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INTRODUCTION Some clinicians believe second sets of tympanostomy tubes extrude more quickly than first sets. STUDY DESIGN Retrospective case-control series. METHODS We identified children who were examined 12 months after placement of their second set of tympanostomy tubes and compared them to a similar number of children who were examined 12 months after their first set of tympanostomy tubes. Extrusion was determined by otoscopy, otomicroscopy, and/or tympanometry. RESULTS One hundred eighteen children had 12-month follow-up data available after their first set of tubes, 54 had 12-month follow-up data available for their second set, and 56 had 12-month follow-up data after their first and second sets. A total of 568 tubes were observed. Looking at each tube, second set tubes were significantly more likely to be extruded at 12 months (48%) compared to first set (28%) (P

Objective Oral ibuprofen is believed to be safe and effective after pediatric adenotonsillectomy. There has been little study of its use as a preoperative analgesic. We attempt to document its safety in this setting. Study design Individual case control study. Methods Children who underwent tonsillectomy or adenotonsillectomy from January 2013 to December 2015 did not receive preoperative ibuprofen. Those who underwent tonsillectomy or adenotonsillectomy from January 2016 to December 2017 received oral ibuprofen 7 mg/kg preoperatively. Pre- and postoperative records were reviewed. Intraoperative bleeding > 50 mL or early postoperative bleeding requiring surgical control were outcome measures. Delayed bleeding events were also recorded. Results A total of 217 children met inclusion criteria. Of those, 112 patients did not receive preoperative ibuprofen, and 105 patients did receive preoperative ibuprofen. Mean age was 8.7 years (range: 1-18) in the control/non-ibuprofen cohort and 8.3 years (range: 1-18) in the ibuprofen cohort. No child experienced significant intraoperative or early postoperative bleeding in the non-ibuprofen (95% confidence interval [CI] 0-0.027) or in the ibuprofen cohort (95% CI 0- 0.029). Delayed bleeding rates were similar in both groups. Conclusion In this series, children treated with preoperative ibuprofen did not experience increased bleeding during or soon after tonsillectomy compared to controls. Pain control was not studied in these patients. These favorable safety data argue for a future prospective randomized study of preoperative ibuprofen's effectiveness in reducing pain and opioid requirement after pediatric tonsillectomy. Level of evidence 3b. Laryngoscope, 128:2415-2418, 2018.

Streptococcus pneumoniae displays increased resistance to antibiotic therapy following biofilm formation. A genome-wide search revealed that SP 0320 and SP 0675 (respectively annotated as 5-keto-D-gluconate-5-reductase and glucose dehydrogenase) contain the highest degree of homology to CsgA of Myxococcus xanthus, a signaling factor that promotes cell aggregation and biofilm formation. Single and double SP 0320 and SP 0675 knockout mutants were created in strain BS72; however, no differences were observed in the biofilm-forming phenotypes of mutants compared to the wild type strain. Using the chinchilla model of otitis media and invasive disease, all three mutants exhibited greatly increased virulence compared to the wild type strain (increased pus formation, tympanic membrane rupture, mortality rates). The SP 0320 gene is located in an operon with SP 0317, SP 0318 and SP 0319, which we bioinformatically annotated as being part of the Entner-Doudoroff pathway. Deletion of SP 0317 also resulted in increased mortality in chinchillas; however, mutations in SP 0318 and SP 0319 did not alter the virulence of bacteria compared to the wild type strain. Complementing the SP 0317, SP 0320 and SP 0675 mutant strains reversed the virulence phenotype. We prepared recombinant SP 0317, SP 0318, SP 0320 and SP 0675 proteins and confirmed their functions. These data reveal that disruption of genes involved in the degradation of ketogluconate, the Entner-Doudoroff pathway, and glucose dehydrogenase significantly increase the virulence of bacteria in vivo; two hypothetical models involving virulence triggered by reduced in carbon-flux through the glycolytic pathways are presented.

Oral ibuprofen is believed to be safe and effective after pediatric adenotonsillectomy. There has been little study of its use as a preoperative analgesic. We attempt to document its safety in this setting.Individual case control study.Children who underwent tonsillectomy or adenotonsillectomy from January 2013 to December 2015 did not receive preoperative ibuprofen. Those who underwent tonsillectomy or adenotonsillectomy from January 2016 to December 2017 received oral ibuprofen 7 mg/kg preoperatively. Pre- and postoperative records were reviewed. Intraoperative bleeding 50 mL or early postoperative bleeding requiring surgical control were outcome measures. Delayed bleeding events were also recorded.A total of 217 children met inclusion criteria. Of those, 112 patients did not receive preoperative ibuprofen, and 105 patients did receive preoperative ibuprofen. Mean age was 8.7 years (range: 1-18) in the control/non-ibuprofen cohort and 8.3 years (range: 1-18) in the ibuprofen cohort. No child experienced significant intraoperative or early postoperative bleeding in the non-ibuprofen (95% confidence interval [CI] 0-0.027) or in the ibuprofen cohort (95% CI 0- 0.029). Delayed bleeding rates were similar in both groups.In this series, children treated with preoperative ibuprofen did not experience increased bleeding during or soon after tonsillectomy compared to controls. Pain control was not studied in these patients. These favorable safety data argue for a future prospective randomized study of preoperative ibuprofen's effectiveness in reducing pain and opioid requirement after pediatric tonsillectomy.3b. Laryngoscope, 128:2415-2418, 2018.

In biomedical research, it is imperative to differentiate chance variation from truth before we generalize what we see in a sample of subjects to the wider population. For decades, we have relied on null hypothesis significance testing, where we calculate P values for our data to decide whether to reject a null hypothesis. This methodology is subject to substantial misinterpretation and errant conclusions. Instead of working backward by calculating the probability of our data if the null hypothesis were true, Bayesian statistics allow us instead to work forward, calculating the probability of our hypothesis given the available data. This methodology gives us a mathematical means of incorporating our "prior probabilities" from previous study data (if any) to produce new "posterior probabilities." Bayesian statistics tell us how confidently we should believe what we believe. It is time to embrace and encourage their use in our otolaryngology research.

Background There are limited data on the benefits of surgical tumor resection plus stereotactic radiosurgery (SRS) in comparison with SRS alone for patients with oligometastatic brain disease. Objective To determine the benefit of adding resection to SRS. Methods We reviewed 162 consecutive patients with oligometastatic brain disease, who underwent surgical tumor resection and SRS boost (n = 49) or SRS alone (n = 113). Patients receiving prior whole brain radiation therapy were excluded. Factors related to patient survival and time-to-local recurrence (TTLR) were determined by Cox regression. The effect of complete resection + SRS boost on survival was further explored by propensity score matching. Results The average age of the cohort was 65.3 years, it was 49.4% female, and included 260 brain tumors, of which 119 tumors were single. Seventy-three brain tumors recurred (28%). TTLR was related to radiation-sensitive pathology (hazards ratio [HR] = 0.34, P = .001), treatment volume (HR = 1.078/mL, P = .002), and complete tumor resection (HR = 0.37, P = .015). Factors related to survival were age (HR = 1.21/decade, P = .037), Eastern Cooperative Oncology Group performance score (HR = 1.9, P = .001), and complete surgical resection (HR = 0.55, P = .01). Propensity score matched analysis of complete surgical resection + SRS boost (n = 40) vs SRS alone (n = 80) yielded nearly identical survival results (HR = 0.52, P = .030) compared with the initial unmatched sample. Incomplete tumor resection had both median survival and TTLR equivalent to SRS alone. Conclusion Complete surgical resection + SRS boost is associated with improved survival and reduced likelihood of local tumor recurrence in comparison with SRS alone. Incomplete resection did not improve survival or TTLR compared with SRS alone.

To determine the prevalence of chronic ear disease in HIV+, highly active anti-retroviral therapy (HAART)-treated children and compare this to the prevalence in healthy children of similar age living in a similar setting.From previous clinical work in Ethiopia, we suspected that chronic middle ear disease was common both in the general pediatric population and especially among children with HIV/AIDS. Few studies have examined the prevalence of chronic ear disease in HIV + children, particularly in those treated with HAART.Full examination of the head and neck was performed by otolaryngologists. This including cleaning of cerumen, otoscopy and microscopic otoscopy when needed. Patient's medical records were reviewed. Presence or absence of tympanic membrane (TM) perforation (unilateral or bilateral), tympanosclerosis, TM atrophy, otorrhea and/or cholesteatoma was documented.112 HIV+ and 162 healthy (HIVU) children were included. Prevalence of TM perforations was 17% in the HIV + infected versus 3% in the HIVU (Fisher's-Exact-Test; OR: 7.2, 95% CI 2.5-20, p-value0.0001). Presence of unilateral TM perforations was 12% in the HIV + population and 2% in the HIVU population (Fisher's-Exact-Test; OR: 6.8, 95% CI 2.0-22, p-value 0.002). The presence of bilateral perforations was 4% in the HIV + population and 1% in the HIVU population (Fisher's-Exact-Test; OR: 6.8, 95% CI 1.1-42, p-value 0.088). In the HIV + cohort, 2% were diagnosed with cholesteatoma compared to 0% in the HIVU population (95% CI HIV+ 0.002-0.06; HIVU 0.0-0.02) and 8% of HIV + subjects had active middle ear discharge, compared to 0% in the HIVU population (95% CI HIV+ 0.04-0.1; HIVU 0.0-0.02). Neither tympanosclerosis nor tympanic membrane atrophy was more frequent in the HIV + population compared to the HIVU population. Persistent or recurrent TM perforation was not more frequent in children with prior tympanoplasty in the HIV + population compared to the HIVU population.TM perforations are significantly more common in HAART-treated HIV + children than in healthy, age-matched HIVU population. Otorrhea and cholesteatoma were found only in the HIV + cohort.

To report the authors' experience with hydroxyapatite cement (HAC) cranioplasty and analyze the material's long-term safety and efficacy in repairing translabyrinthine skull-base defects by examining adverse events, specifically cerebrospinal fluid (CSF) leaks and surgical site infections.Retrospective case-control study (primary study arm); prospective cross-sectional study of patients not examined within the last 5 years (secondary arm).tertiary-care neurotology private practice and academic practice (two centers).Hydroxyapatite cement implanted following translabyrinthine approach, with or without fat graft, was included. Combined approaches were excluded. Implant-associated adverse events were defined as 1) CSF leaks requiring reoperation or spinal drainage, and (2) infections requiring reoperation. Patients not examined within 5 years were interviewed by telephone to update their condition. Incidence of adverse events was compared to published data for translabyrinthine cranioplasty using fat graft alone. Implant survival analysis was performed.The study cohort included 369 HAC implants in the same number of patients. There were seven CSF leaks and seven infections. Combined (n = 14) incidence of adverse events was 3.8% (2.09%, 6.28%). Compared to fat graft alone, the adverse events associated with HAC were fewer (P0.001). Up to 15 years (5,475 days), HAC cement maintained 95% adverse event-free survival. There were no cases of meningitis.Cranioplasty using HAC with autologous fat following translabyrinthine skull-base surgery is safer and more effective than fat graft alone, up to 15 years after surgery.4. Laryngoscope, 127:2120-2125, 2017.

Bacteria can grow as individual, planktonic organisms or as complex biofilm communities that are more resistant to treatment. This review was designed to systematically search to identify recent laboratory studies on eradication of biofilms in otolaryngologic infections to highlight promising advances in biofilm treatment.A systematic electronic literature search of Medline/PubMed, CINHAL, and Web of Science was conducted for articles describing the treatment of biofilm infections in ear, nose, and throat (ENT) diseases through March 2010. English-language articles and articles with an English abstract that focused on biofilm treatment were considered for review.Each included article was reviewed by one of the authors for study design, treatment intervention, and outcome. Data from in vitro and animal studies were considered separately from human studies.A total of 30 articles were identified for this review, including 5 studies that included a human treatment component. In general, antibiotics were relatively ineffective for eradicating biofilm infections. Markedly higher antibiotic dosages were required to reduce biofilm presence compared with doses that were effective in eradicating planktonic bacteria. Mupirocin irrigation, gentian violet, and thiamphenicol glycinate acetylcysteine effectively eradicated biofilms. Physical disruption, surfactants, and probiotics were also shown to be beneficial in both nonhuman and human studies.Eradicating ENT biofilms is difficult when treating single-organism or mixed flora biofilms. Antibiotic therapy is often ineffective against biofilms, and clinical treatment may need to focus on nonantibiotic therapies that reduce, disrupt, or eradicate ENT biofilms.

Staphylococcus epidermidis is a clinically important opportunistic pathogen that forms biofilm infections on nearly all types of indwelling medical devices. The biofilm forming capability of S. epidermidis has been linked to the presence of the ica operon in the genome, and the amount of biofilm formation measured by the crystal violet (CV) adherence assay. Six S. epidermidis strains were characterized for their ica status using PCR, and their biofilm forming ability over 6 days, using the CV assay and a flow cell system. Ica-negative strains characterized as 'negative for biofilm formation' based on the CV assay were demonstrated to form strongly attached biofilms after 6 days. However, the biofilms were not as extensive as the ica-positive strains. It was concluded that ica is not required for biofilm formation, nor is the 24-h CV assay generalizable for predicting the 6-day biofilm-forming ability for all S. epidermidis strains.

Objective Investigations that seek to generalize findings or to understand uncommon diseases must be conducted at multiple centers. This study describes the process of obtaining regulatory approval for a minimal risk genetic study in a multi-center setting as undertaken by the Recurrent Respiratory Papillomatosis (RRP) Task Force. Study design and setting Sequential cohort of American children’s hospitals. A single protocol was submitted to each Institutional Review Board (IRB). Results Documentation was prepared for 14 IRBs over 2.5 years. The median time between enlistment and approval at the first 8 sites was 15 months. Institutions varied considerably in their requirements and in the issues that were raised. Protocols were submitted sequentially and accumulated experience was used in the preparation of applications to subsequent IRBs. Nevertheless, there was no correlation between the accumulated experience and the number of issues that were raised. Conclusion Despite uniform federal standards, all local IRBs required unique and individualized submissions. For multicenter studies, investigators should seriously consider the establishment of cooperative authorization agreements. On a simpler level, a standardized format for applications needs to be adopted nationwide. EBM rating: B-3b

Background Venipuncture (phlebotomy) is an obstacle to subject recruitment and ongoing participation in cohort studies investigating human papillomavirus (HPV) infection. Anti-HPV antibodies are not only detected in serum but also in oral fluid. Objectives To evaluate if oral fluid specimens can be used in lieu of blood specimens for determining HPV antibody status. Study design One hundred and seven paired oral fluid and blood specimens from female university students were tested in a HPV16 ELISA and compared to sexual history and serial genital HPV16 DNA status. Results ELISA results were in agreement in 97% (104/107) of paired sera and oral fluid. Of six women with positive anti-HPV16 serum samples, only three had positive oral fluid specimens. However, the specificity of the oral fluid test was 100% compared to the blood test. Conclusions Detection of antibodies in oral fluid correlated with antibodies but was less sensitive than sera. A larger validation study is required to fully characterize the oral fluid assay.

I thank Celis-Moralis and colleagues for the important and interesting study on active commuting.1 I find the all cause mortality to be the most meaningful finding. I had …

This study describes the patterns of perioperative antimicrobial use by otolaryngologists during common otolaryngologic surgical procedures. Through the American Academy of Otolaryngology--Head and Neck Surgery Infectious Diseases Committee, a survey was developed to assess the current practice patterns regarding the use of perioperative antibiotics in otolaryngology. A total of 6903 surveys were sent out; 458 were fully or partially completed, and a total of 442 responses were included in the final analysis. Most physicians reported routinely prescribing antibiotics either preoperatively or postoperatively for 12 of the 17 procedures included in the questionnaire despite providers agreeing that there is not enough evidence to support their use. The most common procedure for which antibiotics were prescribed was laryngectomy (91.1%). Antibiotic use is a common practice during the perioperative period for otolaryngologic procedures; however, there is a discrepancy between utilization and evidence of benefit.

Objective: To determine the relationship between coin size, coin location, patient age, and patient weight and likelihood of coin passage through the esophagus following pediatric coin ingestion. A secondary objective is to test the hypothesis that coin denomination can be determined based on radiographic appearance. Methods: A retrospective review was performed of all children seen and evaluated for coin ingestion at a single institution over a 25-month period. Outcome measures included the number of coins that were retained in the esophagus, and the number that passed. Various factors were assessed for their predictive value in judging outcome in coin ingestion cases. Results: Nineteen percent of patients (15/79) in the study passed their ingested coins. Coin denomination could be accurately determined on every patient that had a standard AP or lateral X-ray film. These findings were marked when compared with the lack of reliability of history in determining coin denomination. Patients who passed coins were as a group older (4.6 vs. 3.2 year, P =0.04), but did not differ significantly by weight (19.5 vs. 15.4 kg, P =0.07) from those that retained the coins. Coins located at the gastroesophageal junction had a significantly higher passage rate than coins located elsewhere in the esophagus (89 vs. 8.2%, P 0.01). Coin size was not predictive of coin passage ( P =0.7 by χ 2 ). Conclusions: Radiographic assessment of coin denomination is reliable, but in this study could not be used to predict coin passage. Patient age and coin location at the gastroesophageal junction, however, do correlate with this event.

Amylin is normally secreted in a regulated fashion by the pancreatic β-cells in parallel with insulin and has been reported to have bone-conserving properties. Type I diabetes mellitus results in a low-turnover osteopenia in the presence of decreased amylin, which is in contrast to type II diabetes where less bone loss, in the presence of high amylin levels, occurs. We investigated the effects of amylin on bone mineral metabolism in normal and dibetic (streptozotocin-induced) rats, in order to ascertain whether amylin would modify the streptozotocin-induced diabetic osteopenia. Tenweek-old male Sprague-Dawley rats were randomized as follows: group A (n=18) received normal saline; group B (n=18) received amylin; group C, diabetic rats (n=23), received normal saline; and group D, diabetic rats (n=23), received amylin. Amylin (100 pmol/100 g b. w.) was administered by a daily subcutaneus injection. Double calceinlabeled tibiae were removed for histomorphometric analysis followed sacrifice on day 19. Results showed no difference in blood ionized calcium between groups. Blood glucose remained above 600 mg/dl in the diabetic animals and was not affected by the administration of amylin. Serum osteocalcin, insulin-like growth factor-1 (IGF-1), parathyroid hormone (PTH), and 1,25 dihydroxyvitamin D [1,25(OH)2D] were significantly lower in the diabetic rats compared with control group A by day 19. Amylin produced higher levels of serum osteocalcin in group B on day 9 (P

To describe and compare the intraoperative blood loss in children who underwent tonsillectomy and/or adenoidectomy during a transition from using electrocautery to a microdebrider.Retrospective case series of a single pediatric otolaryngologist at an urban general hospital. Patients aged 2-20 years who had tonsillectomy, adenoidectomy, or adenotonsillectomy over a 12 month period were included. Tonsillectomy was performed by microdebrider or electrocautery and adenoidectomy was performed by microdebrider, curette, or suction electrocautery. Total intraoperative blood loss was measured and compared between surgical techniques.Of the 148 patients, 109 had tonsillectomy with or without adenoidectomy and 39 had adenoidectomy alone. The mean blood loss was 47 ml or 1.8 ± 1.6 ml/kg and the maximum blood loss was 11 ml/kg. Adenoid curette and adenoid microdebrider yielded similar blood loss but were associated with more bleeding than suction electrocautery (P0.05). Microdebrider tonsillectomy yielded more blood loss than electrocautery tonsillectomy (mean of 2.6 ± 2.2 ml/kg versus 1.2 ± 1.2 ml/kg, P=0.0002). Eighteen percent of adenotonsillectomy patients lost greater than 5% of calculated circulating blood volume (95% CI, 9.8-26). Linear regression models did not show an association between the amount of blood loss and patient age, clinical indication, or the surgeon's experience with the microdebrider (P0.05).Microdebrider tonsillectomy is associated with more intraoperative bleeding than electrocautery tonsillectomy. Approximately twice as much blood was lost with the microdebrider, but the absolute increase was insignificant from a hemodynamic perspective.

Interferon-gamma (IFN gamma) in vitro inhibits both bone resorption and bone formation, resulting in a net decrease in bone turnover. In vivo administration of cyclosporin A (CsA) produces accelerated bone remodeling with resultant bone loss. The aim of this study was to investigate whether administration of IFN gamma to rats would favorably modify the high turnover osteopenia caused by CsA. Thirty-six male Sprague-Dawley rats were randomized into 4 equal groups to receive either CsA (15 mg/kg.day) or vehicle by gavage and IFN gamma (10(6) IU/kg.day) or vehicle by ip injection for 8 days. Group 1 received CsA vehicle plus IFN gamma vehicle; group 2 received CsA plus IFN gamma vehicle; group 3 received CsA vehicle plus IFN-gamma; group 4 received CsA plus IFN gamma. Blood was sampled on days 0, 4, and 8 for measurement of ionized calcium (Ca2+), PTH, 1,25-dihydroxyvitamin D, and bone gla protein. Tibiae were removed on day 8 after double tetracycline labeling for histomorphometric analysis. Ca2+ and PTH levels were similar in all groups during the study period. Rats receiving CsA (groups 2 and 4) had elevated levels of 1,25-dihydroxyvitamin D and bone gla protein, whereas rats receiving IFN gamma alone (group 3) had no change in levels of these parameters. Bone histomorphometry revealed that treatment with CsA and/or IFN gamma (groups 2-4) caused an increase in bone resorption surface and a decrease in some parameters of bone formation, resulting in a net loss of bone volume. Thus, IFN gamma failed to influence the osteopenia caused by CsA and on its own had adverse effects on bone in vivo. These results demonstrate that immune-mediating agents have opposing actions in vitro as compared to in vivo.

Objective: 1) Describe the intraoperative blood loss during adenotonsillectomy in a pediatric population during the transition to a new technique. 2) Confirm or refute the suspicion that intraoperative blood loss is greater when using the microdebrider than when using traditional curette and electrocautery techniques.Method: Retrospective evaluation of 148 children who underwent tonsillectomy and/or adenoidectomy by a single surgeon transitioning from curette and electrocautery to microdebrider in 2008. Primary outcome was percentage of circulating blood volume lost. Surgical methods were compared by multiple linear regression and chi-squared to evaluate incidence of “bleeders” (>5% loss).Results: Median age was 6.8 years (range, 2.2-20.6 years). 84 had adenotonsillectomy, 39 had adenoidectomy, and 25 had tonsillectomy. Blood loss ranged from 0% to 15% (median = 2%) blood volume. Sixteen of 148 patients (11%) lost >5% blood volume. A total of 2.3% of blood volume was lost with adenoidectomy by curette or ...

We report on the comparative genomics and characterization of the virulence phenotypes of four S. pneumoniae strains that belong to the multidrug resistant clone PMEN1 (Spain(23F) ST81). Strains SV35-T23 and SV36-T3 were recovered in 1996 from the nasopharynx of patients at an AIDS hospice in New York. Strain SV36-T3 expressed capsule type 3 which is unusual for this clone and represents the product of an in vivo capsular switch event. A third PMEN1 isolate - PN4595-T23 - was recovered in 1996 from the nasopharynx of a child attending day care in Portugal, and a fourth strain - ATCC700669 - was originally isolated from a patient with pneumococcal disease in Spain in 1984. We compared the genomes among four PMEN1 strains and 47 previously sequenced pneumococcal isolates for gene possession differences and allelic variations within core genes. In contrast to the 47 strains - representing a variety of clonal types - the four PMEN1 strains grouped closely together, demonstrating high genomic conservation within this lineage relative to the rest of the species. In the four PMEN1 strains allelic and gene possession differences were clustered into 18 genomic regions including the capsule, the blp bacteriocins, erythromycin resistance, the MM1-2008 prophage and multiple cell wall anchored proteins. In spite of their genomic similarity, the high resolution chinchilla model was able to detect variations in virulence properties of the PMEN1 strains highlighting how small genic or allelic variation can lead to significant changes in pathogenicity and making this set of strains ideal for the identification of novel virulence determinants.

Only seven cases of a sphenoid mucocele occurring after transsphenoidal hypophysectomy have been previously reported in the world literature. In this article, we report a new case, which occurred in a 67-year-old man. The sphenoid sinus mucocele developed 12 years following transsphenoidal hypophysectomy and adjunctive radiotherapy. The patient was successfully managed with incision and drainage. Although transsphenoidal hypophysectomy is a common operation, this particular complication appears to be rare or at least under-reported. Sphenoid sinus mucocele deserves consideration in the differential diagnosis of a sphenoidal parasellar mass in a patient who has undergone an earlier transsphenoidal hypophysectomy.

Background RRP is a devastating disease in which papillomas in the airway cause hoarseness and breathing difficulty. The disease is caused by human papillomavirus (HPV) 6 or 11 and is very variable. Patients undergo multiple surgeries to maintain a patent airway and in order to communicate vocally. Several small studies have been published in which most have noted that HPV 11 is associated with a more aggressive course. Methodology/Principal Findings Papilloma biopsies were taken from patients undergoing surgical treatment of RRP and were subjected to HPV typing. 118 patients with juvenile-onset RRP with at least 1 year of clinical data and infected with a single HPV type were analyzed. HPV 11 was encountered in 40% of the patients. By our definition, most of the patients in the sample (81%) had run an aggressive course. The odds of a patient with HPV 11 running an aggressive course were 3.9 times higher than that of patients with HPV 6 (Fisher's exact p = 0.017). However, clinical course was more closely associated with age of the patient (at diagnosis and at the time of the current surgery) than with HPV type. Patients with HPV 11 were diagnosed at a younger age (2.4y) than were those with HPV 6 (3.4y) (p = 0.014). Both by multiple linear regression and by multiple logistic regression HPV type was only weakly associated with metrics of disease course when simultaneously accounting for age. Conclusions/Significance Abstract The course of RRP is variable and a quarter of the variability can be accounted for by the age of the patient. HPV 11 is more closely associated with a younger age at diagnosis than it is associated with an aggressive clinical course. These data suggest that there are factors other than HPV type and age of the patient that determine disease course.

Background Streptococcus pneumoniae [Sp] infection is associated with local and systemic disease. Our current understanding of the differential contributions of genetic strain variation, serotype, and host response to disease phenotype is incomplete. Using the chinchilla model of otitis media [OM] we investigated the disease phenotype generated by the laboratory strain TIGR4 and each of thirteen clinical strains (BS68-75, BS290, BS291, BS293, BS436 and BS437); eleven of the thirteen strains have been genomically sequenced. Methodology/Principal Findings For each strain 100 colony forming units were injected bilaterally into the tympanic bullae of 6 young adult chinchillas under general anesthesia. All animals were examined daily for local and systemic disease by a blinded observer. Pneumatic otoscopy was used to evaluate local disease, and behavioral assessments served as the measure of systemic disease. Virulence scoring was performed using a 4-point scale to assess four clinical parameters [severity and rapidity of local disease onset; and severity and rapidity of systemic disease onset] during a 10-day evaluation period. Highly significant variation was observed among the strains in their ability to cause disease and moribundity. Conclusions/Significance As expected, there was a significant correlation between the rapidity of systemic disease onset and severity of systemic disease; however, there was little correlation between the severity of otoscopic changes and severity of systemic disease. Importantly, it was observed that different strains of the same serotype produced as broad an array of disease phenotypes as did strains of different serotypes. We attribute these phenotypic differences among the strains to the high degree of genomic plasticity that we have previously documented.

Background The nontypeable Haemophilus influenzae (NTHi) are associated with a spectrum of respiratory mucosal infections including: acute otitis media (AOM); chronic otitis media with effusion (COME); otorrhea; locally invasive diseases such as mastoiditis; as well as a range of systemic disease states, suggesting a wide range of virulence phenotypes. Genomic studies have demonstrated that each clinical strain contains a unique genic distribution from a population-based supragenome, the distributed genome hypothesis. These diverse clinical and genotypic findings suggest that each NTHi strain possesses a unique set of virulence factors that contributes to the course of the disease. Results The local and systemic virulence patterns of ten genomically characterized low-passage clinical NTHi strains (PittAA – PittJJ) obtained from children with COME or otorrhea were stratified using the chinchilla model of otitis media (OM). Each isolate was used to bilaterally inoculate six animals and thereafter clinical assessments were carried out daily for 8 days by blinded observers. There was no statistical difference in the time it took for any of the 10 NTHi strains to induce otologic (local) disease with respect to any or all of the other strains, however the differences in time to maximal local disease and the severity of local disease were both significant between the strains. Parameters of systemic disease indicated that the strains were not all equivalent: time to development of the systemic disease, maximal systemic scores and mortality were all statistically different among the strains. PittGG induced 100% mortality while PittBB, PittCC, and PittEE produced no mortality. Overall Pitt GG, PittII, and Pitt FF produced the most rapid and most severe local and systemic disease. A post hoc determination of the clinical origins of the 10 NTHi strains revealed that these three strains were of otorrheic origin, whereas the other 7 were from patients with COME. Conclusion Collectively these data suggest that the chinchilla OM model is useful for discriminating between otorrheic and COME NTHi strains as to their disease-producing potential in humans, and combined with whole genome analyses, point the way towards identifying classes of virulence genes.

Human immunodeficiency virus (HIV) infection represents a major global health problem, with HIV now recognized as the fourth leading cause of death on a worldwide basis. One approach to developing effective anti- HIV interventions is to identify and understand the molecular mechanisms by which natural genetic variations provide protection from infection or disease progression. This approach can be used to identify human gene alleles that confer resistance or increased susceptibility to HIV infection. To date, however, this approach has been underutilized in the African population and all HIV-resistance alleles that have been described have been identified by evaluating candidate genes. This limited approach is based upon a researcher's assumption that those genes that will provide the host with a benefit can be predicted, a priori, but it does not provide for a large scale systematic screen of all possible candidate genes. Nonetheless, this method has met with some success in identifying HIV-resistance genes, mostly among the white population. The lack of a comprehensive genetic approach, both in terms of the populations studied and the percentage of the genome investigated, likely explains why all of the HIV-restriction alleles identified to date fall within two gene families, and why no resistance genes have been identified among black Africans. It is likely, as with any complex trait, that most protective alleles will provide only partial HIV resistance. Thus, HIV resistance in most persons likely arises through a QTL (quantitative trait loci) mechanism meaning that protection is a polygenic trait. This feature coupled with interpopulation genetic heterogeneity makes the candidate gene mapping approach a daunting task. A comprehensive genome-wide case-control allelic association study in the African population will maximize our chances of identifying new targets for the development of new therapeutics that have the promise of benefiting all persons infected with HIV.

Summary Objective Epidermodysplasia verruciformis is a skin disease characterized by abnormal susceptibility to human papilloma viruses. Recently four mutations in the Epidermodysplasia verruciformis 1 gene ( EVER1 , also known as TMC6 ) have been associated with the disease. Because of the phenotypic similarity between Epidermodysplasia verruciformis and recurrent respiratory papillomatosis, we decided to investigate whether any of these mutations accounts for the susceptibility to human papilloma viruses in subjects with recurrent respiratory papillomatosis (RRP). Methods Allele-specific PCR and restriction fragment length polymorphisms (RFLPs) were employed for genotyping a cohort of 101 patients with recurrent respiratory papillomatosis. Results None of these four mutations were found in the studied subjects. Conclusion The absence of these mutations in RRP patients might indicate that EVER 1 alleles are not associated with susceptibility to RRP, or that other, as yet unidentified, mutations in the Epidermodysplasia verruciformis 1 gene, might account for the susceptibility to RRP.

Objectives: To determine the host genes that govern susceptibility to recurrent respiratory papillomatosis (RRP). RRP is caused by human papillomavirus (HPV) 6 and 11. Millions of babies are exposed during the birthing process, but relatively few develop the disease and the aggressiveness of the course is highly variable. Genetically encoded host susceptibility is postulated. Determining the host genes that govern susceptibility will enhance our understanding not only of RRP but also of host-viral interaction in general. Study Design: A genome-wide association study on familial triads consisting of an RRP-affected child and his or her parents. Using the HapMap data from the human genome project, we will identify those alleles that are over-transmitted by the parents to their affected offspring as compared to those alleles that are under-transmitted. Methods: Approximately 400 patients and their parents will be recruited through a collaboration between the Center for Genomic Sciences and the RRP Task Force. DNA will be extracted from blood specimens and viral typing will be performed on biopsy specimens. Patients will be genotyped using single nucleotide polymorphism (SNP) markers and compared to their respective parents' genotype using the transmission disequilibrium test. Both a genome scan and a candidate gene approach will be utilized. Results: Institutional Review Board authorization has been obtained at three hospitals and the process is underway at 18 more. Patient and parent recruitment has begun. Specimens have been forwarded to Pittsburgh, Pennsylvania, where the DNA has been extracted and is being stored. Conclusions: A novel approach combining a nationwide patient resource and the mapping power of the subcentimorgan human haplotype map has been developed to elucidate the biological mechanisms of RRP by determining the genetically encoded susceptibilities of host-virus interaction.

Objectives Improving the quality of pediatric healthcare in the developing world poses some formidable challenges. Surgical missions aim to improve the lot of individual children, but do little to alter the wellness of the majority. Methods Members of the American Society of Pediatric Otolaryngology (ASPO) are working in coordination with existing programs - universities, mission hospitals and non-governmental organizations - with a focus on public health and education of local physicians. Results We have completed our first four visits to Ethiopia, teaching, performing surgery and building relationships. Conclusions We hope that by moving from the traditional surgical mission format to a long-term, integrated educational effort we can enhance otolaryngic care for children in Sub-Saharan Africa.

There are limited data on the benefits of surgical tumor excision plus stereotactic radiosurgery (SRS) in comparison to SRS alone for patients with oligometastatic brain disease. We conducted a retrospective cohort study of 162 consecutive patients, who were whole brain radiation therapy (WBXRT) naïve, undergoing tumor excision + SRS boost (n = 49) or SRS alone (n = 113). Factors related to patient survival were determined by Cox regression; time-to-recurrence (TTR) was analyzed similarly but with robust residuals to account for clustering. The effect of complete resection + SRS boost on survival was further explored by 2:1 propensity score matching. The average age of the cohort was 65.3 years, 49.4% female, and included 260 brain tumors of which 119 tumors were solitary. Surgical patients were younger, had better performance status and less burden of disease compared to SRS alone. None of these differences were statistically significant; RPA class was significantly better in the SRS + boost group (p = .003, Mann-Whitney). Seventy three tumors recurred (28%). Factors related to TTR were radiation sensitive pathology (non renal or melanoma) (hazard ratio (HR) =.345, p = .0014), treatment volume (HR = 1.078, p = .0022) and complete tumor resection (HR = .371, p = .015). Factors related to survival were age (HR = 1.02, p = .037), ECOG performance score (HR= 1.9, p = .0001) and complete surgical excision (HR = .55, p = .01). Propensity score matched analysis of complete resection + SRS boost (n = 40) versus SRS alone (n = 80) yielded nearly identical results of improved survival with complete resection (HR = .52, p = .03). Incomplete tumor resection had both median survival and TTR equivalent to SRS alone. Complete surgical excision + SRS boost is associated with improved survival and reduced likelihood of tumor recurrence in comparison to SRS alone. These results were replicated in the propensity score analysis which matched surgical and non-surgical patients on factors known to influence outcome. Incomplete resection did not improve survival or TTR compared to SRS alone.

OBJECTIVE: The goal was to determine the incidence of symptomatic adenoidal regrowth after adenoidectomy.STUDY DESIGN: A cross-sectional follow-up study was done in a randomly selected group of 175 children who had undergone adenoidectomy 2 to 5 years earlier. Nasopharyngoscopy was performed in those children who still had symptoms of nasal obstruction.SETTING: All surgery was performed at an academic hospital-based practice in the northeastern United States by a single surgeon using a consistent operative technique.RESULTS: Forty-six (26%) patients had nasal airway obstruction symptoms at follow-up. Of the 35 who agreed to undergo nasopharyngoscopy, not a single one had adenoids occupying more than 40% of the nasopharynx, and most (71%) were found to have either no or only trace amounts of adenoidal tissue (usually in the pharyngeal recess).CONCLUSION: Adenoids rarely, if ever, regrow enough to cause symptoms of nasal obstruction after adenoidectomy that includes visualization and electrocautery of the a...

Immunosuppression with cyclosporin A (CsA) is effective in a number of immune-mediated diseases and in preventing rejection following organ transplantation. We have repeatedly demonstrated that CsA in the rat model produces accelerated bone remodelling with net bone loss, best characterized in trabecular bone. IGF-I holds promise as a treatment for various osteopenic conditions. Although currently a subject of much controversy, various studies have suggested that in vivo it is anabolic to cortical as well as trabecular bone. The purpose of this study was, in part, to further characterize the effects of CsA and IGF-I on trabecular and cortical bone, and to see whether systemic IGF-I is able to modulate CsA's deleterious skeletal effects. Sixty 10 week-old, male, Sprague-Dawley rats were randomized to receive the following daily for 3 weeks: (1) CsA vehicle (veh) per os (po) + recombinant human (rh) IGF-1 veh subcutaneously (sc); (2) CsA 15 mg/kg po + rhIGF-I-veh; (3) CsA-veh + rhIGF-I 200 microg/kg sc; (4) CsA-veh + rhIGF-I 600 microg/kg sc; (5) CsA 15 mg/kg + rhIGF-I 200 microg/kg, and (6) CsA 15 mg/kg + rhIGF-I 600 microg/kg. Rats were weighed and venous blood was sampled serially for determination of glucose, ionized calcium (Ca2+), PTH, vitamin D, and osteocalcin. Following sacrifice on day 20, histomorphometry was performed on double calcein-labeled tibial metaphysis and diaphysis. All rats receiving CsA had elevated levels of blood glucose and osteocalcin by day 9 and vitamin D at day 20. PTH was similar in all groups, and Ca2+ was only raised in the CsA and CsA + IGF-I 200 microg/kg groups. Rats receiving IGF-I 200 microg/kg and IGF-I 600 microg/kg gained more weight than either vehicle- or CsA-treated animals, attesting to IGF-1's anabolic properties. CsA caused severe trabecular bone loss, not prevented by IGF-I; it even further increased the eroded surface. CsA and IGF-I had little effect on cortical bone volume or marrow area. IGF-I increased endocortical matrix synthesis, as evidenced by the increases in the percent endocortical osteoid perimeter, an effect negated by the addition of CsA. This experiment demonstrates that trabecular bone is more susceptible than cortical bone to the deleterious effects of CsA and indicates little role for IGF-1 in the pathophysiology or treatment of CsA-induced bone disease at the given doses and duration of treatment.

The T lymphocyte suppressor, cyclosporin A, has been shown to cause high turnover osteoporosis. We postulated that cyclosporin A may exert its effects via the T cell rather than direct activity on bone. In this study we administered cyclosporin A (15 mg/kg.day by gavage) to 11 10-week-old Rowett athymic nude rats and to 12 age-matched immunocompetent Sprague-Dawley rats. Placebo was administered to control groups (n = 12 for both). After 28 days of treatment, the Sprague-Dawley rats displayed high turnover bone loss, but the nude rats were largely unaffected by the drug. Sprague-Dawley treated rats had less than half the percent trabecular area of their controls as measured at the secondary spongiosa of the proximal tibial metaphysis (P < 0.001; strain by treatment, P = 0.007). The same pattern was evident for trabecular number, separation, and thickness (strain by treatment, P = 0.034, P = 0.001, and P = 0.021, respectively). Only the Sprague-Dawley rats had an elevated percent eroded perimeter and an elevated bone area referent bone formation rate (strain by treatment, P = 0.002 and P = 0.0003, respectively). Mass, glucose, ionized calcium, PTH, osteocalcin, 1,25-dihydroxyvitamin D, and creatinine all responded similarly to cyclosporin A regardless of strain. T Lymphocytes thus appear to be a prerequisite for the development of cyclosporin A-induced osteopenia.

The immune and skeletal systems are known to interact. We have repeatedly shown that in contrast to in vitro data, the administration of T lymphocyte immunosuppressants, such as cyclosporin A, leads to an increase in bone resorption and a high turnover osteopenia. The purpose of this study was to characterize the bone metabolism of the T lymphocyte deficient Rowett athymic homozygous (rnu/rnu) nude rat. We wished to determine whether these rats share the bone abnormalities of cyclosporin A-treated rats. Eleven 10-week-old Sprague-Dawley rats and 12 similarly aged nude rats were studied over a 4-week period. Metaphyseal cancellous bone histomorphometry was similar in the two groups of rats and only differed with regard to percentage eroded perimeter (lower in nude rats, p = 0.0008) and longitudinal growth rate (49% lower in nude rats, p < 0.001). The nude rats had less body mass (p < 0.001) but nevertheless gained the same percentage of their body weight over the study period. The athymic rats had lower levels of serum, 1,25-dihydroxyvitamin D (p < 0.014) and serum osteocalcin(p < 0.009), and at the age of 14 weeks the nude rats had lower concentrations of serum creatinine (p = 0.001) and blood ionized calcium (p = 0.0002), yet serum PTH was similar throughout. RNA isolated from the contralateral tibias revealed that the nude group had lower steady-state levels of osteocalcin mRNA despite similar rates of bone formation. In its entirety, the data suggest that T cell deficiency per se is not necessarily associated with high turnover osteopenia.

Immunosuppressant therpay is associated with osteoporosis both clinically, post-transplantation, and experimentally. In rats, cyclosporin A (CsA) and FK506 induce a state of high turnover rapid bone loss. After 14 days of administration in immunosuppressive doses, the more recently discovered immunosuppressant, rapamycin, resulted in no change of cancellous bone volume. A longer study over 28 days has now been carried out; contrasting the new drug with CsA and FK506. Sixty, 10-week-old Sprague-Dawley rats were randomly divided into five groups of 12 rats each. The first group served as an aging control. The remaining four groups received, by daily gavage, a combined vehicle placebo, CsA 15 mg/kg, FK506 5 mg/kg, and rapamycin 2.5 mg/kg, respectively. CsA- and FK506-treated rats, but not those treated with rapamycin, demonstrated high turnover osteoporosis with raised serum 1,25(OH)2D (p0.05) and elevated serum osteocalcin (p0.05). The trabecular bone area was decreased by 66% (p0.01) in the CsA group and 56% (p0.05) in the FK506-treated group compared with the control animals. The CsA- and the rapamycin-treated groups failed to gain weight and developed severe hyperglycemia (20 mmol/l, p0.001) by day 14 but which largely resolved by day 28. Unlike the groups treated with CsA and FK506, rapamycin-treated rats had no loss of trabecular bone volume but there was increased modeling and remodeling and a decreased longitudinal growth rate. Rapamycin may thus confer a distinct advantage over the established immunosuppressants in not reducing bone volume in the short term.(ABSTRACT TRUNCATED AT 250 WORDS)

The immunosuppressant agent cyclosporin A (CsA) induces a high turnover osteopenic state, while the effect on bone of the antimetabolite azathioprine, a drug often used in conjunction with CsA in transplant patients, is less clear. This study was therefore designed to investigate the outcome of azathioprine administration, with reference to CsA, on bone mineral metabolism using the rat model. Four groups of 10-week-old male Sprague-Dawley rats (12 per group) were randomly allocated to receive by daily gavage for a 28-day period: (1) no treatment (control group); (2) azathioprine 1.5 mg/kg bw; (3) CsA 15 mg/kg bw; and (4) a combination of azathioprine and CsA, as described above. Rats were weighed and blood assayed serially for osteocalcin, ionized calcium, 1,25-dihydroxyvitamin D (1,25(OH)2VitD), and parathyroid hormone (PTH). Tibiae were removed following sacrifice on day 28 after double calcein labeling for histomorphometric analysis. Immunosuppressant groups were compared with nontreated control. We confirmed our previous findings that CsA induces a state of high turnover bone loss which is accompanied by a diminished gain in body weight (p < 0.01) and elevated serum osteocalcin (p < 0.001) and 1,25(OH)2VitD levels (p < 0.001). Azathioprine treatment alone did not alter ionized calcium, 1,25(OH)2VitD, or PTH levels. However, there was biochemical evidence of impaired osteoblastic activity as seen by decreased osteocalcin values on days 14 and 28 (p < 0.001). Azathioprine caused no loss of bone volume nor any deviation from the norm in mineral apposition rate, bone formation rate, or longitudinal bone growth. All three treatment groups showed an increased recruitment of osteoclasts to the bone surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Two multidrug resistant strains of Streptococcus pneumoniae – SV35-T23 (capsular type 23F) and SV36-T3 (capsular type 3) were recovered from the nasopharynx of two adult patients during an outbreak of pneumococcal disease in a New York hospital in 1996. Both strains belonged to the pandemic lineage PMEN1 but they differed strikingly in virulence when tested in the mouse model of IP infection: as few as 1000 CFU of SV36 killed all mice within 24 hours after inoculation while SV35-T23 was avirulent. Whole genome sequencing (WGS) of the two isolates was performed (i) to test if these two isolates belonging to the same clonal type and recovered from an identical epidemiological scenario only differed in their capsular genes? and (ii) to test if the vast difference in virulence between the strains was mostly – or exclusively – due to the type III capsule. WGS demonstrated extensive differences between the two isolates including over 2500 single nucleotide polymorphisms in core genes and also differences in 36 genetic determinants: 25 of which were unique to SV35-T23 and 11 unique to strain SV36-T3. Nineteen of these differences were capsular genes and 9 bacteriocin genes. Using genetic transformation in the laboratory, the capsular region of SV35-T23 was replaced by the type 3 capsular genes from SV36-T3 to generate the recombinant SV35-T3* which was as virulent as the parental strain SV36-T3* in the murine model and the type 3 capsule was the major virulence factor in the chinchilla model as well. On the other hand, a careful comparison of strains SV36-T3 and the laboratory constructed SV35-T3* in the chinchilla model suggested that some additional determinants present in SV36 but not in the laboratory recombinant may also contribute to the progression of middle ear disease. The nature of this determinants remains to be identified.

Two multidrug resistant strains of Streptococcus pneumoniae - SV35-T23 (capsular type 23F) and SV36-T3 (capsular type 3) were recovered from the nasopharynx of two adult patients during an outbreak of pneumococcal disease in a New York hospital in 1996. Both strains belonged to the pandemic lineage PMEN1 but they differed strikingly in virulence when tested in the mouse model of IP infection: as few as 1000 CFU of SV36 killed all mice within 24 hours after inoculation while SV35-T23 was avirulent.Whole genome sequencing (WGS) of the two isolates was performed (i) to test if these two isolates belonging to the same clonal type and recovered from an identical epidemiological scenario only differed in their capsular genes? and (ii) to test if the vast difference in virulence between the strains was mostly - or exclusively - due to the type III capsule. WGS demonstrated extensive differences between the two isolates including over 2500 single nucleotide polymorphisms in core genes and also differences in 36 genetic determinants: 25 of which were unique to SV35-T23 and 11 unique to strain SV36-T3. Nineteen of these differences were capsular genes and 9 bacteriocin genes.Using genetic transformation in the laboratory, the capsular region of SV35-T23 was replaced by the type 3 capsular genes from SV36-T3 to generate the recombinant SV35-T3* which was as virulent as the parental strain SV36-T3* in the murine model and the type 3 capsule was the major virulence factor in the chinchilla model as well. On the other hand, a careful comparison of strains SV36-T3 and the laboratory constructed SV35-T3* in the chinchilla model suggested that some additional determinants present in SV36 but not in the laboratory recombinant may also contribute to the progression of middle ear disease. The nature of this determinants remains to be identified.

We report on the comparative genomics and characterization of the virulence phenotypes of four S. pneumoniae strains that belong to the multidrug resistant clone PMEN1 (Spain(23F) ST81). Strains SV35-T23 and SV36-T3 were recovered in 1996 from the nasopharynx of patients at an AIDS hospice in New York. Strain SV36-T3 expressed capsule type 3 which is unusual for this clone and represents the product of an in vivo capsular switch event. A third PMEN1 isolate - PN4595-T23 - was recovered in 1996 from the nasopharynx of a child attending day care in Portugal, and a fourth strain - ATCC700669 - was originally isolated from a patient with pneumococcal disease in Spain in 1984. We compared the genomes among four PMEN1 strains and 47 previously sequenced pneumococcal isolates for gene possession differences and allelic variations within core genes. In contrast to the 47 strains - representing a variety of clonal types - the four PMEN1 strains grouped closely together, demonstrating high genomic conservation within this lineage relative to the rest of the species. In the four PMEN1 strains allelic and gene possession differences were clustered into 18 genomic regions including the capsule, the blp bacteriocins, erythromycin resistance, the MM1-2008 prophage and multiple cell wall anchored proteins. In spite of their genomic similarity, the high resolution chinchilla model was able to detect variations in virulence properties of the PMEN1 strains highlighting how small genic or allelic variation can lead to significant changes in pathogenicity and making this set of strains ideal for the identification of novel virulence determinants.

BACKGROUND: Streptococcus pneumoniae [Sp] infection is associated with local and systemic disease. Our current understanding of the differential contributions of genetic strain variation, serotype, and host response to disease phenotype is incomplete. Using the chinchilla model of otitis media [OM] we investigated the disease phenotype generated by the laboratory strain TIGR4 and each of thirteen clinical strains (BS68-75, BS290, BS291, BS293, BS436 and BS437); eleven of the thirteen strains have been genomically sequenced. METHODOLOGY/PRINCIPAL FINDINGS: For each strain 100 colony forming units were injected bilaterally into the tympanic bullae of 6 young adult chinchillas under general anesthesia. All animals were examined daily for local and systemic disease by a blinded observer. Pneumatic otoscopy was used to evaluate local disease, and behavioral assessments served as the measure of systemic disease. Virulence scoring was performed using a 4-point scale to assess four clinical parameters [severity and rapidity of local disease onset; and severity and rapidity of systemic disease onset] during a 10-day evaluation period. Highly significant variation was observed among the strains in their ability to cause disease and moribundity. CONCLUSIONS/SIGNIFICANCE: As expected, there was a significant correlation between the rapidity of systemic disease onset and severity of systemic disease; however, there was little correlation between the severity of otoscopic changes and severity of systemic disease. Importantly, it was observed that different strains of the same serotype produced as broad an array of disease phenotypes as did strains of different serotypes. We attribute these phenotypic differences among the strains to the high degree of genomic plasticity that we have previously documented.