16 mL of whole blood was processed using the methods as described in Example 1 and amplified as either 4 individual subsamples (4 mL each; dots) or 1 single combined sample (16 mL; squares). The amplified DNA product was processed and sequenced with Oxford Nanopore Technology flow cells. “M:H” is the ratio of microbial (“M”; e.g., bacterial) reads to human (“H”) reads from each individual amplified reaction. The mean is displayed as a horizontal bar when available.
FIG. IB shows the CFU / mL plating control range of Klebsiella pneumoniae spiked into each of the 12 donor replicates.
FIG. 1C shows the average reads generated for all subsamples compared to the combined samples.
FIG. ID shows the average assembly coverage for all subsamples compared to the combined samples. DETAILED DESCRIPTION
The present invention relates to improved methods that, in some embodiments, can (i) highly enrich for microbial cells for detection, (ii) rapidly recover microbial cells directly from whole blood at low (e.g., < 5) CFU/mL levels, (iii) agnostically amplify microbial DNA, and (iv) sequence the amplified genetic material with quick turnaround times (TATs). Moreover, the methods can, in some embodiments, produce DNA sequencing data with a significantly enriched proportion of pathogen DNA (e.g., > 25%). This enriched pathogen sequencing data makes it possible to provide faster TATs while providing whole genome coverage for high confidence AMR profiling using a bacteremia diagnostic device.
Principles of the Invention
The present invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are merely illustrative of certain preferred or exemplary embodiments. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention.
Bacteremia is currently the leading cause of in-hospital deaths in the United States, yet the standard, culture-based diagnostic methods can require several days to identify microbial species within a sample from a subject, and often include false negatives. Other methods, such as polymerase- and hybridization-based assays, provide more rapid identification of microbial species than culture-based methods; however, there are inherent limitations to the breadth of species these methods can identify, and same-day AMR profiling remains difficult to achieve. A critical reason for this is the relative rarity of microbial DNA in a sample (e.g., a blood sample) taken from a human subject with bacteremia or another microbial infection. For example, at 1 CFU/mL, human DNA is up to a billion times more abundant than microbial DNA. This relative rarity makes the use of sequencing-based methods to identify microbial DNA difficult, slow, and cost-inefficient.
To address the small quantity of microbial DNA present in a clinical sample taken from a subject with bacteremia or another microbial infection, a possible strategy is to simply increase the sample size. A larger sample would naturally lead to a greater abundance of microbial DNA, which can theoretically enhance the specificity of targeted-amplification methods such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), etc. However, larger sample volumes (e.g., > 50 mL) require increased handling of the sample, which can lead to contamination and added expense, and obtaining larger samples from the subject may be difficult, or not possible. Larger sample volumes may also be harmful to the subject and thus be clinically unacceptable. Larger sample sizes also lead to a greater amount of human DNA, and therefore do not improve the relative abundance of microbiakhuman DNA per volume of sample. Thus, for a sample preparation process with a defined efficiency of human cell/DNA depletion, using a larger sample would not yield superior results, because the greater amount of human DNA would negate the benefits obtained from having a greater amount of microbial DNA.
The present invention provides an alternative way to address both the small quantity and small percentage of microbial DNA present in a clinical sample taken from a subject with bacteremia. Counterintuitively, the present invention describes a method of increasing the relative abundance of one or more microbial species in a clinical sample from a human subject by dividing the clinical sample into subsamples. Instead of combining samples for a quantitatively larger microbial DNA input, Example 1 surprisingly demonstrates that the relative abundance of microbiakhuman DNA can be increased per sample using smaller subsamples, instead of a combined sample. Unexpectedly, this increase was achieved without negatively impacting overall percent genome coverage. Smaller subsamples are additionally desirable for the reasons discussed above; namely, smaller samples are cheaper to obtain and test, easier to handle, and faster to process, and accordingly facilitate the isolation of microbial DNA from a biological sample (e.g., blood) for rapid detection and species-specific treatment of bacteremia.
As a non-limiting and hypothetical example to describe how subsampling may increase the relative abundance of microbiakhuman DNA, consider that one sample of human blood may contain, for example, 16 “units” of microbial DNA. Dividing that clinical sample into four subsamples, the expected value would be 4 units of microbial DNA per subsample. Indeed, if the size of the clinical sample is sufficiently large and/or the percentage of microbial DNA is sufficiently high, the distribution of the microbial DNA units per subsample might be expected to be 4, 4, 4, and 4 units (z.e., 4 units of microbial DNA per subsample). But for relatively small clinical samples and/or relatively low percentages of microbial DNA, the random distribution of the microbial DNA units would have greater variance, and might result in 6, 8, 1, and 1 units (z.e., 6 units of microbial DNA in subsample A, 8 units in subsample B, 1 unit in subsample C, and 1 unit in subsample D). The abundance of human DNA in each subsample can be separately determined, or may be assumed to be constant because of its relatively greater abundance and correspondingly lower variance.
In the latter hypothetical, assuming a constant abundance of human DNA in each subsample, the relative abundance of microbiakhuman DNA would be increased in subsample A (by 50%) and subsample B (by 100%), and decreased in subsamples C and D (by 75%). Thus, by dividing the sample into subsamples, the amount of microbial DNA present in each subsample will vary from subsample to subsample, and therefore the relative abundance of microbiakhuman DNA present in at least one subsample is likely to be increased relative to the other subsamples.
Accordingly, aspects of the invention provide methods of determining the presence of a microbial infection in a subject. In some embodiments, the method comprises the following steps: (a) obtaining a clinical sample from the subject; (b) dividing the clinical sample into at least two subsamples; (c) subjecting the clinical sample prior to step (b), and/or the subsamples after step (b), to at least one method of enrichment for microbial DNA; and (d) determining the abundance of microbial DNA in the enriched subsamples. In some embodiments, an abundance of microbial DNA greater than zero in at least one subsample indicates the presence of a microbial infection. In some embodiments, an abundance of microbial DNA of zero in all subsamples indicates that a microbial infection has not been detected.
Aspects of the invention contemplate a method of identifying a microbial infection in a subject. In some embodiments, the method comprises the following steps: (a) obtaining a clinical sample from the subject; (b) dividing the clinical sample into at least two subsamples; (c) subjecting the clinical sample prior to step (b), and/or the subsamples after step (b), to at least one method of enrichment for microbial DNA; and (d) analyzing the DNA from the subsamples. In some embodiments, the method comprises the following steps: (a) obtaining a clinical sample from the subject; (b) dividing the clinical sample into at least two subsamples; (c) subjecting the clinical sample prior to step (b), and/or the subsamples after step (b), to at least one method of enrichment for microbial DNA; (d) determining the abundance of microbial DNA in the enriched subsamples; (e) (i) if at least one subsample has an abundance of microbial DNA greater than zero, selecting at least one subsample with a higher abundance of microbial DNA relative to at least one unselected subsample, or (e) (ii) if no subsample has an abundance of microbial DNA greater than zero, determining that the presence of a microbial infection has not been identified; and (f) if at least one subsample has an abundance of microbial DNA greater than zero, analyzing the microbial DNA from the subsamples selected in (e) (i). In some embodiments of either of the foregoing methods, the microbial infection is identified based upon the analyzed DNA.
In the statistically highly improbable event that all subsamples have the same abundance of microbial DNA, the samples can be combined, and then subsampled again until variance between the subsamples is detected.
Definitions
All scientific and technical terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of any conflict, the present specification, including definitions, will control. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent or later-developed techniques which would be apparent to one of skill in the art. In order to more clearly and concisely describe the subject matter which is the invention, the following definitions are provided for certain terms which are used in the specification and appended claims.
As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells.
As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and/or” and not the exclusive sense of “either/or.”
As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for a variable that is inherently discrete, the variable can be equal to any integer value within the numerical range, including the endpoints of the range. Similarly, for a variable that is inherently continuous, the variable can be equal to any real value within the numerical range, including the endpoints of the range. As an example, and without limitation, a variable that is described as having values between 0 and 2 can take the values 0, 1, or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values 0 and
Figure imgf000011_0001
2 if the variable is inherently continuous.
As used herein, an “agnostic”, “global”, or “species non-specific” amplification refers to amplification of DNA (e.g., microbial DNA, human DNA, or both) which uses primer sequences which are not limited to a single species but, rather, are characteristic of two or more species. Thus, the amplification is agnostic with respect to the multiple species within the DNA being amplified because no particular species is targeted or, in some embodiments, can be targeted. For example, amplification methods using primers directed to 16S rRNA sequences that are conserved across many bacterial species can agnostically or species non- specifically enrich for multiple bacterial 16S rRNA sequences. Similarly, some agnostic methods can amplify all or most sequences in a population of nucleic acids (e.g., using a mixture of random hexamer primers).
As used herein, “amplification” refers to the process of increasing the number of copies of a specific nucleotide sequence in a population of nucleic acids by templatedependent and polymerase-dependent chemical synthesis. Methods of amplification include, but are not limited to, polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), reverse-transcriptase PCR (RT-PCR), degenerate oligonucleotide PCR, primer extension pre-amplification, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HD A), transcription mediated amplification (TMA), and recombinase polymerase amplification (RPA). In some embodiments, the nucleic acid amplification is PCR, qPCR, RT-PCR, degenerate oligonucleotide PCR, primer extension pre-amplification, LAMP, RT-LAMP, SDA, HDA, TMA, or RPA.
As used herein, “bacteria” are single-celled microbes of the kingdom Prokaryota. Of particular interest in the methods of the invention are human pathogenic bacteria species, and more particularly those species associated with bacteremia in humans. In some embodiments, the bacteria are Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Enterococcus faecalis , Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, or any other species associated with bacteremia.
As used herein, “bacteremia” refers to the presence of bacteria in the blood. In some embodiments, the bacteria present in the blood are infectious bacteria that cause disease in a host. However, bacteremia can also include non-pathogenic bacteria.
As used herein, a “clinical sample” is a biological sample obtained from a subject. A clinical sample may be directly obtained from the subject (e.g., by collecting the sample from the subject), or may be received indirectly from another person or entity (e.g., a healthcare provider or reference laboratory). A step of “obtaining” can include obtaining directly or indirectly.
As used herein, “enrichment” refers to the increase in the proportion of one population of DNA (e.g., microbial DNA) relative to a second population of DNA (e.g., human DNA) in a mixed sample comprising at least two populations of DNA. Such an increase in proportion may be achieved by increasing the abundance of the first population of DNA, and/or by decreasing the abundance of the second population of DNA. In some embodiments, enrichment comprises a method of non-amplification enrichment. Methods of non-amplification enrichment may include, for example, centrifugation, immunoaffinity purification, filtration, and selective lysis (e.g., to remove human cells and/or human DNA). In some embodiments, enrichment comprises a method of amplification. Such amplification may in some embodiments be global or species-specific.
As used herein, “host DNA” refers to DNA derived from a host (e.g., a human patient or subject), and “non-host DNA” refers to DNA derived from a microbe.
As used herein, the term “mammal” refers to a warm-blooded vertebrate that is distinguished by the possession of hair or fur, the secretion of milk by females to nourish the young, and the birth of live young.
As used herein, the terms “microbe” and “microbial” refer to a microorganism that requires a microscope to be visualized. Non-limiting examples of microbes include: bacteria, archaea, fungi, protists, viruses, and microscopic animals. Pathogenic microbes are capable of causing disease in a host organism.
As used herein, a “mixed sample” or a “mixed clinical sample” is a sample that comprises DNA from at least two sources. In some embodiments, the mixed sample comprises a first population and a second population of nucleic acids. In some embodiments the first population of nucleic acids is mammalian DNA (e.g., human DNA) and the second population of nucleic acids is microbial DNA. In some embodiments, the first population of nucleic acids is host DNA (e.g., patient DNA) and the second population of nucleic acids is non-host DNA. In some embodiments, the first population and the second population of nucleic acids are both microbial DNA. In some embodiments, the first population and second population of nucleic acids are both bacterial DNA.
As used herein, the term “relative abundance” refers to the abundance of a nucleotide sequence in a first population of DNA relative to a second population of DNA. The relative abundance can be calculated by dividing (a) the abundance of the nucleotide sequence in the first population by (b) the abundance of the nucleotide sequence in the second population. The abundance of the nucleotide sequence in each population can be estimated by dividing (a) the amount of DNA (e.g., number of bp) in the population corresponding to the nucleotide sequence (including duplicate copies) by (b) the total amount of DNA (e.g., number of bp) in the population. The relative abundance can be approximated based upon knowledge of whole or partial genome sequences or experimentally. If the abundance of the second population of DNA is assumed to be constant (or substantially constant), the relative abundance can be estimated based on the abundance of the first population.
As used herein, “sequencing” refers to a method for determining the nucleotide sequence of a polynucleotide (e.g.. a genomic DNA sequence). Preferably, sequencing methods include as non-limiting examples whole genome sequencing (WGS), next generation sequencing (NGS) methods, in which clonally amplified DNA templates or single DNA molecules are sequenced in a massively parallel fashion, or third generation sequencing technology such as nanopore-based methods, in which nucleic acid molecules are carried through a pore and the resulting ionic current change is measured and converted to nucleotide sequences (e.g., as described in Volkerding et al. (2010), Clin. Chem., 55:641-658; Metzker (2010), Nature Rev. 11:31-46; Minervini et al. (2020), Frontiers in Genetics 11; Petersen et al. (2019), J. Clin. Microbio. 58(1)). In some embodiments, the NGS or third generation sequencing achieves at least 75% coverage of the microbial genome. Such coverage may, in some embodiments, be achieved in less than 3 hours.
As used herein, “species non-specific microbial DNA” refers to microbial DNA having a sequence found in two or more microbial species, but not found in human DNA.
As used herein, “subsamples” are two or more samples that have been derived from a single, original sample. “Clinical subsamples” are subsamples that have been divided from a single, original clinical sample obtained from a subject (e.g., a blood, CSF, joint fluid, or abscess sample).
As used herein, “substantially equal” refers to a first measurement (e.g., a volume) that differs from a second measurement by a value less than about 1 percent. In certain embodiments, the first measurement differs from the second measurement by less than 0.9 percent, 0.8 percent, less than 0.7 percent, less than 0.6 percent, less than 0.5 percent, less than 0.4 percent, less than 0.3 percent, less than 0.2 percent, or less than 0.1 percent.
As used herein, “Whole Genome Amplification (WGA)” refers to a process whereby DNA sequences present in a sample are amplified to provide multiple copies of the genome that the sequences represent. WGA generally refers to a method for amplification of a limited DNA sample in a non-specific manner in order to generate a new sample that has a higher DNA concentration. Degenerate oligonucleotide-primed PCR (DOP), primer extension PCR (PEP), and multiple displacement amplification (MDA) are examples of whole genome amplification methods. In some embodiments, WGA is a method of global amplification. As used herein, “Whole Genome Sequencing (WGS)” refers to a process whereby the DNA originating from the entire genome of an organism, for example, humans, dogs, mice, viruses or bacteria, is sequenced. It is not necessary that the entire genome actually be sequenced. The WGS methods of the invention are those sequencing methods that, when applied to a sample of genomic DNA, are capable of obtaining the sequence of substantially the entire genome. WGS can be performed using any next-generation or third generation sequencing technology known in the art. In some embodiments, WGS is a method of global sequencing.
Incorporation by Reference
The patent, scientific, and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued U.S. patents, allowed applications, published, and pending patent applications, and other references, including database citations for nucleic acid and protein sequences, that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.
Clinical Samples
In some aspects, the present invention provides methods for determining the presence of and/or identifying one or more microbial species present in a clinical sample obtained from a mammalian subject, typically a human subject.
In some embodiments, clinical samples are obtained directly or indirectly from human subjects. In other embodiments, the clinical samples are obtained from non-human mammalian subjects. In some embodiments, the non-human mammalian subjects are companion animals such as dogs or cats; agricultural animals such as cows, pigs, sheep, goats or horses; or common laboratory animals such as rodents, rabbits or non-human primates.
In some embodiments, the clinical sample is obtained or derived from blood, joint fluid, abscess fluid, serum, sputum, mucus, saliva, wound drainage, urine, stool, lymph, lavage, cerebral- spinal fluid (CSF), or any fluid aspirate or tissue extraction of human and/or other mammalian origin. In some embodiments, the clinical sample is obtained or derived from blood. In some embodiments, the clinical sample is obtained or derived from CSF. In some embodiments, the clinical sample is obtained or derived from joint fluid (e.g., a prosthetic joint). In some embodiments, the clinical sample is obtained or derived from tissue abscess fluid. In some embodiments, the clinical sample comprises a quantity of DNA including from between 1 and 108 genomes (e.g., human, microbial, and any other genomes) per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1 and 10 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1 and 102 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1 and 103 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1 and 104 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1 and 105 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1 and 106 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1 and 107 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 103 and 105 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 105 and 106 genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000 , 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 50,000,000, 60,000,000, 70,000,000, 80,000,000, 90,000,000, or 100,000,000 genomes per milliliter, or any value contained therein.
In some embodiments, the clinical sample comprises a quantity of DNA including from between 0.1 and 104 microbial genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 0.1 and 10 microbial genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 0.1 and 102 microbial genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 0.1 and 103 microbial genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 102 and 103 microbial genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 103 and 104 microbial genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 microbial genomes per milliliter, or any value contained therein. In some embodiments, the clinical sample comprises an abundance of microbial DNA comprising no more than 10 genomic copies per milliliter of clinical sample volume prior to enrichment or determining abundance.
In some embodiments, the clinical sample comprises a quantity of DNA including from between 200 and 40,000,000 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 200 and 400 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 200 and 4,000 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 200 and 40,000 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 200 and 105 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including between 200 and 106 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 200 and 107 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 200 and 20,000,000 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including between 200 and 30,000,000 human genomes per milliliter. In some embodiments, the clinical sample comprises DNA including from between 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, or 40,000,000 human genomes per milliliter, or any value contained therein. In some embodiments, the clinical sample comprises DNA including from between 8,000,000 to 22,000,000 human genomes per milliliter (e.g., 8,000,000, 9,000,000, 10,000,000, 11,000,000, 12,000,000, 13,000,000, 14,000,000, 15,000,000, 16,000,000, 17,000,000, 18,000,000, 19,000,000, 20,000,000, 21,000,000, or 22,000,000 human genomes per milliliter, or any value contained therein).
In some embodiments, the subject has, or is suspected of having, a microbial infection (e.g., bacteremia). In some embodiments, the clinical sample comprises DNA from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, or more microbial species.
In some embodiments, the clinical sample is directly obtained by a person practicing the methods of the present invention. In some embodiments, the clinical sample is obtained indirectly by a person practicing the methods of the present invention. For example, in some embodiments, the clinical sample may be directly obtained from a subject, by either the subject or by a physician, physician’s assistant, nurse, laboratory technician or other healthcare personnel, and then may be indirectly obtained by a person practicing the methods of the present invention. As used herein, “obtaining” a clinical sample encompasses both directly and indirectly obtaining the sample according to the description herein.
Methods for obtaining clinical samples from a subject are known in the art, and may include, for example, identifying the patient prior to collecting a sample (e.g., by checking identification, armbands, etc.), labelling collection containers with appropriate patient identifiers in the presence of the patient, using at least two patient identifiers to label each container, sterilizing the collection site, drawing the samples into collection tubes in the proper sequence (e.g., blood culture tubes; coagulation tubes; serum tubes with or without clot activator, and with or without gel; heparin tubes, with or without gel plasma separator; EDTA tubes; oxalate and fluoride tubes; etc.), inverting the collection tubes end-to-end (/'.<?., gentle inversion) multiple times (e.g., 10 times) after collection, using proper collection containers, not transferring samples into secondary containers, delivering samples to the laboratory promptly after collection and/or processing the samples promptly after collection, avoiding hemolysis, drawing a first “flush” syringe prior to collecting the sample from a line, etc.
In some embodiments, a clinical sample comprises anywhere between 0.1 mL and 50 mL of sample material. In some embodiments, a clinical sample comprises 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 45, 40, 45, or 50 mL, or any value included therein, such as 1.1, 1.2, 1.3, etc., mL of sample material. In some specific embodiments, a clinical sample comprises 16 mL of sample material.
Methods for handling and storing clinical samples are known in the art, and are described, for example, in Redrup et al. (2016), AAPS J. 18(2):290-93. In some embodiments, clinical samples are stored at room temperature (e.g., -20-25 °C). In some embodiments, clinical samples are refrigerated (e.g., -2-8 °C, including 4 °C). In some embodiments, clinical samples are frozen (e.g., less than or equal to 0 °C).
In some embodiments, clinical samples are stored in sample containers. A variety of different sample containers are used in the art, and may be adapted to different types of clinical samples. For example, commonly used blood collection containers, blood culture bottles, plasma tubes, and blood culture media may include heparin, including lithium heparin and/or sodium heparin (e.g., Cat. Nos. 364960, 366667, 367871, 367878, 367884, 367886, 367960, 367961, 367962, and 367964 Vacutainer® collection tubes, BD Biosciences, Franklin Lakes, NJ), SPS (e.g., Cat. No. 364960 Vacutainer® collection tubes, Cat. Nos. 442022 and 442023, BACTEC™ PLUS media, BD Biosciences, Franklin Lakes, NJ), ACD (Cat. Nos. 364816 and 364606 Vacutainer® collection tubes, BD Biosciences, Franklin Lakes, NJ), citrate (Cat. Nos. 363083 and 363080 Vacutainer® collection tubes, BD Biosciences, Franklin Lakes, NJ), sodium citrate (Cat. Nos. 369714 and 367947 Vacutainer® collection tubes, BD Biosciences, Franklin Lakes, NJ), or potassium EDTA (e.g., Cat. Nos. 367855, 367842, 367899, and 368589, Vacutainer® Plus Plastic K2EDTA Tubes, BD Biosciences, Franklin Lakes, NJ).
It will be appreciated that, in many cases, the most commonly used and commercially available sample containers are pre-loaded with preservatives and/or anticoagulants (e.g., sodium polyanetholesulfonate (SPS), heparin, lithium heparin, sodium heparin, citrate, sodium citrate, acid citrate dextrose (ACD), hyaluronate, dermatan sulfate polyanion, EDTA, potassium EDTA (K2EDTA), and chondroitin D-glucuronate anion) that may have the unintended effect of acting as nucleic acid amplification inhibitors during genetic identification or analysis of the various nucleic acids present in a sample (see, e.g., Fredericks and Reiman (1998), J. Clin. Microbiol. 36(10): 2810-16; Qian et al. (2001), J. Clin. Microbiol. 39(10): 3578-85; and Regan et al. (2012), J. Mol. Diagn. 14(2): 120-29). In addition to such additives, blood components such as hemoglobin, lactoferrin, heme, and immunoglobulins can also interfere with nucleic acid amplification procedures. Such inhibition of and interference with amplification methods can be reduced without substantially harming the integrity or quality of the samples by adding proteinases (e.g., proteinase K) to the clinical blood samples, as described in US Patent No. 10,544,446, issued January 28, 2020, and International Patent Application No. PCT/US 2020/020275, published on September 3, 2020 under International Publication Number WO2020/176822, the entire disclosures of which are hereby incorporated by reference in their entirety.
Subsampling of Clinical Samples
The methods of the invention comprise dividing a clinical sample into two or more subsamples. In some embodiments, the clinical sample is approximately evenly divided such that each subsample contains an equal volume of clinical sample material. In some embodiments, the subsamples comprise substantially equal volumes. In some embodiments, the clinical sample is unevenly divided, either intentionally or unintentionally, such that at least one subsample contains an unequal volume of clinical sample material relative to the other subsamples. Methods for dividing a clinical sample would be readily apparent to those skilled in the art. Briefly, a clinical sample may be divided into subsamples by aliquoting certain amounts of sample material from a first container into two or more secondary containers (e.g.. test tubes, vials, etc.). Methods for aliquoting may be performed by a human or by a machine, and include, for example, pouring, dispensing from a syringe, or dispensing from a pipette.
In some embodiments, subsamples are handled and stored using the same methods that would be practiced in the handling and storage of similar clinical samples, as known by those of skill in the art and described herein.
In some embodiments, a clinical sample is divided into two or more subsamples. In some embodiments, a clinical sample is divided into between 2 and 1,000 subsamples. In some embodiments, a clinical sample is divided into between 2 and 10 subsamples. In some embodiments, a clinical sample is divided into between 2 and 20 subsamples. In some embodiments, a clinical sample is divided into between 2 and 50 subsamples. In some embodiments, a clinical sample is divided into between 2 and 100 subsamples. In some embodiments, a clinical sample is divided into between 2 and 500 subsamples. In some embodiments, a clinical sample is divided into 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 subsamples. In some embodiments, a clinical sample is divided into 4 subsamples. In some embodiments, a clinical sample is divided into at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 subsamples.
In some embodiments, a 16 mL clinical sample is divided into 4 subsamples, each with a volume of approximately 4 mL. In some embodiments, the subsamples each comprise at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mL.
In some embodiments, the abundance of microbial DNA present in each of the two or more divided subsamples is determined, and one or more of the divided subsamples is selected for further analysis.
In some embodiments, subsamples (e.g.. selected subsamples) are further divided into two or more subsamples. In some embodiments, a subsample is divided into between 2 and 1,000 subsamples. In some embodiments, a subsample is divided into between 2 and 10 subsamples. In some embodiments, a subsample is divided into between 2 and 20 subsamples. In some embodiments, a subsample is divided into between 2 and 50 subsamples. In some embodiments, a subsample is divided into between 2 and 100 subsamples. In some embodiments, a subsample is divided into between 2 and 500 subsamples. In some embodiments, a subsample is divided into 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 subsamples. In some embodiments, the abundance of microbial DNA present in each of the two or more further divided subsamples is determined, and one or more of the further divided subsamples is selected for further analysis.
In some embodiments, at least two selected subsamples are recombined (e.g., pooled) to create one or more combined selected subsamples. The step of pooling may be performed before or after various steps of the methods of the present invention. In some embodiments, the step of pooling is performed prior to detecting and/or identifying one or more specific microbial species based upon the presence of species- specific microbial DNA present in at least one selected subsample. In some embodiments, the step of pooling is performed prior to subjecting the selected subsample to a qualitative second analysis to identify one or more specific microbial species within the sub sample.
Subsamples may be pooled in order to create combined selected subsamples which contain a higher relative abundance of microbial DNA than was present in one or more nonpooled subsamples, or than was present in the original clinical sample. In some embodiments, the selected subsamples chosen for the step of pooling are those with a higher relative abundance of microbial DNA than any of the non-selected subsamples. In some embodiments, the selected subsamples have a 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%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,
43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%,
118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%,
131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%,
144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%,
157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%,
170%, 171%, 172%, 173%, 174%, 175%, 176%, 178%, 179%, 180%, 181%, 182%, 183%,
184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%, 193%, 194%, 195%, 196%,
197%, 198%, 199%, 200%, 250%, 300%, 400%, 500%, 1,000%, etc. higher relative abundance of microbial DNA relative to the subsamples not chosen for the step of pooling. In some embodiments, one or more secondary clinical samples (e.g., clinical samples created from the pooling of selected subsamples) are further divided into two or more tertiary subsamples (e.g., subsamples created upon dividing the pooled secondary clinical samples) according to the subsampling methods described herein. In some embodiments, two or more secondary subsamples are re-combined (e.g.. re-pooled) to create one or more tertiary clinical samples (e.g., clinical samples created from the pooling of selected secondary subsamples). It will be understood by those of skill in the art that the steps of dividing and pooling can be repeated one time, two times, three times, or more than three times, according to the methods described herein. In some embodiments, the subsamples (e.g., subsamples, secondary subsamples, tertiary subsamples, etc.) or clinical samples (e.g., clinical samples, secondary clinical samples, tertiary clinical samples, etc.) undergo the amplification, detection, and/or identification steps according to the methods of the present invention.
In embodiments where one or more selected clinical subsamples are each further divided into two or more subsamples, the abundance of microbial DNA present in each of the two or more divided subsamples may be determined, and one or more of the divided subsamples may be selected for further analysis.
Enriching for Microbial DNA
Certain methods of the invention include a step of enriching for microbial DNA. Such enrichment may occur before and/or after the step of subsampling (e.g., before and/or after step (b)). In some embodiments, the clinical sample is subjected to at least one method of enrichment for microbial DNA before the step of subsampling. In some embodiments, the clinical sample is subjected to at least one method of enrichment for microbial DNA after the step of subsampling. In some embodiments, the clinical sample is subjected to at least one method of enrichment for microbial DNA before and after the step of subsampling. In some embodiments, the methods of the invention therefore involve processing (e.g., enriching) the clinical sample to alter the abundance of certain cells and/or DNA present in the sample prior to determining the abundance of microbial DNA in the enriched subsamples.
It will be understood that enriching for microbial DNA may be achieved by increasing the abundance of microbial cells and/or DNA present in the clinical sample or subsample (e.g., by a method of species-specific enrichment, such as by species-specific amplification), and/or by decreasing the abundance of human cells and/or DNA present in the clinical sample or subsample (e.g., by a method of non-amplification enrichment, such as selective lysis). In some embodiments, the method of enrichment comprises a method of nonamplification enrichment. In some embodiments, the method of non-amplification enrichment comprises reducing of the abundance of human cells present in the clinical sample or subsample. In some embodiments, the method of non-amplification enrichment comprises reducing of the abundance of human DNA present in the clinical sample or subsample.
Reducing the abundance of human cells and/or human DNA present in the clinical sample or subsample, while not proportionately reducing the abundance of microbial cells and/or microbial DNA present in the clinical sample or subsample, results in a higher ratio of microbiakhuman cells and/or microbiakhuman DNA present in the sample (e.g., M:H) and thereby enriches for microbial DNA.
Various methods of enrichment for reducing the abundance of a population of certain cells and/or DNA (e.g., human cells and/or human DNA) in a clinical sample are known in the art, and may in some embodiments be used in combination with the methods of the present invention to increase the M:H ratio present in the sample. In some embodiments, the method of non-amplification enrichment includes at least one method selected from the group consisting of centrifugation, immunoaffinity purification, filtration, and selective lysis to remove human cells. Centrifugation may in some embodiments include differential centrifugation to separate one population of cells and/or DNA from another population based on size (see, e.g., Amasia and Madou (2010), Bioanalysis, 2(10):1701-10; Pitt et al. (2019), Biotech. Prog. 36(1)). Selective lysis may in some embodiments be applied to one population of cells and/or DNA using detergents such as Triton X-100, Tween 20, Tween 80, or saponin (see, e.g., Hasan et al. (2016), J. Clin. Microbio. 54(4): 919-27; Shehadul Islam et al. (2017), Micromachines 8(3): 83), or using proteins targeting human cells and/or human DNA (see, e.g., O’Grady et al., US Pat. Pub. No. 20190316113A1). In some embodiments, the method of non-amplification enrichment comprises a combination of selective lysis and DNase I (see, e.g., Charalampous et al. (2019), Nat. Biotech. 'NK 'y.'l 83-92), or any other combination of the foregoing.
Although physical and chemical approaches, such as those described above, can be effective in reducing the abundance of human chromosomal DNA, human mitochondrial DNA (mtDNA) has proven more difficult to remove. In some embodiments, therefore, the abundance of human mtDNA is reduced using blocking oligonucleotides that can specifically reduce the amplification of undesired mtDNA sequences. Such sample processing methods and blocking oligonucleotides are described in International Patent Application No. PCT/US2018/056598, published on April 25, 2019 as WO2019/079656, the entire disclosure of which is incorporated herein in its entirety.
In some embodiments, these sample processing methods result in at least a 50% increase in relative abundance of a targeted population of nucleic acids (e.g., microbial, bacterial, or non-host) in clinical samples and/or subsamples, relative to clinical samples and/or subsamples which have not undergone the sample processing methods. In some embodiments, there is at least a 60%, at least a 70%, at least an 80%, at least a 90%, at least a 95%, at least a 99%, or at least a 99.99% increase in the relative abundance of a targeted population of nucleic acids (e.g., microbial, bacterial, or non-host) in clinical samples and/or subsamples which have undergone the sample processing methods, relative to clinical samples and/or subsamples which have not undergone the sample processing methods. In some embodiments, there is a 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%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,
45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,
61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,
77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%,
120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%,
133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%,
146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%,
159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%,
172%, 173%, 174%, 175%, 176%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%,
186%, 187%, 188%, 189%, 190%, 191%, 192%, 193%, 194%, 195%, 196%, 197%, 198%,
199%, 200%, 250%, 300%, 400%, 500%, 1,000%, etc., increase in the relative abundance of a targeted population of nucleic acids (e.g., microbial, bacterial, or non-host) in clinical samples and/or subsamples which have undergone the sample processing methods, relative to clinical samples and/or subsamples which have not undergone the sample processing methods.
In some embodiments, there is at least a 50% decrease in the relative abundance of a non-targeted population of nucleic acids (e.g., mammalian, human, or host) in clinical samples and/or subsamples which have undergone the sample processing methods, relative to clinical samples and/or subsamples which have not undergone the sample processing methods. In some embodiments, there is at least a 60%, at least a 70%, at least an 80%, at least a 90%, at least a 95%, at least a 99%, or at least a 99.99% decrease in the relative abundance of a non-targeted population of nucleic acids (e.g., mammalian, human, or host) in clinical samples and/or subsamples which have undergone the sample processing methods, relative to clinical samples and/or subsamples which have not undergone the sample processing methods. In some embodiments, there is a 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%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%,
117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%,
130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%,
143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%,
156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%,
169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 178%, 179%, 180%, 181%, 182%,
183%, 184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%, 193%, 194%, 195%,
196%, 197%, 198%, 199%, 200%, 250%, 300%, 400%, 500%, 1,000%, etc., decrease in the relative abundance of a non-targeted population of nucleic acids (e.g., mammalian, human, or host) in clinical samples and/or subsamples which have undergone the sample processing methods, relative to clinical samples and/or subsamples which have not undergone the sample processing methods.
In some embodiments, after the presence of human cells and/or human DNA is reduced according to any of the sample processing methods described herein, at least one subsample will have a higher relative abundance of microbial DNA (e.g., an increased ratio of M:H) than other subsamples. In some embodiments, at least one subsample with a higher relative abundance of microbial DNA, relative to the other subsamples, is selected for further analysis, such as, for example, amplification, sequencing, and/or identification of microbial species present in the sample, etc. In some embodiments, at least one selected subsample has a higher relative abundance of microbial DNA relative to at least one non-selected subsample. In some embodiments, the method of enrichment comprises a method of global amplification. It will be understood that methods of global amplification would encompass the amplification of microbial DNA, if such DNA is present in the clinical sample or subsample. In some embodiments, the method of global amplification amplifies human and microbial DNA present in the clinical sample or subsample. In some embodiments, the method of global amplification comprises a method selected from the group consisting of polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), reversetranscriptase PCR (RT-PCR), degenerate oligonucleotide PCR, primer extension preamplification, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HD A), transcription mediated amplification (TMA), and recombinase polymerase amplification (RPA).
Determining the Abundance of Microbial DNA in Subsamples
Certain methods of the invention include a step in which the abundance of microbial DNA present in the subsamples is determined. In some embodiments, this step may include species-specific amplification (e.g., species-specific enrichment) of microbial DNA in each subsample, quantification of the amount of microbial DNA in each subsample, and/or calculation of the abundance of microbial DNA, or relative abundance of microbial DNA : host DNA, in each subsample.
Methods of determining the abundance of a cellular or DNA population are known in the art. Briefly, in some embodiments, the abundance of microbial DNA present in the subsamples can be determined using whole genome sequencing (WGS) or targeted 16S sequencing (see, e.g., Xia et al. (2011), PLoS ONE 6(12), p.e27992), hybridization assays (e.g., sandwich hybridization assays, competitive hybridization assays, hybridization-ligation assays, dual ligation hybridization assays, nuclease hybridization assays, etc.) (see, e.g., Wu et al. (2006), Ana/. Biochem. 353(1): 22-29), polymerase-based amplification assays (e.g., quantitative polymerase chain reaction (qPCR), loop mediated isothermal amplification (LAMP), reverse transcriptase LAMP (RT-LAMP), shotgun sequencing) (see, e.g., Ivy, et al. (2018), J. Clin. Microbiol. 56(9): e00402-18), or recombinase polymerase amplification (RPA) (see, e.g., Zhang et al. (2011), App. and Environ. Microbiol. 77(18): 6495-6501; Vasileva Wand, et al. (2018), J. Gen. Virol. 99(8): 1012-26), as described in the art and elsewhere herein. In some embodiments, the polymerase-based assay is selected from the group consisting of quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP), reverse transcriptase LAMP (RT-LAMP), helicase dependent amplification (HD A), and recombinase polymerase amplification (RPA) based assays. In some embodiments, the hybridization-based assay is selected from the group consisting of microarray, southern blot, and northern blot-based assays.
In some embodiments, determining the abundance of microbial DNA comprises species-specific amplification of microbial DNA. It will be understood that methods of species-specific amplification would encompass the amplification of microbial DNA, if such DNA is present in the clinical sample or subsample. In some embodiments, the method of species-specific amplification amplifies microbial DNA present in the clinical sample or subsample. In some embodiments, the microbial DNA is amplified by a method selected from the group consisting of: polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), reverse-transcriptase PCR (RT-PCR), degenerate oligonucleotide PCR, primer extension pre-amplification, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HD A), transcription mediated amplification (TMA), and recombinase polymerase amplification (RPA).
Detecting Microbial Species in Subsamples
Certain methods of the invention include a step of analyzing the DNA from the subsamples. The analyzed DNA may in some embodiments be microbial DNA, human DNA, or both. Thus, in some embodiments, analyzing the DNA comprises analyzing human and/or microbial DNA, if such DNA is present in the subsamples.
In some embodiments, analyzing the DNA (e.g.. microbial and/or human DNA) comprises whole genome sequencing (WGS). In some embodiments, WGS employs the DNA sequencing technology of Helicos True Single Molecule Sequencing (tSMS) (e.g., as described in Harris et al. (2008), Science 320:106-09). In some embodiments, WGS employs the DNA sequencing technology of 454 sequencing (Roche) (e.g., as described in Margulies et al. (2005), Nature 437:376-380). In some embodiments, WGS employs the DNA sequencing technology of nanopore sequencing (e.g., as described in Soni and Meller (2007), Clin. Chem. 53:1996-2001). Nanopore sequencing DNA analysis techniques are being industrially developed by various companies, including Oxford Nanopore Technologies (Oxford, United Kingdom). In some embodiments, WGS employs the DNA sequencing technology of chemical-sensitive field effect transistor (chemFET) array (e.g., as described in U.S. Patent Application Publication No. 2009/0026082). In some embodiments, WGS employs the DNA sequencing technology of Halcyon Molecular's method that uses transmission electron microscopy (TEM) (e.g., as described in PCT Patent Publication No. W02009/046445). In some embodiments, WGS employs parallel sequencing of millions of DNA fragments using Illumina’s sequencing-by- synthesis and reversible terminator-based sequencing chemistry (e.g., as described in Bentley et al. (2009), Nature 6:53-59). In some embodiments, WGS employs the DNA sequencing technology of sequencing-by-ligation (see, e.g., Ho et al. (2011), BMC Genom. 12:598), which is available commercially as SOLiD™ technology (Applied Biosystems). In some embodiments, WGS employs single molecule, real-time (SMRT) DNA sequencing (see, e.g., Ardui et al. (2018), Nucl. Acids Res. 46(5):2159-68), which is available commercially as SMRT™ sequencing technology of Pacific Biosciences. In some embodiments, WGS employs the DNA sequencing technology of Ion Torrent single molecule sequencing (see, e.g., Buermans et al. (2014), Biochem. Biophys. Acta - Mol. Basis of Dis. 1842( 10): 1932-41 ; Quail et al. (2012), BMC Genom. 13:341), which is available commercially from ThermoFisher Scientific.
In some embodiments, analyzing the DNA identifies the DNA of one or more specific microbial species which may be present in a subsample. Thus, in some embodiments, one or more specific microbial species are identified by analyzing differences in polymorphic sequences that are present in the mixed clinical sample or subsample. Differences in polymorphic microbial sequences present in samples comprising a mixture of DNA from two or more different microbial genomes may be identified by any of a variety of methods known in the art.
For example, WGS that employs next generation sequencing technologies (NGS) in which clonally amplified DNA templates or single DNA molecules are sequenced in a massively parallel fashion (see, e.g., Volkerding et al. (2009), Clin. Chem. 55:641-658; Metzker (2010), Nature Rev. 11:31-46; Xia et al. (2011), PLoS ONE 6(12), p.e27992; Minervini, C., et al., (2020), Frontiers in Genetics, 11; Petersen, L., et al., (2019), J. Clin. Microbio., 58(1)) can provide the most definitive identification of species-specific microbial sequences. In addition to high-throughput sequence information, NGS provides digital quantitative information, in that each sequence read is a countable “sequence tag” representing an individual clonal DNA template or a single DNA molecule. The sequencing technologies of NGS include pyro sequencing, sequencing-by- synthesis with reversible dye terminators, sequencing by oligonucleotide probe ligation, real time sequencing, and biological or solid state nanopore sequencing.
Such NGS technologies are known in the art, and some are commercially available, such as the sequencing-by-hybridization platform from Affymetrix, Inc. (Sunnyvale, Calif.), the sequencing-by-synthesis platforms from 454 Life Sciences (Bradford, Conn.), Illumina/Solexa (Hayward, Calif.), Helicos Biosciences (Cambridge, Mass.), and the sequencing-by-ligation platform from Applied Biosystems (Foster City, Calif.), as described below. In addition to the single molecule sequencing performed using sequencing-by- synthesis of Helicos Biosciences, other single molecule sequencing technologies are encompassed by the method of the invention and include the SMRT™ technology of Pacific Biosciences, the Ion Torrent™ technology, Hyb & Seq™ NGS Technology (Nanostring Technologies, Inc.), and nanopore sequencing being developed by, for example, Oxford Nanopore Technologies.
Alternatively, Sanger sequencing, including automated Sanger sequencing, can be employed in the methods of the invention, despite being considered a ‘first generation’ technology. Additional sequencing methods that comprise the use of nucleic acid imaging technologies, such as atomic force microscopy (AFM) or transmission electron microscopy (TEM), may also be used.
Other methods known in the art include, for example, hybridization assays (e.g., sandwich hybridization assays, competitive hybridization assays, hybridization-ligation assays, dual ligation hybridization assays, nuclease hybridization assays, etc.) (see, e.g., Wu et al. (2006), Ana/. Biochem. 353(1): 22-29), polymerase-based amplification assays (e.g., quantitative polymerase chain reaction (qPCR), loop mediated isothermal amplification (LAMP), reverse transcriptase LAMP (RT-LAMP), helicase dependent amplification (HDA) or recombinase polymerase amplification (RPA)) (see, e.g., Zhang et al. (2011), App. and Environ. Microbiol. 77(18): 6495-6501; Vasileva Wand et al. (2018), J. Gen. Virol. 99(8): 1012-26), microarrays (see, e.g., Rajilic-Stojanovic et al. (2009), Environ. Microbio. 11(7): 1736-51), southern blots (see, e.g., Ryschkewitsch et al. (2004), J. Virol. Meth. 121(2): 217- 21; Hill et al. (1996), Anal. Biochem. 235(1): 44-48), northern blots (see, e.g., Taniguchi et al. (2001), Genomics 71(1): 34-39), or any combination of the foregoing, as described in the art and elsewhere herein.
Accordingly, in some embodiments, analyzing the DNA comprises using a molecular- or sequencing-based method to identify the microbial infection. In some embodiments, analyzing the DNA comprises using a molecular- or sequencing-based method to identify one or more genus, species, or sub-species strains. In some embodiments, the molecular- or sequencing-based method is selected from the group consisting of quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP), reverse transcriptase LAMP (RT-LAMP), helicase dependent amplification (HDA), recombinase polymerase amplification (RPA), next generation sequencing (NGS), third generation sequencing, microarrays, southern blot, and northern blot. In some embodiments, third generation sequencing comprises nanopore -based methods, in which nucleic acid molecules are carried through a protein pore and the resulting ionic current change is measured and converted to nucleotide sequences (e.g., as described in Volkerding et al. (2010), Clin. Chem. 55:641-658; Metzker (2010), Nature Rev. 11:31-46; Minervini et al. (2020), Frontiers in Genetics 11; Petersen et al. (2019), J. Clin. Microbio. 58(1)). Examples of third generation sequencing methods may be found, for example, in Schadt et al. (2010), Hum. Mol. Gen. 19(R2): R227-240.
In some embodiments, the NGS or third generation sequencing achieves at least 75% coverage of the microbial genome. In some embodiments, the NGS or third generation sequencing achieves at least 75% coverage of the microbial genome in less than 3 hours.
Further methods of identifying specific microbial species which are known in the art may include, for example, k-mer matching-based approaches (see, e.g., Ye et al. (2019), Cell 178(4):779-94), alignment-based approaches, and commercially-available tools.
K-mer matching-based approaches rely on the unique subsequences of a sequence of length “k”. K-mer matching-based approaches are known in the art, and include, for example, Kraken (see, e.g., Wood and Salzberg (2014), Genome Bio. 15(3):R46), Kraken2 (see, e.g., Wood et al. (2019), Genome Bio. 20(1)), Bracken (see, e.g., Lu et al. (2017), Peer J Comp. Sci. 3:el04), KrakenUniq (see, e.g., Breitwieser et al. (2018), Genome Bio. 19(1)), and Centrifuge (see, e.g., Kim et al. (2016), Genome Res. 26(12): 1721-29).
Alignment-based approaches involve aligning a sequence of interest (e.g., one belonging to the amplified and detected microbial species) to a reference genome (e.g., a bacterial genome). Alignment-based approaches are known in the art, and may include, for example SURPI (see, e.g., Naccache et al. (2014), Genome Res. 24(7): 1180-92), MEGAN- LR (see, e.g., Huson et al. (2018), Bio. Direct 13(1)); Segata et al. (2012), Nat. Meth. 8: 811— 14), and LORCAN (see, e.g., Neuenschwander et al. (2020), J. Clin. Microbio. 58(6)).
Other methods for identifying microbial species are commercially available, and may include, for example, identification services such as those offered by CosmosID® (Rockville, MD), One Codex (San Francisco, CA), and IDbyDNA, Inc. (Salt Lake City, UT).
Identifying the specific species of microbes present in a clinical sample or subsample which has been subjected to the methods of the present disclosure allows for the appropriate diagnosis and treatment of microbial infections, including bacteremia. While broad- spectrum antibiotics may be used to treat infections upon the detection of microbes in the clinical sample, species- specific treatment is preferred not only for enhanced treatment efficacy, but also to mitigate the risk of developing antibiotic-resistant bacteria in the patient (see, e.g., Ventola (2015), Pharma. Therap. 40(4): 277-83).
In some embodiments, analyzing the DNA comprises generating an Antimicrobial Resistance Sensitivity (AMR/S) profile using a computational algorithm for the microbial species. In some embodiments, the AMR/S profile is generated using a polymerase- or hybridization-based assay, and/or a computational algorithm. Polymerase- and hybridizationbased assays are described elsewhere herein. The generation of an AMR/S profile may in some embodiments be useful for the treatment of bacteremia, for example by allowing for strain-specific treatment. In some embodiments, the methods of the present invention are used to determine the antibiotic susceptibility of a microbial infection (e.g., bacteremia).
In some embodiments, the microbial DNA sequence data resulting from the methods of the present invention can be used to determine antibiotic susceptibility through a “rulebased” computational algorithm by cross referencing against known genetic antibiotic determinants as described in the art (see, e.g., Su et al. (2018), J. Clin. Microbio. 57(3)). In some embodiments, the microbial DNA sequence data resulting from the methods of the present invention can be used to determine antibiotic susceptibility through a “model-based” computational algorithm (e.g., a machine learning or statistical modeling approach) as described in the art (see, e.g., Su et al. (2018), supra). Any number of databases may be used to construct either rule-based or model-based computational algorithms, including CARD, ResFinder, ARG-ANNOT, ARDB, MEGARes, Resfams, RAST, BARRGD, etc.
Nucleic Acid Amplification
The methods of the present invention include one or more steps suitable for amplifying a nucleic acid population. Non-limiting examples of nucleic acid amplification techniques known in the art include: polymerase chain reaction (PCR), quantitative PCR (qPCR), reverse-transcriptase PCR (RT-PCR), degenerate oligonucleotide PCR, primer extension pre-amplification (PEP), strand displacement amplification (SDA), helicase dependent amplification (HD A), recombinase polymerase amplification (RPA), loop- mediated isothermal amplification (LAMP), reverse transcriptase LAMP (RT-LAMP), and transcription mediated amplification (TMA).
Amplification of one population of nucleic acids by any of the previously mentioned methods requires a primer and a polymerase. As used herein, a “primer” is an oligonucleotide that is complementary to a sequence in the population of nucleic acids to be amplified. As used herein, “complementary” refers to the ability of a nucleotide sequence to base-pair with another nucleotide sequence. Base-pairing may be by Watson-Crick base pairing, Hoogsteen base pairing, or any other method of base-pairing known in the art. As used herein, a “polymerase” is an enzyme that synthesizes relatively long stretches of nucleic acids, including DNA and RNA, by extending a primer. Polymerases utilize an existing nucleic acid strand as a template for nucleic acid synthesis and typically employ Watson- Crick base pairing to select the correct nucleotide to add to the growing nucleic acid strands, wherein adenine (A) pairs with thymine (T), A pairs with uracil (U), and cytosine (C) pairs with guanosine (G). Non-limiting examples of polymerases include eukaryotic polymerases such as polymerase alpha, polymerase delta, and polymerase epsilon; bacterial polymerases such as Thermits aquaticus (Taq), Deep Vent, and Therminator; RNA polymerases such as RNA polymerase I and RNA polymerase II; and strand-displacing polymerases.
As used herein, a “strand-displacing polymerase” refers to an enzyme which separates the strands of a DNA double helix as it extends a primer template. In some embodiments, the strand displacement polymerase is selected from the group consisting of: phi29 polymerase; Bst DNA polymerase, large fragment™; Bsu DNA polymerase, large fragment™; Deep Vent DNA polymerase®; Deep Vent (exo) DNA polymerase®; Klenow fragment; DNA polymerase I, large fragment; M-MuLV reverse transcriptase; Therminator DNA polymerase®; Vent DNA polymerase®; Vent (exo) DNA polymerase®; and SD polymerase. As used herein, “phi29” refers to the replicative polymerase from the Bacillus subtilis phage phi29. The phi29 polymerase has exceptional strand displacement and processive synthesis properties, as well as an inherent 3’— >5’ exonuclease proofreading ability.
In some embodiments, nucleic acid amplification is whole genome amplification (WGA). Different and novel approaches to WGA have been developed since its inception in 1992, including degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). MDA by phi29 DNA polymerase has become a preferred method because it utilizes isothermal amplification in which phi29 strand displaces the double stranded DNA it encounters during DNA synthesis.
A key aspect of WGA by phi29 is usage of random hexamer oligonucleotides to amplify all possible genomic sequences. While this is crucial for an unbiased DNA amplification approach, the use of random hexamers becomes a liability when dealing with samples of pathogenic microbial DNA isolated from a human biological sample due to undesired human nucleic acid contaminants. The human genome is approximately 1,000 times larger than the average microbial genome, which presents a daunting challenge when sequencing pathogenic microbes that are vastly outnumbered by human cells.
In some embodiments, microbial nucleic acids present in the clinical sample obtained from the subject are species non- specific ally amplified (e.g., nucleic acids from two or more microbial species present in the clinical sample are amplified). In some embodiments, the step of amplification occurs before the step of subsampling. Thus, in some embodiments, the sample is processed before subsampling to species non- specifically amplify microbial DNA in the sample. In some embodiments, the step of amplification occurs after the step of subsampling. In some embodiments, the subsamples are processed after subsampling to species non- specifically amplify microbial DNA in each subsample.
In some embodiments, the microbial nucleic acids present in the clinical sample obtained from the subject are species-specifically amplified (e.g., nucleic acids from a single microbial species present in the clinical sample are amplified). In some embodiments, the step of amplification occurs before the step of subsampling. Thus, in some embodiments, the sample is processed before subsampling to species-specifically amplify microbial DNA in the sample. In some embodiments, the step of amplification occurs after the step of subsampling. In some embodiments, the subsamples are processed after subsampling to species-specifically amplify microbial DNA in each sub sample.
Pathogenic Microbes
In some aspects, the present invention provides improved methods for determining the presence of and/or identifying a microbial infection in a subject. In some embodiments, the microbial infection is determined and/or identified by the detection of one or more microbial species present in a clinical sample or subsamples using the methods described herein.
In some embodiments, the one or more microbial species comprise one or more of a bacterium, a virus, a fungus, a protist, or a yeast. In some embodiments, the one or more microbial species comprise one or more bacterial species. Although some bacteria are normally present in healthy mammals, disruption of the normal balance between the bacteria and the human host, or the presence of abnormal or pathogenic bacteria within the host, can lead to infection.
Staphylococcus aureus (S. aureus) is a bacterium that is normally present in the human body and is frequently found in the nose, respiratory tract, and on the skin. Although S. aureus is not always pathogenic, it is a common cause of skin infections including abscesses, respiratory infections, and food poisoning. The common method of treating
S. aureus infections is using antibiotics, although the emergence of antibiotic -resistant strains of S. aureus such as Methicillin-Resistant S. aureus (MRS A) and Vancomycin-Resistant S. aureus (VRSA) have become worldwide clinical health challenges.
Staphylococcus epidermidis (S. epidermidis) is a bacterium that is normally present in the human body, where it is frequently found on the skin. Although S. epidermidis is not generally pathogenic, subjects with compromised immune systems are at risk of developing S. epidermidis infections, and S. epidermidis poses a particular threat to subjects with surgical implants because it can grow on plastic surfaces and spread to the human body.
S. epidermidis strains are often resistant to antibiotics, including rifamycin, fluoroquinolones, gentamicin, tetracycline, clindamycin, and sulfonamides.
Streptococcus agalactiae ( S. agalactiae ) is a bacterium that is generally not pathogenic and can be found in the gastrointestinal and genitourinary tract in up to 30% of humans. Pathogenic infections due to S. agalactiae are of concern for neonates and immunocompromised individuals. S. agalactiae infections in adults can be life-threatening and include bacteremia, soft-tissue infections, osteomyelitis, endocarditis, and meningitis. S. agalactiae is increasingly resistant to clindamycin and erythromycin.
Enterococcus faecalis (E. faecalis) is a bacterium that inhabits the gastrointestinal tracts of humans and other mammals. However, E. faecalis can cause endocarditis, septicemia, urinary tract infections, and meningitis. E. faecalis infections can be lifethreatening, particularly when the E. faecalis is resistant to treatment with gentamicin and vancomycin.
Enterococcus f aecium (E. faecium) is a bacterium that inhabits the gastrointestinal tracts of humans and other mammals, but it may also be pathogenic, resulting in diseases such as meningitis and endocarditis. E. faecium infections can be life-threatening, particularly when the E. faecium is resistant to treatment with vancomycin.
Escherichia coli (E. coli) is a bacterium that inhabits the gastrointestinal tracts of humans and other mammals, but it may also be pathogenic, resulting in conditions such as gastroenteritis, urinary tract infections, neonatal meningitis, hemorrhagic colitis, and bacteremia. E. coli is increasingly resistant to multiple antibiotics, including fluoroquinolones, cephalosporins, and carbapenems.
Klebsiella pneumoniae ( K. pneumoniae ) is a bacterium that is normally found in the mouth, skin, and intestines of humans and other mammals. However, it can cause destructive changes to mammalian (e.g., human) lungs if inhaled, particularly to alveoli. K. pneumoniae infections are generally seen in subjects with a compromised immune system, including subjects with diabetes, alcoholism, cancer, liver disease, chronic obstructive pulmonary diseases, glucocorticoid therapy, and renal failure. K. pneumoniae is increasingly resistant to multiple antibiotics, including fluoroquinolones, cephalosporins, tetracycline, chloramphenicol, carbapenems, and trimethoprim/sulfamethoxazole.
In some embodiments, the microbe is a pathogenic microbe. In some embodiments, the microbe is a bacterium. In some embodiments, the bacterium is associated with bacteremia. In some embodiments, the bacterium is S. aureus, S. epidermidis, S. agalactiae, E. faecalis, E. faecium, E. coli, K. pneumoniae, or any other bacterial species associated with clinical infection.
In some embodiments, methods of the present invention are used to identify a pathogenic microbe selected from: Achromobacter spp., Acinetobacter calcoaceticus/baumannii complex, Acinetobacter haemolyticus, Acinetobacter junii, Acinetobacter radioresistens, Acinetobacter ursingii, Acinetobacter Iwoffii, Actinomyces israelii, Actinomyces meyeri, Actinomyces naeslundii, Actinomyces neuii, Actinomyces odontolyticus, Actinomyces pyogenes, Actinomyces viscosus, Aerococcus urinae, Aerococcus viridans, Aeromonas spp., Alcaligenes faecalis, Alcaligenes xylosoxidans, Alpha hemolytic streptococcus, Arcanobacterium haemolyticum, Aspergillus spp., Bacillus spp., Bacteriodes fragilis, Bartonella Quintana, Blastocystis hominis, Bordetella spp., Borrelia spp., Brevundimonas diminuta, Brevundimonas vesicularis Brucella spp., Burkholderia spp., Burkholderia cepacia, Burkholderia cepacia complex, Burkholderia gladioli, Burkholderia multivorans, Burkholderia pseudomallei, Burkholderia vietnamiensis, Campylobacter spp., Cedecea davisae, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chryseobacterium indologenes, Citrobacter spp., Citrobacter amalonaticus, Citrobacter farmer, Citrobacter freundii complex, Citrobacter koseri, Citrobacter sedlakii, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Coagulase negative staphylococcus, Corynebacterium diphtheriae, Corynebacterium pseudotuberculosis, Corynebacterium ulcerans, Coxiella spp., Cronobacter sakazakii, Delftia acidovorans, Dermabacter hominis, Edwardsiella tarda, Ehrlichia spp., Eikenella corrodens, Enterobacter spp., Enterobacter aerogenes, Enterobacter cancerogenus, Enterobacter cloacae complex, Enter obius vermicularis, Enterococcus spp., Enterococcus avium, Enterococcus casseliflavus, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus raffinosus, Escherichia coli, Francisella spp., Fusarium spp., Gemella spp., Granulicatella adiacens, Group A streptococcus, Group B salmonella, Group B streptococcus, Group Cl salmonella, Group C2 salmonella, Group C streptococcus, Group D salmonella, Group G salmonella, Group G streptococcus, Haemophilus influenza, Haemophilus parainfluenzae, Hafnia alvei, Helicobacter spp., Kingella kingae, Klebsiella spp., Klebsiella granulomatis, Klebsiella oxytoca, Klebsiella pneumoniae, Kluyvera spp., Kocuria kristinae, Lactobacillus spp., Leclercia adecarboxylata, Legionella pneumophila, Leishmania spp., Leptospira spp., Leuconostoc pseudomesenteroides, Listeria monocytogenes, Micrococcus luteus, Moraxella catarrhalis, Morganella morganii, Mycobacterium abscessus, Mycobacterium chimaera, Mycobacterium fortuitum, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma genitalium, Mycoplasma pneumoniae, Neisseria meningitidis, Neisseria gonorrhoeae, Nocardia spp., Ochrobactrum anthropic, Orientia tsutsugamushi, Pandoraea spp., Pantoea spp., Pantoea agglomerans, Paracoccus yeei, Pasteur ella canis, Pasteurella multocida, Pediococcus, Peptostreptococcus, Plesiomonas shigelloides, Prevotella spp., Propionibacterium spp., Propionibacterium acnes, Proteus spp., Proteus mirabilis, Proteus penneri, Proteus vulgaris, Providencia rettgeri, Providencia stuartii, Pseudomonas spp., Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, Rahnella aquatilis, Ralstonia spp., Raoultella spp., Raoultella ornithinolytica, Raoultella planticola, Rickettsia prowazekii, Rickettsia typhi, Roseomonas gilardii, Rothia mucilaginosa, Salmonella spp., Scedosporium spp., Serratia spp., Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Shigella flexneri, Shigella sonnei, Sphingobacterium spp., Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus intermedins, Staphylococcus lugdunensis, Staphylococcus pasteuri, Staphylococcus saccharolyticus, Staphylococcus saprophyticus, Staphylococcus schleiferi, Staphylococcus simulans, Staphylococcus warneri, Stenotrophomonas maltophilia, Streptococcus spp., Streptococcus agalactiae, Streptococcus anginosus, Streptococcus canis, Streptococcus constellatus, Streptococcus dysgalactiae, Streptococcus intermedins, Streptococcus parasanguinis, Streptococcus pneumoniae, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus vestibularis, Treponema spp., Trichophyton spp., Trichosporon asahii, Trueperella bernardiae, Tsukamurella tyrosinosolvens, Ureaplasma spp., Vibrio cholerae, Vibrio parahaemolyticus, Weiss ella confuse, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yokenella regensburgei, Aspergillus fumigatus, Candida albicans, Candida auris, Candida dubliniensis, Candida glabrata, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, Pneumocystis, Penicillium, Fusarium, Microsporum spp., Mucormycosis, Histoplasma, Blastomyces, Coccidioides, Trichophyton, Trichosporon spp., Microsporum, Pneumocystis jiroveci, Epidermophyton, Curvularia, Saccharomyces, Sporothrix, Microsporidia, Adenoviruses, Alphavirus, Arbovirus, Astrovirus, Bocaviruses, Bunyaviridae, Chikungunya virus, Coronavirus, Coxsackievirus, Cytomegalovirus, Dengue, Echovirus, Ebola, Enterovirus, Epstein-Barr virus, Flaviviridae, Foot-and-mouth disease virus, Hantavirus, Hepatitis A, Hepatitis B , Hepatitis C, Hepatitis E, Herpes simplex virus, Human cytomegalovirus, Human herpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, Human immunodeficiency virus, Human papillomaviruses, Human polyomaviruses, Human T-cell lymphotrophic virus, Influenza, Lassa virus, Marburg virus, Measles virus, Metapneumovirus, Molluscipoxvirus, Morbillivirus, Mumps, Nipah virus, Norovirus, Parainfluenza, Parechovirus, Parvovirus B19, Poliovirus, Polyomavirus, Poxviruses, Rabies virus, Respiratory syncytial virus, Rhinovirus, Rotavirus, Rubella virus, Rubivirus, Sapovirus, Togaviridae, Tick-borne Encephalitis virus, Usutu virus, Vaccinia virus, Varicella zoster virus, Variola virus, West Nile virus, yellow fever virus, and Zika virus.
In some embodiments, the microbial infection comprises microbes having one or more resistance genes, virulence genes, single nucleotide polymorphisms, accessory genomes, plasmids, and/or recombination regions. In some embodiments, one or more detected resistance or virulence genes are amplified. In some embodiments, the one or more detected resistance or virulence genes are amplified by polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP), helicase dependent amplification (HD A), or recombinase polymerase amplification (RPA).
In some embodiments, the methods of the present invention are used to select an appropriate treatment for a pathogenic microbe infection. In some embodiments, a clinical sample is obtained from a subject having or suspected of having a pathogenic microbial infection. In some embodiments, the infection is a bacteremia. In some embodiments, the appropriate treatment is treatment with an antibiotic. Non-limiting examples of antibiotics include: vancomycin, bacterium, methicillin, ceftobiprole, ceftaroline, dalbacancin, daptomycin, fusidic acid, linezolid, mupirocin, oritavancin, tedzolid, telavancin, tetracycline, amoxicillin, penicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole/trimethoprim, and levofloxacin. In some embodiments, the infection is a viral infection. In some embodiments, the appropriate treatment is an anti-viral. Non-limiting examples of anti-virals include: abacavir, acyclovir, balavir, cidofovir, darunavir, entecavir, famciclovir, ganciclovir, ostellamivir, penciclovir, and zalcitabine. In some embodiments, the infection is a fungal infection. In some embodiments, the appropriate treatment is an anti-fungal. Non-limiting examples of anti-fungals include: amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, rimocidin, bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconzaole, and anidulafungin.
EXAMPLES
Example 1 - Subsampling results in a quicker and more effective microbial enrichment process.
To determine the benefit of processing 16 mL of patient sample as 4 subsamples versus as an individual sample, whole blood samples from healthy donors were collected in two SPS vacutainers (BD Cat # 364960) and processed in the following fashion.
Blood from two vacutainers (approximately 20 mL total of blood) was combined and 4 x 4 mL of blood was aliquoted into 4 x 15 mL conical tubes containing 8 to 16 mL of 1 x TBS (20 mM Tris-Cl, pH 7.0, 150 mM NaCl). To mix, the tubes were then inverted. To simulate a bacteremic blood sample, bacteria Klebsiella pneumoniae') was added or “spiked” into the blood samples at a final concentration of 2-3 colony forming units (CFU) / mL of blood. The bacteremic blood samples were centrifuged at low speeds (for example, 500g to 700g for 3 to 7 minutes) in order to compact the red blood cells (RBC) at the bottom of the tube and maximize bacterial retention in the supernatant. The maximal amount of the first supernatant (SI) from each tube was aspirated and the process was repeated after diluting the RBC with the same amount of 1 x TBS used previously. All supernatant-containing subsamples were then treated with Proteinase K (NEB P8107), as described in US Patent No. 10,544,446, to remove SPS carry over and subsequent molecular amplification inhibition. Detergents commonly used for selective lysis, such as Tween-20 (Sigma Aldrich P9416) and Saponin (Sigma Aldrich Cat. #47036) were used at a final concentration between 0.1 and 2% along with DNase I (see, e.g., Charalampous et al. (2019), Nat. Biotechnol. 37 , 783-92; Street et al. (2019), J. Clin. Microbio. 58(3); Hasan et al. (2016), J. Clin. Microbio. 54(4); Shehadul Islam et al. (2017), Micromachines 8(3): 83) to efficiently deplete human DNA. The detergent concentration used was informed by the specific bacteria’s viability in the detergent used. To isolate the bacteria, samples were spun down (for example, 2000g to 3000g for 10 to 20 minutes) to remove most of the supernatant. Subsequent washes were performed with TBST (1 x TBS + 0.1% Tween 20) and spun down at 10,000g for 3 minutes to collect the bacterial pellet.
The concentrated sample was then subjected to a modified REPLI-g Single Cell protocol (Qiagen Cat. #150345) to lyse the collected cells. Samples were resuspended in 4.8 pL of 1 x PBS and 3.6 pL of D2 buffer was added. The samples were then incubated at 65°C for 10 minutes. 3.6 pL of STOP buffer was added post-incubation.
5 pL from each 12 pL lysate was used for a 25 pL REPLI-g Single Cell MDA reaction and another 5pL was combined with the rest of the donor subsamples (4 x 5 pL) for a lOOpL REPLI-g Single Cell MDA reaction. Each REPLI-g SC reaction was supplemented with 2% BSA (Sigma Aldrich Cat # A7638) + 2% DMSO (Sigma Aldrich Cat # D8418) and amplified for 4 hours.
Amplified DNA samples were quantified with the Qubit dsDNA HS kit (Thermo Fisher Cat #Q32854) and Qubit Fluorometer 3.0 (Thermo Fisher). 1 pg of each amplified DNA sample was used for library preparation with the SQK-LSK109 library preparation kit from Oxford Nanopore Technology (ONT) and sequenced with nanopore flow cells. The resulting sequencing data was base-called / demultiplexed by ONT’s Guppy Basecaller (v3.3.O). Sequencing files for each barcode were combined into a single file and processed through the following pipeline: each read was classified using Kraken, sequences were aligned to both a human reference genome and to the internal DZD reference genome assembly for that strain using minimap 2 (v2.9; see, e.g., Li (2018), Minimap2: Pairwise alignment for nucleotide sequences, Bioinformatics 34(18): 3094-3100), and alignment statistics were computed using minimap2 (v2.9) and SAMtools (vl.3.1; see, e.g., Li et al. (2009), 1000 Genome Project Data Processing Subgroup, The Sequence Alignment/Map format and SAMtools, Bioinformatics 25(16): 2078-79). M:H ratios were measured by dividing the megabases classified to the pathogen of interest to the megabases classified to the human genome.
In FIG. 1A, analysis of the sequencing results shows the ratio of microbial (bacterial) to human (M:H ratio) reads in the subsamples. The results show that each replicate had at least 1 and up to 3 subsample(s) with a higher M:H ratio as compared to the combined sample. A higher M:H ratio means a larger fraction of microbial sequences per given sample or subsample, which effectively decreases the necessary sequencing time for a bacteremia diagnostics device and results in a shorter sample-to-result time. In a scenario where a diagnostic device has one hour to generate data for roughly > 25 x K. pneumoniae genome coverage (e.g., > 140 Mb), a data output rate of 500 Mb per hour, and is working with low bacterial load samples (FIG. IB), 100% of the subsamples met those criteria, versus only 66.7% of the combined sample replicates. This example shows that subsampling results in a microbial enrichment process to generate a higher ratio of useful microbial data when working with low microbial load blood samples. A comparison of the genome coverage between the subsamples (4 mL) and the combined samples (16 mL) also shows that there is no significant difference in resulting genome coverage with similar number of total sequencing reads generated, despite using a fraction of the total microbial input (FIGs. 1C and ID).
The enrichment process utilized techniques as described in U.S. Patent No. 10,544,446, which is expressly incorporated by reference herein in its entirety.
Example 2 - Subsampling methods increase speed and accuracy of determining presence of and/or identifying microbial infection.
The human genome comprises approximately 3.3 x 109 bp of DNA and, therefore, a diploid human cell comprises approximately 6.6 x 109 bp. A standard clinical sample of blood drawn directly from a patient will include approximately 4 million white blood cells per milliliter (mL) and, therefore, 1 mL of blood can be expected to contain roughly 2.64 x 1016 bp of human DNA.
In contrast, the pathogenic bacteria present in human blood have much smaller genomes, and are likely to be present in much lower numbers. For example, the genomes of the bacteria commonly isolated in sepsis or bacteremia typically range between 2 x 106 to 6 x 106 bp (e.g., S. pyogenes'. 1.8 mbp, S. aureus'. 2.8 mbp, E. coli: 4.6 mbp, Klebsiella spp.'. 5.5 mbp, P. aeruginosa'. 6.3 mbp). The number of bacteria present in a clinical sample from a sepsis patient is usually less than 50 cells/mL, commonly less than 10 cells/mL, and often less than 1 cell/mL. Thus, assuming genomes of 2-6 x 106 bp and numbers of cells between 1-50 cells/mL, the bacterial DNA present in a clinical sample from a sepsis patient may typically range from 2-300 x 106 bp/mL.
Therefore, the initial ratio of bacterial DNA (2-300 x 106 bp/mL) to human DNA (2.64 x 1016 bp/mL) may be expected to range from about 7.6 x 10“ 11 to 1.1 x 10’8, which makes detection of bacterial DNA exceedingly difficult.
However, using existing techniques for selectively removing human cells and human DNA from blood samples (e.g., removal of cells by lysis and centrifugation), it is possible to reduce the amount of human DNA to about 1 x 10’9 of the original amount, or 2.64 x 107 bp/mL, and thereby enrich the sample for bacterial DNA relative to human DNA. After such enrichment, the ratio of bacterial DNA (2-300 x 106 bp/mL) to human DNA (2.64 x 107 bp/mL) may be expected to range from about 7.6 x IO’2 to 11.
Thus, for example, a single 16 mL clinical sample of blood is obtained from a patient. The sample is divided in 4 subsamples. Each subsample is subjected to standard techniques (e.g., lysis, centrifugation, etc.) for reducing human cells and human DNA by a factor of 109.
The abundance of bacterial DNA in each sample is separately determined using standard techniques (e.g., next generation sequencing, third generation sequencing, or estimated via targeted PCR amplification). It is found that sample 1 contains 4.0 x 106 bp/ml, sample 2 contains 1.0 x 106 bp/ml, sample 3 contains 3.0 x 106 bp/ml, and sample 4 contains 8.0 x 106 bp/ml of bacterial DNA. The average abundance of bacterial DNA across the four samples is 4 x 106, but the abundance in sample 4 is twice that, at 8.0 x 106. Assuming the abundance of human DNA (after enrichment, as described above), is 2.64 x 107, the ratio of bacteriakhuman DNA in sample 4 is 0.30.
If the goal is to sequence at least 100 million bases of bacterial DNA, by subsampling, the amount of DNA to be sequenced is reduced 2-fold by choosing to sequence only one of four samples instead of all four, and the relative abundance of bacteriakhuman DNA is increased 2-fold by choosing subsample 4, which has twice the bacteriakhuman DNA ratio of the original clinical sample.
Although particular embodiments of the invention have been illustrated by the foregoing exemplary embodiments, it should be understood that the examples are illustrative only and not intended to be limiting. One of skill in the art will recognize that methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, and numerous changes in the details of implementation of the disclosed subject matter may be made without departing from the spirit and scope of the disclosed subject matter. The scope of the invention is limited only by the claims.

Claims

CLAIMS What is claimed is:
1. A method of determining the presence of a microbial infection in a subject, the method comprising:
(a) obtaining a clinical sample from the subject;
(b) dividing the clinical sample into at least two subsamples;
(c) subjecting the clinical sample prior to step (b), and/or the subsamples after step
(b), to at least one method of enrichment for microbial DNA; and
(d) determining the abundance of microbial DNA in the enriched subsamples; wherein an abundance of microbial DNA greater than zero in at least one subsample indicates the presence of a microbial infection and an abundance of microbial DNA of zero in all subsamples indicates that a microbial infection has not been detected.
2. A method of identifying a microbial infection in a subject, the method comprising:
(a) obtaining a clinical sample from the subject;
(b) dividing the clinical sample into at least two subsamples;
(c) subjecting the clinical sample prior to step (b), and/or the subsamples after step
(b), to at least one method of enrichment for microbial DNA; and
(d) analyzing the DNA from the subsamples; wherein the microbial infection is identified based upon the analyzed DNA.
3. A method of identifying a microbial infection in a subject, the method comprising:
(a) obtaining a clinical sample from the subject;
(b) dividing the clinical sample into at least two subsamples;
(c) subjecting the clinical sample prior to step (b), and/or the subsamples after step (b), to at least one method of enrichment for microbial DNA;
(d) determining the abundance of microbial DNA in the enriched subsamples;
(e) (i) if at least one subsample has an abundance of microbial DNA greater than zero, selecting at least one sub sample with a higher abundance of microbial DNA relative to at least one unselected subsample; or
(ii) if no subsample has an abundance of microbial DNA greater than zero, determining that the presence of a microbial infection has not been identified; and (f) if at least one subsample has an abundance of microbial DNA greater than zero, analyzing the microbial DNA from the subsamples selected in (e)(i); wherein the microbial infection is identified based upon the analyzed microbial DNA.
4. The method of claim 1 or claim 3, wherein determining the abundance of microbial DNA comprises species-specific amplification of microbial DNA.
5. The method of claim 4, wherein the microbial DNA is amplified by a method selected from the group consisting of: polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), reverse-transcriptase PCR (RT-PCR), degenerate oligonucleotide PCR, primer extension pre-amplification, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HD A), transcription mediated amplification (TMA), and recombinase polymerase amplification (RPA).
6. The method of claim 1 or claim 3, wherein determining the abundance of microbial DNA comprises whole genome sequencing.
7. The method of any of claims 1 or 3-5, wherein determining the abundance of microbial DNA comprises a polymerase-based or hybridization-based assay.
8. The method of claim 7, wherein the polymerase-based or hybridization-based assay is selected from the group consisting of: quantitative polymerase chain reaction (qPCR), loop- mediated isothermal amplification (LAMP), reverse transcriptase LAMP (RT-LAMP), recombinase polymerase amplification (RPA), microarrays, southern blot, and northern blot.
9. The method of any one of claims 1-8, wherein the microbial DNA comprises DNA from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, or more microbial species.
10. The method of any one of claims 1-9, wherein the clinical sample comprises an abundance of microbial DNA comprising no more than 10 genomic copies per mL of clinical sample volume prior to enrichment or determining abundance.
11. The method of any one of claims 1-10, wherein the clinical sample is subjected to at least one method of enrichment for microbial DNA before step (b).
12. The method of any one of claims 1-11, wherein the subsamples are subjected to at least one method of enrichment for microbial DNA after step (b).
13. The method of claim 11 or claim 12, wherein the method of enrichment comprises a method of non-amplification enrichment.
14. The method of claim 13, wherein the method of non-amplification enrichment comprises reducing of the abundance of human cells present in the clinical sample or subsample.
15. The method of claim 13, wherein the method of non-amplification enrichment comprises reducing the abundance of human DNA present in the clinical sample or subsample.
16. The method of claim 14 or claim 15, wherein the method of non-amplification enrichment includes at least one method selected from the group consisting of: centrifugation, immunoaffinity purification, filtration, and selective lysis to remove human cells and/or human DNA.
17. The method of any one of claims 1-12, wherein the method of enrichment comprises a method of global amplification.
18. The method of claim 17, wherein the method of global amplification comprises a method selected from the group consisting of: polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), reverse-transcriptase PCR (RT-PCR), degenerate oligonucleotide PCR, primer extension pre-amplification, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HD A), transcription mediated amplification (TMA), and recombinase polymerase amplification (RPA).
19. The method of claim 17 or claim 18, wherein the method of global amplification amplifies human and/or microbial DNA present in the clinical sample or subsample.
20. The method of any one of claims 2-19, wherein analyzing the DNA comprises analyzing human and/or microbial DNA present in the subsamples.
21. The method of any one of claims 2-20, wherein analyzing the DNA comprises whole genome sequencing.
22. The method of any one of claims 2-21, wherein analyzing the DNA comprises generating an Antimicrobial Resistance Sensitivity (AMR/S) profile using a computational algorithm for the microbial species.
23. The method of any one of claims 2-22, wherein analyzing the DNA comprises using a molecular- or sequencing-based method to identify the microbial infection.
24. The method of any one of claims 2-23, wherein analyzing the DNA comprises using a molecular- or sequencing-based method to identify one or more genus, species, or subspecies strains.
25. The method of claim 23 or claim 24, wherein the molecular- or sequencing -based method is selected from the group consisting of: quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP), reverse transcriptase LAMP (RT- LAMP), recombinase polymerase amplification (RPA), microarrays, southern blot, northern blot, next generation sequencing (NGS), and third generation sequencing.
26. The method of claim 25, wherein the NGS or third generation sequencing achieves at least 75% coverage of the microbial genome.
27. The method of claim 26, wherein the NGS or third generation sequencing achieves at least 75% coverage of the microbial genome in less than 3 hours.
28. The method of any one of claims 1-27, wherein the microbial infection comprises microbes having one or more resistance genes, virulence genes, single nucleotide polymorphisms, accessory genomes, plasmids, and/or recombination regions.
29. The method of claim 28, wherein one or more detected resistance or virulence genes are amplified.
30. The method of claim 29, wherein the one or more detected resistance or virulence genes are amplified by polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP), or recombinase polymerase amplification (RPA).
31. The method of any one of claims 1-30, wherein the clinical sample of step (b) is divided into 4 subsamples.
32. The method of any one of claims 1-30, wherein the clinical sample of step (b) is divided into at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 subsamples.
33. The method of any one of claims 1-32, wherein the subsamples comprise substantially equal volumes.
34. The method of claim 33, wherein the subsamples each comprise at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mL.
35. The method of any one of claims 1-34, wherein the clinical sample is a blood sample.
36. The method of any one of claims 1-34, wherein the clinical sample is a cerebrospinal fluid (CSF) sample.
37. The method of any one of claims 1-34, wherein the clinical sample is a joint fluid, abscess fluid, serum, lymph, urine, stool, or sputum sample.
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