Jussi Jero

Detection of Rhinovirus, Respiratory Syncytial Virus, and Coronavirus Infections in Acute Otitis Media by Reverse Transcriptase Polymerase Chain Reaction

Objective. To determine the frequencies of human rhinovirus (HRV), respiratory syncytial virus (RSV), and coronavirus (HCV) infection in children with acute otitis media (AOM). Methods. Middle ear fluids (MEF) collected by tympanocentesis and nasopharyngeal aspirates (NPA) at the time of the AOM diagnosis were examined by reverse transcriptase polymerase chain reaction for HRV, RSV, and HCV RNA. Patients. Ninety-two children aged 3 months to 7 years during a 1-year period. Results. Virus RNA was detected in a total of 69 children (75%) and in 44 MEF samples (48%) and 57 NPA samples (62%) at the time of AOM diagnosis. HRV RNA was detected in both MEF and NPA in 18 (20%), in MEF alone in 4 (4%), and in NPA alone in 10 (11%). RSV was detected in both MEF and NPA in 12 (13%), in MEF alone in 5 (5%), and in NPA alone in 9 (10%). HCV RNA was detected in both MEF and NPA in 5 (5%), in MEF alone in 2 (2%), and in NPA alone in 9 (10%). Dual viral infections were detected in 5% of children. HRV and RSV were detected simultaneously in 2 MEF samples and in 2 NPA samples; RSV and HCV were detected in 1 NPA sample. Bacterial pathogens were detected in 56 (62%) MEF from 91 children. Viral RNA was detected in 20 (57%) MEF of 35 bacteria-negative and in 25 (45%) of 56 bacteria-positive MEF samples. No important differences in the risk of treatment failure, relapse, or occurrence of late secretory otitis media were noted between children with virus-positive and virus-negative MEF aspirates. Conclusion. These findings highlight the importance of common respiratory viruses, particularly HRV and RSV, in predisposing to and causing AOM in young children.

Cochlear Gene Delivery through an Intact Round Window Membrane in Mouse

Cochlear gene transfer studies in animal models have utilized mainly two delivery methods: direct injection through the round window membrane (RWM) or intracochlear infusion through a cochleostomy. However, the surgical trauma, inflammation, and hearing loss associated with these methods lead us to investigate a less invasive delivery method. Herein, we studied the feasibility of a vector transgene-soaked gelatin sponge, Gelfoam, for transgene delivery into the mouse cochlea through an intact RWM. The Gelfoam absorbed with liposomes and adenovirus, but not with adeno-associated virus (AAV), was successful in mediating transgene expression across an intact RWM in a variety of cochlear tissues. The Gelfoam technique proved to be an easy, atraumatic, and effective, but vector-dependent, method of delivering transgenes through an intact RWM. Compared with the more invasive gene delivery methods, this technique represents a safer and a more clinically viable route of cochlear gene delivery in humans.

A surgical approach appropriate for targeted cochlear gene therapy in the mouse.

Therapeutic manipulations of the mammalian cochlea, including cochlear gene transfer, have been predominantly studied using the guinea pig as the experimental model. With the significant developments in mouse genomics and the availability of mutant strains of mice with well-characterized hearing loss, the mouse justifiably will be the preferred animal model for therapeutic manipulations. However, the potential advantages of the mouse model have not been fully realized due to the surgical difficulty of accessing its small cochlea. This study describes a ventral approach, instead of the routinely used postauricular approach in other rodents, for accessing the mouse middle and inner ear, and its application in cochlear gene transfer. This ventral approach enabled rapid and direct delivery of liposome-transgene complex to the mouse inner ear while avoiding blood loss, facial nerve morbidity, and mortality. Transgene expression at 3 days was detected in Reissners membrane, spiral limbus, spiral ligament, and spiral ganglion cells, in a pattern similar to that previously described in the guinea pig. The successful access and delivery of material to the mouse cochlea and the replication of gene expression seen in the guinea pig demonstrated in this study should promote the use of the mouse in future studies investigating targeted cochlear therapy.

Limited Cone-Beam Computed Tomography Imaging of the Middle Ear: A Comparison with Multislice Helical Computed Tomography

Purpose: To determine the applicability of cone-beam computed tomography (CBCT) in otological imaging, and to compare its accuracy with the routinely used multislice helical CT (MSCT) for imaging of the middle- and inner-ear areas. Material and Methods: Thirteen unoperated human cadaver temporal bones were imaged with CBCT and MSCT. Sixteen landmarks of the middle and adjacent inner ear were evaluated and compared for their conspicuity according to a modified Likert scale. Total scores and scores for subgroups including landmarks of specific clinical interest were also compared. Results: No significant differences were found between the imaging techniques or subgroups when scores of individual structures were compared. While the middle ear itself was visible in all cases with CBCT, parts of the inner ear were “cut off” in four cases due to the limited field of view. For the same reason, the evaluation of the whole mastoid was not possible with CBCT. The cochlear and vestibular aqueducts were not visualized in either CT techniques. The contrast-to-noise ratio was more than 50% lower in CBCT than in MSCT, but still adequate for diagnostic task. Conclusion: CBCT proved to be at least as accurate as routinely used MSCT in revealing the clinically and surgically important middle-ear structures. The results show that high-quality imaging of the middle ear is possible with the current CBCT device.

Acute complications of otitis media in adults

Objectives:  To establish the incidence, current treatment and outcome of adult patients with acute intratemporal and intracranial complications of otitis media (OM).

The Use of Preyer's Reflex in Evaluation of Hearing in Mice

Preyers reflex, the elicitation of startle response to auditory stimuli, has been widely used for the evaluation of hearing in rodents and other animals. Surprisingly, however, the sensitivity and specificity of Preyers reflex in the assessment of hearing has not been adequately studied. The aim of this study was to investigate the utility of Preyers reflex in the evaluation of auditory function in mice. Forty-six adult albino mice on an FVB background with variable hearing loss were used for this study. Two different methods for eliciting a Preyers reflex were tested: a handclap and a sharp metallic sound. The reflex was considered positive when a rapid movement of the whole body of the animal was clearly noticed. Thereafter, the mice underwent auditory brain stem response (ABR) testing with broadband clicks. The presence or absence of Preyers reflex was compared with the corresponding ABR thresholds. Five of the 46 animals studied (11%) showed a negative Preyers reflex, while the remaining 41 animals demonstrated a positive Preyers reflex. There was no difference between the abilities of the two different stimuli to elicit a Preyers reflex. The click-evoked ABR thresholds in the test animals varied between 8 and 136 (mean 50) dB sound pressure level (SPL). Preyers reflex was positive in all animals with an ABR threshold of < or = 76 dB SPL, but was absent in animals with an ABR threshold of > or = 81 dB SPL. Preyers reflex is effective for identifying profound sensorineural hearing loss in experimental mice, but is insensitive for detecting less severe auditory dysfunction. For definitive hearing assessment, and for defining the hearing thresholds. objective electroacoustical methods such as ABR should be used.

Polymerase chain reaction-based detection of rhinovirus, respiratory syncytial virus, and coronavirus in otitis media with effusion.

Abstract Objectives: To study the association of human rhinovirus (HRV), respiratory syncytial virus (RSV), and human coronavirus infections in children aged 6 months to 12 years with otitis media with effusion (OME). To determine how long HRV RNA can be detected after HRV infection. Methods: Middle ear effusion (MEE) samples collected at the time of tympanostomy tube placement from 100 children with OME were examined. Viral RNA was detected by reverse-transcriptase polymerase chain reaction. For HRV the results were compared with virus isolation in cell culture. In vitro studies of the persistence of HRV infectivity and RNA were conducted by combining ~105 median cell culture infectious doses of HRV with pooled MEE at 37°C and assaying serial samples for 12 weeks. Results: Virus RNA was detected in 30 children. HRV was detected by reverse-transcriptase polymerase chain reaction in 19 children with OME and by virus isolation in 5 children. RSV RNA was found in 8 and HCV in 3 children with OME. No dual viral infection was found. Bacterial pathogens were isolated from 35 MEE samples and were associated with viral RNA in 11 cases, most often with HRV (9 cases). Under in vitro conditions, HRV culture positivity declined rapidly (<2 days), but RNA was detectable for up to 8 weeks. Conclusions: These results suggest that virus infection, particularly HRV infection, either alone or concurrent with bacteria, is present in a larger percentage of children with OME than previously suspected. It remains to be determined how often the presence of viral RNA in MEE represents persistent RNA, ongoing viral replication, or recurrent infection. (J Pediatr 1998;133:390-4)

Aquaporin-2 expression in the mammalian cochlea and investigation of its role in Meniere's disease.

The expression pattern of aquaporin-2 (AQP2), a vasopressin regulated member of the aquaporin gene family, in the cochlea and its potential role in Menieres disease was investigated. RT-PCR screen of multiple rat tissues identified AQP2 transcripts in the cochlea, testis and kidney and an absence of tissue-specific splice variants. The level of AQP2 transcript in the cochlea was 10-fold lower relative to its expression in the testis and kidney. Western blot analysis demonstrated a single, 29 kDa band in the membrane fractions from cochlea, testis and the kidney. In the rat and mouse cochlea, AQP2 was expressed in the structures bordering the endolymph, including Reissners membrane, the organ of Corti, inner and outer sulcus cells and the spiral limbus. A mutation screen of AQP2 in 12 individuals with Menieres disease did not identify any sequence alterations or mutations within the four coding exons of AQP2 and their intron-exon junctions. The physiological role of AQP2 in water transport and its expression pattern in the cochlea suggests an important role for AQP2 in fluid homeostasis of the inner ear; however, its role in the pathogenesis in Menieres disease remains to be established.

Bacterial adherence to titanium surface coated with human serum albumin.

Hypothesis: An albumin coating on titanium implants will inhibit bacterial adhesion on the implant surface. Background: Bacterial, protein, and platelet adhesion on otologic implants and tympanostomy tubes is a major reason for implant sequelae and can eventually lead to implant removal. The role of albumin coating of the implant in prevention of protein adhesion on implant surface has already been tested by the authors. In the present study the authors examined the in vitro adherence of Staphylococcus aureus and Pseudomonas aeruginosa on an albumin-coated and uncoated titanium surface. Methods: Human serum albumin (HSA)-coated and uncoated titanium surfaces were exposed to viable S. aureus and P. aeruginosa and, after washings, photographed by fluorescence microscopy to quantify the adhered bacteria, which was stained with acridine orange. Results: Bacteria in the suspension adhered at a significantly lesser rate to the coated surfaces than to the uncoated surfaces, with overall bacterial adhesion dependent on bacterial concentration. Binding of S. aureus on HSA-coated surfaces was inhibited significantly (from 82 to 95% depending on concentration). Binding of P. aeruginosa was inhibited from 29 to 37%. Conclusion: Because albumin coating can reduce bacterial adherence on titanium surfaces in vitro, reduction is possible in bacterial contamination and infection of the HSA-coated titanium implant in vivo.

The clinical role of Alloiococcus otitidis in otitis media with effusion

OBJECTIVE To investigate the presence of Alloiococcus otitidis (A. otitidis) in MEEs from patients with otitis media with effusion (OME) using PCR and to correlate the findings with the clinical picture of children with OME for assessing the clinical role of A. otitidis in OME. METHODS Bacterial culture and PCR were used to detect A. otitidis, Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in MEE samples from 123 patients with OME. The culture and PCR results and the clinical picture of the patients were compared. RESULTS Bacteria were cultured in 55 (45%) of the 123 MEEs, and major pathogens (S. pneumoniae, H. influenzae and M. catarrhalis) were found in 40 (33%); A. otitidis was not found in culture. PCR of the MEEs yielded positive results for one or more of the four tested pathogens in 108 (88%) of the samples and 25 (20%) were positive for A. otitidis. The effusions that persisted 3 months or longer had a higher prevalence of A. otitidis than those with shorter durations (P=0.03). A. otitidis was found to be more often positive in PCR in mucoid MEEs than in mucoserous MEEs (30 vs. 9%; P=0.015). CONCLUSIONS While A. otitidis is extremely difficult to detect with bacterial culture, PCR provides a sensitive and specific means for detecting it. A. otitidis is associated with a more prolonged course and mucoid MEEs in OME. Thus, its existence seems to be related to a more chronic stage of OME, but its pathogenic potential should be the subject of further investigation.