Quantitative SARS-CoV-2 Serology in

Children With Multisystem

Inflammatory Syndrome (MIS-C)
Christina A. Rostad, MD,a,e Ann Chahroudi, MD, PhD,a,b,e Grace Mantus, BS,a,e Stacey A. Lapp, MS, MPH,a,e
Mehgan Teherani, MD, MS,a,e Lisa Macoy, MSN,a,e Keiko M. Tarquinio, MD,a Rajit K. Basu, MD, MS,a Carol Kao, MD,a,e
W. Matthew Linam, MD,a,e Matthew G. Zimmerman, BS,a,d,e Pei-Yong Shi, PhD,f Vineet D. Menachery, PhD,g
Matthew E. Oster, MD, MPH,a Srilatha Edupuganti, MD,c Evan J. Anderson, MD,a,c,e Mehul S. Suthar, PhD,a,b,d,e
Jens Wrammert, PhD,a,b,e Preeti Jaggi, MDa,e

OBJECTIVES: We aimed to measure severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) abstract
serological responses in children hospitalized with multisystem inflammatory syndrome in children
(MIS-C) compared with those with coronavirus disease 2019 (COVID-19), those with Kawasaki disease
(KD), and hospitalized pediatric controls.
METHODS: From March 17, 2020, to May 26, 2020, we prospectively identified hospitalized children with
MIS-C (n = 10), symptomatic COVID-19 (n = 10), and KD (n = 5) and hospitalized controls (n = 4) at
Children’s Healthcare of Atlanta. With institutional review board approval, we obtained prospective and
residual blood samples from these children and measured SARS-CoV-2 spike receptor-binding domain
(RBD) immunoglobulin M and immunoglobulin G (IgG), full-length spike IgG, and nucleocapsid protein
antibodies using quantitative enzyme-linked immunosorbent assays and SARS-CoV-2 neutralizing
antibodies using live-virus focus-reduction neutralization assays. We statistically compared the log-
transformed antibody titers among groups and performed linear regression analyses.
RESULTS: All children with MIS-C had high titers of SARS-CoV-2 RBD IgG antibodies, which correlated with
full-length spike IgG antibodies (R2 = 0.956; P , .001), nucleocapsid protein antibodies (R2 = 0.846; P ,
.001), and neutralizing antibodies (R2 = 0.667; P , .001). Children with MIS-C had significantly higher
SARS-CoV-2 RBD IgG antibody titers (geometric mean titer 6800; 95% confidence interval 3495–13 231)
than children with COVID-19 (geometric mean titer 626; 95% confidence interval 251–1563; P , .001),
children with KD (geometric mean titer 124; 95% confidence interval 91–170; P , .001), and
hospitalized controls (geometric mean titer 85; P , .001). All children with MIS-C also had detectable
RBD immunoglobulin M antibodies, indicating recent SARS-CoV-2 infection. RBD IgG titers correlated
with the erythrocyte sedimentation rate (R2 = 0.512; P , .046) and with hospital (R2 = 0.548; P = .014)
and ICU lengths of stay (R2 = 0.590; P = .010).
CONCLUSIONS: Quantitative SARS-CoV-2 serology may have a role in establishing the diagnosis of MIS-C,
distinguishing it from similar clinical entities, and stratifying risk for adverse outcomes.

a WHAT’S KNOWN ON THIS SUBJECT: Although the clinical features of a multisystem

Department of Pediatrics, Emory University and Children’s Healthcare of Atlanta, Atlanta, Georgia; bEmory inflammatory syndrome in children (MIS-C) associated with coronavirus disease
Vaccine Center and cDepartment of Medicine, School of Medicine and dYerkes National Primate Research Center, 2019 have been recently described, the serological features of MIS-C are unknown.
Emory University, Atlanta, Georgia; eCenter for Childhood Infections and Vaccines, Children’s Healthcare of Atlanta
and School of Medicine, Emory University, Atlanta, Georgia; and fDepartments of Biochemistry and Molecular WHAT THIS STUDY ADDS: In this case series, all hospitalized children with MIS-C
Biology and gMicrobiology and Immunology, The University of Texas Medical Branch, Galveston, Texas had significantly higher severe acute respiratory syndrome coronavirus 2 binding
and neutralizing antibodies than children with coronavirus disease 2019 or
Kawasaki disease. Severe acute respiratory syndrome coronavirus 2 antibodies
Drs Rostad, Chahroudi, and Jaggi conceived and designed the study, contributed to data acquisition
correlated with metrics of systemic inflammation and clinical outcomes,
and analysis, and drafted the manuscript; Dr Teherani, Mrs Macoy, and Drs Tarquinio, Basu, Kao, suggesting diagnostic and prognostic value.
Linam, Oster, Edupuganti, and Anderson contributed to clinical data acquisition for the study and
critically reviewed and revised the manuscript for important intellectual content; Drs Wrammert,
Suthar, Shi, and Menachery, Ms Mantus, Mr Lapp, and Mr Zimmerman contributed to laboratory To cite: Rostad CA, Chahroudi A, Mantus G, et al. Quantitative
data acquisition and analysis and critically reviewed and revised the manuscript for important SARS-CoV-2 Serology in Children With Multisystem Inflammatory
intellectual content; and all authors approved the final manuscript as submitted and agree to be Syndrome (MIS-C). Pediatrics. 2020;146(6):e2020018242
accountable for all aspects of the work.

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PEDIATRICS Volume 146, number 6, December 2020:e2020018242 ARTICLE
The majority of severe acute (CHOA) from March 17, 2020, to given). These laboratory tests were
respiratory syndrome coronavirus 2 May 26, 2020, were eligible selected because they have been
(SARS-CoV-2) infections in children for enrollment for prospective associated with MIS-C in previous
are either mild or asymptomatic, and/or residual blood collection. reports.6,7 The majority of children
with a small subset requiring Residual samples consisted of in our cohort did not have
hospitalization.1 However, plasma or sera leftover from laboratory data available for serum
reports of a severe inflammatory clinical laboratory tests. With troponin, brain-natriuretic peptide,
syndrome in children after the institutional review board procalcitonin, prothrombin or
onset of SARS-CoV-2 transmission approval, at the time of enrollment, partial thromboplastin time,
have recently emerged in areas prospective samples were interleukin 6, or soluble interleukin
with a high prevalence of collected when parental consent 2R, so these were not included in
coronavirus disease 2019 was possible and residual samples the analysis.
(COVID-19).2–8 Many of these were obtained if consent was not
Treatment data abstracted
children have had clinical features possible. Demographic, clinical,
included the administration
similar to Kawasaki disease laboratory, treatment, and outcome
of IVIg, systemic corticosteroids,
(KD),6,7,9,10 and they have had data were obtained through
convalescent plasma, remdesivir,
high rates of hemodynamic abstraction of the electronic
immunomodulators (eg,
instability, myocardial medical record and were recorded
anakinra, tocilizumab), and
involvement,11 and respiratory on a standardized case report
hydroxychloroquine. Patient
failure requiring intensive form. Data abstracted included
outcome data abstracted
care–level support. On May 14, age, sex, self-reported race and
included the duration of
2020, the US Centers for Disease ethnicity, BMI (which was
hospitalization, admission to
Control and Prevention (CDC) determined at the time of
and duration of ICU care,
released a health advisory and hospitalization for all children
requirement for inotropes
a preliminary case definition for $2 years of age), underlying
or vasopressors (any) for shock,
this clinical entity, which they medical conditions, and
the development of respiratory
termed multisystem inflammatory presenting signs and symptoms.
failure (defined as requiring
syndrome in children (MIS-C) Gastrointestinal symptoms
high-flow nasal cannula or
associated with COVID-19.12 were defined as any reported
greater respiratory support),
Although the clinical features abdominal pain, nausea, vomiting,
and death.
of MIS-C have been described, or diarrhea at the time of
the quantitative SARS-CoV-2 presentation. Respiratory
Definition of Cohorts
serological responses in these symptoms were defined as any
children in comparison with upper respiratory (nasal congestion MIS-C
children hospitalized with or rhinorrhea) or lower respiratory Children who met the CDC
COVID-19 are unknown. In sign or symptom (cough, chest case definition12 were classified
this study, we measured pain, dyspnea, difficulty breathing, as having MIS-C if they had
antibodies to several immunogens hypoxia, or hypercarbia) at the SARS-CoV-2 nucleocapsid
of SARS-CoV-2, the spike protein time of presentation. protein antibodies detected
receptor-binding domain (RBD), by a clinician-ordered qualitative,
the full-length spike protein, and Laboratory values abstracted commercially available
the nucleocapsid protein, and included the maximum white assay (Abbott Laboratories,
neutralizing antibodies in children blood cell count; minimum Chicago, IL) or if they had
hospitalized with MIS-C compared absolute lymphocyte count; nasopharyngeal detection of
with those with COVID-19, those minimum platelet count; minimum SARS-CoV-2 by real-time
with KD, and other hospitalized serum sodium, maximum polymerase chain reaction
controls. alanine aminotransferase, and (RT-PCR). The molecular test
maximum C-reactive protein (CRP) performed at CHOA for inpatients
level; maximum erythrocyte during this time period was the
METHODS sedimentation rate (ESR), DiaSorin Molecular Simplexa
and maximum D-dimer level COVID-19 Direct RT-PCR assay,
Setting and Patient Population obtained before administration which has an analytical sensitivity
Children and young adults of intravenous immunoglobulin or limit of detection (LOD) of 500
(0–21 years of age) hospitalized at (IVIg) (if given) or during the viral copies per mL when specimens
Children’s Healthcare of Atlanta hospitalization (if IVIg was not are collected by using

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2 ROSTAD et al
a nasopharyngeal swab, with near read at 490 nm. Absorbance GraphPad Prism version 8.0.
perfect specificity for distinguishing curves were generated by Frequencies were compared by
from endemic human using nonlinear regression using x2 tests, and continuous
coronaviruses.13,14 analysis, and end point titers variables were compared by using
were interpolated from curves one-way analysis of variance
COVID-19 by using a baseline value (ANOVA) with Tukey’s post hoc
Symptomatic hospitalized calculated from the pooled analysis. Linear regression was
children who tested positive sera of 8 healthy controls. performed on log-transformed
for SARS-CoV-2 by nasopharyngeal The assay LOD was 100, and antibody titers, and the coefficient
RT-PCR and did not meet undetectable titers were assigned of determination (R 2) values
the CDC case definition for a value of 85. were determined. P values #.05
MIS-C were classified as having were considered statistically
COVID-19. Focus-Reduction Neutralization significant.
Assays
KD An infectious clone of the full-length
mNeonGreen SARS-CoV-2
RESULTS
Children were classified as having
complete (n = 3) or incomplete (n = (2019-nCoV/USA_WA1/2020) Baseline Characteristics
2) KD by using American Heart was generated, as previously
During the study period, a total of
Association criteria.15 described.18 Serially diluted
66 children were hospitalized at
patient serum or plasma and
Hospitalized Controls CHOA with either MIS-C (n = 12) or
virus were combined and
COVID-19 (n = 54). We enrolled 10
Additional contemporaneous incubated at 37°C for 1 hour.
children with MIS-C, 10 children
hospitalized children who The antibody-virus mixture was
with COVID-19, 5 children with KD
were evaluated for possible aliquoted onto a monolayer of
(Table 1), and 4 hospitalized
COVID-19 or MIS-C but tested VeroE6 cells and incubated at
controls (Supplemental Table 2)
negative for SARS-CoV-2 by 37°C for 1 hour. The inoculum
who had sera available for analysis.
clinician-ordered nasopharyngeal was removed, and medium
Two of the children with MIS-C had
RT-PCR and measurement supplemented with 1%
concurrently positive SARS-CoV-2
of qualitative nucleocapsid methylcellulose was added.
nasopharyngeal RT-PCR results, and
protein antibodies were included Plates were incubated at 37°C
their clinical presentation of MIS-C
as controls. for 24 hours and were then
appeared to coincide with
fixed with 2% paraformaldehyde.
SARS-CoV-2 Enzyme-Linked symptomatic COVID-19. Children
Green fluorescent foci were
Immunosorbent Assays with MIS-C had a median age of
visualized by using an enzyme-
8.5 years (interquartile range [IQR]
We previously described the linked immune absorbent spot
6.5–12 years) (Table 1,
cloning, expression, and purification reader (Cellular Technology
Supplemental Fig 4A), and the
of a recombinant form of the Limited, Shaker Heights, OH).
majority were of male sex, Black
spike RBD from SARS-CoV-2.16 Green fluorescent protein–based
race, and non-Hispanic ethnicity
We measured anti-RBD focus-reduction neutralization test
and were previously healthy
antibodies using enzyme-linked (FRNT50) curves were generated by
with a normal BMI (Supplemental
immunosorbent assays (ELISAs) using nonlinear regression analysis.
Fig 4B). In contrast, 8 (80%)
in duplicate, as described.16 We Titers were expressed as the serum
children with COVID-19 had
similarly performed ELISAs for dilution at; which fluorescent foci
an underlying medical
the full-length spike (HexaPro,17 were reduced by 50% (FRNT50). The
comorbidity, and 3 (30%) were
courtesy of Jason McLellan) and assay LOD was defined by the
immunocompromised. Two of these
nucleocapsid proteins (Sino starting dilution of serum samples,
children were receiving induction
Biological, Wayne, PA). Secondary which was either 1:20 or 1:40. For
chemotherapy for new diagnoses of
antibodies used for development graphing purposes, the LOD was
acute myelogenous leukemia, and 1
included anti-human-IgG-HRP, anti- represented as 40, and all
child was receiving consolidation
human-IgM-HRP, and anti-human- undetectable titers were assigned
chemotherapy for
HRP (Jackson ImmunoResearch a value of 20.
rhabdomyosarcoma.
Laboratories, Inc, West Grove,
PA). Plates were developed Statistical Methods The distinguishing laboratory
by using o-Phenylenediamine Descriptive statistics and statistical features of MIS-C included
substrate, and absorbance was comparisons were made by using elevated D-dimer levels,

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PEDIATRICS Volume 146, number 6, December 2020 3
TABLE 1 Characteristics of Children With MIS-C, COVID-19, and KD
MIS-C (n = 10) COVID-19 (n = 10) KD (n = 5) P
Demographic characteristics
Age, y, median (IQR) 8.5 (6.5–12) 16 (2.7–18.3) 3 (1–6.5) 0.017
Sex, female, n (%) 4 (40) 5 (50) 2 (40) 0.885
Race, n (%)
Black 6 (60) 4 (40) 2 (40) 0.537
White 3 (30) 6 (60) 3 (60)
Other 1 (10) 0 (0) 0 (0)
Ethnicity, n (%)
Hispanic 2 (20) 5 (50) 1 (20) 0.289
Clinical characteristics
BMI, median (IQR) 18.8 (17.7–24.4) 26.8 (18.5–31) 17 (14.1–18.7) 0.08
Underlying medical condition (any), n (%)a 3 (30) 8 (80) 0 (0) 0.007
Immunocompromised 0 (0) 3 (30) 0 (0)
Respiratory 3 (30) 2 (20) 0 (0)
Other 0 (0) 3 (30) 0 (0)
Respiratory symptoms at presentation, n (%) 5 (50) 5 (50) 1 (20) 0.482
Gastrointestinal symptoms at presentation, n (%) 10 (100) 6 (60) 4 (80) 0.082
Laboratory valuesb
White blood cell count, cells 3 103/µL, median (IQR) 7.9 (6.1–14.0) 19.8 (13.6–24.1) 16.6 (15.2–17.8) 0.117
Absolute lymphocyte count, cells per µL, median (IQR) 623 (437–768) 1330 (579–1889) 2932 (942–3324) 0.021
Platelet count, cells 3 103/µL, median (IQR) 115 (105–131) 249 (30–282) 343 (326–431) 0.01
Sodium, mmol/L, median (IQR) 133 (129–136) 136 (135–137) 136 (134–138) 0.126
Alanine aminotransferase, U/L, median (IQR) 97 (82.5–136) 61 (37–124) 79 (18–82) 0.579
CRP, mg/dL, median (IQR) 15.1 (11.5–17.7) 8.1 (4.9–11.8) 19.9 (17.7–20.2) 0.124
ESR, mm/h, median (IQR) 67 (42.3–71.5) 32 (23.5–70) 79 (53–114) 0.453
D-dimer,cng/mL, median (IQR) 2183 (1735–3732) 1301 (843–1735) 2792 0.034
Treatments and outcomes
IVIg, n (%) 10 (100) 0 (0) 5 (100) ,0.001
Corticosteroids, n (%) 5 (50) 0 (0) 0 (0) 0.003
Duration of hospitalization, d, median (IQR) 9 (7.3–11.5) 17 (4–48) 4 (2–5) 0.073
ICU admission, n (%) 10 (100) 5 (50) 1 (20) 0.005
Duration of ICU, d, median (IQR) 7 (5.3–8.8) 3 (0–15) 0 (0) 0.28
Respiratory failure (requiring HFNC or greater), n (%) 7 (70%) 5 (50%) 1 (20%) 0.186
Shock (inotropes or vasopressors), n (%) 10 (100) 2 (20) 0 (0) ,0.001
Statistical comparisons were made by using 1-way ANOVA for continuous variables and x2 tests for categorical variables. P values #.05 were considered statistically significant. HFNC,
high-flow nasal cannula.
a Subjects may have .1 underlying medical condition.
b Laboratory values represent the maximum white blood cell count, minimum absolute lymphocyte count, minimum platelet count, minimum serum sodium level, maximum alanine

aminotransferase level, maximum CRP level, maximum ESR, and maximum D-dimer level obtained before administration of IVIg (if given) or during hospitalization (if IVIg not given).
c Only 9 children with COVID-19 and 1 child with KD had D-dimer tests performed. The P value for D-dimer represents a 2-tailed Student’s t test for comparing COVID-19 and MIS-C.

absolute lymphopenia, and received corticosteroids (Table 1, late-onset cardiovascular

thrombocytopenia. Supplemental Table 3). All children complications after hospital
Gastrointestinal symptoms with MIS-C and KD were ultimately discharge.
were predominant at the time of discharged from the hospital.
presentation, although half of However, 2 children with COVID-19 Serological Responses
the children with MIS-C also had and acute myelogenous leukemia All children with MIS-C and
respiratory symptoms. Six (60%) died of presumed bacterial sepsis the majority of children with
children developed myocardial after prolonged hospitalizations (48 COVID-19 (90%) had detectable
dysfunction, and 1 child (10%) and 74 days). Another child with immunoglobulin G (IgG) antibodies
developed a borderline COVID-19 remained hospitalized to SARS-CoV-2 spike protein RBD
coronary ectasia. Children with MIS- for management of underlying (Fig 1A). The child with COVID-19
C were significantly more likely to comorbidities at the time of who had undetectable antibodies
require admission to the ICU (P = publication. Of the 9 children had only mild respiratory
.005) and to have shock requiring with MIS-C who had $3 weeks symptoms. In contrast, children with
inotropes or vasopressors (P , of outpatient follow-up data KD had minimally reactive antibodies,
.001) compared with children with available, all had resolution or near and all hospitalized controls had
COVID-19 or KD. All children with resolution of their clinical signs and undetectable antibody responses.
MIS-C received IVIg, and half symptoms. None had developed When statistically compared, children

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4 ROSTAD et al
 trigger of KD in the absence of clinical
findings of MIS-C.
Live-virus neutralization assays
were performed by using an
mNeonGreen fluorescently labeled
SARS-CoV-2 virus (Fig 1C). Whereas
all children with MIS-C had
detectable neutralization titers to
SARS-CoV-2, only 30% of children
with COVID-19 had neutralizing
antibodies. Children with MIS-C had
significantly higher neutralizing
antibody titers than children with
COVID-19 (P = .006), children with
KD (P = .001), and hospitalized
controls (P = .002). RBD IgG binding
titers strongly correlated with
live-virus neutralization titers,
as determined by linear regression
of the log-transformed titers (R2 =
0.674; P , .001) (Fig 1D). These
data are consistent with previous
studies in which it was found that
the RBD IgG was highly
discriminatory for neutralizing
antibodies in patients with COVID-
19,8 with a sensitivity and
specificity of 97.5% and 98%,
respectively.16
All children with MIS-C and most
FIGURE 1
SARS-CoV-2 spike protein RBD and neutralizing antibodies. A and B, ELISAs to SARS-CoV-2 RBD children with COVID-19 also had
expressed as IgG (A) and IgM (B) end point titers. C, SARS-CoV-2 neutralizing antibodies expressed as detectable antibodies to the SARS-
FRNT50. D, Correlation between log-transformed RBD IgG titers and FRNT50. R2, coefficient of de- CoV-2 full-length spike and
termination. * P # .05; ** P # .01; *** P # .001; **** P # .0001.
nucleocapsid proteins (Fig 2 A
and B). Children with MIS-C had
with MIS-C had significantly higher titers than hospitalized control significantly higher full-length spike
SARS-CoV-2 RBD IgG titers than patients, there were not statistical and nucleocapsid antibody titers
children with COVID-19, children with differences between the other groups. than children with COVID-19,
KD, and other hospitalized controls Interestingly, 1 child with incomplete children with KD, and hospitalized
(P , .001 for each comparison). KD had a high RBD IgM titer and controls (P , .001 for all
Although the timing of sample a negative IgG titer result. Although comparisons). Both spike and
collection after the onset of illness was this child had tested negative for nucleocapsid antibody titers
not standardized (Supplemental Fig 5), SARS-CoV-2 by nasopharyngeal RT- correlated highly with RBD
the days from symptom onset did not PCR and qualitative nucleocapsid IgG antibody titers (R2 = 0.956
differ significantly among children antibody assay (Abbott Laboratories), [P , .001] and R2 = 0.846 [P , .001],
with MIS-C and COVID-19 (P = .816). her parent did report that she had respectively) (Fig 2 C and D), which
a respiratory illness 2 weeks implied that the immune responses
We observed that all children with preceding her presentation. The child observed were broadly reactive to
MIS-C and most children with COVID- did not meet the case definition of SARS-CoV-2 and not specific to
19 (80%) also had detectable MIS-C because she did not have a particular antigen. Interestingly,
immunoglobulin M (IgM) antibodies multiorgan involvement. all children with KD had low-level
to SARS-CoV-2 RBD (Fig 1B). Nevertheless, her positive IgM titer seropositivity to the nucleocapsid
Although children with MIS-C had result suggests that acute SARS-CoV-2 protein. This finding may have been
significantly higher IgM antibody infection may have been an infectious attributable to the receipt of IVIg

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PEDIATRICS Volume 146, number 6, December 2020 5
because the nucleocapsid protein
shares greater amino acid identity
with the circulating endemic human
coronavirus strains, resulting in
a greater degree of antibody cross-
reactivity compared with the spike
protein.19

We next performed linear regression

analyses to identify correlations
between RBD IgG antibody titers and
MIS-C clinical and laboratory features.
We found that RBD IgG antibody
titers correlated positively with the
peak ESR obtained before IVIg
administration (n = 8; R2 = 0.512;
P = .046) (Fig 1E) but not with
peak CRP values (R2 = 0.026; P =
.655; data not shown). RBD IgG
antibody titers also correlated
with total hospital length of stay
(LOS) (n = 10; R2 = 0.548; P = .014;
data not shown) and ICU LOS
(n = 10; R2 = 0.590; P = .010)
(Fig 1F), which were collinear
outcome variables. Because all
children with MIS-C in our cohort
required ICU admission and
inotropes or vasopressors for FIGURE 2
SARS-CoV-2 spike and nucleocapsid protein antibodies. A and B, ELISAs to SARS-CoV-2 full-length
shock, and no children with MIS-C spike IgG end point titers (A) and nucleocapsid protein total antibody end point titers (B). C and D,
died, the correlation between Correlation between log-transformed RBD IgG titers and spike IgG end point titers (C) or nucleo-
antibody titers and these patient capsid protein end point titers (D) by linear regression. *** P # .001; **** P # .0001.
outcomes could not be ascertained
(Fig 3). SARS-CoV-2 RBD IgG strongly of SARS-CoV-2 full-length
correlated with metrics of systemic spike and nucleocapsid protein
inflammation (ESR) and clinical antibodies compared with other
DISCUSSION outcomes (hospital and ICU cohorts, indicating a broad and
We demonstrated that all children LOS). Children with MIS-C also robust immune response to
with MIS-C in our cohort had demonstrated higher levels SARS-CoV-2. Nevertheless,
high titers of SARS-CoV-2 RBD
IgG antibodies, which correlated
with live-virus neutralization.
Children with MIS-C also had
significantly higher RBD antibody
titers than children with COVID-19,
children with KD, or hospitalized
controls. These results suggest
that quantitative SARS-CoV-2
RBD IgG serology may be helpful
in establishing the diagnosis of
MIS-C and distinguishing it from
other syndromes with similar
clinical appearances, such as FIGURE 3
KD. Quantitative serology may Serological correlations in children with MIS-C. A and B, Correlation between RBD IgG end point
also have prognostic value because titers and ESR (A) (millimeters per hour; n = 8) and ICU LOS (B) (n = 10) in children with MIS-C.

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6 ROSTAD et al
the RBD antigen may be well CoV-2 seropositivity (98%). In children with high viral loads may
suited for quantitative antibody contrast, children in class 2 (29.6%) develop robust immune responses,
measurements because of its had a high frequency of respiratory even in the absence of clinical
relative ease of expression, disease (76.3%) and SARS-CoV-2 RT- symptoms.
potent immunogenicity,8,20 PCR positivity (84.0%), indicating
The magnitude of antibody responses
and lack of cross-reactivity that their disease manifestations
also correlate with the timing from
with endemic coronavirus coincided with and may have been
SARS-CoV-2 exposure and symptom
strains.8,21 primarily attributable to COVID-19.
onset. Although the incubation period
Children in class 3 (34.7%) had
Quantitative serology may also for COVID-19 has a median time of 4
clinical features more closely
provide clues about MIS-C disease to 5 days from exposure to symptom
resembling KD, including younger
pathogenesis and the timing onset,29 the window period between
age, higher frequencies of rash
of MIS-C symptom onset after SARS-CoV-2 exposure and the
and mucocutaneous involvement,
SARS-CoV-2 infection. To date, development MIS-C has not been well
and milder clinical disease course.23
the pathogenesis of MIS-C is established. In the minority of
On the basis of these descriptions,
incompletely understood. Its temporal children who recall preceding viral
it is likely that 8 (80%) children
association after peak SARS-CoV-2 symptoms, the number of days to
in our cohort would have been
transmission in certain regions has led MIS-C onset has ranged from 6 to
categorized as having class
some to hypothesize that MIS-C is 79.6,7,10 This wide range likely
1 MIS-C, whereas 2 (20%) children
attributable to postinfectious immune impacts SARS-CoV-2 antibody titers,
would have been categorized
dysregulation and which are known to increase by day 7
hyperinflammation.22 No children as having class 2 MIS-C. This and plateau at day 14 after onset of
with MIS-C in our study recalled implies that the serological COVID-19 symptoms.27 Further
having an antecedent febrile or findings in our study may be longitudinal studies are needed to
respiratory illness. However, all had most useful in distinguishing determine if convalescent antibody
detectable RBD IgM antibody titers, children with class 1 MIS-C and titers in children recovering from
indicating a recent SARS-CoV-2 identifying children with class COVID-19 rise to similar levels
infection. Interestingly, RBD IgM 2 MIS-C at risk for severe disease. as those in children with MIS-C.
antibodies did not correlate with Differentiating these clinical These data would help distinguish
any clinical parameters, and the and laboratory phenotypes is whether the magnitude of antibody
majority of children with MIS-C necessary for risk stratification titers observed in children with MIS-C
had negative SARS-CoV-2 because of the differences in represented an exaggerated immune
nasopharyngeal RT-PCR results, clinical management and patient response to SARS-CoV-2, a delayed
suggesting that active SARS-CoV-2 outcomes. complication of COVID-19, or
infection was not driving MIS-C a combination of both.
Serological studies in adults have
pathogenesis in the majority of
demonstrated that SARS-CoV-2 Our study has several limitations,
patients in our cohort. However, 2
antibody titers correlate with including a small sample size. Mild
children had positive SARS-CoV-2
COVID-19 disease severity.24–27 or moderate clinical phenotypes of
nasopharyngeal RT-PCR results
Thus, it seems paradoxical that MIS-C may have been under-
with concurrent respiratory
children in our study without represented because of a lack of
symptoms at the time of their
preceding viral symptoms recognition early during the
MIS-C presentations, suggesting
consistent with acute SARS-CoV-2 pandemic. This underscores the need
that MIS-C can coincide with acute
infection would develop such for collaboration among pediatric
COVID-19.
robust immune responses as centers to corroborate clinical and
Recently, Godfred-Cato et al23 demonstrated by high binding laboratory findings associated with
differentiated 3 distinct and neutralizing antibody titers. varying clinical phenotypes of MIS-C
categorizations of MIS-C in 570 US Recent data have suggested that in larger patient cohorts. Second,
children using a statistical modeling children with mild to moderate detection of SARS-CoV-2 binding and
technique that grouped cases on the COVID-19 symptoms can harbor neutralizing antibodies in children
basis of their underlying clinical significantly higher nasopharyngeal with MIS-C demonstrates association
similarities. Children in class 1 SARS-CoV-2 viral loads than but not necessarily causation. Also,
(35.6%) had severe clinical adults with similar symptoms, because the seroprevalence of SARS-
manifestations with a high frequency as represented by low RT-PCR CoV-2 antibodies increases, the
of multiorgan involvement, shock, cycle threshold values.28 This clinical significance of these
cardiovascular disease, and SARS- raises the possibility that some antibodies may eventually become

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PEDIATRICS Volume 146, number 6, December 2020 7
obscured. Several patients with MIS-C quantitative SARS-CoV-2 RBD
had specimens drawn after antibody titers may have a role in
ABBREVIATIONS
administration of IVIg, which establishing the diagnosis of MIS-C, ANOVA: analysis of variance
conceivably could have affected distinguishing it from other similar CDC: US Centers for Disease Con-
SARS-CoV-2 antibody titers. However, clinical entities, and stratifying risk trol and Prevention
4 of 5 patients with KD also received for adverse outcomes. CHOA: Children’s Healthcare of
IVIg before specimen collection, with Atlanta
negligible effects on RBD titers. COVID-19: coronavirus disease 2019
Although RBD IgG antibody titers CRP: C-reactive protein
ACKNOWLEDGMENTS
correlated well with the ESR, they did ELISA: enzyme-linked immuno-
not correlate with the acute-phase We thank our colleagues in clinical sorbent assay
reactant CRP. This may have been, in care, the clinical and research ESR: erythrocyte sedimentation rate
part, attributable to the timing of laboratories, and the clinical FRNT50: focus-reduction neutrali-
sample collection and the response research nurses and coordinators, zation test
kinetics of ESR to systemic without whom this study would not IgG: immunoglobulin G
inflammation; however, the ESR can have been possible. We thank Dr IgM: immunoglobulin M
be affected by multiple factors, and Jason McLellan from the Department IQR: interquartile range
these observations warrant further of Molecular Biosciences, The IVIg: Intravenous immunoglobulin
investigation. University of Texas at Austin, for KD: Kawasaki disease
kindly providing the HexaPro pre- LOD: limit of detection
fusion stabilized full-length spike LOS: length of stay
CONCLUSIONS protein. We thank our colleagues in MIS-C: multisystem inflammatory
We found that all children with radiology, Drs Bradley S. Rostad and syndrome in children
MIS-C in our cohort had high Sarah S. Milla, who collaboratively RBD: receptor-binding domain
titers of SARS-CoV-2 RBD IgG interpreted radiographic images to RT-PCR: real-time polymerase
antibodies, which correlated with contribute to early drafts of this chain reaction
viral neutralization. RBD IgG article. We thank all the study SARS-CoV-2: severe acute respira-
antibodies also correlated with participants and their families, and tory syndrome
metrics of systemic inflammation and we thank our families for their coronavirus 2
clinical outcomes. Thus, measuring steadfast support.

DOI: https://doi.org/10.1542/peds.2020-018242
Accepted for publication Aug 27, 2020
Address correspondence to Preeti Jaggi, MD, Department of Pediatrics, School of Medicine, Emory University, 2015 Uppergate Dr NE, Atlanta, GA 30322.
E-mail: preeti.jaggi@emory.edu
PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275).
Copyright © 2020 by the American Academy of Pediatrics
FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose.
FUNDING: Supported by the Center for Childhood Infections and Vaccines of Children’s Healthcare of Atlanta and Emory University, the Georgia Research Alliance,
a Synergy Award from Emory University School of Medicine, and a Fast Grant from Emergent Ventures at the Mercatus Center at George Mason University. The
Center for Childhood Infections and Vaccines, the Georgia Research Alliance, George Mason University, and Emory University had no role in the design or conduct of
this study.
POTENTIAL CONFLICT OF INTEREST: Dr Anderson has received personal fees from AbbVie and Pfizer for consulting; Dr Rostad receives royalties unrelated to this
article to Emory University from Meissa Vaccines, Inc; Drs Anderson and Rostad’s institution receives funds to conduct clinical research unrelated to this article
from MedImmune, Regeneron, PaxVax, Pfizer, GlaxoSmithKline, Merck, Novavax, Sanofi-Pasteur, and Micron; Drs Menachery and Shi coinvented the reverse genetics
system used to generate the live severe acute respiratory syndrome coronavirus 2 virus described in this article (a patent has been applied for this technology, and
it has been licensed for commercial use); the other authors have indicated they have no potential conflicts of interest to disclose

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PEDIATRICS Volume 146, number 6, December 2020 9
Quantitative SARS-CoV-2 Serology in Children With Multisystem Inflammatory
Syndrome (MIS-C)
Christina A. Rostad, Ann Chahroudi, Grace Mantus, Stacey A. Lapp, Mehgan
Teherani, Lisa Macoy, Keiko M. Tarquinio, Rajit K. Basu, Carol Kao, W. Matthew
Linam, Matthew G. Zimmerman, Pei-Yong Shi, Vineet D. Menachery, Matthew E.
Oster, Srilatha Edupuganti, Evan J. Anderson, Mehul S. Suthar, Jens Wrammert and
Preeti Jaggi
Pediatrics 2020;146;
DOI: 10.1542/peds.2020-018242 originally published online September 2, 2020;

Updated Information & including high resolution figures, can be found at:
Services http://pediatrics.aappublications.org/content/146/6/e2020018242
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Quantitative SARS-CoV-2 Serology in Children With Multisystem Inflammatory
Syndrome (MIS-C)
Christina A. Rostad, Ann Chahroudi, Grace Mantus, Stacey A. Lapp, Mehgan
Teherani, Lisa Macoy, Keiko M. Tarquinio, Rajit K. Basu, Carol Kao, W. Matthew
Linam, Matthew G. Zimmerman, Pei-Yong Shi, Vineet D. Menachery, Matthew E.
Oster, Srilatha Edupuganti, Evan J. Anderson, Mehul S. Suthar, Jens Wrammert and
Preeti Jaggi
Pediatrics 2020;146;
DOI: 10.1542/peds.2020-018242 originally published online September 2, 2020;

The online version of this article, along with updated information and services, is
located on the World Wide Web at:
http://pediatrics.aappublications.org/content/146/6/e2020018242

Data Supplement at:

http://pediatrics.aappublications.org/content/suppl/2020/11/11/peds.2020-018242.DCSupplemental

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