Smell Outcome and Seropositivity Titre in Post-COVID-19 Patients

In late December 2019, coronavirus disease 2019 (COVID-19) erupted formidably out of Wuhan city, Hubei Province, China. The major clinical symptoms of COVID-19 bear a resemblance to any other respiratory illnesses, i.e., fever and dry cough. Anosmia (loss of smell) and ageusia (loss of taste) have also been noted as early and sometimes the only symptoms [1]. In addition, as high as 78% of individuals are asymptomatic [2]. This is corroborated by a recent study done by Zahedi et al. [3], which found that 77.4% out of 98.8% self-perceiving asymptomatic COVID-19 patients had impaired smell. Studies showed that individuals with smell disorders tend also to have a taste disorder, suggesting a probable interrelation between the two [4].

Seroconversion is the transition point of viral infection to when antibodies of the virus become present in the blood. According to the WHO, seronegative condition is where no antibodies are in the serum, or they are present but below the limit of detection. Seropositive condition is when antibodies can be detected in serum samples. Similar to common acute viral infections, the antibody profile against the SARS-CoV has a typical pattern of immunoglobulin M (IgM) and immunoglobulin G (IgG) production. Determining the optimal time points for the collection of patient specimens increases the efficacy of diagnostic antibody testing [5].

The SARS-specific IgM antibody levels rise during the first week after SARS-CoV-2 infection, peak at 2 weeks, and disappear at the end of week 12. IgG was detectable after 1 week and was maintained at a high level for an extended period, even over 48 days [6]. IgM provides the first line of humoural immunity defence, after which high‐affinity IgG responses are initiated and play a key role in long‐term immune memory.

In a study by Iannuzzi et al. [7], 30 patients underwent a quantitative olfactory test performed with the Sniffin’ Sticks test (SST) considering olfactory threshold (T), odour discrimination (D), and odour identification (I). Results were presented as a composite TDI score (range 1–48) that was used to define functional anosmia (TDI ≤ 16.5), hyposmia (16.5 < TDI < 30.5), or functionally normal ability to smell (TDI ≥ 30.5). Patients were tested during hospitalization and 2 months post-onset of symptoms. During the hospitalization, the overall TDI score indicated that 10% were diagnosed with anosmia and more than 50% were hyposmic. Almost all patients showed a significant improvement at around 1 month for all the parts of the SST except for odour identification. At 1 month, none of the subjects were still diagnosed with anosmia. Anosmic subjects improved more than hyposmic and normosmic subjects.

Moein et al. reported that 59 COVID-19 patients (98%) were experiencing at least some hyposmia: 8 (13%) with mild microsmia, 16 (27%) with moderate microsmia, 20 (33%) with severe microsmia, and 15 (20%) with anosmia. Literature also suggests a higher prevalence of anosmia in mild-moderate disease and in younger age groups [8, 9], particularly the age group of 20–39 years, which showed a tendency to be associated with a longer persistence of anosmia [10].

Nasal respiratory epithelial cells and olfactory epithelial support cells have been shown to express high levels of ACE2 proteins used by the SARS-Cov2 virus. It has the potential of microinvasion to the target receptor which is expressed on the olfactory bulb and the epithelial cell of the oral mucosa [11].

RT PCR is a diagnostic test to detect COVID-19. This polymerase chain reaction testing looks for the presence of the virus’ genetic material (RNA) on a nasal or throat swab and more recently via saliva. These tests can tell whether someone has an active, current infection by magnifying viral material presence or absence in the samples [12]. SARS-CoV-2 can initially be detected 1-2 days prior to the onset of symptoms and can persist for 7–12 days in moderate cases and up to 2 weeks in severe cases. The PCR method may not detect the virus in the very early stage of infection or late stage when the viral load is very low [13].

In general, there are 3 types of antibodies (IgA, IgM, and IgG) that will be produced in response to the infection. The immune system typically produces IgM soon after infection as the first line of defence during viral infections, and IgG is generated later and persists in the body longer than IgM. This contributes to long-term immunity and immunological memory. IgA is another type of antibody, typically found in mucous membranes, that can be produced in high quantities during infections [14].

SARS-CoV-2 infection, like other pathogen infections, induces the production of IgM and IgG antibodies, which are the most useful for assessing antibody response. However, unlike the typical pattern of increasing IgM in the acute phase followed by IgG, in SARS-CoV-2 infection, these antibodies arise nearly simultaneously in serum (CDC 2020). There is a significant inter-individual difference in the levels and chronological appearance of antibodies in COVID-19 cases, but median seroconversion timing has been observed at approximately 2 weeks [14, 15] with the seroconversion rate ranging from 90% to 100% [14, 16, 17].

In a recent study, of 535 plasma samples taken from 173 patients with SARS-CoV-2, the median seroconversion time for total antibody (Ab), IgM, and then IgG was day 11, day 12, and day 14, separately. The presence of antibodies was <40% among patients within 1 week since onset and rapidly increased to 100.0% (Ab), 94.3% (IgM), and 79.8% (IgG) 15 days after onset [14]. This study aimed to investigate the smell outcome and seropositivity titre (IgG) in post-COVID-19 patients.

A prospective cohort study was conducted in UKMMC between January 2021 and June 2022 in the ORL clinic UKMMC. Ethical approval was obtained from the Institutional Review Board (IRB) of Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Malaysia. All participants provided informed consent for study participation.

Sniffin’s Sticks test during acute COVID infection was obtained from previous study and recorded as baseline. Sniffin’s Sticks test was repeated within 12 months after discharge to the same patients; together with msQOD-NS, mSS-SIT and ELISA tests were performed. All patients were screened for their eligibility in participating in the study. Participants included all patients who tested positive for COVID-19 with other mild illnesses.

The inclusion criteria were 18 years and above of age, post-COVID-19 infection (laboratory confirmed), able to undergo a smell test, able to complete a questionnaire, and agreed to undergo a serology blood test. Our exclusion criteria were pregnancy, acute respiratory tract infection, underlying sinonasal diseases, and allergic rhinitis. Each patient who consented and fulfilled the criteria was enrolled in this study. Each one of them underwent the Malaysian version Sniffin’ Sticks identification smell test (mSS-SIT); Malay short version of the Questionnaire of Olfactory Disorders-Negative Statements (msQOD-NS) questionnaire; and serology SARS-CoV-2 antibodies blood test. The score was calculated for each test.

• 1.

  Malay short version of Questionnaire of Olfactory Disorder-Negative Statements (msQOD-NS): original sQOD-NS is a 7-item patient-reported outcome questionnaire including social, eating, annoyance, and anxiety questions. Items are rated on a scale of 0–3, with higher scores reflecting better olfactory-specific QOL. The total score ranges from 0 (severe impact on QoL) to 21 (no impact on QoL). The item and total scores of sQOD-NS significantly differ between patients with presumed anosmia at the time of the assessment and those with presumed hyposmia or without olfactory dysfunction (*p = 0.001) [18].

Malaysia is well known for its multi-ethnicity and using the Malay language as a universal communication tool at least among locals. In order to assist locals to understand this questionnaire better, we used Malay version of sQOD-NS questionnaire in this study [19].

• 2.

  Sniffin’ Sticks identification smell test (Burghardt^®, Wedel, Germany): in this study, we evaluated patient odour identification using the cultural adapted Malaysian version of Sniffin’ Sticks smell identification test (mSS-SIT) [19‒21]. It was carried out in a properly ventilated room with the use of odourless gloves. The subjects should neither have eaten nor drank anything other than plain water 15 min prior to the test. This rule extends also to smoking and the use of nasal topical medication or chewing gum. Sixteen pens with different odours were presented to the patient to smell and for each odour, a list of 4 options was given to the patient to choose, with the tip of the pen placed approximately 2 cm in front of the nostrils for 2 s. The interval between odour presentations is 20 s. Using multiple forced-choice designs, the subjects identify the correct odorant from a list of four descriptors that includes one correct answer and three distractors. One mark will be given for each correctly identified odorant, with a total score ranging from 0 to 16. Participants were grouped as indicative of normosmia if they achieved a score of >12, hyposmia for 9–12, and anosmia if they scored <9 [20]. The Sniffin’ Sticks test was done during active COVID-19 and repeated on same patients once they have recovered from COVID-19.

• 3.

  Serology testing: in vitro quantitative determination of SARS-CoV-2 antibodies is performed using Elecsys Anti-SARS-CoV-2 S via ROCHE COBAS e601 platform which uses electrochemiluminescence immunoassay for the detection of total antibodies against spike (S) protein receptor-binding domain in patients’ serum samples. The cut-off for antibody response was 0.8 U/mL, above which the sample is considered positive. Quantitative determination is performed via a calibration curve in which the instrument is specifically generated by 2-point calibration and a master curve provided by the manufacturer. The target of this study is to identify olfactory outcomes following COVID-19 infection and to evaluate the score of sQOD-NS (mean score) in patients. This also includes smell identification scores taken using the Sniffin’ Sticks test (mean). The seropositivity titre (IgG) response using automated serology method also will be measured. SPSS version 26.0 (IBM, Armonk, NY, USA) was used for the analysis. Descriptive analysis was done to describe the background characteristics of the study subjects. All categorical data presented in frequency and percentage while numerical data will be expressed in mean ± standard deviation for normally distributed data or median and interquartile range for non-normally distributed data.

At baseline and post-COVID-19 infection of odour identification tests for Sniffin’ Sticks test, results were compared using non-parametric Wilcoxon signed-ranks test and the t test. p < 0.05 was considered statistically significant.

Paired T test will be used to compare symptoms (mean msQOD-NS) between 2 arms if the data were distributed normally; otherwise, Wilcoxon signed test will be used. p < 0.05 was considered statistically significant.

A total of 31 patients were enrolled in the study with the age ranged from 22 to 82 years with a median age of 45 years (IQR = 30.0) and most of them were males with 61.3% and 38.7% were female. The patient’s olfactory function was assessed by performing the Malaysian version of the Sniffin’ Sticks smell identification test (SS-SIT), at 2 time points of the disease, during the hospitalization, when patients had tested positive for SARS-CoV-2 (during COVID-19), and after they had tested negative (after COVID-19) with earliest was at 2 months and latest at 11 months.

During COVID-19, 2 of our patients were anosmia (6.5%), 22 (70.9%) were hyposmia, and 7 (22.6%) were normosmia. We repeated SS-SIT on these same patients after COVID-19, and none of these subjects were hyposmia or anosmia as they achieved a score >12. Highest number of scores were 16 (48.4%, n = 15) followed by 15 (32.3%, n = 10) and 14 (19.4%, n = 6). This supports our hypothesis that the smell outcome in post-COVID-19 patients will be back to normal (p < 0.001) (Table 1).

We evaluated our patients who had recovered from COVID-19 using Malay version of sQOD-NS questionnaire. Items are rated on a scale of 0–3. The total score ranges from 0 (severe impact on quality of life) to 21 (no impact on quality of life). All our patients had scored 21, meaning no impact on quality of life as they had regained their normal olfactory function (Table 2).

Evaluating the IgG level in this study is one of our main objectives. In this study, the median value of IgG antibody less than 6 months post-COVID-19 was 122.90 (IQR = 114.720) with a minimum value of 5.60 and a maximum of 185.10. The median value of IgG antibody 6–12 months post-COVID-19 was 23.55 (IQR = 63.775) with a minimum value of 1.55 and a maximum value of 100.80. IgG antibody levels gradually decreased over time till 6 months and remained stable up to 12 months (Table 3). Spearman correlation was used to measure the correlation of smell score and COVID-19 seropositivity titre, which shows there is no correlation between smell test and seropositivity titre COVID-19 (Table 4).

During the COVID-19 pandemic, it has been reported that the smell test is more sensitive in detecting anosmia and hyposmia in comparison to self-reporting or taking a medical history [22] making the smell test an appropriate test to use compared to several reports addressing the degree of the impairments by using self-reported surveys that may be unable to precisely characterize the degree of loss in the absence of objective olfactory testing [23‒25]. We were able to directly assess the olfactory abilities of patients, by performing the Malaysian version of Sniffin’ Sticks smell identification (SS-SIT) at 2 time points of the disease. During active COVID-19, 77.4% were with reduced olfactory ability where 70.9% were hyposmia and 6.5% were anosmia. More than half were hypsomic during COVID-19; this result we obtained is similar to Iannuzzi et al. [7] where more than 50% were hyposmic and 10% were anosmia and also another study by Le Bon et al. [26] where anosmic patients were reported as 8%. Another study by Lechien et al. [27] also using the Sniffin’ Sticks smell test on 86 patients with COVID-19 found a very similar percentage of participants with olfactory deficits (62%) in total; however, they obtained a higher number of anosmic patient with 48% and only 14% hyposmic. Other studies used different olfactory tests and found different percentages; highly variable was the proportion of anosmic and hyposmic participants [9, 28]. We repeated the same Sniffin’ Sticks identification smell test earliest at 2 months post-COVID-19. None of the subjects were still diagnosed with anosmia or hyposmia; all patients recovered 100%. This is also supported by another study where nearly 80% of patients showed improvement in loss of sense of smell within a few weeks of onset, with recovery appearing to plateau after 3 weeks. Therefore, we could conclude that after 2 months from the onset of the disease, there was a full recovery of the sense of smell. Olfactory recovery was found to occur as early as 7 days, with most patients recovering olfaction within 30 days [29].

In a study by Chary et al. [30], 64% of the patients achieved complete recovery at day 15 with a median recovery time of 15 days (4–27 days) after olfactory onset. This is also similar to a study by Lee et al. [10] where most patients with anosmia recovered within 3 weeks with a median time to recovery of 7 days for both symptoms. A study by Printza et al. [31] found that most of the patients (88%) recovered their sense of smell by 2 months (23%); the olfactory loss lasted longer than a month.

We also did a subjective assessment of our patients who had recovered from COVID-19 using the sQOD-NS questionnaire. All our patients had maximum scoring as they had regained their normal olfactory function (Table 2). The mean age of our population was 45.0 years, and 61.3% were males. Males were significantly more affected by olfactory dysfunctions than females. This is similar to the study done by Shah, Naveed Nazir et al. [32]. This finding was in contrast to the many other studies conducted earlier [30, 33‒35]. The three main determinants proposed to explain male-female disparities in SARS-CoV-2 infection are differences in immune function associated with the X chromosome, the effects of sex hormones, and gender-related behavioural and sociocultural differences [36]. In addition, oestrogen and progesterone might have favourable impacts on peripheral or central olfactory region stem cells, which could delay olfactory decline in women. Moreover, the neural function has a propensity to diminish more rapidly in men as compared to women [36]. The above reason also is the cause for the difference in mortality between men and women and those women are either less prone to develop severe complications or are less likely to die because of severe complications [37].

Immunological responses take longer to appear, and it is evident that both IgA and IgM decline rapidly throughout infection with median seroconversion times IgG of 12 days after symptom onset or 20 days after exposure [38]. Study by Isho, Baweleta et al. [39], on the antibody dynamics tracked IgG up to 115 days after symptom onset in serum and saliva, with peak IgG levels attained by 16–30 days after onset of symptom. Other studies have found that the protective effect may last only 1-2 years after coronavirus infection [40]. Chen et al. followed up with patients with COVID-19 for 100 days and found that IgG levels dramatically decreased 3-4 months after symptom onset. These results were in agreement with Xiao et al. [41] findings, in that the IgG antibodies in most patients with COVID-19 can last for at least 12 months after discharge. IgG titres decreased significantly in the first 6 months and then remained stable in the subsequent 6 months. Our results were on the same page with their findings, in that the IgG levels gradually decreased over time till 11 months, and then it raised and remained stable till 12 months and stayed low but relatively stable.

The antibody kinetics are positively associated with the severity of the disease: the more severe the symptoms are, the longer the antibody-detectable duration is [42]. 95% mildly symptomatic individuals developed IgG response and rose to 100% by day 30. In contrast, individuals infected with SARS-CoV-2 but who remained asymptomatic developed antibody responses significantly less frequently, with only 45% positive for IgG by day 30 after infection. These results confirmed immune responses are generated following COVID-19 who developed the mildly symptomatic illness. However, those with the asymptomatic infection do not respond or have lower antibody levels [43]. It is unknown how long IgG responses last or if they provide protection against future SARS-CoV-2 infection. While whether the antibodies can protect patients from reinfection requires further study [41].

From this study, we can acknowledge that smell outcome in post-COVID-19 patients will be back to normal, and patients will regain their normal olfactory function. Another important data we obtain is that IgG persists in the body till 12 months post-COVID-19 infection, and this is important for immunity and immunological memory.

It should be noted that there were some limitations in this study as this is a pilot study in Malaysia. First, all the cases were collected in a single hospital and the sample size was relatively small. Second, there were no critical cases enrolled in this study; therefore, no correlation between severity of infection with level of IgG antibody can be obtained. Thirdly, a longer follow‐up period might have yielded the time period when IgG level started to decline and what is level of IgG in the body to prevent a COVID-19 reinfection. Lastly, this study was before the emergence of Omicron strain as we know that different COVID strains exhibit varying levels of olfactory impairment.

From this study, we know that IgG persists in the body till 12 months post-COVID-19 infection. As this is a pilot study in Malaysia, further study is needed in determining the severity of infection with level of IgG, when IgG level starts to decline, and whether they confer protection against subsequent SARS-CoV-2 reinfection.

We are thankful to the volunteers for their participation in the study. We acknowledge the assistance of the staff members of UKM, especially Anasuhah Musa, University Kebangsaan Malaysia medical laboratory technologist, assisting in data collection and laboratory handling of serum samples. We also thank Premaa Supramaniam from Clinical Research Centre, Hospital Raja Permaisuri Bainun, Ipoh, Perak, in data analysis for this study.

The research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. This study protocol was approved by the Ethics Committee (Faculty of Medicine Research & Innovation Secretariat of UKM), approval number UKM FPR. SPI. 800-2/28/89 and project code FF-2021-057. Written informed consent was obtained from each patient prior to participation in this study.

The author and the co-authors declare that they have no conflict of interest.

This study is funded by Universiti Kebangsaan Malaysia with project code FF-2021-057.

Dr. Shalina Kaur performed the literature search, collected data, analysed data, and drafted the work. Prof. Dr. Salina conceptualized, designed the study, edited the manuscript, and reviewed the manuscript. Prof. Aneeza Khairiyah and Prof. Farah Dayana conceptualized, designed the study, and acquired data. Dr. Siti Norlia and Prof. Noor Zetti performed the manuscript preparation, running the serology sample and analysing the result. All the authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work.

All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.