Is COVID-19 severity unrelated to antinuclear antibodies?

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces an overreaction of the immune system, resulting in the production of auto-antibodies. Several studies have reported that autoantibodies are prevalent in COVID-19 patients. In our study, antinuclear antibodies were evaluated in patients with COVID-19. We examined 131 sera from patients (>17-year-old) with confirmed COVID-19. Samples were collected prior to receiving any medication and antinuclear antibodies (ANA) levels were measured by the indirect immunofluorescence (IIF) method. Furthermore, the immunoblotting test was used to determine the presence of anti-nuclear antigen antibodies. The IIF-ANA test was positive in 36.4% (48/131) of patients. Overall, non-ICU patients had higher IIF-ANA titers than ICU patients. In particular, ICU patients had fewer nuclear, cytoplasmic, and mitotic IIF-ANA antibodies than non-ICU patients. In conclusion, COVID-19 patients frequently have ANA possibly reflecting the immune dysregulation due to several damaged cells by SARS-CoV-2 virus.

1. Introduction

Although vaccinations and isolation practices have been applied around the world, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues its effects. This infection can cause mild to severe symptoms in different patients [1]. As the immune system tries to remove the virus, it sometimes confronts the body in a manner that stimulates immune and non-immune cells, which can lead to hyperstimulation of the immune system [2] Although, neutralizing antibodies have a protective effect against SARS-CoV2 virus [3], humoral immunity is perturbed in COVID-19 [4]. In susceptible individuals, antibodies to viral proteins of SARS-CoV-2 infection might trigger autoimmune disease since they cross-react with autoimmune targets [5]. There is evidence that the SARS-CoV-2 virus can cause autoimmune disorders in genetically predisposed patients, based on reports of inflammatory/autoimmune symptoms and the detection of circulating antibodies in a subgroup of infected patients [6]. Several studies have reported that autoantibodies are prevalent in COVID-19 patients (21.3%–64%), but larger studies are needed to investigate their relationship with disease outcome [7,8,9].

Our study aimed to evaluate the presence of antinuclear antibodies (ANA) in patients with COVID-19 and to examine ANA frequency and their clinical significance in COVID-19 pneumonia. Each COVID-19 patient who had positive test for fluorescent ANA (FANA) was also examined for the FANA patterns.

2. Objective or hypothesis

In studies conducted on patients with COVID-19, several antibodies were found in these patients, including ANA [8,10], anti-interferon (IFN) antibodies [11], and anti-melanoma differentiation-associated oritein-5 (MDA5) antibodies [12]. We examined the presence of ANA in patients with COVID-19.

• The question was whether ANA positivity rates are higher among COVID-19 patients admitted to ICU than those not admitted to ICU?

• Comparison of ANA titer between ICU and non-ICU patients.

• Do ICU and non-ICU hospitalized COVID-19 patients have different FANA patterns?

• Do patients with ANA have autoantibodies against specific anti-nuclear antigens?

• ANA positivity rates are higher among COVID-19 patients admitted to ICUs than those not admitted to ICUs?

3. Material and method

3.1. Study design and participants

This cross-sectional study included 131 patients (>17-year-old) whose COVID-19 diagnosis was confirmed by a SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) test. Indeed, this test has been performed on patients attending the hospital for the first time with COVID-19 symptoms. After obtaining an informed consent from patients, 5 cc peripheral venous blood samples were taken from patients with confirmed COVID-19 and centrifuged for 10–15 min at 1200g and then transferred to gel-coagulant tubes for up to two hours. Afterwards, serum samples were stored at −80 °C. Spoiled samples and samples with insufficient clinical information or from patients with autoimmune diseases, pregnancy, and chronic viral diseases were excluded.

COVID-19 severity was classified according to the following definitions [13]:

1. Non-ICU Patients (Mild/Moderate): Patients with (SpO2) ≥94% in ambient air at sea level, who are not hospitalized.

2. ICU Patients (Critical/Severe): Patients with SpO2 < 94% in ambient air at sea level, who are hospitalized.

3.2. Ethical considerations

This study was performed following the World Medical Association Declaration of Helsinki (Ethical Principles for Medical Research Involving Human Subjects, amended in 2013. The study was approved by the Ethics Committee of the Ministry of Health and Medical Education of I.R.Iran (IR.NIMAD.REC.1399.041). Written informed consent was obtained from all the subjects in this study.

3.3. ANA indirect immunofluorescence (IIF) testing and evaluation

ANA determination was performed using the indirect immunofluorescence (IIF) method with the HEp-20-10 liver biochip (Monkey) (Euroimmune AG, Luebeck, Germany) kit at a dilution of 1:10 according to the manufacturer's recommendation in the collected samples. Briefly, the serum patients were prepared in dilution 1:10 with PBS (Phosphate buffer solution) also, positive and negative controls were run with each test daily. The evaluation was performed by using a fluorescence microscope (Eurostar III Plus) to observe the BIOCHIP slides under fluorescent and the results were classified into positive or negative patterns. This test reveals much more information than the mere absence or presence of autoantibodies, that is, the level of the antibody as well as the HEp-2 IIFA pattern The fluorescence intensity of the positive control was assumed as 1:640, also The fluorescence intensity was scored at x 400, semi-quantitatively from 1:100 to 1:640 In this process, an evaluation was performed considering International Consensus on ANA Patterns (ICAP) standards [14] Each HEp-2 IIFA pattern was ascribed an alphanumeric code from AC-1 to AC-29. The 29 patterns are divided into four groups: nuclear patterns, cytoplasmic, mitotic, and multiple patterns.

3.4. Analyzing the profile of autoantibodies using immunoblotting

Those samples, whose titers were higher than 1:100 were subjected to the ANA Profile 23 (IgG) Euroimmun test to determine against which nuclear antigen the antibody was produced. In fact, in the manual and instructions that come with the kit, samples with an antibody titer above 1:100 are considered diagnostic, whereas samples from 1:100 are considered weakly positive. For the confirmation and differentiation of auto-antibodies, the serum of patients, which was positive by indirect immunofluorescence assay was further analyzed by Line immunoassay. The test was performed according to the manufacturer's instructions [15]. Briefly, the immunoblot strips coated with 23 highly purified antigens (nRNP/ Sm, Sm, SS-A, Ro-52, SS-B, Scl-70, PM-Scl100, PM-Scl75, RP11, RP155, gp210, PCNA, DFS70, CENP-A, CENP-B, Sp-100, PML, dsDNA, Nucleosomes, Histones, Mi-2α, Mi-2β, Ku) were incubated with patient serum samples which were diluted in 1:101 with PBS (Phosphate buffer solution).

If the samples contain specific antibodies against purified antigens coated on the strip, they are bound and found by using Alkaline phosphatase-labeled anti-human IgG (enzyme conjugate). Then the dried strips were evaluated by comparing the intensity of the reaction with the positive control line by Euroimmun scan software.

3.5. Statistical analysis

The t-test was used for independent samples with normally distributed values. The Chi-Square test was used to compare categorical data. IBM SPSS Statistics 26 was used for statistical analysis. Data are presented as mean, number (N), and percentage (%). P < 0,05 was considered statistically significant.

4. Results

The study included 81 non-ICU patients and 50 ICU patients. The mean age of non-ICU patients was 55 years (range: 21–90 years) and the mean age of ICU patients were 64 years (range: 17–92 years). As seen in Table 1 , the patients in the ICU have a mean age that is higher than the non-ICU patients, which affects the severity of the disease. Moreover, women made up 28% of non-ICU patients, while men made up 72%. In ICU patients, 31.3% of patients were women and 68.8% of patients were men. Although, a statistical test showed that sex did not affect the patient severity of the disease. Furthermore, a comparison of the comorbidities among the two groups is shown in Table 1. Also, no significant difference was observed in terms of comorbid conditions in the two groups. (Table 2 ).

Table 1.

Demographic characteristics.

	Non-ICU	ICU
Age	Mean	Mean	P.Value
	55	64	0.002⁎
Gender	N(%)	N(%)
Female	23(28)	15(31.3)
			0.699
Male	59(72)	34(68.8)

An age comparison was conducted with two groups of COVID-19 patients using a T-test; 0/002*. In both groups of COVID-19 patients, the Chi-square test did not reveal a significant difference in the number of males and females. A person's age affected the severity of the disease, while their gender had no effect.

Table 2.

Prevalence of comorbid conditions In COVID-19 patients.

		No-ICU (N = 81)	ICU Care (N = 50)
Comorbid Conditions		N(%)	N(%)
HTN	No	53(72.6)	27(56.3)
	Yes	20(27.4)	21(43.8)
DM	No	56(76.7)	30(62.5)
	Yes	17(23.3)	18(37.5)
Cancer	No	71(97.3)	46(95.8)
	Yes	2(2.7)	2(4.2)
Heart Disease	No	60(82.2)	36(75)
	Yes	13(17.8)	12(25)
Renal Disease	No	68(93.2)	40(83.3)
	Yes	5(6.8)	8(16.7)
Respiratory Disease	No	66(90.4)	43(89.6)
	Yes	7(9.6)	5(10.4)
Thyroid Disease	No	71(97.3)	42(87.5)
	Yes	2(2.7)	6(12.5)

Abbreviation. HTN; Hypertension. DM; Diabetes Mellitus.

In 63.35% (N: 83/131) of the patients' sera, the IIF-ANA test was negative. In other word, the anti-nuclear antibody has not seen in 59.75% of non-ICU patients (N:49/82), and 69.38% of ICU patients (N:34/49); however, 41% (N: 48/131) had positive results (Table 3 ). In addition, FANA test results in the Mild, Moderate, Severe, and Critical groups of COVID-19 are shown in Table 3.

Table 3.

Results with FANA test non-ICU patients ICU patients.

		Mild		Moderate		Severe		Critical
	Result	N	%	N	%	N	%	N	%	p.value
FANA	Positive	8	34.8%	25	42.4%	7	50.0%	8	20.6%
	Negative	15	65.2%	34	57.6%	7	50.0%	27	79.4%	0.125

The Chi-square test was used to compare the results of patients in the ICU with those in the non-ICU, and no significant differences were found; 0/125.

A titer of 1:100 was detected in 45% of ICU patients (N:15/33) and 50% of non-ICU (N:7/15) patients. Also, in 21.4% of ICU patients(N:3/15) and 30.3% (N:10/33) of non-ICU patients, a titer of 1:160 was reported. Among ICU patients and non-ICU patients, the anti-nuclear antibody titer was 1:320 in 28.6%(N:4/15) and 21.2%(N:7/33), respectively. There was just a non-ICU patient who had 1:640 titer of ANA (Table 4 ).

Table 4.

Anti-nuclear antibody in COVID-19 patients: significance.

	Non- ICU		ICU
Titer	N	%	N	%	P.value
1:100	15	45.5%	7	50.0%
1:160	10	30.3%	3	21.4%	0.807
1:320	7	21.2%	4	28.6%
1:640	1	3.0%	0	0.0%

Both ICU and non-ICU positive samples were examined for ANA and found to have an intensity of 1:100 to 1:160, 1:320 to 1:640. Two groups were compared using Chi-square test and no significant difference was found between the two groups; 0/807.

A type of pattern of positive samples was also investigated in this study. As it has shown in Table 5 . ICU patients had fewer nuclear, cytoplasmic, and mitotic patterns than non-ICU patients. Furthermore, three of the patterns described above are shown in Fig. 1 taken by a fluorescent microscope.

Table 5.

3 Common ANA patterns in COVID-19 patients.

Using descriptive statistics, three nuclear, cytoplasmic, and mitotic patterns were found in ICU and non-ICU individuals, with all three patterns being higher in non-ICU patients.

Fig. 1.

The ANA patterns of three COVID-19 patients.

These three photos show three patterns depending on how the ANA binds; a. Nuclear Pattern; nucleus of the cell and mitotic chromatin are stained because ANA have been produced against it. b. Cytoplasmic pattern; It resulted from antibodies against cytoplasmic components. c. Mitotic Pattern; Spindle fibers between the poles were stained in mitotic cells due to autoantibodies produced against them.

By conducting an immunoblotting test on 25 patients whose ANA titers were reported to be above 1 in 100, it was determined that 36% of the patients did not produce ANA against a specific antigen. Also, 28% of patients have produced anti-Ro-52 antibody,28% anti-Mi2-beta antibody,12% PM-SCL75, and 4% anti- gp210 antibody (Fig. 2).

Fig. 2.

ANAs are produced against which of the nuclear antigens.

5. Discussion

We investigated the type, intensity and presence of ANA in two groups of COVID-19 patients, including those hospitalized in ICU care units and those not.

Our study found that 41% (N: 48/131) of COVID-19 patients had ANA by IIF test and according to our results, the number of non-ICU patients with ANA was higher than that of ICU patients in each titer (Table 4). Three main patterns also appeared to be common among non-ICU patients (Table 5).

There are several studies on anti-nuclear antibody response in COVID-19 patients are not yet at satisfactory level [7,8,16]. Results of our study differed from those of the study to be discussed. In this study the IIF-ANA test was positive in 18% (N = 9/50) of the patients [17] also our study included 50 samples, whereas this study included 7 samples admitted to the ICU. We report three main groups of IIF-ANA test patterns - nuclear, cytoplasmic, and mitotic (Table 5) - in order to avoid data scattering; however, this study reported IIF-ANA test patterns in >3 main groups. According to this study in patients with acute COVID-19, ANAs were typically higher in titers and predominantly in nucleolar patterns; albeit, the number of non-ICU patients with titers higher than 1/160 in our study was higher than that of ICU patients and compared to non-ICU patients, ICU patients had fewer nuclear, cytoplasmic, and mitotic patterns. in both studies the statistical tests showed that gender had no effect on patient severity of disease.

A prospective study conducted by [17] that, 33 consecutive COVID-19-associated pneumonia patients were studied and the presence and role of autoantibodies was analyzed. ANAs were positive for 11 patients (33%). In fact, the number of the participants was so limited. Nuclear patterns have just been observed in patients with ANA positivity as in the study above. This issue can be justified by the few numbers of samples. Also, they did not report the intensity and titer of ANA.in our study, samples were taken on the first day of the patient's visit to conduct laboratory tests.

It has been observed that some elderly people are also positive for ANA due to their age as one of the factors affecting the increase of ANA [18] in study conducted in Italy [19] 35.6% of the COVID-19 patients were ANA positive. Age is one of the important factors that contributed to this result, since all patients are over 66 years old. Participants in this study are between the ages of 17 and 92 (Table 1), because age selection is a problem since most sick patients are older and age affects the severity of the infection (Table 1).

According to another study, which included 27 patients in the ICU and 37 non-ICU patients, (N:16/64) 58.3% of the patients had ANA positivity, and 12 of the 16 patients were in the ICU. Although, 41% (N: 48/131) had positive results (Table 3) and (N:33/48) were non-ICU patients. Indeed, our study includes almost twice as many non-ICU patients as ICU patients, which is one of the challenges. Furthermore, different methods to check for ANA in patient serum can cause differences in results. For instance, [20] study of 64 COVID-19 patients found that 25% were Ena positive. Most were in ICU care.

According to our study, ANA had no significant association with COVID-19 disease severity, even though this study included more samples than previous studies. What is more, our results need to be confirmed by further larger scale studies to define the role of autoantibodies and their association.

Although ANA in SARS-CoV-2 infection was not related to the severity of the disease, the follow-up in connection with the patients of this study can provide us with useful information about the stability or reduction of this autoantibody titer and the percentage of autoimmune diseases in these people in the future.

6. Conclusion

As a consequence of the larger number of samples in this study, we identified three patterns (nuclear, cytoplasmic, mitotic) among COVID-19 patients. The results of this study revealed that there was no significant difference between the ICU and non-ICU groups in terms of the presence of ANA and their severity. Antinuclear antibodies are not associated with COVID-19 severity.

Declaration of Competing Interest

All authors declared no competing interests for this work.

Data availability

Data will be made available on request.