1. Introduction
Infection by SARS-CoV-2 results in the development of mild to serious symptoms in most individuals, with severity being dependent on age and comorbidities [
1,
2,
3]. However, there is mounting evidence that a significant number of infected individuals are asymptomatic [
4,
5]. An asymptomatic SARS-CoV-2 infection refers to the situation where no clinical signs or symptoms, nor imaging abnormalities, are apparent in an individual who is confirmed to be infected with the virus by reverse transcriptase-polymerase chain reaction (RT-PCR) [
6]. Given that asymptomatic patients appear to constitute a significant portion of infections [
7], understanding the factors that are associated with being asymptomatic is important.
Japan has not been spared from the COVID-19 pandemic, and as of 18 January 2021, there have been 326,208 cases of the disease reported in the country [
8]. Hokkaido was the first Japanese prefecture to be affected by COVID-19 with the first reported case being a tourist from Wuhan, China, a 40-year-old female who had the COVID-19 symptoms (fever, cough, fatigue, and pneumonia) on 26 January [
8]. A State of Emergency was declared on 28 February when the number of cumulative confirmed cases reached 65. Issuance of a stay-at-home order, as well as the implementation of social distancing and active contact tracing measures called “cluster countermeasure” by the local government [
9], resulted in a quick reduction in new cases by mid-March. With the disease seemingly under control, the government lifted the State of Emergency on 17 March. However, soon thereafter new cases began to surge again, particularly after the 3-day Equinox holiday between 20 and 22 March [
10]. On 14 April, the government declared a State of Emergency for the second time. Although the State of Emergency was re-lifted on 25 May and the number of cases was no longer surging at the same rate, the prefecture remained designated as one “under special precautions” (
Figure 1) [
11].
Since the beginning of the COVID-19 outbreak, the Japanese government has been publishing the information of each individual who was tested positive for the virus. The current study leveraged registry data from Hokkaido between mid-February and mid-July in order to investigate the viral transmission and clinical characteristics of COVID-19. The registry data published by the government were particularly detailed in the early stage of the pandemic, including the period covered under the current study. The data contain rich information about the transmission paths as a result of the government’s intensive contract tracing effort [
9,
12,
13].
4. Discussion
The current study analyzed data from 1269 (674 females and 595 males) individuals who were PCR-positive for SARS-CoV-2. Some of our findings confirmed the observations of previous studies: Fever was the most prevalent symptom, with 81% females and 88% of male subjects showing it. Fatigue and cough were the next most prevalent symptoms with approximately 40% of female and male patients possessing them. These findings are reported in prior studies including the one by Larsen and colleagues [
17]. Our study additionally identified the four sets of co-occurring symptoms including (i) fever and fatigue; (ii) pharyngitis and rhinitis; (iii) ageusia (loss of taste) and anosmia (loss of smell); and (iv) nausea, vomiting, and diarrhea. With dyspnea as an indicator of severity, our results suggested that severe disease was more frequently observed in patients who had nausea, vomiting, and diarrhea. This interesting correlation may be due to the higher level of viremia and subsequent multi-organ infection, including the gastrointestinal (GI) tract. The presence of nausea and vomiting suggests that upper GI involvement and that of diarrhea, lower GI infection, both are consistent with a disseminated disease as a result of viremia. Moreover, males were more susceptible to developing severe disease, although it is unknown whether or not this sex difference is hormonally driven. One study from China reports a similar finding. Tian et al. reviewed data from 15 clinical studies and case reports to investigate the GI features of COVID-19 in adult and pediatric patients [
18]. They report that the proportion of patients with GI symptoms was higher among severe patients than in non-severe patients. Another single-hospital study from Wuhan, China, analyzed data from 206 patients with mild disease and GI symptoms. The study reports that these patients are more likely to have a positive test result for viral RNA in stool and to have a longer duration before viral clearance. The results of both these studies are consistent with our findings and suggest that GI symptoms may be suggestive of more disseminated diseases, although treatments, including antibiotics, and corticosteroids could also have an impact on the gastrointestinal mucosa and result in GI symptoms. The presence of GI symptoms in younger individuals has been observed previously [
19,
20,
21], and it is speculated that a weaker respiratory immune response in this age group may be the reason for their mild respiratory symptoms when compared to adults [
18]. The presence of anosmia and ageusia suggests that the virus affects the olfactory epithelium, as well as taste buds, both of which have cilia that act as the receptors for specific molecules that we taste and smell, respectively.
Moreove, consistent with prior findings [
4,
22], a significant number (~20%) of infected patients were found to be asymptomatic carriers in our study. Interestingly, however, there were significantly more female asymptomatic patients than males in our study. There are a handful of prior studies suggesting this. Williamson and colleagues analyzed COVID-19 death records in the UK and reported that men had a significantly higher risk of mortality with an HR of 1.59 [
23]. Yang et al. studied 78 patients in Wuhan, China, and reported that being female and younger increased the likelihood of being asymptomatic [
24]. Additionally, a meta-analysis by Kronbichler et al. demonstrated that while abnormal radiological findings could be observed in a large proportion of asymptomatic patients, asymptomatic patients with normal radiological findings were more likely to be female and younger [
25]. Although it is currently unclear why females are more likely to be asymptomatic, a hormonal effect on T-cells may be involved. This possibility is supported by Wu et al., who reported that pregnant women are more likely to be asymptomatic [
26]. In this study, four out of eight pregnant women were asymptomatic before delivery but became symptomatic after delivery, suggesting that the change in hormones and immune system during the pregnancy may play a role in the onsets of symptoms.
In contrast to Yang et al. and Kronbichler et al., our data did not suggest that younger (female) patients were more likely to be asymptomatic. Rather, our data demonstrated that the likelihood of being asymptomatic increased with age in the Hokkaido population. In relation to this, in a study of 98 PCR-positive COVID-19 patients, Takahashi and colleagues observed differences in T cell responses and innate immune cytokine levels in male and female patients, respectively, that correlated with progression to more severe disease. Their patient population, however, did not have a broad age distribution, nor was an assessment of initial symptoms included in their analysis [
27]. As such, no conclusions about age and the presence or absence of symptoms could be reached.
The observations by Wu et al., and Takahashi et al., potentially suggest that sex hormones may be involved in the differences between disease manifestation in male and female COVID-19 patients. A recent hypothesis by Roche and Roche [
28] and Garvin et al. [
29], suggesting a role for the interconnected bradykinin and renin-angiotensin system (RAS), offers a plausible explanation for the observed sex-dependent differences. Estrogen is known to downregulate angiotensin-converting enzyme (ACE), which not only catalyzes the conversion of angiotensin I to the vasoconstrictor angiotensin II but also breaks down bradykinin [
30]. Thus, a decrease in estrogen levels after menopause could result in higher levels of ACE and lower levels of bradykinin in tissues, resulting in a less intense inflammatory response and a reduction in the risk of a “bradykinin storm” triggered by SARS-CoV2. As bradykinin is pro-inflammatory, the lower levels may explain our observations. Moreover, enhanced angiotensin-converting enzyme 2 (ACE2) immunostaining activity during pregnancy has been reported in animal studies [
31,
32]. Bradykinin (BK-1-9 or BK) and its active metabolite [des-Arg
9]-BK (BK-1-8 or DABK) bind to two different G protein-coupled receptors: the B1 receptor (BKB1R), for which its main ligand is DABK, and the B2 receptor (BKB2R), for which its ligand is BK. ACE2 has been shown to inactivate DABK by cleaving a single terminal amino acid from the peptide [
33]. Inhibition of BKB1R activity decreases pulmonary neutrophil infiltration following LPS exposure and decreases the influx of neutrophils in response to endotoxin [
33]. The elevated levels of ACE2 during pregnancy [
34] may result in lower DABK levels and, thus, explain why pregnant women are more likely to be asymptomatic [
20] or show mild symptoms.
As expected, asymptomatic patients in our study were more likely to transmit the virus in our study. Interestingly, however, these patients who became infected by asymptomatic patients were also likely to be asymptomatic. This may be the result of a lower viral load in asymptomatic individuals. Luo and colleagues also observed that transmission risk increased with the severity of the disease, suggesting a higher viral load in patients with more severe symptoms [
35]. Other studies, however, have found similar viral loads in symptomatic and asymptomatic patients, although the number of patients analyzed was small [
23]. Unfortunately, in our study, no viral samples were available; therefore, it was impossible to determine the possible effect of viral load or the presence of variants in explaining asymptomatic to asymptomatic transmissions. The length of time between PCR testing and the onset of symptoms was also positively associated with the transmission in our study, presumably due to the maintenance of social contacts during this period. Furthermore, male patients were 82% more likely to transmit the virus, regardless of being symptomatic or asymptomatic. This may be either due to a higher viral load in males or more likely due to behavioral or physical differences in the anatomy of the upper respiratory tract, including the mouth, which may result in a greater release of the virus in droplets or aerosol.
Of significant interest is the presence of nine cases of COVID-19 reinfection in the study population. Earlier evidence in smaller-scale retrospective clinical studies suggested that reinfection by the coronavirus is possible. Ye and colleagues studied 55 COVID-19 patients with pneumonia, out of which 9% had a second episode of COVID-19 [
36]. Case reports from China (
n = 2) [
37] and Italy (
n = 1) [
38] also confirmed this. In our study, the prevalence of the virus reinfection was 1%, significantly smaller than that reported in Ye et al. Due to the aforementioned limitation regarding the availability of viral samples, it is unclear whether these cases of reinfection were caused by the same variant of SARS-CoV-2 or a different one. The data, however, suggest that in most reinfection cases in the current study, the symptoms of the first infection were similar to those of the second, raising the possibility that the first and second infection episodes were caused by different variants of the virus.
Finally, the current study is a retrospective secondary data analysis; thus, some of the relevant information, particularly the patients’ data on comorbidities, were not available in a consistently analyzable fashion. The authors were also unable to ensure either the accuracy or the completeness of the data. In this sense, some of the results, especially the findings concerning viral transmission networks, are subject to systematic bias if contact tracing was performed disproportionately in specific cases or cohorts. While the most up-to-date guideline published by the Japanese government still requests that all individuals who were in “close contact” with the confirmed cases be subject to an “initial (PCR) screening test” [
14], the level of compliance with such a guideline is unknown, especially in light of the recent resurge in COVID-19 cases in the country. That being said, we believe that the guideline was adhered to in Hokkaido in the period between February and July and that further investigations on the patterns of viral transmissions and symptomology in the networks are warranted. Such investigations are deemed particularly valid as new variants of the virus continue to emerge.