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.