Next-Generation Sequencing Combined with Specific PCR Assays To Determine the Bacterial 16S rRNA Gene Profiles of Middle Ear Fluid Collected from Children with Acute Otitis Media.

The aim of the study was to analyze the bacteriome of acute otitis media with a novel modification of next-generation sequencing techniques. Outpatient children with acute otitis media were enrolled in the study, and middle ear fluids were collected during 90 episodes from 79 subjects aged 5 to 42 months (median age, 19 months). The bacteriome profiles of middle ear fluid samples were determined by a nested-PCR amplification of the 16S rRNA gene (V4 region), followed by mass sequencing. The profiling results were compared to the results of specific PCR assays targeting selected prevalent pathogens. Bacteriome profiling using nested amplification of low-volume samples was aided by a bioinformatic subtraction of signal contaminants from the recombinant polymerase, achieving a sensitivity slightly lower than that of specific PCR detection. Streptococcus pneumoniae was detected in 28 (31%) samples, Haemophilus influenzae in 24 (27%), Moraxella catarrhalis in 18 (20%), Staphylococcus spp. in 21 (23%), Turicella otitidis in 5 (5.6%), Alloiococcus otitidis in 3 (3.3%), and other bacteria in 14 (16%) using bacteriome profiling. S. pneumoniae was the dominant pathogen in 14 (16%) samples, H. influenzae in 15 (17%), M. catarrhalis in 5 (5.6%), T. otitidis in 2, and Staphylococcus auricularis in 2. Weaker signals of Prevotella melaninogenica, Veillonella dispar, and Veillonella montpellierensis were noted in several samples. Fourteen samples (16%) were not explainable by bacterial pathogens; novel causative agents were not detected. In conclusion, unbiased bacteriome profiling helped in depicting the true mutual quantitative ratios of ear bacteria, but at present, its complicated protocol impedes its routine clinical use. IMPORTANCE Although S. pneumoniae, H. influenzae, and M. catarrhalis have been long established as the most important pathogens in acute otitis media using culture and specific PCR assays, the knowledge of their mutual quantitative relations and possible roles of other bacteria is incomplete. The advent of unbiased bacteriome 16S rRNA gene profiling has allowed the detection of nearly all bacteria present in the sample, and it helps in depicting their mutual quantitative ratios. Due to the difficulties in performing mass sequencing in low-volume samples, only a few bacteriome-profiling studies of otitis media have been published, all limited to cases of chronic otitis media. Here, we present a study on samples obtained from young children with acute otitis media, successfully using a strategy of nested PCR coupled with mass sequencing, and demonstrate that the method can confer quantitative information hardly obtainable by other methods.

Human enterovirus and rhinovirus infections are associated with otitis media in a prospective birth cohort study.

BACKGROUND: Human enteroviruses (HEVs) and rhinoviruses (HRVs) have been linked to acute otitis media (AOM). OBJECTIVES: The present study evaluates the aforementioned association in a birth cohort setting. STUDY DESIGN: The cohort included 286 healthy infants (191 boys) followed from birth up to the age of 2 years in the Type 1 Diabetes Prediction and Prevention study in Finland. Stool samples were collected monthly and analyzed for the presence of HRV and HEV RNA using RT-PCR. Clinical symptoms were recorded by a questionnaire every 3-6 months. RESULTS: Altogether 610 AOM episodes were reported during the follow-up. 9.8% of the stool samples were positive for HRV and 6.8% for HEV. HRV positivity peaked at the age of 3-6 months declining gradually after this age, whereas HEV positivity peaked later, at the age of 12-24 months. The risk of AOM was increased in children who were HEV positive at least once at the age of 6-12 months (OR 2.2 [95%CI 1.1-4.2], P=0.023) or who were HRV positive at least once at the age of 18-24 months (OR 2.3 [95%CI 1.0-5.2], P=0.042). Having an older sibling, short breast-feeding and maternal smoking during pregnancy were also significantly associated with AOM. CONCLUSIONS: HRV and HEV infections are frequent during the first months of life. The observed trend for increased risk of AOM in HRV and HEV positive children is in line with the results from hospital series suggesting that these viruses may play an independent role in the pathogenesis of AOM.

Moraxella catarrhalis Might Be More Common than Expected in Acute Otitis Media in Young Finnish Children.

According to studies based on bacterial cultures of middle ear fluids, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis have been the most common pathogens in acute otitis media. However, bacterial culture can be affected by reduced viability or suboptimal growth of bacteria. PCR detects bacterial DNA from samples with greater sensitivity than culture. In the present study, we analyzed the middle ear pathogens with both conventional culture and semiquantitative real-time PCR in 90 middle ear fluid samples obtained from children aged 5 to 42 months during acute otitis media episodes. Samples were tested for the presence of S. pneumoniae, H. influenzae, M. catarrhalis, Alloiococcus otitidis, Staphylococcus aureus, and Pseudomonas aeruginosa One or more bacterial pathogens were detected in 42 (47%) samples with culture and in 69 (77%) samples with PCR. According to PCR analysis, M. catarrhalis results were positive in 42 (47%) samples, H. influenzae in 30 (33%), S. pneumoniae in 27 (30%), A. otitidis in 6 (6.7%), S. aureus in 5 (5.6%), and P. aeruginosa in 1 (1.1%). Multibacterial etiology was seen in 34 (38%) samples, and M. catarrhalis was detected in most (85%) of those cases. Fifteen signals for M. catarrhalis were strong, suggesting a highly probable etiological role of the pathogen. In conclusion, even though M. catarrhalis is often a part of mixed flora in acute otitis media, a considerable proportion of cases may be primarily attributable to this pathogen.

Antibiotic resistance in pathogens causing acute otitis media in Finnish children.

INTRODUCTION: Microbiology and susceptibility of middle ear pathogens in children change over time and antibiotic resistance is increasing globally. For the clinicians it is important to be up to date about the resistance situation when considering antibiotic treatment in acute otitis media (AOM). In this study we analysed the resistance profile of AOM pathogens in out-patient children in Finland. METHODS AND MATERIALS: A total of 41 culture positive middle ear fluid (MEF) samples were analysed for bacteria and the presence of antibiotic resistant strains. The samples were obtained from children aged six - 39 months who participated in the otitis media trial during one year period. RESULTS: The most common pathogen was Haemophilus influenzae 17 (40%), followed by Streptococcus pneumoniae in 15 (35%) and Moraxella catarrhalis in 8 (19%). Other pathogens were detected in 3 (7%) of cases. Antibiotic resistance or diminished sensitivity was seen in 63% of the detected bacteria and 28% of pathogens produced beta-lactamase. CONCLUSIONS: Antibiotic resistant bacterial strains causing AOM have increased in Finland. Nevertheless, most of the pathogens (72%) were sensitive to amoxicillin, which is still recommended as the first line antibiotic in the treatment of AOM.

The Experience of Treating Drooling with Repeated Botulinum Toxin Injections.

Botulinum toxin A (BTX-A) injections to the salivary glands are effective in the treatment of drooling, and complications are rare. However, there are only a few previous reports on the long-term use of BTX-A injections. This study retrospectively analyzes our experience of treating drooling with repeated BTX-A injections in patients with neurodegenerative diseases. All patients who received repeated BTX-A injections to the submandibular glands at Tampere University Hospital in 2004-2013 were included in the analysis. Six patients, aged from 6 to 21 years, were included in the study, and a total of 41 bilateral BTX-A injections were administered to their submandibular glands. The average number of injections per patient was 6 (range: 3-11). The average interval between the injections was 9.8 months (range: 4-18), and 95% (39/41) of the injections were performed with good response. The complication rate of the BTX-A injections was 2.4% (1/41), since one of the patients had swallowing problems after an injection. BTX-A injections to the submandibular glands are effective and have a low morbidity rate, and repeated injections can be recommended as long-term treatment of drooling.

Human parechovirus as a minor cause of acute otitis media in children.

Human parechoviruses (HPeVs) cause mild upper respiratory infections, gastrointestinal symptoms, central nervous system infections and some studies have linked them with acute otitis media (AOM). The aim of the present study was to study further the role of HPeV infections in AOM by detecting these viruses directly from middle ear fluid (MEF), respiratory and stool samples collected from children during AOM episodes. A total of 91 MEF samples, 98 nasal swab (NS) samples and 92 stool samples were collected during 100 AOM episodes in a total of 87 children aged between five to 42 months. All specimens were analyzed by real time RT-PCR for the presence of HPeV RNA. HPeV infection was diagnosed in 12 (14%) patients. HPeV RNA was detected in altogether 13 samples, including four MEF samples, three NS samples and six stool samples. One patient was positive in both stool and MEF samples. The results suggest that HPeV may play a role in some AOM cases, but it is not a major cause of AOM in children.

Retrospective analysis of a combined endoscopic and transcutaneous technique for the management of parotid salivary gland stones.

Sialendoscopy is used in the diagnosis and treatment of various symptoms relating to the salivary gland, e.g. chronic swelling or obstruction and inflammation of the salivary duct. Small intraductal stones can be removed with various instruments during sialendoscopy, whereas larger ones can be fragmented with extracorporeal shockwave lithotripsy or laser. However, 5-10% of the patients with parotid stones cannot be treated with these methods. In patients with large impacted stones or stones in a hilus area, a combined endoscopic and transcutaneous technique can be employed. The stone is approached endoscopically, a skin flap is raised over or a small incision is made through the illuminated area, and the stone is removed by an external route with minimal morbidity. This retrospective study analysed the cases of 8 patients treated using the combined technique, 6 of whom became symptom free. Superficial parotidectomy was performed in 1 patient. No complications were observed, and ductal stents were not used. The average diameter of the stones was 7.6 mm (range 7.0-10.2). The combined technique is recommended for the removal of large and impacted intraductal stones in the parotid gland. No major complications have been reported.

[Combined technique in the removal of parotid gland stones]. / Yhdistelmätekniikka korvasylkirauhasen kivien poistossa.

Sialendoscopy is used in the diagnostics and treatment of salivary gland swelling. Small intraductal stones can be removed with various instruments during sialendoscopy. In cases with larger fixed stones a combined technique can be applied. The stone is approached endoscopically, skin flap is raised or a small incision is made through the illuminated area and the stone is removed via the external route with minimal morbidity. In this series five out of seven patients treated by the combined technique became symptomless. Superficial parotidectomy was performed on one patient. The combined technique is recommended in the removal of stones that are large, fixed in the duct or located in the gland's hilus.

Temporal relationship between human parechovirus 1 infection and otitis media in young children.

BACKGROUND: Human parechovirus (HPeV) 1 is a common virus that infects almost everyone during childhood. Because clinical symptoms are poorly documented, we evaluated the symptoms associated with HPeV1 infection in a cohort of children followed prospectively from birth at 3-month intervals. METHODS: Symptoms such as fever, cough, those of the common cold, otitis media, and gastroenteritis were determined from hospital records and from questionnaires administered to the parents of 59 children during regular study visits. HPeV1 infections were diagnosed by measuring neutralizing antibodies in follow-up serum samples. Additionally, HPeV RNA was analyzed in middle ear fluid (MEF) and nasopharyngeal aspirate samples from 33 patients with otitis media by reverse-transcription polymerase chain reaction. RESULTS: Otitis media showed a clear association with HPeV1 infection-it developed in 50% of the 3-month follow-up periods that yielded evidence for HPeV1 infection but in only 14% of the HPeV1-negative periods (odds ratio [OR], 6.14 [95% confidence interval {CI}, 2.75-13.77]). In children with recurring otitis media, MEF samples were positive for HPeV in 15% of episodes. Cough was also associated with HPeV1 infection, but this association was weaker (OR, 3.67 [95% CI, 1.66-8.09]). Other symptoms were not linked to HPeV1 infection. CONCLUSIONS: HPeV1 infections are common in childhood and may cause otitis media and cough.

Stapes surgery: a 32-year follow-up.

Despite a widespread use of stapes surgery, little is known about the long-term durability of hearing results. The present study provides data over a long time frame (32 years) on hearing changes following surgical treatment. During a 10-year period (1965-1975) stapes surgery was performed in 322 consecutive patients in Tampere University Hospital, Finland. Postoperative evaluation was performed in the same hospital during the years 2001 and 2002 in 58 patients (41 females, 17 males). Of these 58 patients, 68 ears were operated decades ago (stapedoplasty in 67 ears and stapes mobilization in one ear). The mean follow-up time at the latest visit was 32.9 years. At the long-term follow-up, we found that the operative result was very well maintained: 37% of the patients had air-bone gap closure less than 10 dB. Most of the patients (83%) reached postoperative (6-12 months) air-bone gap closure less than 20 dB. The result was maintained by 75% of the patients. Stapes surgery gives a rise to a better social life for the hearing-impaired patient and delays the need for hearing aid in most patients.