WO2018156664A1 - Methods and systems for microbial genetic test - Google Patents

Methods and systems for microbial genetic test Download PDF

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WO2018156664A1
WO2018156664A1 PCT/US2018/019063 US2018019063W WO2018156664A1 WO 2018156664 A1 WO2018156664 A1 WO 2018156664A1 US 2018019063 W US2018019063 W US 2018019063W WO 2018156664 A1 WO2018156664 A1 WO 2018156664A1
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study
culture
wound
specimen
patients
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PCT/US2018/019063
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French (fr)
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Tanya MORENO
Kulvinder Kaur
Andria DEL TREDICI
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Millennium Health, LLC
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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
    • C12Q1/6876—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
    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • microbial genetic test in particular the presence or absence of microbes in biological samples obtained from wounds (e.g., swabs) and other tissues.
  • improved methods are needed for a rapid and complete identification of microorganisms in a wound.
  • Such methods can allow for the targeted treatment of a patient's wound, yielding improved outcomes.
  • More rapid microbial identification and diagnosis of a patient's wound infections can also significantly shorten the time required to heal wounds, promote targeted antibiotic therapy, as well as other advantages, including accelerated transition from inpatient to outpatient, a decrease in the potential of repeated surgeries, and a likely reduction in hospital readmission rates.
  • Disclosed herein are methods and systems generally related to a rapid, accurate, and more comprehensive identification of microorganisms in a biological sample such as those obtained from wounds.
  • the methods and systems are useful for rapidly detecting the presence or absence of a plurality of microorganisms from other biological samples such as a blood sample, skin sample, a saliva sample, or a urine sample.
  • Such methods and systems allow for the identification of microorganisms from a small amount of clinical specimen, as well as the qualitative representation of the microorganisms contributing to a polymicrobial infection.
  • the methods and systems disclosed herein enable the precision treatment of a subject's infection (e.g., wound infection), which may lead to shortening the healing time of the infection, e.g., a chronic wound infection.
  • a subject's infection e.g., wound infection
  • a method for detecting the presence or absence of a plurality of microorganisms in a biological sample.
  • the method includes: (a) obtaining a biological sample from a subject in need thereof, wherein the biological sample may, or is suspected to, contain one or more microorganisms; (b) providing a plurality of primer pairs, wherein each primer pair is specifically designed to amplify a target sequence of a microorganism listed in one or more of Tables 1-11; and (c) performing real-time PCR or digital PCR using the plurality of primers, thereby detecting the presence or absence of the plurality of microorganisms listed in one or more of Tables 1-11.
  • the target sequence can include antibiotic resistance genes which may be shared by more than one microorganism.
  • the target sequence may also include virulence factor genes.
  • the plurality of primer pairs are designed to target and/or detect all microorganisms, antibiotic resistance genes and virulence factor genes listed in one or more of Tables 1-11.
  • the method can further include, prior to step (c), extracting nucleic acids from the biological sample.
  • the biological sample or the one or more microorganisms therein are not subject to a step of in vitro culturing.
  • a further aspect relates to a plurality of primer pairs, wherein each primer pair is specifically designed to amplify a target nucleotide sequence within a microorganism, antibiotic resistance gene or virulence factor gene listed in one or more of Tables 1-11, wherein the plurality of primer pairs together target and/or detect all microorganisms, antibiotic resistance genes and virulence factor genes listed in one or more of Tables 1-11.
  • the system or kit includes: (a) a plurality of primer pairs, wherein each primer pair is specifically designed to amplify a target sequence of a microorganism listed in one or more of Tables 1-11; and (b) reagents for performing real-time PCR or digital PCR using the plurality of primers, thereby detecting the presence or absence of the plurality of microorganisms listed in one or more of Tables 1-11.
  • the system or kit can further include reagents for extracting nucleic acids from the biological sample.
  • the plurality of primer pairs are designed to target and/or detect all microorganisms listed in one or more of Tables 1-11.
  • FIG. 1 A shows an overview of the general steps involved in an exemplary method for the identification of the microorganism community.
  • FIG. IB shows exemplary real-time PCR amplification curves.
  • FIG. 1C shows a report of interpreted results from an exemplary real-time PCR run.
  • FIG. 2 depicts the relative representation of different organisms in a specimen over time showing that Device 2 (DxWound) stably captures the microbiome of the wound, while
  • Device 1 standard culture transport media results in large variation in the microbiome.
  • FIG. 3 shows DNA yield from different specimen types.
  • FIG. 4 depicts a bar graph showing the number of microbes identified per patient in a pilot study of 106 wound specimens.
  • FIG. 5 depicts a bar graph showing the number of antibiotic resistance genes identified in patient samples in a pilot study of 106 wound specimens.
  • Disclosed herein are methods and systems that can be used to rapidly and accurately detect the presence or absence of microorganisms (including antibiotic resistance genes and/or virulence factor genes thereof) in a biological sample.
  • the methods and systems disclosed herein provide a comprehensive snapshot of the microbiome of a biological sample.
  • the methods and systems disclosed herein are useful for treating microbial infections through accurate identification of the microorganisms present. Definitions
  • WGT Wild Genetic Test
  • DxWound refers more to the systems.
  • present disclosure in certain embodiments refers to the method in the context of wounds, one of ordinary skill in the art would readily understand that the methods and kits disclosed herein are applicable to other biological samples such as skin, buccal swab, tissue biopsy, bone, nails, plasma, serum, whole blood and blood components, saliva, urine, tears, seminal fluid, vaginal fluids and other fluids and tissues, including paraffin embedded tissues or other tissues collected and preserved in the course of, e.g., a forensic investigation.
  • antibiotic resistance genes describes genes present in bacteria that may mediate resistance to a specific antibiotic or class of antibiotics.
  • methods provided herein can be used to detect several antibiotic resistance genes associated with bacteria that typically cause skin and soft tissue infections (SSTIs). The detection of these genes using methods provided herein suggests the presence of bacteria that may be resistant to specific antibiotics or classes of antibiotics.
  • sample refers to any composition containing or presumed to contain nucleic acid (e.g., DNA) from the microbiota of an individual.
  • nucleic acid e.g., DNA
  • any type of body sample may be used, including without limitation, skin, buccal swab, wound swab, tissue biopsy, bone biopsy, nails, plasma, serum, whole blood and blood components, saliva, urine, tears, seminal fluid, vaginal fluids and other fluids and tissues, including paraffin embedded tissues or other tissues collected and preserved in the course of, e.g., a forensic investigation.
  • DNA can be extracted from various samples using methods known in the art.
  • primer refers to an oligonucleotide that acts as a point of initiation of DNA synthesis in a PCR reaction.
  • a primer is usually about 15 to about 40 nucleotides in length (or shorter or longer) and typically includes at least one target-hybridized region that is at least substantially complementary to the target sequence.
  • probe refers to an oligonucleotide that hybridizes to a target sequence on a target nucleic acid.
  • Target sequence refers to a region of nucleic acid that is to be analyzed and comprises the site of interest.
  • the probe can additionally contain a quencher and a dye (e.g., fluorescent). When the probes are intact, fluorescence is suppressed because the quencher dyes are in the proximity of the reporter dyes. At least two probes can be included in one TaqMan® assay.
  • TaqMan® assay or "TaqMan® chemistry” as used in the context of realtime PCR is a technology that exploits the 5 '-3' nuclease activity of Taq DNA polymerase to allow direct detection of the PCR product by the release of a fluorescent signal from a probe as a result of PCR.
  • the TaqMan® assay permits discrimination between different genomic sequences.
  • a typical TaqMan assay includes a Taq DNA polymerase, dNTPs, a pair of primers designed to amplify a target region, and a probe which will hybridize within the target region, mixed with DNA samples and subjected to PCR (e.g., real-time PCR that can detect and measure fluorescence).
  • Taq DNA polymerase refers to a heat stable enzyme used in the polymerase chain reaction (PCR) to amplify target DNA. It was discovered in bacterium Thermus aquaticus and hence the name. It should be noted that other polymerases having 5 '-3' nuclease activity can also be used to replace the Taq DNA polymerase in TaqMan assays.
  • NGS Next Generation Sequencing
  • Roche Genome Analyzer-based sequencing
  • Ion torrent Proton / PGM sequencing, Oxford Nanopore, PacBio, and ABI-SOLiD-based sequencing (Applied Biosystems).
  • the basic chemistry relies on the fact that DNA polymerase catalyzes the incorporation of dNTPs into a DNA polymer during sequential cycles of DNA synthesis.
  • the nucleotide being incorporated is either identified by an optical signal (e.g., fluorophore excitation) or change in pH (incorporation of each base releases a proton).
  • an optical signal e.g., fluorophore excitation
  • pH incorporation of each base releases a proton.
  • the critical difference is that, instead of sequencing a single DNA fragment at a time, NGS extends the process across of multiple fragments in a parallel fashion, greatly enhancing sequencing speed and accuracy.
  • nucleic acid refers to polymers of nucleotides (e.g., ribonucleotides or deoxyribonucleotides) both natural and non-natural. The term is not limited by length (e.g., number of monomers) of the polymer.
  • a nucleic acid may be single-stranded or double-stranded and will generally contain 5 '-3' phosphodiester bonds, although in some cases, nucleotide analogs may have other linkages.
  • complementary and complementarity refer to nucleic acids (e.g., a sequence of nucleotides) related by the base-pairing rules.
  • sequence 5'-A-G-T- 3' is complementary to the sequence 3'-T- C-A-5'.
  • Complementarity may be "partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.
  • these conventional techniques not the least is cultivation bias. It has been well known that fewer than 2% of all known bacterial species can be routinely cultured in the clinical microbiology laboratory, and only a subset of these microorganisms are able to grow within the 24 hour period prescribed for most cultures (Tatum, O.L. et al., Advances in Wound Care, Volume 1, Number 3, pages 115- 119).
  • other microbes such as fungi, e.g., Candida, are rarely identified in the routine laboratory culture procedures.
  • fungi can be part of the microbial community of a wound, it is critical to detect their presence and recognize their contribution to a wound.
  • anaerobic bacteria which have been identified as a component of recalcitrant wounds (Tatum, O.L. et al., Advances in Wound Care, Volume 1, Number 3, pages 115-119), are difficult to propagate in the laboratory without specialized collection methods, growth media, and environmental control.
  • some microorganisms are known to efficiently grow and outcompete other species in laboratory conditions. Thus, routine laboratory culture procedures have a selection bias of one species over another, leading to potentially to misguided treatments and less than desirable outcomes.
  • the methods and systems disclosed herein allow for rapid, consistent, and accurate identification of the microorganism community present from a biological sample. These methods of microorganism detection thus provide a snapshot of the microorganisms in the wound at the time of sample collection. This is highly desirable, as such methods can allow clinicians to better manage the healing of the wound, thereby improving the prognosis for patients.
  • blood and common exogenous wound treatments e.g., lidocaine, antibiotic creams, dressings, etc., can be successfully removed during the DNA extraction process and therefore do not interfere with the detection methods disclosed herein.
  • Another significant advantage provided by the methods and systems disclosed herein is the ability to detect the microorganism community within 48 hours from presentation.
  • One study suggested that as many as 16.6% of acute skin and soft tissue infections (SSTIs), 34.1% of chronic or ulcerative infections, and 26.7% of surgical site infections had initial treatment failure. It has been shown that antimicrobial therapy which is not targeted to the causative pathogen within 48 hours of presentation is an independent risk factor for treatment failure.
  • This type of data contributed to the U.S. Food and Drug Administration (FDA)'s decision to revise its guidance for the evaluation of clinical response to skin infections to earlier time points of 48 to 72 hours after initiation of therapy.
  • FDA U.S. Food and Drug Administration
  • Using the methods and systems disclosed herein it is possible to detect the microorganism community within 48 hours from presentation. Clinicians can use these results to help them quickly target antibiotic therapy, ideally within that important 48 hour window.
  • a method for detecting the presence or absence of a plurality of microorganisms in a biological sample can include: (a) obtaining a biological sample from a subject in need thereof, wherein the biological sample may, or is suspected to, contain one or more microorganisms; (b) providing a plurality of primer pairs, wherein each primer pair is specifically designed to amplify a target sequence of a microorganism listed in one or more of Tables 1-11; and (c) performing real-time PCR using the plurality of primers, thereby detecting the presence or absence of the plurality of microorganisms listed in one or more of Tables 1-11.
  • the system or kit includes: (a) a plurality of primer pairs, wherein each primer pair is specifically designed to amplify a target sequence of a microorganism listed in one or more of Tables 1-11; and (b) reagents for performing real-time PCR or digital PCR using the plurality of primers, thereby detecting the presence or absence of the plurality of microorganisms listed in one or more of Tables 1-11.
  • the system or kit can further include reagents for extracting nucleic acids from the biological sample.
  • the plurality of primer pairs are designed to target and/or detect all microorganisms listed in one or more of Tables 1-11.
  • FIG. 1 A is an overview of the general steps involved in an exemplary method disclosed herein related to the identification of the microorganism community.
  • various specimen such as blood, wound swab, urine, tissue biopsies and biorepositories can be collected by routine clinical methods.
  • the specimen can be processed (e.g., by inactivating nucleases) and/or stored (e.g., at a temperature lower than the room temperature) to maintain stability.
  • pathogen nucleic acids can be extracted from the specimen using extraction methods known in the art and/or commercially available kits (e.g., miniprep kits), following a step of tissue disruption depending on the type of the specimen (e.g., using QIAGEN
  • molecular analysis of a plurality of specimens simultaneously can then be performed at step (4) using, e.g., real-time PCR or other quantitative PCR methods such as digital PCR.
  • real-time PCR or other quantitative PCR methods such as digital PCR.
  • digital PCR e.g., digital PCR.
  • One example is the Fluidigm Biomark HD System with the 96.96 Dynamic Array Gene Expression Chip.
  • step (5) various positive and negative controls can be included for quality control purpose (step (5)).
  • Exemplary run controls (designed to ensure that the assay is running properly) include but are not limited to:
  • Isolation negative control to verify that the nucleic acid extraction/isolation reagents are not contaminated. Purified water can be used here.
  • Isolation positive control to verify that nucleic acid extraction/isolation has occurred successfully.
  • a standard culture of one or more types of microbes can be used.
  • Purified water can be used here.
  • Microbial Plasmid Pool to verify that the PCR assays are performing as expected.
  • One or more plasmids can be used, each plasmid predesigned to represent one microbe to be detected by the PCR assay.
  • PPC Positive PCR Control
  • a "Pan Bacterial" assay can be designed to detect all bacterial species.
  • universal primers can be designed to amplify the 16S rRNA region for all or most bacterial species of interest (see, e.g., Nature Biotech 31, 814-821
  • the "Pan Bacterial" assay can be used as a control to determine whether there are detectable levels of bacteria present in a specimen.
  • An amplification signal for the Pan Bacterial assay in the absence of a signal for a species specific assay indicates that bacteria which are not currently present in the DxWound panel are present in the specimen (e.g., Corynebacterium). If a signal for a species specific assay is detected, co-amplifi cation of the Pan Bacterial assay is expected. If no amplification signal is detected for the Pan Bacterial assay, the etiology of the wound is unlikely to be attributed to bacterial infection.
  • a "Pan Fungal” assay can also be included as a control to determine whether there are detectable levels of fungi present in a specimen.
  • universal primers to amplify an evolutionarily conserved sequence region in representative fungal species (Candida spp. and/or Aspergillus spp.) present on the DxWound panel.
  • the assay can be used as a confirmatory control if an amplification signal for a specific fungal species is detected.
  • Co-amplification of the assay for the specific fungal species and the Pan Fungal control assay is required to confirm the presence of a DxWound Candida spp. or Aspergillus spp in the specimen.
  • a human gDNA assay can also be included as a control to determine whether there are detectable levels of DNA present in a specimen.
  • primers to amplify representative human housekeeping genes can be used here.
  • the assay can be used as a confirmatory control to verify that the sample was successfully handled from the time of specimen collection through data acquisition.
  • FIG. IB shows exemplary real-time PCR amplification curves, in which exponential amplification begins from, e.g., cycle number 10-20 in positive controls and in specimens where pathogens are present. In contrast, in negative controls and in specimens where pathogens are absent, no amplification would occur.
  • Ct value the number of cycles required for the fluorescent signal to cross the threshold (i.e., exceeds background level) can be obtained for each amplification curve.
  • a heat map can be generated that color codes Ct values of an exemplary real-time PCR run, with a plurality of samples targeting a plurality of microbes. Different colors represent how early or late (in Ct value) amplification occurs, suggesting the amount of specific microbial DNA present in the corresponding samples.
  • the more microbial DNA is present the earlier amplification occurs and the lighter the color is on the heat map.
  • a report can be generated at step (7), FIG. 1 A.
  • such report can include sample name, assay name (microbe species and other targets), Ct value, assay quality control (QC) status, positive control Ct value (e.g., pan-bacteria control), negative control Ct value, and final interpreted result (in the form of positive (POS), negative (NEG) or unable to determine (UTD)).
  • POS positive
  • NEG negative
  • a PCR-based Dx Wound assay can be used to analyze microbial DNA using species-specific DNA sequences, such as 16S rRNA sequences for bacterial detection.
  • species-specific DNA sequences such as 16S rRNA sequences for bacterial detection.
  • sequences specific to virulence gene and antibiotic resistance genes e.g., mecA can also be analyzed.
  • DxWound can utilize a swab for sample collection.
  • the tests can be performed on a sample taken directly from the site of infection, e.g., wound.
  • the swab sample can be collected in an inactivating solution that kills the microorganisms at the same time as protecting the microbial DNA, thus preserving the wound microbiome in time at the point of specimen collection.
  • DNA is detected directly from the patient specimen without culture enrichment.
  • microbes/targets that can be detected using the methods and systems disclosed herein are shown in Tables 1-11.
  • VIM-7 Antibiotic Resistance Gene hla Virulence Factor lukF Virulence Factor spa Virulence Factor ermA Antibiotic Resistance Gene ermB Antibiotic Resistance Gene mefA Antibiotic Resistance Gene
  • Bacteria Bacteroides fragilis Bacteria Bacteroides fragilis
  • Bacteria Bacteroides fragilis Bacteria Bacteroides fragilis
  • DxWound can be used to detect Staphylococcus aureus (S. aureus), which is the most common pathogen associated with skin and soft tissue infections (SSTIs).
  • S. aureus Staphylococcus aureus
  • SSTIs skin and soft tissue infections
  • DxWound can accurately and sensitively identify certain Staphylococcal species, including Staphylococcus aureus, Staphylococcus lugdunensis, Staphylococcus epidermidis and other Coagulase-Negative Staphylococci (CoNS), along with the antibiotic resistance gene mecA and the virulence gene lukF-P V (Panton- Valentine Leukocidin virulence factor, PVL).
  • Staphylococcus aureus Staphylococcus aureus
  • Staphylococcus lugdunensis Staphylococcus epidermidis
  • CoNS Coagulase-Negative Staphyloc
  • An antibiotic resistance gene is a gene present in bacteria that mediates resistance to a specific antibiotic or class of antibiotics.
  • mecA is an antibiotic resistance gene associated with Staphylococcus species. The mecA gene mediates oxacillin/methicillin resistance.
  • MRSA Methicillin-resistant Staphylococcus aureus
  • MRSA has been tied to increased hospital lengths of stay, increased healthcare costs, is an independent risk factor for mortality and is now predictably resistant to all B-lactam antibiotics except ceftaroline. As such, MRSA is considered a serious threat by the CDC.
  • S. aureus can also express virulence factors that are associated with increased infection severity.
  • the presence of the PVL virulence factor in S. aureus is associated with infections that are more likely to lead to necrosis and more likely to require surgical treatment.
  • PVL is a virulence factor encoded by two genes: lukS-PV and lukF-PV.
  • the DxWound Staphylococcal tests can be useful for any patient suspected of having a Staphylococcal skin and soft tissue infection (SSTI) based on the clinician's determination of medical necessity.
  • the DxWound tests can identify S. aureus and other Staphylococcus spp directly from a wound sample without the need for culture enrichment.
  • the presence of mecA may indicate resistance to oxacillin/methicillin and all B-lactams with the exception of cephalosporins with anti-MRSA activity. When S. aureus and mecA are both detected, then MRSA may be present.
  • the presence of PVL may suggest increased infection severity and the potential need for surgical intervention. In a patient with clinical signs and symptoms of infection, these Staphylococcal tests can help clinicians quickly target antibiotic therapy.
  • DxWound can be used for antibiotic resistance gene testing.
  • Antibiotic resistance is considered by the CDC to be a major public health threat in the United States. Patients with antibiotic resistant infections have higher morbidity and mortality, including longer hospital stays with increased costs to the healthcare system. To help mitigate the consequences of antibiotic resistance, the CDC has called for improved antibiotic stewardship and the development of new diagnostic tests.
  • Antibiotic resistance genes are genes present in bacteria that mediate resistance to a specific antibiotic or class of antibiotics. DxWound can be used to test for antibiotic resistance genes associated with bacteria that typically cause skin and soft tissue infections (SSTIs). The detection of these genes suggests the presence of bacteria that may be resistant to specific antibiotics or classes of antibiotics (Table 7).
  • the mecA gene is associated with Staphylococcus species, including Staphylococcus aureus, Staphylococcus lugdunensis and Staphylococcus epidermidis.
  • the mecA gene mediates oxacillin/methicillin resistance and is associated with resistance to most B-lactams with the exception of cephalosporins with anti-MRSA activity.
  • Methicillin-resistant Staphylococcus aureus (MRS A) is a major public health threat.
  • the carbapenemase genes generally mediate resistance to B-lactams, including carbapenems.
  • the carbapenemase genes included in DxWound have been found in
  • Pseudomonas and Enterobacteriaceae such as Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Proteus mirabilis, Proteus vulgaris.
  • the carbapenemase genes have also been found less frequently in other bacteria genera. Carbapenemase-producing organisms are rare but considered a major public health threat.
  • ESBL extended-spectrum B-lactamase
  • DxWound The extended-spectrum B-lactamase (ESBL) genes included in DxWound are commonly associated with Enterobacteriaceae, but have also been found in other bacteria genera.
  • the ESBL genes generally confer resistance to B-lactams except cephamycins and carbapenems.
  • ESBL-producing Enterobacteriaceae are a major public health threat.
  • Enterococcus and in a broad range of other bacterial genera. They mediate resistance to macrolides (e.g., erythromycin), lincosamides (e.g., clindamycin), and type B streptogramins.
  • macrolides e.g., erythromycin
  • lincosamides e.g., clindamycin
  • type B streptogramins e.g., erythromycin-resistant Group B Streptococcus and Erythromycin-resistant Group A Streptococcus are public health threats. For ermB testing, in order to identify potential clindamycin-resistant Group B Streptococcus and erythromycin-resistant Group A
  • Streptococcus, Enterococcus faecium and Enterococcus faecalis tests may also be useful, since ermB is commonly found in Enterococcus species.
  • vanA and vanB mediate resistance to vancomycin and are associated with Enterococcus and very rarely with Staphylococcus species.
  • Vancomycin-resistant Enterococcus (VRE) and Vancomycin-resistant Staphylococcus aureus (VRSA) are public health threats.
  • DxWound tests can indicate whether specific antibiotic resistance genes (e.g., Table 8) are detected in a biological sample (e.g., wound specimen).
  • the resulting report can provide information about the association of the detected antibiotic resistance gene to the specific antibiotic or antibiotic classes that may be affected.
  • the report can indicate when a potential antibiotic resistant organism may be present, based on the detection of specific organisms with the antibiotic resistance genes. For example, when mecA is detected, the report states that if the wound is also positive for Staphylococcus aureus, then MRSA may be present.
  • the report states that if the wound is also positive for Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, or Proteus mirabilis/vulgaris, then an ESBL-producing Enterobactericeae may be present.
  • DxWound tests can be used to detect various targets (e.g., Table 9) in a biological sample suspected of infection.
  • the targets can be selected based on their clinical relevance and/or frequency (Tables 10-12).
  • a method of identifying targets such as organisms, antibiotic resistance genes, and virulence genes that, if detected, would be useful for treatment decisions for infections such as SSTI includes obtaining a clinical score and a frequency score for organisms, antibiotic resistance genes, and virulence genes of interest.
  • Frequency score identifies prevalence of the tested item in infection sites such as wounds/SSTI, according to published studies.
  • Clinical score is a measure of the pathogenicity of the organism, based on the published evidence. The highest clinical score (1) is applied when major public health organizations have identified the organism as an antimicrobial-resistant public health threat. The clinical and frequency score are used to ensure the clinical validity of the PCR-based tests.
  • the target organisms, antibiotic resistance genes, and virulence genes can be selected according to one or more of the following criteria: (a) at least one of the clinical score and frequency score is 4 or lower; and/or (b) the combined clinical and frequency score is 9 or lower.
  • Table 9 Exemplary Wound Genetic Test Menu
  • Candida krusei Candida lusitaniae Citrobacter koseri Diptheroids/Corynebacterium spp.
  • Streptococcus pneumoniae Streptococcus sanguinis (Viridans) Table 10: Exemplary DxWound Organism Clinical and Frequency Scores
  • Table 11 Exemplary DxWound Organism Clinical and Frequency Scores
  • a "Look-Up" table and associated algorithm specific for each microbe can be generated to report the presence ("positive”) or absence ("negative") of the microbe, or, in the unlikely event, inconclusiveness (“unable to determine”) of the assay due to various experimental errors or abnormalities.
  • the Look-Up tables can include a combination of all possible results for a single sample or panel of samples.
  • the software compares the amplification results from the specimen sample and various control samples to the algorithm to generate a call on whether the microorganism is present or not. By analyzing the combination of all the amplification results displayed on the Look-Up table for a given sample or panel of samples, the presence or absence of any microorganism from a wound may be detected.
  • a Look-Up table can be developed for each microbial target, considering the positive and negative controls used.
  • a Look-Up table for a particular bacterium can include inputs such as one or more targets for that bacterium (e.g., commercially available kits, bacterium-specific genes or transcripts), one or more pan bacteria control (positive control for all bacteria such as the 16S rRNA region) and one or more pan fungi control (positive control for fungi).
  • molecular analysis of microbial genetic test can be performed by realtime PCR.
  • Real-time Polymerase Chain Reaction PCR is the ability to monitor the progress of the PCR as it occurs (i.e., in real time). Data is therefore collected throughout the PCR process, rather than at the end of the PCR.
  • reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed.
  • chemistries can be used to detect PCR products— TaqMan® chemistry or SYBRTM Green dye chemistry.
  • TaqMan® chemistry one may utilize an
  • oligonucleotide probe labeled with a reporter dye (e.g., fluorescent dye such as FAM or VIC) at the 5' end of the probe and a quencher dye (e.g., nonfluorescent quencher) at the 3' end of the probe.
  • a reporter dye e.g., fluorescent dye such as FAM or VIC
  • a quencher dye e.g., nonfluorescent quencher
  • the proximity of the quencher to the intact probe maintains a low fluorescence for the reporter.
  • the 5' nuclease activity of DNA polymerase cleaves the probe, and separates the dye and quencher, resulting in an increase in fluorescence of the reporter. Accumulation of PCR product is detected directly by monitoring the increase in fluorescence of the reporter dye.
  • the 5' nuclease activity of DNA polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target and is amplified during PCR.
  • the probe is designed to straddle a target sequence position and hybridize to the nucleic acid molecule only if a particular sequence is present.
  • Genotyping is performed using oligonucleotide primers and probes.
  • Oligonucleotides may be synthesized and prepared by any suitable methods (such as chemical synthesis), which are known in the art. Oligonucleotides may also be conveniently available through commercial sources. One of the skilled artisans would optimize and identify primers flanking the region of interest in a PCR reaction. Commercially available primers may be used to amplify a particular gene of interest for a particular sequence. A number of computer programs (e.g., Primer-Express) are readily available to design optimal primer/probe sets. It will be apparent to one of skill in the art that the primers and probes based on the nucleic acid information provided (or publically available with accession numbers) can be prepared accordingly .
  • the labeled probes are used to hybridize within the amplified region during the amplification reaction.
  • the probes are modified so as to prevent them from acting as primers for amplification.
  • the detection probe is labeled with two dyes, one capable of quenching the fluorescence of the other dy e when in proximity.
  • One dy e is attached to the 5' terminus of the probe and the other is attached to an internal site or the 3' terminus, so that quenching occurs when the probe is in a hybridized state.
  • Primers and probes can be designed based on the target sequence. Algorithms for designing primers and probes are widely used and readily available in the art (e.g., Primer-BLAST available at www.ncbi.nlm.nih.gov/tools/primer-blast/). In essence any primer(s) suitable for amplifying a target sequence in a polymerase chain reaction can be used. In some embodiments it may be desirable to keep maximum amplicon size under 400 bp (e.g., 50-150 bp). The probe(s) should be designed to target a region flanked by the forward and reverse primer pair, with suitable length (e.g., 18-22 bases but can be longer or shorter) and proper dye labeling as discussed herein.
  • suitable length e.g., 18-22 bases but can be longer or shorter
  • primer pair sequences may be a "best fit" amongst several aligned sequences, thus they need not be fully complementary to the hybridization region of any one of the sequences in the alignment.
  • a primer may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., for example, a loop structure or a hairpin structure).
  • the primers may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with a target nucleic acid of interest.
  • an extent of variation of 70% to 100%, or any range falling within, of the sequence identity is possible relative to the specific primer sequences disclosed herein.
  • Percent homology, sequence identity or complementarity can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison WI), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appi. Math., 1981 , 2, 482-489).
  • complementarity of primers with respect to the conserved priming regions of target nucleic acid is between about 70% and about 80%.
  • homology, sequence identity or complementarity is between about 80% and about 90%.
  • homology, sequence identity or complementarity is at least 90%, at least 92%, at least 94%, at least.
  • the primers used herein comprise at least. 70%, at least 75%, at least 80%, at least. 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99%, or 100% (or any range falling within) sequence identity with the primer sequences specifically disclosed herein.
  • real-time PGR can then be performed on any suitable real-time PGR machine such as those manufactured by Fluidigni or Applied Biosystems that can record fluorescence level in real-time.
  • Example 1 A Retrospective Database Study on Association of Microorganism Identification Time Using Culture-Based Test with Antimicrobial Treatment, Clinical Outcomes, and Financial Charges for Patients with Skin and Soft Tissue Infections
  • SSTIs are common and their prevalence has increased significantly since the mid-1990s [1].
  • the increasing prevalence of SSTIs demands effective and efficient treatment and management of SSTIs. Effective treatment requires rapid and accurate identification of microbial pathogens and their resistance patterns.
  • microorganism detection Those methods are suboptimal in providing rapid identification and susceptibility testing [2]. They involve the use of selective media to culture targeted aerobic or anaerobic microbial pathogens. The process to facilitate the incubation of microbial pathogens takes a minimum of 24-48 hours to yield qualitative and semi-quantitative assessments of the cultures [3]. Although anaerobic bacteria often constitute a significant proportion of the total microflora in SSTIs, their culture and isolation is prolonged and more resource demanding than investigations of aerobic bacteria, and consequently, anaerobic microbiology is often excluded from a routine analysis [3] . Such delays in providing definitive microorganism diagnosis make it difficult for the treating clinician to narrow antibiotic therapy to correctly treat the infection
  • Rapid and accurate identification of microbial pathogens in patients with SSTIs and infected chronic wounds may provide improved outcomes through more timely and pathogen specific treatment [2].
  • Benefits can include more efficient utilization of antimicrobial medications, more rapid resolution of infection, faster healing, and a reduction in patient mortality, as well as reduced tissue and limb loss [2] [4] [5] [6].
  • This in turn can provide enhanced health economic outcomes, such as reduced expenditure from use of fewer antimicrobials, reduction in the potential for microbial resistance development, subsequent avoidance of hospitalizations, shorter length of stay for hospitalized patients, reduction in total cost of care, fewer surgeries, and fewer hospital readmissions [7] [8] [9].
  • [0074] Here we propose a database outcomes research study to explore relationships between the timing of reporting of culture results, antimicrobial treatment, microbial identification, and clinical outcomes. We expect that findings from this study can provide data inputs for future studies.
  • results from this study may be used to develop a rapid diagnostic test for SSTI/wound care, including assays, reporting algorithms and test report format.
  • a retrospective study can be conducted using patient electronic medical records (EMR) and associated hospital charge master data (CMD) to achieve the objectives of the study.
  • EMR electronic medical records
  • CMD hospital charge master data
  • EMR has been used since the early 80' s. Its legacy system is TDS, and is still in use to retrieve data entered prior to 2009. A current EMR system is called Quest (Quality, Excellence & Safety through Technology), Allscripts' Sunrise Clinical Manager.
  • the Subject ID (SID) in the data dictionary is a recorded ID (recoded by bioinformatics personnel). It is impossible to link the extracted data back to the subject in EMR and CMD by using the SID.
  • T IDX the Index Time
  • PKI protected health information
  • SAS® 9.4 or later, or other suitable software, can be used to generate all analyses, tables, listings, and figures.
  • the number of observations, mean, standard deviation, median, minimum, and maximum values can be provided for all continuous measurements. For categorical data, the number and percentage in each category can be reported. Missing values for
  • categorical/ordinal data can be counted in a separate category.
  • the frequency and percentage of patients with specific culture-based test ordered can be reported by each individual microorganism, polymicrobial, or class (anaerobes, aerobes, gram positive, and gram negative).
  • the frequency and percentage of patients with a specific culture-based test result can be summarized as each individual microorganism, polymicrobial infection, or class (anaerobes, aerobes, gram positive, and gram negative).
  • T s be the specimen collection time
  • T R S T be the culture-based test result time of completion
  • DAY samp i e be the Days of specimen collection from the index time (see Section 4.3)
  • DAY RST be the days of culture- based results from the index time
  • CUL time be the days of culture-based results from specimen collection time.
  • DAYsam p ie , DAY R S T, and CUL time can be summarized by individual microorganisms or polymicrobial infection.
  • Antimicrobial treatment can be analyzed at the generic name level.
  • polymicrobial infection or class (anaerobes, aerobes, gram positive, gram negative).
  • the dosage and route of administration can be noted.
  • the first antimicrobial treatment prior to or after hospital admission can be denoted as Drugi (The start time T 1 of Drugi can be before or after the hospital admission).
  • the corresponding start time for those treatment can be denoted as STRT TMl, STRT TM2, and STRT TM3 etc.
  • Kaplan-Meier survival curves can be used to display the Time to the 1st antimicrobial switch (STRT TM2) overall and by each individual microorganism or polymicrobial infection. Survival probability estimates can be calculated at continuous time points up to the maximum discharge time to be observed in the data overall and by each individual microorganism or polymicrobial infection.
  • the number and proportion of patients in each level of the category variables can be can be summarized by bins of CUL time and each individual microorganism or polymicrobial infection detected.
  • Scatter plots can be generated to display the relationship between culture test result time (original or bin) and the first antimicrobial treatment time (STRT TMl, original or binning data) by each individual microorganism or polymicrobial infection, and by antimicrobial generic name and each individual microorganism or polymicrobial infection.
  • STT TMl first antimicrobial treatment time
  • antimicrobial treatment appropriateness can be defined.
  • the study endpoints for outcomes and hospital charges can be summarized and compared between groups of different antimicrobial treatment appropriateness using generalized linear model for continuous outpoints and logistic regression model for category outpoints with antimicrobial treatment appropriateness, CUL time, and other factors or covariates such as wound type, comorbidity, age, etc. as predictors.
  • the data provided to MH can include only a limited, de-identified data set.
  • Co-morbidities can be limited by specific disease effects on tissue integrity and perfusion, mobility, compliance, nutrition and risk for infection.
  • Example 3 A Single-Center Observational Study to Assess the Use of PCR in Pathogen Identification Compared to Culture-Based Test Methods and the Potential Impact on Antimicrobial Treatment, Clinical Outcomes, and Hospital Charges in Patients with Skin and Soft Tissue Infections
  • the purpose of the study is to assess the use of PCR in pathogen identification compared to culture based test methods and the potential impact of using PCR on antimicrobial use, clinical outcomes, and financial charges in patients with skin and soft tissue infections (SSTIs).
  • SSTIs skin and soft tissue infections
  • SSTIs are a common problem for patients in the hospital setting and their prevalence has increased significantly since the mid-1990s.
  • the increasing prevalence of SSTIs demands improving the efficacy and efficiency in the treatment and management of those infections.
  • Effective treatment requires rapid and accurate identification of microbial pathogens and their resistance patterns.
  • the traditional standard of testing e.g. culture based methods for pathogenic
  • microorganisms in SSTIs is suboptimal in providing rapid identification and susceptibility testing [1].
  • Traditional methods involve the use of selective media to culture aerobic and anaerobic bacteria and fungi.
  • the process to facilitate the incubation of microbial pathogens takes a minimum of 24-48 to yield qualitative and semi-quantitative assessments of the cultures [2].
  • anaerobic bacteria often constitute a significant proportion of the total microflora in wounds, their culture and isolation is prolonged and more resource demanding than cultures of aerobic bacteria, and consequently, anaerobic microbiology is often excluded from a routine analysis [2].
  • Such delays in providing definitive microbial diagnosis make it difficult for the treating clinician to narrow antibiotic therapy to correctly treat the infection and reduce antibiotic resistance.
  • Rapid and accurate identification of microbial pathogens in patients with SSTIs and infected chronic wounds can potentially provide improved outcomes through more timely and pathogen specific treatment.
  • Benefits can include more efficient utilization of antimicrobial medications, more rapid resolution of infection, faster healing, and a reduction in patient mortality, as well as reduced tissue and limb loss. This in turn can provide enhanced health economic outcomes, such as reduced expenditure from use of fewer antimicrobials, reduction in the potential for microbial resistance development, subsequent avoidance of hospitalizations, shorter length of stay for hospitalized patients, reduction in total cost of care, fewer surgeries, and fewer hospital readmissions.
  • PCR based methods may provide similar benefit and could be a potential game changer in the pathogen identification in patients with SSTIs and lead to improving the efficacy and efficiency in the treatment and management of those infections.
  • This study aims to quantify the unmet need in the detection of the pathogens that cause skin and soft tissue infections and to create an economic simulation based on the timing of initiation of antimicrobial therapy and availability of culture results.
  • the study can also assess the use of molecular methods in pathogen identification compared to culture-based test method and the potential impact of using molecular methods on antimicrobial treatment, clinical outcomes, and financial charges in patients with SSTIs. 3 Objectives of the Study
  • Patients who meet the inclusion criteria at screening can be approached for the possibility of enrolling in the study.
  • Patients who sign the informed consent form (ICF) can be enrolled for information and specimen collection.
  • Two specimens can be collected from each patient per specimen collection event.
  • Specimen B can be shipped to Millennium Health for testing using the PCR based assay and Specimen A can be tested using culture-based method at study site's clinical pathology laboratory as part of standard of care. If a subject has more than one culture test ordered during their initial hospitalization or is readmitted to study site with an SSTI, then more than one specimen collection event can occur per subject.
  • the anticipated duration of the study is 12 months with an expected enrollment period of 6 months.
  • the enrollment period can be 6 months in order to capture a sufficient number of specimen types.
  • Patient has an order for a skin or soft tissue infection culture
  • the subject may withdraw their participation at any time after enrollment, upon which their SSTI specimen can be destroyed and any collected electronic information can be removed from the study database. However, if the subject withdraws after statistical analysis for the study has started, the patient's SSTI specimen can be destroyed, but the sponsor (Millennium Health) can not have any obligation to destroy summary statistics that include data obtained from the patient's sample and study data. 5 Study Procedures:
  • Subjects can be screened on the basis of the criteria in section 4.4.
  • the study nurse can confirm via the enrollment log that the subject has not been previously enrolled in the study. If the subject had been previously enrolled in the study, the nurse can proceed directly to specimen collection since the patient had already consented.
  • the study nurse can use the same Subject ID that the subject had been assigned when they were originally enrolled in the study and can indicate which Specimen Collection Event occurred (i.e. Specimen Collection Event 1 is the first time specimens are collected from a subject, Specimen Collection Event 2 is the second time a specimen is collected from the same site).
  • a subject has not been previously enrolled and are deemed to be eligible for participation, they can be educated on the study procedures, risks, and the molecular test used to analyze the specimen, and the voluntary nature of study participation. If the patient is interested in participating in the study, they can be asked to sign an ICF. This informed consent can be discussed with the study nurse who can be available for questions.
  • Case Report Form(s) can be collected for each subject enrolled in the study. The following information can be collected:
  • the study can be conducted on swab specimens tested using MH's standard clinical laboratory procedures.
  • a study nurse can approach the patient and screen for eligibility. The study nurse can consent the prospective subject, if eligible, and can collect the specimen(s). An additional swab can be collected for analysis at Millennium Health in addition to the swab that was collected for analysis using culture methods. Both specimens can be collected from the same location of the SSTI, at the same time. If a subject has more than one SSTI on their body eligible for inclusion, the study nurse can select only one SSTI to collect the two specimens from and all other SSTI locations can be excluded from the study. The nurse may collect any additional specimens from the other SSTI locations per standard of care.
  • specimen A can be sent to the study site's clinical pathology laboratory for testing using standard culture procedures and analysis.
  • specimen B can be sent to Millennium Health using a provided collection device along with a study specific requisition form. The requisition form can be filled out according to the instructions provided to the study team and can be sent with Specimen B to Millennium Health.
  • the information listed in section 5.2 can also be collected during the consent process and recorded on a single Case Report Form (CR ). If a patient does not consent to participate in the study, the study nurse can only collect the specimen to be cultured at study site's clinical pathology laboratory and no further information or interaction with the patient regarding the study can occur.
  • CR Case Report Form
  • Specimen B can be shipped to Millennium Health for analysis and specimen A can be analyzed and cultured at study site's clinical pathology laboratory using standard methods according the physician's order. All patient identifiable information can be removed from specimen B before it is sent to Millennium Health.
  • Specimens and their corresponding requisition forms that can be sent to Millennium Health can be packaged and shipped in accordance with the designated commercial carrier's requirements for clinical samples. Each specimen B can be shipped on a daily basis to Millennium Health, unless otherwise indicated by the study sponsor. Designated study personnel can ship specimens to
  • the study nurse can access the subject's EMR to fill out the final portion of the CRF.
  • the study nurse or designated study team member can send copies of completed CRFs to Millennium Health on a weekly basis.
  • Each specimen shipped to Millennium Health must have an accompanying completed CRF (shipped separately), including specimens from additional specimen collection events.
  • CRFs must be returned for samples that were unable to be tested in the hospital due to any error in handling, failure to generate a result, or other unexpected event.
  • Each specimen's extracted microbial DNA once examined with WGT, may be used specifically and strictly for research purposes as part of the Millennium Health WGT Biobank & Database for a period of 5 years.
  • the DNA samples can be stored at Millennium Health in a freezer with restricted, employee only access.
  • Master data can be stored in a secure database for up to 6 years to be maintained at Millennium Health.
  • Each patient's WGT results can be stored in Millennium Health's proprietary laboratory information system (MLIS) indefinitely per company policy and may be used for future research purposes at Millennium Health. Confidentiality can be strictly maintained.
  • MLIS laboratory information system
  • a subset of the EMR data and associated Hospital Charge Master Data for the enrolled patients can be extracted as specified in the Data Dictionary in Appendix A and can include data 30 days post discharge for each subject.
  • a de-identified data set can be included in the subset of data and each subject can only be identified by the SID assigned upon enrollment, which can link the associated EMR, Charge Master Data, culture results, and WGT results to that subject.
  • the de-identified data set used in the study can be pulled by study site Clinical Informatics department using the Honest Broker process.
  • study site can observe and record information on patients who provide informed consent. Data can be recorded and entered, compiled and analyzed to determine the key outcomes previously defined. Confidentiality can be strictly maintained.
  • the study personnel can also be trained to avoid recording any personal identifiable information on the case report forms by inadvertence. Any clinical and charge data can be sent to Millennium Health for analysis every month until the completion of the study.
  • the patient's WGT results and culture results can be merged with the patient's EMR and Charge Master data using the assigned SID and SID B.
  • Only the on-site study team can have access to patients' coded private information and, therefore, can be responsible for maintaining the privacy of patients and protecting the confidentiality of patients' identifiable information.
  • the study team at Millennium Health can have no access to any patient PHI. 5.5 Subject and Specimen Labeling
  • specimens can be collected from each enrolled subject during each specimen collection event and subsequent specimen collection events (i.e. in the case of a subject having more than one culture ordered for their SSTI during their hospitalization period or if a subject was readmitted to the study site during the study enrollment period).
  • a designated study team member can keep and record specimen collection entry into the log file.
  • the designated person can assign the next SID to specimen B and can fill the medical record number with the subject's medical record number in the EMR, print, and sign his/her name.
  • the study team member can attach the pre-printed label with the SID on Specimen B.
  • the SID is the sequential number from 00001 to 99999. If a subject has an additional specimen collected at a 2 nd , 3 rd , etc. specimen collection event, the team member collecting the specimens can add a dash, then the number of the Specimen Collection Event after the dash.
  • specimen collection event number is >1
  • the study nurse can refer to the prior CRF recorded during specimen collection event number 1 to ensure that the additional specimens are taken from the same location as the original collection.
  • the study team member can apply pre-printed labels with the Subject ID to specimen B. If the specimen collection event is a second, third, etc. event, the study team member can write in the specimen collection event number following the SID (e.g. B0002-2) on the label that is applied to the specimen.
  • SID e.g. B0002-2
  • the study team can send the Specimen Collection Log file to the Clinical Informatics team once per week.
  • study site transfers the EMR and Charge Master data to Millennium Health
  • the medical record number in the EMR and Charge Master data can be replaced with the SID.
  • the specimen collection log file with the medical record number removed can be provided to Millennium Health together with the de-identified, de-identified EMR data set and charge master data (See Appendix A for the data to be extracted from the EMR and Charge Master files) at least 30 days post discharge.
  • Study site can transfer the EMR and Charge Master data to Millennium Health every month until the completion of the study and at least 30 days post discharge of the last patient enrolled.
  • a collection device comprised of:
  • Transport media formulated to maintain the integrity of DNA present in the clinical specimen.
  • a DNA preparation process comprised of:
  • An aliquot of the DNA extracted from the microorganisms in the specimen may be tested using sequence analysis.
  • the specimen can be prepared for sequencing using published standard operating procedure and analyzed with specialized software for microorganism specification.
  • One or more qPCR protocols comprised of:
  • a pre-determined list of target DNA sequences which may include organism identification sequences, antibiotic resistance sequences, virulence factor sequences.
  • a data interpretation pipeline comprised of:
  • SAS® 9.4 or later can be used to generate all analyses, tables, listings, and figures. If not otherwise specified, the number of observations, mean, standard deviation, median, minimum, and maximum values can be provided for all continuous measurements. For categorical data, the number and percentage in each category can be reported. Missing values for categorical/ordinal data can be counted in a separate category.
  • the first pair of specimen collection time (hour 0) is the baseline for this study.
  • the baseline time is denoted as T BL.
  • PCR based test results on specimen B and culture-based test results on specimen A at the baseline can be used for the comparison of the test characteristic.
  • Antimicrobial treatment can be analyzed at the generic name level.
  • polymicrobial infection or class (anaerobes, aerobes, gram positive, gram negative).
  • the dosage and route of administration can be noted.
  • the first antimicrobial treatment prior to or after hospital admission can be denoted as Drugi (The start time T 1 of Drugi can be before or after the hospital admission).
  • Time from specimen collection to the Drugi use (STRT TMl) can be summarized overall and by antimicrobial generic name.
  • Drug 2 The subsequent administration of any antimicrobial different from Drugi (see Section 6.4.2) can be denoted as Drug 2; the subsequent administration of any antimicrobial different from Drug 2 can be denoted as Drug 3 etc.
  • Drug 3 the subsequent administration of any antimicrobial different from Drug 2
  • Any change from Drug x to Drug x+ i is an antimicrobial switch.
  • Kaplan-Meier survival curves can be used to display the Time to the 1st antimicrobial switch (STRT TM2) overall and by each individual microorganism or polymicrobial infection. Survival probability estimates can be calculated at continuous time points up to the maximum discharge time to be observed in the data overall and by each individual microorganism or polymicrobial infection.
  • scatter plots can be generated to display the relationship between CUL time (original or bin) and each of the endpoints (original or binning data) by each individual microorganism or polymicrobial detected.
  • the number and proportion of patients in each level of the category variables can be can be summarized by bins of CUL time and each individual microorganism or polymicrobial infection detected.
  • Scatter plots can be generated to display the relationship between culture test result time (original or bin) and the first antimicrobial treatment time (STRT TMl, original or binning data) by each individual microorganism or polymicrobial infection, and by antimicrobial generic name and each individual microorganism or polymicrobial infection.
  • STT TMl first antimicrobial treatment time
  • antimicrobial treatment appropriateness By examining the microorganisms/susceptibility detected by both the WGT test and culture test and the antimicrobial treatments that the patients received, antimicrobial treatment appropriateness can be defined.
  • the study endpoints for outcomes and hospital charges can be summarized and compared between groups of different antimicrobial treatment appropriateness using generalized linear model for continuous outpoints and logistic regression model for category outpoints with antimicrobial treatment appropriateness, CUL time, and other factors or covariates such as wound type,
  • Descriptive statistics for outcomes and hospital charge can be calculated by virulence factor group (with/without virulence factors) detected by WGT.
  • Box plots or bar chart can also be created for outcomes and hospital charge.
  • Millennium Health recognizes the growing problems of antibiotic overuse, antibiotic resistance, and pathogen identification.
  • the Millennium Health DxWound product analyzes DNA isolated from microbes present in various types of wounds using real-time polymerase chain reaction (PCR) technology.
  • the isolated DNA is used to identify pathogens (i.e. bacterial and fungal organisms) present as mono- or polycultures within chronic wounds, as well as antimicrobial resistance signatures and virulence factors that may be carried by the microbes.
  • pathogens i.e. bacterial and fungal organisms
  • This product may complement and in some clinical cases, replace, current techniques involving culturing of infected specimens followed by susceptibility testing.
  • the product can be offered as a single test that can be used to detect the presence of >20 bacterial and fungal pathogens in a single specimen.
  • Rapid identification of microbes, along with antibiotic resistance signatures and virulence factors can allow for faster, more precise selection of antibiotic therapies that are most likely to effectively treat the infections and help to reduce antibiotic waste, delays in treatment of needed antibiotics, and the use of expensive antibiotics. This has significant potential to improve patient quality and healthcare outcomes.
  • MH's Health Outcomes and Economic Study aims to quantify the unmet needs in the current standard of care for the identification of pathogens that cause wound and surgical site infections.
  • the multi-phase study can measure the value of the test using a number of economic and clinical outcomes, including the potential for reduction in cost of hospital related care and complications, antibiotic spend, incidence rates of antibiotic waste, and improvement in quality of care.
  • microorganisms could solve unmet needs in the identification and treatment of wound and surgical site infections in the hospital environment.
  • MH can compare its molecular diagnostic test to the current gold standard of care to assess the value based on the clinical and economic outcomes listed above.
  • the first phase is a pilot study designed to evaluate the assay as a method for identifying a pre-selected list of potential pathogens, virulence factors, and antimicrobial resistance genes in specimens obtained from wounds.
  • the study can measure the accuracy rate of pathogen identification for the WGT vs the culture method as well as to define the logistics for preserving, transporting, and analyzing the specimens for future phases of the study.
  • This phase is exploratory in nature and the results can be used to design and power the BEVI and retrospective portions of the study.
  • Phase two involves a BIM and Supporting Retrospective Study that can both focus on subsets of patients where MH can demonstrate the greatest unmet need with the highest potential to benefit from the new technology.
  • the BEVI can be designed to allow hospital administrators and decision makers to interactively explore their costs of care for patients with wound infections and demonstrate potential savings if they were to use MH's PCR based test as a supplement to the standard of care.
  • the retrospective study can serve as means to provide clinical and economic outcomes data for the BIM.
  • Phase three can include an evidence-based, prospective study that can incorporate the genetic test into the standard provision of care.
  • the prospective study can randomize patients with wound infections into either a "research arm” or a "control arm”: the control arm can identify the wound infection using current standard of care techniques and the research arm can identify the pathogen using the PCR based test in addition to the standard of care.
  • the study can evaluate the clinical decisions made by clinicians in each arm and additional clinical and economic outcomes can be measured, including hospital related care and complications, antibiotic spend, incidence rates of antibiotic waste, and quality of care measures.
  • MH has developed a molecular genetic test for organism identification, antibiotic resistance, and virulence factor identificationthat is targeted toward wound and surgical site infections. This development requires testing on clinical samples in order to refine, optimize and validate the test. Therefore, MH is in need of patient samples to compare the results of the PCR based test to the current gold-standard method of testing.
  • Specimens used in the pilot study can be obtained from patients admitted who have any wound and/or surgical site infections.
  • the specimens can be collected using the current standard of care, however MH can want to know what technique was used to collect the specimen and what type of wound it was taken from. MH can obtain an additional specimen collected from patients during the course of normal care for testing at MH on the PCR assay. MH can also obtain the culture results and compare these to the results from the genetic test. MH would like to obtain 200 samples with no patient identifiable information for this purpose.
  • BIM Budget Impact Model
  • the BIM can include subsets of patients who would have the potential to benefit from the new technology. For example, such patients could include those who have compromised immune systems, complex surgeries, or resistant diabetic infections (2).
  • the list of patient groups can be identified through a literature review of studies that evaluate patients who are at high risk of developing a wound or surgical site infection and which pathogens would most likely cause those infections.
  • the BIM can incorporate incidence rates obtained from a company that collects prescription and healthcare data, such as IMS, into the model to demonstrate value for outcomes such as reduced overall hospital related costs, readmission rates, and spending on expensive antibiotics.
  • the Retrospective study can be determined by the results from Phase 1.
  • the Retrospective Study can serve as a means to provide clinical and outcomes data for use in the BIM.
  • the Retrospective Study phase can also select subsets of patients where the need to have an accurate and rapid wound microbial identification test is the greatest and for whom the test can demonstrate the maximum economic value. Similar to the Pilot Phase, MH can obtain the unused remnants of wound and surgical site infection specimens from patients admitted to the study site. The specimens can be collected using the current standard of care and can be split into three parts, A, B, and C; Part A can be analyzed using the culture method, Part B can be analyzed using MH's wound infection genetic test, and Part C can be stored for future testing if there is a variation between the results from Parts A and B. MH can be evaluating the accuracy and turn-around-time of each method. The study can also use electronic medical records (EMR) to retrospectively measure incidence rates of avoidable waste of antibiotic use, cost of hospital related care, complications, treatment patters, and quality care metrics for each patient.
  • EMR electronic medical records
  • Immunosuppressed patients e.g. cancer, HIV, hepatitis, etc.
  • the third phase of the study can be comprised of an evidence-based, prospective study that can be designed based on the results of the Retrospective Phase and Budget Impact Model.
  • This phase can incorporate genomic testing into the standard provision of care as a supplement to the culture method. It can provide a high level of evidence to demonstrate that MH's wound and surgical site infection can provide a more rapid, precise method of pathogen identification and can help physicians to select the most effective antibiotic therapies.
  • the prospective study can likely comprise of the same subsets of patients that can be enrolled in the Retrospective Study and can include informed consent.
  • the current design methodology would randomize patients with wound infections into either a "research arm” or a "control arm”: the control arm can identify the wound infection using current standard of care techniques and the research arm can use PCR based testing in addition to the standard of care.
  • the final study design and methodology can warrant further exploration and discussion following the completion of the BIM and Retrospective Studies.
  • the study can measure any differences in clinical decisions making between both arms.
  • Several clinical outcomes that might be analyzed between the two groups could include how often the clinicians initially chose the most effective antibiotic, was there a reduction in the use of broad- spectrum antibiotics, and did the PCR based test help to avoid unneeded antibiotic therapy.
  • Additional primary economic and clinical outcomes that can be measured can be determined by the results from the BIM and Retrospective studies, but can likely include hospital related care and complications, antibiotic spend, incidence rates of antibiotic waste, and quality of care measures.
  • a specimen collection kit can be provided to participating sites for wound sample collection.
  • the specimen collection kit can comprise of a microbiology specimen bag containing:
  • Wound or other specimens can be collected using the Disposable Dermal Curette.
  • the specimen can be placed in the Lysis Tube and the lid secured in place.
  • the collected specimen can be placed in the microbiology specimen bag.
  • the appropriate fields can be completed in the accessioning form.
  • the form can be placed in the document specific pocket of the specimen bag.
  • the specimens can be shipped to MH, according to standard specimen handling protocols.
  • the specimen can be accessioned at MH and stored at 4°C until sample processing.
  • Pathogen DNA can be extracted from the specimen
  • Fluidigm Biomark HD System Fluidigm, San Francisco, CA
  • 96.96 Dynamic Array Gene Expression Chip
  • Beta-Mercaptoethanol (Sigma- Aldrich, St. Louis MO) to 19,900 DNA Binding Buffer.
  • Step (13) Transfer the eluted DNA from Step (13) to a prepared Spin Filter in a clean 1.5 mL microcentrifuge tube. Loosely cap the Spin Filter and centrifuge at 8,000 x g for 1 minute. This is your final microbial DNA eluted product.
  • IFC Integrated Fluidic Circuit
  • CTX-M-1 Antibiotic resistance gene
  • VRE vanA (VRE) Antibiotic resistance gene
  • FIG. 1 A An overview of the general steps involved in the methods disclosed herein related to the identification of the microorganism community is provided in FIG. 1 A. These general overview steps may be referred to as the "Wound Genetic Test” (WGT) herein.
  • WT Wild Genetic Test
  • the first step is specimen collection.
  • Samples of specimens may be collected from any source of the body (e.g., blood, skin, urine etc.).
  • the location of skin samples may come from the ankle, forehead, hand, head, lower back, retroauricular crease, toe web, intranasal, knee crook, groin, neck, elbow crook, or perianal location.
  • Wound specimens may be collected using a variety of devices, such as a sterile swab or syringe (i.e., swab collection kit).
  • the next step is DNA extraction (FIG. 1 A).
  • DNA can be extracted from the pathogenic microorganisms in a specimen using commercially available DNA Miniprep Kits. Real-Time PCR assays may be performed using commercially available kits. Data can then be generated and analyzed using Fluidigm Biomark HD System on the 96.96 Dynamic Array Gene Expression Chip.
  • Millennium Health Microbial Community Standard is a control developed at Millennium Health which contains representative microorganisms. Gram positive and gram negative bacterial strains are represented, as well as both aerobes and anaerobes and fungal species (i.e.
  • the strains present in this control represent the most common microorganisms found in skin and soft tissue infections (SSTIs).
  • SSTIs skin and soft tissue infections
  • the control is used to ensure that the DNA from all different types of SSTI relevant microorganisms is effectively extracted during sample preparation.
  • the control is used with every batch of specimens collected (samples) to establish that the extracted DNA from the microorganism represents the microorganisms present in the isolated wound sample.
  • Isolation Negative Control can be DNase/RNase Free Water.
  • NIC Negative Isolation Control
  • Isolation Negative Control Isolation Negative Control
  • IPC Isolation Positive Control
  • Additional controls include the following: (a) No Template Control using Microbial DNA- Free Water (Qiagen, Venlo, The Netherlands); this control can verify that the assay reagents are not contaminated; (b) Microbial Plasmid Pool ("Millennium Health Plasmid Pool (Millennium Health, San Diego CA)), which is used to verify that the assays are performing as expected; (c) Positive PCR Control, such as PPC (Qiagen, Venlo, The Netherlands), which is used to verify that the samples do not contain anything that might inhibit PCR amplification; and (d) A human gDNA assay can also be included as a control to determine whether there are detectable levels of DNA present in a specimen.
  • DNA can be extracted using procedures known in the art.
  • One of the central premises of the DxWound product is the ability to provide a real-time snapshot of the microbial content of a wound without the need for artificially inflating the microbes by, for example, growing them up on a petri dish.
  • care should be taken to reduce any bias introduced by the different growth rates and growth requirements of different microorganisms, and minimize any bias introduced by microorganisms competing for resources after sample collection, which can artificially inflate the presence of some microorganisms whilst diminishing others.
  • the extracted DNA can be used directly in RT-PCR without the need for an intermediate amplification step. This enables the whole process to be completed in less than 8 hours, which is a key benefit as it enables the physician to customize their treatment decisions to the microbial flora in the wound earlier.
  • the samples can then be prepared according to standard protocol for running on the 96.96 Dynamic ArrayTM Gene Expression Chip IFC. Assays curated for inclusion on the WGT panel (e.g., one or more of Tables 1-11) can be added to the chip. Generally, at least three assays contribute to any final result: (a) the microorganism (bacterial, fungal assay), and (b) at least two control assays. Data can then be generated using Fluidigm Biomark HD System on the 96.96 Dynamic Array Gene Expression Chip. Approximately 96 input samples are analyzed per chip on the Fluidigm Biomark HD System, which includes approximately 80 specimen samples and 16 control samples. For each specimen sample on the chip, a plurality of primers can be added to each sample that have specific sequences directed to different microorganisms. One or more Pan Bacterial Controls and one or more Pan Fungal Controls can also be included.
  • Assays curated for inclusion on the WGT panel e.g., one or more of Tables 1-11
  • the resulting data is then initially analyzed using the Fluidigm Real-Time PCR Analysis Software, and then using "Look-Up" tables specific for each microbe, by evaluating amplification curves against control samples within a defined reportable range. If there is an amplification signal within a defined reportable range, then it means that the primers were able to detect the presence of a given microorganism for that particular microorganism species. If there is no amplification signal within a defined reportable range, then it means that no microorganism was able to be detected for that particular species.
  • the amplification results are then analyzed on Look-Up tables that comprise algorithms that consist of a combination of all possible results for a single sample or panel of samples.
  • the software compares the amplification results from the specimen sample to the algorithm to generate a call on whether the microorganism is present or not.
  • the presence or absence of any microorganism from a wound may be detected.
  • a Look-Up table can be developed for each microbial target, considering the positive and negative controls used.
  • An exemplary Look-Up table for 5 , aeruginosa is shown in Table 14 below.
  • "Code” represents the output of the software, along with the phenotype and genotype. Inputs include
  • pan bacteria positive control for bacteria
  • pan Candida positive control for fungi
  • wound samples were obtained from either patients located at the Palomar Health or Pomerado Health outpatient wound clinics. In general, patients had to first meet the inclusion criteria. Next, specimens from wounds of a variety of locations were collected. Such locations included the ankle, forehead, hand, head, lower back, retroauricular crease, toe web, intranasal, knee crook, groin, neck, elbow crook, or perianal location. Specimens were then shipped to Millennium Health and stored at either room temperature or -20°C. Subsequently, the DNA was extracted and stored for future use. The DNA was also quantified and compared based on different collection devices (see below) to determine the optimal device for collecting samples.
  • Participating physicians enrolled subjects who met the inclusion criteria and consented to the wound study. Next, they proceed to collect the following samples from the subject: (a) a swab before debridement (SB); next, the physician would debride the wound and (b) collect tissue for standard care; (c) then, the physician would collect a swab after debridement (SA); and lastly, (d) the physician would collect a tissue sample (TC).
  • a total of four samples were collected for each enrolled subject. Three out of the four samples were sent to Millennium Health (MH) for testing and analysis. One sample was sent to a hospital lab for routine, traditional culture. The separation of samples in this format allowed a comparison between the traditional method of analyzing microorganisms in a wound sample, and the molecular methods disclosed herein related to the identification of microorganisms in a wound directly from a biological sample.
  • Phase I The purpose of the first phase was to use two different devices for sample collection and compare the ability to extract DNA in high quantity and quality, test the extracted DNA samples (preliminary WGT samples), and evaluate whether one device was superior to the other in yielding high amounts of microorganism DNA from wound samples.
  • 75 samples were collected in Device 1.
  • Device 1 has traditionally been utilized as a collection device for wound samples because it is known to preserve bacterial samples for future growth in the lab and maintain relative sample stability when shipped or transported to a different location for analysis.
  • Device 1 has components that preserve the sample for shipping or future growth in a lab. Specifically, the liquid Amies medium bacteriology swab collection system was used as Device 1. Also, 75 samples were collected in Device 2.
  • Device 2 has traditionally been utilized to inactivate infectious agents and preserves the genetic integrity and expression profiles of samples.
  • Device 2 has components that inactivate the organisms and/or nucleases (e.g., using chelating agents such as EDTA) present in the sample, but preserves the DNA and RNA for future analysis.
  • chelating agents such as EDTA
  • NGS is used to sequence and determine the microbial composition in samples collected and stored in Device 1 and Device 2 overtime (at day 0, day 3 and day 10, FIG. 2). As shown in FIG. 2, the samples collected using Device 2 preserve the sample consistently over time. Device 2 effectively inactivates the microbes at the time of specimen collection to ensure an unbiased representation of the microbial content of the wound.
  • the purpose of the second phase of the wound study of this example was to further optimize the extraction method and PCR testing panel, determine specimen/kit characteristics (stability, storage, interfering substances etc.), and define performance characteristics (accuracy, sensitivity, specificity, robustness etc.).
  • specimen/kit characteristics quality, storage, interfering substances etc.
  • performance characteristics accuracy, sensitivity, specificity, robustness etc.
  • SB, SA, and TC sample types were collected per subject as described above (SB, SA, and TC), and the sample collection continued in the device of choice from Phase I.
  • the overall goal of this phase was to finalize the WGT test and methods and perform quality/control analysis.
  • NGS next generation sequencing
  • Tables 17-18 show the identification of targets that are present on the WGT Panel using the culture method.
  • Table 18 shows the identification of targets that are not on WGT Panel (because these targets are not currently recognized as wound pathogens) and other phenotypical results using the culture method.
  • the WGT method was analyzed for several parameters, including: component accuracy and precision using known isolates (Table 19), WGT findings by organism type (Table 20), WGT findings per subject (FIG. 4), the number of antibiotic resistance genes (Abrx) found per subject (FIG. 5), swab versus tissue WGT findings concordance (Table 26), WGT concordance to clinical findings (Tables 21-24), as well as key unexpected findings and messages (Table 30 and Table 31).
  • the WGT method was successfully used to detect microbes in clinical specimens.
  • 106 subjects were analyzed with matched culture and WGT samples, and of these, 60% of subjects had polymicrobial wounds.
  • Table 20 below provides a summary of the number of microbes per organism type as analyzed by the WGT method. Each listed microbe in the middle column is a clinically relevant organism. G+: Gram positive. G-: Gram negative. Table 20. WGT Results
  • results from the WGT microbial identification assays were considered. These include 34 microbial targets, 106 subjects (swabs and tissues), and 3,604 data points analyzed. Table 22 shows the rate of concordance between the culture approach and the WGT method (mm: mismatch). 55 findings were present in culture only as they are not included on the WGT panel for lack of clinical relevance. For targets that are included on the WGT panel, the WGT method was able to detect an additional 65 incidents of microbes that were missed by the culture method.
  • WGT Vancomycin-resi stance genes along with Enterococcus faecalis, indicating a possible finding of Vancomycin-resistant Enterococcus (VRE).
  • VRE Vancomycin-resistant Enterococcus
  • the results obtained from the WGT method have a significantly improved performance, intersection, concordance, speciation, and is able to detect missing and additional findings.
  • the WGT method has sensitive, accurate and precise detection on known microbial isolates.
  • the WGT panel is shown to cover all or substantially all organisms identified by culture in the pilot study, with 96.4% or greater concordance to culture results.
  • the WGT method also speciated 61% of genus-level culture findings, identified microbes in 62% of patients with "mixed flora" culture findings, including 3 with possible VRE, identified additional microbes missed by culture in 43% of patients with culture-positive results, and detected culture errors in 5.4% of patients.

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Abstract

Disclosed herein are methods and systems for detecting the presence or absence of a plurality of microorganisms in a biological sample. The method can include: (a) obtaining a biological sample from a subject in need thereof, wherein the biological sample contains one or more microorganisms; (b) providing a plurality of primer pairs, wherein each primer pair is specifically designed to amplify a target sequence of a microorganism from a plurality of microorganisms; and (c) performing real-time PCR or digital PCR using the plurality of primers, thereby detecting the presence or absence of the plurality of microorganisms.

Description

METHODS AND SYSTEMS FOR MICROBIAL GENETIC TEST
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S. Provisional Patent
Application Nos. 62/461,688 filed February 21, 2017; 62/465, 111 filed February 28, 2017; 62/551,515 filed August 29, 2017 and 62/565,335 filed September 29, 2017, the disclosures of which applications are incorporated herein by reference in their entirety.
FIELD
[0002] Disclosed herein are methods and systems for microbial genetic test, in particular the presence or absence of microbes in biological samples obtained from wounds (e.g., swabs) and other tissues.
BACKGROUND
[0003] Many types of wounds represent a major healthcare challenge. According to a 2009 National Institutes of Health study, approximately 6.5 million patients in the United States suffered from chronic wounds with treatment costs exceeding $25 billion annually. Poor vascular supply and infection are some of the causes of a chronic wound. For example, in the United States, the diabetic foot ulcer is a major contributor to leg amputations because the body loses the ability to deliver sufficient oxygen to the wound, which is necessary for healing. Also, wounds may be difficult to heal because of the presence of many microbes within the wound site. Bacterial populations cooperate to promote their own survival, leading to the chronic nature of the infection. Some chronic wounds are also associated as a primary contributing factor in hundreds of thousands of annual deaths and billions of dollars in direct medical costs annually.
[0004] Conventional laboratory methods for detecting microorganisms in wounds have many significant limitations. One reason for this derives from the nature of classical culture techniques. Classical laboratory culture techniques are typically only able to detect (as isolates) those few organisms that grow relatively quickly and easily in laboratory media. However, chronic wounds are not typically infections comprising one or two microorganisms. Rather, they are generally polymicrobial infections having many different bacterial species and fungi, sometimes dozens. Culture methods often misrepresent the contribution of individual bacterial and/or fungal species present in a wound's bioburden because of the bias created by the culture process. That is, many bacteria thrive in wound environments, but fail to propagate in culture. Conventional laboratory methods also have significant limitations due to the delay in microbial diagnosis because of slow growing cultures. Such a delayed diagnosis may lead to sepsis, surgery, and/or sequential amputations. In addition, anaerobic bacteria require very specific culture conditions which must be requested from the physician in a hypothesis driven approach and can up to 10 days to grow. Thus, conventional laboratory methods do not provide an accurate representation of the microorganisms present within a wound. As a result, the current clinical dependence on conventional laboratory methods may lead to a treatment decision that doesn't reflect the wound's ecology and contributes to the chronic nature of the wound.
[0005] Thus, improved methods are needed for a rapid and complete identification of microorganisms in a wound. Such methods can allow for the targeted treatment of a patient's wound, yielding improved outcomes. More rapid microbial identification and diagnosis of a patient's wound infections can also significantly shorten the time required to heal wounds, promote targeted antibiotic therapy, as well as other advantages, including accelerated transition from inpatient to outpatient, a decrease in the potential of repeated surgeries, and a likely reduction in hospital readmission rates.
[0006] Accordingly, disclosed herein are methods and systems that address the need to rapidly diagnose various microorganisms in wounds, especially those organisms such as anaerobes which are challenging to propagate using traditional culture based methods.
SUMMARY
[0007] Disclosed herein are methods and systems generally related to a rapid, accurate, and more comprehensive identification of microorganisms in a biological sample such as those obtained from wounds. The methods and systems are useful for rapidly detecting the presence or absence of a plurality of microorganisms from other biological samples such as a blood sample, skin sample, a saliva sample, or a urine sample. Such methods and systems allow for the identification of microorganisms from a small amount of clinical specimen, as well as the qualitative representation of the microorganisms contributing to a polymicrobial infection.
Furthermore, the methods and systems disclosed herein enable the precision treatment of a subject's infection (e.g., wound infection), which may lead to shortening the healing time of the infection, e.g., a chronic wound infection.
[0008] In one aspect, a method is provided for detecting the presence or absence of a plurality of microorganisms in a biological sample. The method includes: (a) obtaining a biological sample from a subject in need thereof, wherein the biological sample may, or is suspected to, contain one or more microorganisms; (b) providing a plurality of primer pairs, wherein each primer pair is specifically designed to amplify a target sequence of a microorganism listed in one or more of Tables 1-11; and (c) performing real-time PCR or digital PCR using the plurality of primers, thereby detecting the presence or absence of the plurality of microorganisms listed in one or more of Tables 1-11.
[0009] In various embodiments, the target sequence can include antibiotic resistance genes which may be shared by more than one microorganism. The target sequence may also include virulence factor genes. In various embodiments, the plurality of primer pairs are designed to target and/or detect all microorganisms, antibiotic resistance genes and virulence factor genes listed in one or more of Tables 1-11. In some embodiments, the method can further include, prior to step (c), extracting nucleic acids from the biological sample. In certain embodiments, the biological sample or the one or more microorganisms therein are not subject to a step of in vitro culturing.
[0010] A further aspect relates to a plurality of primer pairs, wherein each primer pair is specifically designed to amplify a target nucleotide sequence within a microorganism, antibiotic resistance gene or virulence factor gene listed in one or more of Tables 1-11, wherein the plurality of primer pairs together target and/or detect all microorganisms, antibiotic resistance genes and virulence factor genes listed in one or more of Tables 1-11.
[0011] Another aspect relates to systems and/or kits for detecting the presence or absence of a plurality of microorganisms in a biological sample. The system or kit includes: (a) a plurality of primer pairs, wherein each primer pair is specifically designed to amplify a target sequence of a microorganism listed in one or more of Tables 1-11; and (b) reagents for performing real-time PCR or digital PCR using the plurality of primers, thereby detecting the presence or absence of the plurality of microorganisms listed in one or more of Tables 1-11. In some embodiments, the system or kit can further include reagents for extracting nucleic acids from the biological sample. In various embodiments, the plurality of primer pairs are designed to target and/or detect all microorganisms listed in one or more of Tables 1-11.
[0012] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages can be apparent from the description and drawings, and from the claims.
BRIEF DESCRIPTION OF DRAWINGS
[0013] The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and the examples, serve to explain the principles and implementations of the disclosure.
[0014] FIG. 1 A shows an overview of the general steps involved in an exemplary method for the identification of the microorganism community.
[0015] FIG. IB shows exemplary real-time PCR amplification curves.
[0016] FIG. 1C shows a report of interpreted results from an exemplary real-time PCR run.
[0017] FIG. 2 depicts the relative representation of different organisms in a specimen over time showing that Device 2 (DxWound) stably captures the microbiome of the wound, while
Device 1 (standard culture transport media) results in large variation in the microbiome.
[0018] FIG. 3 shows DNA yield from different specimen types.
[0019] FIG. 4 depicts a bar graph showing the number of microbes identified per patient in a pilot study of 106 wound specimens.
[0020] FIG. 5 depicts a bar graph showing the number of antibiotic resistance genes identified in patient samples in a pilot study of 106 wound specimens.
DETAILED DESCRIPTION
[0021] Disclosed herein are methods and systems that can be used to rapidly and accurately detect the presence or absence of microorganisms (including antibiotic resistance genes and/or virulence factor genes thereof) in a biological sample. In some embodiments, the methods and systems disclosed herein provide a comprehensive snapshot of the microbiome of a biological sample. The methods and systems disclosed herein are useful for treating microbial infections through accurate identification of the microorganisms present. Definitions
[0022] Various terms used throughout this specification shall have the definitions set forth herein.
[0023] The terms "Wound Genetic Test" (WGT) or "DxWound" can be used interchangeably and refer to the methods and systems disclosed herein for detecting the presence or absence of microorganisms in a biological sample. In some embodiments, WGT is used to more
specifically describe the methods while DxWound refers more to the systems. It should be noted that while the present disclosure in certain embodiments refers to the method in the context of wounds, one of ordinary skill in the art would readily understand that the methods and kits disclosed herein are applicable to other biological samples such as skin, buccal swab, tissue biopsy, bone, nails, plasma, serum, whole blood and blood components, saliva, urine, tears, seminal fluid, vaginal fluids and other fluids and tissues, including paraffin embedded tissues or other tissues collected and preserved in the course of, e.g., a forensic investigation.
[0024] The term "antibiotic resistance genes" describes genes present in bacteria that may mediate resistance to a specific antibiotic or class of antibiotics. In some embodiments, methods provided herein can be used to detect several antibiotic resistance genes associated with bacteria that typically cause skin and soft tissue infections (SSTIs). The detection of these genes using methods provided herein suggests the presence of bacteria that may be resistant to specific antibiotics or classes of antibiotics.
[0025] The term "biological sample," "sample" or "specimen" refers to any composition containing or presumed to contain nucleic acid (e.g., DNA) from the microbiota of an individual. In the context of the present disclosure, any type of body sample may be used, including without limitation, skin, buccal swab, wound swab, tissue biopsy, bone biopsy, nails, plasma, serum, whole blood and blood components, saliva, urine, tears, seminal fluid, vaginal fluids and other fluids and tissues, including paraffin embedded tissues or other tissues collected and preserved in the course of, e.g., a forensic investigation. DNA can be extracted from various samples using methods known in the art.
[0026] The term "primer" refers to an oligonucleotide that acts as a point of initiation of DNA synthesis in a PCR reaction. A primer is usually about 15 to about 40 nucleotides in length (or shorter or longer) and typically includes at least one target-hybridized region that is at least substantially complementary to the target sequence.
[0027] The term "probe" refers to an oligonucleotide that hybridizes to a target sequence on a target nucleic acid. Target sequence refers to a region of nucleic acid that is to be analyzed and comprises the site of interest. For TaqMan® assays, the probe can additionally contain a quencher and a dye (e.g., fluorescent). When the probes are intact, fluorescence is suppressed because the quencher dyes are in the proximity of the reporter dyes. At least two probes can be included in one TaqMan® assay.
[0028] The term "TaqMan® assay" or "TaqMan® chemistry" as used in the context of realtime PCR is a technology that exploits the 5 '-3' nuclease activity of Taq DNA polymerase to allow direct detection of the PCR product by the release of a fluorescent signal from a probe as a result of PCR. The TaqMan® assay permits discrimination between different genomic sequences. A typical TaqMan assay includes a Taq DNA polymerase, dNTPs, a pair of primers designed to amplify a target region, and a probe which will hybridize within the target region, mixed with DNA samples and subjected to PCR (e.g., real-time PCR that can detect and measure fluorescence). In the PCR annealing step, the probe hybridizes to the targeted site. During PCR extension, the reporter and quencher dyes are released due to the 5' nuclease activity of the Taq polymerase, resulting in an increased characteristic fluorescence of the reporter dye. Exonuclease activity only happens on perfectly hybridized probes, since a probe containing a mismatched base will not be recognized by the Taq polymerase. "Taq DNA polymerase" refers to a heat stable enzyme used in the polymerase chain reaction (PCR) to amplify target DNA. It was discovered in bacterium Thermus aquaticus and hence the name. It should be noted that other polymerases having 5 '-3' nuclease activity can also be used to replace the Taq DNA polymerase in TaqMan assays.
[0029] "Next Generation Sequencing" or "NGS" can include one or more of 454-based sequencing (Roche), Genome Analyzer-based sequencing (Illumina/Solexa), Ion torrent: Proton / PGM sequencing, Oxford Nanopore, PacBio, and ABI-SOLiD-based sequencing (Applied Biosystems). The basic chemistry relies on the fact that DNA polymerase catalyzes the incorporation of dNTPs into a DNA polymer during sequential cycles of DNA synthesis. During each cycle, the nucleotide being incorporated is either identified by an optical signal (e.g., fluorophore excitation) or change in pH (incorporation of each base releases a proton). Compared to conventional sequencing such as Sanger sequencing, the critical difference is that, instead of sequencing a single DNA fragment at a time, NGS extends the process across of multiple fragments in a parallel fashion, greatly enhancing sequencing speed and accuracy.
[0030] The term "nucleic acid" refers to polymers of nucleotides (e.g., ribonucleotides or deoxyribonucleotides) both natural and non-natural. The term is not limited by length (e.g., number of monomers) of the polymer. A nucleic acid may be single-stranded or double-stranded and will generally contain 5 '-3' phosphodiester bonds, although in some cases, nucleotide analogs may have other linkages.
[0031] The terms "complementary" and "complementarity" refer to nucleic acids (e.g., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence 5'-A-G-T- 3' is complementary to the sequence 3'-T- C-A-5'. Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.
[0032] Conventional molecular biology, microbiology, and recombinant DNA techniques including sequencing techniques are well known among those skilled in the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein "Sambrook, et ai., 1989"); DNA Cloning: A Practical Approach, Volumes I and ίί (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. ( 1985)); Transcription And Translation (B. D. Hames & S, J. Higgins, eds, (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986));
Immobilized Cells And Enzymes (IRL Press, ( 1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel, et al . (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
[0033] The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. "Consisting essentially of means inclusion of the items listed thereafter and which is open to unlisted items that do not materially affect the basic and novel properties of the invention.
[0034] Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for the use of the ordinal term) to distinguish the claim elements.
Microbial Genetic Test
[0035] Identifying microorganisms in a sample conventionally requires performing routine laboratory culture procedures. However, there are many limitations to these conventional techniques, not the least is cultivation bias. It has been well known that fewer than 2% of all known bacterial species can be routinely cultured in the clinical microbiology laboratory, and only a subset of these microorganisms are able to grow within the 24 hour period prescribed for most cultures (Tatum, O.L. et al., Advances in Wound Care, Volume 1, Number 3, pages 115- 119). Moreover, other microbes such as fungi, e.g., Candida, are rarely identified in the routine laboratory culture procedures. As fungi can be part of the microbial community of a wound, it is critical to detect their presence and recognize their contribution to a wound. Also, anaerobic bacteria, which have been identified as a component of recalcitrant wounds (Tatum, O.L. et al., Advances in Wound Care, Volume 1, Number 3, pages 115-119), are difficult to propagate in the laboratory without specialized collection methods, growth media, and environmental control. Furthermore, some microorganisms are known to efficiently grow and outcompete other species in laboratory conditions. Thus, routine laboratory culture procedures have a selection bias of one species over another, leading to potentially to misguided treatments and less than desirable outcomes.
[0036] These routine laboratory culture procedures currently used to diagnose bacterial colonization and infection are not only time consuming, but do not reveal the microbiological communities present within the samples because classical culture methods lack sensitivity in polymicrobial environments (Bowler P. G. et al., Int J Dermatol 1999; 38: 573-578).
[0037] While PCR methods have been used recently to detect a molecular signature for a pathogen, they have limitations as well (Gontcharova V, et al., Open Microbiol J 2010; 4:8; Wolcott R.D. et al., BMC Microbiol 2009; 9: 226; Wolcott R.D. et al., J Wound Care 2009; 18: 317). For example, there are many known inhibitory contaminants in wound samples that are derived from sources such as the blood. Iron in the blood is a known PCR inhibitor, and therefore, traditional PCR approaches are unlikely to completely and accurately reveal microorganism community present in a sample.
[0038] In contrast to conventional methods, the methods and systems disclosed herein allow for rapid, consistent, and accurate identification of the microorganism community present from a biological sample. These methods of microorganism detection thus provide a snapshot of the microorganisms in the wound at the time of sample collection. This is highly desirable, as such methods can allow clinicians to better manage the healing of the wound, thereby improving the prognosis for patients. In addition, it has been surprisingly shown that blood and common exogenous wound treatments (e.g., lidocaine, antibiotic creams, dressings, etc.,) can be successfully removed during the DNA extraction process and therefore do not interfere with the detection methods disclosed herein.
[0039] Another significant advantage provided by the methods and systems disclosed herein is the ability to detect the microorganism community within 48 hours from presentation. One study suggested that as many as 16.6% of acute skin and soft tissue infections (SSTIs), 34.1% of chronic or ulcerative infections, and 26.7% of surgical site infections had initial treatment failure. It has been shown that antimicrobial therapy which is not targeted to the causative pathogen within 48 hours of presentation is an independent risk factor for treatment failure. This type of data contributed to the U.S. Food and Drug Administration (FDA)'s decision to revise its guidance for the evaluation of clinical response to skin infections to earlier time points of 48 to 72 hours after initiation of therapy. Using the methods and systems disclosed herein, it is possible to detect the microorganism community within 48 hours from presentation. Clinicians can use these results to help them quickly target antibiotic therapy, ideally within that important 48 hour window.
[0040] In one aspect, disclosed herein is a method for detecting the presence or absence of a plurality of microorganisms in a biological sample. The method can include: (a) obtaining a biological sample from a subject in need thereof, wherein the biological sample may, or is suspected to, contain one or more microorganisms; (b) providing a plurality of primer pairs, wherein each primer pair is specifically designed to amplify a target sequence of a microorganism listed in one or more of Tables 1-11; and (c) performing real-time PCR using the plurality of primers, thereby detecting the presence or absence of the plurality of microorganisms listed in one or more of Tables 1-11.
[0041] Another aspect relates to systems and/or kits for detecting the presence or absence of a plurality of microorganisms in a biological sample. The system or kit includes: (a) a plurality of primer pairs, wherein each primer pair is specifically designed to amplify a target sequence of a microorganism listed in one or more of Tables 1-11; and (b) reagents for performing real-time PCR or digital PCR using the plurality of primers, thereby detecting the presence or absence of the plurality of microorganisms listed in one or more of Tables 1-11. In some embodiments, the system or kit can further include reagents for extracting nucleic acids from the biological sample. In various embodiments, the plurality of primer pairs are designed to target and/or detect all microorganisms listed in one or more of Tables 1-11.
[0042] FIG. 1 A is an overview of the general steps involved in an exemplary method disclosed herein related to the identification of the microorganism community. In step (1), various specimen such as blood, wound swab, urine, tissue biopsies and biorepositories can be collected by routine clinical methods. At step (2), the specimen can be processed (e.g., by inactivating nucleases) and/or stored (e.g., at a temperature lower than the room temperature) to maintain stability. At step (3), pathogen nucleic acids can be extracted from the specimen using extraction methods known in the art and/or commercially available kits (e.g., miniprep kits), following a step of tissue disruption depending on the type of the specimen (e.g., using QIAGEN
TissueLyser II).
[0043] After extraction of nucleic acids, molecular analysis of a plurality of specimens simultaneously can then be performed at step (4) using, e.g., real-time PCR or other quantitative PCR methods such as digital PCR. One example is the Fluidigm Biomark HD System with the 96.96 Dynamic Array Gene Expression Chip.
[0044] In parallel with step (4), various positive and negative controls can be included for quality control purpose (step (5)). Exemplary run controls (designed to ensure that the assay is running properly) include but are not limited to:
1. Isolation negative control, to verify that the nucleic acid extraction/isolation reagents are not contaminated. Purified water can be used here. 2. Isolation positive control, to verify that nucleic acid extraction/isolation has occurred successfully. A standard culture of one or more types of microbes can be used.
3. No Template Control (NTC), to verify that the PCR assay reagents are not
contaminated. Purified water can be used here.
4. Microbial Plasmid Pool, to verify that the PCR assays are performing as expected. One or more plasmids can be used, each plasmid predesigned to represent one microbe to be detected by the PCR assay.
5. Positive PCR Control (PPC), to verify that the samples do not contain anything that might inhibit PCR amplification.
[0045] In addition to the above run controls, additional controls can also be included in each assay as a control for the overall test. A "Pan Bacterial" assay can be designed to detect all bacterial species. For example, universal primers can be designed to amplify the 16S rRNA region for all or most bacterial species of interest (see, e.g., Nature Biotech 31, 814-821
(2013)). This assay amplifies a range of bacteria in a species independent manner. The "Pan Bacterial" assay can be used as a control to determine whether there are detectable levels of bacteria present in a specimen. An amplification signal for the Pan Bacterial assay in the absence of a signal for a species specific assay indicates that bacteria which are not currently present in the DxWound panel are present in the specimen (e.g., Corynebacterium). If a signal for a species specific assay is detected, co-amplifi cation of the Pan Bacterial assay is expected. If no amplification signal is detected for the Pan Bacterial assay, the etiology of the wound is unlikely to be attributed to bacterial infection.
[0046] A "Pan Fungal" assay can also be included as a control to determine whether there are detectable levels of fungi present in a specimen. For example, universal primers to amplify an evolutionarily conserved sequence region in representative fungal species (Candida spp. and/or Aspergillus spp.) present on the DxWound panel. The assay can be used as a confirmatory control if an amplification signal for a specific fungal species is detected. Co-amplification of the assay for the specific fungal species and the Pan Fungal control assay is required to confirm the presence of a DxWound Candida spp. or Aspergillus spp in the specimen.
[0047] A human gDNA assay can also be included as a control to determine whether there are detectable levels of DNA present in a specimen. For example, primers to amplify representative human housekeeping genes can be used here. The assay can be used as a confirmatory control to verify that the sample was successfully handled from the time of specimen collection through data acquisition.
[0048] Thereafter, data analysis can be performed at step (6), FIG. 1 A using, e.g., the Fluidigm Real-Time PCR Analysis Software by evaluating amplification signal against control samples within a defined reportable range. FIG. IB shows exemplary real-time PCR amplification curves, in which exponential amplification begins from, e.g., cycle number 10-20 in positive controls and in specimens where pathogens are present. In contrast, in negative controls and in specimens where pathogens are absent, no amplification would occur. Ct value, the number of cycles required for the fluorescent signal to cross the threshold (i.e., exceeds background level) can be obtained for each amplification curve. A heat map can be generated that color codes Ct values of an exemplary real-time PCR run, with a plurality of samples targeting a plurality of microbes. Different colors represent how early or late (in Ct value) amplification occurs, suggesting the amount of specific microbial DNA present in the corresponding samples.
Generally, the more microbial DNA is present, the earlier amplification occurs and the lighter the color is on the heat map.
[0049] Finally, a report can be generated at step (7), FIG. 1 A. As shown in FIG. 1C, such report can include sample name, assay name (microbe species and other targets), Ct value, assay quality control (QC) status, positive control Ct value (e.g., pan-bacteria control), negative control Ct value, and final interpreted result (in the form of positive (POS), negative (NEG) or unable to determine (UTD)).
[0050] In one embodiment, a PCR-based Dx Wound assay can be used to analyze microbial DNA using species-specific DNA sequences, such as 16S rRNA sequences for bacterial detection. In addition, sequences specific to virulence gene and antibiotic resistance genes (e.g., mecA) can also be analyzed.
[0051] In some embodiments, DxWound can utilize a swab for sample collection. The tests can be performed on a sample taken directly from the site of infection, e.g., wound. The swab sample can be collected in an inactivating solution that kills the microorganisms at the same time as protecting the microbial DNA, thus preserving the wound microbiome in time at the point of specimen collection. DNA is detected directly from the patient specimen without culture enrichment. WGT Microbes
[0052] Exemplary microbes/targets that can be detected using the methods and systems disclosed herein are shown in Tables 1-11.
Table 1. Exemplary Wound Genetic Test Menu
Figure imgf000015_0001
carbapenemase (IMP)
carbapenemase (SME)
extended and broad spectrum β-lactamase (SHV) extended spectrum β-lactamase (CTX-M group 1) extended spectrum β-lactamase (CTX-M group 9) Macrolide Lincosamide Streptogramin (ermA) Macrolide Lincosamide Streptogramin (ermB) Macrolide Lincosamide Streptogramin (me/A)
Virulence a-toxin (hid)
factors Panton-Valentine leukocidin (lukF)
Table 2: Exemplary Wound Genetic Test Menu
Figure imgf000016_0001
Streptococcus agalactiae Bacterial Strain ID
Streptococcus pyogenes Bacterial Strain ID
Aspergillus flavus Fungal Strain ID
Aspergillus fumigatus Fungal Strain ID
Aspergillus niger Fungal Strain ID
Candida albicans Fungal Strain ID
Candida glabrata Fungal Strain ID
Candida krusei Fungal Strain ID
Candida parapsilosis Fungal Strain ID
Candida tropicalis Fungal Strain ID
CTX-M-1 group Antibiotic Resistance Gene
CTX-M-8 group Antibiotic Resistance Gene
CTX-M-9 group Antibiotic Resistance Gene
IMP-1 group Antibiotic Resistance Gene
IMP- 12 group Antibiotic Resistance Gene
IMP-2 group Antibiotic Resistance Gene
IMP-5 group Antibiotic Resistance Gene
KPC Antibiotic Resistance Gene mecA Antibiotic Resistance Gene DM Antibiotic Resistance Gene
OXA-48 Group Antibiotic Resistance Gene
SHV Antibiotic Resistance Gene
SHVQ56D) Antibiotic Resistance Gene
SHV(156G) Antibiotic Resistance Gene
SHV(238G240E) Antibiotic Resistance Gene
SHV(238G240K) Antibiotic Resistance Gene
SHV(238S240E) Antibiotic Resistance Gene
SHV(238S240K) Antibiotic Resistance Gene vanA (VRE) Antibiotic Resistance Gene vanB Antibiotic Resistance Gene vanC Antibiotic Resistance Gene
VIM-1 group Antibiotic Resistance Gene
VIM- 13 Antibiotic Resistance Gene
VIM-7 Antibiotic Resistance Gene hla Virulence Factor lukF Virulence Factor spa Virulence Factor ermA Antibiotic Resistance Gene ermB Antibiotic Resistance Gene mefA Antibiotic Resistance Gene
SME Antibiotic Resistance Gene Table 3. Exem lar Wound Genetic Test Menu
Figure imgf000018_0001
Table 4 Exem lar Wound Genetic Test Menu
Figure imgf000018_0002
Bacteria Clostridium septicum
Bacteria Bacteroides fragilis
Bacteria Prevotella intermedia
Fungi Candida albicans
Fungi Candida glabrata
Fungi Candida parapsilosis
Antibiotic Resistance Gene mecA
Antibiotic Resistance Gene vanA
Antibiotic Resistance Gene vanB
Table 5. Exemplary Wound Genetic Test Menu
Type Assay target
Bacteria Acinetobacter baumannii
Bacteria Citrobacter freundii
Bacteria Enterobacter aerogenes
Bacteria Enterobacter cloacae
Bacteria Escherichia coli
Bacteria Proteus mirabilis/vulgaris
Bacteria Pseudomonas aeruginosa
Bacteria Enterococcus faecalis
Bacteria Enterococcus faecium
Bacteria Mycobacterium abscessus
Bacteria Mycobacterium chelonae
Bacteria Staphylococcus aureus
Bacteria Streptococcus agalactiae (Group B)
Bacteria Streptococcus pyogenes (Group A)
Bacteria Clostridium perfringens
Bacteria Clostridium septicum
Bacteria Bacteroides fragilis
Bacteria Prevotella intermedia
Fungi Candida albicans
Fungi Candida glabrata
Fungi Candida parapsilosis
Antibiotic Resistance Gene mecA
Antibiotic Resistance Gene vanA
Antibiotic Resistance Gene vanB
Table 6. Exemplary Wound Genetic Test Menu
AEROBIC BACTERIA, GRAM-POSITIVE
Enterococcus faecalis
Enterococcus faecium Mycobacterium abscessus
Mycobacterium chelonae
Staphylococcus aureus
Staphylococcus lugdunensis (Coagulase-Negative)
Streptococcus agalactiae (Group B)
Streptococcus pyogenes (Group A)
STAPHYLOCOCCAL VIRULENCE GENE
lukF-PV (Panton- Valentine Leukocidin, PVL)
AEROBIC BACTERIA, GRAM-NEGATIVE
Acinetobacter baumannii
Citrobacter freundii
Enterobacter aerogenes
Enterobacter cloacae
Escherichia coli
Proteus mirabilis/vulgaris
Pseudomonas aeruginosa
ANAEROBIC BACTERIA, GRAM-POSITIVE
Clostridium perfringens
Clostridium septicum
ANAEROBIC BACTERIA, GRAM-NEGATIVE
Bacteroides fragilis
Prevotella intermedia
Prevotella oralis
FUNGI
Aspergillus falvus
Aspergillus fumigatus
Aspergillus niger
Candida albicans
Candida glabrata
Candida parapsilosis
Candida tropicalis
ANTIBIOTIC RESISTANCE GENES
Carbapenamase
IMP
KPC DM
OXA-48
SME
VIM
Extended-Spectrum 6-Lactamase
CTX-M
SHV
Macrolide-Lincosamide-Streptogramin B Resistance ermA
ermB Oxacillin/Methicillin Resistance
mecA
Vancomycin Resistance
vanA
vanB
[0053] In some embodiments, DxWound can be used to detect Staphylococcus aureus (S. aureus), which is the most common pathogen associated with skin and soft tissue infections (SSTIs). For example, DxWound can accurately and sensitively identify certain Staphylococcal species, including Staphylococcus aureus, Staphylococcus lugdunensis, Staphylococcus epidermidis and other Coagulase-Negative Staphylococci (CoNS), along with the antibiotic resistance gene mecA and the virulence gene lukF-P V (Panton- Valentine Leukocidin virulence factor, PVL).
[0054] An antibiotic resistance gene is a gene present in bacteria that mediates resistance to a specific antibiotic or class of antibiotics. mecA is an antibiotic resistance gene associated with Staphylococcus species. The mecA gene mediates oxacillin/methicillin resistance. When mecA is present in Staphylococcus aureus, then Methicillin-resistant Staphylococcus aureus (MRSA) may be present. In one study, almost 80% of positive SSTI cultures detected S. aureus, and about 50% of those were MRSA. MRSA has been tied to increased hospital lengths of stay, increased healthcare costs, is an independent risk factor for mortality and is now predictably resistant to all B-lactam antibiotics except ceftaroline. As such, MRSA is considered a serious threat by the CDC.
[0055] S. aureus can also express virulence factors that are associated with increased infection severity. The presence of the PVL virulence factor in S. aureus is associated with infections that are more likely to lead to necrosis and more likely to require surgical treatment. PVL is a virulence factor encoded by two genes: lukS-PV and lukF-PV.
[0056] The DxWound Staphylococcal tests can be useful for any patient suspected of having a Staphylococcal skin and soft tissue infection (SSTI) based on the clinician's determination of medical necessity. The DxWound tests can identify S. aureus and other Staphylococcus spp directly from a wound sample without the need for culture enrichment. The presence of mecA may indicate resistance to oxacillin/methicillin and all B-lactams with the exception of cephalosporins with anti-MRSA activity. When S. aureus and mecA are both detected, then MRSA may be present. The presence of PVL may suggest increased infection severity and the potential need for surgical intervention. In a patient with clinical signs and symptoms of infection, these Staphylococcal tests can help clinicians quickly target antibiotic therapy.
[0057] In some embodiments, DxWound can be used for antibiotic resistance gene testing. Antibiotic resistance is considered by the CDC to be a major public health threat in the United States. Patients with antibiotic resistant infections have higher morbidity and mortality, including longer hospital stays with increased costs to the healthcare system. To help mitigate the consequences of antibiotic resistance, the CDC has called for improved antibiotic stewardship and the development of new diagnostic tests.
[0058] Antibiotic resistance genes are genes present in bacteria that mediate resistance to a specific antibiotic or class of antibiotics. DxWound can be used to test for antibiotic resistance genes associated with bacteria that typically cause skin and soft tissue infections (SSTIs). The detection of these genes suggests the presence of bacteria that may be resistant to specific antibiotics or classes of antibiotics (Table 7).
[0059] For example, as discussed above, the mecA gene is associated with Staphylococcus species, including Staphylococcus aureus, Staphylococcus lugdunensis and Staphylococcus epidermidis. The mecA gene mediates oxacillin/methicillin resistance and is associated with resistance to most B-lactams with the exception of cephalosporins with anti-MRSA activity. Methicillin-resistant Staphylococcus aureus (MRS A) is a major public health threat.
[0060] The carbapenemase genes generally mediate resistance to B-lactams, including carbapenems. The carbapenemase genes included in DxWound have been found in
Pseudomonas and Enterobacteriaceae , such as Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Proteus mirabilis, Proteus vulgaris. The carbapenemase genes have also been found less frequently in other bacteria genera. Carbapenemase-producing organisms are rare but considered a major public health threat.
[0061] The extended-spectrum B-lactamase (ESBL) genes included in DxWound are commonly associated with Enterobacteriaceae, but have also been found in other bacteria genera. The ESBL genes generally confer resistance to B-lactams except cephamycins and carbapenems. ESBL-producing Enterobacteriaceae are a major public health threat.
[0062] ermA and ermB genes have been found in Staphylococcus, Streptococcus,
Enterococcus, and in a broad range of other bacterial genera. They mediate resistance to macrolides (e.g., erythromycin), lincosamides (e.g., clindamycin), and type B streptogramins. Clindamycin-resistant Group B Streptococcus and Erythromycin-resistant Group A Streptococcus are public health threats. For ermB testing, in order to identify potential clindamycin-resistant Group B Streptococcus and erythromycin-resistant Group A
Streptococcus, Enterococcus faecium and Enterococcus faecalis tests may also be useful, since ermB is commonly found in Enterococcus species.
[0063] vanA and vanB mediate resistance to vancomycin and are associated with Enterococcus and very rarely with Staphylococcus species. Vancomycin-resistant Enterococcus (VRE) and Vancomycin-resistant Staphylococcus aureus (VRSA) are public health threats.
[0064] DxWound tests can indicate whether specific antibiotic resistance genes (e.g., Table 8) are detected in a biological sample (e.g., wound specimen). The resulting report can provide information about the association of the detected antibiotic resistance gene to the specific antibiotic or antibiotic classes that may be affected. In addition, the report can indicate when a potential antibiotic resistant organism may be present, based on the detection of specific organisms with the antibiotic resistance genes. For example, when mecA is detected, the report states that if the wound is also positive for Staphylococcus aureus, then MRSA may be present. In another example, when KPC is detected, the report states that if the wound is also positive for Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, or Proteus mirabilis/vulgaris, then an ESBL-producing Enterobactericeae may be present.
Clinically, these test results can help clinicians quickly target antibiotic therapy.
Figure imgf000024_0001
Table 8. Exemplary Antibiotic Resistance Gene Tests
Figure imgf000025_0001
[0065] In summary, DxWound tests can be used to detect various targets (e.g., Table 9) in a biological sample suspected of infection. In some embodiments, the targets can be selected based on their clinical relevance and/or frequency (Tables 10-12).
[0066] In some embodiments, provided herein a method of identifying targets such as organisms, antibiotic resistance genes, and virulence genes that, if detected, would be useful for treatment decisions for infections such as SSTI. This method includes obtaining a clinical score and a frequency score for organisms, antibiotic resistance genes, and virulence genes of interest. Frequency score identifies prevalence of the tested item in infection sites such as wounds/SSTI, according to published studies. Clinical score is a measure of the pathogenicity of the organism, based on the published evidence. The highest clinical score (1) is applied when major public health organizations have identified the organism as an antimicrobial-resistant public health threat. The clinical and frequency score are used to ensure the clinical validity of the PCR-based tests.
[0067] In some embodiments, the target organisms, antibiotic resistance genes, and virulence genes can be selected according to one or more of the following criteria: (a) at least one of the clinical score and frequency score is 4 or lower; and/or (b) the combined clinical and frequency score is 9 or lower. Table 9: Exemplary Wound Genetic Test Menu
Figure imgf000026_0001
Streptococcus spp Microrganisms
Alpha Hemolytic Streptococcus Anaerococcus prevotii Aspergillus terreus Bacteroides fragilis group Bacteroides distasonis* Bacteroides ovatus Bacteroides thetaiotaomicron Bacteroides uniformis Bacteroides vulgatus Beta Hemolytic Streptococcus (Group F)
Candida dubliniensis
Candida krusei Candida lusitaniae Citrobacter koseri Diptheroids/Corynebacterium spp.
Eikenella corrodens
Finegoldia magna Fusobacterium spp.
Klebsiella oxytoca Klebsiella pneumoniae
Klebsiella spp.
Morganella morganii Mycobacterium fortuitum
Mycobacterium marinum Neisseria Meningitidis Peptoniphilus asaccharolyticus Peptostreptococcus anaerobius Prevotella spp.
Propionibacterium acnes Serratia marcescens Staphylococcus capitis (Coagulase-Negative)
Staphylococcus epidermidis (Coagulase-Negative)
Stenotrophomonas maltophilia
Streptococcus dysgalactiae subsp. equisimilis (Group G)
Streptococcus intermedius (Viridans)
Streptococcus pneumoniae Streptococcus sanguinis (Viridans) Table 10: Exemplary DxWound Organism Clinical and Frequency Scores
Figure imgf000028_0001
Table 11 : Exemplary DxWound Organism Clinical and Frequency Scores
Figure imgf000029_0001
Table 12: Explanation of DxWound Organism Clinical and Frequency Scores
Figure imgf000029_0002
4: Evidence for pathogenicity in SSTI, but not part of categories 1-3 4: 1-5%
5: not of major importance 5: <1%
(When more than one score applies to a microbe, the lowest score is
displayed)
[0068] It should be noted that for the microbes disclosed herein, a "Look-Up" table and associated algorithm specific for each microbe can be generated to report the presence ("positive") or absence ("negative") of the microbe, or, in the unlikely event, inconclusiveness ("unable to determine") of the assay due to various experimental errors or abnormalities. The Look-Up tables can include a combination of all possible results for a single sample or panel of samples. The software compares the amplification results from the specimen sample and various control samples to the algorithm to generate a call on whether the microorganism is present or not. By analyzing the combination of all the amplification results displayed on the Look-Up table for a given sample or panel of samples, the presence or absence of any microorganism from a wound may be detected.
[0069] A Look-Up table can be developed for each microbial target, considering the positive and negative controls used. For example, a Look-Up table for a particular bacterium can include inputs such as one or more targets for that bacterium (e.g., commercially available kits, bacterium-specific genes or transcripts), one or more pan bacteria control (positive control for all bacteria such as the 16S rRNA region) and one or more pan fungi control (positive control for fungi).
Real-time PCR
[0048] In some embodiments, molecular analysis of microbial genetic test can be performed by realtime PCR. Real-time Polymerase Chain Reaction (PCR) is the ability to monitor the progress of the PCR as it occurs (i.e., in real time). Data is therefore collected throughout the PCR process, rather than at the end of the PCR. In real-time PCR, reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed.
[0049] Generally, two types of chemistries can be used to detect PCR products— TaqMan® chemistry or SYBR™ Green dye chemistry. In TaqMan® chemistry, one may utilize an
oligonucleotide probe labeled with a reporter dye (e.g., fluorescent dye such as FAM or VIC) at the 5' end of the probe and a quencher dye (e.g., nonfluorescent quencher) at the 3' end of the probe. The proximity of the quencher to the intact probe maintains a low fluorescence for the reporter. During the PGR reaction, the 5' nuclease activity of DNA polymerase cleaves the probe, and separates the dye and quencher, resulting in an increase in fluorescence of the reporter. Accumulation of PCR product is detected directly by monitoring the increase in fluorescence of the reporter dye. The 5' nuclease activity of DNA polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target and is amplified during PCR. The probe is designed to straddle a target sequence position and hybridize to the nucleic acid molecule only if a particular sequence is present.
[0050] Genotyping is performed using oligonucleotide primers and probes. Oligonucleotides may be synthesized and prepared by any suitable methods (such as chemical synthesis), which are known in the art. Oligonucleotides may also be conveniently available through commercial sources. One of the skilled artisans would optimize and identify primers flanking the region of interest in a PCR reaction. Commercially available primers may be used to amplify a particular gene of interest for a particular sequence. A number of computer programs (e.g., Primer-Express) are readily available to design optimal primer/probe sets. It will be apparent to one of skill in the art that the primers and probes based on the nucleic acid information provided (or publically available with accession numbers) can be prepared accordingly .
[0051] Methods for labeling of probes are known in the art. The labeled probes are used to hybridize within the amplified region during the amplification reaction. The probes are modified so as to prevent them from acting as primers for amplification. The detection probe is labeled with two dyes, one capable of quenching the fluorescence of the other dy e when in proximity. One dy e is attached to the 5' terminus of the probe and the other is attached to an internal site or the 3' terminus, so that quenching occurs when the probe is in a hybridized state.
[0052] Primers and probes can be designed based on the target sequence. Algorithms for designing primers and probes are widely used and readily available in the art (e.g., Primer-BLAST available at www.ncbi.nlm.nih.gov/tools/primer-blast/). In essence any primer(s) suitable for amplifying a target sequence in a polymerase chain reaction can be used. In some embodiments it may be desirable to keep maximum amplicon size under 400 bp (e.g., 50-150 bp). The probe(s) should be designed to target a region flanked by the forward and reverse primer pair, with suitable length (e.g., 18-22 bases but can be longer or shorter) and proper dye labeling as discussed herein.
[0053] In addition, one of ordinary skill in the art of design of primers will recognize that a given primer need not hybridize with 100% complementarity to prime the synthesis of a complementary nucleic acid strand. Primer pair sequences may be a "best fit" amongst several aligned sequences, thus they need not be fully complementary to the hybridization region of any one of the sequences in the alignment. Moreover, a primer may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., for example, a loop structure or a hairpin structure). The primers may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with a target nucleic acid of interest. Thus, in some embodiments, an extent of variation of 70% to 100%, or any range falling within, of the sequence identity is possible relative to the specific primer sequences disclosed herein.
[0054] To illustrate, determination of sequence identity is described in the following example. A primer of 20 nucleobases in length which has 18 identical residues and 2 non-identical residues to another 20 nucleobase primer has 18/20 = 0.9 or 90% sequence identity. In another example, a primer 15 nucleobases in length having ail residues identical to a 15 nucleobase segment of primer 20 nucleobases in length would have 15/20 = 0.75 or 75% sequence identity with the 20 nucleobase primer. Percent identity need not be a whole number, for example when a 28 consecutive nucleobase primer is completely identical to a 31 consecutive nucleobase primer (28/31 = 0.9032 or 90.3% identical). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison WI), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appi. Math., 1981 , 2, 482-489). In some embodiments, complementarity of primers with respect to the conserved priming regions of target nucleic acid, is between about 70% and about 80%. In other embodiments, homology, sequence identity or complementarity, is between about 80% and about 90%. In yet other embodiments, homology, sequence identity or complementarity, is at least 90%, at least 92%, at least 94%, at least. 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100%. In some embodiments, the primers used herein comprise at least. 70%, at least 75%, at least 80%, at least. 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99%, or 100% (or any range falling within) sequence identity with the primer sequences specifically disclosed herein.
[0055] With primers and probes designed and synthesized, real-time PGR can then be performed on any suitable real-time PGR machine such as those manufactured by Fluidigni or Applied Biosystems that can record fluorescence level in real-time.
EXAMPLES Example 1: A Retrospective Database Study on Association of Microorganism Identification Time Using Culture-Based Test with Antimicrobial Treatment, Clinical Outcomes, and Financial Charges for Patients with Skin and Soft Tissue Infections
1. Purpose of the Study
[0070] To explore the association between microorganism identification time using culture-based testing and antimicrobial treatment, clinical outcomes, and financial charges for patients with skin and soft tissue infections (SSTIs).
2. Background and Rationale
[0071] SSTIs are common and their prevalence has increased significantly since the mid-1990s [1]. The increasing prevalence of SSTIs demands effective and efficient treatment and management of SSTIs. Effective treatment requires rapid and accurate identification of microbial pathogens and their resistance patterns.
[0072] However, the current standard-of-care depends on culture-based test methods for
microorganism detection. Those methods are suboptimal in providing rapid identification and susceptibility testing [2]. They involve the use of selective media to culture targeted aerobic or anaerobic microbial pathogens. The process to facilitate the incubation of microbial pathogens takes a minimum of 24-48 hours to yield qualitative and semi-quantitative assessments of the cultures [3]. Although anaerobic bacteria often constitute a significant proportion of the total microflora in SSTIs, their culture and isolation is prolonged and more resource demanding than investigations of aerobic bacteria, and consequently, anaerobic microbiology is often excluded from a routine analysis [3] . Such delays in providing definitive microorganism diagnosis make it difficult for the treating clinician to narrow antibiotic therapy to correctly treat the infection
[0073] Rapid and accurate identification of microbial pathogens in patients with SSTIs and infected chronic wounds may provide improved outcomes through more timely and pathogen specific treatment [2]. Benefits can include more efficient utilization of antimicrobial medications, more rapid resolution of infection, faster healing, and a reduction in patient mortality, as well as reduced tissue and limb loss [2] [4] [5] [6]. This in turn can provide enhanced health economic outcomes, such as reduced expenditure from use of fewer antimicrobials, reduction in the potential for microbial resistance development, subsequent avoidance of hospitalizations, shorter length of stay for hospitalized patients, reduction in total cost of care, fewer surgeries, and fewer hospital readmissions [7] [8] [9]. [0074] Here we propose a database outcomes research study to explore relationships between the timing of reporting of culture results, antimicrobial treatment, microbial identification, and clinical outcomes. We expect that findings from this study can provide data inputs for future studies.
[0075] The results from this study may be used to develop a rapid diagnostic test for SSTI/wound care, including assays, reporting algorithms and test report format.
3. Objectives of the study
[0076] The objectives of this study are:
1. To explore the relationships among timeliness of availability of culture results, detected microorganism/susceptibility, antimicrobial treatment, and study endpoints listed in Section 4.2.2. a) To calculate the proportion of patients with SSTIs, who had monomicrobial or polymicrobial results reported, out of those who had specific culture tests ordered
b) To observe the timing of reporting of culture-based test results.
c) To examine antimicrobial treatment patterns in patients with SSTIs.
2. To identify the subset of subjects (for example, community acquired vs. hospital acquired, subjects with certain co-morbidities, surgical-site infections etc.) for whom more rapid organism identification might lead to improved clinical and economic outcomes as outlined in Section 4.2.2.
4. Study Design
[0077] A retrospective study can be conducted using patient electronic medical records (EMR) and associated hospital charge master data (CMD) to achieve the objectives of the study.
4.1. Data Source
[0078] EMR has been used since the early 80' s. Its legacy system is TDS, and is still in use to retrieve data entered prior to 2009. A current EMR system is called Quest (Quality, Excellence & Safety through Technology), Allscripts' Sunrise Clinical Manager.
[0079] The Subject ID (SID) in the data dictionary is a recorded ID (recoded by bioinformatics personnel). It is impossible to link the extracted data back to the subject in EMR and CMD by using the SID.
4.2. Endpoints
4.2.1. Endpoints for culture test results and treatment
1. Proportion of microorganism/susceptibility or their combinations detected using culture- based methods.
2. All culture test results, including Gram stain, preliminary, final, and all results from samples sent to external laboratories. 3. Time to any culture test result (days) including Gram stain, preliminary, and final.
4. Time (hours) from specimen collection to any antimicrobial treatment.
5. Antimicrobial treatment patterns
4.2.2. Endpoints for outcomes and hospital charge
1. Length of stay (LOS)
2. 30-day readmission rates
3. Total cost of inpatient care for hospitalization (does not include cost of hospitalization for readmission)
4. Mortality during hospitalizations
5. Rates of amputation
6. Proportion of patients with SSTIs reported as cured and those reported as deteriorated.
7. Cost of antibiotic treatment
4.3. Patient Selection
[0080] Patients are eligible for inclusion in the study if the following inclusion criteria are met:
1. Inpatients with SSTIs, as identified by diagnosis code. The first hospitalization with a SSTI is defined as the Index Event and the admission time for the index event is defined as the Index Time (TIDX). Because no protected health information (PHI) information can be included in the data to be transferred to MH, Tmx can be set to 0. All other time can be defined as hours from TIDX.
2. Patients who are 18 years of age or older at the index time.
3. Patients who have at least one culture-based test result for microorganism/susceptibility (detected/not detected) during the index event.
5. Statistical Method
[0081] SAS® 9.4 or later, or other suitable software, can be used to generate all analyses, tables, listings, and figures.
[0082] If not otherwise specified, the number of observations, mean, standard deviation, median, minimum, and maximum values can be provided for all continuous measurements. For categorical data, the number and percentage in each category can be reported. Missing values for
categorical/ordinal data can be counted in a separate category.
[0083] Since this is an exploratory study, different methods can be explored and reported together with the associated results. The primary analyses can be listed in following 5.x subsections. 5.1. Proportion of patients with specific culture-based test results reported out of those who had specific culture tests ordered
[0084] The frequency and percentage of patients with specific culture-based test ordered can be reported by each individual microorganism, polymicrobial, or class (anaerobes, aerobes, gram positive, and gram negative).
[0085] The frequency and percentage of patients with a specific culture-based test result (e.g., positive/negative) can be summarized as each individual microorganism, polymicrobial infection, or class (anaerobes, aerobes, gram positive, and gram negative).
5.2. Time needed for reporting culture-based test result
Figure imgf000036_0001
i ::v>; T5 ' RST
[0086] The time for reporting of each culture-based test result can be identified. Let Ts be the specimen collection time, TRST be the culture-based test result time of completion, DAYsampie be the Days of specimen collection from the index time (see Section 4.3), DAYRST be the days of culture- based results from the index time, and CUL time be the days of culture-based results from specimen collection time. The picture above describes the relationship of those aforementioned time variables.
[0087] DAYsampie, DAYRST, and CUL time can be summarized by individual microorganisms or polymicrobial infection.
5.3. Patterns of antimicrobial treatment
[0088] Antimicrobial treatment can be analyzed at the generic name level.
5.3.1. Number and proportion of patients treated with antimicrobial
[0089] Number and proportion of patients treated with any antimicrobial (including prior to or after the hospital admission) can be summarized overall, by each individual microorganism, by
polymicrobial infection, or class (anaerobes, aerobes, gram positive, gram negative). The dosage and route of administration can be noted.
5.3.2. Time (hours) from specimen collection to the first antimicrobial treatment
[0090] The first antimicrobial treatment prior to or after hospital admission can be denoted as Drugi (The start time T1 of Drugi can be before or after the hospital admission). Time from specimen collection to the Drugi use (STRT_TMl=Ti - Ts) can be summarized overall and by antimicrobial generic name.
5.3.3. Antimicrobial switch
[0091] The subsequent administration of any antimicrobial different from Drugi (see Section 5.3.2) can be denoted as Drug2; the subsequent administration of any antimicrobial different from Drug2 (see Section 5.3.2) can be denoted as Drug3; etc. Any change from Drugxto Drugx+i is an antimicrobial switch.
[0092] The corresponding start time for those treatment can be denoted as STRT TMl, STRT TM2, and STRT TM3 etc.
[0093] Kaplan-Meier survival curves can be used to display the Time to the 1st antimicrobial switch (STRT TM2) overall and by each individual microorganism or polymicrobial infection. Survival probability estimates can be calculated at continuous time points up to the maximum discharge time to be observed in the data overall and by each individual microorganism or polymicrobial infection.
[0094] The number and proportion of patients can be summarized for each switch pattern (no switch, Drugi-> Drug2; Drugi-> Drug2 .> Drug3; etc.) by each individual microorganism or polymicrobial infection.
[0095] Changes in dosage and/or route of administration can also be included in the analysis.
5.4. Relationships among timing of availability of culture results, detected microorganism/susceptibility, antimicrobial treatment, and study endpoints listed in Section 4.2.2.
5.4.1. Relationship among timing of availability of culture results, detected microorganism/susceptibility, and study endpoints for outcomes and hospital charge
[0096] For continuous endpoints, scatter plots can be generated to display the relationship between
CUL time (original or bin) and each of the endpoints (original or binning data) by each individual microorganism or polymicrobial infection detected.
[0097] Generalized linear regression models for the continuous endpoints can be explored, the predictors to be included can be CUL time and other factors or covariates such as comorbidity, age, etc.
[0098] For category endpoints, the number and proportion of patients in each level of the category variables can be can be summarized by bins of CUL time and each individual microorganism or polymicrobial infection detected.
[0099] Logistics regression models for the category endpoints can be explored, the predictors to be included can be CUL time and other factors or covariates such as comorbidity, age, etc. 5.4.2. Relationship among timing of availability of culture results, detected microorganism/susceptibility, and antimicrobial treatment
[00100] Scatter plots can be generated to display the relationship between culture test result time (original or bin) and the first antimicrobial treatment time (STRT TMl, original or binning data) by each individual microorganism or polymicrobial infection, and by antimicrobial generic name and each individual microorganism or polymicrobial infection.
[00101] Time difference between the first antimicrobial treatment and the culture test result time (= STRT TMl - CUL time) can be summarized by each individual microorganism or polymicrobial infection, and by antimicrobial generic name and each individual microorganism or polymicrobial infection.
[00102] For those with an antimicrobial switch, the above analyses can be conducted to show the relationship between culture test result time and the first antimicrobial treatment switch time.
5.4.3. Impact of appropriate antimicrobial treatment on study endpoints for outcomes and hospital charge
[00103] By examining the microorganisms/susceptibility detected by culture test and the
antimicrobial treatments that the patients received, antimicrobial treatment appropriateness can be defined. The study endpoints for outcomes and hospital charges can be summarized and compared between groups of different antimicrobial treatment appropriateness using generalized linear model for continuous outpoints and logistic regression model for category outpoints with antimicrobial treatment appropriateness, CUL time, and other factors or covariates such as wound type, comorbidity, age, etc. as predictors.
6. Risk Analysis
[00104] As this study involves only the retrospective collection and analysis of medical record and charge master information, there are no specific physical risks or side effects associated with subjects' participation in this study. The data provided to MH can include only a limited, de-identified data set.
7. References
[1] A. N. Amin et cl, "Hospitalist Perspective on the Treatment of Skin and Soft Tissue Infections," Mayo Clin Proc, vol. 89, no. 10, pp. 1436-1451, 2014.
[2] K. A. Bauer et al, "Review of Rapid Diagnostic Tests Used by Antimicrobial Stewardship Programs," Clinical Infectious Diseases, vol. 59, no. suppl 3, 2014, doi: 10.1093/cid/ciu547.
[3] P. G. Bowler et al, "Wound Microbiology and Associated Approaches to Wound Management," clinical microbiology Review, vol. 14, no. 2, pp. 244-269, Jan. 2001, doi: 10.1128/cmr.
[4] S. Dowd et cl, "Molecular diagnostics and personalised medicine in wound care: assessment of outcomes, "journal of wound care, vol. 20, no. 5, pp. 232-241, May 2011.
[5] R. D. Wolcott et cl, "Healing and healing rates of chronic wounds in the age of molecular pathogen diagnostics," Journal of Wound Care, vol. 19, no. 7, pp. 272-278, 7/2010.
[6] O. L. TATUM et cl, "Wound Healing Finally Enters the Age of Molecular," ADVANCES IN WOUND CARE, vol. 1, no. 3, pp. 115-119, 2012.
[7] K. A. Bauer et al, "An Antimicrobial Stewardship Program's Impact with Rapid Polymerase Chain Reaction Methicillin-Resistant Staphylococcus aureus/S. aureus Blood Culture Test in Patients with S. aureus Bacteremia," Clinical Infectious Disease, vol. 51, no. 9, pp. 1074-1080, 2010 doi: l 0.1086/656623.
[8] D. T. Nguyen et cl, "Real-Time PCR Testing for mecA Reduces Vancomycin Usage and Length of Hospitalization for Patients Infected with Methicillin-Sensitive Staphylococci," Journal of clinical microbiology, vol. 48, no. 3, pp. 785-790, 2010.
[9] H. Hatoum et cl, "The attributable clinical and economic burden of skin and skin structure infections in hospitalized patients: a matched cohort study," Diagnostic Microbiology & Infectious Disease, vol. 64, no. 3, pp. 305-310, 2009.
[10] J. H. Melendez et cl, "Real-time PCR assays compared to culture-based approaches for identification of aerobic bacteria in chronic wounds," Clinical Microbiology and Infection, vol. 16, no. 12, pp. 1762-1769, 2010.
[11] J. Vincent et al, "Rapid diagnosis of infection in the critically ill, a multicenter study of molecular detection in bloodstream infections,," Crit Care Med. 2015;43(11):2283-91, vol. 43, no. 11, pp. 2283-91, 2015 . doi: 10.1097/CCM.0000000000001249.
[12] A. Bacconi et al, "Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood," Journal of Clinical Microbiology, vol. 52, no. 9, pp. 3164-3174, September 2014.
[13] J. Bartlett, "John Bartlett's Game Changers in Infectious Disease: 2011," Medscape, 26 Oct. 2011, www.medscape.com/viewarticle/751959.
Example 2: Potential Covariates for Hybrid Study
[00105] Patient History
A. How long has wound been present
B. Treatment history to date
C. What types of health-care providers have been involved in the management of the wound
D. History of previous wounds
E. Type of wound
[00106] Co-morbidities - Patient's capacity to heal can be limited by specific disease effects on tissue integrity and perfusion, mobility, compliance, nutrition and risk for infection.
A. Diabetes
1. abnormal glucose levels are not compatible with wound healing
2. decreased sensation in feet high risk for breakdown B. Vascular
1. Coronary Artery Disease - decreased circulating oxygen
2. Congestive Heart Failure - edema in lower extremities
3. Peripheral Vascular Disease - poor perfusion
C. Cancer
1. Radiation - high risk or may cause skin breakdown
2. Antineoplastic medications impair wound healing
D. Autoimmune diseases
[00107] Systemic factors affecting healing
A. Age
B. Vital signs
C. Polymicrobial or single microbe infection
D. Medications - Any immunosuppressants (i.e Prednisone, Tamoxifen, non-steroidal antiinflammatory drugs)
E. Lab data - CBC, comprehensive metabolic panel, c reactive protein, erythrocyte sedimentation rate, hgb Ale (if available), lymphocytes, T-cell count, CD A, cellular immunity
F. Nutritional Deficiencies
1. Weight loss (>10% involuntary weight loss in previous 6 months)
Example 3: A Single-Center Observational Study to Assess the Use of PCR in Pathogen Identification Compared to Culture-Based Test Methods and the Potential Impact on Antimicrobial Treatment, Clinical Outcomes, and Hospital Charges in Patients with Skin and Soft Tissue Infections
1 Purpose of the Study
[00108] The purpose of the study is to assess the use of PCR in pathogen identification compared to culture based test methods and the potential impact of using PCR on antimicrobial use, clinical outcomes, and financial charges in patients with skin and soft tissue infections (SSTIs).
2 Background and Rationale
[00109] SSTIs are a common problem for patients in the hospital setting and their prevalence has increased significantly since the mid-1990s. The increasing prevalence of SSTIs demands improving the efficacy and efficiency in the treatment and management of those infections. Effective treatment requires rapid and accurate identification of microbial pathogens and their resistance patterns. [00110] The traditional standard of testing (e.g. culture based methods) for pathogenic
microorganisms in SSTIs is suboptimal in providing rapid identification and susceptibility testing [1]. Traditional methods involve the use of selective media to culture aerobic and anaerobic bacteria and fungi. The process to facilitate the incubation of microbial pathogens takes a minimum of 24-48 to yield qualitative and semi-quantitative assessments of the cultures [2]. Although anaerobic bacteria often constitute a significant proportion of the total microflora in wounds, their culture and isolation is prolonged and more resource demanding than cultures of aerobic bacteria, and consequently, anaerobic microbiology is often excluded from a routine analysis [2]. Such delays in providing definitive microbial diagnosis make it difficult for the treating clinician to narrow antibiotic therapy to correctly treat the infection and reduce antibiotic resistance.
[00111] Rapid and accurate identification of microbial pathogens in patients with SSTIs and infected chronic wounds can potentially provide improved outcomes through more timely and pathogen specific treatment. Benefits can include more efficient utilization of antimicrobial medications, more rapid resolution of infection, faster healing, and a reduction in patient mortality, as well as reduced tissue and limb loss. This in turn can provide enhanced health economic outcomes, such as reduced expenditure from use of fewer antimicrobials, reduction in the potential for microbial resistance development, subsequent avoidance of hospitalizations, shorter length of stay for hospitalized patients, reduction in total cost of care, fewer surgeries, and fewer hospital readmissions.
[00112] PCR based methods may provide similar benefit and could be a potential game changer in the pathogen identification in patients with SSTIs and lead to improving the efficacy and efficiency in the treatment and management of those infections.
[00113] To meet the unmet need in the treatment and management of patients with wound and SSTIs, Millennium Health is developing a PCR based method using taqman-type assays (a molecular genetic assays method which may be referred to as a Wound Genetic Test, WGT) to detect microorganisms, antibiotic resistance genes, and virulence factors commonly occurring in patients with wounds and SSTIs which were pre-selected to constitute Millennium Health's WGT panel.
[00114] This study aims to quantify the unmet need in the detection of the pathogens that cause skin and soft tissue infections and to create an economic simulation based on the timing of initiation of antimicrobial therapy and availability of culture results. The study can also assess the use of molecular methods in pathogen identification compared to culture-based test method and the potential impact of using molecular methods on antimicrobial treatment, clinical outcomes, and financial charges in patients with SSTIs. 3 Objectives of the Study
[00115] Primary Objectives
1. To compare the performance characteristics of WGT with that of culture-based tests used in standard-of-care for patients with SSTIs as measured by sensitivity, specificity, agreement and timing of the results.
2. To examine the antimicrobial treatment patterns (e.g. time from specimen collection to first antimicrobial treatment, switch pattern of antimicrobial treatment) in patients with SSTIs.
3. To explore the relationship between timing of availability of culture results, antimicrobial treatment, and study endpoints listed in Section 4.2.
4. To explore if the existence of virulence factors as detected by WGT impacts the study outcomes.
Secondary Objective
1. Comparison of culture and WGT results from additional specimens collected from the same subject.
4 Study Design:
4.1 General Study Design.
[00116] This is a prospective, single-center observational study that can take place at a study site to assess the use of PCR based assays in pathogen identification compared to a culture-based test method and the potential impact of using PCR based assays on antimicrobial treatment, clinical outcomes, and financial charges in patients with SSTIs. Other than specimen collection, this study can have no intervention that impacts the standard practice of medicine at the study site.
[00117] Patients who meet the inclusion criteria at screening can be approached for the possibility of enrolling in the study. Patients who sign the informed consent form (ICF) can be enrolled for information and specimen collection. Two specimens (specimen A and specimen B) can be collected from each patient per specimen collection event.
[00118] Specimen B can be shipped to Millennium Health for testing using the PCR based assay and Specimen A can be tested using culture-based method at study site's clinical pathology laboratory as part of standard of care. If a subject has more than one culture test ordered during their initial hospitalization or is readmitted to study site with an SSTI, then more than one specimen collection event can occur per subject.
4.2 Outcomes and Cost Endpoints
[00119] The outcomes and cost endpoints are: 1. Sensitivity, specificity, and agreement of WGT and culture results
2. Time (hours) from baseline (see section 6.2) to the final test results
3. Time (hours) from baseline (see section 6.2) to first antimicrobial treatment
4. Difference between time of availability of WGT and culture test results
5. Length of stay (LOS)
6. 30-day readmission rates
7. Total cost of inpatient care for hospitalization (does not include cost of hospitalization for readmission)
8. Antimicrobial treatment patterns
9. Mortality during hospitalizations
10. Rates of amputation
4.3 Anticipated Duration of the Study:
[00120] The anticipated duration of the study is 12 months with an expected enrollment period of 6 months. We estimate that the enrollment period can be 6 months in order to capture a sufficient number of specimen types.
4.4 Study Population
[00121] Patients are eligible for the study if the following criteria are met.
4.4.1 Inclusion criteria
a. All patients who present with a skin and soft tissue infection
b. Patient or legal representative has given written informed consent
c. Patient has an order for a skin or soft tissue infection culture
d. Patients must be 18 years of age or older
4.4.2 Exclusion criteria
a. Patients from whom an additional sample of their infected skin or soft tissue cannot be provided to Millennium Health for testing at the time of collection
4.5 Subj ect Withdrawal
[00122] The subject may withdraw their participation at any time after enrollment, upon which their SSTI specimen can be destroyed and any collected electronic information can be removed from the study database. However, if the subject withdraws after statistical analysis for the study has started, the patient's SSTI specimen can be destroyed, but the sponsor (Millennium Health) can not have any obligation to destroy summary statistics that include data obtained from the patient's sample and study data. 5 Study Procedures:
5.1 Screening and Enrollment Procedures
[00123] Subjects can be screened on the basis of the criteria in section 4.4. The study nurse can confirm via the enrollment log that the subject has not been previously enrolled in the study. If the subject had been previously enrolled in the study, the nurse can proceed directly to specimen collection since the patient had already consented. The study nurse can use the same Subject ID that the subject had been assigned when they were originally enrolled in the study and can indicate which Specimen Collection Event occurred (i.e. Specimen Collection Event 1 is the first time specimens are collected from a subject, Specimen Collection Event 2 is the second time a specimen is collected from the same site).
[00124] If a subject has not been previously enrolled and are deemed to be eligible for participation, they can be educated on the study procedures, risks, and the molecular test used to analyze the specimen, and the voluntary nature of study participation. If the patient is interested in participating in the study, they can be asked to sign an ICF. This informed consent can be discussed with the study nurse who can be available for questions.
5.2 Information Collection
[00125] Case Report Form(s) can be collected for each subject enrolled in the study. The following information can be collected:
Baseline information collected upon enrollment
1. Date and time of specimen collection
2. SSTI characteristics
3. SSTI location on body
4. Any antimicrobial therapy within 30 days prior to hospitalization
5. Any hospitalizations within the previous 30 days
6. Any prior treatment of SSTI before hospitalization
7. Any antimicrobial therapy initiated during hospitalization prior to specimen collection
Information collected after final culture results are available in subject's EMR
1. Final culture results from Specimen A (results must be marked final in EMR)
5.3 Specimen Collection and Shipment
[00126] The study can be conducted on swab specimens tested using MH's standard clinical laboratory procedures. [00127] Once an order for an SSTI culture has been placed, a study nurse can approach the patient and screen for eligibility. The study nurse can consent the prospective subject, if eligible, and can collect the specimen(s). An additional swab can be collected for analysis at Millennium Health in addition to the swab that was collected for analysis using culture methods. Both specimens can be collected from the same location of the SSTI, at the same time. If a subject has more than one SSTI on their body eligible for inclusion, the study nurse can select only one SSTI to collect the two specimens from and all other SSTI locations can be excluded from the study. The nurse may collect any additional specimens from the other SSTI locations per standard of care.
[00128] The two specimens can be designated in this study as specimen A and specimen B (See section 5.5 for additional specimen labeling information). Specimen A can be sent to the study site's clinical pathology laboratory for testing using standard culture procedures and analysis. Specimen B can be sent to Millennium Health using a provided collection device along with a study specific requisition form. The requisition form can be filled out according to the instructions provided to the study team and can be sent with Specimen B to Millennium Health.
[00129] In addition to specimen collection, the information listed in section 5.2 can also be collected during the consent process and recorded on a single Case Report Form (CR ). If a patient does not consent to participate in the study, the study nurse can only collect the specimen to be cultured at study site's clinical pathology laboratory and no further information or interaction with the patient regarding the study can occur.
[00130] Specimen B can be shipped to Millennium Health for analysis and specimen A can be analyzed and cultured at study site's clinical pathology laboratory using standard methods according the physician's order. All patient identifiable information can be removed from specimen B before it is sent to Millennium Health.
[00131] Specimens and their corresponding requisition forms that can be sent to Millennium Health can be packaged and shipped in accordance with the designated commercial carrier's requirements for clinical samples. Each specimen B can be shipped on a daily basis to Millennium Health, unless otherwise indicated by the study sponsor. Designated study personnel can ship specimens to
Millennium Health using a courier service in packaging provided by the study sponsor.
[00132] Following completion of the analysis of specimen A at the study site and the final report is uploaded to the subject's EMR, the study nurse can access the subject's EMR to fill out the final portion of the CRF. The study nurse or designated study team member can send copies of completed CRFs to Millennium Health on a weekly basis. Each specimen shipped to Millennium Health must have an accompanying completed CRF (shipped separately), including specimens from additional specimen collection events. In addition, CRFs must be returned for samples that were unable to be tested in the hospital due to any error in handling, failure to generate a result, or other unexpected event.
[00133] In the event that Millennium personnel receive a specimen or CRF results that contains any patient identifiable information, the specimen and patient information can be destroyed. Only study staff at study site can have access to patients' coded private information and, therefore, can be responsible for maintaining the privacy of patients and protecting the confidentiality of patients' identifiable information.
[00134] Each specimen's extracted microbial DNA, once examined with WGT, may be used specifically and strictly for research purposes as part of the Millennium Health WGT Biobank & Database for a period of 5 years. The DNA samples can be stored at Millennium Health in a freezer with restricted, employee only access.
[00135] As this research involves the patient's de-identified EMR data set and Hospital Charge Master data can be stored in a secure database for up to 6 years to be maintained at Millennium Health. Each patient's WGT results can be stored in Millennium Health's proprietary laboratory information system (MLIS) indefinitely per company policy and may be used for future research purposes at Millennium Health. Confidentiality can be strictly maintained.
[00136] All subjects' biological specimens can be destroyed by Millennium Health after the WGT analysis has been completed and the specimens are no longer needed. All original specimen swabs can be destroyed following completion of the study.
5.4 EMR and Charge Master Data
[00137] A subset of the EMR data and associated Hospital Charge Master Data for the enrolled patients can be extracted as specified in the Data Dictionary in Appendix A and can include data 30 days post discharge for each subject. A de-identified data set can be included in the subset of data and each subject can only be identified by the SID assigned upon enrollment, which can link the associated EMR, Charge Master Data, culture results, and WGT results to that subject.
[00138] The de-identified data set used in the study can be pulled by study site Clinical Informatics department using the Honest Broker process. During the study, study site can observe and record information on patients who provide informed consent. Data can be recorded and entered, compiled and analyzed to determine the key outcomes previously defined. Confidentiality can be strictly maintained. The study personnel can also be trained to avoid recording any personal identifiable information on the case report forms by inadvertence. Any clinical and charge data can be sent to Millennium Health for analysis every month until the completion of the study.
[00139] Upon receiving the de-identified data set, the patient's WGT results and culture results can be merged with the patient's EMR and Charge Master data using the assigned SID and SID B. Only the on-site study team can have access to patients' coded private information and, therefore, can be responsible for maintaining the privacy of patients and protecting the confidentiality of patients' identifiable information. The study team at Millennium Health can have no access to any patient PHI. 5.5 Subject and Specimen Labeling
[00140] Two specimens (specimen A and specimen B) can be collected from each enrolled subject during each specimen collection event and subsequent specimen collection events (i.e. in the case of a subject having more than one culture ordered for their SSTI during their hospitalization period or if a subject was readmitted to the study site during the study enrollment period).
[00141] A designated study team member can keep and record specimen collection entry into the log file. When a subject is enrolled and the specimens are to be collected, the designated person can assign the next SID to specimen B and can fill the medical record number with the subject's medical record number in the EMR, print, and sign his/her name. The study team member can attach the pre-printed label with the SID on Specimen B. The SID is the sequential number from 00001 to 99999. If a subject has an additional specimen collected at a 2nd, 3rd, etc. specimen collection event, the team member collecting the specimens can add a dash, then the number of the Specimen Collection Event after the dash. This can be recorded in the Specimen Collection Log and on the labels that are placed specimens A and B. If the specimen collection event number is >1, the study nurse can refer to the prior CRF recorded during specimen collection event number 1 to ensure that the additional specimens are taken from the same location as the original collection.
[00142] The study team member can apply pre-printed labels with the Subject ID to specimen B. If the specimen collection event is a second, third, etc. event, the study team member can write in the specimen collection event number following the SID (e.g. B0002-2) on the label that is applied to the specimen.
[00143] The study team can send the Specimen Collection Log file to the Clinical Informatics team once per week.
[00144] When study site transfers the EMR and Charge Master data to Millennium Health, the medical record number in the EMR and Charge Master data can be replaced with the SID. The specimen collection log file with the medical record number removed can be provided to Millennium Health together with the de-identified, de-identified EMR data set and charge master data (See Appendix A for the data to be extracted from the EMR and Charge Master files) at least 30 days post discharge. Study site can transfer the EMR and Charge Master data to Millennium Health every month until the completion of the study and at least 30 days post discharge of the last patient enrolled.
5.6 Study Treatment or Diagnostic Product Procedures.
[00145] No clinical decisions can be made based on the results generated by Millennium Health's PCR based results and patients can be treated according to the current standard of care defined by their physicians.
5.6.1 WGT Description
1. A collection device comprised of:
(1) Transport media formulated to maintain the integrity of DNA present in the clinical specimen.
(2) A vessel to hold the clinical specimen with transport solution
(3) A label with the subject identifier and collection data
2. A DNA extraction process: see Example 5 for details.
3. A DNA preparation process comprised of:
(1) An assay to measure DNA concentration in the sample.
(2) An aliquot of the DNA extracted from the microorganisms in the specimen may be tested using sequence analysis. The specimen can be prepared for sequencing using published standard operating procedure and analyzed with specialized software for microorganism specification.
4. One or more qPCR protocols comprised of:
(1) A pre-determined list of target DNA sequences which may include organism identification sequences, antibiotic resistance sequences, virulence factor sequences.
(2) An enrichment PCR which amplifies these specific sequences.
(3) One or more TaqMan assays for each specific sequence
(4) An instrument with which to detect the amplification curve of the TaqMan assays.
(5) A data review process in which a qualified user operates the data analysis software packaged with the detection instrument to label samples, label assays, set cycle thresholds for each assay and export labels with Ct values as text.
5. A data interpretation pipeline comprised of:
(1) An algorithm to interpret the Ct values exported for each sample-assay combination and determine the presence or absence of each assay target in each sample.
(2) An algorithm to qualify controls used in each run. A clinical report Statistical Method
[00146] SAS® 9.4 or later can be used to generate all analyses, tables, listings, and figures. If not otherwise specified, the number of observations, mean, standard deviation, median, minimum, and maximum values can be provided for all continuous measurements. For categorical data, the number and percentage in each category can be reported. Missing values for categorical/ordinal data can be counted in a separate category.
6.1 Analysis population
[00147] All enrolled patients from whom the first pair of specimens are collected with enough material from the same SSTI location at the same time. Culture results are available for the testing on the specimen sent to study site's lab.
6.2 Baseline
[00148] The first pair of specimen collection time (hour 0) is the baseline for this study. The baseline time is denoted as TBL.
6.3 Characteristics of WGT compared to culture-based method
6.3.1 Performance characteristics
[00149] PCR based test results on specimen B and culture-based test results on specimen A at the baseline can be used for the comparison of the test characteristic.
[00150] For each combination of those bacteria/fungi/ microbial genes any one of Tables 1-11, the combinations of the possible results are listed and denoted as below.
Figure imgf000049_0001
[00151] The numbers and proportions of pairs of specimens in each of above cells can be summarized.
[00152] WGT Sensitivity (=nn/ni.) and specificity (=n00/n0.) compared to culture-based test can be calculated.
[00153] Agreement (= (nu + n00)/n .) and the two-sided 95% confidence interval can be provided.
[00154] Cohen's kappa can also be reported. 6.3.2 Operational characteristics
[00155] The time (hours) from baseline to the final test results for both the PCR based (WGT time) and culture-based test (CUL time) for the specimens collected at baseline can be analyzed
descriptively.
[00156] The descriptive statistics of the time difference (hours) of the two test results
(Test_T_DIFF=CUL_time - WGT time) can also be provided together with its 95% two-sided confidence interval.
6.4 Patterns of antimicrobial treatment
[00157] Antimicrobial treatment can be analyzed at the generic name level.
6.4.1 Number and proportion of patients treated with antimicrobial
[00158] Number and proportion of patients treated with any antimicrobial (including prior to or after the hospital admission) can be summarized overall, by each individual microorganism, by
polymicrobial infection, or class (anaerobes, aerobes, gram positive, gram negative). The dosage and route of administration can be noted.
6.4.2 Time (hours) from specimen collection to the first antimicrobial treatment
[00159] The first antimicrobial treatment prior to or after hospital admission can be denoted as Drugi (The start time T1 of Drugi can be before or after the hospital admission). Time from specimen collection to the Drugi use (STRT TMl) can be summarized overall and by antimicrobial generic name.
6.4.3 Antimicrobial switch
[00160] The subsequent administration of any antimicrobial different from Drugi (see Section 6.4.2) can be denoted as Drug2; the subsequent administration of any antimicrobial different from Drug2 can be denoted as Drug3 etc. Any change from Drugxto Drugx+i is an antimicrobial switch.
[00161] The corresponding start time for those treatments can be denoted as STRT TMl,
STRT TM2, and STRT TM3 etc.
[00162] Kaplan-Meier survival curves can be used to display the Time to the 1st antimicrobial switch (STRT TM2) overall and by each individual microorganism or polymicrobial infection. Survival probability estimates can be calculated at continuous time points up to the maximum discharge time to be observed in the data overall and by each individual microorganism or polymicrobial infection.
[00163] The number and proportion of patients can be summarized for each switch pattern (no switch, Drugi.> Drug2; Drugi.> Drug2 _> Drug3; etc.) by each individual microorganism or polymicrobial infection. [00164] Changes in dosage and/or route of administration can also be included in the analysis.
6.5 Relationships among timing of availability of culture results, detected microorganism/susceptibility, antimicrobial treatment, and study endpoints listed in Section 4.2.2
6.5.1 Relationship among timing of availability of culture results, detected microorganism/susceptibility, and study endpoints for outcomes and hospital charge
[00165] For continuous endpoints, scatter plots can be generated to display the relationship between CUL time (original or bin) and each of the endpoints (original or binning data) by each individual microorganism or polymicrobial detected.
[00166] Generalized linear regression models for the continuous endpoints can be explored, the predictors to be included can be CUL time and other factors or covariates such as comorbidity, age, etc.
[00167] For category endpoints, the number and proportion of patients in each level of the category variables can be can be summarized by bins of CUL time and each individual microorganism or polymicrobial infection detected.
[00168] Logistics regression models for the category endpoints can be explored, the predictors to be included can be CUL time and other factors or covariates such as comorbidity, age, etc.
6.5.2 Relationship among timing of availability of culture results, detected microorganism/susceptibility, and antimicrobial treatment
[00169] Scatter plots can be generated to display the relationship between culture test result time (original or bin) and the first antimicrobial treatment time (STRT TMl, original or binning data) by each individual microorganism or polymicrobial infection, and by antimicrobial generic name and each individual microorganism or polymicrobial infection.
[00170] Time difference between the first antimicrobial treatment and the culture test result time (= STRT TMl - CUL time) can be summarized by each individual microorganism or polymicrobial infection, and by antimicrobial generic name and each individual microorganism or polymicrobial infection.
[00171] For those with an antimicrobial switch, the above analyses can be conducted to show the relationship between culture test result time and the first antimicrobial treatment switch time.
6.5.3 Impact of appropriate antimicrobial treatment on study endpoints for outcomes and hospital charge
[00172] By examining the microorganisms/susceptibility detected by both the WGT test and culture test and the antimicrobial treatments that the patients received, antimicrobial treatment appropriateness can be defined. The study endpoints for outcomes and hospital charges can be summarized and compared between groups of different antimicrobial treatment appropriateness using generalized linear model for continuous outpoints and logistic regression model for category outpoints with antimicrobial treatment appropriateness, CUL time, and other factors or covariates such as wound type,
comorbidity, age, etc. as predictors.
6.5.4 Impacts of the existence of virulence factors as detected by WGT on study endpoints for outcomes and hospital charge
[00173] Descriptive statistics for outcomes and hospital charge can be calculated by virulence factor group (with/without virulence factors) detected by WGT.
[00174] Box plots or bar chart can also be created for outcomes and hospital charge.
[00175] Generalized linear regression models or logistics regression models can be explored with virulence factor group etc. as a predictor.
7 References
1. Bauer, K. A. et al. "Review of Rapid Diagnostic Tests Used by Antimicrobial Stewardship Programs." Clinical Infectious Diseases, vol. 59, no. suppl 3, 2014, doi: 10.1093/cid/ciu547.
2. Bowler, P. G. et al. "Wound Microbiology and Associated Approaches to Wound Management." Clinical Microbiology Reviews, vol. 14, no. 2, Jan. 2001, pp. 244-269. doi: 10.1128/cmr.14.2.244-269.2001.
3. Vincent, Jean-Louis et al. "Rapid Diagnosis of Infection in the Critically 111, a Multicenter Study of Molecular Detection in Bloodstream Infections, Pneumonia, and Sterile Site Infections*." Critical Care Medicine, vol. 43, no. 11, 2015, pp. 2283-2291. doi: 10.1097/ccm.0000000000001249.
4. Bauer, Karri A. et al. "An Antimicrobial Stewardship Program's Impact with Rapid Polymerase Chain Reaction MethicillinDResistant Staphylococcus Aureus / S. Aureus Blood Culture Test in Patients with S. Aureus Bacteremia." Clinical Infectious Diseases, vol. 51, no. 9, 2010, pp. 1074-1080. doi: 10.1086/656623.
5. Bartlett, John. "John Bartlett's Game Changers in Infectious Disease: 2011." Medscape, 26 Oct. 2011, www.medscape.com/viewarticle/751959.
Example 4: Millennium Health Wound Infection Test Health Economics and Outcomes Study 1. Introduction:
[00176] Current "gold standard" techniques for detecting microbial infections in wounds require incubation and growth of organisms in predetermined/selective culture media under controlled laboratory conditions. Such culturing techniques can be error-prone and unreliable due to their low sensitivity for some pathogens. Moreover, because selective growth media is required, testing must be hypothesis-driven rather than a generalized approach. Additionally, turn-around time (TAT) for culture results can be slow, often requiring days or sometimes weeks, particularly for polymicrobial infections composed of microbes with dramatically different growth rates. In a study involving six U.S. hospitals in 2009 and 2010, only 59 percent of patients received appropriate cultures, and by the fifth day of therapy, 66 percent of antimicrobial therapy regimes were unchanged, despite negative cultures in 58 percent of patients. In 2010, 56 percent of hospitalized patients in 323 hospitals across the United States received an antibiotic during their stay, often broad-spectrum agents. Among patients who received an antibiotic, 37 percent of treatments could have been improved, primarily through better use of diagnostic tests. This challenge leads to potential inappropriate use of antibiotics which leads to issues with resistance development, increasedcost, and medical errors.
[00177] Millennium Health recognizes the growing problems of antibiotic overuse, antibiotic resistance, and pathogen identification. The Millennium Health DxWound product analyzes DNA isolated from microbes present in various types of wounds using real-time polymerase chain reaction (PCR) technology. The isolated DNA is used to identify pathogens (i.e. bacterial and fungal organisms) present as mono- or polycultures within chronic wounds, as well as antimicrobial resistance signatures and virulence factors that may be carried by the microbes. This product may complement and in some clinical cases, replace, current techniques involving culturing of infected specimens followed by susceptibility testing. The product can be offered as a single test that can be used to detect the presence of >20 bacterial and fungal pathogens in a single specimen. Rapid identification of microbes, along with antibiotic resistance signatures and virulence factors can allow for faster, more precise selection of antibiotic therapies that are most likely to effectively treat the infections and help to reduce antibiotic waste, delays in treatment of needed antibiotics, and the use of expensive antibiotics. This has significant potential to improve patient quality and healthcare outcomes.
[00178] MH's Health Outcomes and Economic Study aims to quantify the unmet needs in the current standard of care for the identification of pathogens that cause wound and surgical site infections. The multi-phase study can measure the value of the test using a number of economic and clinical outcomes, including the potential for reduction in cost of hospital related care and complications, antibiotic spend, incidence rates of antibiotic waste, and improvement in quality of care.
2. Multi-Phase Study Objectives:
[00179] The study can help to determine if MH's molecular diagnostic test for pathogenic
microorganisms could solve unmet needs in the identification and treatment of wound and surgical site infections in the hospital environment. MH can compare its molecular diagnostic test to the current gold standard of care to assess the value based on the clinical and economic outcomes listed above.
[00180] The study can take place in three phases:
1. Pilot Phase 2. Budget Impact Model (BIM) Phase and Supporting Retrospective Study
3. Prospective Phase
[00181] The first phase is a pilot study designed to evaluate the assay as a method for identifying a pre-selected list of potential pathogens, virulence factors, and antimicrobial resistance genes in specimens obtained from wounds. The study can measure the accuracy rate of pathogen identification for the WGT vs the culture method as well as to define the logistics for preserving, transporting, and analyzing the specimens for future phases of the study. This phase is exploratory in nature and the results can be used to design and power the BEVI and retrospective portions of the study.
[00182] Phase two involves a BIM and Supporting Retrospective Study that can both focus on subsets of patients where MH can demonstrate the greatest unmet need with the highest potential to benefit from the new technology. The BEVI can be designed to allow hospital administrators and decision makers to interactively explore their costs of care for patients with wound infections and demonstrate potential savings if they were to use MH's PCR based test as a supplement to the standard of care. The retrospective study can serve as means to provide clinical and economic outcomes data for the BIM.
[00183] Phase three can include an evidence-based, prospective study that can incorporate the genetic test into the standard provision of care. The prospective study can randomize patients with wound infections into either a "research arm" or a "control arm": the control arm can identify the wound infection using current standard of care techniques and the research arm can identify the pathogen using the PCR based test in addition to the standard of care. The study can evaluate the clinical decisions made by clinicians in each arm and additional clinical and economic outcomes can be measured, including hospital related care and complications, antibiotic spend, incidence rates of antibiotic waste, and quality of care measures.
3. Research Methodology for Multiphase Study:
3.1 Pilot Phase
[00184] Objectives: Optimize and validate the equivalency of the WGT methods in comparison with the current standard microbiological culturing techniques that are used to identify and characterize potential disease causing pathogens in patients with wound and surgical site infections. MH has developed a molecular genetic test for organism identification, antibiotic resistance, and virulence factor identificationthat is targeted toward wound and surgical site infections. This development requires testing on clinical samples in order to refine, optimize and validate the test. Therefore, MH is in need of patient samples to compare the results of the PCR based test to the current gold-standard method of testing. [00185] Specimens used in the pilot study can be obtained from patients admitted who have any wound and/or surgical site infections. The specimens can be collected using the current standard of care, however MH can want to know what technique was used to collect the specimen and what type of wound it was taken from. MH can obtain an additional specimen collected from patients during the course of normal care for testing at MH on the PCR assay. MH can also obtain the culture results and compare these to the results from the genetic test. MH would like to obtain 200 samples with no patient identifiable information for this purpose.
3.2 BIM and Supporting Retrospective Study Phase:
Objectives:
BIM
■ Provide a framework to evaluate the financial impact if the PCR test were to be used in the hospital setting
■ Provide decision makers with the ability to interactively compare costs and potential for savings between different scenarios involving the use of the standard of care and the PCR test.
Supporting Retrospective Study
■ Provide clinical and economic outcomes data for use in the BFM
■ Retrospectively identify opportunities to avoid waste and improve patient care with the use of PCR based testing and evaluate the test's effects on the costs of hospital related care and complications
■ Retrospectively evaluate if MH's test could more accurately and rapidly determine a more effective therapeutic course than the culture method
■ If possible individualize the model outcomes based on input variable unique to the local hospital or setting
[00186] The Budget Impact Model (BIM) can be developed in parallel to the Supporting
Retrospective Study and it can allow hospital decision makers to quantify their financial exposure if they were to use MH's PCR based test as a supplement to the standard of care in pathogen
identification of wound infections. The BIM can include subsets of patients who would have the potential to benefit from the new technology. For example, such patients could include those who have compromised immune systems, complex surgeries, or resistant diabetic infections (2). The list of patient groups can be identified through a literature review of studies that evaluate patients who are at high risk of developing a wound or surgical site infection and which pathogens would most likely cause those infections. The BIM can incorporate incidence rates obtained from a company that collects prescription and healthcare data, such as IMS, into the model to demonstrate value for outcomes such as reduced overall hospital related costs, readmission rates, and spending on expensive antibiotics. The outcomes that can be measured in the BIM and The Retrospective study can be determined by the results from Phase 1. The Retrospective Study can serve as a means to provide clinical and outcomes data for use in the BIM.
[00187] The Retrospective Study phase can also select subsets of patients where the need to have an accurate and rapid wound microbial identification test is the greatest and for whom the test can demonstrate the maximum economic value. Similar to the Pilot Phase, MH can obtain the unused remnants of wound and surgical site infection specimens from patients admitted to the study site. The specimens can be collected using the current standard of care and can be split into three parts, A, B, and C; Part A can be analyzed using the culture method, Part B can be analyzed using MH's wound infection genetic test, and Part C can be stored for future testing if there is a variation between the results from Parts A and B. MH can be evaluating the accuracy and turn-around-time of each method. The study can also use electronic medical records (EMR) to retrospectively measure incidence rates of avoidable waste of antibiotic use, cost of hospital related care, complications, treatment patters, and quality care metrics for each patient.
[00188] Possible List of Inclusion Criteria:
• Patients admitted
• Immunosuppressed patients (e.g. cancer, HIV, hepatitis, etc.)
• Diabetic foot ulcers
• Chronic wounds
• Pathogens with positive culture results that are unresponsive to single or multiple courses of antibiotics
[00189] Possible List of Exclusion Criteria:
• Patients with wounds being treated in an outpatient setting
• Patients for whom culturing may be difficult
• Patients with an identified secondary infection
3.3 Prospective Phase
Objectives:
Examine the impact, if any, on clinical decision making of the MH test vs. culture.
Evaluate the impact, if any, on antimicrobial prescriptions and clinical outcomes Prospectively demonstrate the economic value of the PCR based test
[00190] The third phase of the study can be comprised of an evidence-based, prospective study that can be designed based on the results of the Retrospective Phase and Budget Impact Model. This phase can incorporate genomic testing into the standard provision of care as a supplement to the culture method. It can provide a high level of evidence to demonstrate that MH's wound and surgical site infection can provide a more rapid, precise method of pathogen identification and can help physicians to select the most effective antibiotic therapies.
[00191] The prospective study can likely comprise of the same subsets of patients that can be enrolled in the Retrospective Study and can include informed consent. The current design methodology would randomize patients with wound infections into either a "research arm" or a "control arm": the control arm can identify the wound infection using current standard of care techniques and the research arm can use PCR based testing in addition to the standard of care. The final study design and methodology can warrant further exploration and discussion following the completion of the BIM and Retrospective Studies.
[00192] The study can measure any differences in clinical decisions making between both arms. Several clinical outcomes that might be analyzed between the two groups could include how often the clinicians initially chose the most effective antibiotic, was there a reduction in the use of broad- spectrum antibiotics, and did the PCR based test help to avoid unneeded antibiotic therapy. Additional primary economic and clinical outcomes that can be measured can be determined by the results from the BIM and Retrospective studies, but can likely include hospital related care and complications, antibiotic spend, incidence rates of antibiotic waste, and quality of care measures.
Example 5. DNA Extraction, QC, Quantification and Normalization
[00193] Prepare the Pre-Amplification master mix:
Figure imgf000057_0001
[00194] Add 7.5 μL· of master mix into each well of a 96 well plate.
[00195] Add 2.5 μΐ^ of the specimens (or control) to the appropriate well.
[00196] Seal the plate, vortex and briefly centrifuge.
[00197] Thermal cycling conditions: 9 cycles
Temperature 95°C 95°C 60°C 4°C
Time 10 minutes 15 seconds 4 minutes Hold
[00198] After the Pre-Amplification program has completed cycling, add 40 μL· IX TE to each well.
[00199] Prime a 96.96 Dynamic Array™ Genotyping Chip on the IFC Controller.
Prepare the Sample Master Mix:
Figure imgf000058_0001
[00200] Add 5.8 μΐ. of Sample Master Mix to each well of a 96 well plate.
[00201] Add 4.2 μΐ. of the diluted, pre-amplification PCR product to each well.
[00202] Seal the plate, vortex for 10 mins and briefly centrifuge.
[00203] When the 96.96 Dynamic Array™ Genotyping Chip IFC has completed priming, add 6 μΐ. of the lOx Qiagen qPCR Microbial DNA assays to each Assay inlet.
[00204] Add 6 μΐ^ of the Sample Master Mix and pre-amplification PCR product mix to each Sample inlet.
[00205] Load chip onto the IFC Controller and run the 'Load Mix' (136x) script.
[00206] When the script "Load Mix" (136x) is finished, remove the loaded chip from the IFC
Controller and run on the Biomark Gene Expression PCR cycle.
[00207] After the gene expression read is complete, run and EndPoint cycle on the Biomark.
Example 6: Exemplary WGT Protocol
1. Specimen Handling and Shipping
[00208] A specimen collection kit can be provided to participating sites for wound sample collection. The specimen collection kit can comprise of a microbiology specimen bag containing:
i. Sample accessioning form
ii. Swab
iii. Disposable Dermal Curette
iv. Buffer [00209] Wound or other specimens can be collected using the Disposable Dermal Curette. The specimen can be placed in the Lysis Tube and the lid secured in place. The collected specimen can be placed in the microbiology specimen bag. The appropriate fields can be completed in the accessioning form. The form can be placed in the document specific pocket of the specimen bag. The specimens can be shipped to MH, according to standard specimen handling protocols. The specimen can be accessioned at MH and stored at 4°C until sample processing.
2. Procedure Guidelines
Pathogen DNA can be extracted from the specimen
Data can be generated using Fluidigm Biomark HD System (Fluidigm, San Francisco, CA) on the 96.96 Dynamic Array Gene Expression Chip
Unless specified, all buffers and components required can be provided in the kits used.
3. DNA Extraction
(1) Place the DNase/RNase free water used for the final elution at 60°C
(2) Add 100 μΐ. Beta-Mercaptoethanol (Sigma- Aldrich, St. Louis MO) to 19,900 DNA Binding Buffer.
(3) Disrupt the specimen in aTissueLyser fitted with a 2 mL tube holder assembly.
(4) Remove the Lysis Tube from the tissue lyser and centrifuge
(5) Snap off the base of a Spin Filter and insert into a collection tube (one per sample).
(6) Transfer supernatant from the Lysis Tube to the Spin Filter and centrifuge
(7) Place filtrate in a clean new 1.7 mL Eppendorf tube.
(8) Transfer the remaining supernatant from the Lysis Tube to the Spin Filter and centrifuge
(9) Add this filtrate to the filtrate already transferred to the clean new 1.7 mL Eppendorf tube.
(10) Add DNA Binding Buffer to the filtrate (from steps 6 and 8).
(11) Transfer 800 μΕ of the mixture from Step (7) to a Column in a new collection tube and centrifuge Discard the flow through
(12) Repeat Step 10 until all filtrate has been passed through the column.
(13) Add 400 μΕ DNA Wash Buffer 1 to the Column in a new collection tube and centrifuge at 10,000 x g for 1 minute. Discard the flow through.
(14) Add 700 μL· DNA Wash Buffer 2 to the Column in a collection tube and centrifuge at 10,000 x g for 1 minute. Discard the flow through.
(15) Add 200 μΕ DNA Wash Buffer 2 to the Column in a collection tube and centrifuge at 10,000 x g for 1 minute. (16) Transfer the Column to a clean 1.5 mL microcentrifuge tube and add 50 μL· heated DNase/RNase Free Water directly to the column matrix and incubate for 1 minute. Centrifuge at 10,000 x g for 1 minute to elute the DNA.
(17) Prepare the Spin Filters, one per sample: Snap off the base of the Spin Filter and place into a new collection tube. Centrifuge at 8,000 x g for 3 minutes. Discard the flow through.
(18) Transfer the eluted DNA from Step (13) to a prepared Spin Filter in a clean 1.5 mL microcentrifuge tube. Loosely cap the Spin Filter and centrifuge at 8,000 x g for 1 minute. This is your final microbial DNA eluted product.
4. Sample Processing
Prime the 96.96 Dynamic Array™ Gene Expression Chip IFC.
Inject Control Line Fluid into each accumulator on the Chip plate.
Place the chip into the Integrated Fluidic Circuit (IFC) Controller
Using the IFC Controller software, run the script "Chip Prime" (138X for 96.96 Chips) to prime the control line fluid into the chip.
5. Prepare the Sample Master Mix
[00210] Mix the components listed below in a 1.7 mL vial:
Figure imgf000060_0001
Add 5.8 μΕ Sample Master Mix to each well of a 96 well plate
Add 4.2 μΐ^ final microbial DNA eluted product to the aliquoted Sample Master Mix.
Add positive and negative controls samples to pre-determined wells.
Votex the plate for >5 minutes at 2,000 rpm
6. Prepare the lOx Assay Plate
Add equal volume Assay Loading Reagent to each 20x pre-prepared Microbial DNA qPCR Assay. Plate onto a 96 well plate in a predetermined assay plate layout.
See Table 18 for Assay list and Assay Plate well coordinates.
7. Loading of Assays and Samples in to Chip using the IFC
Retrieve the primed chip from the IFC Controller Add 6 μΐ. of each sample from the Sample Master Mix plate into the appropriate wells of a 96.96 Dynamic Array™ Gene Expression Chip IFC.
Add 6 μΐ^ from the lOx Assay Plate into the appropriate wells of a 96.96 Dynamic Array™ Gene Expression Chip IFC.
Return the loaded chip to the IFC Controller
Using the IFC Controller software, run the Script "Load Mix" (136x) to load the samples and assays into the chip.
When the script "Load Mix" (136x) is finished, remove the loaded chip from the IFC Controller.
8. Generate Amplification Data on Biomark
Launch the Biomark HD Data Collection Software
Click "Start a New Run."
Remove protective "Blue Film" underneath the chip before loading.
Place the chip into the instrument tray.
Set the run parameters for GE WGT in the BioMark software.
Start Run
9. Data Analysis
Analyze the data in the Fluidigm Real-Time PCR Analysis Software
Define the ' Sample Setup' and 'Detector Setup' fields using the appropriate sample and assay plate maps
Generate amplification curve data by clicking the "Analyze' button.
Determine the quality of the run by evaluating the passive ROX signal, the positive and negative control samples and the Pan Assays.
Evaluate amplification curves for each sample against each assay.
Determine whether the amplification curve is indicative of a positive signal for a given assay using a predetermined set of evaluation criteria related to amplification efficiency and CT value.
Report results for each sample.
Table 13 : Assay List and Type
Figure imgf000061_0001
Bacteroides fragilis Bacterial Strain ID
Citrobacter freundii Bacterial Strain ID
Clostridium perfringens Bacterial Strain ID
Clostridium septicum Bacterial Strain ID
Enterobacter/Klebsiella spp Bacterial Strain ID
Enterococcus faecalis Bacterial Strain ID
Enterococcus faecium Bacterial Strain ID
Escherichia/Shigella spp Bacterial Strain ID
Kingella kingae Bacterial Strain ID
Mycobacterium abscessus Bacterial Strain ID
Mycobacterium chelonae Bacterial Strain ID
Mycobacterium fortuitum Bacterial Strain ID
Prevotella bivia Bacterial Strain ID
Prevotella buccalis Bacterial Strain ID
Prevotella disiens Bacterial Strain ID
Prevotella intermedia Bacterial Strain ID
Prevotella loescheii Bacterial Strain ID
Prevotella melaninogenica Bacterial Strain ID
Prevotella oralis Bacterial Strain ID
Prevotella spp. Bacterial Strain ID
Prevotella nigrescens Bacterial Strain ID
Proteus spp Bacterial Strain ID
Pseudomonas aeruginosa Bacterial Strain ID
Staphylococcus aureus Bacterial Strain ID
Staphylococcus epidermidis Bacterial Strain ID
Staphylococcus lugdunensis Bacterial Strain ID
Streptococcus agalactiae Bacterial Strain ID
Streptococcus pyogenes Bacterial Strain ID
Aspergillus flavus Fungal Strain ID
Aspergillus fumigatus Fungal Strain ID
Aspergillus niger Fungal Strain ID
Candida albicans Fungal Strain ID
Candida glabrata Fungal Strain ID
Candida krusei Fungal Strain ID
Candida parapsilosis Fungal Strain ID
Candida tropicalis Fungal Strain ID
CTX-M-1 group Antibiotic resistance gene
CTX-M-8 group Antibiotic resistance gene
CTX-M-9 group Antibiotic resistance gene
IMP-1 group Antibiotic resistance gene
IMP- 12 group Antibiotic resistance gene
IMP-2 group Antibiotic resistance gene
IMP-5 group Antibiotic resistance gene KPC Antibiotic resistance gene
mecA Antibiotic resistance gene
NDM Antibiotic resistance gene
OXA-48 Group Antibiotic resistance gene
SHV Antibiotic resistance gene
SHV(156D) Antibiotic resistance gene
SHVQ 56G) Antibiotic resistance gene
SHV(238G240E) Antibiotic resistance gene
SHV(238G240K) Antibiotic resistance gene
SHV(238S240E) Antibiotic resistance gene
SHV(238S240K) Antibiotic resistance gene
vanA (VRE) Antibiotic resistance gene
vanB Antibiotic resistance gene
vanC Antibiotic resistance gene
VIM-1 group Antibiotic resistance gene
VIM- 13 Antibiotic resistance gene
VIM-7 Antibiotic resistance gene
hla Virulence Factor
lukF Virulence Factor
spa Virulence Factor
ermA Antibiotic resistance gene
ermB Antibiotic resistance gene
mefA Antibiotic resistance gene
SME Antibiotic resistance gene
Example 7: Exemplary Wound Genetic Test
[00211] An overview of the general steps involved in the methods disclosed herein related to the identification of the microorganism community is provided in FIG. 1 A. These general overview steps may be referred to as the "Wound Genetic Test" (WGT) herein.
[00212] The first step is specimen collection. Samples of specimens may be collected from any source of the body (e.g., blood, skin, urine etc.). For example, the location of skin samples may come from the ankle, forehead, hand, head, lower back, retroauricular crease, toe web, intranasal, knee crook, groin, neck, elbow crook, or perianal location. Wound specimens may be collected using a variety of devices, such as a sterile swab or syringe (i.e., swab collection kit).
[00213] The next step is DNA extraction (FIG. 1 A). An overview of the steps necessary for microbial DNA isolation using the DNA Miniprep kit for swab samples collected from the Collection Tube are outlined below. In general, DNA can be extracted from the pathogenic microorganisms in a specimen using commercially available DNA Miniprep Kits. Real-Time PCR assays may be performed using commercially available kits. Data can then be generated and analyzed using Fluidigm Biomark HD System on the 96.96 Dynamic Array Gene Expression Chip.
Materials and Equipment
[00214] Materials:
• DNA/RNA Collection Tube w/ Swab
• DNA MiniPrep Kit
• Beta-mercaptoethanol
• Millennium Health Microbial Community Standard
[00215] Note that Millennium Health Microbial Community Standard is a control developed at Millennium Health which contains representative microorganisms. Gram positive and gram negative bacterial strains are represented, as well as both aerobes and anaerobes and fungal species (i.e.
Candida). The strains present in this control represent the most common microorganisms found in skin and soft tissue infections (SSTIs). The control is used to ensure that the DNA from all different types of SSTI relevant microorganisms is effectively extracted during sample preparation. The control is used with every batch of specimens collected (samples) to establish that the extracted DNA from the microorganism represents the microorganisms present in the isolated wound sample.
[00216] Equipment:
• Microfuge 22R Centrifuge (or equivalent)
• Dry Bath Incubator
• TissueLyser
• Controls:
[00217] One isolation negative control for each Isolation Group to verify that the isolation reagents are not contaminated. This Isolation Negative Control (INC) can be DNase/RNase Free Water.
Negative Isolation Control (NIC) and Isolation Negative Control (INC) both refer to the same control sample and are used interchangeably.
[00218] One isolation positive control for each Isolation Group to verify that isolation has occurred successfully. This control can also be used during analysis to verify that the Pan Bacteria and represented assays are functioning properly. This Isolation Positive Control (IPC) can be the
Millennium Health Microbial Community Standard, which is prepared in-house.). The IPC is used to verify that the isolation occurred successfully.
[00219] Additional controls include the following: (a) No Template Control using Microbial DNA- Free Water (Qiagen, Venlo, The Netherlands); this control can verify that the assay reagents are not contaminated; (b) Microbial Plasmid Pool ("Millennium Health Plasmid Pool (Millennium Health, San Diego CA)), which is used to verify that the assays are performing as expected; (c) Positive PCR Control, such as PPC (Qiagen, Venlo, The Netherlands), which is used to verify that the samples do not contain anything that might inhibit PCR amplification; and (d) A human gDNA assay can also be included as a control to determine whether there are detectable levels of DNA present in a specimen.
[00220] DNA can be extracted using procedures known in the art. One of the central premises of the DxWound product is the ability to provide a real-time snapshot of the microbial content of a wound without the need for artificially inflating the microbes by, for example, growing them up on a petri dish. During DNA extraction, care should be taken to reduce any bias introduced by the different growth rates and growth requirements of different microorganisms, and minimize any bias introduced by microorganisms competing for resources after sample collection, which can artificially inflate the presence of some microorganisms whilst diminishing others.
[00221] The extracted DNA can be used directly in RT-PCR without the need for an intermediate amplification step. This enables the whole process to be completed in less than 8 hours, which is a key benefit as it enables the physician to customize their treatment decisions to the microbial flora in the wound earlier.
Sample Processing and Data Analysis
[00222] The samples can then be prepared according to standard protocol for running on the 96.96 Dynamic Array™ Gene Expression Chip IFC. Assays curated for inclusion on the WGT panel (e.g., one or more of Tables 1-11) can be added to the chip. Generally, at least three assays contribute to any final result: (a) the microorganism (bacterial, fungal assay), and (b) at least two control assays. Data can then be generated using Fluidigm Biomark HD System on the 96.96 Dynamic Array Gene Expression Chip. Approximately 96 input samples are analyzed per chip on the Fluidigm Biomark HD System, which includes approximately 80 specimen samples and 16 control samples. For each specimen sample on the chip, a plurality of primers can be added to each sample that have specific sequences directed to different microorganisms. One or more Pan Bacterial Controls and one or more Pan Fungal Controls can also be included.
[00223] The resulting data is then initially analyzed using the Fluidigm Real-Time PCR Analysis Software, and then using "Look-Up" tables specific for each microbe, by evaluating amplification curves against control samples within a defined reportable range. If there is an amplification signal within a defined reportable range, then it means that the primers were able to detect the presence of a given microorganism for that particular microorganism species. If there is no amplification signal within a defined reportable range, then it means that no microorganism was able to be detected for that particular species. The amplification results are then analyzed on Look-Up tables that comprise algorithms that consist of a combination of all possible results for a single sample or panel of samples. The software compares the amplification results from the specimen sample to the algorithm to generate a call on whether the microorganism is present or not. By analyzing the combination of all the amplification results displayed on the Look-Up table for a given sample or panel of samples, the presence or absence of any microorganism from a wound may be detected.
[00224] A Look-Up table can be developed for each microbial target, considering the positive and negative controls used. An exemplary Look-Up table for 5, aeruginosa is shown in Table 14 below. "Code" represents the output of the software, along with the phenotype and genotype. Inputs include
P. aeruginosa, pan bacteria (positive control for bacteria) and pan Candida and Aspergillus (positive control for fungi).
Table 14: P. aeruginosa Look-Up Table
Figure imgf000066_0001
Example 8: Wound Sample Acquisition, Molecular Analysis, and Culture Results
[00225] In the wound study of this example, wound samples were obtained from either patients located at the Palomar Health or Pomerado Health outpatient wound clinics. In general, patients had to first meet the inclusion criteria. Next, specimens from wounds of a variety of locations were collected. Such locations included the ankle, forehead, hand, head, lower back, retroauricular crease, toe web, intranasal, knee crook, groin, neck, elbow crook, or perianal location. Specimens were then shipped to Millennium Health and stored at either room temperature or -20°C. Subsequently, the DNA was extracted and stored for future use. The DNA was also quantified and compared based on different collection devices (see below) to determine the optimal device for collecting samples. Next, molecular testing was undertaken using Millennium Health's WGT panel and NGS platforms. Real-time PCR assays were then performed and the data was further analyzed using Fluidigm Biomark HD System on the 96.96 Dynamic Array Gene Expression Chip and associated software. Using Look-Up tables that comprise algorithms of different combinations of possible results for a single sample or panel of samples, the software compares the amplification results from the specimen sample(s) to the algorithm to generate a call on whether the microorganism is present or not. The culture results were then recorded, identifying the presence or absence of microorganisms in the sample, and reported to the physician.
Study Design
[00226] For the study design, several criteria had to be met for inclusion. Patients who met the inclusion criteria at screening were consenting subjects who were experiencing wounds that were being treated by a physician where standard of care included wound samples being collected and sent for culture. Patients who did not meet the inclusion criteria were excluded at screening, and therefore, may have been experiencing a wound, but wound samples were not cultured. Patients who were excluded included incapacitated patients, and/or patients who were unable to give consent.
Approach
[00227] Participating physicians enrolled subjects who met the inclusion criteria and consented to the wound study. Next, they proceed to collect the following samples from the subject: (a) a swab before debridement (SB); next, the physician would debride the wound and (b) collect tissue for standard care; (c) then, the physician would collect a swab after debridement (SA); and lastly, (d) the physician would collect a tissue sample (TC). A total of four samples were collected for each enrolled subject. Three out of the four samples were sent to Millennium Health (MH) for testing and analysis. One sample was sent to a hospital lab for routine, traditional culture. The separation of samples in this format allowed a comparison between the traditional method of analyzing microorganisms in a wound sample, and the molecular methods disclosed herein related to the identification of microorganisms in a wound directly from a biological sample.
Phases
[00228] Next, two phases of the wound study took place to analyze specimen collection, storage, and stability.
Phase I [00229] The purpose of the first phase was to use two different devices for sample collection and compare the ability to extract DNA in high quantity and quality, test the extracted DNA samples (preliminary WGT samples), and evaluate whether one device was superior to the other in yielding high amounts of microorganism DNA from wound samples. In the first phase, 75 samples were collected in Device 1. Device 1 has traditionally been utilized as a collection device for wound samples because it is known to preserve bacterial samples for future growth in the lab and maintain relative sample stability when shipped or transported to a different location for analysis. Device 1 has components that preserve the sample for shipping or future growth in a lab. Specifically, the liquid Amies medium bacteriology swab collection system was used as Device 1. Also, 75 samples were collected in Device 2. Device 2 has traditionally been utilized to inactivate infectious agents and preserves the genetic integrity and expression profiles of samples. Device 2 has components that inactivate the organisms and/or nucleases (e.g., using chelating agents such as EDTA) present in the sample, but preserves the DNA and RNA for future analysis.
[00230] NGS is used to sequence and determine the microbial composition in samples collected and stored in Device 1 and Device 2 overtime (at day 0, day 3 and day 10, FIG. 2). As shown in FIG. 2, the samples collected using Device 2 preserve the sample consistently over time. Device 2 effectively inactivates the microbes at the time of specimen collection to ensure an unbiased representation of the microbial content of the wound.
Phase II
[00231] The purpose of the second phase of the wound study of this example was to further optimize the extraction method and PCR testing panel, determine specimen/kit characteristics (stability, storage, interfering substances etc.), and define performance characteristics (accuracy, sensitivity, specificity, robustness etc.). In this phase, the same sample types were collected per subject as described above (SB, SA, and TC), and the sample collection continued in the device of choice from Phase I. The overall goal of this phase was to finalize the WGT test and methods and perform quality/control analysis.
[00232] Initially, different specimen types (SB, SA, and TC) were subject to the DNA extraction method disclosed herein, and the DNA yield was determined and summarized in FIG. 3. The majority of specimens contained about 0.01 to 1.0 ng/ul DNA.
[00233] Then, DNA concentrations from various locations on healthy donors A-E (Table 15) were compared to clinical wound samples (Table 16). It was found that bacterial DNA quantity is significantly higher (e.g., about 10-1000 times higher) in wound samples than healthy samples. Table 15. DNA concentration in healthy skin samples
Figure imgf000069_0001
Table 16. DNA concentration in clinical wound samples
Figure imgf000069_0002
[00234] Next, samples from 106 patients with matched traditional culture and WGT results were included for comparison. Traditional culture results were generated using the protocols outlined in the Clinical and Laboratory Standards Institute (CLSI) guidelines M35A2E and M100S26E. The WGT results were generated using the WGT methods disclosed herein. Confirmation data was generated by next generation sequencing (NGS). NGS data was used for to provide additional information on the microbial community in samples and compared to WGT results. NGS data for bacterial identification was available for 102 of the 106 patient samples and NGS data for fungal species
identification/confirmation and analysis is ongoing. Altogether, these data demonstrate the accuracy, efficiency and specificity of the WGT method. [00235] Identification of various microbial targets using the culture method is summarized in Tables 17-18 below. Table 17 shows the identification of targets that are present on the WGT Panel using the culture method. Table 18 shows the identification of targets that are not on WGT Panel (because these targets are not currently recognized as wound pathogens) and other phenotypical results using the culture method.
Table 17. Identification of WGT Panel Targets using Culture method
TYPE COUNT
Bacteroides
fragilis 7
Candida 3
MRSA 37
Staphylococcus
19 aureus
Staphylococcus
species 7
Prevotella 7
Proteus 4
Pseudomonas 14
Streptococcus
15 species
Enterococcus
5 species
E coli 3
Figure imgf000071_0001
Table 15 Identification of Targets not on WGT Panel and other results using Culture method
Figure imgf000072_0001
[00236] Next, the WGT method was analyzed for several parameters, including: component accuracy and precision using known isolates (Table 19), WGT findings by organism type (Table 20), WGT findings per subject (FIG. 4), the number of antibiotic resistance genes (Abrx) found per subject (FIG. 5), swab versus tissue WGT findings concordance (Table 26), WGT concordance to clinical findings (Tables 21-24), as well as key unexpected findings and messages (Table 30 and Table 31).
[00237] As shown in Table 19 below, using the WGT method, most known clinically relevant microbe targets were detected in established lab isolates in an accurate and precise manner.
Table 19. Detection of isolates using WGT
Figure imgf000073_0001
[00238] Furthermore, the WGT method was successfully used to detect microbes in clinical specimens. In this experiment, 106 subjects were analyzed with matched culture and WGT samples, and of these, 60% of subjects had polymicrobial wounds. Table 20 below provides a summary of the number of microbes per organism type as analyzed by the WGT method. Each listed microbe in the middle column is a clinically relevant organism. G+: Gram positive. G-: Gram negative. Table 20. WGT Results
Figure imgf000074_0001
[00239] Next, clinical specimens from chronic wound patients were analyzed using WGT (FIG. 4 and FIG. 5). The clinical specimens were collected from patients who had been seen by a physician at a wound clinic because the wound was not healing. As shown in FIG. 4, 67% of patients had 1-4 microbes in their samples, and 14.2% of patients had >5 microbes in their samples. Furthermore, as shown in FIG. 5, 55.6% of patients had zero antibiotic resistance (AbxR) genes, 36.8% of patients had 1-2 AbxR genes, and 7.5% of patients had 3-4 AbxR genes.
[00240] Next, concordance for WGT results produced by swab (before or after) and tissue biopsy was calculated for individual patients for their matched samples (Table 21). Conventional wisdom held by many clinicians as well as standard clinical practice is that a tissue biopsy and swab sample should be taken after debridement of a wound to accurately assess whether there is an infection, since microbes present in the surface dusts and debridement are likely background noises that do not represent wound pathogens. Surprisingly, it was found here that the WGT results from the swab were concordant with results obtained from the tissue for about 97% of the time (8,410 data points; 145 sample pairs). This suggests that the overall microbial composition is the same between TC, SA and SB. Table 21. Swab vs tissue concordance
Figure imgf000075_0001
[00241] Next, results from the WGT microbial identification assays were considered. These include 34 microbial targets, 106 subjects (swabs and tissues), and 3,604 data points analyzed. Table 22 shows the rate of concordance between the culture approach and the WGT method (mm: mismatch). 55 findings were present in culture only as they are not included on the WGT panel for lack of clinical relevance. For targets that are included on the WGT panel, the WGT method was able to detect an additional 65 incidents of microbes that were missed by the culture method.
Table 22. Concordance between culture and WGT
Figure imgf000075_0002
[00242] One of the key findings is that the culture report identified only to a genus or group level, whereas the WGT test provided additional clarification of the results by detecting the species in 61% of cases (11/18) (Table 23). The ability to speciate using the WGT method is important to treat species-based infections. For example, in Candida infections, treatment options differ between species. Table 23. Speciation by WGT
Figure imgf000076_0001
[00243] Another key finding was the ability of WGT to detect missed or masked findings from the culture method. Amongst 42 patients with "mixed flora" culture results, WGT identified specific organisms in 26 (62%) of them (Table 24). These identified organisms commonly carry antibiotic resistance (e.g. Escherichia coli, Enterococcus faecalis). For example, in three of the patients in Table 24, the WGT method identified Vancomycin-resi stance genes along with Enterococcus faecalis, indicating a possible finding of Vancomycin-resistant Enterococcus (VRE).
Table 24. WGT identification of masked findings from culture method
Figure imgf000076_0002
[00244] As shown in Table 25, in 93 patients with culture-positive wounds, WGT identified additional microbes not reported by culture in 40 patients (43%). In 5 patients, WGT detected culture results (5.4%). Table 25. WGT identification of missed findings from culture method
Figure imgf000077_0001
[00245] In summary, the results obtained from the WGT method have a significantly improved performance, intersection, concordance, speciation, and is able to detect missing and additional findings. Specifically, the WGT method has sensitive, accurate and precise detection on known microbial isolates. The WGT panel is shown to cover all or substantially all organisms identified by culture in the pilot study, with 96.4% or greater concordance to culture results. The WGT method also speciated 61% of genus-level culture findings, identified microbes in 62% of patients with "mixed flora" culture findings, including 3 with possible VRE, identified additional microbes missed by culture in 43% of patients with culture-positive results, and detected culture errors in 5.4% of patients.
OTHER EMBODIMENTS
[00246] From the foregoing description, it can be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
[00247] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
INCORPORATION BY REFERENCE
[00248] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

1. A method for detecting the presence or absence of a plurality of microorganisms in a biological sample, the method comprising:
(a) obtaining a biological sample from a subject in need thereof, wherein the biological sample is suspected to contain one or more microorganisms,
(b) providing a plurality of primer pairs, wherein each primer pair is specifically designed to amplify a target nucleotide sequence within a microorganism listed in one or more of Tables 1- 11 ; and
(c) performing real-time PCR using the plurality of primers, thereby detecting, at the time of obtaining the biological sample in step (a), the presence or absence of the plurality of microorganisms listed in one or more of Tables 1-11.
2. The method of claim 1, wherein the target nucleotide sequence comprises antibiotic resistance and/or virulence factor gene.
3. The method of claim 2, wherein the plurality of primer pairs are designed to target and/or detect all microorganisms, antibiotic resistance genes and virulence factor genes listed in one or more of Tables 1-11.
4. The method of claim 1, further comprising, prior to step (c), extracting nucleic acids of the microorganisms from the biological sample.
5. The method of claim 1, wherein the biological sample or the one or more microorganisms therein are not subject to a step of in vitro culturing.
6. A plurality of primer pairs, wherein each primer pair is specifically designed to amplify a target nucleotide sequence within a microorganism, antibiotic resistance gene or virulence factor gene listed in one or more of Tables 1-11, wherein the plurality of primer pairs together target and/or detect all microorganisms, antibiotic resistance genes and virulence factor genes listed in one or more of Tables 1-11.
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