Direct SARS-CoV-2 infection of the human inner ear may underlie COVID-19-associated audiovestibular dysfunction

Background COVID-19 is a pandemic respiratory and vascular disease caused by SARS-CoV-2 virus. There is a growing number of sensory deficits associated with COVID-19 and molecular mechanisms underlying these deficits are incompletely understood. Methods We report a series of ten COVID-19 patients with audiovestibular symptoms such as hearing loss, vestibular dysfunction and tinnitus. To investigate the causal relationship between SARS-CoV-2 and audiovestibular dysfunction, we examine human inner ear tissue, human inner ear in vitro cellular models, and mouse inner ear tissue. Results We demonstrate that adult human inner ear tissue co-expresses the angiotensin-converting enzyme 2 (ACE2) receptor for SARS-CoV-2 virus, and the transmembrane protease serine 2 (TMPRSS2) and FURIN cofactors required for virus entry. Furthermore, hair cells and Schwann cells in explanted human vestibular tissue can be infected by SARS-CoV-2, as demonstrated by confocal microscopy. We establish three human induced pluripotent stem cell (hiPSC)-derived in vitro models of the inner ear for infection: two-dimensional otic prosensory cells (OPCs) and Schwann cell precursors (SCPs), and three-dimensional inner ear organoids. Both OPCs and SCPs express ACE2, TMPRSS2, and FURIN, with lower ACE2 and FURIN expression in SCPs. OPCs are permissive to SARS-CoV-2 infection; lower infection rates exist in isogenic SCPs. The inner ear organoids show that hair cells express ACE2 and are targets for SARS-CoV-2. Conclusions Our results provide mechanistic explanations of audiovestibular dysfunction in COVID-19 patients and introduce hiPSC-derived systems for studying infectious human otologic disease.

the right side (Fig. 1a). He was started on a 60 mg prednisone burst for 4 days, with taper (40 mg PO for 4 days, 20 mg PO for 2 days, 10 mg PO for 2 days). COVID-19 testing was ordered (based on qRT-PCR of a nasopharyngeal swab) and came back positive. Subsequent MRI demonstrated mild maxillary and ethmoid thickening, and normal 7 th and 8 th nerve complexes.
During a one month, telemedicine follow up, the patient reported no change in his unilateral hearing loss and tinnitus, and no additional symptoms. Three months after hearing loss onset, audiometric testing showed slightly worse hearing loss in the low and mid frequencies with recovery in the high frequencies (Fig. 1a). The patient opted against receiving an intratympanic dexamethasone injection.

Patient #3
A 44-year-old Latin-American man with previous history of alcohol-induced dizziness presented with severe vertigo. No associated fever, anosmia, or sore throat. His vertigo was associated with fluctuating sense of imbalance and nausea which coincided with recent airplane travel over a 3-week period. At a 7-day follow up, the patient describes worsening nausea and dizziness, and the onset of tinnitus and hearing loss in the left ear. Audiometric testing revealed bilateral asymmetric sensorineural hearing loss, worse on the left side (Fig. 1a). He was found to have severe gait instability and right beating nystagmus. No facial weakness. He started with vestibular physical therapy and began on a 60mg prednisone burst with anticipated taper after 10 days. Subsequent MRI and MRA did not reveal any cause for the audiovestibular symptoms. Two-weeks later he began experiencing myalgias, anosmia, cough, sore throat, fever, and ultimately tested positive for COVID-19 (based on qRT-PCR of a nasopharyngeal swab). During a telemedicine visit three days after COVID diagnosis, he complained of left ear pain without an obvious change in hearing. There were no skin lesions or facial weakness. He was started on oral cefpodoxime. During a follow up visit 4 months later he reported his balance was almost fully restored and he did not have vertigo. Ongoing left-sided, high-pitched nonpulsatile tinnitus has persisted and he noted no noticeable difference in hearing between the ears. Objectively, there was mild improvement in the left ear from the initial to the 4 month follow up audiogram with persistent high frequency hearing loss, worse in the left ear (Fig. 1a).

Patient #4
A 68-year-old black female with no previous medical history other than fibrocystic breast disease presented with sore throat, dyspnea, cough, and fever. She had no associated hearing loss or anosmia. COVID-19 testing was positive (based on qRT-PCR of a nasopharyngeal swab). She was started on doxycycline and albuterol before returning to the emergency department 3 days later with worsening dyspnea, cough, and fever. She was admitted for supportive treatment and intubated after 5 days due to progressive tachypnea and hypoxia. She was extubated after 12 days and remained in the hospital for additional supportive care for 2 more weeks until discharged. She did not receive any ototoxic medications while hospitalized.
She was first evaluated for sudden hearing loss and right buzzing tinnitus almost 2 months after being taken off the ventilator. Physical examination revealed normal appearing ear canals and tympanic membranes. Audiometric testing showed profound right-sided SNHL (Fig. 1a). She returned 7 days later with sustained vertigo that lasted approximately 10-14 days.
Subsequent MRI of the posterior fossa excluded any retrocochlear pathology to account for her hearing loss and vertigo. During a follow up examination, around 3 and half months after initial sudden SNHL, her hearing showed no improvement (Fig. 1a). Hearing in the contralateral ear also showed no change or decline. A few weeks after follow-up, quantitative IgG testing resulted in a positive antibody concentration.

Patient #5
A 31 year-old white previously healthy Talmud student with no prior otologic history and no active medical problems presented with a sudden right-sided hearing loss. He and his family (wife and two children) were infected with COVID-19 as were many in their Orthodox Jewish community. For the first 10 days before the sudden hearing loss, he experienced what he described as "extreme weakness and fatigue." He was febrile with a temperature of 101°F (38.3°C). He was symptom-free for one day and then he developed a fever of 102°F (38.9°C) and recurrence of his fatigue and weakness. Two days later he experienced right-sided hearing loss, tinnitus, gravitational receptor (otolithic) dysfunction type of vertigo, ageusia and anosmia.
He also experienced chest tightness, but no dyspnea. He did not experience true rotational vertigo. While inpatient COVID-19 testing was available at the time of his infection, outpatient COVID-19 testing was not yet available. Physical examination revealed normal appearing ear canals and tympanic membranes with aerated middle ears bilaterally. His audiogram demonstrated normal left ear hearing and profound right sensorineural hearing loss. He was started on 40 mg prednisone, tapering by 10 mg every three days. Subsequent MRI of the internal auditory canals revealed no retrocochlear pathology to explain his hearing loss. Ten days after starting on prednisone, he received intratympanic dexamethasone (24 mg/mL). There was no response to the oral and intratympanic corticosteroids (Fig. 1a). One month after the onset of his symptoms, COVID-19 testing, based on qRT-PCR of a nasopharyngeal swab, was negative. Two months after the onset of his symptoms COVID-19 quantitative IgG testing showed a concentration of 85.1 AU/mL (normal range 0-15.0 AU/mL). There was no improvement of his profound hearing loss or tinnitus and no improvement of his aguesia and anosmia. He perceived no chronic vestibular dysfunction.

Patient #6
A 22 year-old previously healthy white woman with no prior otologic history and no active medical problems presented with a sudden right-sided hearing loss and rotational vertigo. She and her family (both parents and three siblings) were infected with COVID-19 as were many in their Orthodox Jewish community. For the first 6 days before the sudden hearing loss, tinnitus and vertigo, she experienced what she described as "extreme fatigue and headache." She was febrile with a temperature of 103°F (39.4°C). On the seventh day of her illness she experienced right-sided hearing loss, tinnitus, and rotational vertigo. She also experienced mild dyspnea and coughing. The rotational vertigo was unrelenting and lasted two days. Aside from her hearing loss and tinnitus, her symptoms resolved on day 12. Physical examination revealed normal appearing ear canals and tympanic membranes with aerated middle ears bilaterally. She was started on 40 mg prednisone, tapering by 10 mg every three days. Intratympanic dexamethasone injection was offered, but she declined. Her audiometry revealed a right-sided profound hearing loss with no measurable speech discrimination ability (Fig. 1a). While inpatient COVID-19 testing was available at the time of her infection, outpatient COVID-19 testing was not yet available. Subsequent MRI of the internal auditory canals revealed no retrocochlear pathology to explain her sudden hearing loss, tinnitus and vertigo. There was no response to the oral corticosteroids. Nine weeks after the onset of her symptoms, COVID-19 testing, based on qRT-PCR of a nasopharyngeal swab, was negative and COVID-19 quantitative IgG testing showed a concentration of 93.4 AU/mL (normal range 0-15.0 AU/mL). There was no improvement of her hearing loss or tinnitus. She perceived no chronic vestibular dysfunction.

Patient #7
A 27 year-old previously healthy white man living in New York City with no prior otologic history and no active medical problems presented with a history of recent sudden left-sided hearing loss and rotational vertigo. For the first 6 days before the sudden hearing loss, tinnitus and vertigo, he experienced what he described as "extreme fatigue with intense headache." He had no ageusia or anosmia. He was febrile with a temperature of 102°F (38.9°C) and experienced repeated shaking and chills. COVID-19 testing, using a qRT-PCR of a nasopharyngeal swab sample, was positive. On the seventh day of his illness he experienced worsening left-sided hearing loss, tinnitus, and rotational vertigo. He had no facial nerve dysfunction. He also experienced mild dyspnea and coughing. The rotational vertigo was unrelenting and lasted two days. Physical examination revealed normal appearing ear canals and tympanic membranes with aerated middle ears bilaterally. Subsequent MRI of the internal auditory canals revealed diffuse contrast uptake and enhancement of the left vestibule, cochlear and vestibular nerves, geniculate ganglion and facial nerve (Fig. 1b). He was started on 60 mg prednisone, tapering by 10 mg every three days. Intratympanic dexamethasone injection was offered, but he declined.
Aside from his hearing loss and tinnitus (Fig. 1a), his symptoms resolved on day 12. Initially, there was no response to the oral corticosteroids. At 4 months follow-up his audiogram showed recovery of his hearing loss (Fig. 1a) and he perceived no vestibular dysfunction.

Patient #8
A 72 year-old previously healthy retired white woman presented with bilateral sudden hearing loss and tinnitus. She had several medical problems including essential hypertension, glaucoma and gastroesophageal reflux. For the first 5 days before the sudden hearing loss and tinnitus, she experienced what she described as "extreme fatigue." She was afebrile throughout her COVID-19 infection. On the fifth day of her illness she experienced bilateral sudden hearing loss and tinnitus. She had no dyspnea, coughing, headache, gastroesophageal disturbance, ageusia or anosmia. Aside from her hearing loss and tinnitus, her fatigue resolved on day 12. Physical examination revealed normal appearing ear canals and tympanic membranes with aerated middle ears bilaterally. She was started on azithromycin 500 mg initially then 250 mg each day for 4 days, and 60 mg prednisone for 7 days, then tapering by 10 mg every day. Intratympanic dexamethasone injection was offered, but she declined. Her initial audiometry revealed bilateral downsloping severe to profound hearing loss with impaired speech discrimination ability (Fig.   1a). Subsequent MRI of the internal auditory canals revealed no retrocochlear pathology to explain her bilateral sudden hearing loss and tinnitus. There was no response to the oral corticosteroids and azithromycin. One month after the onset of her symptoms, COVID-19 quantitative IgG testing was positive. At 4 months after her infection there was mild improvement of her auditory thresholds and no change in her speech discrimination ability (Fig.   1a).

Patient #9
A 46 year-old previously healthy Asian Indian woman with no prior otologic history and no active medical problems presented with a sudden left-sided high frequency hearing loss and tinnitus.
Her parents both contracted COVID-19, confirmed by PCR testing. She moved into her parent's home to care for them and 7 days later developed cough and influenza-like upper respiratory tract infection symptoms consistent with COVID-19 infection. She had no anosmia or dysgeusia.
Because her parents had PCR confirmed COVID-19 infection, she did not leave their home while ill for COVID-19 testing. On day 7 of her infection she developed left-sided sudden hearing loss and tinnitus. She also felt left aural pressure that was not relieved by performing the Valsalva maneuver. Her audiogram (Fig. 1a) showed a high frequency sensorineural hearing loss. She was started on oral prednisone 50 mg daily for 3 days, followed by 10 mg taper every 3 days. She was offered intratympanic dexamethasone, but declined this intervention. There was no response to the oral glucocorticoid. Magnetic resonance imaging of the internal auditory canals revealed no retrocochlear pathology to explain her sudden hearing loss and tinnitus.
Nasopharyngeal swab SARS-CoV-2 PCR testing was negative 14 days after her initial symptoms. Her SARS-CoV-2 IgG antibody testing was positive two months after her initial COVID-19 symptoms. Audiometric testing two months after her initial testing showed no improvement or worsening of her hearing (Fig. 1a).

Patient #10
A 61 year-old previously healthy white man with no prior otologic history and no active medical problems presented with right aural pressure, increased tinnitus and episodic true rotational vertigo. His father-in-law contracted COVID-19 while living in a nursing home. His family decided to take him into their home to care for him. Three days after his father-in-law passed away, the patient, his wife and two adult children all became ill. Initially, the patient experienced extreme fatigue and a sense of heaviness. He was mildly febrile with a temperature of 100°F (37.8°C). COVID-19 testing, using a qRT-PCR of a nasopharyngeal swab sample, was positive.
By the fifth day he began experiencing labored breathing and a sensation in his chest that lasted two weeks. He had no anosmia or dysgeusia. Three weeks after the onset of his COVID-19 symptoms he also felt right aural pressure that was not relieved by performing the Valsalva maneuver. He did not perceive a change of his hearing, but his right tinnitus amplitude increased and became bothersome to him. He then had his first episode of true rotational vertigo, nausea and vomiting that lasted 3 hours. His audiogram (Fig. 1a) showed bilateral, right greater than left, down-sloping sensorineural hearing loss. Comparison to a pre-COVID-19 audiogram revealed that he had additional bilateral sensorineural hearing loss. The right aural pressure, tinnitus and episodic true rotational vertigo persisted. He had mild reduction of these symptoms with lowering his daily sodium intake to 1,500-2,000 mg. He was started on oral prednisone 60 mg each day for 3 days and tapering the dose by 10 mg every 3 days. There was a positive response to the oral corticosteroids. During the first 2 weeks of this 18 day course, he noted reduction of his aural pressure and tinnitus with decreased frequency and intensity of his episodic true rotational vertigo. However, as his corticosteroid dose declined his aural pressure and tinnitus increased again. Subsequent magnetic resonance imaging (MRI) of the internal auditory canals revealed no retrocochlear pathology. Audiometric testing 3 months after his initial testing showed mild improvement of his hearing (Fig. 1a). His vestibular function tests showed no vestibular asymmetries in otolithic function or rotational receptor function. Over time, he has experienced continued reduction of his aural pressure and tinnitus and complete resolution of his episodic true rotational vertigo.