Anita Mahadevan-Jansen - US grants

9806047
Mahadevan-Jansen
The objective of this research is to construct and characterize two new probes to be used in surgery for the detection of brain tumor tissue (surface probe) and pre-cancerous tissue in the ovary (endoscopic probe). The feasibility of the probe design will be evaluated using a tabletop experimental system and standard materials. Final probes will be tested in vivo using an appropriate animal model. This research is appropriate for the POWRE program because it will permit the principal investigator to establish an independent research program following a maternity leave of absence for 6 months before starting her position as a research associate.
***

DESCRIPTION (Verbatim Adapted from Applicant's Abstract): Research has shown removing maximum amount of tumor with minimal sacrifice to normal tissues is the key to improving the survival rate of brain tumors. Thus, there is a greater need for an intra-operative tool, which effectively detects tumor margins in real time and provides a sub-millimeter spatial resolution for guidance of tumor resection. Optical spectroscopy can provide such a tool as it has the advantage of providing automated, real-time, non-intrusive diagnosis with high sensitivity and spatial resolution. Fluorescence and diffuse reflectance spectra were acquired from normal and various types of tumor brain tissues in vitro of about 20 patients. Based on the spectral differences, diagnostic algorithms developed showed that fluorescence could differentiate normal white and gray matter from primary tumors with 97 percent sensitivity. Fluorescence alone was insufficient in separating normal brain tissues from secondary tumors; combining diffuse reflectance with fluorescence yielded 97 percent sensitivity for this discrimination. Following the success of these studies, a pilot study of 21 patients was successfully performed. Preliminary in vivo results showed that tumor margin tissues can be differentiated from normal tissues with a sensitivity and specificity of 83 percent and 85 percent respectively using fluorescence and diffuse reflectance spectra. In this proposal, we plan to develop autofluorescence in combination with diffuse reflectance spectroscopy for intra-operative brain tumor and tumor margin detection in real-time to guide tumor resection. To achieve this goal, the following specific aims are proposed; (1) Characterize tissue fluorescence and diffuse reflectance signatures of brain tissues in vivo. (2) Develop diagnostic algorithms that separate normal and tumor tissues from tumor margins. (3) Study the basis of observed differences in the spectral characteristics using microspectroscopy, cyto-chemical analysis, and modeling. (4) Conduct retrospective and prospective evaluation of the algorithms developed to obtain estimates of their performance. (5) Assess the feasibility of using optical spectroscopy during stereotactic procedures and verify the performance capability of this technique for brain tumor demarcation. (6) Develop (a) software interface to implement and automate data acquisition and diagnosis that provides real-time feedback to the surgeon for therapy guidance and (b) next-generation clinical spectroscopic system to reduce the scale but not the accuracy of the spectroscopic system. This research will have tremendous impact on the future of tumor resection as upon the successful development of the proposed research, this can be translated to the application of other organ systems such as prostate and ovary.

It was estimated that 4,400 deaths would occur in the United States alone from this disease and 12,900 new cases of invasive cervical cancer would be diagnosed in 2001. Existing screening and detection techniques, the Pap smear and colposcopy, have several deficiencies that prevent efficient management of an otherwise controllable disease. An automated diagnostic with improved sensitivity and specificity that could allow for a "See and Treat" protocol would significantly improve the management of the disease. Optical spectroscopy can provide automated, fast and non-intrusive characterization of normal and non-normal tissues. In particular, Raman spectroscopy can be used to provide accurate differential diagnosis of early disease. Preliminary results indicate the potential of using Raman spectroscopy for the diagnosis of cervical precancers and to translate its application for the detection of vulvar disease. In particular, in vitro studies show that Raman spectroscopy can differentiate between cervical precancers and all other tissue categories with a sensitivity and specificity of 91 percent and 90 percent, significantly better than fluorescence spectroscopy. More importantly, the results from the initial 13 patients studied in vivo, indicate that 1 it is possible to measure Raman spectra from cervical tissue in vivo and (2) Raman spectroscopy can identify cervical lesions in vivo I (with spectra similar to that observed in vitro). Thus this proposal seeks to develop a real-time, optical method for the differential diagnosis of cervical precancerous lesions by providing real-time, automated, non-intrusive information of the tissue biochemistry and pathology. In addition, this proposal seeks to extend the capability of this technique to include vulvar disease. The specific aims of the proposed project are as follows; (1) Characterize Raman signatures of cervical tissues in vivo. (2) Develop diagnostic algorithms that separate normal and non- precancerous tissues from precancerous tissues. (3) Study the basis of observed differences in the spectral characteristics using microspectroscopy, cyto-chemical analysis, and modeling. (4) Conduct retrospective and prospective evaluation of the algorithms developed to obtain estimates of their performance. (5) Assess the feasibility of using optical spectroscopy for vulvar disease and verify the performance capability of this technique for vulvar precancer detection. (6) Develop software interface to implement and automate data acquisition and provide real-time diagnosis and to develop a compact clinical Raman system to reduce the scale of the system while maintaining its accuracy. These objectives when achieved will yield a method of obtaining real-time, non intrusive detection of cervical precancers that will facilitate the immediate management of the disease with high sensitivity and specificity. In addition, the potential of translating the application of Raman spectroscopy to other organs and in the vulva, in particular, will be assessed.

DESCRIPTION (provided by applicant): In this proposal, the design, assembly and testing of a dual resolution image-guided confocal Raman microspectrometer in a compact instrument is proposed. The proposed system will consist of a handheld probe capable of providing three modes of information. (1) Color video imaging - will provide gross tissue imaging ( about 5mm x 5mm) and thus will guide the selection of interesting areas that require a closer look, (2) Bright-field confocal microscopy - will provide depth resolved morphological mapping from the center of the selected region ( about 300 mu m x 300 mu m) at a cellular level with a single point resolution of 0.75 mu m and (3) Raman microspectroscopy - will provide depth resolved biochemical evaluation of the selected region (same as confocal) with a resolution around 5-10 mu m to be optimized in the R21 phase based on signal to noise. The handheld probe will be coupled to a spectrometer and detector configuration that can compile, process and display all information required for tissue characterization in a compact instrument for clinical use as well research investigation. The instrument proposed here will be optimized for the detection of early skin cancer in a real-time, non-invasive manner as a diagnostic as well as surgical guidance tool.The specific aims of the feasibility phase (R21) of the proposed project are as follows; (1) Design and build a bench-top HIRM probe and instrument,. (2) Characterize Roman signatures of skin tissues in vitro and assess the feasibility of measuring it in vivo (3) Evaluate the parameters necessary to build a miniature HIRM device (4) Design the miniature HIRM probe and system based on the specifications determined in Aim (3). These objectives when achieved will show feasibility of the proposed work, determine the specifications and thus yield a design of a commercially viable, clinical useful handheld image-guided confocal Raman microspectrometer optimized for skin cancer detection.Once designed, the probe and system will be constructed and tested in the R33 phase of the proposal. The specific aims of the development phase of tile proposal are as follows; (1) Build and test the handheld probe and instrument using tissue phantoms and in vitro skin samples, debug system and modify design, (2) Characterize in vivo skin Raman spectra using the bench-top probe on 30 skin cancer patients and 30 normal volunteers, (3) Evaluate the basis for the differences seen in live tissue sections using the confocal Raman microspectrometer. (4) Use the final handheld probe prototype for skin tissue studies in vivo in 30 patients, (5) Develop diagnostic algorithms that distinguish between the various types of malignant and non-malignant skin tissues.The development of the proposed work will have a significant impact on health care by providing the potential for complete disease management with a single compact, sensitive detection tool. With the success of this technique, Raman spectroscopy can be applied to other organ sites such as the prostate and ovary as well as other diseases thus impacting a broader field in medicine.

DESCRIPTION (provided by applicant): The American Cancer Society recommends that the best way to find skin cancers early is to recognize changes in existing skin lesions or the appearance of new lesions by regular self-examination. One way to facilitate this routine examination of skin lesions would be the availability of an easy-to-use, non-invasive device that can scan the body in a relatively short time. Such a device could be utilized through the general physician's office or the dermatologist's office. Thus a novel tool that provides spatial, structural as well as biochemical information about tissue disease state is proposed directed initially towards skin cancer management. This revised proposal seeks to develop a handheld combined confocal Raman spectroscopy and confocal imaging device that, can provide real-time, morphologic as well as biochemical information of skin lesions, potentially identifying these lesions as normal, benign and malignant for diagnosis of the disease. Raman spectroscopy is a purely biochemical technique and yields little information about the tissue microstructure. Confocal imaging could provide spatial as well as structural information that together with confocal Raman spectroscopy could yield information about tissue structure as well as biochemistry allowing for complete diagnosis. Thus this proposal is focused on the development of a clinical, confocal Raman instrument with confocal imaging as well as bright-field imaging capability for the differential diagnosis of skin lesions by providing real-time, automated, non-intrusive spectral as well as spatial information about the tissue biochemistry as well as structure. The steps needed to implement such a device will be pursued independently. The specific aims of the proposed project are as follows;Specific Aim (1): Develop and test a compact handheld Raman probe with video imaging capability. Specific Aim (2) Develop and test a handheld confocal imaging device at video rate: Specific Aim (3): Develop and test an integrated handheld confocal Raman/scanning confocal imaging handheld device. Specific Aim (4): Test signatures of normal and malignant skin tissues in vivo. Specific Aim (5): Understand the basis and dynamics of the malignancy associated changes observed in malignant tissues in regions away from the disease. The pilot clinical studies planned in this proposal will be conducted at the Vanderbilt University Medical Center and the Vanderbilt Clinic. The development of the proposed work will have a significant impact on health care by providing the potential for complete disease management with a single compact, sensitive detection tool. With the success of this technique, this multi-modal instrument can be applied to other organ sites such as the prostate and ovary as well as other diseases impacting a broader field in medicine.

[unreadable] DESCRIPTION (provided by applicant): The incidence of skin cancer has been on the rise in recent years and as in most cancers, early diagnosis and thorough treatment (i.e., complete resection) are the keys to gain a favorable prognosis. Current diagnostic methods for skin cancers rely on physical examination in conjunction with skin biopsy, methods that have limited accuracy in early diagnosis. Hence there is considerable interest in developing a non-invasive diagnostic and screening tool, which can accurately recognize skin cancer in its early stages. Such a tool would not only detect early signs of malignancy, it will also reduce unnecessary diagnostic surgical procedures, facilitate complete excision of skin cancers by accurately delineating the skin cancer margins prior to and during the surgery, and allow treated patients to be monitored for recurrence. In this proposal we present a method that combines Optical Coherence Tomography (OCT), an imaging technique that visualizes tissue microstructure, with Raman spectroscopy, a spectroscopy technique that provides a tissue biochemical map in a single portable instrument for the effective detection of skin lesions. The real-time non-invasive nature of this combined technique can enable a "see and treat" protocol that would decrease the number of return clinic visits and patient anxiety as the long waits for histopathology diagnoses would be eliminated. Thus, the technology has the potential to increase access to underserved populations where follow-up to screening may not be available. The technology poses no known risks to the patient, and therefore could be a safe alternative to current screening methods. [unreadable] PUBLIC HEALTH RELEVANCE: The incidence of skin cancer has been on the rise in recent years and as in most cancers, early diagnosis and thorough treatment (i.e., complete resection) are the keys to gain a favorable prognosis. Current diagnostic methods for skin cancers rely on physical examinations in conjunction with skin biopsy, methods that have limited accuracy for early diagnosis. Hence there is considerable interest in developing a non- invasive diagnostic and screening tool, which can accurately recognize skin cancer in its early stages. Such a tool would not only detect early signs of malignancy, it could also reduce unnecessary diagnostic surgical procedures, facilitate complete excision of skin cancers by accurately delineating the skin cancer margins prior to and during the surgery, and allow treated patients to be monitored for recurrence. Initial reports have identified the practical limitations of non-invasive optical devices using either imaging or spectroscopy to evaluate the diverse clinical appearance of normal skin, benign lesions, and malignancy. In this proposal, we describe the design of a device that is specifically geared towards thorough non-invasive analysis of both the underlying microstructure and biochemical composition of the skin. The challenge presented by the variability of skin and skin cancers present the ideal test organ and pathology for the proposed multi-modal device. This R21 proposal seeks to validate the hypothesis that a device that combines morphological imaging with optical coherence tomography (OCT) and biochemical evaluation with Raman spectroscopy (RS) increases the diagnostic information available to the physician and could improve the diagnostic performance of each technique alone. The feasibility phase of this project is designed towards the development of a combined RSOCT system and the validation of such a combined imaging method for the detection of skin cancers. The specific aims are designed towards quantifiable and achievable milestones that will demonstrate the potential of such an approach towards cancer diagnosis in general and skin cancer detection in particular. Specific Aim #1: Develop and test a benchtop combined RS-OCT system: A benchtop version of the combined system will be developed on the platform of a Fourier Domain-OCT spectral radar system consisting of two sources but a single detector and sample arm. The performance of both OCT and RS will each be compared with benchmark values of existing in vivo systems. Specific Aim #2: Develop an RSOCT handheld probe for in vivo application: In order to test the utility of such a multi-modal system in vivo, a handheld probe will be developed and the entire system made portable to allow for flexibility of measurements from hard to reach lesion sites. The performance of both OCT and RS will each be compared with benchmark values of the benchtop system. Specific Aim #3: Conduct a pilot study on 30 patients with suspected basal cell carcinomas (BCCs) to demonstrate the utility RS-OCT: A small pilot study will evaluate the validity of the combined device in clinical practice where lesions suspected of BCCs will be assessed with RS alone, OCT alone, and combined RS-OCT. The ability of RS to discriminate between normal benign and malignant lesions will be compared with OCT guided RS. The specific aims described above will demonstrate the potential of RSOCT for the diagnosis and possible screening of skin cancers. The proposed work will have a significant impact on health care by providing the potential for complete disease management with a single detection tool. This would result in improved patient care and cost-effectiveness. With the success of this technique, RSOCT can be applied to other organ sites such as the breast, prostate and ovary as well as other diseases thus impacting a broader field in medicine. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cervical cancer is the second most common malignancy among women worldwide. It is estimated that in 2007, 3,670 deaths will occur in the United States alone from this disease and 11,150 new cases of invasive cervical cancer will be diagnosed. Although early detection of cervical precancer has played a central role in reducing the mortality associated with this disease over the last 50 years, the incidence of pre-invasive squamous carcinoma of the cervix. When cervical cancers are detected early, they are highly curable. Existing screening and detection techniques, the Pap smear and colposcopy, have several deficiencies that prevent efficient management of an otherwise controllable disease. An automated diagnostic method with improved sensitivity and specificity that could allow for a "See and Treat" protocol would significantly improve the management of the disease. This proposal seeks to prove the hypothesis that Raman spectroscopy, can provide differential diagnosis of cervical precancers, both high and low grade from inflammation, squamous metaplasia and normal areas of the cervix. The main objective of this proposal is to characterize and validate that low grade lesions can be differentiate from high grade lesions as well as the normal cervix and that this diagnosis can be performed regardless of the menopausal status of the patient. This will be accomplished by conducting a large clinical investigation, collecting Raman spectra from both normal and diseases cervix, separating them by histological category and classifying them via a multi-class discrimination algorithm. Additionally, with the development of a unique model for tissue spectral signatures, we seek to use this model to understand the disease process as it applies to optical spectroscopy. Finally, the system will be updated to be as compact as possible and the software developed to be "easy" (one click collection and classification of a spectra) for clinical use. The results of the proposed work will have a significant impact on health care by providing the potential for complete disease management with a single detection tool. This would result in improved patient care and cost-effectiveness. With the success of this technique, Raman spectroscopy can be applied to other organ sites such as the prostate and ovary as well as other diseases thus impacting a broader field in medicine. PUBLIC HEALTH RELEVANCE: Cervical cancer is the second most common type of cancer among women worldwide. Early detection of cervical precancer is the key to proper treatment of the disease before it becomes a cancer. Methods of detecting cervical precancer used today are limited in their ability to identify low and high grade disease from other non-normal conditions. This proposal presents a method for detecting cervical precancers in real time to guide treatment. Raman spectroscopy has the potential to be a more accurate and less expensive method of screening and diagnosing cervical dysplasia. The ability of Raman spectroscopy to enable a "see and treat" protocol would decrease the number of return clinic visits and patient anxiety as the long waits for histopathology diagnoses would be eliminated. Thus, the technology has the potential to increase access to underserved populations where follow-up to screening may not be available. The technology poses no known risks to the patient, and therefore could be a safe alternative to current screening methods.

DESCRIPTION (provided by applicant): Our understanding of cervical remodeling during pregnancy and labor is incomplete, partly due to the lack of in vivo studies on the biochemical changes that occur in the cervix over the course of pregnancy. Elucidation of the mechanisms for cervical ripening could be used to predict the onset of preterm labor. Until recently, in vivo research methods were too invasive to be used as discovery tools, particularly in women who present with preterm labor. This proposal will use in vivo Raman spectroscopy, an optical technique that is sensitive to collagen content, collagen structure, hydration, lipids, proteins, ad other biomolecules to non-invasively investigate the biochemistry of the cervix throughout pregnancy. Using fiber optic in vivo Raman spectroscopy, we recently found significant differences in Raman spectra in at least four important peaks during the course of pregnancy in mice, including discrete signatures for lipids, collagen, amide bonds, and enriched amino acids (proline, tyrosine). Computational analysis of these spectra yielded predictive algorithms with 94% classification accuracy for stage of pregnancy. Studies performed in 2-hour windows at the end of pregnancy identified spectra predictive for the timing of parturition. This approach provides a detailed real-time biomolecular map of cervical ripening that is currently unavailable by other means. In this proposal, we hypothesize that the different mechanisms of premature cervical ripening have unique Raman spectral signatures that correspond to underlying biochemical and mechanical changes that precede preterm birth, which can be detected in vivo. Two Specific Aims are proposed: 1) Determine spectral changes in the cervix of mice with normal and abnormal pregnancy and parturition; 2) Identify specific mediators of cervical remodeling by comparing Raman spectra to mechanical and biochemical changes in the ex vivo cervix during normal and abnormal parturition. Raman spectroscopy has primarily been used for detection of disease. Collaboration between our reproductive biology and bioengineering groups will capitalize on our expertise in Raman analysis of cervical tissues to study dynamic changes in cervix composition during pregnancy. Key elements in cervical biochemistry will be identified. In vivo Raman spectroscopy will be combined with biomechanical studies and imaging mass spectrometry, a powerful tool for in situ proteomic analysis, to examine mice with premature or delayed cervical remodeling. Together, these highly innovative approaches will generate in-depth profiles of cervical biology that will translate into novel non-invasive methods to detect impending premature birth in women.

DESCRIPTION (provided by applicant): Our understanding of cervical remodeling during pregnancy and labor is incomplete, partly due to the lack of in vivo studies on the biochemical changes that occur in the cervix over the course of pregnancy. Elucidation of the mechanisms for cervical ripening could be used to predict the onset of preterm labor. Until recently, in vivo research methods were too invasive to be used as discovery tools, particularly in women who present with preterm labor. This proposal will use in vivo Raman spectroscopy, an optical technique that is sensitive to collagen content, collagen structure, hydration, lipids, proteins, ad other biomolecules to non-invasively investigate the biochemistry of the cervix throughout pregnancy. Using fiber optic in vivo Raman spectroscopy, we recently found significant differences in Raman spectra in at least four important peaks during the course of pregnancy in mice, including discrete signatures for lipids, collagen, amide bonds, and enriched amino acids (proline, tyrosine). Computational analysis of these spectra yielded predictive algorithms with 94% classification accuracy for stage of pregnancy. Studies performed in 2-hour windows at the end of pregnancy identified spectra predictive for the timing of parturition. This approach provides a detailed real-time biomolecular map of cervical ripening that is currently unavailable by other means. In this proposal, we hypothesize that the different mechanisms of premature cervical ripening have unique Raman spectral signatures that correspond to underlying biochemical and mechanical changes that precede preterm birth, which can be detected in vivo. Two Specific Aims are proposed: 1) Determine spectral changes in the cervix of mice with normal and abnormal pregnancy and parturition; 2) Identify specific mediators of cervical remodeling by comparing Raman spectra to mechanical and biochemical changes in the ex vivo cervix during normal and abnormal parturition. Raman spectroscopy has primarily been used for detection of disease. Collaboration between our reproductive biology and bioengineering groups will capitalize on our expertise in Raman analysis of cervical tissues to study dynamic changes in cervix composition during pregnancy. Key elements in cervical biochemistry will be identified. In vivo Raman spectroscopy will be combined with biomechanical studies and imaging mass spectrometry, a powerful tool for in situ proteomic analysis, to examine mice with premature or delayed cervical remodeling. Together, these highly innovative approaches will generate in-depth profiles of cervical biology that will translate into novel non-invasive methods to detect impending premature birth in women.

PROJECT SUMMARY This proposal is based on an Academic-Industrial partnership between Vanderbilt University and Anasys Instruments Inc. that seeks to successfully bring a new imaging capability in endocrine cancer surgery to the end-user? the surgeon, for the intraoperative identification of the parathyroid gland. Here we propose to develop optical imaging in the near infrared for the real time visualization of relevant structures in the neck during endocrine surgery ? an application and a technology that has no predicate Thyroid and parathyroid diseases rely on surgery for definitive treatment. In these over 100,000 surgeries performed each year in the US, parathyroid glands are difficult to distinguish from the thyroid and surrounding tissues in the neck, due to its small size and variability in position. Complications occur when the parathyroid is accidentally injured or removed during thyroidectomies or only partially removed in the case of parathyroidectomies. In the former, hypoparathyroidsim and hypocalcemia can occur, resulting in serious long term effects. The incidence of hypocalcemia is reported to occur in 9-21% of total thyroidectomies. In fact, hypocalcemia is the most common cause of malpractice litigation after endocrine surgery. Therefore, there is a critical need for a sensitive tool that can identify the parathyroid glands intraoperatively, regardless of disease state. Vanderbilt and Anasys jointly propose the clinical translation of near infrared autofluorescence to improve patient outcome in endocrine cancer surgeries. The scientific innovation is in the application of an optical technique for surgical guidance that is not focused on disease detection but on anatomical identification of a physiological organ regardless of its disease state with near 100% accuracy. The technique of near infrared fluorescence (particularly in the wavelength range proposed here) has never been commercially implemented using natural biological fluorophores as the contrast agent. Further, combining fluorescence imaging with laser speckle imaging with tissue overlay for real-time assessment of the anatomy and function of the parathyroid gland adds to the innovation of this work. The specific aims of the proposed project are as follows: (1) Validate the fluorescence of tissues in the neck during endocrine surgery (2) Evaluate the basis of the observed NIR fluorescence. (3) Develop a combined fluorescence and laser speckle imaging system for clinical implementation. (4) Validate the performance of NIR fluorescence prospectively. These objectives, when achieved, will yield two systems ready for commercial translation that can provide real-time optical feedback to reduce the likelihood of long and short term side effects associated with accidental or incomplete removal of parathyroid tissue.

PROJECT SUMMARY Otitis media (middle ear infection) is a highly prevalent disease, especially in young children. It has been documented that more than 75% of children will have at least one episode of otitis media (OM) by age 3, and many children will have recurrent or chronic OM. In most cases, diagnosis of an ear infection is performed based on the appearance of the tympanic membrane and presence of fluid via an otoscope. In the absence of a method to identify bacterial infections, broad-spectrum antibiotics are prescribed without definitive knowledge of whether i) an active infection present, and ii) whether it is a bacterial infection (versus a viral infection). Previous clinical data also suggests that antibiotic therapy is only effective for one-third of OM patients, with two thirds of cases likely caused by antibiotic resistant bacteria, or more likely, viral pathogens, resulting in a misdirected expense of more than $700 million, annually. Therefore, there is a clear and urgent need for a point-of-care diagnostic tool to determine the presence of an active infection and whether an infection is bacterial in origin. Further, if we can distinguish the bacterial species present, unnecessary administration of broad-spectrum antibiotics can be eliminated, thus improving the efficacy of treatment and management of this highly prevalent disease. This proposal seeks to fulfill this unmet need by developing integrated Raman Spectroscopy-Optical Coherence Tomography (RSOCT) for the real-time detection and bacterial differentiation of pathological microorganisms in the middle ear. The scientific premise of the proposed research is that OCT can be used for image-guided placement of the RS probing beam, as well as visualization of any biofilm affixed to the tympanic membrane which would be indicative of a bacterial infection. This imaging system will be coupled with Raman spectroscopy for a more direct assessment of any biofilm and/or effusion by determining the presence of OM-causing bacteria and speciation via its biochemical fingerprint. The overall objective of the project will be accomplished by technological innovation and integration, followed by a series of systematic in vitro, ex vivo, and in vivo human studies with rigorous data analysis methods outlined in the specific aims. This research will have a profound impact on how we diagnose and care for these common ear infections. Most importantly, this project will provide critical data (identifying specific bacterial species) to monitor the development of antibiotic resistance, to reduce the overuse of antibiotics, and ultimately, to efficiently improve the health of patients.