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. 2022 Sep 28;17(9):e0275295. doi: 10.1371/journal.pone.0275295

Hygienic behaviors during the COVID-19 pandemic may decrease immunoglobulin G levels: Implications for Kawasaki disease

Hiromi Yamaguchi 1,2, Masaaki Hirata 1, Kuniya Hatakeyama 1, Ichiro Yamane 1, Hisashi Endo 1, Hiroe Okubo 1, Yoshimi Nishimura 1, Yoshiro Nagao 1,*
Editor: Ghulam Md Ashraf3
PMCID: PMC9518924  PMID: 36170286

Abstract

Background

Due to the coronavirus disease 2019 (COVID-19) pandemic, hygienic behaviors became a new norm since January 2020. The hygiene hypothesis predicts that an excessively hygienic environment may adversely affect human health.

Objective

We quantified the effect of COVID-19 on immunological parameters linked to the hygiene hypothesis.

Methods

We examined age-specific levels of total nonspecific immunoglobulin G (IgG) and IgE in individuals who visited Fukuoka Tokushukai Hospital between 2010 and 2021. Pre-COVID (2010–2019) and COVID (2020–2021) periods were compared.

Results

IgG levels steadily decreased throughout Pre-COVID period. IgG levels fell abruptly from the pre-COVID period to the COVID period in all age groups (P = 0.0271, < 0.3 years; P = 0.0096, 0.3–5 years; P = 0.0074, ≥ 5 years). The declines in IgG in < 0.3 years and that in ≥ 5 years accelerated during the COVID period. IgE levels were seasonal, but did not change noticeably from the pre-COVID to COVID period. IgG levels recorded for patients with Kawasaki disease (KD) (mean 709 mg/dL) were significantly lower than for matched control subjects (826 mg/dL) (P<0.0001).

Discussion

Hygienic behaviors during the COVID-19 outbreak decreased the chance of infection, which may explain the decreases in IgG levels in children and adults. Neonatal IgG declined, possibly because of the decrease in maternal IgG.

Conclusion

Hygienic behaviors decreased the IgG levels in all age groups, from neonates to adults. This downturn in IgG may lead to vulnerability to infections as well as to KD.

Introduction

The new variant coronavirus (SARS-CoV-2) was reportedly brought to Japan in January, 2020 [1]. Following the global and domestic spread of coronavirus disease 2019 (COVID-19), the Japanese public voluntarily took precautional measures, such as wearing face coverings, and distancing from each other. In March 2020, 67% of the Japanese wore face masks in public places, at a higher rate than in Western countries (e.g. 42% in Spain, 17% in the US and 6% in the UK) [2]. The Japanese government declared a state of emergency repeatedly and requested that the public refrain from non-urgent travel and gatherings. More rigorous regulations were enforced in populated prefectures including Fukuoka prefecture (population 5 million), where our hospital is located.

It is hypothesized that the strict hygiene regulations practiced during the COVID-19 pandemic may affect microbiota that inhabit humans and cause immunological problems [3]. This concern is based upon the long-held hypothesis that as the environment becomes more hygienic, decreasing exposure to infections can adversely affect human health [4]. One study found that hygienic conditions are correlated with an increased risk for inflammatory bowel disease [5]. It was suggested that targeted hygiene against pathogens and sharing essential microbes are both important [6].

Serum Immunoglobulin E (IgE) is an indicator of allergic propensity. Numerous studies revealed that insufficient exposure to microbial diversity during the early days of life induces high IgE levels, which subsequently leads to autoimmune and allergic disorders [7–14]. In contrast to IgE, whose role in protective immunity is not fully understood, Immunoglobulin G (IgG) is a critical mediator of infection immunity. However, there are few studies of IgG in the context of the hygiene hypothesis. In wild animals, nonspecific total IgG levels are higher than in captive animals [15, 16]. Nonspecific total IgG, which is frequently measured in laboratory tests in Japan, could potentially be a useful indicator of nonspecific infection immunity.

The hygiene hypothesis has been proposed as a possible explanation for Kawasaki disease (KD) [17–19]. KD is a febrile pediatric illness with mucocutaneous manifestations. KD most frequently affects children < 5 years of age in Japan and in many other regions/countries [20–22]. Importantly, KD is associated with high rates of potentially fatal coronary complications [23–25]. Although KD was first reported in 1967 [26], its etiology remains unknown. Numerous study results suggest that KD has an infectious etiology. For example, in Japan, KD incidence has a bimodal seasonality that is identical to pediatric viral infections [27]. KD is rare at < 3 months of age [28], which indicates the presence of maternal immunity. KD cases cluster in space and time [29–31]. B cell selection in patients with KD is consistent with an infectious origin rather than an autoimmune origin [32]. Meanwhile, a genetic preposition to KD has been found [33–36]. Therefore, it is widely accepted that KD is triggered when a genetically susceptible individual is infected by an as yet undetermined microbe(s) [37].

In this study, we examined whether the rigorous hygiene regulations imposed during the COVID-19 pandemic affected immunity in the human population. We also attempted to develop a hypothesis about the relationship between the change in population immunity and health outcomes, in particular for KD epidemiology.

Results

Human movement and distancing in the study area

Fig 1 shows that people in Fukuoka prefecture avoided public places and workplaces to the greatest degree through the first half of 2020. This avoidance culminated in May 2020 with a > 50% reduction in visits to transit stations. Throughout 2020 and 2021, the degree of this indicator fluctuated, but remained 20% below baseline. People started pre-emptive distancing well before states of emergency were declared.

Fig 1. Indicators of community mobility in Fukuoka prefecture.

Fig 1

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Daily data for Fukuoka prefecture was downloaded from the Community Mobility Report [38]. Time spent in residential areas, workplaces, and transit stations were represented as percentages compared to baseline (3 January– 6 February 2020); values were smoothed using the Lowess method and a 3-day bandwidth.

Measurement of IgG and IgE

Between January 2010 and December 2021, total nonspecific IgG and IgE were tested 33,107 and 6,530 times, respectively. To avoid the effects of duplicate sampling on the analysis, we used only the first measurement for each individual (19,744 and 5,433 individuals for IgG and IgE, respectively. Table 1).

Table 1. Numbers of individuals for whom total nonspecific IgG or IgE were measured between 2010 and 2021.

IgG IgE
Year < 0.3 y 0.3–5 y ≥ 5 y Total < 5 y 5–20 y ≥ 20 y Total
2010 660 133 473 1266 90 111 105 306
2011 583 73 454 1110 126 82 236 444
2012 650 122 681 1453 269 109 244 622
2013 643 167 535 1345 318 74 128 520
2014 688 189 725 1602 268 103 129 500
2015 743 314 873 1930 243 105 133 481
2016 807 361 1063 2231 190 88 92 370
2017 727 249 865 1841 169 82 87 338
2018 602 326 699 1627 177 69 171 417
2019 613 322 784 1719 140 89 228 457
2020 413 229 980 1622 101 78 246 425
2021 353 348 1297 1998 144 91 318 553
Total 7482 2833 9428 19744 2235 1081 2117 5433
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IgG and age

IgG level had a mostly positive correlation with age (Spearman’s R = 0.3962, P < 0.0001, n = 19,744, Fig 2A). IgG levels declined from birth to 3–4 months of age; this change reflected waning maternal immunity (Fig 2B). Subsequently, IgG levels steeply surged up to 5 years of age. Based on this result, in the analysis of IgG we classified individuals into three age groups: infants and neonates < 0.3 years; children between 0.3 and 5 years; individuals ≥ 5 years.

Fig 2. IgG and age.

Fig 2

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Nonspecific total IgG values, which were tested between January 2010 and December 2021 in Fukuoka Tokushukai Hospital, were plotted against ages of individuals, for all age groups (a), and for individuals < 10 years of age (b). The red lines represent the results for Lowess smoothing with a 14-day bandwidth.

Temporal shift in IgG

Annual IgG levels declined over the years in all age groups (Fig 3). There was a statistically significant decrease in IgG from 2019 to 2020 in all age groups (P = 0.0096 in < 0.3 y; P = 0.0271 between 0.3 and 5 y; P = 0.0074 in ≥ 5 y, Mann-Whitney-Wilcoxon test) (Fig 3A). Fig 3(B) presents this result using a finer temporal resolution; IgG declined dramatically throughout 2020 in early infants (< 0.3 year) and in individuals > 5 years. These findings were supported by the results of regression analyses (Table 2): the downward temporal trends in IgG were statistically significant in all age three groups during the pre-COVID period (i.e., 2010–2019). In contrast, the decline in IgG was significant only in early infants and neonates (< 0.3 year) and individuals ≥ 5 years during the COVID period (i.e., 2020–2021). This temporal trend was significant even after including age in the multivariate analysis. The results for the multivariate coefficients (Table 2) indicated that IgG decreased by 2.0 mg/dL (< 0.3 years), 9.2 mg/dL (0.3–5 years), and 14 mg/dL (≥ 5 years) per year, during the pre-COVID period. During the COVID period, IgG declined annually by 43 mg/dL (< 0.3 years) and 48 mg/dL (≥ 5 years). IgG did not decrease significantly in the 0.3–5 years age group during the COVID period. We repeated these analyses after transforming IgG data into a normal distribution using the Box-Cox method [39], and qualitatively validated the results (S1 Table). Subdivision of the age class ≥5 years showed that, during the COVID period, the IgG declined by 42.1 mg/dL per year in 5–50 years age group (P = 0.0138, n = 1,325) and 42.5 mg/dL per year in ≥50 years (P = 0.1521, n = 952) (S2 Table).

Fig 3. Temporal changes in IgG.

Fig 3

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(a) IgG values averaged for each year are presented for three age groups of individuals of < 0.3 years (diamond), 0.3–5 years (triangle) and ≥ 5 years (square). Statistical comparisons between IgG values reported in one year and those reported in an adjacent year were performed using sum of rank tests (i.e., Mann-Whitney-Wilcoxon test). * P<0.05, ** P<0.01, *** P<0.001. (b) IgG values from these three age groups were smoothed using the Lowess method with a 14-day bandwidth.

Table 2. Linear regression coefficients to explain IgG in different age groups.

Period < 0.3 years 0.3–5 years ≥ 5 years
1. Pre-COVID (2010–2019) n = 6716 n = 2256 n = 7152
1.1 Univariate Coefficient P Coefficient P Coefficient P
Time* (years) - 4.81 P = 0.0002 - 10.0 P<0.0001 - 16.8 P<0.0001
Adjusted R2 0.1843 P = 0.0002 0.0077 P<0.0001 0.0070 P<0.0001
1.2. Multivariate Coefficient P Coefficient P Coefficient P
Time (years) - 2.01 P = 0.0895 - 9.2 P<0.0001 - 13.6 P<0.0001
Age† (years) - 2770 P<0.0001 94 P<0.0001 5.18 P<0.0001
Adjusted R2 0.1784 P<0.0001 0.1841 P<0.0001 0.0656 P<0.0001
2. COVID (2020–2021) n = 766 n = 577 n = 2277
2.1 Univariate Coefficient P Coefficient P Coefficient P
Time (years) -82.1 P<0.0001 4.23 P = 0.8261 - 48.8 P = 0.0039
Adjusted R2 0.0305 P<0.0001 -0.0017 P = 0.8261 0.0032 P = 0.0039
2.2. Multivariate Coefficient P Coefficient P Coefficient P
Time (years) - 42.6 P = 0.0029 13.1 P = 0.4395 -48.1 P = 0.0029
Age (years) - 2455 P<0.0001 99.4 P<0.0001 5.14 P<0.0001
Adjusted R2 0.3096 P<0.0001 0.2292 P<0.0001 0.0849 P<0.0001
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*Time that elapsed from the beginning of study period to the date of IgG measurement (in years).

† Age at the date of IgG measurement. For both time and age, the minimal temporal resolution used was day, while the unit of time is expressed in year.

The results presented in Fig 3(B) suggested that IgG fluctuated over time. Periodicity analysis found that the cycle of this periodicity was much longer than 2.5 years (S1 Fig). Because the study period was only 12 years, we did not consider effects of this fluctuation in the statistical analyses.

IgE and age

The correlation of IgE values with age was significant (Spearman’s R = 0.5368, P<0.0001, n = 2,988). IgE is not transferred transplancentally. IgE steeply increased as age increased, up to 20 years of age. Therefore, for the analyses of IgE, individuals were classified into three age classes: < 5 years, 5–20 years, and ≥ 20 years.

Temporal shift in IgE

When IgE levels were smoothed over the time in the analysis, there was seasonality in all age classes (Fig 4). To identify periodicities, we performed periodogram analysis of each age class (Fig 5). Periodicity with a one-year cyclicity (i.e., seasonality) was found in children < 5 years of age. However, seasonality was more obscure in the older classes. We used regression analysis to identify temporal trends in IgE. A linear combination of sine and cosine transformations of time was included in multivariate analyses to represent seasonality. Only the results of the multivariate analysis performed for the < 5 years age class and in the pre-COVID period indicated that there was a significantly negative temporal trend (Table 3). A multivariate analysis revealed that the sine term had a significant correlation with IgE, which indicated the presence of seasonality. We repeated these regression analyses by transforming IgE values into a normal distribution [39], and validated the results qualitatively (S3 Table). Taken together, we did not find unequivocally that COVID-19 affected IgE in the population (S2 Fig).

Fig 4. Temporal changes in IgE.

Fig 4

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IgE values were recorded for each age group, (a) < 5 years, (b) 5–20 years, and (c) ≥ 20 years, and were smoothed using the Lowess method (bandwidth = 14 days).

Fig 5. Periodicity of IgE.

Fig 5

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IgE values were averaged for each quarter of the year, in each age group. A periodogram was generated from this time-series data. A periodicity with a cycle of one year (i.e., seasonality) was found (arrow heads) for children < 5 years of age (a). This seasonality was more obscure in individuals between 5 and 20 years of age (b), and < 20 years of age (c).

Table 3. Regression analysis to explain IgE in different age groups.

< 5 years 5–20 years ≥ 20 years
1. Pre-COVID (2010–2019) n = 1990 n = 912 n = 1553
1.1 Univariate Coefficient P Coefficient P Coefficient P
Time (year) -7.47 P = 0.1285 -9.97 P = 0.5106 3.93 P = 0.9009
Adjusted R2 0.0007 P = 0.1285 -0.0006 P = 0.5106 -0.0006 P = 0.9009
1.2. Multivariate Coefficient P Coefficient P Coefficient P
Time (year)* -10.1 P = 0.0335 -12.0 P = 0.4287 5.46 P = 0.8628
Age (year) † 113 P<0.0001 33.0 P = 0.0052 -5.14 P = 0.3134
sin(2π×Time) -57.8 P = 0.0005 -158 P = 0.0087 -297 P = 0.0257
cos(2π×Time) -19.0 P = 0.2693 34.8 P = 0.5828 -250 P = 0.0726
Adjusted R2 0.0756 P<0.0001 0.0125 P = 0.0039 0.0030 P = 0.0721
2. COVID (2020–2021) n = 245 n = 169 n = 564
2.1 Univariate Coefficient P Coefficient P Coefficient P
Time 106 P = 0.1704 -69.3 P = 0.7262 -304 P = 0.2625
Adjusted R2 0.0036 P = 0.1704 -0.0052 P = 0.7262 0.0005 P = 0.2625
2.2. Multivariate Coefficient P Coefficient P Coefficient P
Time (year)* 33.3 P = 0.6663 -187 P = 0.3660 -235 P = 0.4063
Age (year) † 143 P<0.0001 108 P = 0.0002 -9.93 P = 0.2000
sin(2π×Time) -133 P = 0.0246 -146 P = 0.3710 174 P = 0.4367
cos(2π×Time) -107 P = 0.0697 320 P = 0.0410 -48.9 P = 0.8167
Adjusted R2 0.0965 P<0.0001 0.0779 P = 0.0017 -0.0009 P = 0.4771
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*Time elapsed from the beginning of study period to the date of IgE measurement (by year).

† Age at date of IgE measurement. For both time and age, the minimal temporal resolution was day, while the unit of time is expressed in year.

Comparison of IgG between patients with KD and matched control individuals

IgG levels were compared between 314 children with KD and an equivalent number of matched control children (Fig 6). In both groups, the prevalence of female subjects was 40.8%. The mean age at IgG testing was 2.63 years for the group of patients with KD and 2.67 years for the matched control group. The mean IgG was 709 mg/dL (95% confidence interval: 682–736 mg/dL) for the group of patients with KD and 826 mg/dL (789–862 mg/dL) for the matched control group (P<0.0001, Wilcoxon’s matched-pairs signed-ranks test).

Fig 6. IgG distributions of patients with KD and matched control individuals.

Fig 6

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Patients with KD were selected from the pre-COVID period (2010–2019). Each control individual was matched to a patient with KD in terms of sex, age, and date of IgG testing. IgG values in patients with KD were significantly lower than those in control individuals.

To consider the possibility that this lower IgG in KD patients may have biased our previous analysis, we repeated the regression analysis (Table 2) by excluding the patients with KD. However, the result was not affected qualitatively (S4 Table).

Discussion

We examined whether changes in hygiene behavior during the COVID-19 outbreak affected nonspecific immunity represented by total IgE and IgG. IgE levels presented seasonality, being consistent with previous reports [40, 41]. However, there was no unambiguous result which supported presence of temporal trend in IgE or effect of COVID-19 on IgE levels. In contrast, IgG decreased slowly but steadily since at least 2010. The rate of decrease in IgG abruptly accelerated in 2020, coinciding with the COVID-19 pandemic. This drop in IgG was most prominent in populations > 5 years of age and in neonates and early infants < 0.3 years of age. These results suggested that hygienic behaviors (e.g., face coverings and distancing) reduced opportunities for infection with microbes, leading to the decline in IgG. Because the mean half-life of IgG is 21 days [42], lack of infection would most likely decrease the IgG without delay. The mother’s IgG is transferred to the fetus during the last few months of pregnancy [43]. The IgG levels in mothers and in umbilical cord blood are highly correlated [44]. Therefore, a downturn in maternal IgG would be reflected in a change in neonatal IgG. In contrast, children between 0.3 years and 5 years would likely not have been affected by the reduction in infection because they were not required to wear face coverings during the COVID-19 outbreak in Japan. Interestingly, there was a significant IgG decline in all age groups from 2016 to 2017 (Fig 3A). This decrease may have been related to an increase of the face mask production in Japan which occurred in 2015 [45].

The KD incidence in Japan decreased by 35% in 2020 from the pre-COVID years [46], implying that the hygiene behaviors may have affected this illness. Therefore, it would be worthwhile to discuss the potential impact of the decreasing IgG on KD. The number of KD cases has been increasing rapidly in Japan and in many developed countries/regions [47, 48]. This constant increase in developed countries prompted discussion about the hygiene hypothesis [17]. Consistent with the hypothesis, KD risk is positively correlated with higher income, urbanization, and smaller family size [18, 49]. Lee hypothesized that the etiology of KD is dysregulated early B cell development under reduced microbial exposure [19]. Study findings indicate that before intravenous immunoglobulin (IVIG) administration, serum IgG levels in patients with KD are lower than in individuals without KD [50]. To our knowledge, our study was the first to quantify this phenomenon using accurately matched control subjects. Whether this low IgG level in patients with KD is a consequence of the systemic inflammation associated with KD or a predisposing factor for development of the illness, or both, remains unknown. Although IVIG is the mainstay of KD treatment, the mechanism for how IVIG cures KD is unknown. It was hypothesized that the IgG in IVIG suppresses excessive immune reactions or neutralizes causative microbes, or both [51]. A low serum IgG level before the first IVIG treatment predicts unresponsiveness to the treatment [50, 52], possibly because IVIG fails to sufficiently elevate the IgG level [53]. A lower level of IgG after the initial IVIG administration predicts a risk of coronary aneurysm [54–56]. Therefore, it is likely that a child with lower IgG may be more prone to develop KD (or at least complicated KD) than children with higher IgG, when triggered by one or more unknown aetiologic agents. This hypothesis may explain why preterm birth is associated an elevated risk for KD [57], because IgG levels in preterm babies are lower than in term babies (S3 Fig) [43]. In a sense, the "window" of pediatric ages at which children are vulnerable to KD (or at least IVIG-refractory KD) may correspond to a hypothetical threshold of IgG (Fig 7). This study revealed that IgG levels in all age groups constantly decreased over at least a decade, and this downward trend accelerated during the COVID-19 outbreak. This finding suggested that the "window" of age vulnerable to KD expands into both older and younger age groups. This mechanism may also explain why the age distribution of KD in Japan has expanded in both directions since 1970 [49]. In 2020, the number of patients with KD decreased in Japan [46, 58]. However, our window hypothesis predicts that the incidence of KD may increase after a transient decrease, in a way that has been observed for other infectious diseases [59].

Fig 7. Hypothesis that explains and predicts expansion of ages of KD due to decreased IgG levels.

Fig 7

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The force of infection is high in a less hygienic environment (red). As the environment becomes more hygienic, the force of infection becomes lower (blue). Under an assumption that KD occurs more frequently under a hypothetical threshold of IgG, the age window for KD risk expands into both younger and older ages.

The results of one study suggested that a decline in the total fertility rate in Japan caused an increase in KD incidence, with a lag of 15 years [49] (S4 Fig). Total fertility rate has also been found to be a surrogate for force of infection of pediatric infectious diseases [27]. Based on these findings, we hypothesized that a decline in the force of infection increased KD incidence after a long delay. The rate of decrease in IgG predicted in this study may seem too small to cause any change in KD epidemiology. However, this very slow rate may explain why it takes 15 years for a decline in fertility rate (and hence, in the force of infection) to increase KD incidence [49].

The new hygienic norm has become prevalent in developed and developing countries. This behavioral change may decrease IgG levels in diverse regions, and result in unexpected consequences for human health. For example, KD may be expanded into previously unaffected regions and ages. Because of decreasing IgG in children, the current standard dose of IVIG (2 g/kg) may become insufficient for an increasing number of patients with KD.

This study had some limitations. The data were derived retrospectively from a single hospital and may not necessarily represent a general, healthy population. The time elapsed during the COVID-19 period was only 2 years. This short period limited the statistical power to detect any temporal trends from this period or to identify a change in the clinical picture of KD, compared with the pre-COVID era. In our dataset, 50.1% of infants < 0.3 years of age were neonates admitted to the neonatal intensive care unit (NICU). However, the proportion of extremely preterm and/or low birthweight babies admitted to the NICU steadily decreased in recent years (S5 Fig). Because these babies have had a limited duration to receive maternal IgG transplacentally [43], their IgG levels are low (S3 Fig). Therefore, the analysis most likely underestimated the rate in IgG decrease in the population < 0.3 years of age. Despite these caveats, we identified a significant downturn in the IgG in this youngest population. This result supported the robustness of our result.

Although indiscriminate extinction of commensal organisms and common harmless microbes would likely induce unfavorable effects on regulatory immunity [60], targeted hygiene should still be maintained [61]. The magnitude of distancing varies greatly between countries/regions. These heterogeneities provide an opportunity to correlate socio-behavioral changes to human immunological parameters (e.g. total IgG) and health outcomes. The results may provide information that can be used to develop effective targeted hygiene measures. Future multi-region long-term studies are warranted to predict the effects of the global COVID pandemic on health problems, including KD.

Materials and methods

Ethics statement

This study was approved by the Ethics Committee for Fukuoka Tokushukai Hospital, with reference 220302. The Ethics Committee waived the requirement for informed consent because of the retrospective nature of this study. All data were fully anonymized before the analysis.

Community mobility

Indicators for time spent in categorized locations, which started 15 February 2020, were downloaded from "COVID-19 Community Mobility Reports–Google" [38]. The baseline represented a median value for day of the week, recorded between 3 January and 6 February 2020. We examined three categories for location (i.e., transit stations, workplace, and residential area), smoothed over time using the Lowess method.

Study site and period

We extracted clinical records and laboratory data records from the electronic medical recording system of Fukuoka Tokushukai Hospital. This system began operation in August 2009. We used the dates from January 2010 to December 2021. Medical records for 896,381 individuals were stored in the system on December 2021.

IgG measurement

IgG was measured using a Clinical Analyzer 7180® (Hitachi High-Tech, Japan) until August 2014. A TBA-c16000® analyzer (Canon Medical Systems, Japan) was used thereafter. Measurement accuracy was validated once per year by the Medical Society of Fukuoka Prefecture (MSFP). At each validation, the laboratory of our hospital measured two to three samples delivered from the MSFP and returned the values to the MSFP. During the study period, our hospital passed all the validations with "A-level" scores. This result indicated that the results were within 2 standard deviations of values reported from all hospital laboratories in Fukuoka prefecture.

IgE measurement

Total nonspecific IgE was measured by BML Inc. (Tokyo, Japan) until March 2015, and by the SRL Corporation (Tokyo, Japan) thereafter.

Regression analysis to detect temporal trends

We used univariate or multivariate linear regression analysis to identify temporal trends in IgG and IgE. Day was the level of resolution for the age and time variables, while unit of time is expressed in year.

Statistical test to compare IgG between adjacent years

We compared IgG and IgE values between adjacent years (e.g., 2009 vs 2010) using Mann-Whitney-Wilcoxon tests.

Periodicity analysis

The values for IgG or IgE were averaged for each quarter of each year, in each age class. The resulting time-series data were analyzed using the periodogram method to identify periodicity [62].

Comparison of IgG between patients with KD and matched control children

We screened 476 patients with KD who had their first clinical diagnosis for KD, between 2010 and 2019 (i.e., pre-COVID era), and for whom IgG was tested before initiation of IVIG treatment. Here, we selected patients with KD from only the pre-COVID period to exclude possible SARS-CoV-2-induced illness, which mimics KD [63–65]. IgG levels were compared between children with KD and the control children. A control child without KD was matched to a case with KD, in terms of sex, age, and date of IgG measurement. The difference in age and that in date of IgG measurement between a patient with KD and a matched control subject were both within 6 months. Using in-house software [66], we matched 314 patients with KD to an equivalent number of control children.

Statistical software

We used Stata/SE 13.1 (TX, USA) for the statistical analysis. Statistical significance was defined as P<0.05.

Datasets

The datasets used in the present study are available from https://www.kaggle.com/datasets/yoshironagao/hygienic-behaviors-during-the-covid-stata-files.

Supporting information

S1 Fig. IgG periodicity.

IgG values were averaged for each quarter of each year between 2010 and 2021, for each age group. Periodograms were created for these time-series datasets. Seasonality (i.e., one-year cycles) was not found for each age group.

(EPS)

Click here for additional data file. (1.2MB, eps)
S2 Fig. Temporal change in IgE.

IgE values were averaged for each year, in individual age groups. IgE values recorded in one year were compared to those in the adjacent year using rank sum tests (i.e., Mann-Whitney-Wilcoxon tests).

(EPS)

Click here for additional data file. (1.1MB, eps)
S3 Fig. IgG and gestational age in neonatal intensive care unit (NICU).

Between 2010 and 2021, 3,739 neonates were admitted to the NICU. Among these neonates, this number includes 3,638 neonates for whom IgG was measured within 7 days from birth. IgG was highly correlated with gestational age (adjusted R2 = 0.5447, P<0.0001).

(EPS)

Click here for additional data file. (948.1KB, eps)
S4 Fig. Temporal negative correlation between total fertility rate and KD incidence.

The annual incidence of KD reported from 47 prefectures in Japan was correlated strongly with the total fertility rate (TFR) recorded 15 years previously (a). In a prefecture where KD incidence was high, the TFR was low 15 years previously (b). Ref. [49] was modified.

(EPS)

Click here for additional data file. (1.3MB, eps)
S5 Fig. Birthweights and gestational ages in neonatal intensive care unit of Fukuoka Tokushukai Hospital.

The mean values for birthweights and gestational ages in neonates who were admitted to the neonatal intensive care unit (NICU) increased. This increase was due to a change in NICU policy.

(EPS)

Click here for additional data file. (823.5KB, eps)
S1 Table. Linear regression coefficients to explain normalized IgG in different age groups.

(DOCX)

Click here for additional data file. (17.7KB, docx)
S2 Table. Liner regression coefficients to explain IgG in four age groups.

(DOCX)

Click here for additional data file. (27.1KB, docx)
S3 Table. Regression analysis to explain normalized IgE in different age groups.

(DOCX)

Click here for additional data file. (18.9KB, docx)
S4 Table. Regression coefficients which explain IgG, after excluding Kawasaki disease cases.

(DOCX)

Click here for additional data file. (18KB, docx)

Acknowledgments

Funding sources

We are grateful to Teppei Nagai, Kazuma Shibata, Akihito Hori, Shinichiro Yamauchi and Hiroshi Manabe for their assistance in data preparation and statistical analysis. There was no funding source.

Data Availability

The data underlying the results presented in the study are available from Kaggle (https://www.kaggle.com/datasets/yoshironagao/hygienic-behaviors-during-the-covid-stata-files).

Funding Statement

The author(s) received no specific funding for this work.

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Decision Letter 0

Ghulam Md Ashraf

27 Jul 2022

PONE-D-22-10882Hygienic behaviors during the COVID-19 pandemic may decrease immunoglobulin G levels: implications for Kawasaki diseasePLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Interesting concept study. It merits careful thinking but also raises questions regarding “regular” wearing of face covering masks in general.

Here is one main question to the authors: The Japanese society is famously known to adopt the wearing of face masks in general especially in public transportation. And this is way before COVID-19 pandemic. It is also known that KD is endemic at the highest level in Japan (followed by other eastern Asian nations). The incidence in Asian descents in their home countries as well as abroad actually supports to a great extent the genetic preposition of Asians in general and Japanese in particular to KD. The following questions therefore inherently follow: 1) how much does the face mask wearing (before COVID-19) predisposes Japanese children to attract KD from the environment perspective; 2) what is the typical school-aged (or preschool) Japanese child exposure to “polluents” and “microbes” compared to Western nations from the standpoint of wearing face masks in general (before COVID-19) – was it common but perhaps to a lesser widespread use before COVID-19 pandemic? ; 3) How do the Ig levels in Japanese children compare to Japanese descent children living in Western countries (where face masks or not common practice in normal years)?

Pleasae incorporate aspects from the above in Intro, limitations and Discussion

Back to the paper itself now.

Nice concept and interesting data source.

There should be a reviewer with deep knowledge in Public health and general populational immunology background reviewing this paper. My knowledge in KD can justifies my overall appreciation of the paper as is, but would limit me to give a sound science-based objective feed-back in the setting.

Excellent paper in my opinion in general however.

Reviewer #2: Title- Hygienic behaviors during the COVID-19 pandemic may decrease immunoglobulin G levels: implications for Kawasaki disease

In this study, Yamaguchi et al. investigated if rigorous hygiene regulations imposed during the COVID-19 pandemic affected immunity in the human population. They also investigated a potential relationship between the change in population immunity and Kawasaki disease (KD) epidemiology. Given the seriousness of the COVID pandemic, I believe such studies are essential and can add to the knowledge pool that is slowly building toward understanding the disease. The authors have done a lot of good work. However, before the manuscript is accepted for publication, authors need to address the following queries and make significant changes.

1. Foremost concern is that the manuscript is exceptionally long. The authors have included so much information in the introduction section, which ideally belongs to the discussion section. Please make the entire manuscript short and to the point. The introduction section alone has 6-7 paragraphs over-explaining everything and distracting from what they actually did and what was the rationale behind the work.

2. This study is conducted using data between January 2010 and December 2021. They mentioned that the study was approved by the ethics committee. Do they have consent from individuals whose data is used for the work? It is an important concern, and they will need to clearly define it in the M&M section.

3. The age groups authors selected for IgG analysis are infants and neonates < 0.3 years; children between 0.3 and 5 years; individuals ≥ 5 years. The rationale they gave is that IgG levels steeply surged up to 5 years of age. However, in Fig. 2a, it looks like there is almost a steady increase in IgG till > 80 years. The age groups are imbalanced. Keeping a 6-year-old child and a 70-year-old individual in the same group is not the right way of doing the analysis. Kindly break down age groups from >5 years into smaller groups.

4. In Fig. 3a, the authors show a gradual decrease in IgG from 2010 to 2021. There are years like 2016-2017, where there is a significant reduction in all age groups. What was the reason for this observation? They need to include this in the discussion as this observation is similar to COVID years. This is diluting the COVID-specific IgG decline the authors are trying to mention in their study. It is making it look like that in this region before COVID as well, there were similar observations.

5. Writing is poor, with many repetitive statements throughout the manuscript. For eg. “…the public refrain from non-urgent travel and gatherings. Schools closed.” School closed? Two work sentences? They could have merged this in the previous statement.

Reviewer #3: I read with deep interest--this manuscript entitled 'Hygienic behaviors during the COVID-19 pandemic may decrease immunoglobulin G levels: implications for Kawasaki disease" by Yamaguchi et al. The manuscript is sound; the topic is of interest; and the manuscript is carefully written.

This is among the very few manuscripts that I believe could be published in the present form.

It can be allowed to be published in its present form if its similarity check (e.g. by iThenticate or any other program) is within acceptable limits as per journal policies.

I congratulate the authors.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Shazi Shakil

**********

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PLoS One. 2022 Sep 28;17(9):e0275295. doi: 10.1371/journal.pone.0275295.r002

Author response to Decision Letter 0

26 Aug 2022

Your reference: PONE-D-22-10882

Manuscript title: Hygienic behaviors during the COVID-19 pandemic may decrease immunoglobulin G levels: implications for Kawasaki disease

Dr Emily Chenette

Editor-in-Chief

PLOS ONE

Dr Ghulam Md Ashraf

Academic Editor

PLOS ONE

Dear Dr Chenette and Dr Ashraf,

Thank you very much for assigning the three excellent reviewers to our manuscript. We are aware that, under the present circumstances, finding experienced reviewers is increasingly difficult.

We would like to respond to the useful comments from these three Reviewers, and the Editorial Office as follows:

Comment from the Editorial Office:

Editorial comment 1: Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”)…Your ethics statement should only appear in the Methods section of your manuscript.

Response to Editorial Comment 1: We detailed the “Ethics Statement” paragraph in the Methods section (Line 366-), as in:

Ethics statement. This study was approved by the Ethics Committee for Fukuoka Tokushukai Hospital, with reference 220302. The Ethics Committee waived the requirement for informed consent because of the retrospective nature of this study. All data were fully anonymized before the analysis.

We also amended the Ethics Statement field of the Submission form, accordingly.

Editorial Comment 2. We note that you have included the phrase “data not shown” in your manuscript.

Response to Editorial Comment 2. We deleted the phrase “data not shown”

Editorial Comment 3. We note that Figures 8 and S5 in your submission contain [map/satellite] images which may be copyrighted.

Response to Editorial Comment 3. These figures were deleted.

Editorial Comment 4. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response to Editorial Comment 4. We replaced

Y Nagao, C Urabe, H Nakamura, N Hatano. Predicting the characteristics of the aetiological agent for Kawasaki disease from other paediatric infectious diseases in Japan. Epidemiol Infect. 2016;144(3):478-492.

with

Y Nagao, C Urabe, H Nakamura, N Hatano. Predicting the characteristics of the aetiological agent for Kawasaki disease from other paediatric infectious diseases in Japan. Epidemiol Infect. 2016;144(3):478-492. Erratum in p. 493.

Comments from the Reviewers

Reviewer #1: Interesting concept study. It merits careful thinking but also raises questions regarding “regular” wearing of face covering masks in general.

We appreciated that Reviewer #1 found our manuscript interesting.

1) how much does the face mask wearing (before COVID-19) predisposes Japanese children to attract KD from the environment perspective;

We have been unable to find a literature which specifically estimated the effect of face mask wearing in reducing the exposure to the KD agent(s) “before COVID-19”. However, the KD incidence decreased in 2020 substantially in Japan as compared to the pre-COVID era. This indicates that the hygienic measures (including face masks) are effective temporarily, in reducing the exposure to KD agent(s). We mentioned this in the Discussion section (Lines 241-), as in:

The KD incidence in Japan decreased by 35% in 2020 from the pre-COVID years [46], implying that the hygiene behaviors may have affected this illness. Therefore, it would be worthwhile to discuss the potential impact of the decreasing IgG on KD.

2) what is the typical school-aged (or preschool) Japanese child exposure to “polluents” and “microbes” compared to Western nations from the standpoint of wearing face masks in general (before COVID-19) – was it common but perhaps to a lesser widespread use before COVID-19 pandemic? ;

To our knowledge, no study (at least written in English) compared microbial exposure between the Japanese and the Western people, to say nothing of children. However, we agree with the Reviewer #1 that this is an important topic which should be studied. The COVID outbreak drew attention to this topic, as we mentioned in the Introduction section (lines 49-):

In March 2020, 67% of the Japanese wore face masks in public places, at a higher rate than in Western countries (e.g. 42% in Spain, 17% in the US and 6% in the UK) [2].

3) How do the Ig levels in Japanese children compare to Japanese descent children living in Western countries (where face masks or not common practice in normal years)?

Unfortunately, we failed to identify a study which compared the IgG levels between Japanese children and Japanese-descent children in Western Countries. However, this would make an interesting research question which is relevant to the elucidation of the pathophysiology of Kawasaki Disease. We encouraged future studies toward this direction as in Lines 314- in the Discussion section:

These heterogeneities provide an opportunity to correlate socio-behavioral changes to human immunological parameters (e.g. total IgG) and health outcomes.

Back to the paper itself now.

Nice concept and interesting data source.

My knowledge in KD can justifies my overall appreciation of the paper as is,

Excellent paper in my opinion in general however.

We are honored by all of these positive comments from the Reviewer #1. Thank you very much.

Reviewer #2:

Given the seriousness of the COVID pandemic, I believe such studies are essential and can add to the knowledge pool that is slowly building toward understanding the disease. The authors have done a lot of good work.

We are grateful to this Reviewer #2 for recognizing the significance of our study. Thank you very much.

1. Foremost concern is that the manuscript is exceptionally long. The authors have included so much information in the introduction section, which ideally belongs to the discussion section. Please make the entire manuscript short and to the point. The introduction section alone has 6-7 paragraphs over-explaining everything.

Following this important advice, we omitted redundant information and moved some paragraphs from the Introduction section to the Discussion section. As a result, the Introduction section was shortened into 5 paragraphs.

2. This study is conducted using data between January 2010 and December 2021. They mentioned that the study was approved by the ethics committee. Do they have consent from individuals whose data is used for the work? It is an important concern, and they will need to clearly define it in the M&M section.

We agree with this Reviewer #3 that research ethics is the most important part of a study which involves human subjects. We detailed the process of ethical clearance in Lines 366- in the Methods section as in:

Ethics statement. This study was approved by the Ethics Committee for Fukuoka Tokushukai Hospital, with reference 220302. The Ethics Committee waived the requirement for informed consent because of the retrospective nature of this study. All data were fully anonymized before the analysis.

3. The age groups authors selected for IgG analysis are infants and neonates < 0.3 years; children between 0.3 and 5 years; individuals ≥ 5 years. The rationale they gave is that IgG levels steeply surged up to 5 years of age. However, in Fig. 2a, it looks like there is almost a steady increase in IgG till > 80 years. The age groups are imbalanced. Keeping a 6-year-old child and a 70-year-old individual in the same group is not the right way of doing the analysis. Kindly break down age groups from >5 years into smaller groups.

Following this useful suggestion by Reviewer #2, we conducted an analysis which broke down the age groups (Lines 141- in the Results section):

Subdivision of the age class ≥5 years showed that, during the COVID period, the IgG declined by 42.1 mg/dL per year in 5-50 years age group (P=0.0138, n=1,325) and 42.5 mg/dL per year in ≥50 years (P=0.1521, n=952) (S2 Table).

4. In Fig. 3a, the authors show a gradual decrease in IgG from 2010 to 2021. There are years like 2016-2017, where there is a significant reduction in all age groups. What was the reason for this observation? They need to include this in the discussion as this observation is similar to COVID years. This is diluting the COVID-specific IgG decline the authors are trying to mention in their study. It is making it look like that in this region before COVID as well, there were similar observations.

We appreciated it that this Reviewer #2 read our manuscript very carefully. We mentioned this interesting observation in Lines 238- in the Discussion section:

Interestingly, there was a significant IgG decline in all age groups from 2016 to 2017 (Figure 3 a). This decrease may have been related to an increase of the face mask production in Japan, which occurred in 2015 [45].

5. Writing is poor, with many repetitive statements throughout the manuscript. For eg. “…the public refrain from non-urgent travel and gatherings. Schools closed.” School closed? Two work sentences?

Following this important advice, we maximally deleted repetitive statements. The sentence “School closed” was deleted.

Please let us thank once again to the Reviewer #2, whose comments improved our manuscript greatly.

Reviewer #3 (Dr Shazi Shakil): I read with deep interest--this manuscript... The manuscript is sound; the topic is of interest; and the manuscript is carefully written.

This is among the very few manuscripts that I believe could be published in the present form.

It can be allowed to be published in its present form if its similarity check (e.g. by iThenticate or any other program) is within acceptable limits as per journal policies.

I congratulate the authors.

We are very grateful to all these encouraging comments from the Reviewer #3.

Dr Shazi Shakil, Thank you very much.

Attachment

Submitted filename: IgG_IgE_Kawasaki_disease_PLOS_ONE_IgG_rebuttal.docx

Click here for additional data file. (26.7KB, docx)

Decision Letter 1

Ghulam Md Ashraf

13 Sep 2022

Hygienic behaviors during the COVID-19 pandemic may decrease immunoglobulin G levels: implications for Kawasaki disease

PONE-D-22-10882R1

Dear Dr. Nagao,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Ghulam Md Ashraf, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Excellent paper, looking forward to see it published soon.

ONE HUNDRED CHARACTERS REQUIRED ATTAINED

Reviewer #2: (No Response)

Reviewer #3: As far as my part of comments are concerned; I feel the manuscript is acceptable now.

I congratulate the authors.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: SHAZI SHAKIL

**********

Acceptance letter

Ghulam Md Ashraf

19 Sep 2022

PONE-D-22-10882R1

Hygienic behaviors during the COVID-19 pandemic may decrease immunoglobulin G levels: implications for Kawasaki disease

Dear Dr. Nagao:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Ghulam Md Ashraf

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. IgG periodicity.

    IgG values were averaged for each quarter of each year between 2010 and 2021, for each age group. Periodograms were created for these time-series datasets. Seasonality (i.e., one-year cycles) was not found for each age group.

    (EPS)

    Click here for additional data file. (1.2MB, eps)
    S2 Fig. Temporal change in IgE.

    IgE values were averaged for each year, in individual age groups. IgE values recorded in one year were compared to those in the adjacent year using rank sum tests (i.e., Mann-Whitney-Wilcoxon tests).

    (EPS)

    Click here for additional data file. (1.1MB, eps)
    S3 Fig. IgG and gestational age in neonatal intensive care unit (NICU).

    Between 2010 and 2021, 3,739 neonates were admitted to the NICU. Among these neonates, this number includes 3,638 neonates for whom IgG was measured within 7 days from birth. IgG was highly correlated with gestational age (adjusted R2 = 0.5447, P<0.0001).

    (EPS)

    Click here for additional data file. (948.1KB, eps)
    S4 Fig. Temporal negative correlation between total fertility rate and KD incidence.

    The annual incidence of KD reported from 47 prefectures in Japan was correlated strongly with the total fertility rate (TFR) recorded 15 years previously (a). In a prefecture where KD incidence was high, the TFR was low 15 years previously (b). Ref. [49] was modified.

    (EPS)

    Click here for additional data file. (1.3MB, eps)
    S5 Fig. Birthweights and gestational ages in neonatal intensive care unit of Fukuoka Tokushukai Hospital.

    The mean values for birthweights and gestational ages in neonates who were admitted to the neonatal intensive care unit (NICU) increased. This increase was due to a change in NICU policy.

    (EPS)

    Click here for additional data file. (823.5KB, eps)
    S1 Table. Linear regression coefficients to explain normalized IgG in different age groups.

    (DOCX)

    Click here for additional data file. (17.7KB, docx)
    S2 Table. Liner regression coefficients to explain IgG in four age groups.

    (DOCX)

    Click here for additional data file. (27.1KB, docx)
    S3 Table. Regression analysis to explain normalized IgE in different age groups.

    (DOCX)

    Click here for additional data file. (18.9KB, docx)
    S4 Table. Regression coefficients which explain IgG, after excluding Kawasaki disease cases.

    (DOCX)

    Click here for additional data file. (18KB, docx)
    Attachment

    Submitted filename: IgG_IgE_Kawasaki_disease_PLOS_ONE_IgG_rebuttal.docx

    Click here for additional data file. (26.7KB, docx)

    Data Availability Statement

    The data underlying the results presented in the study are available from Kaggle (https://www.kaggle.com/datasets/yoshironagao/hygienic-behaviors-during-the-covid-stata-files).

    Articles from PLoS ONE are provided here courtesy of PLOS

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