Prolonged SARS-CoV-2 Infection in Patients Receiving Anti-CD20 Monoclonal Antibodies: A Diagnostic Challenged by Negative Nasopharyngeal RT-PCR and Successful Treatment with COVID-19 High-Titer Convalescent Plasma

We highlighted in this current paper similar prolonged respiratory presentation with COVID-19 pneumonia in four severely immunocompromised patients currently being treated with anti-CD20 monoclonal antibodies (mAbs), such as ocrelizumab and rituximab, for multiple sclerosis or rheumatoid polyarthritis. Real-time reverse transcription-polymerase chain reaction on a nasopharyngeal swab specimen was negative in all patients. SARS-CoV-2 infection was confirmed from bronchoalveolar lavage fluid. A high titer of post-vaccine COVID-19 convalescent plasma was administered with complete recovery in all patients.


Key Bullet Points
Immunocompromised patients with B-cell depletion agents are at risk for persistent SARS-CoV-2 infection.
Negative tests for SARS-CoV-2 pneumonia based on RT-PCR cannot rule out this diagnosis.We reported a case series of prolonged SARS-CoV-2 infection (with negative nasopharyngeal RT-PCR result) in patients receiving anti-CD20 mAbs who recovered after administration of high-titer post-vaccine COVID-19 convalescent plasma.
COVID-19 convalescent plasma is a promising approach through the transfer of neutralizing antibodies specific to SARS-CoV-2 in patients with B-cell immunosuppression and persistent viral shedding.
In this particular population of patients undergoing anti-CD20 therapy, the T-cell response is conserved [6][7][8], present also against variants [9], and detectable by commercial tests [10].'Prolonged' SARS-CoV-2 infection was defined as confirmed COVID-19 with symptoms persisting for 1 month or more.We reported herein four cases of confirmed prolonged SARS-CoV-2 infection (despite negative nasopharyngeal RT-PCR result) in patients receiving anti-CD20 mAbs who recovered after administration of high-titer postvaccine COVID-19 convalescent plasma (CCP).

Results
Three of four patients were females with a median age of 54 (43-65) years.All patients were receiving anti-CD20 mAbs, such as ocrelizumab and rituximab, for multiple sclerosis (n = 3) and rheumatoid polyarthritis (n = 1), respectively.Symptoms onset began 48 (33-58) days before hospitalization with fever and upper respiratory symptoms (Figure 1 and Table 1).
In this particular population of patients undergoing anti-CD20 therapy, the T-cell response is conserved [6][7][8] , present also against variants [9], and detectable by commercial tests [10].'Prolonged' SARS-CoV-2 infection was defined as confirmed COVID-19 with symptoms persisting for 1 month or more.We reported herein four cases of confirmed prolonged SARS-CoV-2 infection (despite negative nasopharyngeal RT-PCR result) in patients receiving anti-CD20 mAbs who recovered after administration of high-titer post-vaccine COVID-19 convalescent plasma (CCP).

Results
Three of four patients were females with a median age of 54 (43-65] years.All patients were receiving anti-CD20 mAbs, such as ocrelizumab and rituximab, for multiple sclerosis (n = 3) and rheumatoid polyarthritis (n = 1), respectively.Symptoms onset began 48 (33-58) days before hospitalization with fever and upper respiratory symptoms (Figure 1 and Table 1).Real-time reverse transcription-polymerase chain reaction (RT-PCR) on nasopharyngeal swab specimen (TaqPath™ COVID-19 CE-IVD RT-PCR Kit, ThermoFisher Scientific, Waltham, MA, USA) and SARS-CoV-2 serologies (chimiluminescence, Liaison ® XL) for IgG (anti-RBD, anti-NTD, anti-Interface) were negative in all patients.Pulmonary computed tomography (CT) scans showed bilateral ground-glass opacities in all patients (Figure 2).SARS-CoV-2 infection was confirmed from bronchoalveolar lavage (BAL) fluid by performing DiagCORE (Maryland, USA) ® Respiratory Panel 2-SAT Dx.CCP was administered in all patients (a total of four units) with a complete recovery without relapse after a mean of 73 days of follow-up.

Discussion
This case series suggested (i) the use of lower respiratory samples such as choalveolar lavage (BAL) is required in patients with anti-CD20 agents, in case of c examination and imaging finding suggesting SARS-CoV-2 infection and (ii) using C a good alternative treatment for patients receiving anti-CD20 agents (such as o zumab and rituxumab) who experience prolonged SARS-CoV-2 infection.
In our patients, repeated nasopharyngeal swabs were negative for SARS-RT-PCR, but these negative results on nasopharyngeal swabs are clearly insuffici rule out COVID-19 diagnosis [2].We strongly recommend the use of lower respi tract specimen testing for the direct detection of SARS-CoV-2 infection in immun pressed patients, in case of clinical and/or radiological suspicion [11].Morandi evaluated the additional value of lower respiratory tract sampling in the diagno COVID-19 in patients with clinically suspected SARS-CoV-2 infection with ava chest CT scan and at least two negative nasopharyngeal swab RT-PCR tes SARS-CoV-2 [12].A correlation was found between SARS-CoV2 detection on the respiratory tract and the presence of a cough as well as with typical CT features.Th domains of anti-CD20 mAbs, such as ocrelizumab and rituxumab, target CD20+ B phocytes, causing selective depletion of circulating B cells through natural cell-mediated, antibody-dependent cellular cytotoxicity, complement-dependent toxicity, and antibody-triggered apoptosis.In all our patients who received B-cell d tion therapy, prolonged SARS-CoV-2 viral shedding was reported with a mean days, which was consistent with medical literature data [1].Recently, remdesivi nirmatrelvir/ritonavir combined with sotrovimab were suggested as treatment in s

Case 1
A 43-year-old female with a past history of rheumatoid arthritis treated with rituximab presented with daily fever for two months, chills, and respiratory symptoms such as dypnea.Respiratory and neurologic examinations were normal and RT-PCR SARS-CoV-2 in a nasopharyngeal sample was negative.Antimicrobial empiric treatment was started intravenously with no clinical response.On day 5, the patient developed hypoxemia which needed oxygen therapy flow at 4 L/min and was transferred to an intensive care unit.Given concern for prolonged SARS-CoV-2 infection, induced BAL was performed and COVID-19 was diagnosed from results of a RT-PCR panel.Treatment began with oral steroids (dexamethasone, 6 mg daily), then intravenous (IV) tocilizumab (one injection of 8 mg) and remdesivir IV (200 mg was administered, followed by 100 mg daily for a total of five days) with no response and the persistence of hypoxemia.A high-titer of Omicron CCP (four units) was administered with total regression of symptoms.

Case 2
A 65-year-old female with a past history of multiple sclerosis treated with ocrelizumab sought care for a persistent non-productive cough and dyspnea four months after a SARS-CoV-2 infection.Pulmonary auscultation found unilateral crackling sounds, confirmed on imaging findings.Although RT-SARS-CoV-2 PCR on nasopharyngeal swab was negative, positive PCR results were obtained from the BAL sample, with persistent presence of the SARS-CoV-2 in the lower respiratory tract.She received only IV high-titer Omicron CCP (>8000 IU).A significant clinical improvement was observed, followed by complete resolution of the cough and dyspnea without relapse after 6 months of follow-up.

Case 3
A 48-year-old woman with a history of multiple sclerosis treated with ocrelizumab sought care for a fever which had persisted for 3 months and respiratory symptoms such as a dry cough and dyspnea.Supportive treatment was initiated (analgesics, antitussives) with empirical antimicrobial drugs (amoxicillin/clavulanate, 3 g by IV infusion per day) with no recovery.On admission, she was febrile and had unilateral crackling sounds on pulmonary auscultation.Laboratory findings revealed a high C-reactive protein (CRP) level of 146 mg/L.Nasopharyngeal PCR and serological assay for SARS-CoV-2 were negative.However, we performed a PCR respiratory panel on BAL which detected SARS-CoV-2 RNA.Administrations of four units of high-titer CCP (>8000 IU) were carried out, with a total symptom disappearance and CRP control decrease to 13 mg/L, with no evidence of relapse afterwards.

Case 4
A 61-year-old male, receiving ocrelizumab for multiple sclerosis for 6 months, presented to the emergency room with an approximate 2 months history of daily fever and cough.He received two empiric courses of antibiotics (spiramycine once and amoxicillinclavulanate twice) for presumed community-acquired pneumonia.A nasopharyngeal swab for SARS-CoV-2 was negative.COVID-19 was diagnosed from the results of RT-PCR on BAL, and CT thoracic imaging (Figure 2).After CCP, oral nirmatrelvir/ritonavir for five days and IV remdesevir (200 mg on the first day, then 100 mg/day for two days) administration outcome was favorable with rapid improvement of general condition and respiratory symptoms.

Discussion
This case series suggested (i) the use of lower respiratory samples such as bronchoalveolar lavage (BAL) is required in patients with anti-CD20 agents, in case of clinical examination and imaging finding suggesting SARS-CoV-2 infection and (ii) using CCP is a good alternative treatment for patients receiving anti-CD20 agents (such as ocrelizumab and rituxumab) who experience prolonged SARS-CoV-2 infection.
In our patients, repeated nasopharyngeal swabs were negative for SARS-CoV2 RT-PCR, but these negative results on nasopharyngeal swabs are clearly insufficient to rule out COVID-19 diagnosis [2].We strongly recommend the use of lower respiratory tract specimen testing for the direct detection of SARS-CoV-2 infection in immunosuppressed patients, in case of clinical and/or radiological suspicion [11].Morandi et al. evaluated the additional value of lower respiratory tract sampling in the diagnosis of COVID-19 in patients with clinically suspected SARS-CoV-2 infection with available chest CT scan and at least two negative nasopharyngeal swab RT-PCR tests for SARS-CoV-2 [12].A correlation was found between SARS-CoV2 detection on the lower respiratory tract and the presence of a cough as well as with typical CT features.The Fab domains of anti-CD20 mAbs, such as ocrelizumab and rituxumab, target CD20+ B lymphocytes, causing selective depletion of circulating B cells through natural killer cell-mediated, antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-triggered apoptosis.In all our patients who received B-cell depletion therapy, prolonged SARS-CoV-2 viral shedding was reported with a mean of 48 days, which was consistent with medical literature data [1].Recently, remdesivir and nirmatrelvir/ritonavir combined with sotrovimab were suggested as treatment in several cases of COVID-19 patients with prolonged clinical symptoms and viral shedding [13][14][15], but also in pre-and post-exposure prophylaxis of COVID-19 [16].
The efficacy of CCP to treat long-standing COVID-19 in patients with B-Cell depletion was discussed [4,17].In our paper, patient 2 and 3 were treated exclusively with high-titer post-vaccine CCP with a complete recovery.Scott et al. suggested that spike-protein proteolytic antibodies in CCP contribute to SARS-CoV-2 neutralization [18].Cognasse et al. also demonstrated that CCP exhibited moderately increased inflammatory markers on endothelial cells, neutralizing auto-Abs to type I IFNs compared to the control plasma with no discernible differences in ex vivo bioactivity [3].CCP therapy with robust Fc-effector antiviral functions can serve as secondary defense when neutralization is compromised [19].Given the increasingly recognized role of T-cell responses in protection against severe COVID-19, the circulation of SARS-CoV-2 variants, and the potential implementation of novel vaccines, it becomes imperative to continuously monitor T-cell responses [20,21].
Currently, the use of CCP is permitted in France as an off-label indication, and it implies authorization by 'The National Reference Multidisciplinary Team'.CCP therapy seems to be a promising approach through the transfer of neutralizing antibodies specific to SARS-CoV-2 in patients with B-cell immunosuppression and prolonged COVID-19.Further studies are required to clarify therapeutic management strategies for immunocompromised patients receiving B-cell depletion therapy who experience prolonged COVID-19.

Viruses 2023 ,Figure 2 .
Figure 2. Thoracic computed tomography scan in patients with B-cell depletion and prol SARS-CoV-2 infection.(A) Ground-glass interstitial syndrome with a crazy paving, predom on the left upper lobe, and alveolar condensation in the right lower lobe with pleural effusio tient 1).(B) Bilateral ground-glass opacities with sequelae lesions (Patient 2).(C) Subp ground-glass opacities with a basal distribution (Patient 3).(D) Multiple bilateral f ground-glass opacities, affecting all lobes except the right upper lobe (Patient 4).

Figure 2 .
Figure 2. Thoracic computed tomography scan in patients with B-cell depletion and prolonged SARS-CoV-2 infection.(A) Ground-glass interstitial syndrome with a crazy paving, predominant on the left upper lobe, and alveolar condensation in the right lower lobe with pleural effusion (Patient 1).(B) Bilateral ground-glass opacities with sequelae lesions (Patient 2).(C) Subpleural ground-glass opacities with a basal distribution (Patient 3).(D) Multiple bilateral foci of ground-glass opacities, affecting all lobes except the right upper lobe (Patient 4).

Table 1 .
Cont.We administered four units of high-titer post-vaccine Omicron COVID-19 convalescent plasma in all patients (two units on day 1 and two units on day 2).*** BAL SARS-CoV-2 RT-PCR follow-up after convalescent plasma administration were not performed in any patients regarding the complete recovery.