Materials and methods

2.1 Participants

The strengthening of the reporting of observational studies in epidemiology (STROBE) statement was used for reporting the study. Between October 2021 and October 2022, two groups were included in the study to investigate MRS data in cases with COVID-19 related anosmia and those with normal olfactory function.
The exclusion criteria for both groups were as follows: (i) history of neurological, psychiatric, sino-nasal disorders, sinus or olfactory disorders; or (ii) current Symptoms of a rhinovirus Infection. (iii) any evidence may suggest other possible pathophysiology for the anosmia in two anosmic groups.
Normal healthy volunteers with an age between 18-60 were considered as group 1 and included in the study if they had no exclusion criteria. All healthy controls underwent the Iran smell identification test (Ir-SIT) to confirm normal olfactory function.
COVID-19 related anosmia patients (Group 2) were recruited based on the following criteria: (i) COVID-19 infection and subsequent anosmia; (ii) documented positive SARS-CoV-2 tests (direct antigen detection or reverse transcriptase-polymerase chain reaction) on nasopharyngeal swab specimen before the onset of anosmia; (iii) persisting the anosmia for at least three months; (iv) fulfillment of criteria for anosmia based on Ir-SIT; 12 (v) excluding all possible pathological etiologies by CT-scan and structural MRI; and (vi) age between 18-60 years.
Institutional review board (IRB) approval and patient informed consent was obtained. All the visits, physical examinations, and MRS acquisitions were performed concerning health protocols against COVID-19.

2.2 Iran smell identification test (Ir-SIT)

Iran Smell Identification Test (Ir-SIT) is a Modified Version of the University of Pennsylvania Smell Identification Test (UPSIT) for the Iranian Population 13. The cut point for the anosmia was 9. A score between 10-13 and 14-18 is considered severe and mild microsomia, respectively. A score between 19-24 indicates that the participant has a normal olfactory function. Patients with COVID-19 related anosmia were completely anosmic. All participants in the control group were utterly normosmic (Table 1).

2.3 MRS protocol

Clinical evaluation was performed at a 1.5 Tesla Siemens scanner using an eight-channel receive-only head coil. A conventional 3-dimensional brain image (sagittal T1 MPRAGE, TR/TE = 1800/3.5, the field of view (FOV) = 256 ×2 56 × 160 mm3, Resolution = 1 × 1 × 1 mm3) as a reference image for the volume of interest (VOI) positioning was performed for all patients before MRS sequence. For single-voxel spectroscopy (SVS), MRS was obtained using a point-resolved spectroscopy (PRESS) sequence. Four 2×2×2 cm3 voxels were located on the ACC, DLPFC, IC, and VMPFC. One 2×2×1 cm3 voxel was located at OFC to accurately estimate cerebral cortex neurometabolites concentration and to prevent the effect of surrounding bone tissue on the spectroscopy. The voxels were carefully placed to avoid the subcutaneous fat, skull, vasculature, arachnoid space, and cerebrospinal fluid. Manual shimming was performed for all acquisitions. The parameters were set as TR/TE=1500/135 and NEX=128. Six saturation bands were placed around the VOI to suppress the outer volume signals. The average time for each MRS duration was 25 ± 2 minutes (5 minutes for each region).

2.4 MRS data processing

Data were pre-processed by the administration of a water removal algorithm for the reference offset of 4.65 ppm to remove the residual water signals. SVS raw data were fitted using TARQUIN (version 4.3.10)14. Targeted approaches selected a predefined group of metabolites such as NAA, Cho, and Cr for peak fitting and metabolite concentration. The metabolite ratios of NAA to Cr (NAA/Cr), NAA to Cho (NAA/Cho), and Cho to Cr (Cho/Cr) were measured by dividing the metabolite values in the same spectrum for each ROI.

2.5 Statistical analysis

Statistical analyses were performed using SPSS version 26. Shapiro-Wilk test was used to determine the normality of MRS data distribution, including absolute and relative values of neurometabolites. The relative values of neurometabolites were calculated by dividing the arbitrary unit (au.) levels of NAA/Cr, NAA/Cho, and Cho/Cr.
Parametric independent t-test and non-parametric Mann-Whitney test were carried out for the comparison of quantitative variables with and without normal distribution between groups. Repeated measured ANOVA was used to determine the effect of the ROI and spectroscopic data on the anosmia as well as the interaction between MRS data in different brain regions. P values less than 0.05 were considered as significant.