Characterization of bacteria causing acute otitis media using Raman microspectroscopy.

Otitis media (OM) is a prevalent disease that is the most frequent cause of physician visits and prescription of antibiotics for children. Current methods to diagnose OM and differentiate between the two main types of OM, acute otitis media (AOM) and otitis media with effusion (OME), rely on interpreting symptoms that may overlap between them. Since AOM requires antibiotic treatment and OME does not, there is a clinical need to distinguish between AOM and OME to determine whether antibiotic treatment is necessary and guide future prescriptions. We used an optical spectroscopy technique, Raman spectroscopy (RS), to identify and characterize the biochemical features of the three main pathogens that cause AOM in vitro. A Renishaw inVia confocal Raman microscope at 785 nm was used to spectrally investigate the Raman signatures of Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae. Biochemical features or biomarkers important for classification of each bacterial species were identified and yielded a 97% accuracy of discrimination. To test the effectiveness of Raman-based bacterial classification in a clinical sample, human middle ear effusion (MEE) from patients affected by recurrent AOM was collected, cultured, and measured using RS. The probability of bacterial involvement from each of the three main bacteria that cause AOM was determined from the clinical MEE samples. These results suggest the potential of utilizing RS to aid in accurately diagnosing AOM and providing physicians with bacterial identification to guide treatment.

Prevention of anterior glottic restenosis in a canine model with topical mitomycin-C.

To develop a model for recurrent anterior glottic stenosis and to test the efficacy of topical mitomycin-C in preventing restenosis, we induced anterior glottic stenosis with a CO2 laser in 5 dogs. In 3 dogs, recurrence was established after surgical lysis. Subsequently, the 3 dogs received a single topical 3-minute treatment with a 1% solution of mitomycin-C after a second surgical lysis. In a parallel experiment, the other 2 dogs received a single topical 3-minute treatment with a 1% solution of mitomycin-C after the initial surgical lysis. An anterior glottic web was induced in all 5 dogs with the CO2 laser. The 3 dogs experienced restenosis at the anterior glottis after surgical lysis alone. Mitomycin-C prevented anterior glottic restenosis in 2 of the 3 dogs treated twice and in both of the dogs treated once (p = .02). We conclude that a recurrent stenosis of the anterior glottis may be induced reproducibly in the canine model with the CO2 laser. Application of topical mitomycin-C after lysis of an anterior glottic stenosis produces a statistically significant reduction in the rate of restenosis as compared to surgical lysis alone.

Human nasopharyngeal-associated lymphoreticular tissues. Functional analysis of subepithelial and intraepithelial B and T cells from adenoids and tonsils.

Subepithelial and intraepithelial lymphocytes of human adenoids and tonsils were characterized and directly compared to determine the potential contribution of these tissues to mucosal and systemic immune responses. The distribution of T and B cell subsets, cytokine patterns, and antibody (Ab) isotype profiles were similar for adenoids and tonsils. Both tissues contained predominantly B cells ( approximately 65%), approximately 5% macrophages, and 30% CD3(+) T cells. The T cells were primarily of the CD4(+) subset ( approximately 80%). Tonsillar intraepithelial lymphocytes were also enriched in B cells. The analysis of dispersed cells revealed a higher frequency of cells secreting IgG than IgA and the predominant Ig subclass profiles were IgG1 > IgG3 and IgA1 > IgA2, respectively. In situ analysis also revealed higher numbers of IgG- than IgA-positive cells. These IgG-positive cells were present in the epithelium and in the subepithelial zones of both tonsils and adenoids. Mitogen-triggered T cells from tonsils and adenoids produced both Th1- and Th2-type cytokines, clearly exhibiting their pluripotentiality for support of cell-mediated and Ab responses. Interestingly, antigen-specific T cells produced interferon-gamma and lower levels of interleukin-5. These results suggest that adenoids and tonsils of the nasopharyngeal-associated lymphoreticular tissues represent a distinct component of the mucosal-associated lymphoreticular tissues with features of both systemic and mucosal compartments.

Noninvasive fluorescent identification of bacteria causing acute otitis media in a chinchilla model.

OBJECTIVES: To evaluate a noninvasive method of bacterial identification via fluorescence spectroscopy in the setting of acute otitis media in a chinchilla model. STUDY DESIGN: Prospective. METHODS: For each chinchilla, transbullar inoculation with Streptococcus pneumoniae, Haemophilus influenzae, or Staphylococcus aureus was performed bilaterally and clinical infection was determined by otoscopy. An optical fiber coupled to a spectrofluorometer allowed for the delivery of an excitation wavelength to the inflamed tympanic membrane and the acquisition of the resulting emission signal. Sequential emission spectra obtained over a range of excitation wavelengths were assembled by a computer algorithm, and a single, three-dimensional plot was created for each test ear. Similarly, plots from the healthy external auditory canal (EAC) were also recorded. Twelve animals were used to establish a library of four reference plots representing the three bacteria and the EAC. Of the 24 ears available for study, 10 were excluded from analysis because of lack of clinical infection or presence of tympanic membrane perforation with purulent drainage. From four additional animals, four samples from ears infected with the above bacteria and three samples of the EAC served as unknowns. The unknown plots were analyzed by an investigator blinded to their identity. RESULTS: Using a multiple correlation of the unknown to the reference plots, seven of seven samples were correctly identified. CONCLUSIONS: We were able to establish a prototype method for the noninvasive identification of a limited library of pathogens in a chinchilla model of acute otitis media.

Preservation of function and histologic appearance in the injured glottis with topical mitomycin-C.

OBJECTIVE: To evaluate the functional and histological effects of a single application of topical mitomycin-C after laser injury in the posterior canine glottis. STUDY DESIGN: A prospective, randomized study of 16 canines. METHODS: A supersaturated (1%) solution of topical mitomycin-C was applied to a unilateral, laser-induced injury near the cricoarytenoid joint in eight dogs. The mitomycin-soaked pledget was placed immediately after induction of the injury and was left in contact with exposed cartilage for 3 minutes. The opposite side was not injured to provide an internal control. In eight additional dogs, the same laser injury was allowed to heal untreated. After 6 weeks, the animals were sacrificed and their larynges harvested. Arytenoid adduction sutures were placed bilaterally, and the force required to bring the vocal folds to midline was measured for each side using tensiometry. Gross and microscopic histological analysis was performed. Statistical analysis was accomplished using a two-tailed Student t test of unpaired samples, and the Wilcoxon Signed Rank Test where appropriate. RESULTS: The mitomycin-C treated larynges demonstrated improved cricoarytenoid joint mobility (P = .007), decreased granulation tissue development (P = .03), and complete prevention of secondary "vocal granuloma" formation (P = .0004) when compared with eight dogs with identical laser injuries allowed to heal untreated. No complications were noted. CONCLUSIONS: This study demonstrates functional preservation and improved histological appearance of the injured glottis after a single treatment with topical mitomycin-C. Potential applications of these findings include prophylactic use of topical mitomycin-C on glottic insults that commonly progress from granulation tissue formation to scarring and decreased vocal fold function.

Ablation of Teflon granulomas in the canine larynx with the free-electron laser.

This study develops a canine model for the treatment of laryngeal Teflon granulomas and demonstrates endoscopic ablation using the free-electron laser (FEL) set at a wavelength of 8.5 microm. Laryngeal Teflon granulomas may cause dysphonia and airway obstruction, and they are difficult to remove. The infrared absorption spectrum of Teflon reveals a strong absorption peak centered at 8.5 microm. In this study, 12 dogs had the right vocal cord injected with Teflon paste. Two months later, Teflon granuloma formation was confirmed histologically. Laser incisions into the granulomas were performed at 3 different wavelengths: 7.4 microm (FEL), 8.5 microm (FEL), and 10.6 microm (carbon dioxide laser). Histopathologic analysis was performed at 1 week and 6 weeks after the laser incisions. The FEL at the 8.5-microm wavelength was found to optimally ablate the Teflon granulomas, but the granulomas persisted in the specimens treated with 7.4 microm (FEL) and 10.6 microm (carbon dioxide laser).

Ablation of teflon granuloma with the free-electron laser emitting in the eight- to nine-micron range.

In this study, we developed a rat model for Teflon granuloma and used this model to evaluate the removal of the granuloma at laser wavelengths at which Teflon has a maximal absorption. Twenty-four Teflon granulomas were created in 12 rats, and the gross and histologic effects from laser incision at four different wavelengths (8.25, 8.5, 8.75, and 10.6 microm) were evaluated acutely and at 7 and 14 days postoperatively. Polytetrafluoroethylene, or Teflon, is a relatively inert substance that has been used over the past 4 decades for endoscopic injection into the thyroarytenoid muscle of the larynx for the purposes of laryngeal rehabilitation in cases of unilateral vocal fold paralysis or incomplete glottic closure. In certain cases in which formation of granulomatous reaction to the Teflon occurs, patients may have significant dysphonia or airway compromise. Once Teflon has infiltrated the surrounding tissue planes, it is exceedingly difficult to remove endoscopically. Endoscopic removal of this granuloma is usually attempted with the carbon dioxide (CO2) laser and has had limited success. Examination of the infrared absorption spectrum of polytetrafluoroethylene reveals strong absorption in the mid-infrared region in the 8- to 9-microm range, with minimal absorption at 10.6 microm. Therefore, this absorption spectrum predicts a more efficient vaporization of Teflon at wavelengths near 8.5 microm. Using the free-electron laser to generate 8.25-, 8.5-, and 8.75-microm laser light, we found Teflon granuloma ablation was far superior to CO2 laser ablation at 10.6 microm. The 8.25-, 8.5-, and 8.75-microm wavelengths selectively ablated Teflon granuloma with minimal to no collateral thermal injury to tissue. The differences in thermal effects observed while actually using the lasers were confirmed histologically.

Interstitial Nd:YAG photocoagulation for vascular malformations and hemangiomas in childhood.

BACKGROUND: Vascular malformations and cavernous hemangiomas are common in childhood. Although cavernous hemangiomas may resolve spontaneously, aggressive intervention is required when their growth could damage vital adjacent structures, such as the orbit, nose, or tongue. OBJECTIVE: To evaluate the efficacy of interstitial Nd:YAG photocoagulation as an adjunct to intralesional and systemic corticosteroids for treatment of hemangiomas and vascular malformations that had failed to respond to other therapies. DESIGN: Prospective, nonrandomized trial. SETTING: Two referral practices of facial plastic and reconstructive surgery in tertiary care, academic medical centers. PATIENTS: Ten consecutive pediatric patients with either hemangioma or vascular malformation of the head and neck. INTERVENTION: Laser photocoagulation with an interstitial technique. The Nd:YAG fiber was introduced into the lesion via a 14-gauge angiocatheter needle, and the laser fiber was advanced as coagulation proceeded within the tissue. MAIN OUTCOME MEASURES: Decrease in the area of the target lesion, amount of energy applied, and number of treatments required to achieve reduction in size. RESULTS: Long-term follow-up demonstrated regression of the lesion in all 10 patients with good cosmetic results. The range of reduction in size was 20% to 98%. No reexpansion of the lesions was noted after a mean follow-up of 13 months. CONCLUSIONS: Interstitial photocoagulation of hemangiomas and vascular malformations is an effective treatment for carefully selected patients. When properly applied, this technique can achieve reduction in the size of these lesions without compromising cosmesis.

Diagnosis and management of pediatric laryngeal stenosis.

Pediatric laryngeal stenosis presents diagnostic and management challenges. A team approach involving multiple pediatric subspecialties is desirable. Management includes conservative endoscopic and aggressive open surgical intervention. Deciding which approach is appropriate presents a diagnostic dilemma. The long-term prognosis for most children is very good, and the successes are emotionally rewarding.

Noninvasive optical diagnosis of bacteria causing otitis media.

Currently, the identification of the bacteria responsible for acute otitis media requires a painful invasive procedure: tympanocentesis. To develop a rapid and noninvasive technique for bacterial diagnosis, the fluorescence profiles of four common pathogens and the optical characteristics of the tympanic membrane have been investigated. Each bacterium produces a unique in vitro fluorescence profile when measured in a saline suspension. Also, spectrally resolved transmission measurements from the chinchilla tympanic membrane demonstrate an optical window that will transmit sufficient light for in vivo measurement of the fluorescence profiles. Thus, we have established the precept for a fluorescence-based bacterial diagnosis technique to be used in otitis media. This paper presents the theory, optical data, and a discussion of the device engineering involved in the technique.

Clinical applications of lasers in otolaryngology--head and neck surgery.

The use of lasers in otolaryngology--head and neck surgery is described from the invention of the laser in 1960, through the current uses of the laser, and concludes with a summary for the future directions of laser surgery. The various lasers, including the argon, the KTP, and the carbon dioxide lasers used in otolaryngology, are briefly described. The applications of lasers in the larynx, sinuses, and the ear are separately covered, as well as pediatric otolaryngology. In addition to a brief description of the procedure, the complications and limitations are given. Anesthetic considerations are also covered.

Bacteria identification of otitis media with fluorescence spectroscopy.

We have investigated the fluorescence profiles of four common pathogens: S. pneumoniae, S. aureus, M. catarrhalis, and H. influenzae. The steady-state auto fluorescence spectra of bacteria are measured as a function of the incident light from 200 to 700 nm. The spectra for each bacterium are combined into a fluorescence profile or fluorescence finger print. Each bacterium produces a unique in vitro fluorescence profile when measured in a saline suspension. The profiles are reproducible. Suspensions of a bacterial strain, where the identification is not known, can be correctly matched to a small library of previously measured fluorescence profiles using a linear least-squares fitting algorithm. In addition, we have measured the fluorescence and absorption spectrum of the tympanic membrane removed from a chinchilla. The optical properties of the tympanic membrane and the least-squares identification process form precept for a non-invasive, fluorescence based bacterial diagnosis technique to be used in otitis media.

Carbon dioxide laser serial microtrapdoor flap excision of subglottic stenosis.

The recent development of microspot micromanipulators for the carbon dioxide laser with 250-microns spot sizes has greatly facilitated the extension of endolaryngeal procedures to children and neonates. It is possible to accurately develop microtrapdoor flaps within the limited exposures in the pediatric subglottis. We have used serial microtrapdoor flaps in the excision of subglottic scar tissue in eight children. In five children, the stenosis was improved with relief of stridor. In three children, the flaps were used as an adjunct to laryngotracheoplasty. The surgical techniques of this procedure are detailed in this report.

Soft-tissue complications of laser surgery for recurrent respiratory papillomatosis.

Twenty-two patients with recurrent respiratory papillomatosis underwent 105 carbon dioxide (CO2) laser microlaryngoscopic and tracheobronchoscopic operations from July 1986 through February 1990. All soft-tissue complications, whether intraoperative or delayed secondary to laser surgery, were retrospectively analyzed. The intraoperative laser-related soft-tissue complication rate was zero. Two of the 22 patients acquired slight unilateral true vocal cord scar tissue and 1 patient developed a small posterior laryngeal web. The delayed soft-tissue complication rate was 13.6%, which compares favorably with published reports of 28.7% and 45%. This low complication rate has resulted from the selection of appropriate CO2 laser emission parameters and the use of the microspot micromanipulator, which help minimize lateral and/or deep thermal damage at the site of laser impact.

Advanced microspot microslad for the CO2 laser.

New advances in instrumentation have facilitated the development of a second generation carbon dioxide (CO2) laser microspot micromanipulator. The 710 Acuspot has unique advantages over the previous generation of microspots. The compact design is easier to handle and has attachment points for sterile draping. The unit produces a spot size of 250 microns at a 400-mm focal length and 160 microns at 250 mm; the maximum de-focus is 3.2 mm at all focal lengths. An innovative dichroic mirror allows use of the laser unit's own HeNe laser as the aiming beam, eliminating possible aiming error introduced with a virtual image-aiming system. The dichroic mirror also allows better light transmission, resulting in a brighter field of view. As with first generation microspots, the laser beam path is coincident with the microscope optical path, eliminating parallax; this feature has been especially advantageous in pediatric and otologic cases. Having used this unit for 6 months on more than 50 patients, we now consider the use of a microspot to be our delivery system of choice for most microlaryngeal laser surgical applications.

Neonatal and pediatric microsubglottiscope set.

A microsubglottiscope for infants and children has been developed for microlaryngeal surgery in the glottic and subglottic regions of the larynx. The universal handle allows selection of several sizes of blades to be used according to the patient's anatomic limitations. The tips of these blades have been designed specifically to give exposure to the subglottis and upper trachea under microscopic magnification. A needle adapter for jet ventilation has been included. The authors have used this instrument in young infants and children for diagnosis and for laser resection of subglottic stenosis, hemangioma, and suprastomal granuloma, and have found the exposure to be superior to that obtained with conventional microlaryngoscopes.

Porphyrin fluorescence and photosensitization in head and neck cancer.

Indistinct margin demarcation and autofluorescence are problems in fluorescence delineation of porphyrin-containing head and neck squamous cell cancer (HNSCC). We studied the time course and in vivo localization of porphyrin fluorescence in a squamous cell cancer hamster model and in human HNSCC. After intravenous injection, a gradient in fluorescence intensity developed rapidly until tumors fluoresced above a lower-intensity mucosal background. Hamster tumor and ulcerated HNSCC without porphyrin injection demonstrated autofluorescence grossly indistinguishable from fluorescence in porphyrin-injected tumors. However, fluorescence microscopy revealed autofluorescence to be a surface phenomenon and showed injected porphyrin localized in tumor stroma. We conclude that autofluorescence must be considered when interpreting porphyrin fluorescence. In addition, empirically designed photodynamic therapy can be effective in selected HNSCC. Data from animal experiments provide useful guidelines for the delivery of this therapy.