key: cord-0302458-jdxu3rcy
authors: Han, W. G. H.; Swart, A.; Bonacic Marinovic, A.; Eggink, D.; Reimerink, J.; Wijsman, L. A.; van der Veer, B.; van den Brink, S.; van den Brandt, A.-M.; Brouwer, F.; Hoogerwerf, M.; the Dutch FFX-COVID-19 research group,; Reukers, D. F. M.; Rots, N.; Reusken, C.; Meijer, A.
title: SARS-CoV-2 RNA and antibody dynamics in a Dutch household study with dense sampling frame
date: 2021-10-06
journal: nan
DOI: 10.1101/2021.10.06.21263384
sha: 6bd53bfeea8b164717d6d0fa7dd5b070c880a841
doc_id: 302458
cord_uid: jdxu3rcy

This study investigated the dynamics of SARS-CoV-2 infection and diagnostics in household members of different ages and with different symptom severity after SARS-CoV-2 exposure during the early phase of the pandemic. Households with a SARS-CoV-2 confirmed positive case and at least one child in the Netherlands were followed for 6 weeks. Naso (NP)- and oropharyngeal (OP) swabs, oral fluid and feces specimens were analyzed for SARS-CoV-2 RNA and serum for SARS-CoV-2-specific antibodies. The dynamics of the presence of viral RNA and the serological response was modeled to determine the sampling time-frame and sample type with the highest sensitivity to confirm or reject a SARS-CoV-2 diagnosis. Transmission of SARS-CoV-2 between adults and children within a household was correlated with symptom severity of index cases. In children higher viral loads compared to adults were detected at symptom onset. Early in infection, higher viral loads were detected in NP and OP specimens, while RNA in especially feces were longer detectable. SARS-CoV-2-specific antibodies have a 90% probability of detection from 7 days (total Ig) and 18 days (IgG) since symptom onset. In conclusion this study has shown that on average, children carry higher loads of virus as compared to adults early after infection. For highest probability of detection in SARS-CoV-2 diagnostics early in infection, RT-PCR on NP and OP specimens are more sensitive than on oral fluid and feces. For SARS-CoV-2 diagnostics late after infection, RT-PCR on feces specimens and serology are more valuable.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly across the world 56 since January 2020 [1] . In the Netherlands, the first COVID-19 (the syndrome caused by SARS-CoV-2) 57 case was detected on 27 February 2020. From March until May 2020, the Dutch government 58 mandated a partial lockdown. This included social distancing, self-quarantine and self-isolation orders, 59 closing of schools, bars and restaurants, and urging people to work from home [2] . Yet, households 60 are close-contact settings with high probability of (pre/a-symptomatic) transmission of SARS-CoV-2 61 after introduction of the virus. In this period, a prospective cohort study was performed including 55 62 complete households with a RT-PCR-confirmed SARS-CoV-2 positive case (index case) and at least one 63 child below 18 years of age. All household contacts were tested as soon as possible after a SARS-CoV-64 2 infection in the household was identified. At multiple timepoints, various clinical samples were 65 collected for molecular and serological diagnostics. Using a dense sampling strategy, SARS-CoV-2 66 transmission and kinetics of diagnostic parameters could be closely monitored within the households. 67

Earlier we described that the estimated Secondary Attack Rate (SAR) in this cohort that was high (35% 68 in children, 51% in adults), with reduced susceptibility of children compared to adolescents and adults 69 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted October 6, 2021. ; https://doi.org/10.1101/2021.10.06.21263384 doi: medRxiv preprint categories a generalized linear model (GLM) for count data with Poisson family and exponential link 105 was set up with interactions between the transmission category and severity classes. 106 107 Molecular diagnostics 108 Nasopharyngeal swabs (NP) and oropharyngeal swabs (OP) were collected in gelatin-lactalbumin-109 yeast (GLY) viral transport medium (Mediaproducts BV, Groningen, The Netherlands), transported to 110 the laboratory in a cooling box and stored at maximum a few days at 4° C until being processed for 111 RT-PCR. Feces specimens were self-collected by the patient and send to the laboratory by regular mail, 112 stored frozen at -20° C until being processed for RT-PCR. Oral fluid specimens were collected with a 113 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint This assay is a double-antigen sandwich ELISA using the recombinant receptor-binding domain of 131 SARS-CoV-2 as antigen. Optical density (OD) is measured at 450 nm and the antibody OD ratio for each 132 sample is calculated as the ratio of the OD of that sample to the reading of a calibrator (included in 133 the kit). 134 Sera were tested for the presence of IgG antibodies reactive with the SARS-CoV-2 S1 and SARS-135

CoV-2 N antigens in a protein microarray, in duplicate 2-fold serial dilutions starting at 1:20, essentially 136 as described previously [8] . For each antigen, a 4-parameter loglogistic calibration curve was 137 generated. Antibody titers (EC50 value) were defined as the interpolated serum dilution that gave a 138 fluorescence intensity of 50% of the corresponding calibration curve. Raw data were processed with 139 the R 4.04 statistical software as described previously [9] . 140 141

All available RT-PCR outcomes (Table 1) is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted October 6, 2021. The dynamics of serology cannot be assumed to be linear as is the case for Ct-values. Rather, 168 seronegative individuals have a titer (OD ratio for Wantai or EC50 for protein microarray) varying 169 around a low value, and seropositive individuals have a titer varying around a high value. In the case 170 of the ELISA-test and microarray-based assays used in the current study, we find that a cut-off value 171 to distinguish seropositives and seronegatives works well, since the two components are well 172 separated ( Figure S1 ). Using the cut-off values 1 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted October 6, 2021. ; https://doi.org/10.1101/2021.10.06.21263384 doi: medRxiv preprint we consider differences between Ct-values of less than one as not meaningful. For changes in Ct-value 185 per day (the slope) we choose [-1/7, 1/7], which means that we consider differences between Ct-186 values of less than one per week as not meaningful. For serology detection probability (dps) our ROPE 187 interval is [-2,2], which means that we consider differences between days of less than 2 as not 188 meaningful. The ROPE is compared to the 89% highest posterior density interval (HDI). When the ROPE 189 contains the HDI, no meaningful difference exists, when the ROPE is completely outside of the HDI, 190 there is a difference, when the ROPE and HDI overlap we withhold a decision because of too high 191 uncertainty. 192

193

A total of 242 participants from 55 complete households were included in this study. The number of 196 analyses performed per assay and specimen type at the various timepoints with the day of the first 197 home visit (so the start of the study within the particular household) defined as day 1 are described in 198 Table 1 and Table S1 . To identify different transmission patterns, we visualized SARS-CoV-2 infection 199 detection by the different assays and specimen types per participant and household in heatmaps. We is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted October 6, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted October 6, 2021. per day) ( Figure 5C ). The relevance of these findings is uncertain as there is partial overlap between 258 the ROPE and HDI ( Figure S2 -B1 to E1 and B2 to E2). Overall, as a most likely estimate, the estimated 259 viral load is higher in NP and OP swabs compared to oral fuid until 21.1 dps and 19.0 dps, respectively 260 ( Figure S4-B and D) , and compared to feces until 10.2 dps and 9.7 dps, respectively ( Figure is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted October 6, 2021. ; https://doi.org/10.1101/2021.10.06.21263384 doi: medRxiv preprint (90% detection until 27 dps), compared to NP, OP and oral fluid specimens (90% detection until 19.4, 263 20.3 and 22.7 dps, respectively) ( Figure 5D and Figure S5 -C, E and F). Similar trends are shown for 50% 264 and 10% detection probability ( Figure S5 and S6) . We could not find a clear correlation in severity of 265 symptoms and the dynamics of the SARS-CoV-2 RNA detection (data not shown). 266 Furthermore, we investigated the dynamics of SARS-CoV-2 antibody detection ( Figure 6 ). The 267

Wantai assay (total Ig) demonstrated a higher sensitivity for detection of anti-S1 antibodies than the 268 micro-array (IgG) as the probability for detection was earlier using Wantai upon onset of illness ( Figure  269 6A and S7-A). The dps at which 90% detection probability was reached for Wantai was 7.1 compared 270 to 16.9 and 18 for Nucleoprotein (N)-and S1-protein microarray respectively ( Figure 6B ). The protein 271 microarray for S1 and N had comparable sensitivity, in line with a previous study [8] . The probability 272 of detecting N-specific IgG antibodies in children was delayed by 3.0 days (at 90% probability 273 detection) versus adults ( Figure 6C and D), while this was not the case for detection of S1-specific 274 antibodies ( Figure 6C ). The relevance of this finding is uncertain as there is much overlap between the 275 distributions in children and adults ( Figure S8 ). The N-specific IgG antibody titers (at visit 3; 276 convalescent phase) were not significantly lower in children compared to adults ( Figure S9 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted October 6, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted October 6, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted October 6, 2021. ; https://doi.org/10.1101/2021.10.06.21263384 doi: medRxiv preprint respiratory specimens and that beyond 10 dps, feces sampling may be preferred [22, 24, 25] . Although 341 SARS-CoV-2 RNA can remain present in respiratory and feces specimens for a long time, the duration 342 of presence of viable virus is relatively short-lived [26, 27] . Alternatively, from a week after symptom 343 onset testing for the presence of SARS-CoV-2 Spike-specific total Ig using Wantai ELISA can confirm a 344 recent or past SARS-CoV-2 infection. SARS-CoV-2 diagnostics using protein microarray detecting SARS-345

CoV-2 Spike-and Nucleoprotein-specific IgG antibodies, is useful 2 weeks after infection or symptom 346 onset. 347

The infection dynamics of SARS-CoV-2 may be influenced by characteristics of the tested 348 population, such as age and the severity of COVID-19. We, however, could not find a clear correlation 349 in severity of symptoms or age and the RNA and SARS-CoV-2-specific antibody kinetics, although our 350 study may be underpowered to detect these differences (data not shown). Children in general report 351 milder symptoms compared to adults (Table S1) (higher viral loads) at the day of symptom onset compared to adults, while the decay in viral load was 357 comparable ( Figure 5 ) . This suggests that if children become infected with SARS-CoV-2, they can carry 358 high loads of virus for a longer time compared to adults. Therefore, children are potentially longer 359 infectious than adults after symptom onset. Whether this observation holds for new Variants of 360 concern (VOC) e.a. delta, warrants further investigation. No clear differences between adults and 361 children were found in the dynamics of SARS-CoV-2 serology, yet the detection of N-specific antibodies 362 seems slightly delayed in children compared to adults ( Figure 6C, D and S8) . A study showed a reduced 363 breadth of anti-SARS-CoV-2-specific antibodies, predominantly generating IgG antibodies specific for 364 the S protein but not the N protein in children compared to adults [31] . Whether this has 365 consequences for the development of immunity to SARS-CoV-2 is not yet clear. 366 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted October 6, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted October 6, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted October 6, 2021. ; https://doi.org/10.1101/2021.10.06.21263384 doi: medRxiv preprint (Table 1) were incuded in these analyses. Probability SARS-CoV-2-specific antibody 512 detection (A) and average dps from when the Wantai (Spike-specific IgM and IgG), microarray S1 513 (Spike-specific IgG) and microarray N (Nucleoprotein-specific IgG) assays have at least 90% detection 514 probability (B) for all ages. C) Probability SARS-CoV-2-specific antibody detection by Wantai, 515 microarray S1 and microarray N in adults and children D) Average dps from when microarray N has at 516 least 90% detection probability for adults and children. The shadows in (C) and (D) indicate the 95% 517 Bayesian confidence interval. 518 519 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted October 6, 2021. ; Table 1 . Schedule of administering questionnaires, symptom diaries and home visits for sampling. The numbers in the table indicate the amount of analyzed specimens in 242 participants. 1 A naso-and oropharyngeal swab was not collected for the index case at the first home visit, as these persons were already swabbed a few days before and tested SARS-CoV-2 positive. 

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