Quantum Dots for Diagnostic Imaging

Abstract

Methods for detecting disease in a patient are disclosed. The methods involve administering to the patient a quantum dot-analyte conjugate, which includes an analyte that binds to a marker for the disease in the patient's gastrointestinal tract. The analyte is conjugated to a quantum dot having a characteristic emission wavelength. Using an endoscopic modality, a physician can illuminate portions of the patient's gastrointestinal tract and detect the presence of the marker based on emission of the quantum dot. Also disclosed are methods of predicting a response to a treatment in a patient.

Claims

1. A mixture of at least two different quantum-dot analyte conjugates adapted for administration to a patient in order to discriminate between possible gastrointestinal diseases in the patient, the mixture of at least two different quantum-dot analyte conjugates comprising: (1) a first quantum dot-analyte conjugate comprising a first analyte capable of binding to a marker for a first possible disease in the patient's gastrointestinal tract, wherein the first quantum-dot analyte conjugate includes a first quantum dot having emission at a first characteristic wavelength when illuminated at an excitatory wavelength in the patient's gastrointestinal tract; (2) a second quantum dot-analyte conjugate comprising a second analyte capable of binding to a marker for a second possible disease in the patient's gastrointestinal tract, wherein the second quantum-dot analyte conjugate includes a second quantum dot having emission at a second characteristic wavelength that is different from the first wavelength when illuminated at the excitatory wavelength in the patient's gastrointestinal tract; wherein at least one of the first and second analytes binds to and identifies a presence of disease related immune cells in the gastrointestinal tract of the patient and at least one of the analytes is an antibody selected from anti-IFN, anti-IL1, anti-CD11b, anti-alpha-4 integrin, anti-B220, anti-IL3, anti-IL5, and anti-addressin.

2. The mixture of claim 1, wherein the first and second quantum dots comprise indium phosphide, an alloy of indium phosphide or a doped derivative of indium phosphide.

3. The mixture of claim 1, wherein the mixture is adapted for oral administration.

4. The mixture of claim 1, wherein the mixture is adapted for administration as a suppository or intravenously.

5. The mixture of claim 1, wherein the first quantum-dot analyte conjugate comprises a first analyte specific for binding to a bacteria, virus or parasite, and the second quantum-dot analyte conjugate comprises a second analyte that binds to and identifies the presence of disease related immune cells in the gastrointestinal tract of the patient.

6. The mixture of claim 5, wherein the bacteria is a Mycobacterium.

7. The mixture of claim 1, wherein the first quantum-dot analyte conjugate comprises a first analyte specific for binding to a marker that identifies an elevated level of macrophages or neutrophils in the patient's gastrointestinal tract, and the second quantum-dot analyte conjugate comprises a second analyte specific for binding to a marker that identifies an elevated level of eosinophils in the patient's gastrointestinal tract.

8. The mixture of claim 1, wherein the first quantum-dot analyte conjugate comprises a first analyte specific for binding to a marker that identifies malignant cells in the patient's gastrointestinal tract, and the second quantum-dot analyte conjugate comprises a second analyte that binds to and identifies the presence of disease related immune cells in the gastrointestinal tract of the patient.

9. A mixture of at least two different quantum-dot analyte conjugates adapted for administration to a patient in order to discriminate between possible gastrointestinal diseases in the patient, the mixture of at least two different quantum-dot analyte conjugates comprising: (1) a first quantum dot-analyte conjugate comprising a first analyte capable of binding to a marker for a first possible disease in the patient's gastrointestinal tract, wherein the first quantum-dot analyte conjugate includes a first quantum dot having emission at a first characteristic wavelength when illuminated at an excitatory wavelength in the patient's gastrointestinal tract; (2) a second quantum dot-analyte conjugate comprising a second analyte capable of binding to a marker for a second possible disease in the patient's gastrointestinal tract, wherein the second quantum-dot analyte conjugate includes a second quantum dot having emission at a second characteristic wavelength that is different from the first wavelength when illuminated at the excitatory wavelength in the patient's gastrointestinal tract; wherein the mixture comprises a first quantum dot-analyte conjugate specific for binding to one of cell-type specific markers TNF, INF, IL1, CD11b, alpha-4 integrin, anti-B220, IL3, IL5, myeloperoxidase, and addressin, and a second quantum dot-analyte conjugate specific for binding to another of cell-type specific markers TNF, IFN, IL1, CD11b, alpha-4 integrin, anti-B220, IL3, IL5, myeloperoxidase, and addressin.

10. The mixture of claim 9, wherein the first and second quantum dots comprise indium phosphide, an alloy of indium phosphide or a doped derivative of indium phosphide.

11. The mixture of claim 9, wherein the mixture is adapted for oral administration.

12. The mixture of claim 9, wherein the mixture is adapted for administration as a suppository or intravenously.

13. A method of treating a patient having a gastrointestinal disease, the method comprising: administering an oral mixture comprising first emission color quantum dot conjugates to one of a group of cell-type specific markers selected from INF, INF, IL1, CD11b, alpha-4 integrin, anti-B220, IL3, IL5, myeloperoxidase, and addressin, and second emission color quantum dot conjugates to a different one of the group of cell-type specific markers; illuminating the patient's gastrointestinal tract and determining a relative fluorescence of the first emission color quantum dot conjugates and the second emission color quantum dot conjugates; and selecting treatment of the patient based on the highest relative fluorescence intensities of the first and second emission color quantum dots.

14. The method of claim 13, wherein the first emission color quantum dots are conjugated to anti-TNF antibodies and the second emission color quantum dots are conjugated to anti-IL1 antibodies.

15. The method of claim 13, wherein the first and second emission color quantum dot conjugates comprise quantum dots formed of indium phosphide, an alloy of indium phosphide or a doped derivative of indium phosphide.

16. The method of claim 13, wherein the illuminating of the patient's gastrointestinal tract and determining a relative fluorescence of the first and second emission color quantum dot conjugates is performed by capsule endoscopy.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 is a diagram illustrating the quantum dot imaging technique. For example, to distinguish between two conditions, X and Y, after administering the quantum dot contrast agent, the underlying cause of the inflammation can be distinguished by the color the inflamed bowel wall fluoresces upon irradiation by a capsule endoscope.

[0033] FIG. 2 is a flow chart showing the process for detecting the presence of disease markers using quantum dots, as described herein.

DESCRIPTION

[0034] Labelling of biological entities with QDs has been reported in the prior art. A given size population of QDs will fluoresce at a specific wavelength, which has been used in the detection and diagnosis of medical conditions via in vivo animal studies. The fluorescence wavelength (colour) of QDs depends on the particle size. The present disclosure expands on prior art imaging techniques, by combining two or more size populations of QDs, each population (colour) being labelled with moieties that will selectively bind to a disease marker that is present in one condition but is not observed in the condition(s) that are detectable using the other size population(s) of labelled QDs. By combining the technique with capsule endoscopic imaging, the labelled biological entities can be observed by their fluorescence upon illumination by the endoscopic light source. The technique offers the potential to distinguish between two or more differential diagnoses by the colour that the tissue fluoresces upon illumination. When combined with clinical findings (such as symptoms), this may enable patients to be diagnosed where the location of their condition has previously proved challenging to biopsy, such as areas of the small bowel beyond the reach of standard endoscopes.

[0035] Error! Reference source not found. summarizes some inflammatory conditions that can affect the small bowel and the histological presentations used to assist in making a diagnosis when a biopsy is taken. Where applicable, strategies that could potentially be developed to label these histological observations with QDs, within the scope of the present method, are included.

TABLE-US-00001 TABLE 1 Inflammatory conditions and diagnostic strategies. HISTOLOGICAL CONDITION OBSERVATIONS QD LABELLING STRATEGY (Marker) REF. CROHN'S small granulomas granulomas - collection of macrophages - 1, 2 DISEASE (CD) label e.g. with cytokine antibodies or CD11b antibody no caseation Mycobacterium wouldn't be present - no 3, 4 fluorescence in the presence of a mycobacterium-specific QD label deep ulceration n/a - observed macroscopically inflammation targeting crypt inflammation is characterized by 5 crypt cells neutrophils - label with anti-myeloperoxidase mucosal changes distal mucosal changes observable macroscopically 1, 2 to sites with granulomas granulomas - collection of macrophages - label e.g. with cytokine antibodies or CD11b antibody GASTRO- acid-fast bacilli QD bacterial labelling 3, 4 INTESTINAL multiple/large/confluent label macrophages, e.g. with cytokine 1, 2 TUBERCULOSIS granulomas antibodies or CD11b antibody - (GITB) larger/multiple regions of fluorescence should be observed caseating necrosis caused by Mycobacterium - label bacteria 3, 4 with QDs epithelioid histocytes activated macrophages - label e.g. with 1, 2 cytokine antibodies or CD11b antibody BEHET'S vasculitis involving DISEASE (BD) small/medium-sized vessels lymphocytic infiltrate labelling of lymphocytes - conjugate to 2 amine-reactive B220 antibody. dense perivascular inflammatory cells located around blood 1, 2, 5 infiltrate vessels - observable by labelling of leukocytes ULCERATIVE villous atropy n/a - observed macroscopically JEJUNITIS (UJ) crypt hyperplasia epithelial lymphocytes labelling of lymphocytes - conjugate to 2 amine-reactive B220 antibody. eosinophil and plasma eosinophils develop in the presence of 6 cell infiltration of the interleukin-3 and -5 - label with QD- lamina propria conjugated IL-3 or IL-5 antibodies biopsy may be required to confirm location of eosinophils enterocyte pleomorphism fibrovascular granulation tissue between crypt bases and muscularis mucosae LUPUS eosinophils in deeper eosinophils develop in the presence of 6 ENTERITIS (LE) areas of the lamina interleukin-3 and -5 - label with QD- propria between the tip conjugated IL-3 or IL-5 antibodies biopsy of the mucosal glands may be required to confirm location of and muscularis mucosa eosinophils eosinophils and eosinophils develop in the presence of 6 exocytosis in the interleukin-3 and -5 - label with QD- mucosal glandular conjugated IL-3 or IL-5 antibodies biopsy epithelium may be required to confirm location of eosinophils

Detection of Bacteria Using Quantum Dots

[0036] QDs can be used for the detection of bacteria, e.g. Mycobacterium tuberculosis in the case of gastrointestinal tuberculosis (GITB). Edgar et al. proposed a method of detecting slow growing bacterial strains such as Mycobacterium. [R. Edgar, M. McKinstry, J. Hwang, A. B. Oppenheim, R. A. Fekete, G. Giulian, C. Merril, K. Nagashima and S. Adhya, Proc. Natl. Acad. Sci., 103 (2006) 4841] The method, which was successfully demonstrated for the detection of E. coli, involved engineering a phage (virus that infects bacteria) displaying a peptide that can be biotinylated, bound to its outer shell. Streptavidin-coated QDs were conjugated to the phage. In the presence of bacteria sensitive to the phage, the phage would infect the bacteria, producing progeny virions that could be biotinylated by a protein in each bacterium. This technique enabled a high degree of amplification to occur for each bacterium present, accelerating the rate at which detection could occur. Thus, by conjugating streptavidin-coated QDs to a phage that invades M. tuberculosis, the method can detect GITB during capsule endoscopy.

Detection of White Blood Cells Using Quantum Dots

[0037] White blood cells (WBCs), or leukocytes, are the components of the blood that act as part of the body's immune system to defend itself from attack by harmful species such as bacteria, viruses, parasites, and foreign bodies. In autoimmune diseases, the immune system fails to recognize its own tissue, instead attacking it as if the tissue were a foreign species. High levels of WBCs are typically observed in and around inflamed tissue, though one or more types of WBC may be characteristically expressed by a particular condition. For instance, in conditions affecting the small bowel, high levels of macrophages and neutrophils (characteristic of granulomas and crypt inflammation) may be expressed in Crohn's disease, while over-expression of eosinophils is typical of lupus enteritis.

[0038] Activated white blood cells express certain proteins or enzymes, many of which have a known antibody. By conjugating QDs to the appropriate antibody, the method can be developed to detect high levels of specific types of WBCs in situ from the color that they fluoresce.

[0039] As summarized in Error! Reference source not found., granulomas are observed of a number of small bowel conditions. Granuloma size and distribution from biopsy sites are often used to differentiate between diagnoses. Since granulomas are collections of macrophages, one method for their detection would be via macrophage labelling. Certain cytokines (cell signaling proteins), e.g. TNF- and INF-, are secreted by macrophages. Monoclonal antibody therapies, targeting cytokines, have recently generated high interest, and as such a number of cytokine antibodies are commercially available. Conjugation of appropriate cytokine antibodies to QDs could be exploited as a method of detection of macrophages in vivo.

[0040] Yuan et al. conjugated human anti-rabbit TNF- antibody to CdTe QDs by forming an acrylamide bond between carboxyl groups capping the QDs and amino groups of the TNF- antibody. [L. Yuan, X. Hua, Y. Wu, X. Pan and S. Liu, Anal. Chem., 83 (2011) 6800] Prior to this, the CdTe QDs were implanted onto the surface of polymer-functionalized silica nanospheres. Once conjugated with the cytokine antibodies, the QD-polymer functionalized silica nanosphere labels were used to detect TNF- antibodies via electrochemiluminescence and square-wave voltammetry measurements.

[0041] Integrin M is a protein expressed by macrophage cells. The CD11b antibody targets macrophage cells that express integrin M. Jennings et al. used fluorescent CD11b-nanoparticle conjugates to detect macrophage cells in mouse spleen tissue in vitro. [T. L. Jennings, R. C. Triulzi, G. Tao, Z. E. St. Louis and S. G. Becker-Catania, Sensors, 11 (2011) 10570]. CD11b is sulphydryl-reactive, so was conjugated with maleimide-capped QDs via a thioether bond between sulphydryl groups, formed by reduction of disulphide links on the antibody, and the maleimide ligand. In the same publication, leukocyte detection was also reported via QD conjugation to an amine-reactive antibody, B220, which targets B-cells (lymphocytes). Conjugation was achieved by first modifying the antibody with a heterobiofunctional crosslinking molecule that targets functionalities commonly found in proteins, forming a hydrazine functionality that would ligate to 4-formylbenzene-capped nanoparticles via a bis-aryl hydrazine bond.

[0042] Eosinophils, which are typically over-expressed in certain autoimmune diseases including systemic lupus erythematosus, are by definition eosinophilic, i.e. can be stained by the fluorescent dye eosin. Thus, eosinophils in biopsy samples are typically detected via staining with eosin. Eosinophil development occurs in the presence of interleukin-3 (IL-3) and interleukin-5 (IL-5) cytokines. As such, elevated levels of eosinophils in the GI tract, as may be observed in lupus enteritis, could potentially be detected using QDs conjugated to interleukin antibodies. A method to conjugate rabbit anti-IL-3 to colloidal gold nanoparticles for the detection of IL-3 using fluorescence immunoassay techniques has previously been described by Potkov et al. [L. Potkov, F. Frando, M. Bambouskov and P. Drber, J. Immunological Methods 371 (2011) 38] It is therefore be possible to conjugate colloidal QDs to anti-IL-3 for the detection of IL-3, as an indicator of the presence of eosinophils, in vivo.

[0043] Crypt cell inflammation, as observed in Crohn's disease, is typically accompanied by over-expression of activated neutrophils. QD labelling of activated neutrophils has been described by Hoshino et al., via the conjugation of anti-myeloperoxidase antibodies to fluorescent nanoparticles. [A. Hoshino, T. Nagao, A. Nakasuga, A. Ishida-Okawara, K. Suzuki, M. Yasuhara and K. Yamamoto, IEEE Trans. Nanobiosci., 6 (2007) 341] The myeloperoxidase enzyme is expressed on the surface of activated neutrophils, so the technique was found to selectively detect activated neutrophils, without binding to inactivated neutrophils.

[0044] Thus, in the method described herein, by preparing a contrast agent that combines a number of different QD labels, each type of QD biomarker having a different size population of QDs and therefore a different fluorescence wavelength, it is possible to distinguish between two or more potential causes of inflammation by the color(s) inflamed tissue in the small bowel fluoresces upon irradiation by a capsule endoscope.

Detection of Immune Cells Using Quantum Dots

[0045] Immune cells, many of which have a known antibody, are over-expressed at sites of inflammation. The type of immune cells released depends on the nature of the underlying cause of inflammation, with one or more varieties of immune cell being characteristic markers of a specific condition. Thus, labelling of immune cells using QD-immune cell antibody conjugates could be exploited as a method of their detection in vivo.

[0046] A further embodiment involves the employment of the QD imaging technique to assess a patient's potential response to one of more monoclonal antibody therapies. Monoclonal antibody therapies target over-expressed immune cells that are responsible for the inflammatory reaction in certain diseases with the aim of damping the body's immune response. A wide range of monoclonal antibody therapies have now been developed, many of which are licensed for the treatment of disease. As such, a large number of monoclonal antibodies are commercially available. Manipulation of the surface functionalization of QDs such that they can be conjugated to monoclonal antibodies used in disease treatment may offer a means to detect whether a patient will respond to that treatment; if the QD-antibody conjugate binds to the site of inflammation the treatment may be of benefit, whereas if no fluorescence is observed the antibodies are unlikely to target the site of inflammation and therefore the treatment may not be a worthwhile option.

[0047] TNF- is a cytokine that is predominantly produced by activated macrophages. Monoclonal antibody therapies targeting TNF- include infliximab and adalimumab, both of which cost in excess of 1000 per treatment. TNF- has previously been labelled with QDs. [L. Yuan, X. Hua, Y. Wu, X. Pan and S. Liu, Anal. Chem., 83 (2011) 6800] CdTe QD-polymer-functionalized silica nanospheres were bonded to anti-rabbit TNF- antibodies, to detect TNF- using electrochemiluminescence and square-wave voltammetry measurements. Similarly, the binding of anti-TNF- antibodies to the surface of QDs or QD polymer beads can be used to detect TNF- in vivo. This can be used as an indicator of macrophage activity, which in turn may be proportional to the size and distribution of granulomas. For assessment of a patient's potential response to anti-TNF- antibody therapy, by administering a contrast agent comprising QDs conjugated with anti-TNF- antibodies prior to a capsule endoscopy, an assessment as to whether high levels of TNF- are being expressed at sites of inflammation, and therefore whether the patient is likely to benefit from anti-TNF- antibody therapy, can be made.

[0048] Another cytokine, INF-, acts as a macrophage-activating factor. Binding anti-INF- to QDs could be used to detect presence of granulomas (macrophages).

[0049] The cytokine IL-1, catabolin, is produced by activated macrophages, monocytes, fibroblasts and dendritic cells. Its therapeutic antibody, canakinumab, is licensed for the treatment of a number of autoimmune diseases, with further clinical trials in progress to assess its potential in the treatment of other conditions. Conjugation of QDs with canakinumab can therefore be used to detect the presence of activated macrophages, monocytes, fibroblasts and/or dendritic cells at the site of inflammation, and/or to assess a patient's potential response to treatment with canakinumab.

[0050] Within the scope of the present disclosure, by way of example, a contrast agent comprising a first color of QDs conjugated to anti-TNF- antibodies and a second color of QDs conjugated to canakinumab (in appropriate proportions such that their adherence to inflamed tissue is approximately equal) can be administered to a patient prior to capsule endoscopy. The patient's potential response to treatment with one or other of the therapies can hence be evaluated by the color(s) that the inflamed bowel tissue fluoresces upon irradiation by the capsule endoscope, with the relative fluorescence intensities acting as a predictor of which, if either, of the two therapies is likely to be the most effective. The method is not restricted to the comparison of two antibody therapies; any number of QD-antibody conjugates can be incorporated into the contrast agent providing that their fluorescence wavelengths are sufficiently distinguishable by the naked eye.

QD Preparation

[0051] For use in vivo, the QD contrast agent should be non-toxic and emit in the visible region of the electromagnetic spectrum. This can be achieved using many types of semiconductor material (toxic or otherwise, providing the nanoparticles are appropriately functionalized to render them non-toxic in vivo). For example, III-V-based QDs, such as InP-based QDs (including alloys and doped derivatives thereof), may be employed. Recent evidence claims that the cytotoxicity of InP-based QDs is reduced upon decomposition in vivo, suggesting that the material would be safe for use in contrast agents administered to humans. [H. Chibli, L. Carlina, S. Park, N. M. Dimitrijevic and J. L. Nadeau, Nanoscale 3 (2011) 2553] An example of suitable commercially available QD material is CFQD quantum dots (Nanoco Technologies, UK). In some embodiments, the QD cores are capped with one or more shell layers of a wider band gap material, which may include one or more compositionally graded alloys, to eliminate surface defects and dangling bonds, thus improving the QD optical properties. Examples include, but are not restricted to, InP/ZnS, InP/ZnS/ZnO, or InP/ZnSe1-xSx.

[0052] Conjugation of QDs to antibodies or other analytes depends on functionalization of the nanoparticles with ligands containing moieties that are able to bind to accessible functionalities in the antibody. Methods to alter the surface functionality of QDs are well known in the prior art, and include ligand exchange procedures and polymerization techniques.

[0053] If specific particle sizes are required, e.g. larger than the QD regime so as to adhere only to cell membranes rather than penetrating into cells, QDs can be incorporated into polymer beads. The beads can then be functionalized to conjugate to the desired analyte. Incorporating QDs into polymer beads in this fashion may also provide an effective means to achieve aqueous compatibility, as described in the applicant's co-pending US patent application 2010/0059721, which is hereby incorporated by reference in its entirety.

Contrast Agent Preparation

[0054] After selecting two or more desired QD-analyte conjugates, a contrast agent may be prepared by mixing the labelled QDs in appropriate concentrations such that their relative affinity to their respective target cells are equal, i.e. for a given area of inflamed tissue, approximately equal fluorescence intensities would be observed by the naked eye regardless of the QD-analyte conjugate (color) with which it is labelled. The observed fluorescence intensity should take into consideration the human eye's spectral response in low light intensity conditions produced by the pulse of light from a capsule endoscope. One skilled in the art will be able to develop a method to quantify the fluorescence intensity from each color of QD-analyte conjugates using computerized software.

[0055] For imaging of the small bowel, in some embodiments the contrast agent is prepared into an oral solution to be ingested by the patient prior to the capsule endoscopy examination. However, the method of administration is not restricted to the oral route; one skilled in the art will realize that other methods of administration, such as a suppository or intravenous injection, may be suitable depending on the tissue or organ to be imaged. Other than the QD-analyte conjugates, the additional components of the contrast agent, which may be added to assist in the delivery of the QD fluorescent labels and/or improve the palatability of the solution, should not fluoresce in the visible region under illumination by the wavelength of light used in the capsule endoscope. All components should be non-toxic. The contrast agent should be designed such that the QD fluorescent labels remain in the small bowel for at least the time it takes to ingest the solution and perform the examination (in the region of ten hours), but should not remain in the body for substantially longer.

[0056] In the presence of the appropriate disease marker, a QD-analyte conjugate will bind to the bowel wall. Where a disease marker is not present in the bowel, the corresponding QD-analyte conjugate will not bind to the bowel wall and will be excreted.

[0057] Following administration of the contrast agent, a capsule endoscopy is performed on the patient to image the small bowel. The QD-analyte conjugate(s) that are bound to the bowel wall are detected by their fluorescence upon irradiation by the capsule endoscope, enabling the presence of one or more specific disease markers to be identified.

[0058] The process of using QDs to detect specific disease markers is described in FIG. 2.

[0059] It is the aim of the present method that the QD-containing contrast agent described herein should enhance the diagnostic capabilities of the capsule endoscopy technique, by labelling specific disease markers with QDs emitting at a distinct wavelength depending on the cause of the underlying disease, to improve in the diagnosis of conditions affecting the small bowel.

[0060] Rather than just using one color of QDs, the present method labels specific analytes with different colored QDs which can help to distinguish between several conditions in a single endoscopy, rather than just a positive or negative answer for a single condition. Further, the precise wavelength tunability of QDs and the ability to control their conjugation by manipulating their surface chemistry offers the potential to design bespoke contrast agents based on the patient's presenting symptoms and other test results.

[0061] The imaging technique is non-invasive, unlike conventional endoscopies where there is a risk of bleeding from a biopsy site. The capsule endoscopy procedure is usually comfortable, with the patient being able to continue with daily activities throughout the test and without a recovery period afterwards. In contrast, conventional endoscopic procedures are uncomfortable, often requiring prolonged periods of sedation and a recovery period in the region of 24 hours.

[0062] When using the contrast agent to assess the potential response to monoclonal antibody therapies, the technique may provide faster access to the most effective treatment, rather than subjecting a patient to sequential courses of different treatments until a satisfactory response is achieved. This may not only save time, reducing the risk of the patient's health deteriorating further, but may also save money since monoclonal antibody therapies are highly expensive, while two or more doses may be required to assess the patient's response. Further, by providing faster access to the most effective treatment, potential side-effects of the ineffective treatment(s) are also avoided.

[0063] In summary, the present method may be of benefit to both the patient and the healthcare provider, by delivering a faster diagnosis and identification of an appropriate treatment.

REFERENCES

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