key: cord-1005314-quk343zo authors: Interiano, Cristina; Muze, Sheicho; Turner, Brian; Gonzalez, Mark; Rogers, Beverly; Jerris, Robert; Weinzierl, Elizabeth; Elkhalifa, Mohamed; Leung-Pineda, Van title: Longitudinal Evaluation of the Abbott Architect SARS-CoV-2 IgM and IgG Assays in a Pediatric Population date: 2021-02-19 journal: Pract Lab Med DOI: 10.1016/j.plabm.2021.e00208 sha: f7575a3dc1d00f26edfae9bc2341386df6f95290 doc_id: 1005314 cord_uid: quk343zo BACKGROUND: Clinical laboratory testing has been an essential part of COVID-19 management. Serology can provide valuable information regarding a patient’s exposure to virus, and may have a larger role to play as vaccines becomes available. Limited data is available on the serological response in pediatric patients. Here we investigate the use of one manufacturer’s commercial assays for detecting IgM and IgG in an exclusively pediatric population. METHODS: Abbott SARS-CoV-2 IgM and IgG assays were performed on an Abbott Architect i1000. For specificity studies, we tested 78 patient specimens collected before the COVID-19 pandemic, and 66 specimens from patients who tested negative for SARS-CoV-2 nucleic acid amplification test (NAAT) during the COVID-19 pandemic. For sensitivity we tested 181 specimens from 41 patients with a positive NAAT result. Precision data was acquired for 20 days. RESULTS: For IgM, the highest qualitative positive agreement with molecular results was observed to be 15 to 30 days after a positive NAAT result or after symptom onset. For IgG, the highest positive agreement was 31-60 days after a positive NAAT result or 61-90 days after the start of symptoms. IgM started to decline 30 days after NAAT results and faded by 90 days. IgG started to decrease 60 days after a positive NAAT result. CONCLUSION: The Abbott IgM and IgG assays have negative agreements of 98.7-100% relative to NAAT results. The IgM and IgG levels assayed by these methods start to decline months after positive molecular results and onset of symptoms in a pediatric population. The recent pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing Coronavirus disease-19 in 2020 has revealed many novel characteristics previously not seen with other coronavirus caused illnesses. Among patients, the symptoms and degree of illness can vary significantly. One particular feature is the discrepancy in how COVID-19 affects the pediatric population as compared to other groups. Children are traditionally more affected by coronavirus infections, however SARS-CoV-2 does not follow that pattern (1) . Clinical laboratory testing has played a crucial role in managing this illness, from molecular assays used to diagnose acute infections to serological assays that can inform past exposure. At (12) . In this article, we evaluated the performance of two serological assays made by Abbott, to detect anti-SARS-CoV-2 IgM and IgG in children. Furthermore, we profiled the longitudinal antibody response to SARS-CoV-2 infection with these methods. To our knowledge, this is a unique study looking at the longitudinal serological response to SARS-CoV-2 infection in a pediatric population using the Abbott methods. All samples tested were residual samples left over from clinical testing. Our presumptive negative samples were derived from 2 groups. One consisted of 78 samples from the pre-COVID-19 era. These specimens were collected from children in the Atlanta area prior to February of 2020. The second group consisted of 66 specimens collected in April and May of 2020 from patients who had a negative RT-PCR result for SARS-CoV-2. A third group of samples was collected from patients who had a positive NAAT result for SARS-CoV-2. For this group, date of symptom onset was derived from information from the medical record. This study was approved by the institutional review board (IRB) at Children's Healthcare of Atlanta. Serology testing was performed by the Abbott Architect i1000 instrument using the Abbott SARS-CoV-2 IgG and the AdviseDx SARS-CoV-2 IgM assays (Abbott Park, IL). Both assays are chemiluminescent microparticle immunoassays that give a qualitative result of "Positive" or "Negative" based on a numerical index value and a cutoff based on the sample result divided by calibrator result (S/C). The IgG assay targets the nucleocapsid protein and has a cutoff value of 1.4. The IgG assay was authorized by the FDA under Emergency Use Authorization (EUA) in April 2020. The IgM assay targets the spike protein and has a cutoff value of 1.0 and was authorized by the FDA under an EUA in October 2020. Nucleic Acid Amplification Test (NAAT) detection of SARS-CoV-2 from nasopharyngeal swabs was performed using the Simplexa COVID-19 Direct assay on the Diasorin Liason MDX thermocycler system (Cypress, CA) targeting the ORF1ab and S genes, for the majority of samples. An alternate assay was used for 2 of the NAAT positives: the Hologic Aptima SARS-CoV-2 (Marlborough, MA) assay targeting the ORF1ab gene. To study the precision of the assay, we ran 2 levels of controls for each assay for 20 days. The results are presented in Table 1 . The negative and positive controls gave the expected results each time and the precision statistics were similar to the manufacturer's data (13, 14) . To assess the specificity of the assays we first assayed 78 samples that were collected prior to the COVID-19 era (before February of 2020) from pediatric patients at our institution. Forty four (56%) were specimens from female patients and 34 (44%) from male patients. The median age of this cohort was 12.5 years old, ranging from 11 days to 19 years old. One specimen was from September of 2017; eleven of the specimens were from January and March of 2018; 50 of the specimens were from March, April, and June to December of 2019, and 2 specimens were from January of 2020. Results are presented in Table 2 . Of the 78 specimens none tested positive for IgG, but one did test positive for IgM. The Index (S/C) for the sample was 1.12 (cutoff is ≥ 1), and the sample was collected 6/25/2019, from a patient visiting the ED with GI symptoms and non-reactive hepatitis B and HIV serology. Specificity of the serology assays was tested on a second cohort of samples from patients who had a negative RT-PCR result for SARS-CoV-2. The leftover samples used for serology from these patients corresponded from same day to 3 days after collection of the NAAT sample. We tested samples from 66 patient samples, consisting of 40 males and 26 females. This cohort had a median age of 5.5 years old ranging from 28 days old to 20 years old, and samples were collected in April and May of 2020. Results are presented in Table 3 . Of the 66 specimens tested in this group, all results were negative for both IgM and IgG. To determine assay performance, we analyzed SARS-CoV-2 IgM and IgG reactivity on samples from pediatric patients who tested positive for SARS-CoV-2 RNA by a NAAT. This cohort was composed of samples from 41 patients with the characteristics presented in Table 4 . The median age of this group was 14 years, ranging from 12 days to 19 years. A total of 181 samples were analyzed for both IgM and IgG. Four of the 41 patients were asymptomatic for COVID-19. Most were samples from inpatients (80%), including the COVID-19 asymptomatic patients. The remaining 8 were outpatients, with 5 of those coming from emergency department visits. We first looked at the positive percent agreement (PPA) between the serologic assays and the molecular NAAT results (Figure 1 Figure 1A) , PPA was 38% and 36% for IgM and IgG, respectively, at 0-7 days, and 80% for both IgM and IgG at 8 to 14 days. For the time period of 15-30 days after a positive NAAT result, PPA peaked at 100% for IgM and 83% for IgG. PPA values for IgM then decreased to 77% for the time between 31-60 days and decreased significantly after 60 days to values of 25% or less. Agreement numbers for IgG stayed between 80% and 89% for the time periods between 8 and 120 days after a positive NAAT result (80%, 83%, 87%, 70% and 89% respectively) and declined to 38% after 120 days after a positive NAAT result. Alternatively the serologic data based on days after symptom onset ( Figure 1B) showed that IgM's PPA peaked in the time period of 15-30 days at 91%, decreased to 85% at 31-60 days and rapidly declined to 36%, 9% and 0% in the three time periods after 60 days. For IgG, the PPA increased steadily from 0-7 days after symptom onset (14%) to 8-14 days (52%) to 15-30 days (71%) to 31-60 days (88%), and peaked at 100% at 61-90 days. The PPA for IgG started to decline after day 90, first to 64% at 91-120 days and then to 17% after 120 days. Next we analyzed the data based on the quantitative index output (Figure 2) . Although the output of both tests is a qualitative result, there is an associated numerical index that increases with higher antibody concentration. We analyzed these numerical indices for the same time groups described above after a positive molecular result (Figure 2A-B We present here our evaluation of Abbott's anti-SARS-CoV-2 IgM and IgG tests with exclusively pediatric samples. Our precision study results yielded results similar to those obtained from the manufacturer based on their package insert information (13, 14) . Our specificity studies showed that on samples from patients with a negative molecular result for SARS-CoV-2 our negative agreement was 100% for both IgM and IgG. When testing samples collected previous to COVID- 19 we found that one specimen collected on 6/25/2019 tested positive for IgM. The Index (S/C) of this specimen was 1.12, just above the cutoff of 1 for the IgM assay. Due to the timing of the specimen, this is likely a false positive result. Our NPA was 98.7 % for the IgM assay based on the pre-COVID-19 cohort and NPA of 99.3% for the overall negative cohort, slightly lower than the 99.56% reported by the manufacturer in their package insert (13) . Pre-COVID-19 specimens showed 100% negative agreement with IgG. Samples were limited to residual specimens, excluding the possibility of a randomized study. For the specificity cohort, we could not evaluate cross reactivity of the assays to other coronaviruses, as we didn't know if any of the specimens had antibodies to non-novel coronaviruses in their systems, especially for the pre-COVID-19 group. We only obtained test results from one manufacturer that targets the nucleocapsid (N) for IgG and spike (S) for IgM, so the concentrations of antibodies to other SARS-CoV-2 antigens is unknown (i.e. IgG against spike protein). Finally, as the majority of positive patients in this study were inpatient (80%), and due to limited clinical information, it is possible that some patients had medical co-morbidities that could affect their immunological response. Comparison of the positivity rate of the NAAT and IgG assays at our institution showed that a first peak in IgG positivity in August followed the first peak in NAAT results in July, and then IgG had a second peak concurrent with NAAT positives in December. Of importance is that the NAAT positive population is not the same as the IgG positive population, as the NAAT group is much larger and would include pre surgical and other outpatients, whereas the IgG positive group is inpatient and much smaller. However, it is noteworthy that there is a temporal relationship between the positivity rate of both tests. When looking at duration of IgM detection in our study group, it starts to decrease by 30 days (19) . From our limited set of data it appears that the pediatric immunological response follows a similar trend to that seen in adult studies. Our results could be helpful in assessing the serological response of children to the COVID-19 infection and to define immunity trends to help design preventive measures for outbreak policies for school children (20) . One interesting unknown is what the relationship is between antibody levels detected with the assays used in this study to actual neutralization of the virus. Recent reports have shown that results of antibody concentrations by commercial platforms may have varied viral neutralization capacities (21, 22) . Other future directions would include assessing the role of these serological assays in assessing the efficacy of SARS-CoV-2 vaccines. However, the IgG assay in this study would not be effective for recipients of vaccines targeting the Spike protein, because it detects antibodies against the nucleocapsid protein. 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The authors wish to thank Kevin Pannell and Wilfred Morales for collecting residual specimens and Randal Schneider for coordinating reagent procurement.