Lack of Antinuclear Antibodies in Convalescent Coronavirus Disease 2019 Patients With Persistent Symptoms

To the Editor—We read with great interest the recent article by Seeßle et al [1] who reported that 77.1% of 96 patients with coronavirus disease 2019 (COVID-19) had persistent symptoms 12 months following acute infection. Of particular interest, antinuclear antibody (ANA) titers were observed to be elevated in 43.6% of participants at this late timepoint. Interestingly, the number of symptoms participants reported was higher in those with a positive ANA, and significantly more females with concentration problems had ANA titers > 1:160. These findings are intriguing given that autoimmunity has been proposed as a potential underlying etiology of postacute sequelae of severe acute respiratory syndrome coronavirus 2 infection (PASC) [2–4].

We established a convalescent severe acute respiratory syndrome coronavirus 2 infection cohort and have collected clinical and laboratory data in participants with and without PASC. We measured ANA titers in a convenience sample of participants approximately 4 and 8 months following initial infection (M4 and M8 timepoints, respectively) using an enzyme-linked immunosorbent assay with reflex to an indirect fluorescent assay using HEp-2 substrate in a commercial reference laboratory (ARUP Laboratories). The screen is designed to detect antibodies against double-stranded DNA, histones, SS-A (Ro), SS-B (La), Smith, Smith/RNP, Scl-70, Jo-1, centromeric proteins, and other HEp-2 cell nuclear antigens.

We tested 46 samples at 4 months and 69 samples 8 months after acute infection from individuals with and without PASC as shown in Table 1. At M4, 57% had at least 1 PASC symptom; at M8, 78% had at least 1 PASC symptom (8 participants had data at both the M4 and M8 timepoints). No ANAs were detected in the M4 participants and only 3 of 69 participants at M8 had a positive ANA screen and reflex indirect fluorescent assay. The participants with detectable ANAs were female and none had been hospitalized or had a known preexisting autoimmune disorder. No participant with a history of autoimmune disease (9.5% in the M4 cohort and 4.3% in the M8 cohort) had detectable ANAs (Table 1). All participants with a positive ANA reported at least 1 symptom, although 2 with higher titers (1:320) had 2 or fewer symptoms, whereas the participant with a low titer (1:80) reported 19 symptoms. Like the prior study, a large proportion (up to 40%) of our PASC cohort reports neurocognitive symptoms 4 and 8 months following acute infection [5].

Table 1.

Demographic and Clinical Information for Participants With and Without a Positive ANA Test at the Month 4 and Month 8 Study Timepoints

	M4 Cohort, alla	M8 Cohort, all	M8 ANA+b	M8 ANA–
Cohort, N (%)	42 (100)	69 (100)	3 (4.3)	66 (95.7)
Days since onset of acute COVID-19, median (min, max)	124.5 (109, 198)	247 (118, 291)	240 (236, 269)	247 (118, 291)
Female, median (min, max)	23 (54.8)	37 (53.6)	3 (100)	34 (51.5)
Age, median (min, max)	49.5 (24, 74)	48 (19, 85)	43 (30, 48)	48 (19, 85)
Race/ethnicity, N (%)c
White	29 (69.0)	36 (52.2)	2 (66.7)	34 (51.5)
Latinx	11 (26.2)	15 (21.7)	0 (0)	15 (22.7)
Asian	2 (4.8)	11 (15.9)	0 (0)	11 (16.7)
Black/African American	0 (0)	4 (5.8)	1 (33.3)	3 (4.5)
Hospitalized, N (%)	10 (23.8)	18 (26.1)	0 (0)	18 (27.3)
Preexisting disease, N (%)
Autoimmune disease	4 (9.5)	3 (4.3)	0 (0)	3 (4.5)
Cancer	2 (4.8)	2 (2.9)	0 (0)	2 (3.0)
Diabetes	5 (11.9)	9 (13.0)	1 (33.3)	8 (12.1)
Heart disease	0 (0)	0 (0)	0 (0)	0 (0)
Hypertension	7 (16.7)	11 (15.9)	1 (33.3)	10 (15.2)
Lung disease	7 (16.7)	13 (18.8)	0 (0)	13 (19.7)
Kidney disease	0 (0)	1 (1.4)	0 (0)	1 (1.5)
N > 1 PASC symptoms, median (min, max)	24 (57.1)	54 (78.2)	3 (100)	51 (77.3)
N > 5 PASC symptoms, N, median (min, max)	15 (35.7)	26 (37.7)	1 (33.3)	25 (37.9)
Number of PASC symptoms, Median (min, max)	2 (0, 18)	2 (0, 23)	2 (1, 19)	2 (0, 23)
Total SARS-CoV-2 antibody levels,d median (min, max)	424 (17, 1942)	568 (2, 10624)	115 (57, 585)	569.5 (2, 10626)

Abbreviations: ANA, antinuclear antibody; COVID-19, coronavirus disease 2019; M4, month 4 study timepoint; M8, month 8 study timepoint; PASC, postacute sequelae of SARS-CoV-2 infection; RBD, receptor-binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

No ANA antibodies at M4 were detected in any participant.

No significant differences were observed between any M8 ANA⁺ and M8 ANA^– groups using the Fisher exact test or Mann-Whitney tests for paired analyses.

Three M8 participants had unknown race/ethnicity.

Anti-RBD antibody levels tested using a Pylon-enhanced fluorescence assay.

The reason for the discrepancies between our cohort and the study by Seeßle et al is not known but raises concerns about the generalizability of the prior observation that a very large proportion of patients recovering from COVID-19, with or without PASC, have persistent ANA elevations 1 year after acute infection. Both our cohort and the participants reported by Seeßle et al included hospitalized and nonhospitalized participants and both males and females. The frequency of ANA positivity (titer > 1:160) in the general population has been estimated to be 5% [6], and we identified 4.3% of convalescent participants with COVID-19 with detectable ANAs despite using similar sensitive and specific laboratory methods. Further studies are warranted, particularly in diverse geographic and sociodemographic populations.

Notes

Financial support. This work was supported by NIH/NIAID 3R01AI141003-03S1 (grant to University of California San Francisco, to T. J. H.); grants from Merck and Co. to University of California San Francisco.

Potential conflicts of interest. T. J. H. reports grants from Merck & Co., NIH/NIAID, Bristol Myers Squibb, and Gilead Biosciences (to University of California San Francisco) outside the submitted work. All other authors report no potential conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.