key: cord-291987-zpkzzldu authors: To, Kelvin KW; Chua, Gilbert T.; Kwok, Ka Li; Wong, Joshua SC; Au, Dennis Chi Yu; Lam, Yuen Yu; Wong, Wilfred HS; Ho, Marco HK; Chan, Godfrey CF; Chui, Celine SL; Li, Xue; Tung, Keith TS; Wong, Rosa S; Tso, Winnie WY; Wong, Ian CK; Wong, Christina SM; Fong, Carol HY; Chan, Kwok Hung; Yuen, Kwok Yung; Ip, Patrick; Kwan, Mike YW title: False-positive SARS-CoV-2 serology in three children with Kawasaki disease date: 2020-07-17 journal: Diagn Microbiol Infect Dis DOI: 10.1016/j.diagmicrobio.2020.115141 sha: doc_id: 291987 cord_uid: zpkzzldu BACKGROUND: Kawasaki disease (KD) is an acute febrile and eruptive disease with systemic vasculitis predominantly affecting young East Asian children. Recent reports showed that children with KD-like disease from KD low prevalence regions had positive SARS-CoV-2 serology despite a negative SARS-CoV-2 polymerase chain reaction (PCR) in respiratory samples. OBJECTIVES: To describe three pediatric Kawasaki Disease patients with false positive SARS-CoV-2 serology. STUDY DESIGN: We retrospectively recruited children with KD diagnosed during the COVID-19 outbreak in Hong Kong. Clinical characteristics and laboratory test results including SARS-CoV-2 PCR results were retrieved. We performed a microparticle-based immunoassay for the detection of IgG against nucleoprotein (NP) and spike protein receptor binding domain (RBD), and a microneutralization assay for the detection of neutralizing antibodies. RESULTS: Three Chinese children with typical KD were identified. They had no epidemiological links with COVID-19 patients and tested negative for SARS-CoV-2 NPA PCR. They were treated with IVIG and aspirin, and were discharged without complications. Subsequently two of them were tested positive against anti-RBD and anti-NP antibodies and one was tested positive against anti- RBD antibodies. However, microneutralization assay showed that neutralizing antibodies were absent, suggesting a false-positive IgG result. CONCLUSION: Detection of neutralizing antibodies is recommended to confirm previous SARS-CoV-2 infection in IgG-positive but PCR-negative patients. Kawasaki disease (KD) is an acute febrile and eruptive disease with systemic vasculitis predominantly affecting young East Asian children. Recent reports showed that children with KD-like disease from KD low prevalence regions had positive SARS-CoV-2 serology despite a negative SARS-CoV-2 polymerase chain reaction (PCR) in respiratory samples. To describe three paediatric Kawasaki Disease patients with false positive SARS-CoV-2 serology. We retrospectively recruited children with KD diagnosed during the COVID-19 outbreak in Hong Kong. Clinical characteristics and laboratory test results including SARS-CoV-2 PCR results were retrieved. We performed a microparticle-based immunoassay for the detection of IgG against nucleoprotein (NP) and spike protein receptor binding domain (RBD), and a microneutralization assay for the detection of neutralizing antibodies. Three Chinese children with typical KD were identified. They had no epidemiological links with COVID-19 patients and tested negative for SARS-CoV-2 NPA PCR. They were treated with IVIG and aspirin, and were discharged without complications. J o u r n a l P r e -p r o o f 6 Kawasaki disease (KD) is an acute systemic vasculitis complicated by coronary aneurysms that predominantly occurs in young East Asian children. Typical symptoms include fever for more than 5 days, exanthema, lymphadenopathy, conjunctival injection, altered oropharyngeal mucosa, and extremity changes. The etiology of KD remains uncertain and cases remain rare. 1 Nevertheless, an upsurge of KD cases in Europe was observed during the Coronavirus Disease 2019 (COVID- 19) pandemic. Out of the ten KD cases reported in children from Bergamo, Italy, two tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by PCR, whereas eight tested positive for SARS-CoV-2 antibodies. 2 A similar cluster of KD cases in children was reported in France during the outbreak, in which 14 children tested positive for SARS-CoV-2 IgG, but only seven children tested positive for SARS-CoV-2 by PCR. As KD is known to be more prevalent in Hong Kong than in Therefore, serological testing for SARS-CoV-2 was offered to Hong Kong children diagnosed with KD between January to April 2020. We aim to describe three paediatric Kawasaki Disease patients diagnosed during the COVID-19 outbreak with false positive SARS-CoV-2 serology. Blood (5 mL) was collected from each patient and serum was obtained for the detection of IgG against SARS-CoV-2 nucleoprotein (NP) and spike protein receptor binding domain (RBD) using a microsphere-based antibody assay as described previously. 5 IgM was not measured in this assay. Briefly, cloning and purification of SARS-CoV-2 NP and spike RBD were performed as described previously. 6 Virus culture and MN assay were performed as previously described. 7 8 Briefly, serum samples (50 L) were prepared in minimum essential medium and mixed with SARS-CoV-2 (50 L) to give a serum dilution of 1:10 and a final virus inoculum of 100 TCID 50 . The serum-virus mixture was incubated at 37C for 1 hour and then added to VeroE6 cells and incubated at 37C and 5% CO 2 for 72 hours. Cytopathic effects were determined under inversion microscopy. The MN antibody titer was determined as the highest dilution showing 50% inhibition of CPE. An MN titer ≥10 was considered positive as described previously. 9 Three Chinese children, who had no epidemiological links with COVID-19 patients were diagnosed with typical KD during the peak of COVID-19 outbreak in Hong Kong (Table 1) 10 11 We believe the false positive SARS-CoV-2 serology results were unrelated to the administration of IVIG for treating KD. First, the time interval between IVIG administration and serology testing ranged between 60 and 90 days, whereas the median half-life of IVIG is 21 days. 12 Second, the IVIG treatment, Intragam® P (CSL Behring Asia Pacific Limited), was manufactured from the pooled plasma of healthy Hong Kong blood donors and had been made months before its use, 13 and therefore unlikely to contain any plasma from donors with COVID-19, as the first case of COVID-19 was diagnosed in Hong Kong in late January 2020. 14 The association between KD and COVID-19 remains unclear. To date, a total of four case series and reports on 40 patients have proposed a possible link between COVID-19 and KD. [15] [16] [17] [18] Although 11 patients were confirmed to have COVID-19 by a positive SARS-CoV-2 PCR in respiratory specimens, the other patients were reported to have positive SARS-CoV-2 serology without confirmation by microneutralization assay (Table 2) . Thus, the possibility that these patients might have false positive results cannot be ruled out. Clinically, not all the patients in these case series presented with KD symptoms reported in cases in Asia, which typically involved younger children under 4 years and commonly included symptoms such as erythema and swelling of BCG scars. 19 Epidemiologically, there was a 30-fold increase in the monthly incidence of KD in Italy during the COVID-19 pandemic, whereas the incidence of KD in Hong Kong remained the same (unpublished data). Furthermore, we reviewed all NPS samples from KD patients admitted to public hospitals in Hong Diagnosis, Treatment, and Long-Term Management of Kawasaki Disease: A Scientific Statement for Health Professionals From the American Heart Association Kawasaki-like disease: emerging complication during the COVID-19 pandemic Epidemiology of Kawasaki disease in Asia, Europe, and the United States Kawasaki disease in Hong Kong Improved detection of antibody against SARS-CoV-2 by microsphere-based antibody assay Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study Seroprevalence of SARS-CoV-2 in Hong Kong and in residents evacuated from Hubei province, China: a multicohort study Consistent detection of 2019 novel coronavirus in saliva SARS-CoV-2 specific antibody responses in COVID-19 patients False-positive IgM antibody tests for cytomegalovirus in patients with acute Epstein-Barr virus infection False-positive serologies for acute hepatitis A and autoimmune hepatitis in a patient with acute Epstein-Barr virus infection Pharmacokinetics of intravenous immunoglobulin: a systematic review The Production Processes and Biological Effects of Intravenous Immunoglobulin From a Sprint to a Marathon in Hong Kong COVID-19 and Kawasaki Disease: Novel Virus and Novel Case An outbreak of severe Kawasaki-like disease at the Italian epicentre of the SARS-CoV-2 epidemic: an observational cohort study Kawasaki-like multisystem inflammatory syndrome in children during the covid-19 pandemic in Paris, France: prospective observational study Hyperinflammatory shock in children during COVID-19 pandemic Causes of Kawasaki Disease-From Past to Present EV/RV -enterovirus/rhinovirus, IVIG -intravenous immunoglobulin, MN -microneutralization assay