Antiphospholipid Antibodies in Patients with Covid-19: Trend Over Time

Purpose Aim of the study was to investigate whether aPL positivity correlated with thrombosis in COVID-19 patients and whether it was transient or persistent. Methods We enrolled COVID-19 patients who underwent aPL tests: Lupus Anticoagulant (LA); IgM, IgG, IgA anticardiolipin antibodies (aCL); and IgM, IgG anti- β 2-Glycoprotein-I antibodies (a β 2GPI). Results Twenty-eight out of 73 (38.4%) patients resulted positive for at least one aPL assay: 32.8% for IgA aCL, 6.8% for IgM aCL and 4.1% for IgM a β 2GPI. No patients tested positive for IgG aPL or LA at the first determination. Seven (9.6%) patients developed thrombotic events during hospitalization, with 4 of them resulting positive for aPL. In patients with thrombotic events during hospitalization the risk of death was increased 9-fold (LR+8.9, p=0.003). Patients with double positivity for aCL and a β 2GPI IgM had a LR+ of 6.3 to have thrombotic events (p=0.012) and a LR+ of 4.9 to have elevated D-dimer levels (p=0.027). In 10 out of 28 positive patients, aPL was detected in a second occasion at least 12-weeks apart and two patients confirmed the positivity. Conclusion Results suggest that double positivity for aCL and a β 2GPI IgM increases the risk of thrombosis in COVID-19, unlike IgA aCL positivity. APL positivity may be persistent, and it is advisable to monitor it over time.


INTRODUCTION
Since the beginning of the SARS-CoV-2 outbreak, antiphospholipid antibodies (aPL), a known thrombotic risk factor, have been studied in COVID-19 patients, in whom thromboembolic events have been associated with poor prognosis [1].
The association between infections and aPL is historically known, causing transient positivity for aPL or representing the so-called "second hit" in causing thrombosis in APS patients [2].
The prevalence of aPL in COVID-19 patients is variable, ranging from 8.3% to 74% [3][4][5][6]. Furthermore, these studies are heterogeneous and difficult to compare: in some studies, aPL has been searched in critical COVID-19 patients, a condition associated with a greater thrombotic risk [5], in others aPL has been tested in patients with varying degrees of COVID-19 severity [7]. In addition, aPL levels are not reported in all studies and LA was sometimes performed in patients on anticoagulant therapy, with the risk of false positivity [8,9]. To date, the pathogenetic role of aPL and the antibodies trend over time are still unknown.
Aim of the present study was to investigate whether aPL positivity correlated with thrombosis in COVID-19 patients and whether it was transient or persistent.

PATIENTS
We included all consecutive COVID-19 patients hospitalized in one of the COVID-19 Units of the Policlinico Umberto I, Sapienza University of Rome from April 1, 2020 to June 7, 2020 in this prospective study. The diagnosis of SARS-CoV-2 infection was confirmed in all the patients by nasopharyngeal swab for SARS-CoV-2 on real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR). Patients'exclusion criteria were: non-COVID-19 patients and patients affected by autoimmune inflammatory rheumatic diseases (AIIRD).

METHODS
IgG and IgM aCL and aβ2-GPI were detected by chemiluminescence assay using Zenit RA Immunoanalyzer (A. Menarini Diagnostics, Florence, Italy), IgA aCL by EliA detection kit by ThermoFisher Scientific (Waltham, USA). LA was analyzed using two coagulation systems, a dilute sensitized activated partial thromboplastin time (aPTT) and a dilute Russell's viper venom time (dRVVT), and then performing a confirm test with reagents and instrumentation by Hemoliance Instrumentation Laboratory, Lexington, MA, USA.

STATISTICAL ANALYSIS
Data are expressed as mean ± standard deviation (S.D.) or median (interquartile range) according to the distribution of the variables. χ2-test or Fisher exact test was used for comparison of categorical variables. P values < 0.05 were considered statistically significant. SPSS 20.0 statistical software package (SPSS Inc., Chicago, IL, USA) was utilized for all statistical tests.

RESULTS
We enrolled 73 hospitalized patients affected by COVID-19 (40 females and 33 males, all Caucasian) with a mean age of 65 years (S.D. 17.6). Clinical and demographic characteristics of COVID-19 are reported in Table   1. The most common symptoms at disease onset were fever (82%), dyspnea (60%) and cough (31.5%); the median time from appearance of the first symptoms to hospital admission and positive nasopharyngeal swab for SARS-CoV-2 on RT-PCR assay was 5 days (IQR 9. Seven out 73 (9.6%) patients developed thrombotic events during hospitalization, among them 4 (57%) tested positive for aPL (Table 2).
We observed that the risk of death was 9-fold higher in patients with thrombotic events during hospitalization [likelihood positive ratio (LR+) 8.9, p=0.003] and that patients with double positivity for IgM aCL and IgM aβ2GPI had a LR+ of 6.3 to have thrombotic events (p=0.012) and a LR+ of 4.9 to have an increase in D-dimer levels (p=0.027). IgA aCL, the most prevalent aPL, did not result associated with thrombosis.

DISCUSSION
The results of the present study confirm that thrombotic events increase the risk of death in COVID-19 patients.
In addition, we showed that, in COVID-19 patients, double positivity for IgM aCL and IgM aβ2GPI increased the risk of thrombotic events. For the first time, in this study, we evaluated the persistence of aPL after 12 weeks since the first determination.
It's well known that infections may lead to transient aPL positivity without clinical manifestations or trigger aPLrelated features. To explain such phenomena various possible mechanisms have been proposed, such as molecular mimicry, post-translational modifications, exposition of self-antigens on the cell surface [10]. A wide variety of infectious agents have been linked to aPL positivity, such as CMV, EBV, HIV, HBV, HCV and parvovirus B19 [11,12]. In a systematic review, aPL have been detected in 293 patients during 50 different infections. Among these patients, 72 patients fulfilled APS criteria, 128 patients reported a thrombotic event in association with a transient aPL positivity and 93 patients did not show any association between aPL and APS-related features [13].
In our study, 38.4% of COVID-19 patients showed positivity for at least one aPL assay at low-medium titer. In agreement with a previous chinese study, the IgA isotype was found to be the most prevalent aPL in our cohort; those authors suggested that SARS-CoV-2 may preferentially induce the IgA isotype of aPL due to SARS-CoV-2 mucosal tropism. [4].
The possible pathogenetic role of aPL in COVID-19 is still far from being clear; we observed that COVID-19 patients with double positivity for IgM aCL and IgM aβ2GPI have a 6-fold increased risk of thrombotic events and a 5-fold increased risk of higher D-dimer plasma levels. Besides, thrombotic events appeared to increase the risk of death (LR+=8.9, p=0.003).
Xiao and colleagues evaluated the dynamic changes in aPL levels in 6 COVID-19 patients over a period of 60 to 77 days from disease onset; the results obtained suggest that aPL levels are fluctuating and they encouraged further studies with a long-term follow-up on COVID-19 patients positive for aPL [4]. Indeed, to date, only 2 case reports have tested aPL on a second occasion after at least three months in aPL positive COVID-19 patients [14,15].
At the best of our knowledge, this is the first study in which aPL-positive COVID-19 patients were re-tested after at least 12 weeks apart. The results of our study suggest that the 20% of COVID-19 patients may show persistent positivity for aPL. Given that aPL is a thrombotic risk factor, it may be appropriate to monitor antibodies trend over time and minimize other thrombotic risk factors in patients with SARS-CoV-2 infection.
The main limitations of our study are represented by the small sample size and the one-center design. Our results should be validated in larger and multicenter studies.

CONCLUSIONS
In conclusion, the results of our study confirm that thrombotic events increase the risk of death in  patients and indicate that double positivity for aCL and aβ2GPI IgM increases the risk of thrombosis in patients, unlike positivity for IgA aCL.
Antiphospholipid antibodies positivity resulted to be persistent in a small percentage of COVID-19 patients, given that aPL is a thrombotic risk factor, it may be appropriate to monitor antibodies trend over time and minimize other thrombotic risk factors in patients with SARS-CoV-2 infection.

Declarations Not applicable
Funding No specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out the work described in this article.

Conflicts of interest 'None declared'
Availability of data and material Data that support the findings of this study are available on request from the corresponding author.
Code availability Research code are available on request from the corresponding author.