Immunohistochemical and qPCR Detection of SARS-CoV-2 in the Human Middle Ear Versus the Nasal Cavity: Case Series

Viral infections have already been implicated with otitis media and sudden sensorineural hearing loss. However, the pathophysiology of COVID-19 as it relates to otologic disorders is not well-defined. With the spread of SARS-CoV-2, it is important to evaluate its colonization of middle ear mucosa. Middle ear and nasal tissue samples for quantitative RT-PCR and histologic evaluations were obtained from post-mortem COVID-19 patients and non-diseased control patients. Here we present evidence that SARS-CoV-2 colonizes the middle ear epithelium and co-localizes with the primary viral receptor, angiotensin-converting enzyme 2 (ACE2). Both middle ear and nasal epithelial cells show relatively high expression of ACE2, required for SARS-CoV-2 entry. The epithelial cell adhesion molecule (EpCAM) was use as a biomarker of epithelia. Furthermore, we found that the viral load in the middle ear is lower than that present in the nasal cavity.


Introduction
COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been linked to acute otitis media (OM) [1], as well as longer term otologic sequelae including hearing loss and tinnitus [2]. In a survey of admitted COVID-19 patients, 1 in 10 reported changes in their hearing [3]. Other respiratory viruses are known to cause OM through increased susceptibility to bacterial OM or, less commonly, direct viral infection of the middle ear (ME) [4,5]. The pathophysiology of COVID-19 associated otologic disorders has not yet been elucidated.
Although the nasal cavity is a known viral entry site, there is little data as to whether the mucosal epithelium lining the ME is infected and if so, the resulting clinical implications.
One prior qPCR study demonstrated the presence of SARS-CoV-2 RNA in the mastoid and ME in autopsy specimens [6], but specific infected tissue tropism was unknown. More information is needed regarding ME colonization, viral distribution, the potential ME effects of COVID-19 and its relationship to viral infection in the nasal cavity.
In this case series, we examined the ME and nasal septal tissue from post-mortem COVID-19 patients for evidence of SARS-CoV-2 colonization. We also evaluated these tissues for expression of the primary viral receptor, angiotensinconverting enzyme 2 (ACE2).

Materials and Methods
Subjects ME and nasal cavity mucosal samples were obtained postmortem from six COVID-19 patients (P1-6) with PCR-confirmed SARS-CoV-2. The median age at death was 65 years (range, 44 to 91 years), and the time from COVID-19 symptom onset to autopsy ranged from 6 to 37 days (see Table 1 for further clinical details). All subjects had acute respiratory distress syndrome (ARDS) and hearing loss was present in two of the six subjects (P1 and P5). PCR-confirmed, COVID-19 negative patients (P7-10) served as controls with tissue obtained during routine otolaryngologic surgeries unrelated to middle ear or nasal mucosa abnormalities.

COVID-19 qPCR
Quantitative RT-PCR for SARS-CoV-2 detection was used per the US Centers for Disease Control panel assay [7]. COVID-19 primers (N1, N2, and N3 -GENEWIZ) plus human GAPDH gene (Qiagen) were used to assess all clinical samples using the StepOnePlus PCR cycler system (Applied Biosystems). A quantitative synthetic SARS-CoV-2 RNA: Spike 3' template (ATCC-VR3276SD) was used as a positive control template. Total RNA was extracted by Trizol/chlorophorm protocol (Invitrogen) and reverse transcribed using SuperScript-III (Invitrogen). The relative expression was normalized to that of human GAPDH gene present using the ΔΔC t method.
Efficiency of primers was determined to be 95-100% by standard curve, and melt curves were used to assure the correct amplicon size. To calculate viral load, the quantitative synthetic COVID-19 template was used to generate a standard curve for quantification [8]. All experiments were performed in triplicates per biological sample with error bars depicting standard error. Statistical significance was determined by Wilcoxon non-parametric test with p < 0.05. Samples with Ct values > 38 or undetectable were considered negative.

Results
SARS-CoV-2 RNA expression in the ME and the nasal cavity are overall increased in COVID-19 subjects compared to non-infected patients control tissue, with higher expression in the nasal cavity compared to the ME (Fig. 1). ME mucosa from individuals P1 and P3 was positive for SARS-CoV-2 RNA by RT-qPCR with cycle thresholds ranging from 29-33. Meanwhile, in the nasal tissue, all three samples were positive for SARS-CoV-2 with cycle thresholds ranging in 23-32. The viral loads (Table 2) in the nasal cavity were significantly higher than in the ME for subjects P1 (2 × 10 5 vs. 2 × 10 2 copies /µl, p < 0.05) and P2 (1 × 10 3 copies /µl vs. ME COVID-19 negative).
There was no statistical difference in viral load for subject P3 (3.8 × 10 2 vs. 1 × 10 2 copies /µl, ME vs. nasal cavity). Histological evaluations of tissue from patients P4, P5 and P6 all showed that expression of SARS-CoV-2 was found in both the ME and nasal septum which co-localized with ACE2 expression (Fig. 2). Expression of ACE2 was detected on surface epithelium identified by EPCAM. Both ME and nasal epithelial cells showed relatively high expression of ACE2 required for SARS-CoV-2 entry. Viral staining was not detected in COVID-19 negative and control ME and nasal samples. Paraffin sections were labeled with DAPI nuclear staining (blue) and the indicated Alexa 488-conjugated secondary antibodies (green) in representative sections in Fig. 2.

Discussion
We demonstrate concomitant viral presence in the ME and nasal cavity, suggesting systemic infection of SARS-CoV-2 in the head and neck region. In the ME viral load due to other upper respiratory tract (URT) viral infections often varies with a range between (10 1 -10 4 copies/µl) [11,12] with higher viral loads typically associated with increasing URT infection severity [12,13]. Looking at COVID-19 studies in other organ systems, viral loads have been reported in the range of 10 1 -10 5 copies/µL in gut and 10 1 -10 3 copies/ µL in blood [14]. Hence SARS-CoV-2 colonizes the ME at comparable viral loads in our preliminary findings although additional samples are required to strengthen these comparisons. Moreover, given the lower viral loads observed in the ME tissue compared to the nasal cavity, we hypothesize that ME viral entry occurs via the eustachian tube, as previously demonstrated by other viral URT infections that lead to otitis media [5,15]. However, circulation-mediated viral entry is also possible. Both the nasal cavity and ME epithelium express ACE2, the receptor known to interact with the SARS-CoV-2 spike protein for cell entry. ME viral infection has been previously shown to contribute to otologic pathology and symptom manifestations such as OM and conductive hearing loss [5]. It is unclear if SARS-CoV-2 may further damage the auditory sensorineural system or if the COVID-19 symptoms of hearing loss and tinnitus may reflect the ototoxic effects of antiviral medications or immune mediators such as cytokines. However, the presence of viral loads in the ME could lead to direct inner ear SARS-CoV-2 infections. It is also possible that the virus triggers inner ear autoimmunity, known to produce hearing loss [16]. In our sample size Expression depicted as log 10 fold difference, normalized to control COVID-19 negative tissue (P7 and P8, septal mucosa). All COVID-19 ME and nasal cavity samples demonstrated statistically higher levels of SARS-CoV-2 RNA expression compared to control except for the ME sample for P2 as indicated by + (p < 0.05). A noninfectious positive control template yielded strong positive results in each assay (data not shown). For subjects P1 and P2, the viral loads in the nasal cavity were higher than those in the ME, as indicated by *(p < 0.05)  otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.