Cochlear Implants International An Interdisciplinary Journal
ISSN: 1467-0100 (Print) 1754-7628 (Online) Journal homepage: http://www.tandfonline.com/loi/ycii20
Meningitis and a safe dexamethasone-eluting intracochlear electrode array Dimitra Stathopoulos, Scott Chambers, Louise Adams, Roy Robins-Browne, Christopher Miller, Ya Lang Enke, Benjamin P C Wei, Stephen O'Leary, Robert Cowan & Carrie Newbold To cite this article: Dimitra Stathopoulos, Scott Chambers, Louise Adams, Roy Robins-Browne, Christopher Miller, Ya Lang Enke, Benjamin P C Wei, Stephen O'Leary, Robert Cowan & Carrie Newbold (2015) Meningitis and a safe dexamethasone-eluting intracochlear electrode array, Cochlear Implants International, 16:4, 201-207 To link to this article: http://dx.doi.org/10.1179/1754762814Y.0000000099
Published online: 15 Oct 2014.
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Date: 23 March 2016, At: 20:15
Original research paper
Meningitis and a safe dexamethasone-eluting intracochlear electrode array Dimitra Stathopoulos 1,2, Scott Chambers 1, Louise Adams 3, Roy Robins-Browne 3, Christopher Miller4, Ya Lang Enke4, Benjamin P C Wei 2, Stephen O’Leary 2, Robert Cowan 1, Carrie Newbold 1,2
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1
The HEARing Cooperative Research Centre, Carlton, Victoria, Australia, 2Department of Otolaryngology, The University of Melbourne, East Melbourne, Victoria, Australia, 3Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Victoria, Australia, 4 Cochlear Limited at Macquarie University, Sydney, New South Wales, Australia Objectives: To evaluate the potential risk of pneumococcal meningitis associated with the use of a dexamethasone-eluting intracochlear electrode array as compared with a control array. Methods: In two phases, adult Hooded–Wistar rats were implanted via the middle ear with an intracochlear array and were inoculated with Streptococcus pneumoniae 5 days post-surgery. Phase I created a dosing curve by implanting five groups (n = 6) with a control array, then inoculating 5 days later with different numbers of S. pneumoniae: 0 CFU, 103 CFU, 104 CFU, 104 CFU repeated, or 105 CFU (colony forming units). A target infection rate of 20% was aimed for and 104 CFU was the closest to this target with 33% infection rate. In phase II, we implanted two groups (n = 10), one with a dexamethasone-eluting array, the other a control array, and both groups were inoculated with 104 CFU of S. pneumoniae 5 days post-surgery. Results: The dexamethasone-eluting array group had a 40% infection rate; the control array group had a 60% infection rate. This difference was not statistically significant with a P value of ≥0.5. Conclusion: The use of a dexamethasone-eluting intracochlear electrode array did not increase the risk of meningitis in rats when inoculated with S. pneumoniae via the middle ear 5 days following implantation. Keywords: Cochlear implant, Meningitis, Streptococcus pneumoniae, Dexamethasone, Corticosteroid, Drug delivery, Rat
Introduction Despite advances made in implant design, software, and surgical technique, cochlear implantation remains an invasive surgical procedure. The introduction of the electrode array into the cochlea initiates an inflammatory response within the cochlea that may lead to development of fibrous tissue or new bone formation, changes in electrical impedance, and a postoperative loss of residual hearing (Bas et al., 2012; Eshraghi and Van De Water, 2006; Kiefer et al., 2004; O’Leary et al., 2013; Huang et al., 2007; Tykocinski et al., 2005). Recent changes in candidature guidelines have meant that many prospective cochlear implant recipients have significant levels of residual acoustic hearing, and it has been shown that the use of acoustic and electric hearing in the same ear can provide significant benefits to speech and Correspondence to: Dimitra Stathopoulos, Department of Otolaryngology, The University of Melbourne, 32 Gisborne St. 2nd Flr. PHW, East Melbourne, Victoria 3002, Australia. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1754762814Y.0000000099
music perception (Incerti et al., 2013; Simpson et al., 2009). Given these findings, preservation of usable residual hearing is an important clinical goal. Anti-inflammatory steroids – such as the glucocorticoid dexamethasone – administered into the scala tympani during cochlear implantation can reduce inflammation and maintain electrical impedance levels (Clark et al., 1995; Paasche et al., 2006; Paasche et al., 2009). Animal studies have investigated residual hearing preservation using a single application of dexamethasone via either local or systemic delivery (Connolly et al., 2010; Eastwood et al., 2010; James et al., 2008; Maini et al., 2009; Chang et al., 2009; Lee et al., 2013). Of the various methods investigated, results have shown that dexamethasone application to the round window 2 hours before surgery provided the most significant benefit (Chang et al., 2009; Lee et al., 2013). Longer-term delivery via mini-osmotic pump has also been shown to sustain auditory brainstem response thresholds (Eshraghi et al., 2007; Vivero et al., 2008).
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Elution of dexamethasone from the surface of an implanted electrode array, if clinically feasible, would appear to be a simple solution for efficient delivery of the therapeutic directly to the site of intended action within the cochlea. Several studies have investigated the potential that such an array might have to reduce the inflammatory response within the cochlea and thereby preserve residual hearing thresholds (Douchement et al., 2015; Niedermeier et al., 2012; Stathopoulos et al., 2014). The foremost concern, however, is that steroid elution from an electrode array is safe and does not suppress the inner ear’s cellular defences against pathogens. In particular, bacterial meningitis is a significant known risk following cochlear implantation, especially for patients with predisposing risk factors such as a history of previous meningitis, head trauma, and inner ear malformation (Cohen, 2004; FDA, 2006; Reefhuis et al., 2003). Pneumococcal meningitis has a high fatality rate at 19–37% and up to 50% of survivors suffer long-term effects including hearing loss, seizures, and cognitive impairment (Kastenbauer and Pfister, 2003; van de Beek et al., 2006; van de Beek et al., 2004; Weisfelt et al., 2006). Previous designs of cochlear implant electrode array have, in the past, been associated with an increased risk of meningitis (FDA, 2006; Reefhuis et al., 2003), making it vital that new electrode array designs incorporating features such as steroid elution are proved safe before clinical application. In a rat model, Wei et al. (2007) showed that the presence of a cochlear implant reduced the threshold of bacteria required to induce pneumococcal meningitis while a cochleostomy alone did not, implying that the primary risk factor was the presence of the foreign body rather than the breach into the cochlea. Several studies have shown a foreign body (e.g. a cochlear implant) can decrease the functionality of inflammatory cells, thus lowering the threshold of bacteria required to induce infection (Fabre et al., 1999; Zimmerli et al., 1984; Zimmerli et al., 1982). This is sufficient justification to test that anti-inflammatories such as dexamethasone will not increase the risk of meningitis. Another consideration is the potential compromise of the development of a robust fibrous tissue seal. Although it is conjectured that development of a mature fibrous tissue seal protects the inner ear from infection, several animal and human studies have shown bacteria and inflammatory cells can track through the mature fibrous tissue sheath around the implanted electrode array (Sudhoff, 2003; Wei et al., 2006, 2007). Niedermeier et al.’s (2012) guinea pig study gave a 5-week recovery period for fibrous tissue seal formation before inoculation with Streptococcus pneumoniae. They found no difference
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in infection rate between dexamethasone-eluting arrays and untreated groups. These previous studies informed our decision to inoculate 5 days post-implantation with an immature tissue seal, as our main concern was the potential compromise of the cellular immune response to the bacteria in the early postelution period. In this study, we assessed the rate of pneumococcal meningitis in rats implanted with either a dexamethasone-eluting array or a non-eluting control array. We used the rat model developed by Wei et al. (2006) and inoculated the rats 5 days after implant with S. pneumoniae as this is the most common cause of meningitis in cochlear implant recipients (Reefhuis et al., 2003; Cohen, 2004).
Materials and methods This study was approved and conducted under the ethical oversight of the Animal Research and Ethics Committee of the Royal Victorian Eye and Ear Hospital under project number 11/242AR. It was conducted in accordance with the ‘Australian code of practice for the care and use of animals for scientific purposes’ (National Health and Medical Research Council, 2013).
Trial experimental designs We implanted silicone dummy arrays unilaterally into the cochleae of adult male Hooded–Wistar rats weighing between 200 and 350 g, followed 5 days later by middle ear inoculation with either a phosphate-buffered saline (PBS) control or S. pneumoniae. The study was split into two phases: Phase I In this phase, we created a dosing curve to determine the number of bacteria required to produce an infection rate of approximately 20% in animals implanted with a non-eluting control array. We allocated 30 rats to five groups (n = 6), each inoculated with 10 μl of PBS containing 103 CFU, 104 CFU, or 105 CFU (colony forming units), or with 10 μl PBS as a control. The fifth group was to receive a higher CFU concentration but, as the upper and lower limits of desired infection rate had been determined, we repeated the 104 CFU inoculum – the closest to our goal of a 20% infection rate – for a more robust result. Phase II In this phase, we allocated 20 rats to two groups (n = 10) to compare the meningitis rate in rats implanted with either a dexamethasone-eluting array or a noneluting control array. Both groups were inoculated with 10 μl of PBS containing 104 CFU of S. pneumoniae.
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Figure 1 Dimensions of the silicone rubber dummy array. Dexamethasone-eluting arrays were identical in size and shape. X shows the approximate location of the trim line, where the array was cut to fit into the bulla.
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Electrode arrays We used moulded silicone rubber dummy arrays, without electrodes or wires, for all implantations (Fig. 1). The arrays in phase I and the control arrays in phase II were silicone rubber only. The experimental arrays in phase II were moulded using silicone rubber mixed with 40% w/w dexamethasone base. All arrays were sterilized with ethylene oxide.
Cochlear implant surgery We performed all surgeries using strict aseptic technique. We induced anaesthesia with 2–3% isofluorane in oxygen and maintained it at 1.5–3% isofluorane at 3–4 l per minute of oxygen. We used local anaesthesia (2% lidocaine hydrochloride) at the incision site and gave analgesia (0.05 mg/kg buprenorphine, diluted 1:10) and antibiotic (10 mg/kg enrofloxacin) post-surgery. We used a post-auricular approach to expose the bulla and drilled a bullostomy to expose the middle ear and the cochlea. To access the cochleostomy site, we cauterized the stapedial artery – located across the round window niche of the cochlea – using a bipolar coagulator. We drilled a cochleostomy below the round window niche and implanted a silicone dummy array into the scala tympani to a depth of 2.5 mm. We then sealed the cochleostomy with fascia and sutured the wound with absorbable monofilament. The silicone dummy array was a non-eluting control for all animals except those in the dexamethasone group in phase II, which received dexamethasone-loaded arrays.
dilutions on chocolate blood agar (CHA) and incubating overnight at 37°C in air with 5% CO2.
Inoculation Five days after cochlear implant surgery, we re-opened the wound. We packed the middle ear with absorbable gelatin surgical sponge and, using a micro-syringe, injected 10 μl of inoculum (or PBS in the phase I control group) into the middle ear. We covered the bullostomy with fascia and re-sutured the wound.
Clinical monitoring After inoculation, we monitored the animals for signs of morbidity over a period of 5 days. We used Table 1, adapted from Wei (2007), to inform a point-based system of determining the time of euthanasia in cases of suspected meningitis. Once an animal scored 10 points or more, it was euthanized.
Specimen collection At termination, we anaesthetized the rats using a 2:1 mixture of ketamine hydrochloride (75 mg/kg) and xylazine hydrochloride (8 mg/kg). Under aseptic laboratory conditions, we exposed the bullostomy to collect middle ear fluid using a sterile needle. Table 1
Scoring guide to assess morbidity of rats
Factor
Score
Alertness
1 2 3
Inoculum We inoculated rats with S. pneumoniae strain 447A (type-2 capsular antigen), originally isolated from a child with meningitis (Dahm, 1994). Bacteria were incubated overnight on horse blood agar at 37°C in air with 5% CO2. Isolated colonies were emulsified in sterile PBS to a concentration of approximately 2–3 × 108 CFU/ml using McFarland equivalence turbidity standards (McFarland, 1907; Miles et al., 1938), and then serially diluted to give the required CFU in 10 μl of inoculum. The viable counts of the inocula were determined retrospectively by plating
Grooming
1 2
Posture
1 2
Movement
1 2 3
Skin temperature
1 2
Criteria Normal, spontaneous Tired but still responding to light, sound, and tactile stimuli Very lethargic with little sustained response to stimuli Normal Abnormal – e.g. scruffy fur, exudate around eyes and nose, porphyrin stains Normal Abnormal – e.g. hunched, curled, head tilt, isolation Normal gait and behaviour Spontaneous but hesitant, careful, slow movement Only with tactile stimuli, unable to move without stumbling or falling Normal >39°C
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Through cardiac puncture, we collected approximately 2 ml of blood, which we inoculated into a BD Bactec™ Peds Plus™/F medium bottle (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA). A lethal dose of pentobarbital sodium (120 mg/kg) was then injected intracardially. We collected cerebrospinal fluid (CSF) from the cisterna magna, incising the skin first to avoid surface contamination. We opened the skull, inspected the meninges for abnormalities, and then collected the dorsal meningeal layer for histological processing. We removed the brain to a sterile petri dish where we took a coronal section for histological processing and another section to incubate in heart infusion broth. We then used a sterile swab to sample the surface of the ventral meningeal layer before removing the tissue for histological processing. We plated the middle ear fluid, CSF, and meningeal swab samples onto CHA and inoculated any remaining fluid into brain heart infusion broth. We embedded brain and meningeal tissue samples in paraffin wax for sectioning. Sections were stained with haematoxylin and eosin, and with Gram’s stain. We used the latter to assess the presence of Gram positive, lanceolate diplococci to confirm a diagnosis of pneumococcal meningitis.
Specimen preparation We incubated all plates, broths, and Bactec™ blood bottles at 37°C for 24 hours. We then assessed the plates as positive or negative for typical green colonies with surrounding green colouration of the medium indicating probable S. pneumoniae. In the case of a negative plate – i.e. no green colonies – we inspected the corresponding broth. If positive for growth, we streaked the broth on CHA, incubated for 24 hours and reassessed for green colonies. We performed the same process for positive brain-inoculated broths. After 24 hours, we sampled positive Bactec™ blood culture bottles for bacteria by plating on CHA, which was incubated for 24 hours to determine the presence of green colonies. Negative Bactec™ blood culture bottles were also sampled and plated on CHA. After incubation for 24 hours, we inspected the plates for green colonies. If the 24-hour sample plate was negative, we took another sample from the bottle and plated it as described above.
develop meningitis) and the event rate for a dexamethasone-eluting array was set at 0.7 (i.e. 7 in 10 animals develop meningitis). With an alpha of 0.05 and power of 0.8, this allowed a sample size of 9 for each group. We increased this number to 10 for convenience.
Results Phase I Around 12 hours following inoculation, we often observed swelling on the implanted side of the head around the jaw and ear. This facial swelling was observed only in rats inoculated with S. pneumoniae, though not all, and never in rats inoculated with PBS. All rats that went on to develop meningitis exhibited this initial swelling but not all rats with facial swelling developed meningitis. In those rats, the swelling often resolved 36–48 hours post-inoculation. Rats inoculated with PBS alone or with 103 CFU of S. pneumoniae did not develop meningitis as determined by histological examination of meningeal tissue for Gram positive lanceolate diplococci. Two of six rats in both groups inoculated with 104 CFU developed meningitis, as did five of six rats inoculated with 105 CFU (Table 2). Viable counts of the inocula showed the actual number of CFUs was consistent with the expected number: 103 CFU = 2.6 × 103 viable count 104 CFU = 2.4 × 104 viable count 105 CFU = 5.2 × 105 viable count 104 CFU (repeat) = 2.7 × 104 viable count.
We chose 104 CFU as the inoculum for phase II as the resultant 33% infection rate in those groups was closest to our desired infection rate of 20%.
Phase II Four of 10 rats implanted with the dexamethasoneeluting array developed meningitis (determined by the presence of Gram positive lanceolate diplococci in meningeal tissue) compared with 6 of 10 implanted with a control array (Table 2; P = 0.66; Fisher’s exact test, two-tailed). Viable counts of the inocula showed consistent colony counts over the three batches of surgery: Batch 1 = 3.5 × 104 CFU viable count Batch 2 = 1.7 × 104 CFU viable count Batch 3 = 3.4 × 104 CFU viable count.
Bacterial isolation from samples Statistical analysis We compared the test and control groups of phase II with Fisher’s exact test. Sample sizes for phase II were developed using a Fisher’s exact test for comparing two proportions, where the event rate for a standard array was set at 0.1 (i.e. 1 in 10 animals
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From each animal terminated with a morbidity score of 10 and later histologically confirmed as meningitic, we cultured distinctive green colonies typical of streptococci from the collected samples. From animals that remained healthy, no typical green colonies were isolated from the meninges,
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Table 2 Meningitis outcomes in phases I and II where (+) indicates a positive result for Gram positive lanceolate diplococci in meningeal tissue and hence a diagnosis of meningitis Phase treatment Group treatment
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Meningitis rate (%)
Phase II (104 CFU)
Phase I (control array) PBS
103 CFU
104 CFU
104 CFU
105 CFU
Control array
Dexamethasone-eluting array
− − − − − −
− − − − − −
+ + − − − −
+ + − − − −
+ + + + + −
0
0
33
33
83
+ + + + + + − − − − 60
+ + + + − − − − − − 40
brain, CSF, or blood cultures except from two animals in phase I, both inoculated with 104 CFU. From these two animals, we isolated green colonies from the middle ear of both, from the blood of one and the CSF of the other. We observed no evidence of meningitis in these two animals, neither in behaviour nor histologically. The middle ear swabs of several healthy S. pneumoniae-inoculated animals yielded green colonies. We did not isolate green colonies from any sample in the phase I group inoculated with PBS only.
Histology We identified Gram positive, lanceolate diplococci in the meninges of all animals terminated with a morbidity score of 10 (Fig. 2). This confirmed our diagnosis of meningitis. We saw no signs of bacterial infection in the tissue of any animal that remained healthy by the appointed termination day.
Discussion We found that implantation with a dexamethasoneeluting array had no effect on meningitis susceptibility compared with a control array, when bacterial inoculation occurred 5 days post-implant. Wei et al. (2007) demonstrated in the rat model that the presence of a cochlear implant decreases the
bacterial threshold required to induce pneumococcal meningitis regardless of inoculation route: haematogenous, inner, or middle ear. Reefhuis et al. (2003) studied perioperative cases of meningitis where bacteria were suspected to have been introduced during surgery, and sporadic cases where 40% of patients also presented with acute otitis media – the bacteria being introduced via the middle and inner ears. For these reasons, we chose to inoculate via the middle ear as the most relevant route for this particular study. We isolated typical green colonies from all samples in meningitic animals, but only from middle ear samples in some healthy animals indicating that S. pneumoniae had not reached the central nervous system nor the bloodstream in detectable numbers. Wei et al. (2006) found rats that did not develop meningitis had cleared the bacteria by 5 days after inoculation. During our study, we observed that rats inoculated with bacteria often presented with localized inflammation and swelling around the jaw and ear 12 hours post-inoculation, regardless of whether they later developed meningitis. We did not see swelling in animals inoculated with PBS or in any animal after the initial cochlear implant surgery. The clinical objective of reducing the growth of excess fibrous tissue and bone by suppressing the
Figure 2 Photomicrograph of Gram stained (A) healthy meningeal tissue and (B) inflamed meningeal tissue from an animal terminated with severe morbidity. An example of the Gram positive lanceolate diplococci found in infected animals is circled in B.
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inflammatory response is contrasted by the necessity for a robust immune system to protect against infection. A mature fibrous tissue seal has been thought to provide a physical protective barrier to the inner ear (Nadol and Eddington, 2004), though several studies have subsequently shown bacteria and inflammatory cells can infiltrate via the cochleostomy seal (Sudhoff, 2003; Wei et al., 2006, 2007). Inoculating guinea pigs with S. pneumoniae 5 weeks postimplant – ensuring a relatively mature tissue seal – Niedermeier et al. (2012) found no difference in the rate of meningitis infection between dexamethasoneeluting arrays and untreated groups. Our decision to inoculate in the early post-elution period was shaped primarily by our concern of a dexamethasone-affected immune response to the bacteria but also considered the longer post-implant period previously investigated in guinea pigs. We note our rat model represented normal cochlear anatomy and did not take into account patients at higher risk of meningitis such as those with history of meningitis, abnormal middle ear anatomy, and cranial trauma, or an immature immune system (Wei, 2007). Nevertheless, the inoculum we used resulted in meningitis in 33–60% of rats.
Conclusion A dexamethasone-eluting intracochlear electrode did not increase the risk of meningitis in rats inoculated with S. pneumoniae via the middle ear, 5 days following implantation.
Acknowledgements We would like to thank Michelle Stirling, Nicole Christie, Sue Pierce, and the staff of the Biological Research Centre, and Prudence Nielsen for histological work.
Disclaimer statements Contributors Dimitra Stathopoulos: design of experiment; acquisition, analysis and interpretation of data; drafting, revision and approval of final article. Scott Chambers: design of experiment; acquisition, analysis and interpretation of data; critical revision of article for content and accuracy; approval of final article. Louise Adams: design of experiment; interpretation of data; critical revision of article for content and accuracy. Roy Robins-Browne: design of experiment; interpretation of data; critical revision of article for content and accuracy. Christopher Miller: concept and design of experiment; critical revision of article for content and accuracy. Ya Lang Enke: concept and design of experiment; critical revision of article for content and accuracy. Ben Wei: design of experiment, critical revision of article for content and accuracy. Stephen O’Leary: design of experiment, critical
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revision of article for content and accuracy. Robert Cowan: design of experiment; critical revision of article for content and accuracy; approval of final article. Carrie Newbold: concept and design design of experiment; acquisition, analysis and interpretation of data; critical revision of article for content and accuracy; approval of final article. Funding We acknowledge the financial support of the HEARing CRC, established and supported under the Cooperative Research Centres Program, an Australian Government initiative. Conflicts of interest None. Ethics approval This study was approved by the Animal Research and Ethics Committee of the Royal Victorian Eye and Ear Hospital under the project number 11/242AR.
References Bas E., Dinh C.T., Garnham C., Polak M., Van de Water T.R. 2012. Conservation of hearing and protection of hair cells in cochlear implant patients’ with residual hearing. Anatomical RecordAdvances in Integrative Anatomy and Evolutionary Biology, 295: 1909–1927. Chang A., Eastwood H., Sly D., James D., Richardson R., O’Leary S. 2009. Factors influencing the efficacy of round window dexamethasone protection of residual hearing post-cochlear implant surgery. Hearing Research, 255: 67–72. Clark G.M., Shute S.A., Shepherd R.K., Carter T.D. 1995. Cochlear implantation: osteoneogenesis, electrode-tissue impedance, and residual hearing. Annals of Otology, Rhinology and Laryngology Supplement, 166: 40–42. Cohen N.L. 2004. Meningitis in cochlear implant recipients: the North American experience. Otology & Neurotology, 25: 275. Connolly T.M., Eastwood H., Kel G., Lisnichuk H., Richardson R., O’Leary S. 2010. Pre-operative intravenous dexamethasone prevents auditory threshold shift in a guinea pig model of cochlear implantation. Audiology and Neuro-Otology, 16: 137–144. Dahm M. 1994. Cochlear implantation in children: labyrinthitis following pneumococcal otitis media in unimplanted and implanted cat cochleas. Acta Otolaryngologica, 114(6): 620–625. Douchement D., Terranti A., Lamblin J., Salleron J., Siepmann F., Siepmann J., et al. 2015. Dexamethasone eluting electrodes for cochlear implantation: effect on residual hearing. Cochlear Implants International, 16(4): 195–200. Eastwood H., Chang A., Kel G., Sly D., Richardson R., O’Leary S.J. 2010. Round window delivery of dexamethasone ameliorates local and remote hearing loss produced by cochlear implantation into the second turn of the guinea pig cochlea. Hearing Research, 265: 25–29. Eshraghi A.A., Adil E., He J., Graves R., Balkany T.J., Van De Water T.R. 2007. Local dexamethasone therapy conserves hearing in an animal model of electrode insertion traumainduced hearing loss. Otology & Neurotology, 28: 842–849. Eshraghi A.A., Van De Water T.R. 2006. Cochlear implantation trauma and noise-induced hearing loss: apoptosis and therapeutic strategies. The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology, 288A: 473–481. Fabre T., Belloc F., Dupuy B., Schappacher M., Soum A., BertrandBarat J., et al. 1999. Polymorphonuclear cell apoptosis in exudates generated by polymers. Journal of Biomedical Material Research A, 44: 429–435. Food and Drug Administration. 2006. FDA public health Web notification: cochlear implant recipients may be at greater risk for meningitis. [viewed 2013 June 12]. Available from: http:// www.fda.gov/MedicalDevices/Safety/AlertsandNotices/Public HealthNotifications/ucm062104.htm. Huang C.Q., Tykocinski M., Stathopoulos D., Cowan R. 2007. Effects of steroids and lubricants on electrical impedance and
Downloaded by [University of California Santa Barbara] at 20:15 23 March 2016
Stathopoulos et al.
tissue response following cochlear implantation. Cochlear Implants International, 8: 123–147. Incerti P.V., Ching T.Y.C., Cowan R. 2013. A systematic review of electric-acoustic stimulation: device fitting ranges, outcomes, and clinical fitting practices. Trends in Amplification, 17: 3–26. James D.P., Eastwood H., Richardson R.T., O’Leary S.J. 2008. Effects of round window dexamethasone on residual hearing in a guinea pig model of cochlear implantation. Audiology and Neuro-Otology, 13: 86–96. Kastenbauer S., Pfister H.W. 2003. Pneumococcal meningitis in adults – spectrum of complications and prognostic factors in a series of 87 cases. Brain, 126: 1015–1025. Kiefer J., Gstoettner W., Baumgartner W., Pok S.M., Tillein J., Ye Q., et al. 2004. Conservation of low-frequency hearing in cochlear implantation. Acta Otolaryngologica, 124: 272–280. Lee J., Ismail H., Lee J.H., Kel G., O’Leary J., Eastwood H., et al. 2013. Effect of both local and systemically administered dexamethasone on long-term hearing and tissue response in a guinea pig model of cochlear implantation. Audiology and Neuro-Otology, 18: 392–405. Maini S., Lisnichuk H., Eastwood H., Pinder D., James D., Richardson R.T., et al. 2009. Targeted therapy of the inner ear. Audiology and Neuro-Otology, 14: 402–410. McFarland J. 1907. The Nephelometer: an instrument for estimating the number of bacteria in suspensions used for calculating the opsonic index and for vaccines. Journal of the American Medical Association, 49: 1176. Miles A.A., Misra S.S., Irwin J.O. 1938. The estimation of the bactericidal power of the blood. Journal of Hygiene (London), 38: 732–749. Nadol J.B., Eddington D.K. 2004. Histologic evaluation of the tissue seal and biologic response around cochlear implant electrodes in the human. Otology & Neurotology, 25: 257–262. National Health and Medical Research Council. 2013. Australian code of practice for the care and use of animals for scientific purposes. n.p. Canberra, Australia: NHMRC. Niedermeier K., Braun S., Fauser C., Kiefer J., Straubinger R.K., Stark T. 2012. A safety evaluation of dexamethasone-releasing cochlear implants: comparative study on the risk of otogenic meningitis after implantation. Acta Otolaryngologica, 132: 1252–1260. O’Leary S., Monksfield P., Kel G., Connolly T., Souter M., Chang A., et al. 2013. Relations between cochlear histopathology and hearing loss in experimental cochlear implantation. Hearing Research, 298: 27–35. Paasche G., Bogel L., Leinung M., Lenarz T., Stover T. 2006. Substance distribution in a cochlea model using different pump rates for cochlear implant drug delivery electrode prototypes. Hearing Research, 212: 74–82. Paasche G., Tasche C., Stover T., Lesinski-Schiedat A., Lenarz T. 2009. The long-term effects of modified electrode surfaces and intracochlear corticosteroids on postoperative impedances
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in cochlear implant patients. Otology & Neurotology, 30: 592–598. Reefhuis J., Honein M.A., Whitney C.G., Chamany S., Mann E.A., Biernath K.R., et al. 2003. Risk of bacterial meningitis in children with cochlear implants. New England Journal of Medicine, 349: 435–445. Simpson A., McDermott H.J., Dowell R.C., Sucher C., Briggs R.J. 2009. Comparison of two frequency-to-electrode maps for acoustic-electric stimulation. International Journal of Audiology, 48: 63–73. Stathopoulos D., Chambers S., Enke Y.L., Timbol G., Risi F., Miller C., et al. 2014. Development of a safe dexamethasoneeluting electrode array for cochlear implantation. Cochlear Implants International, 15(5): 254–263. Sudhoff H. 2003. Temporal bone fracture and latent meningitis: temporal bone histopathology study of the month. Otology & Neurotology, 24: 521. Tykocinski M., Cohen L.T., Cowan R.S. 2005. Measurement and analysis of access resistance and polarization impedance in cochlear implant recipients. Otology & Neurotology, 26: 948–956. van de Beek D., de Gans J., Spanjaard L., Weisfelt M., Reitsma J.B., Vermeulen M. 2004. Clinical features and prognostic factors in adults with bacterial meningitis. New England Journal of Medicine, 351: 1849–1859. van de Beek D., de Gans J., Tunkel A.R., Wijdicks E.F.M. 2006. Community-acquired bacterial meningitis in adults. New England Journal of Medicine, 354: 44–53. Vivero R.J., Joseph D.E., Angeli S., He J., Chen S., Eshraghi A.A., et al. 2008. Dexamethasone base conserves hearing from electrode trauma-induced hearing loss. Laryngoscope, 118: 2028–2035. Wei B.P. 2007. Pneumococcal meningitis post cochlear implantation: risk assessment and prevention. Thesis, University of Melbourne, Melbourne. Wei B.P., Shepherd R.K., Robins-Browne R.M., Clark G.M., O’Leary S.J. 2006. Pneumococcal meningitis: development of a new animal model. Otology & Neurotology, 27: 844–854. Wei B.P.C., Shepherd R.K., Robins-Browne R.M., Clark G.M., O’Leary S.J. 2007. Threshold shift: effects of cochlear implantation on the risk of pneumococcal meningitis. Otolaryngology – Head and Neck Surgery, 136: 589–596. Weisfelt M., van de Beek D., Spanjaard L., Reitsma J.B., de Gans J. 2006. Clinical features, complications, and outcome in adults with pneumococcal meningitis: a prospective case series. Lancet: Neurology, 5: 123–129. Zimmerli W., Lew P.D., Waldvogel F.A. 1984. Pathogenesis of foreign body infection. Evidence for a local granulocyte defect. Journal of Clinical Investigation, 73: 1191–1200. Zimmerli W., Waldvogel F.A., Vaudaux P., Nydegger U.E. 1982. Pathogenesis of foreign body infection: description and characteristics of an animal model. Journal of Infectious Disease, 146: 487–497.
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ISSN: 1467-0100 (Print) 1754-7628 (Online) Journal homepage: http://www.tandfonline.com/loi/ycii20
Meningitis and a safe dexamethasone-eluting intracochlear electrode array Dimitra Stathopoulos, Scott Chambers, Louise Adams, Roy Robins-Browne, Christopher Miller, Ya Lang Enke, Benjamin P C Wei, Stephen O'Leary, Robert Cowan & Carrie Newbold To cite this article: Dimitra Stathopoulos, Scott Chambers, Louise Adams, Roy Robins-Browne, Christopher Miller, Ya Lang Enke, Benjamin P C Wei, Stephen O'Leary, Robert Cowan & Carrie Newbold (2015) Meningitis and a safe dexamethasone-eluting intracochlear electrode array, Cochlear Implants International, 16:4, 201-207 To link to this article: http://dx.doi.org/10.1179/1754762814Y.0000000099
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Date: 23 March 2016, At: 20:15
Original research paper
Meningitis and a safe dexamethasone-eluting intracochlear electrode array Dimitra Stathopoulos 1,2, Scott Chambers 1, Louise Adams 3, Roy Robins-Browne 3, Christopher Miller4, Ya Lang Enke4, Benjamin P C Wei 2, Stephen O’Leary 2, Robert Cowan 1, Carrie Newbold 1,2
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1
The HEARing Cooperative Research Centre, Carlton, Victoria, Australia, 2Department of Otolaryngology, The University of Melbourne, East Melbourne, Victoria, Australia, 3Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Victoria, Australia, 4 Cochlear Limited at Macquarie University, Sydney, New South Wales, Australia Objectives: To evaluate the potential risk of pneumococcal meningitis associated with the use of a dexamethasone-eluting intracochlear electrode array as compared with a control array. Methods: In two phases, adult Hooded–Wistar rats were implanted via the middle ear with an intracochlear array and were inoculated with Streptococcus pneumoniae 5 days post-surgery. Phase I created a dosing curve by implanting five groups (n = 6) with a control array, then inoculating 5 days later with different numbers of S. pneumoniae: 0 CFU, 103 CFU, 104 CFU, 104 CFU repeated, or 105 CFU (colony forming units). A target infection rate of 20% was aimed for and 104 CFU was the closest to this target with 33% infection rate. In phase II, we implanted two groups (n = 10), one with a dexamethasone-eluting array, the other a control array, and both groups were inoculated with 104 CFU of S. pneumoniae 5 days post-surgery. Results: The dexamethasone-eluting array group had a 40% infection rate; the control array group had a 60% infection rate. This difference was not statistically significant with a P value of ≥0.5. Conclusion: The use of a dexamethasone-eluting intracochlear electrode array did not increase the risk of meningitis in rats when inoculated with S. pneumoniae via the middle ear 5 days following implantation. Keywords: Cochlear implant, Meningitis, Streptococcus pneumoniae, Dexamethasone, Corticosteroid, Drug delivery, Rat
Introduction Despite advances made in implant design, software, and surgical technique, cochlear implantation remains an invasive surgical procedure. The introduction of the electrode array into the cochlea initiates an inflammatory response within the cochlea that may lead to development of fibrous tissue or new bone formation, changes in electrical impedance, and a postoperative loss of residual hearing (Bas et al., 2012; Eshraghi and Van De Water, 2006; Kiefer et al., 2004; O’Leary et al., 2013; Huang et al., 2007; Tykocinski et al., 2005). Recent changes in candidature guidelines have meant that many prospective cochlear implant recipients have significant levels of residual acoustic hearing, and it has been shown that the use of acoustic and electric hearing in the same ear can provide significant benefits to speech and Correspondence to: Dimitra Stathopoulos, Department of Otolaryngology, The University of Melbourne, 32 Gisborne St. 2nd Flr. PHW, East Melbourne, Victoria 3002, Australia. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1754762814Y.0000000099
music perception (Incerti et al., 2013; Simpson et al., 2009). Given these findings, preservation of usable residual hearing is an important clinical goal. Anti-inflammatory steroids – such as the glucocorticoid dexamethasone – administered into the scala tympani during cochlear implantation can reduce inflammation and maintain electrical impedance levels (Clark et al., 1995; Paasche et al., 2006; Paasche et al., 2009). Animal studies have investigated residual hearing preservation using a single application of dexamethasone via either local or systemic delivery (Connolly et al., 2010; Eastwood et al., 2010; James et al., 2008; Maini et al., 2009; Chang et al., 2009; Lee et al., 2013). Of the various methods investigated, results have shown that dexamethasone application to the round window 2 hours before surgery provided the most significant benefit (Chang et al., 2009; Lee et al., 2013). Longer-term delivery via mini-osmotic pump has also been shown to sustain auditory brainstem response thresholds (Eshraghi et al., 2007; Vivero et al., 2008).
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Elution of dexamethasone from the surface of an implanted electrode array, if clinically feasible, would appear to be a simple solution for efficient delivery of the therapeutic directly to the site of intended action within the cochlea. Several studies have investigated the potential that such an array might have to reduce the inflammatory response within the cochlea and thereby preserve residual hearing thresholds (Douchement et al., 2015; Niedermeier et al., 2012; Stathopoulos et al., 2014). The foremost concern, however, is that steroid elution from an electrode array is safe and does not suppress the inner ear’s cellular defences against pathogens. In particular, bacterial meningitis is a significant known risk following cochlear implantation, especially for patients with predisposing risk factors such as a history of previous meningitis, head trauma, and inner ear malformation (Cohen, 2004; FDA, 2006; Reefhuis et al., 2003). Pneumococcal meningitis has a high fatality rate at 19–37% and up to 50% of survivors suffer long-term effects including hearing loss, seizures, and cognitive impairment (Kastenbauer and Pfister, 2003; van de Beek et al., 2006; van de Beek et al., 2004; Weisfelt et al., 2006). Previous designs of cochlear implant electrode array have, in the past, been associated with an increased risk of meningitis (FDA, 2006; Reefhuis et al., 2003), making it vital that new electrode array designs incorporating features such as steroid elution are proved safe before clinical application. In a rat model, Wei et al. (2007) showed that the presence of a cochlear implant reduced the threshold of bacteria required to induce pneumococcal meningitis while a cochleostomy alone did not, implying that the primary risk factor was the presence of the foreign body rather than the breach into the cochlea. Several studies have shown a foreign body (e.g. a cochlear implant) can decrease the functionality of inflammatory cells, thus lowering the threshold of bacteria required to induce infection (Fabre et al., 1999; Zimmerli et al., 1984; Zimmerli et al., 1982). This is sufficient justification to test that anti-inflammatories such as dexamethasone will not increase the risk of meningitis. Another consideration is the potential compromise of the development of a robust fibrous tissue seal. Although it is conjectured that development of a mature fibrous tissue seal protects the inner ear from infection, several animal and human studies have shown bacteria and inflammatory cells can track through the mature fibrous tissue sheath around the implanted electrode array (Sudhoff, 2003; Wei et al., 2006, 2007). Niedermeier et al.’s (2012) guinea pig study gave a 5-week recovery period for fibrous tissue seal formation before inoculation with Streptococcus pneumoniae. They found no difference
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in infection rate between dexamethasone-eluting arrays and untreated groups. These previous studies informed our decision to inoculate 5 days post-implantation with an immature tissue seal, as our main concern was the potential compromise of the cellular immune response to the bacteria in the early postelution period. In this study, we assessed the rate of pneumococcal meningitis in rats implanted with either a dexamethasone-eluting array or a non-eluting control array. We used the rat model developed by Wei et al. (2006) and inoculated the rats 5 days after implant with S. pneumoniae as this is the most common cause of meningitis in cochlear implant recipients (Reefhuis et al., 2003; Cohen, 2004).
Materials and methods This study was approved and conducted under the ethical oversight of the Animal Research and Ethics Committee of the Royal Victorian Eye and Ear Hospital under project number 11/242AR. It was conducted in accordance with the ‘Australian code of practice for the care and use of animals for scientific purposes’ (National Health and Medical Research Council, 2013).
Trial experimental designs We implanted silicone dummy arrays unilaterally into the cochleae of adult male Hooded–Wistar rats weighing between 200 and 350 g, followed 5 days later by middle ear inoculation with either a phosphate-buffered saline (PBS) control or S. pneumoniae. The study was split into two phases: Phase I In this phase, we created a dosing curve to determine the number of bacteria required to produce an infection rate of approximately 20% in animals implanted with a non-eluting control array. We allocated 30 rats to five groups (n = 6), each inoculated with 10 μl of PBS containing 103 CFU, 104 CFU, or 105 CFU (colony forming units), or with 10 μl PBS as a control. The fifth group was to receive a higher CFU concentration but, as the upper and lower limits of desired infection rate had been determined, we repeated the 104 CFU inoculum – the closest to our goal of a 20% infection rate – for a more robust result. Phase II In this phase, we allocated 20 rats to two groups (n = 10) to compare the meningitis rate in rats implanted with either a dexamethasone-eluting array or a noneluting control array. Both groups were inoculated with 10 μl of PBS containing 104 CFU of S. pneumoniae.
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Figure 1 Dimensions of the silicone rubber dummy array. Dexamethasone-eluting arrays were identical in size and shape. X shows the approximate location of the trim line, where the array was cut to fit into the bulla.
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Electrode arrays We used moulded silicone rubber dummy arrays, without electrodes or wires, for all implantations (Fig. 1). The arrays in phase I and the control arrays in phase II were silicone rubber only. The experimental arrays in phase II were moulded using silicone rubber mixed with 40% w/w dexamethasone base. All arrays were sterilized with ethylene oxide.
Cochlear implant surgery We performed all surgeries using strict aseptic technique. We induced anaesthesia with 2–3% isofluorane in oxygen and maintained it at 1.5–3% isofluorane at 3–4 l per minute of oxygen. We used local anaesthesia (2% lidocaine hydrochloride) at the incision site and gave analgesia (0.05 mg/kg buprenorphine, diluted 1:10) and antibiotic (10 mg/kg enrofloxacin) post-surgery. We used a post-auricular approach to expose the bulla and drilled a bullostomy to expose the middle ear and the cochlea. To access the cochleostomy site, we cauterized the stapedial artery – located across the round window niche of the cochlea – using a bipolar coagulator. We drilled a cochleostomy below the round window niche and implanted a silicone dummy array into the scala tympani to a depth of 2.5 mm. We then sealed the cochleostomy with fascia and sutured the wound with absorbable monofilament. The silicone dummy array was a non-eluting control for all animals except those in the dexamethasone group in phase II, which received dexamethasone-loaded arrays.
dilutions on chocolate blood agar (CHA) and incubating overnight at 37°C in air with 5% CO2.
Inoculation Five days after cochlear implant surgery, we re-opened the wound. We packed the middle ear with absorbable gelatin surgical sponge and, using a micro-syringe, injected 10 μl of inoculum (or PBS in the phase I control group) into the middle ear. We covered the bullostomy with fascia and re-sutured the wound.
Clinical monitoring After inoculation, we monitored the animals for signs of morbidity over a period of 5 days. We used Table 1, adapted from Wei (2007), to inform a point-based system of determining the time of euthanasia in cases of suspected meningitis. Once an animal scored 10 points or more, it was euthanized.
Specimen collection At termination, we anaesthetized the rats using a 2:1 mixture of ketamine hydrochloride (75 mg/kg) and xylazine hydrochloride (8 mg/kg). Under aseptic laboratory conditions, we exposed the bullostomy to collect middle ear fluid using a sterile needle. Table 1
Scoring guide to assess morbidity of rats
Factor
Score
Alertness
1 2 3
Inoculum We inoculated rats with S. pneumoniae strain 447A (type-2 capsular antigen), originally isolated from a child with meningitis (Dahm, 1994). Bacteria were incubated overnight on horse blood agar at 37°C in air with 5% CO2. Isolated colonies were emulsified in sterile PBS to a concentration of approximately 2–3 × 108 CFU/ml using McFarland equivalence turbidity standards (McFarland, 1907; Miles et al., 1938), and then serially diluted to give the required CFU in 10 μl of inoculum. The viable counts of the inocula were determined retrospectively by plating
Grooming
1 2
Posture
1 2
Movement
1 2 3
Skin temperature
1 2
Criteria Normal, spontaneous Tired but still responding to light, sound, and tactile stimuli Very lethargic with little sustained response to stimuli Normal Abnormal – e.g. scruffy fur, exudate around eyes and nose, porphyrin stains Normal Abnormal – e.g. hunched, curled, head tilt, isolation Normal gait and behaviour Spontaneous but hesitant, careful, slow movement Only with tactile stimuli, unable to move without stumbling or falling Normal >39°C
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Through cardiac puncture, we collected approximately 2 ml of blood, which we inoculated into a BD Bactec™ Peds Plus™/F medium bottle (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA). A lethal dose of pentobarbital sodium (120 mg/kg) was then injected intracardially. We collected cerebrospinal fluid (CSF) from the cisterna magna, incising the skin first to avoid surface contamination. We opened the skull, inspected the meninges for abnormalities, and then collected the dorsal meningeal layer for histological processing. We removed the brain to a sterile petri dish where we took a coronal section for histological processing and another section to incubate in heart infusion broth. We then used a sterile swab to sample the surface of the ventral meningeal layer before removing the tissue for histological processing. We plated the middle ear fluid, CSF, and meningeal swab samples onto CHA and inoculated any remaining fluid into brain heart infusion broth. We embedded brain and meningeal tissue samples in paraffin wax for sectioning. Sections were stained with haematoxylin and eosin, and with Gram’s stain. We used the latter to assess the presence of Gram positive, lanceolate diplococci to confirm a diagnosis of pneumococcal meningitis.
Specimen preparation We incubated all plates, broths, and Bactec™ blood bottles at 37°C for 24 hours. We then assessed the plates as positive or negative for typical green colonies with surrounding green colouration of the medium indicating probable S. pneumoniae. In the case of a negative plate – i.e. no green colonies – we inspected the corresponding broth. If positive for growth, we streaked the broth on CHA, incubated for 24 hours and reassessed for green colonies. We performed the same process for positive brain-inoculated broths. After 24 hours, we sampled positive Bactec™ blood culture bottles for bacteria by plating on CHA, which was incubated for 24 hours to determine the presence of green colonies. Negative Bactec™ blood culture bottles were also sampled and plated on CHA. After incubation for 24 hours, we inspected the plates for green colonies. If the 24-hour sample plate was negative, we took another sample from the bottle and plated it as described above.
develop meningitis) and the event rate for a dexamethasone-eluting array was set at 0.7 (i.e. 7 in 10 animals develop meningitis). With an alpha of 0.05 and power of 0.8, this allowed a sample size of 9 for each group. We increased this number to 10 for convenience.
Results Phase I Around 12 hours following inoculation, we often observed swelling on the implanted side of the head around the jaw and ear. This facial swelling was observed only in rats inoculated with S. pneumoniae, though not all, and never in rats inoculated with PBS. All rats that went on to develop meningitis exhibited this initial swelling but not all rats with facial swelling developed meningitis. In those rats, the swelling often resolved 36–48 hours post-inoculation. Rats inoculated with PBS alone or with 103 CFU of S. pneumoniae did not develop meningitis as determined by histological examination of meningeal tissue for Gram positive lanceolate diplococci. Two of six rats in both groups inoculated with 104 CFU developed meningitis, as did five of six rats inoculated with 105 CFU (Table 2). Viable counts of the inocula showed the actual number of CFUs was consistent with the expected number: 103 CFU = 2.6 × 103 viable count 104 CFU = 2.4 × 104 viable count 105 CFU = 5.2 × 105 viable count 104 CFU (repeat) = 2.7 × 104 viable count.
We chose 104 CFU as the inoculum for phase II as the resultant 33% infection rate in those groups was closest to our desired infection rate of 20%.
Phase II Four of 10 rats implanted with the dexamethasoneeluting array developed meningitis (determined by the presence of Gram positive lanceolate diplococci in meningeal tissue) compared with 6 of 10 implanted with a control array (Table 2; P = 0.66; Fisher’s exact test, two-tailed). Viable counts of the inocula showed consistent colony counts over the three batches of surgery: Batch 1 = 3.5 × 104 CFU viable count Batch 2 = 1.7 × 104 CFU viable count Batch 3 = 3.4 × 104 CFU viable count.
Bacterial isolation from samples Statistical analysis We compared the test and control groups of phase II with Fisher’s exact test. Sample sizes for phase II were developed using a Fisher’s exact test for comparing two proportions, where the event rate for a standard array was set at 0.1 (i.e. 1 in 10 animals
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From each animal terminated with a morbidity score of 10 and later histologically confirmed as meningitic, we cultured distinctive green colonies typical of streptococci from the collected samples. From animals that remained healthy, no typical green colonies were isolated from the meninges,
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Table 2 Meningitis outcomes in phases I and II where (+) indicates a positive result for Gram positive lanceolate diplococci in meningeal tissue and hence a diagnosis of meningitis Phase treatment Group treatment
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Meningitis rate (%)
Phase II (104 CFU)
Phase I (control array) PBS
103 CFU
104 CFU
104 CFU
105 CFU
Control array
Dexamethasone-eluting array
− − − − − −
− − − − − −
+ + − − − −
+ + − − − −
+ + + + + −
0
0
33
33
83
+ + + + + + − − − − 60
+ + + + − − − − − − 40
brain, CSF, or blood cultures except from two animals in phase I, both inoculated with 104 CFU. From these two animals, we isolated green colonies from the middle ear of both, from the blood of one and the CSF of the other. We observed no evidence of meningitis in these two animals, neither in behaviour nor histologically. The middle ear swabs of several healthy S. pneumoniae-inoculated animals yielded green colonies. We did not isolate green colonies from any sample in the phase I group inoculated with PBS only.
Histology We identified Gram positive, lanceolate diplococci in the meninges of all animals terminated with a morbidity score of 10 (Fig. 2). This confirmed our diagnosis of meningitis. We saw no signs of bacterial infection in the tissue of any animal that remained healthy by the appointed termination day.
Discussion We found that implantation with a dexamethasoneeluting array had no effect on meningitis susceptibility compared with a control array, when bacterial inoculation occurred 5 days post-implant. Wei et al. (2007) demonstrated in the rat model that the presence of a cochlear implant decreases the
bacterial threshold required to induce pneumococcal meningitis regardless of inoculation route: haematogenous, inner, or middle ear. Reefhuis et al. (2003) studied perioperative cases of meningitis where bacteria were suspected to have been introduced during surgery, and sporadic cases where 40% of patients also presented with acute otitis media – the bacteria being introduced via the middle and inner ears. For these reasons, we chose to inoculate via the middle ear as the most relevant route for this particular study. We isolated typical green colonies from all samples in meningitic animals, but only from middle ear samples in some healthy animals indicating that S. pneumoniae had not reached the central nervous system nor the bloodstream in detectable numbers. Wei et al. (2006) found rats that did not develop meningitis had cleared the bacteria by 5 days after inoculation. During our study, we observed that rats inoculated with bacteria often presented with localized inflammation and swelling around the jaw and ear 12 hours post-inoculation, regardless of whether they later developed meningitis. We did not see swelling in animals inoculated with PBS or in any animal after the initial cochlear implant surgery. The clinical objective of reducing the growth of excess fibrous tissue and bone by suppressing the
Figure 2 Photomicrograph of Gram stained (A) healthy meningeal tissue and (B) inflamed meningeal tissue from an animal terminated with severe morbidity. An example of the Gram positive lanceolate diplococci found in infected animals is circled in B.
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inflammatory response is contrasted by the necessity for a robust immune system to protect against infection. A mature fibrous tissue seal has been thought to provide a physical protective barrier to the inner ear (Nadol and Eddington, 2004), though several studies have subsequently shown bacteria and inflammatory cells can infiltrate via the cochleostomy seal (Sudhoff, 2003; Wei et al., 2006, 2007). Inoculating guinea pigs with S. pneumoniae 5 weeks postimplant – ensuring a relatively mature tissue seal – Niedermeier et al. (2012) found no difference in the rate of meningitis infection between dexamethasoneeluting arrays and untreated groups. Our decision to inoculate in the early post-elution period was shaped primarily by our concern of a dexamethasone-affected immune response to the bacteria but also considered the longer post-implant period previously investigated in guinea pigs. We note our rat model represented normal cochlear anatomy and did not take into account patients at higher risk of meningitis such as those with history of meningitis, abnormal middle ear anatomy, and cranial trauma, or an immature immune system (Wei, 2007). Nevertheless, the inoculum we used resulted in meningitis in 33–60% of rats.
Conclusion A dexamethasone-eluting intracochlear electrode did not increase the risk of meningitis in rats inoculated with S. pneumoniae via the middle ear, 5 days following implantation.
Acknowledgements We would like to thank Michelle Stirling, Nicole Christie, Sue Pierce, and the staff of the Biological Research Centre, and Prudence Nielsen for histological work.
Disclaimer statements Contributors Dimitra Stathopoulos: design of experiment; acquisition, analysis and interpretation of data; drafting, revision and approval of final article. Scott Chambers: design of experiment; acquisition, analysis and interpretation of data; critical revision of article for content and accuracy; approval of final article. Louise Adams: design of experiment; interpretation of data; critical revision of article for content and accuracy. Roy Robins-Browne: design of experiment; interpretation of data; critical revision of article for content and accuracy. Christopher Miller: concept and design of experiment; critical revision of article for content and accuracy. Ya Lang Enke: concept and design of experiment; critical revision of article for content and accuracy. Ben Wei: design of experiment, critical revision of article for content and accuracy. Stephen O’Leary: design of experiment, critical
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revision of article for content and accuracy. Robert Cowan: design of experiment; critical revision of article for content and accuracy; approval of final article. Carrie Newbold: concept and design design of experiment; acquisition, analysis and interpretation of data; critical revision of article for content and accuracy; approval of final article. Funding We acknowledge the financial support of the HEARing CRC, established and supported under the Cooperative Research Centres Program, an Australian Government initiative. Conflicts of interest None. Ethics approval This study was approved by the Animal Research and Ethics Committee of the Royal Victorian Eye and Ear Hospital under the project number 11/242AR.
References Bas E., Dinh C.T., Garnham C., Polak M., Van de Water T.R. 2012. Conservation of hearing and protection of hair cells in cochlear implant patients’ with residual hearing. Anatomical RecordAdvances in Integrative Anatomy and Evolutionary Biology, 295: 1909–1927. Chang A., Eastwood H., Sly D., James D., Richardson R., O’Leary S. 2009. Factors influencing the efficacy of round window dexamethasone protection of residual hearing post-cochlear implant surgery. Hearing Research, 255: 67–72. Clark G.M., Shute S.A., Shepherd R.K., Carter T.D. 1995. Cochlear implantation: osteoneogenesis, electrode-tissue impedance, and residual hearing. Annals of Otology, Rhinology and Laryngology Supplement, 166: 40–42. Cohen N.L. 2004. Meningitis in cochlear implant recipients: the North American experience. Otology & Neurotology, 25: 275. Connolly T.M., Eastwood H., Kel G., Lisnichuk H., Richardson R., O’Leary S. 2010. Pre-operative intravenous dexamethasone prevents auditory threshold shift in a guinea pig model of cochlear implantation. Audiology and Neuro-Otology, 16: 137–144. Dahm M. 1994. Cochlear implantation in children: labyrinthitis following pneumococcal otitis media in unimplanted and implanted cat cochleas. Acta Otolaryngologica, 114(6): 620–625. Douchement D., Terranti A., Lamblin J., Salleron J., Siepmann F., Siepmann J., et al. 2015. Dexamethasone eluting electrodes for cochlear implantation: effect on residual hearing. Cochlear Implants International, 16(4): 195–200. Eastwood H., Chang A., Kel G., Sly D., Richardson R., O’Leary S.J. 2010. Round window delivery of dexamethasone ameliorates local and remote hearing loss produced by cochlear implantation into the second turn of the guinea pig cochlea. Hearing Research, 265: 25–29. Eshraghi A.A., Adil E., He J., Graves R., Balkany T.J., Van De Water T.R. 2007. Local dexamethasone therapy conserves hearing in an animal model of electrode insertion traumainduced hearing loss. Otology & Neurotology, 28: 842–849. Eshraghi A.A., Van De Water T.R. 2006. Cochlear implantation trauma and noise-induced hearing loss: apoptosis and therapeutic strategies. The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology, 288A: 473–481. Fabre T., Belloc F., Dupuy B., Schappacher M., Soum A., BertrandBarat J., et al. 1999. Polymorphonuclear cell apoptosis in exudates generated by polymers. Journal of Biomedical Material Research A, 44: 429–435. Food and Drug Administration. 2006. FDA public health Web notification: cochlear implant recipients may be at greater risk for meningitis. [viewed 2013 June 12]. Available from: http:// www.fda.gov/MedicalDevices/Safety/AlertsandNotices/Public HealthNotifications/ucm062104.htm. Huang C.Q., Tykocinski M., Stathopoulos D., Cowan R. 2007. Effects of steroids and lubricants on electrical impedance and
Downloaded by [University of California Santa Barbara] at 20:15 23 March 2016
Stathopoulos et al.
tissue response following cochlear implantation. Cochlear Implants International, 8: 123–147. Incerti P.V., Ching T.Y.C., Cowan R. 2013. A systematic review of electric-acoustic stimulation: device fitting ranges, outcomes, and clinical fitting practices. Trends in Amplification, 17: 3–26. James D.P., Eastwood H., Richardson R.T., O’Leary S.J. 2008. Effects of round window dexamethasone on residual hearing in a guinea pig model of cochlear implantation. Audiology and Neuro-Otology, 13: 86–96. Kastenbauer S., Pfister H.W. 2003. Pneumococcal meningitis in adults – spectrum of complications and prognostic factors in a series of 87 cases. Brain, 126: 1015–1025. Kiefer J., Gstoettner W., Baumgartner W., Pok S.M., Tillein J., Ye Q., et al. 2004. Conservation of low-frequency hearing in cochlear implantation. Acta Otolaryngologica, 124: 272–280. Lee J., Ismail H., Lee J.H., Kel G., O’Leary J., Eastwood H., et al. 2013. Effect of both local and systemically administered dexamethasone on long-term hearing and tissue response in a guinea pig model of cochlear implantation. Audiology and Neuro-Otology, 18: 392–405. Maini S., Lisnichuk H., Eastwood H., Pinder D., James D., Richardson R.T., et al. 2009. Targeted therapy of the inner ear. Audiology and Neuro-Otology, 14: 402–410. McFarland J. 1907. The Nephelometer: an instrument for estimating the number of bacteria in suspensions used for calculating the opsonic index and for vaccines. Journal of the American Medical Association, 49: 1176. Miles A.A., Misra S.S., Irwin J.O. 1938. The estimation of the bactericidal power of the blood. Journal of Hygiene (London), 38: 732–749. Nadol J.B., Eddington D.K. 2004. Histologic evaluation of the tissue seal and biologic response around cochlear implant electrodes in the human. Otology & Neurotology, 25: 257–262. National Health and Medical Research Council. 2013. Australian code of practice for the care and use of animals for scientific purposes. n.p. Canberra, Australia: NHMRC. Niedermeier K., Braun S., Fauser C., Kiefer J., Straubinger R.K., Stark T. 2012. A safety evaluation of dexamethasone-releasing cochlear implants: comparative study on the risk of otogenic meningitis after implantation. Acta Otolaryngologica, 132: 1252–1260. O’Leary S., Monksfield P., Kel G., Connolly T., Souter M., Chang A., et al. 2013. Relations between cochlear histopathology and hearing loss in experimental cochlear implantation. Hearing Research, 298: 27–35. Paasche G., Bogel L., Leinung M., Lenarz T., Stover T. 2006. Substance distribution in a cochlea model using different pump rates for cochlear implant drug delivery electrode prototypes. Hearing Research, 212: 74–82. Paasche G., Tasche C., Stover T., Lesinski-Schiedat A., Lenarz T. 2009. The long-term effects of modified electrode surfaces and intracochlear corticosteroids on postoperative impedances
Meningitis and intracochlear electrode array
in cochlear implant patients. Otology & Neurotology, 30: 592–598. Reefhuis J., Honein M.A., Whitney C.G., Chamany S., Mann E.A., Biernath K.R., et al. 2003. Risk of bacterial meningitis in children with cochlear implants. New England Journal of Medicine, 349: 435–445. Simpson A., McDermott H.J., Dowell R.C., Sucher C., Briggs R.J. 2009. Comparison of two frequency-to-electrode maps for acoustic-electric stimulation. International Journal of Audiology, 48: 63–73. Stathopoulos D., Chambers S., Enke Y.L., Timbol G., Risi F., Miller C., et al. 2014. Development of a safe dexamethasoneeluting electrode array for cochlear implantation. Cochlear Implants International, 15(5): 254–263. Sudhoff H. 2003. Temporal bone fracture and latent meningitis: temporal bone histopathology study of the month. Otology & Neurotology, 24: 521. Tykocinski M., Cohen L.T., Cowan R.S. 2005. Measurement and analysis of access resistance and polarization impedance in cochlear implant recipients. Otology & Neurotology, 26: 948–956. van de Beek D., de Gans J., Spanjaard L., Weisfelt M., Reitsma J.B., Vermeulen M. 2004. Clinical features and prognostic factors in adults with bacterial meningitis. New England Journal of Medicine, 351: 1849–1859. van de Beek D., de Gans J., Tunkel A.R., Wijdicks E.F.M. 2006. Community-acquired bacterial meningitis in adults. New England Journal of Medicine, 354: 44–53. Vivero R.J., Joseph D.E., Angeli S., He J., Chen S., Eshraghi A.A., et al. 2008. Dexamethasone base conserves hearing from electrode trauma-induced hearing loss. Laryngoscope, 118: 2028–2035. Wei B.P. 2007. Pneumococcal meningitis post cochlear implantation: risk assessment and prevention. Thesis, University of Melbourne, Melbourne. Wei B.P., Shepherd R.K., Robins-Browne R.M., Clark G.M., O’Leary S.J. 2006. Pneumococcal meningitis: development of a new animal model. Otology & Neurotology, 27: 844–854. Wei B.P.C., Shepherd R.K., Robins-Browne R.M., Clark G.M., O’Leary S.J. 2007. Threshold shift: effects of cochlear implantation on the risk of pneumococcal meningitis. Otolaryngology – Head and Neck Surgery, 136: 589–596. Weisfelt M., van de Beek D., Spanjaard L., Reitsma J.B., de Gans J. 2006. Clinical features, complications, and outcome in adults with pneumococcal meningitis: a prospective case series. Lancet: Neurology, 5: 123–129. Zimmerli W., Lew P.D., Waldvogel F.A. 1984. Pathogenesis of foreign body infection. Evidence for a local granulocyte defect. Journal of Clinical Investigation, 73: 1191–1200. Zimmerli W., Waldvogel F.A., Vaudaux P., Nydegger U.E. 1982. Pathogenesis of foreign body infection: description and characteristics of an animal model. Journal of Infectious Disease, 146: 487–497.
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