Page 1 of 17 Article DOI: https://doi.org/10.3201/eid2709.211024 Predictors of Nonseroconversion after SARS-CoV-2 Infection Appendix Methods Ethics and cohort characteristics The University of Alabama at Birmingham (UAB) COVID-19 convalescent cohort was established in March 2020 at the 1917 Clinic and recruited 72 persons by May 2020. Many participants were UAB employees who were made aware of this cohort after being informed that they had tested positive for COVID-19. Part of the initial phone call included information about the study, which was designed to evaluate immune responses following SARS-CoV-2 infection. Participants also heard about the study by word of mouth from other patients or health care providers. All persons were given information where they could schedule an appointment to provide informed consent and enroll into the study, but they were not asked whether they intended to participate. Thus, the fraction of patients who decided not to enroll is unknown. A potential sampling bias includes a predominance of health care professionals interested in the potency of their antiviral immune responses and persons motivated to advance scientific knowledge about SARS-CoV-2 infection and disease. All participants were enrolled after obtaining written informed consent and approval from the Institutional Review Board (IRB-160125005). Participants had a median age of 40 years (range 20–86 years), were 56% male and 44% female, and had diverse racial/ethnic backgrounds (67% Caucasian, 14% African American, 14% Asian, 5% Latinx). Symptom severity was self- reported, with 0 indicating no symptoms, 1 indicating mild symptoms with little impact on daily activities, 2 indicating moderate symptoms with noticeable impact on daily activities, and 3 indicating severe symptoms with a significant reduction in quality of life (Appendix Table 1). Data on hospital admission and stay were obtained from electronic medical records. Eight of the
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Predictors of Nonseroconversion after SARS-CoV-2 Infection
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Appendix Table 1. Demographic, clinical and laboratory characteristics of 72 persons who recovered from SARS-CoV-2 infection*
ID Age Sex Race/
ethnicity† Nucleic acid test
Symptoms§ Symptom severity¶ Hospitalization#
Antibody tests Date DFOS Ct (Lab)‡ Spike PCR DFOS Seroconversion
CR0001 57 F CC 3/23/20 32 38 (FRL) Neg AA, CO, FA 1 No 41, 60, 119 No CR0003 22 M CC 3/20/20 11 24 (ASL) ND AA, CT, DI, DY, MY 1 No 23, 42, 99 Yes CR0004 54 M CC 3/23/20 7 38 (FRL) ND CO, CT, DY, FA, MY, PD, ST 2 No 16, 35, 86 No CR0005 34 F AA 3/14/20 4 22 (ASL) ND CH, CO, FE, HE, MY, NC, NV, ST 2 No 24, 41, 104 Yes CR0006 26 M CC 3/18/20 4 30 (ASL) Neg AA, CH, CO, CT, DY, FA, FE, HE, MY, NC, NS, NV, PD, RH, ST 2 No 20, 39, 103 No CR0007 28 M CC 3/19/20 2 32 (ASL) ND CH, FA, FE, HE, MY, NC 2 No 20, 38 Yes CR0008 38 F CC 3/18/20 10 34 (FRL) Neg AA, CO, CT, DY, FA, MY, NC 1 No 29 No CR0010 42 F CC 3/16/20 3 NA ND AA, CH, CO, CT, DI, DY, FA, FE, HE, MY, NS, NV, PD 2 No 3, 39, 101 Yes CR0011 64 M CC 3/16/20 3 26 (CoA) ND AA, CH, CO, FA, FE, HE, MY, NC, PD, RH 2 No 25, 39, 48 Yes CR0012 40 M CC 3/18/20 2 27 (ASL) ND AA, CH, CO, CT, DY, FA, HE, MY, NC, NS, PD, RH 2 No 22, 35 Yes CR0014 69 M CC 3/19/20 −7 20 (ASL) ND CO, DI, DY, FA, FE, HE, NS 2 No 14, 28 Yes CR0016 31 F CC 3/30/20 4 37 (FRL) Neg AA, FA, HE, MY 2 No 13, 27, 95 No CR0017 46 F CC 3/27/20 9 33 (FRL) ND AA, CO, NC 1 No 21 Yes CR0019 61 M CC 3/18/20 2 29 (ASL) ND CH, CO, DI, FA, FE, HE, MY, NS, NV, PD 2 No 23, 36, 93 Yes CR0020 39 F CC 3/24/20 4 37 (FRL) Pos CH, CT, DI, DY, FA, FE, HE, MY, NS 2 No 19, 34 No CR0021 76 M CC 3/19/20 7 17 (ASL) ND CO, FA, MY, RH 2 No 28, 40, 96 Yes CR0022 32 M CC 3/21/20 14 37 (FRL) Pos AA, CO, DI, DY, FA, FE, MY, NS, NV, PD, RH 3 Yes 33, 46 No CR0023 46 F CC 3/17/20 4 29 (ASL) ND CH, CO, CT, DI, DY, FA, HE, MY, NS, PD, RH 1 No 27, 47 No CR0024 47 M CC 3/24/20 5 37 (FRL) Neg CH, CO, CT, DY, FA, FE, HE, MY, PD 1 No 22, 36 No CR0025 50 M CC 3/19/20 12 24 (ASL) ND AA, CH, CO, CT, DY, FA, FE, HE, MY 2 No 34, 47, 103 Yes CR0026 30 F AS 3/23/20 5 9 (FRL) ND AA, CO, FA, FE, HE, MY 2 No 26, 34 Yes CR0027 63 M AS 3/18/20 2 16 (ASL) ND AA, CH, CO, DY, FA, FE, HE, MY, PD 2 No 28, 99 Yes CR0028 26 F CC 3/30/20 6 37 (FRL) ND CO, DY, FE, HE, NC, RH 1 No 21 No CR0030 29 M CC 3/25/20 4 19 (FRL) ND AA, CH, CT, DI, DY, FA, FE, HE, MY, NC, NS, NV, RH, ST 2 No 24, 32 Yes CR0032 66 F CC 3/25/20 −3 34 (FRL) ND CO 2 No 20, 35 No CR0033 27 F CC 3/19/20 3 17 (ASL) ND AA, CH, CT, DI, DY, FA, FE, HE, NC, NS, PD, ST 3 Yes 30, 37 Yes CR0035 27 M CC 3/24/20 6 35 (FRL) ND AA, CO, FA, HE, MY, NC 2 No 29, 34, 92 No CR0037 38 M CC 3/16/20 1 NA ND DY, HE, MY 2 No 32, 39, 102 Yes CR0038 56 F AS 3/18/20 4 17 (ASL) ND AA, CO, CT, DY, FA, HE, MY 2 No 33, 40, 95 Yes CR0039 67 M AS 3/19/20 2 22 (ASL) ND AA, CH, CO, CT, DI, DY, FA, FE, HE, MY, NS, NV, PD, ST 2 No 30, 37 Yes CR0042 54 M LA 3/23/20 13 28 (ASL) ND CH, CO, DI, FA, FE, HE, NS, PD, RH 1 No 41, 59 Yes CR0043 86 F CC 3/24/20 9 21 (ASL) ND AA, CT, DI, FA, FE, HE, NC, ST 2 No 37, 45 Yes CR0045 64 F LA 3/27/20 10 NA ND AA, CO, CT, DI, DY, FE, MY 2 No 35, 43 Yes CR0046 29 M CC 3/30/20 3 NA ND AA, CH, CO, DY, FE, MY 3 Yes 24 Yes CR0048 34 M CC 3/19/20 7 22 (CoA) ND CO, FA, FE, HE, PD 1 No 34, 77 Yes CR0050 40 M CC 4/2/20 11 NA ND CT, DY 2 No 31, 38, 95 Yes CR0051 64 F CC 3/27/20 2 36 (ASL) ND DY, FA, FE, HE, MY, PD 2 No 29, 36 No CR0054 49 F LA 3/22/20 10 26 (FRL) ND CH, CO, CT, DI, DY, FA, FE, HE, MY, NS, NV, PD, ST 3 Yes 43, 106 Yes CR0055 62 M AA 4/1/20 10 31 (ASL) ND CO, CT, DY, FE, HE, NC, PD, RH, ST 2 No 33, 40 No CR0057 62 M CC 3/22/20 9 25 (FRL) ND CH, CO, DI, DY, FE, HE, MY, NS, NV 3 Yes (ICU) 45, 97 Yes CR0060 51 M AS 4/2/20 4 32 (FRL) Pos AA, CH, CO, DY, FA, FE, HE, MY, NS, NV, PD 3 No 29, 88 Yes CR0061 50 F LA 3/31/20 2 NA ND AA, CH, CO, CT, DI, DY, FA, FE, HE, MY, NV, PD 2 No 29, 44, 82 Yes CR0062 25 F CC 3/28/20 5 38 (FRL) ND CH, CO, CT, DY, FA, FE, HE, MY, NC, NS, NV, RH 2 No 36, 46 No CR0064 82 M CC 3/23/20 7 NA ND CH, CO, DI, FA, FE, HE, MY, NC, NS, PD, RH 2 No 43, 49 Yes CR0066 72 M CC 4/4/20 14 27 (FRL) Pos AA, CO, DI, DY, FA, HE, MY, NC, RH 3 Yes 39 Yes CR0067 39 M CC 3/13/20 15 29 (ASL) ND AA, CH, CO, CT, DY, FA, FE, MY, NS, PD 2 No 62, 81, 124 Yes CR0068 43 M CC 3/18/20 NA NA ND No symptoms 0 No 43, 93 Yes
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ID Age Sex Race/
ethnicity† Nucleic acid test
Symptoms§ Symptom severity¶ Hospitalization#
Antibody tests Date DFOS Ct (Lab)‡ Spike PCR DFOS Seroconversion
CR0069 37 M CC 3/18/20 20 34 (ASL) ND AA, DI, FA, NC 2 No 62 Yes CR0070 28 F CC 3/23/20 5 35 (FRL) ND CT, DY, FA, FE, HE, MY, NC, PD 2 No 42, 63 No CR0071 54 F AA 4/4/20 14 31 (FRL) Neg AA, CT, DY, FE, HE, NS, NV, PD, RH 3 Yes 40, 94 Yes CR0072 38 F AA 3/28/20 2 33 (FRL) ND CO 1 No 35, 54 No CR0073 69 F CC 3/17/20 5 31 (FRL) ND CH, CO, CT, DY, FA, HE, MY, NC, NS, NV, PD, RH 2 No 49, 92 Yes CR0074 48 F AA 3/31/20 12 22 (FRL) Neg CH, CO, DI, FA, FE, HE, MY, NS, PD 2 No 43 Yes CR0078 40 M AS 3/18/20 16 36 (ASL) ND AA, CH, DI, FA, HE, MY 2 No 60, 102 Yes CR0079 40 M AS 4/7/20 4 21 (ASL) ND AA, HE, PD 2 No 31 Yes CR0082 30 F AS 3/31/20 5 39 (FRL) ND CO, DY, FA 1 No 40, 56 No CR0083 31 F CC 3/28/20 6 36 (FRL) ND AA, CO, CT, DY, FA 1 No 44, 60 No CR0086 68 F CC 3/23/20 10 NA ND AA, CH, CO, DI, DY, FA, FE, HE, MY, NS 2 No 50, 108 Yes CR0087 38 M AA 3/28/20 3 36 (FRL) ND CO 2 No 43, 54 No CR0089 35 M AS 3/23/20 3 31 (FRL) ND DI, FA, FE, MY, NC 2 No 49, 68 No CR0090 35 F CC 3/13/20 17 NA ND CH, CO, CT, DY, FA, FE, HE, MY, NC, NS, NV, PD, RH 2 No 76, 111 No CR0093 30 F CC 3/20/20 9 37 (FRL) ND AA, CH, CO, CT, DY, FA, FE, HE, MY, NS, NV, PD, RH, ST 3 Yes 63, 83 No CR0094 53 F AA 4/2/20 10 NA ND CO, DI, DY, FA, FE, HE, MY, NS, NV, PD 2 No 52, 93 Yes CR0095 20 F AA 3/24/20 3 NA ND AA, CO, FA, FE, HE, MY, NS, PD 2 No 54 Yes CR0098 23 M AA 4/3/20 5 18 (ASL) ND AA, CT, DY, FA, FE, HE, MY, NC, RH 2 No 50 Yes CR0099 34 M AS 3/23/20 2 37 (FRL) ND CH, CO, DI, FA, HE, MY, NV 2 No 58 No CR0100 59 M CC 3/31/20 4 26 (FRL) ND AA, CO, FA, HE 2 No 53 Yes CR0101 37 F CC 3/23/20 6 36 (FRL) ND CH, CO, CT, DI, DY, FA, HE, NC, NS, PD 2 No 63 No CR0102 77 M CC 3/13/20 5 16 (ASL) ND DI, FA, NS, NV 2 No 73 Yes CR0104 47 M AA 4/27/20 11 28 (ASL) ND AA, CO, CT, DI, DY, HE, MY, NC 2 No 36 Yes CR0105 36 M CC 3/21/20 5 NA ND CT, DY, FE 1 No 72 Yes CR0108 34 M CC 5/4/20 NA 37 (ASL) Neg No symptoms 0 No 25 No *SARS-CoV-2, severe acute respiratory syndrome-coronavirus-2; DFOS, days following onset of symptoms (not available for participants CR0068 and CR0108, who were asymptomatic). NA, not available; ND, not done. †CC, Caucasian; AA, African American; AS, Asian; LA, Latinx. ‡Ct, cycle threshold; FRL, Fungal Reference Laboratory; ASL, Assurance Scientific Laboratories; CoA, Children's of Alabama Diagnostic Virology Laboratory. §AA, anosmia/ageusia; CH, chills; CO, cough; CT, chest tightness; DI, diarrhea; DY, dyspnea; FA, fatigue; FE, fever >100.4°F; HE, headache; MY, myalgia; NC, nasal congestion; NS, night sweats; NV, nausea/vomiting; PD, psychataxia/dizziness; RH, rhinorrhea; ST, sore throat (also see Appendix Figure 3). ¶Symptom severity was self-reported, with 0 indicating no symptoms, 1 indicating mild symptoms with little impact on daily activities, 2 indicating moderate symptoms with noticeable impact on daily activities, and 3 indicating severe symptoms with a significant decrease in quality of life. #One hospitalized patient was admitted to the Intensive Care Unit (ICU).
Page 12 of 17
Appendix Table 2. Binding and neutralizing antibody titers in the plasma of 72 persons with confirmed SARS-CoV-2 infection*
CR0095 5/14/20 54 4,253 1,414 553 <100 1.60 <0.20 1.65 133 CR0098 5/18/20 50 4,570 481 437 <100 1.30 0.48 3.60 158 CR0099 5/18/20 58 <100 <100 <100 <100 <0.20 <0.20 0.02 <20 CR0100 5/19/20 53 >312,500 7,543 844 180 25.46 1.70 4.53 1,011 CR0101 5/19/20 63 <100 <100 <100 <100 <0.20 <0.20 0.02 <20 CR0102 5/20/20 73 >312,500 6,423 1,943 <100 16.99 0.78 6.45 2,080 CR0104 5/22/20 36 >312,500 8,838 33,260 234 22.44 28.18 7.20 19,476 CR0105 5/27/20 72 1,822 <100 <100 <100 0.38 0.56 2.14 99 CR0108 5/28/20 25 <100 <100 <100 <100 <0.20 <0.20 0.01 <20 *SARS-CoV-2, severe acute respiratory syndrome-coronavirus-2; DFOS, days following onset of symptoms (for asymptomatic participants CR0068 and CR0108 d following RT-PCR test were used). NA, not available. Positive values are shown in red. †Detection of SARS-CoV-2 binding antibodies; Spike, IgG and IgA ELISA reactivities to a pre-fusion stabilized Wuhan-Hu-1 spike protein, with a cutoff at 100 for endpoint and midpoint titers; RBD, IgM and IgG ELISA reactivities to the receptor binding domain of the Wuhan-Hu-1 spike protein with an arbitrary unit cutoff at 0.2; N, detection of IgG responses to the nucleocapsid protein using the Abbott Architect chemiluminescent microparticle immunoassay (CMIA) with an index cutoff at 1.4. ‡D614G, detection of neutralizing antibodies to the D614G variant of the Wuhan-Hu-1 spike using an HIV-1 based pseudovirus assay (see Appendix Methods).
Page 15 of 17
Appendix Figure 1. Comparison of serologic assays detecting SARS-CoV-2 binding and neutralizing
antibodies and RT-PCR detecting viral RNA. Each subplot depicts the relationship between
measurements from two assays, with half maximal effective concentrations (EC50) plotted for S protein
IgG and IgA ELISA titers, signal-to-cutoff index values (Index) plotted for N protein IgG responses (Abbott
Architect), arbitrary units (AU) plotted for RBD protein IgG and IgM ELISA titers, half maximal inhibitory
doses (ID50) plotted for HIV-1 pseudovirus neutralization titers, and cycle threshold (Ct) values plotted for
RT-PCR as listed in Appendix Tables 1 and 2 (x-axes are labeled at the bottom of the column, while y-
axes are labeled to the left of the row). Each point represents the maximum titer observed for replicate
samples from a given patient and are colored red if any serologic assay for that individual was above the
limit of detection (seropositive) and blue if every assay for that individual was below the limit of detection
(seronegative). Points below the limit of detection are shown at the limit of detection and offset slightly to
aid visualization. The Spearman correlation between the respective assays for seropositive samples is
indicated at the top of each subplot (all serologic assay comparisons p < 0.001; all RT-PCR vs serologic
assays p > 0.4).
Page 16 of 17
Appendix Figure 2. Relationship of race/ethnicity, sex, and disease severity with SARS-CoV-2
seroconversion. Bars indicate the proportion of serologic responders for the category depicted, with lines
indicating the 95% confidence interval for this proportion; p-values are shown for a likelihood ratio test of
a logistic regression predicting seropositivity by demographics after Bonferroni correction for multiple
comparisons.
Appendix Figure 3. Relationship of symptoms and SARS-CoV-2 seroconversion. The proportion of
serologic responders is compared between persons who reported (light shaded bars) versus did not
report (dark shaded bars) one of 16 COVID-19 related symptoms. Vertical lines indicate the 95%
confidence interval for each proportion. Likelihood ratio tests of logistic regressions predicting
seroconversion by symptom were all nonsignificant after Bonferroni correction for 16 multiple
comparisons (all p > 0.2). Bars are arbitrarily colored for ease of visualization.
Page 17 of 17
Appendix Figure 4. Comparison of RT-PCR Ct values relative to the time of RT-PCR and serologic
testing. Ct values of serologic responders (red) and non-responders (blue) are plotted relative to the time
of RT-PCR (A) and serologic (B) testing, measured as days from onset of symptoms (DFOS). For patients
with multiple serologic tests, the day of the last sampling is shown. Overlapping points are offset slightly
in the x-axis to allow visualization.
Appendix Figure 5. Amplification of full-length spike sequences from remnant nasal swab materials. A
highlighter plot of deduced SARS-CoV-2 spike amino acid sequences is shown for amplicons derived
from two serologic responders (CR0060 and CR0066) and two serologic non-responders (CR0020 and
CR0022). Amino acid residues that differ from the Wuhan-Hu-1 reference sequence (listed on top) are
depicted by vertical marks, with an aspartic acid to glycine substitution at position 614 (D614G) identified
in the sequences of all participants and an alanine/threonine mixture at position 67 identified in the
sequences of participant CR0066. All genes contain uninterrupted open reading frames. Ct values
derived from clinical testing of the same nasal swab materials are indicated.