METHODS FOR DETECTING THE PRESENCE OF BACTERIA, FUNGI, PARASITES, AND VIRUSES IN A TEST SAMPLE AND TREATING A PATIENT

Abstract

A method of testing for the presence of an infectious biomarker indicating bacteria, fungi, parasites, or viruses. A test sample comprising cellular and non-cellular material is collected from a patient, the test sample having a first amount of at least one infectious biomarker and a second amount of a host biomarker. The amount of the infectious biomarker(s) in the test sample is determined as is the amount of the host biomarker. The amount of infectious biomarker(s) in the test sample is normalized as a function of the amount of host biomarker therein. The normalizing step is a ratio of the first amount of the infectious biomarker(s) to the second amount of the host biomarker. The test sample is positive for a disease associated with the infectious biomarker(s) if the ratio is above a predetermined value. The normalized result is tracked over time to assess therapeutic response to treatment. A database is provided for interpreting results collected from the patient and for determining predictive sensitivity, specificity, and diagnostic accuracy.

Claims

1. A method of testing for the presence of an infectious biomarker the steps comprising: a) collecting a test sample comprising cellular and non-cellular material from a patient, the sample the test sample comprising a first amount of at least one infectious biomarker and a second amount of a host biomarker; b) determining the first amount of the at least one infectious biomarker in the test sample; c) determining the second amount of the host biomarker in the test sample; and d) normalizing the first amount of at least one infectious biomarker as a function of the second amount of host biomarker therein. e) collecting a test sample comprising of cellular and non-cellular material from a patient and concentrating the sample through precipitation or other methods.

2. The method of testing for the presence of an infectious biomarker in accordance with claim I, wherein the step (d), normalizing the first amount of the infectious biomarker comprises determining a ratio of the first amount of the at least one infectious biomarker to the second amount of the host biomarker.

3. The method of testing for the presence of an infectious biomarker in accordance with claim 2, the steps further comprising: e) identifying the test sample as being positive for a disease associated with the at least one infectious biomarker if the ratio of the first amount of the at least one infectious biomarker to the second amount of the host biomarker is above a predetermined value.

4. The method of testing for the presence of an infectious biomarker in accordance with claim 3, the steps further comprising: f) treating a patient for the disease associated with at least one infectious biomarker.

5. The method of testing for the presence of an infectious biomarker in accordance with claim 4, the steps further comprising: g) graphically displaying the normalized results over the course of time to indicate a response to treatment.

6. The method of testing for the presence of an infectious biomarker in accordance with claim 2, the steps further comprising: e) identifying the test sample as being negative for a disease associated with at least one infectious biomarker if the ratio of the first amount of the at least one infectious biomarker to the second amount of the host biomarker is below the predetermined value.

7. The method of testing for the presence of an infectious biomarker in accordance with claim 2, wherein the normalizing step (d) is performed with an algorithm in the form of one of a set of formulae consisting of: $\frac{\mathrm{host}\mathrm{biomarker}\left(s\right)}{\mathrm{infectious}\mathrm{agent}\mathrm{biomarker}\left(s\right)}\mathrm{and}\frac{\mathrm{infectious}\mathrm{agent}\mathrm{biomarker}\left(s\right)}{\mathrm{host}\mathrm{biomarker}\left(s\right).}$

8. A method of testing for the presence of an infectious biomarker the steps comprising: a) collecting a test sample comprising cellular and non-cellular material from a patient, the test sample comprising a first amount of at least one infectious biomarker and a second amount of a host biomarker; b) providing a database of characterized clinical samples and demographic data; c) determining the first amount of the at least one infectious biomarker in the test sample; d) determining the second amount of the host biomarker in the test sample; e) accessing the database with a numerical value representative of the test sample to determine predictive sensitivity, specificity, and diagnostic accuracy; and f) collecting a test sample comprising of cellular and non-cellular material from a patient and concentrating the sample through precipitation or other methods.

9. The method of testing for the presence of an infectious biomarker in accordance with claim 8, wherein the sensitivity and specificity are determined by a receiver operator characterization (RoC) curve.

10. The method of testing for the presence of an infectious biomarker in accordance with claim 9, wherein the sensitivity and specificity are determined by a statistical method.

11. The method of testing for the presence of an infectious biomarker in accordance with claim 8, the steps further comprising: g) treating a patient for the disease associated with the at least one infectious biomarker.

12. The method of testing for the presence of an infectious biomarker in accordance with claim 8, the steps further comprising: g) identifying the test sample as being positive for a disease associated with at least one infectious biomarker when the sensitivity and specificity meet a predetermined threshold.

13. The method of testing for the presence of an infectious biomarker in accordance with claim 8, the steps further comprising: g) identifying the test sample as being negative for a disease associated with the at least one infectious biomarker if the ratio of the first amount of the at least one infectious biomarker to the second amount of the host biomarker is below a predetermined value.

14. The method of testing for the presence of an infectious biomarker in accordance with claim 8, the steps further comprising: g) predicting the absence of disease and quantifiably associating the prediction on a predetermined wellness scale thereof.

15. The method of testing for the presence of an infectious biomarker in accordance with claim 14, the steps further comprising: h) treating a patient for the disease associated with the at least one infectious biomarker.

16. The method of testing for the presence of an infectious biomarker in accordance with claim 8, wherein the database comprises a set of at least one patient factor consisting of: demographic information, infectious disease, gender, age, biographical ancestry, pregnancy status, treatment, location, zip code, sexual activity, transgender status, number of different sexual partners over a predetermined period of time, sexual preference, sexual practices, smoking status, hormone replacement recipient, menopause, dental darns, contraceptive use, and type of condoms.

17. A method for treating a patient having bacteria, fungi, parasites, or viruses, the steps comprising: a) testing for the presence of an infectious biomarker, the steps comprising: a1) collecting a test sample comprising cellular and non-cellular material from a patient, the test sample comprising a first amount of at least one infectious biomarker and a second amount of a host biomarker: a2) providing a database of characterized clinical samples and demographic data; a3) determining the first amount of the at least one infectious biomarker in the test sample; a4) determining the second amount of the host biomarker in the test sample; and a5) accessing the database with a numerical value representative of the test sample to determine predictive sensitivity, specificity, and diagnostic accuracy; and b) treating the patient for the detected bacteria, fungi, parasites, or virus.

18. The method for treating a patient in accordance with claim 17, wherein the sensitivity and specificity are determined by a receiver operator characterization (RoC) curve.

19. The method of treating a patient in accordance with claim 18, wherein the sensitivity and specificity are determined by a statistical method.

20. The method of treating a patient in accordance with claim 17, the steps further comprising: a6) identifying the test sample as being positive for a disease associated with at least one infectious biomarker when the sensitivity and specificity meet a predetermined threshold.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] A complete understanding of the present invention may be obtained by reference to the accompanying drawings, when considered in conjunction with the subsequent detailed description, in which:

[0032] FIG. 1 is a table of primer and probe sequences for human and some bacteria associated with vaginal bacterial dysbiosis;

[0033] FIG. 2 is a table of primer and probe sequences for the bacterial order Mycoplasmatoales vaginal dysbiosis;

[0034] FIG. 3 is a table of examples of primers and probes for the detection of microorganisms associated with aerobic vaginitis;

[0035] FIG. 4 is a table of examples of primers and probes associated with healthy or vaginal wellness for Lactobacillus sp;

[0036] FIG. 5 is a table of examples of primers and probes for the detection of fungal microorganisms;

[0037] FIG. 6 is a table of examples of primers and probes for the detection of viruses and protozoan associated with vaginal infections;

[0038] FIG. 7 is a graph illustrating a range of DNA collected from patients for testing;

[0039] FIG. 8 is an amplification plot from a clinical specimen of low level detection of Atopobium vaginae DNA with high level detection of a human single copy DNA;

[0040] FIG. 9 is a Receiver Operator Curve (RoC) for an algorithm for Atopobium vaginae without normalization with a host biomarker;

[0041] FIG. 10 is a Receiver Operator Curve (RoC) using a host and infectious organism (e.g., Atopobium vaginae) biomarker algorithm;

[0042] FIGS. 11a and 11b depict graphs showing decreasing levels of infectious agent for serially collected samples with constant levels of a host biomarker with and without normalizing the infectious agent with the host biomarker;

[0043] FIG. 12a is a graph illustrating examples of constant levels of infectious agent without normalization against a host biomarker;

[0044] FIG. 12b is a graph illustrating the inclusion of a host biomarker data algorithm, indicating an increase in the relative amount of infectious agent;

[0045] FIG. 13a is a graph that illustrates decreasing amounts of an infectious agent without normalization with a host biomarker over time;

[0046] FIG. 13b is a graph depicting decreasing levels of an infectious agent over time with the data normalized with a host biomarker algorithm;

[0047] FIG. 14a is a graph illustrating variable infectious agent detection without normalization to variable host biomarker;

[0048] 14b is a graph illustrating algorithm of variable agent normalized by variable levels of host biomarker with diagnostic cutoff designation (flat line);

[0049] FIG. 15 is a portion of a table derived from a database showing sensitivity with specificity;

[0050] FIG. 16 is a flowchart demonstrating the development and implementation of a virtual vaginal wellness database;

[0051] FIG. 17 is a table illustrating examples of results with diagnostic values that are pulled from the virtual database of tested samples.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Although the following detailed description contains specific details for the purposes of illustration, those of ordinary skill in the art will appreciate that variations and alterations to the following details are within the scope of the invention. Accordingly, the exemplary embodiments of the invention described below are set forth without any loss of generality to, and without imposing limitations upon, the claimed invention.

[0053] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that are embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention. Those of ordinary skill in the art will recognize numerous equivalents to the specific apparatus and methods described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0054] The invention is a method of testing for the presence of an infectious biomarker. The amount of the infectious biomarker(s) in the test sample is determined as is the amount of the host biomarker. The test sample is positive for a disease associated with the infectious biomarker(s) if the ratio of the amount of the infectious biomarker(s) to the amount of the host biomarker is above a predetermined value.

[0055] The invention provides methods for diagnosing bacterial vaginosis (BV), aerobic vaginitis (AV), vulvovaginal candidiasis (VVC,), parasite, STI, or viral conditions by quantitatively detecting amplified nucleic acids from microorganisms/viruses and normalizing the data by using quantitative methods for detection of amplified human nucleic acid and/or healthy bacteria. The normalized information is used to confirm specimen adequacy and to determine if a person has bacterial vaginosis (BV), aerobic vaginitis (AV), vulvovaginal candidiasis (VVC), and for STI testing. The normalized data can be used to diagnose individuals suffering from these conditions, to stage therapeutic treatment based on specificity and diagnostic accuracy data and monitor treatment response over time.

[0056] Techniques in molecular biology, cell biology, immunology, microbiology, bioinformatics, database analysis and statistical modeling are used in practicing the inventive method.

[0057] The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

[0058] The term “amplification” or “amplify” means increasing the number of copies of a target nucleic acid. A target may be either DNA or RNA. The examples described here utilize polymerase chain reaction but other methods for amplifying and detecting nucleic acids are known.

[0059] The terms “sensitivity” (also called the true positive rate) measures the proportion of actual positives that are correctly identified as such (e.g., the percentage of sick people who are correctly diagnosed using the test as having bacterial vaginosis, aerobic vaginitis or vulvovaginal candidiasis). The formula used for this determination is:

[00001]$\frac{\mathrm{True}\mathrm{Positive}}{\left(\mathrm{True}\mathrm{Positive}+\mathrm{False}\mathrm{Negatives}\right)}$

[0060] The term “specificity” (also called the true negative rate) measures the proportion of actual negatives that are correctly identified as such (e.g., the percentage of healthy people who are correctly identified as not having bacterial vaginosis, aerobic vaginitis or vulvovaginal candidiasis). The formula used for this determination is:

[00002]$\frac{\mathrm{True}\mathrm{Negative}}{\left(\mathrm{True}\mathrm{Negative}+\mathrm{False}\mathrm{Positive}\right)}$

[0061] “True positive” means sick people correctly identified as sick.

[0062] “False positive” means healthy people incorrectly identified as sick. “True negative” means healthy people correctly identified as healthy. “False negative” means sick people incorrectly identified as healthy.

[0063] “Receiver operator characterization or curve” (RoC) is a graphical plot illustrating the diagnostic ability of a binary classifier system as its discrimination threshold is varied. RoC and other statistical programs are readily available to determining sensitivity and specificity (e.g., see https://www.medcalc.org/contact.php). Alternatively, a graphical description of test performance representing the relationship between the true-positive fraction (sensitivity) and the false-positive fraction (1-specificity) can be provided.

[0064] “Statistical accuracy of measurement” indicates the closeness of agreement between a. measured quantity value and a true quantity value of a measurement and is mathematically defined as:

[00003]$\frac{\left(\mathrm{True}\mathrm{Positive}+\mathrm{True}\mathrm{Negatives}\right)}{\left(\begin{array}{c}\mathrm{True}\mathrm{Positive}+\mathrm{True}\mathrm{Negative}+\\ \mathrm{False}\mathrm{Positive}+\mathrm{False}\mathrm{Negative}\end{array}\right)}$

[0065] “Reference method” is an exactly-defined technique used in association with an internationally agreed reference preparation to provide sufficiently precise and accurate data for assessing the validity of other methods (modified from ICSH).

[0066] “Database” refers to a structured set of data retained on a computer. The database used in this process is known as Access™, provided by the Microsoft Office 365, but other database systems are available. Briefly, residual specimens were retained for microscopic analysis following routine processing for molecular detection. Conventional scoring using microscopy is used to characterize specimens according the Nugent score, Donder, scoring of fungal elements per high power field or manual counting using hemocytometry linked to molecular detection results with patient demographic data. The database includes quantitative data derived from molecular methods.

[0067] The “Ct or threshold cycle value” is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.

[0068] “Host” is defined as the entity from which the specimen is collected.

[0069] “Biomarker” is defined as a specific analyte (DNA, RNA, protein) found in a patient specimen or sample that is useful for measuring the progress of disease or the effects of treatment.

[0070] “Specimen” is defined as discrete portion of a body fluid, breath, hair, or tissue taken for examination, study, or analysis of one or more quantities or properties assumed to apply for the whole (ISO 15189). Alternatively, “specimen” can refer to a discrete portion of a body fluid or tissue taken for examination, study, or analysis of one or more quantities or characteristics to determine the character of the whole.

[0071] A “primary sample collection device” is an apparatus specifically intended by an IVD manufacturer to obtain, contain, and preserve a body fluid or tissue for in vitro diagnostic examination.

[0072] “Analyte component” is represented in the name of a measurable quantity.

[0073] “Liquid with preservative qualities” is defined as a media that deters or prevents the degradation of biomolecules either by inhibiting the growth of bacteria or fungus or killing the organisms.

[0074] “Targeting” is defined as a technique that amplifies a defined region in nucleic acid.

[0075] “Detection” is defined as fluorescent signal that surpasses the background of the threshold.

[0076] “Inconclusive” is defined as a result that is a valid but the interpretation does not yield a definitive answer.

[0077] “Indeterminate” means not definitely or precisely determined or fixed; not leading to a definite end or result. Examples for indeterminate results might result from inhibitors.

[0078] “Diagnosis” is defined as identifying whether a person has bacterial vaginosis (BV), aerobic vaginosis (AV), vulvovaginal candidiasis (VVC), parasitic infections, wellness testing, and/or sexually transmitted infections (STI).

[0079] “Wellness testing” indicates the detection of Lactobacillus clans with or without the detection of other infectious agents, linked to relative sensitivity and specificity.

TABLE-US-00001 TABLE 1 Wellness Testing with Calculations based on Detection of Microorganisms. True False Negatives Positives Not True False in 7-10 Detected Positives Negatives 0-3 Total Analyte Sensitivity Specificity PPV NPV Accuracy Detected 7-10 Detected 0-3 Not detected Analyzed Lactobacillus crispatus 61% 74% 76% 59% 67% 34 96 105 67 302 Lactobacillus gasseri 47% 88% 83% 56% 65% 16 114 80 90 300 Lactobacillus jansenii 61% 69% 71% 59% 65% 40 90 98 62 290 All Lactobacillus sp 60% 92% 81% 80% 80% 8 68 25 17 116 All Lactobacillus exclude positive 82% 92% 75% 94% 90% 3 34 9 2 48 Candida albicans, C. glabrata (mixed infections) All Lactobacillus exclude positive 75% 94% 75% 94% 90% 2 30 6 2 40 Candida albicans, C glab, C pora (mixed infections)

[0080] In practice, a specimen is collected using a solid collection device such as, but not limited to, a flocked swab, brush or broom. The swab is placed against the host's epithelial cell surface and force applied with sweeping motions to collect the sample. The swab is placed in a liquid with preservative properties to stabilized and protect the contents from the swab from degradation. The collected material is allowed to elute from the collecting devices surface and may be vortexed or agitated to encourage the release of materials from the solid collection device's matrix. The contents of the liquid will contain biomarkers from both the host and any infectious entities. The biomarkers are processed for analysis using purification methods. Briefly, an aliquot of the sample is centrifuged to remove the preservative, an extraction control is added to aliquot, and microorganisms are concentrated through salting out procedures. Briefly, a stock solution of 100 ul each of 100% PEG (MW400 W/V), 5M NaCl and extraction control (total 300 ul) is added to 1 ml of clinical specimen, incubated at room temperature for 30 minutes, centrifuged for 2 minutes at 14,000 rpm (16, 873×g) and the supernatant removed and the pellet digested using proteinase K. The nucleic acid is extracted or isolated from the sample. Other processes that do not require precipitation, centrifugation and removal of the preservative may be applied.

[0081] Following purification of the biomarkers, in this case, nucleic acid isolation, the purified nucleic acids are subjected to quantitative polymerase chain reaction (qPCR, q reverse transcriptase PCR, or hybridization) targeting both infectious entities and host cellular contents either simultaneously or in separate reactions. Oligonucleotides in the form of primers and Taqman probes are used to target specific regions of each microbe's genome and the human genome. The fluorescently labeled probe with a quencher molecule is used to detect amplified products. The mixture of primers and probes can be multiplexed or performed separately. The stock concentration (100 μM) of the microorganism's (host's) primers/FAM-labeled probe for each organism. Internal positive control primers and VIC-labeled probe is also added to a master mix to detect inhibition or extraction failure. For a single reaction, the primers/probe mixture is 0.625 μl for each primer, 0.1 μl for each probe, 3.275 of Tris EDTA buffer. The internal positive control is 0.0156 μl for each primer and 0.025 for the probe with 2.4 μl of Tris EDTA. The internal control primer probe mix (2.5 μl per reaction), the microorganism (or host) primer probe mix (3.5 μl), 1 μl of Bovine serum albumin (5 μg/mL) are added to 10 μl of Taqman™ Fast Advanced master mix (catalog number 4444556, ThermoFisher). To this reaction, 3 μl of the extracted sample is added per reaction. The reactions are performed on thermocycler with the following cycling conditions (initial activation at 50° C. for 2 minutes, 90° C. for 20 seconds followed by 39 cycles of 95° C. for 3 sec, 60° C. for 30 S). During the polymerization reaction, the exonuclease activity of the enzyme cleaves the probe, liberating the fluorescent molecule from the quencher molecule. The fluorescent signal is detected in the instrument. The cycle threshold (Ct) is captured by the instrument. Calibrators are included to generate quantitative values. Exemplary primer and probes for the detection of host for the normalization of results for microorganisms and viruses are shown in FIGS. 1-6.

[0082] The detection of these biomarkers is quantified using the cycle threshold (Ct) value. The method provides a method for diagnosing bacterial vaginosis (BV), aerobic vaginitis (AV), vulvovaginal candidiasis (VVC), and/or sexually transmitted infections (STI). The amount of the BV, AV, VVC, and/or STI associated infectious biomarker(s) is normalized (i.e., a ratio of the amount of infectious biomarker(s) to the amount of the host biomarker is calculated). The test sample is identified as being positive for BY, AV, VVC, and/or STI associated with one or more infectious biomarkers if the ratio is above a predetermined value. Likewise, the test sample is identified as being negative for a disease associated with the infectious biomarker(s) if the ratio is below the predetermined value. With STI the quantification may not be diagnostic but could be used to measure response to treatment. Many STI such as chlamydia or gonorrhoeae are difficult to culture.

[0083] The ratio identifies the detection of the infectious entities at levels consistent with morbidity and provides confidence levels for diagnostic interpretation of results.

[0084] In other embodiments, normalizing the first amount of infectious biomarker with the second amount of host biomarker can include comparing the relative infectious and host biomarker amounts with one or more databases of empirical testing data which account for both infectious biomarker and host biomarker information (including patient demographic information such as pregnancy status, geographical location, age, menopause, hormone replacement therapy, and transgender status) in various testing samples. Such empirical data can be used to determine which combinations of infectious and host biomarker amounts can correlate to a positive determination for the bacteria in the testing sample. In some instances, a lack of detection of a host biomarker can be accounted for and used to normalize a testing sample. Such databases can also help determine confidence, specificity, sensitivity, and accuracy values for a given testing sample.

[0085] In the particular embodiments, individual or multiple assays for specific bacteria for BY such as Atopobium vaginae, Gardnerella vaginalis, bacterial vaginosis associated bacterium 2 (BVAB2), Megaspheara type I, Mycoplasma hominis, Mobiluncus curtisii, Ureaplasma urealyticum, Ureaplasma parvum (genotypes) and/or Lactobacillus sp are detected along with the host associated biomarker (e.g., RPP30).

[0086] Specimen collection using collection devices vary significantly between individuals with some individuals aggressively collecting specimens while other individuals prefer a gentle approach. Aggressive collection results in a high concentration and amount of the host biomarker.

[0087] Referring now to FIG. 7, there is shown a range of human biomarkers detected in specimens collected for routine clinical testing. Variability in collection methods results in a range from 0 to more than 5,000 ng/μl of DNA;

[0088] Referring now also to FIG. 8, a representative detection curve is shown for an infectious disease analyte and a host biomarker. The light gray line is the human biomarker collection control with the black line being the infectious agent, specifically Atopobium vaginae. Specimens with high amounts of the host biomarker may produce a sample less accurate (specifically, 52% compared with 82%) with low amounts, as illustrated in Table 2, below.

TABLE-US-00002 TABLE 2 Diagnostic Value with Host Biomarker Quantitative Value High Low Sensitivity 62% 100% Specificity 44%  71% PPV 47%  67% NPV 58% 100% Accuracy 52%  82%

[0089] Additional data employing the detection of a noninfectious (aka host) biomarkers to address variability in collection methods improves diagnostic accuracy of methods used to detect infectious agent biomarkers.

[0090] Referring now also to FIG. 9, there is shown the receiver operator curve (RoC) associated with snore than 1,000 determinations using only a quantitative biomarker associated with a single infectious agent and a reference method for disease. Note the large gap of information for specificity in the graph with no data points below 65% specificity.

[0091] In contrast, FIG. 10 demonstrates a RoC when an algorithm incorporating a quantitative host biomarker is used to normalize data generated from a quantitative biomarker for the infectious agent. Although the RoC illustrates how to use the normalization to generate a specificity number, it should be understood that other RoC curves for vulvovaginal candidiasis and aerobic vaginitis can be used to predict numerical calculations for samples with multiple infectious entities. The gap with interpretative value is eliminated with incorporating the host biomarker in the algorithm for assessing the diagnostic value of the test. RoC analysis was performed using the quantitative analysis of the host biomarker without inclusion of the infectious biomarker data and no correlation with the host biomarker alone with the disease state was identified.

[0092] Referring now to FIGS. 11-14, examples are provided of various interpretations of results with and without normalizing the detection of the infectious agent against the host biomarker. If the collection of host biomarker were constant, the results demonstrate consistent results with and without normalization (FIG. 11). However, as is demonstrated in FIG. 7, collection amounts differ.

[0093] Referring now to FIGS. 12a and 12b, the potential for inaccurate interpretation without normalization is illustrated whereby a provider may consider the patient to have a stable level of infectious agents when the normalized values demonstrate a change of increasing infectious agent load. FIG. 12b illustrates the inclusion of a host biomarker data algorithm, indicating an increase in the relative amount of infectious agent, which may indicate increasing levels of morbidity or failure to respond to treatment.

[0094] FIGS. 13a and 13b illustrate another potential misinterpretation where non-normalized data appear to represent an improvement or decrease in infectious agent that suggests a return to the normal microbiota when the normalized data indicate status. FIG. 13b is a graph similar to that shown in FIG. 13a, following normalization with a biomarker data algorithm, the results demonstrating a static state for an infectious agent.

[0095] FIGS. 14a and 14b illustrate non-normalized data that appear to support a downward trend of infectious agent when the normalized data illustrate variability in infectious agent quantity. FIG. 14b is a graph similar to that shown in FIG. 14a, depicting decreasing levels of an infectious agent over time with the data normalized with a host biomarker algorithm and significant variability occurs. Inclusion of a “diagnostic cutoff line” may guide clinical decisions as to whether additional treatment is necessary. Algorithm values below the diagnostic cutoff would be consider normal results and would not require treatment. A diagnostic cutoff or reference line can be applied that may inform the healthcare provider that values associated with disease (above the diagnostic cutoff or reference line) compared with non-disease states (i.e., values below the diagnostic cutoff or reference line)

[0096] FIG. 15 is a partial table derived from a database showing sensitivity with specificity after applying an algorithm, which information may be used to set reference lines for diagnostic interpretation. This example shows how database and diagnostic values can be customized to analyte or disease population. Specifically, this example demonstrates how the algorithm setting of 1.02 can be used to obtain a sensitivity of 75.4 and a specificity of 94.81.

[0097] In one embodiment, the cycle threshold value was applied to one or more bacteria associated with bacterial vaginosis such as Atopobium vaginae, Gardnerella vaginalis, bacterial vaginosis associated bacterium 2(BVAB2), Megaspheara type 1, Mycoplasma hominis, curtisii, Ureaplasma parvum (genotypes) and Ureaplasma urealyticum and/or Lactobacillus sp. Infectious agent biomarker Ct values greater than a diagnostic cutoff (e.g., 39 Ct) with results of host biomarker (RPP30) greater than the diagnostic cutoff is considered an inadequate sample. This result is reported as indeterminate or inconclusive.

[0098] In another embodiment, the cycle threshold value was applied to one or more bacteria associated with aerobic vaginitis such as Escherichia coil, Group B Streptococcus and other Streptococcus spp., Staphylococcus aureus, and Enterococcus faecalis and/or Lactobacillus sp. Infectious agent biomarker Ct values greater than diagnostic cutoff (e.g., 39 Ct) with results of host biomarker (RPP30) is greater than diagnostic cutoff is considered an inadequate sample. This result is reported as indeterminate or inconclusive.

[0099] In another embodiment, the cycle threshold value was applied to one or more yeast associated with vulvovaginal candidiasis (VVC) such as Candida albicans and/or Candida glabrata and/or Lactobacillus sp. Infectious agent biomarker Ct values of greater than a diagnostic cutoff (e.g., 39 Ct) with results of host biomarker (RPP30) is greater than the diagnostic cutoff is considered an inadequate sample. This result is also reported as indeterminate or inconclusive.

[0100] In another embodiment, the cycle threshold value was applied to one or more bacteria associated with bacterial vaginosis such as Atopobium vaginae, Gardnerella vaginalis, bacterial vaginosis associated bacterium 2 (BVAB2), Megaspheara type 1, Mycoplasma hominis, Mobihalms curtisii, Ureaplasma parvum (genotypes) and Ureaplasma urealyticum and/or Lactobacillus sp. infectious agent biomarker Ct values of less than a diagnostic cutoff (e.g., 39 Ct) with results of host biomarker (RPP30) is less than the diagnostic cutoff is considered an adequate sample and is considered detected or positive.

[0101] In another embodiment, the cycle threshold value was applied to one or more bacteria associated with aerobic vaginitis such as Escherichia coil, Group B Streptococcus and other Streptococcus spp., Staphylococcus aureus, and Enterococcus faecalis and/or Lactobacillus sp. Infectious agent biomarker Ct values less than a diagnostic cutoff (e.g., 39 Ct) with results of host biomarker (RPP30) is less than the diagnostic cutoff is considered an adequate sample and considered detected or positive.

[0102] In another embodiment, the cycle threshold value was applied to one or more yeast associated with vulvovaginal candidiasis (VVC) such as Candida albicans and/or Candida glabrata and/or Lactobacillus sp. Infectious agent biomarker Ct values of less than a diagnostic cutoff (e.g., 39 Ct) with results of host biomarker (RPP 30) is less than the diagnostic cutoff is considered an adequate sample and considered detected or positive.

[0103] In another embodiment, the cycle threshold value was applied to one or more bacteria associated with bacterial vaginosis such as Atopobium vaginae, Gardnerella vaginalis, bacterial vaginosis associated bacterium 2(BVAB2), Megaspheara type I, Mycoplasma hominis, Mobiluncus curtisii, Ureaplasma parvum (genotypes) and Ureaplasma urealyticum and/or Lactobacillus sp. Infectious agent biomarker Ct values of less than a diagnostic cutoff (e.g., 39 Ct) with results of host biomarker (RPP30) is greater than the diagnostic cutoff is considered an adequate sample and is considered detected or positive.

[0104] In another embodiment, the cycle threshold value was applied to one or more bacteria associated with aerobic vaginitis such as Escherichia coil, Group B Streptococcus and other Streptococcus spp., Staphylococcus aureus, and Enterococcus faecalis and/or Lactobacillus sp. Infectious agent biomarker Ct values less than a diagnostic cutoff (e.g., 39 Ct) with results of host biomarker (RPP) is greater than the diagnostic cutoff is considered an adequate sample and considered detected or positive.

[0105] In another embodiment, the cycle threshold value was applied to one or more yeast associated with vulvovaginal candidiasis (VVC) such as Candida albicans and/or Candida glabrata and/or Lactobacillus sp. Infectious agent biomarker Ct values of less than a diagnostic cutoff (e.g., 39 Ct) with results of host biomarker (RPI)30) is greater than the diagnostic cutoff is considered an adequate sample and considered detected or positive.

[0106] In another embodiment, the cycle threshold value was applied to one or more bacteria associated with bacterial vaginosis such as Atopobium vaginae, Gardnerella vaginalis, bacterial vaginosis associated bacterium 2(BVAB2), Megaspheara type I, Mycoplasma hominis, Mobiluncus curtisii, Ureaplasma parvum (genotypes) and Ureaplasma urealyticum and/or Lactobacillus sp. Infectious agent biomarker Ct values of less than a diagnostic cutoff (e.g., 39 Ct) with results of host biomarker (RPP30) is less than the diagnostic cutoff is considered an adequate sample and is considered positive. The results of specimens with multiple positive organisms have associated specificity, sensitivity, and accuracy scores associated with the lab result so to guide diagnosis with confidence.

[0107] In another embodiment, the Ct value was applied to one or more bacteria associated with aerobic vaginitis such as Escherichia coli, Group B Streptococcus and other Streptococcus spp., Staphylococcus aureus, and Enterococcus faecalis and/or Lactobacillus sp. Infectious agent biomarker Ct values of less than a diagnostic cutoff (e.g., 39 Ct) with results of host biomarker (RPP30) is less than the diagnostic cutoff is considered an adequate sample and is considered positive. The results of specimens with multiple positive organisms have associated specificity, sensitivity, and accuracy scores associated with the lab result so to guide diagnosis with confidence.

[0108] In another embodiment, the cycle threshold value was applied to one or more bacteria associated with bacterial vaginosis such as Atopobium vaginae, Gardnerella vaginalis, bacterial vaginosis associated bacterium 2 (BVAB2), Megaspheara type 1, Mycoplasma hominis, Mobiluncus curtisti, Ureaplasma parvum (genotypes) and/or Ureaplasma urealyticum and/or Lactobacillus sp, where the following algorithm for the host to infectious agent biomarker is calculated:

[00004]$\frac{100-\mathrm{Host}\mathrm{Marker}\left(\mathrm{Ct}\right)}{100-\mathrm{infectious}\mathrm{agent}\mathrm{biomarker}\mathrm{Ct}\mathrm{values}}$$\frac{100-\mathrm{Host}\mathrm{Marker}\left(\mathrm{Ct}\right)}{\mathrm{Ave}\left(100-x,100-y,100-z,\mathrm{.Math.}\right)*}*\mathrm{when}\mathrm{infectious}\mathrm{agent}\mathrm{is}\mathrm{detected}\mathrm{at}a\mathrm{given}\mathrm{threshold}$

[0109] The reference range is organism specific and linked to database values identifying confidence levels for specificity, sensitivity, and accuracy of results with diagnosing BV. Results below a threshold (e.g., 50% specificity) were reported as indeterminate or inconclusive.

[0110] In one embodiment, the cycle threshold (Ct) value is applied to one or more bacteria associated with aerobic vaginitis such as Escherichia coli, Group B Streptococcus and other Streptococcus spp., Staphylococcus aureus, and Enterococcus faecalis and/or Lactobacillus sp, where the following algorithm for the host to infectious agent biomarker is calculated:

[00005]$\frac{100-\mathrm{Host}\mathrm{Marker}\left(\mathrm{Ct}\right)}{100-\mathrm{infectious}\mathrm{agent}\mathrm{biomarker}\mathrm{Ct}\mathrm{values}}$$\frac{100-\mathrm{Host}\mathrm{Marker}\left(\mathrm{Ct}\right)}{\mathrm{Ave}\left(100-x,100-y,100-z,\mathrm{.Math.}\right)*}*\mathrm{when}\mathrm{infectious}\mathrm{agent}\mathrm{is}\mathrm{detected}\mathrm{at}a\mathrm{given}\mathrm{threshold}$

[0111] The reference range is organism specific and linked to database values identifying confidence levels for specificity, sensitivity, and accuracy of results with diagnosing AV. Results below a threshold (e.g., 50% specificity) were reported as indeterminate or inconclusive.

[0112] In another embodiment, the cycle threshold (Ct) value was applied to one or more yeasts associated with VVC such as Candida albicans and Candida glabrata and/or Lactobacillus sp. Where the following ratio for the host to infectious agent biomarker is calculated:

[00006]$\frac{100-\mathrm{Host}\mathrm{Marker}\left(\mathrm{Ct}\right)}{\mathrm{Ave}\left(100-x,100-y,100-z,\mathrm{.Math.}\right)*}*\mathrm{when}\mathrm{infectious}\mathrm{agent}\mathrm{is}\mathrm{detected}\mathrm{at}a\mathrm{given}\mathrm{threshold}$

[0113] The reference range is organism specific and linked to database values identifying confidence levels for specificity, sensitivity, and accuracy of results with diagnosing VVC. Results below a threshold (e.g., 50% specificity) were reported as indeterminate or inconclusive.

[0114] In another embodiment, the specificity, sensitivity, and accuracy scores were included in the laboratory result for BV associated infectious agents providing personalized medicine reporting.

[0115] In another embodiment, the ratio/algorithm value for BV was displayed either in a graph or text for each patient providing longitudinal data and tracking of laboratory results over time. This display provided normalized data to track treatment response or failure. (See examples in FIGS. 11-14).

[0116] In another embodiment, the specificity, sensitivity and accuracy scores were included in the laboratory result for AV associated infectious agents providing personalized medicine reporting.

[0117] In another embodiment, the ratio/algorithm value for AV associated biomarkers was displayed either in a graph or text for each patient providing longitudinal data and tracking of laboratory results over time. This display provides normalized data to track treatment response or failure.

[0118] In another embodiment, the specificity, sensitivity and accuracy scores were included in the laboratory result for VVC associated infectious agents providing personalized medicine reporting.

[0119] In another embodiment, the ratio/algorithm value for VVC associated biomarkers was displayed either in a graph or text for each patient providing longitudinal data and tracking of laboratory results over time. This display provides normalized data to track treatment response or failure.

[0120] In another embodiment, where various sexually transmitted infectious agents (e.g., CT/NG, Trichomonas vaginalis, Mycoplasma genitalium) is not detected (e.g., using IVDs tests or other testing methods) have the sample also tested for the host biomarker (e.g., RPP30) was greater than diagnostic cutoff (e.g., 39 Ct) is considered an inadequate sample. This result was reported as indeterminate or inconclusive.

[0121] Addition of wellness score incorporating the detection of Lactobacillus sp can further refine the normalization of the data. It should be understood that the ratio and algorithms including the host biomarker described herein are intended as examples only.

[0122] In some embodiments, the results of a testing sample can be delivered with a confidence rating (i.e., low positive, medium positive, high positive, low negative, medium negative, high negative, etc.). The confidence rating provides insight to the physician or review of the testing results as to the relative confidence or certainty of the diagnosis.

[0123] In some embodiments, the confidence ratings can be based on the ratios of the quantities of the infectious bacteria biomarker to the host biomarker determined from the algorithm. For instance, if the threshold for a positive diagnosis is a ratio of 0.5 or higher, ranges above such a ratio, for instance 0.5-0.7, 0.7-1.0, and >1.0 can be associated with a confidence rating of a low, medium, and high positive, respectively.

[0124] In other embodiments, the confidence rating determination can also take into account actual quantity of a host biomarker and/or infectious biomarker. If a positive or negative diagnosis is achieved, then a physician would likely have more confidence in the diagnosis if the sample size was larger vs if the sample size was smaller. For instance, in the example above, if the calculated ratio in a sample was 0.6, it would be assigned a low positive confidence threshold based on the ratio alone. However, if the physical quantity of host biomarker and/or infectious biomarker were determined to be a large amount, indicative of a large sample size, that determination of a large sample size may affect the confidence rating, such that the confidence rating could be increased to a medium confidence given the larger sample size. Similar analysis between the determined ratios and quantitative values for the host and infectious bacteria biomarkers can be provided for the entire spectrum of potential results and ratio/quantitative data combinations (FIG. 14B). Such confidence thresholds for the different combinations of ratio/quantitative data combinations can be ascertained from empirical data.

[0125] In other embodiments, the confidence and various other statistical interpretations can be based on empiric data from a database.

[0126] FIG. 16 illustrates the development and utilization of information for a virtual vaginal wellness tool, whereby empiric data is captured along with demographic information for samples presenting with similar results.

[0127] FIG. 17 illustrates various diagnostic values associated with specimens containing multiple organisms. Examples of results that would be pulled from virtual database of tested samples. All fields may not be shown in the result for the healthcare provider.

[0128] Thus, although there have been described particular embodiments of the present invention of a new and useful host/infectious disease biomarker testing, it is not intended that such references be construed as limitations upon the scope of this invention.

[0129] All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).

[0130] It should be understood that although the present invention has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the present invention and it will be apparent to one skilled in the art that the present invention may be carried out using a great number of variations of the devices, device components, and method steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a great number of optional composition and processing elements and steps.

[0131] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein.

[0132] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their publication or filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art. For example, when compositions of matter are claimed, it should be understood that compounds known and available in the art prior to Applicant's invention, including compounds for which an enabling disclosure is provided in the references cited herein, are not intended to be included in any composition of matter claims herein.

[0133] As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms “comprising”, “consisting essentially of,” and “consisting of” may be replaced with either of the other two terms. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0134] One of ordinary skill in the art will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0135] Since other modifications and changes varied to fit particular operating requirements and environments will be apparent to those skilled in the art, the invention is not considered limited to the example chosen for purposes of disclosure and covers all changes and modifications which do not constitute departures from the true spirit and scope of this invention. Having thus described the invention, what is desired to be protected by Letters Patent is presented in the subsequently appended claims.