Features predicting treatment failure in pediatric acute otitis media.

OBJECTIVES: To facilitate better antibiotic stewardship, we conducted this clinical trial to identify the prognostic features of treatment failure in pediatric acute otitis media (AOM). STUDY: Design: This is a randomized, parallel-group, open-label, comparative clinical trial. SUBJECTS AND METHODS: Children with AOM and aged between 1 month and 5 years were enrolled. Patients were randomly assigned to receive either amoxicillin alone (70 mg/kg) for five days, or the same with additional clarithromycin (15 mg/kg) for the initial three days. The clinical course of AOM was evaluated based on tympanic membrane scores. Failure of treatment for AOM was confirmed on day 14. Nasal conditions were also assessed by a clinical scoring system for acute rhinosinusitis. RESULTS: Treatment failures occurred in 25 out of 129 (19.4%) children. The ratio of treatment failures by age was significantly higher in children younger than 2 years than in children older than 2 years. The tympanic membrane scores on day 3 (P = 0.0334) and day 5 (P < 0.0001) and acute rhinosinusitis scores on day 5 (P = 0.0004) were higher in failure cases than in cured cases. Multivariate logistic regression analysis indicated significant associations between the treatment failure with tympanic membrane scores and acute rhinosinusitis scores on day 5, and the antimicrobial treatment regimen. CONCLUSIONS: Improvement of acute rhinosinusitis and tympanic membrane scores on day five were important predictive features in failure of treatment for pediatric AOM. These results will be useful when discussing the treatment decisions with the patient's parents.

Molecular epidemiology of nonencapsulated Streptococcus pneumoniae among Japanese children with acute otitis media.

The introduction of pneumococcal conjugate vaccine may change the epidemiology of Streptococcus pneumoniae. The increased prevalence of non-vaccine serotypes as the cause of pneumococcal diseases has already reported in the United States and Europe. However, little attention has been focused on the S. pneumoniae. In this study, nonencapsulated S. pneumoniae were identified in 15 isolates (6.4%) out of 236 pneumococcal strains obtained from the nasopharynges of children with acute otitis media (AOM), in 3 isolates (14.3%) out of 21 strains from acute rhinosinusitis, and in 2 isolates (12.5%) out of 16 nasopharyngeal carriage strains obtained from normal healthy children. Among the 20 nonencapsulated S. pneumoniae isolates, 15 (75.0%) isolates had the pspK gene. Seven sequence types (STs) were identified: ST7502 (5 strains), ST1106 (2 strains), ST7803 (2 strains), ST7786 (1 strain), ST6741 (1 strain), ST7496 (1 strain), and ST8642 (1 strain). Because nonencapsulated S. pneumoniae strains are not targeted by the current available pneumococcal vaccines, these strains will gradually become more common in nasopharyngeal carriage. The increase in colonization and dissemination of these strains would increase the risk of AOM and other systemic pneumococcal diseases against which current vaccines cannot provide protection. Nonencapsulated S. pneumoniae may thus become more prevalent as human pathogen.

Bactericidal effect of tosufloxacin on Haemophilus influenzae internalized in cultured human cells.

Nontypeable Haemophilus influenzae, one of the major causative bacteria for acute otitis media (AOM), is also considered to cause intractable otitis media including prolonged AOM and recurrent AOM in children by the mechanism of internalization of the bacteria into epithelial cells of middle ear mucosa. In this study, we visualized the dynamics of H. infiuenzae internalization in cultured human cells. We also examined the effects of antimicrobials, including a novel quinolone, tosufloxacin, and a cephem antibacterial agent, cefditoren, on H. influenzae internalized in cultured human cells. The results indicated that (1) H. infiuenzae were internalized into human cells, (2) cefditoren has no effect on internalized H. influenzae, and (3) tosufloxacin has a bactericidal action on H. infiuenzae invading human cells. These data strongly support high clinical efficacy of tosuffoxacin on intractable otitis media in children.

Activation of NF-κB via endosomal Toll-like receptor 7 (TLR7) or TLR9 suppresses murine herpesvirus 68 reactivation.

UNLABELLED: In order to understand and possibly treat B-cell malignancies associated with latent gammaherpesvirus infection, it is vital to understand the factors that control the balance between the two transcriptional states of gammaherpesviruses: latency and lytic replication. We used murine gammaherpesvirus 68 (MHV 68) as a model system to investigate how engagement of endosomal Toll-like receptors (TLRs) impacts reactivation from latency in vitro and establishment of latent infection in vivo. We found that treatment with TLR7 ligand R848 or TLR9 ligand CpG oligodeoxynucleotide (ODN) suppresses reactivation of MHV 68 in vitro. These suppressive effects correlated with the ability to activate cellular transcription factor NF-κB. Downregulation of TLR9 by RNA interference in vitro led to a reduction of nuclear levels of NF-κB p65 and consequently to an increase of spontaneous reactivation in cells latently infected with MHV 68, indicating that the TLR9 pathway suppresses spontaneous reactivation events. In vivo, sustained stimulation of TLR7 by repeated R848 treatment led to an increased frequency of infected splenocytes compared to mock-treated control results. Frequencies of infected splenic B cells in tlr7-/- or tlr9-/- mice after establishment of latency did not differ from those seen with their wild-type counterparts. Nevertheless, MHV 68-infected B cells from tlr9-/- mice showed a higher frequency of reactivation than B cells from wild-type or tlr7-/- mice in ex vivo reactivation assays. Thus, we show a suppressive effect of TLR7 or TLR9 triggering on MHV 68 reactivation that correlates with NF-κB activation and that the mere presence of a functional TLR9 signaling pathway contributes to dampen lytic gammaherpesvirus reactivation in infected cells. IMPORTANCE: A hallmark of gammaherpesviruses is their establishment of latency in B cells that is reversible through lytic reactivation. Latency can result in B-cell malignancies. Activation of the innate immune system is thought to contribute to controlling the switch between the transcriptional states of latency and reactivation. Nevertheless, the mechanisms involved are not clear. Here, we show that engagement of Toll-like receptor 7 (TLR7) and TLR9 suppresses reactivation of murine gammaherpesvirus MHV 68 in vitro and that stimulation of TLR7 in vivo increases the frequency of infected cells. TLR7 and TLR9 are innate immunity sensors of nucleic acids localized in endosomes. Additionally, we demonstrate that impairment of TLR9 signaling in latently infected B cells leads to increased reactivation. Thus, activated endosomal TLR7 and TLR9 pathways play an important role in promoting establishment of latent gammaherpesvirus infection. Counteracting signaling of these pathways allows reactivation and could represent treatment targets in gammaherpesvirus-associated malignancies.

Reduction of cortical excitability and increase of thalamic activity in a low-frequency rTMS treatment for chronic tinnitus.

Low-frequency repetitive transcranial magnetic stimulation (rTMS) has received increasing attention for the treatment of tinnitus, but its therapeutic mechanisms are unclear. We performed low-frequency rTMS treatment for a patient with chronic tinnitus and examined changes of cortical excitability and cerebral blood flow using paired-pulse TMS and single-photon emission computed tomography. After the rTMS treatment, tinnitus loudness was decreased, cortical excitability was reduced, and blood flow in the thalamus was increased. Our results suggest that low-frequency rTMS treatment reduces tinnitus loudness by an inhibitory effect on the cortical excitability and a remote activation effect on the thalamus through the corticothalamic networks.

[Evaluation of the Effectiveness and Safety in a Multi-center Clinical Trial of VIBRANT SOUNDBRIDGE in Japan].

Middle ear implants (MEIs) such as the Vibrant Soundbridge (VSB) are attractive and alternative treatments for patients with conductive, sensorineural, and mixed hearing loss who do not benefit from, or who choose not to wear, conventional hearing aids (HAs). Recent studies suggest that MEIs can provide better improvements in functional gain, speech perception, and quality of life than HAs, although there are certain risks associated with the surgery which should be taken into consideration, including facial nerve or chorda tympanic nerve damage, dysfunctions of the middle and inner ears, and future device failure/explantation. In Japan, a multi-center clinical trial of VSB was conducted between 2011-2014. A round window vibroplasty via the transmastoid approach was adopted in the protocol. The bony lip overhanging the round window membrane (RWM) was extensively but very carefully drilled to introduce the Floating Mass Transducer (FMT). Perichondrium sheets were used to stabilize the FMT onto the RWM. According to the audiological criteria, the upper limit of bone conduction should be 45 dB, 50 dB, and 65 dB from 500 Hz to 4, 000 Hz. Twenty-five patients underwent the surgery so far at 13 different medical centers. The age at the surgery was between 26-79 years old, and there were 15 males and 10 females. The cause of conductive or mixed hearing loss was middle ear diseases in 23 cases and congenital aural atresia in two cases. The data concerning on the effectiveness and safety of VSB was collected before the surgery and 20 weeks after the surgery. Significant improvements of free-field Pure Tone Audiogram (PTA) from 250 Hz to 8, 000 Hz were confirmed (p < 0.001). Hearing gain up to 40 dB was achieved in the 1, 000 Hz to 4, 000 Hz range. No deterioration in either air conduction or bone conduction at PTA was noted at 20 weeks after the surgery. Monosyllable speech perception in both quiet and noisy conditions improved significantly (p < 0.001). The speech discrimination score in both quiet and noisy conditions improved significantly too (p < 0.001). In the future, it is likely that there will be an increasing population even in Japan that will meet the criteria for MEIs such as VSB. However, the long-term efficacy and safety of these devices should be established.

[Multicenter Clinical Study of Vibrant Soundbridge in Japan: Analysis of Subjective Questionnaires].

The Vibrant Soundbridge (VSB) is an active middle ear implant with the Floating Mass Transducer (FMT). We performed a multicenter study to study the efficacy of the VSB by means of "the 10 Questionnaire on Hearing 2002" and "the APHAB questionnaire" at 13 hospitals between 2011 and 2013. In all, 23 patients with mixed or conductive hearing loss received VSB implantation by the round window placement technique. These individuals were generally unable to use, or gained little from conventional hearing aids or bone conduction hearing aids. Two questionnaires were administrated before the surgery and 20 weeks after the VSB implantation. Scores on every item of "the 10 Questionnaire on Hearing 2002" showed significant improvement under noise after VSB implantation. On the APHAB, the scores for Ease of Communication, Reverberation, and Background subscales improved significantly after the VSB implantation, while the score for the Aversiveness subscale alone failed to show a positive improvement from the inexperience to the new sound. Analysis of the responses to these subjective questionnaires revealed better results after VSB implantation as compared to the preoperative data. In conclusion, RW vibroplasty with the use of VSB provided subjective benefit in patients with conductive and mixed hearing loss.

Genetic characteristics of Haemophilus influenzae and Streptococcus pneumoniae isolated from children with conjunctivitis-otitis media syndrome.

Acute conjunctivitis is the most common ocular disorders among children and frequently concomitant with acute otitis media (AOM) as conjunctivitis-otitis syndrome. In this study, we evaluated prevalence of causative pathogens and PCR-based genotypes of Haemophilus influenzae and Streptococcus pneumoniae among children with conjunctivitis-otitis media syndrome. Nontypeable H. influenzae (NTHi) is identified most often at 61.8% in conjunctiva exudates followed by S. pneumoniae at 28.2% and Moraxella catarrhalis at 19.1%. Genetic ß-lactamase nonproducing ampicillin resistant (gBLNAR) strains of NTHi and genetic penicillin resistant S. pneumoniae (gPRSP) were identified at 72.1% and at 74.2% among conjunctiva isolates by polymerase chain reaction (PCR), respectively. Pneumococcal strains having either ermB or mefE genes were identified at 93.5% among conjunctiva isolates. The restriction fragment of patterns of 89.7% pairs of H. influenzae isolates and 100% pairs of pneumococcal isolates from conjunctiva exudates, middle ear fluids (MEFs) and nasopharyngeal swabs were identical. In contrast to the previous reports, most prevalent strains from conjunctivitis-otitis media syndrome was BLNAR H. influenzae in this study. The causative pathogen responsible for acute conjunctivitis will be originated from the nasopharynx. In the absence of MEFs one can possibly rely on the nasopharyngeal culture to guide an appropriate treatment.

Minimal biofilm eradication concentration of antimicrobial agents against nontypeable Haemophilus influenzae isolated from middle ear fluids of intractable acute otitis media.

Nontypeable Haemophilus influenzae (NTHi) makes the clinical course of acute otitis media (AOM) intractable by forming a biofilm that may hamper the clearance of the bacteria from middle ear cavity. In this study, we evaluated the minimum biofilm eradication concentration (MBEC) of antimicrobial agents against biofilm-forming NTHi strains. Twelve NTHi strains isolated from middle ear fluids of Japanese children with intractable AOM before antimicrobial treatment were evaluated for MBEC of fluoroquinolones in comparison with those of ß-lactams and macrolides. AMPC and CDTR required much higher concentration, i.e., high MBECs, to suppress the biofilm formation of NTHi. In contrast, fluoroquinolones followed by macrolides showed lower MBECs. MBEC would be a good parameter to infer the efficacies of antimicrobials against NTHi in biofilm.

Compliance with oral antibiotic regimens and associated factors in Japan: compliance survey of multiple oral antibiotics (COSMOS).

BACKGROUND: To provide an overall picture of oral antibiotic use in Japan, we conducted a survey of patients who had been prescribed oral antibiotics. In addition, factors potentially associated with compliance were evaluated. METHODS: General practitioners at 155 GP practices throughout Japan participated in the survey. Questionnaires were collected from 1068 subjects visiting those GP practices (676 females; median age 38 y), with a collection rate of 82.9%. RESULTS: According to this survey, the overall percentage of fully compliant subjects was 74.7%. Subgroup analyses showed that compliance tended to be higher with a shorter duration of prescription and fewer doses per day, and also with a greater ease of understanding of the explanation of treatment provided by the doctor. In multivariate analysis, age, pharyngitis, number of doses per day, duration of prescription, intention to return for follow-up, and ease of understanding the explanation of treatment given by the doctor showed a statistically significant association with compliance. CONCLUSIONS: Based on our survey results, prescribing drugs taken with a minimal number of daily doses in a shorter regimen appears to be an effective strategy for improving compliance. It appears that doctors also need to raise awareness of the importance of taking antibiotics properly by clearly explaining their purpose and significance.

Streptococcus pneumoniae isolates from middle ear fluid and nasopharynx of children with acute otitis media exhibit phase variation.

Pneumococcal phase variation of 37 middle ear and 31 nasopharyngeal isolates obtained from children with acute otitis media was examined in the absence of intervening culture. The fraction of the opaque colonies was significantly higher in middle ear isolates than in nasopharyngeal isolates. The difference is probably the result of the pneumococci adapting to differential selective environments.

[Coblation tonsillectomy under surgical microscopy].

Numbers of tonsillectomies may be decreasing in Japan due to troublesome bleeding involved in managing in a small viewing field and relatively low national health insurance point for tonsillectomy. We found coblation tonsillectomy to have advantages of less bleeding, shorter operating time, and less postoprerative pain than conventional tonsillectomy. We found that 40% of those on whom a coblator was used reported no postoperative throat pain, and noted a quick learning curve in decreasing postoperative bleeding in coblation tonsillectomy.

Tumor-specific mutation and downregulation of ING5 detected in oral squamous cell carcinoma.

Our previous study showed high frequency of allelic loss at chromosome 2q37 region in oral cancer. This location contains several candidate tumor suppressor genes such as PPP1R7, ILKAP, DTYMK and ING5. We previously showed 3 members of inhibitor of growth (ING) family, ING1, ING3 and ING4 as tumor suppressor gene in head and neck cancer. As ING5 shows high homology with other members of ING genes including highly conserved carboxy-terminal plant homeodomain and nuclear localization signal, we first picked up ING5 and examined it as a possible tumor suppressor in oral cancer. For this aim, mutation and mRNA expression status of ING5 in paired normal and oral squamous cell carcinoma samples were examined by reverse transcription polymerase chain reaction (RT-PCR) and sequencing. Three missense mutations located within leucine zipper like (LZL) finger and novel conserved region (NCR) domains in ING5 protein were detected, probably abrogating its normal function. We also found 5 different alternative splicing variants of ING5. Then, we examined mRNA level of ING5 by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) analysis, which demonstrated decreased expression of ING5 mRNA in 61% of the primary tumors as compared to the matched normal samples. In conclusion, tumor-specific mutation and downregulation of ING5 mRNA suggested it as a tumor suppressor gene in oral squamous cell carcinoma.

Expression and mutation analysis of her2 in head and neck squamous cell carcinoma.

We analyzed mutation and expression status of human epidermal growth factor receptor 2 (Her2) in head and neck squamous cell carcinoma (HNSCC) using single strand conformation polymorphism (SSCP) mutation analysis and immunohistochemistry (IHC). Mutations were absent in all 85 cases. Out of 57 cases available for IHC, Her2 protein expression was negative (0) in 40 tumors (70%). Seventeen tumors (29.8%) expressed Her2, among these 13 tumors (22.8%) showed a weak (+1) expression and 4 (7%) showed a moderate expression (+2), none showed a strong (+3) expression. There was not a significant association between expression and any of the patients' clinical variables or prognosis. Our results suggest that Her2 may not be useful as a molecular target in HNSCC.

Frequent allelic loss of Dkk-1 locus (10q11.2) is related with low distant metastasis and better prognosis in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is a frequently occurring cancer worldwide. Dickkopf (Dkk)-1 gene is suggested to function as tumor suppressor gene (TSG) in several kinds of malignancies. In this study, we performed loss of heterozygosity (LOH) analysis of Dkk-1 and examined the correlation between LOH status and clinicopathological parameters for the first time. A pretty high LOH ratio (50%) was detected. Interestingly, in the cases with Dkk-1 retention group showed less distant metastasis and a tendency of longer disease free survival. These results indicate that Dkk-1 can play a role in HNSCC carcinogenesis and it may also be related to distant metastasis.

Establishment of shared antigen reactive cytotoxic T lymphocyte using co-stimulatory molecule introduced autologous cancer cells.

Cytotoxic T lymphocytes (CTLs) play an essential role in immunological responses for tumor rejection. In the past decade, many tumor-associated antigens (TAAs) have been identified predominantly in melanomas. Several clinical trials based on such antigenic peptides with or without adjuvants brought about partially favorable results, suggesting that identification of more immunogenic TAAs is needed. We show here the successful establishment of human leukocyte antigen (HLA)-A24-restricted CTL (TcLHK2 line1) from a pleural effusion of lung cancer patient, using B7.1 (CD80) transduced autologous lung cancer cells as an antigen-presenting cell (APC). TcLHK2 line1 recognized autologous lung adenocarcinoma cell line LHK2 in an HLA-A24-restricted fashion. Moreover, this CTL line also recognized allogeneic HLA-A24-positive lung adenocarcinoma cell line, gastric carcinoma cell line and melanoma cell line. These data raise the possibility that co-stimulatory molecule B7.1 (CD80) plays important role to overcome the immunological tolerance. Furthermore, TcLHK2 line1 is a useful tool for the identification of widely expressed shared antigens restricted by HLA-A24. Further analysis of this CTL and autologous cancer cell line will bring about novel TAAs.

A comparison of conventional and molecular microbiology in detecting differences in pneumococcal colonization in healthy children and children with upper respiratory illness.

Conventional microbiology (CM) and real-time polymerase chain reaction (PCR) were used to determine rate and serotype of pneumococcal nasopharyngeal colonization in healthy children and children with upper respiratory illnesses (URI). One hundred and thirty-six healthy children and 79 children with URI were evaluated. Pneumococcal colonization was detected more often by real-time PCR than CM in healthy children (50% vs. 24%, p

[Mutant prevention concentrations of garenoxacin against Streptococcus pneumoniae isolates from otorhinolaryngological infections].

The minimum inhibitory concentrations (MICs) and the mutant prevention concentrations (MPCs) of garenoxacin (GRNX), were compared to those of levofloxacin (LVFX), and moxifloxacin (MFLX) against 78 Streptococcus pneumoniae isolates from otorhinolaryngological infections in Japan during the period January 2007 to June 2007. The MIC and MPC for 90% of the isolates (MIC90 and MPC90) of GRNX were 0.06 and 0.12 microg/mL, respectively, and were the lower values than LVFX and MFLX MIC90s and MPC90s. The ratios of MPC/MIC of GRNX were the lower values than those of LVFX and MFLX.

[A new scoring system for assessing the severity of adult acute rhinosinusitis].

Treating acute rhinosinusitis requires assessing severity and selecting appropriate antimicrobial agents. In 2006, we developed clinical scoring system for diagnosing and treating acute rhinosinusitis based on three clinical symptoms of rhinorrhea, fever, and facial pain and three nasal findings of characteristics and nasal discharge amount, nasal mucosal swelling and nasal mucosal redness. To verify and update scoring, we studied score-based diagnosis of adult acute rhinosinusitis severity. Prevalence of symptoms such as fever and serous nasal discharge in 95 subjects was low as 8.4% and 3% indicating less useful as evaluation items on the diagnosis. Mucopurulent nasal discharge (r = 0.67), facial pain (r = 0.51), rhinorrhea (r = 0.47), and swelling (r = 0.45) correlated significantly with severity evaluated by attending otolaryngological specialists. Nasal mucosal swelling caused discrepancy between clinical scoring and specialists' assess- ment. Evaluated by multivaliate analysis, factors affecting severity assessment were mucopurulent nasal discharge, facial pain, and rhinorrhea (p < 0.0001), but not swelling (p = 0.49). We concluded that mucopurulent nasal discharge scored 0, 2, or 4, facial pain scored 0, 1, or 2, and rhinorrhea scored 0, 1, or 2 should be used in evaluation in new clinical scoring, classified by severity as mild scored 1-3, moderate scored 4-6, and severe scored 7-8 by evaluating consistency with specialist assessment.

T-lymphocyte maturation-associated protein gene as a candidate metastasis suppressor for head and neck squamous cell carcinomas.

Previous gene expression profiles revealed the T-lymphocyte maturation-associated protein (MAL) gene as being frequently downregulated in head and neck cancer. To define the relationship between the MAL gene and the metastatic process, we evaluated the expression status of the gene in matched primary and metastatic tumors of head and neck cancer by semiquantitative reverse transcription-polymerase chain reaction. Furthermore, we aimed to identify potential genetic and epigenetic mechanisms associated with downregulation of MAL, including loss of heterozygosity (LOH), mutation, and hypermethylation. Thirty-five cell lines of University of Turko squamous cell carcinoma (UT-SCC) series derived from head and neck cancer, including nine pairs from matched primary and metastatic tumors, and 30 pairs of matched primary and metastatic tumor samples were analyzed. Twenty out of 35 (57%) cell lines showed downregulation of MAL expression, whereas no expression was found in 10 cell lines (29%). Considering matched primary and metastatic tumor-derived cell-line pairs, four pairs showed decreased expression only in metastasis-derived cells compared with their primary counterparts. Expression analysis of 21 tissue samples demonstrated decreased or no expression of MAL mRNA in 43% of metastatic tumors compared with matched primary tumors. Relating to mechanisms of downregulation, LOH was observed in 30% of primary tumors and 38% of their metastatic counterparts by a MAL-specific microsatellite marker. Furthermore, we found restoration of MAL mRNA after treatment with demethylating agent (5-aza-2'-deoxycytidine) in 9 (45%) out of 20 cell lines. No mutation was found in UT-SCC cell lines. In conclusion, our findings indicate selective downregulation of MAL expression in metastatic cells, suggesting the MAL gene as a new metastasis-suppressor candidate for head and neck cancer. LOH and hypermethylation appeared to be important mechanisms for inactivation of MAL function.