WO2021239809A1 - Treatment of covid-19 patients - Google Patents

Abstract

The present invention relates to use of plasma exchange therapy using only albumin as replacement for treating Covid-19 patients.

Description

TREATMENT OF COVID-19 PATIENTS

Critically ill patients infected by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) responsible for Coronavirus disease 2019 (Covid-19) may develop acute respiratory distress syndrome (ARDS) and multi-organ failure (1). Systemic host response to infection is thought to be highly involved, based on the observation of high plasma levels of pro-inflammatory cytokines, with interleukin (IL)-6 associated to severity and mortality in Covid-19 patients (2-4). Immune system activation and cytokine release syndrome (CRS) has been pointed out (5). This syndrome is not unique to Covid-19 and has been described in sepsis/septic shock for years with dramatic impact on inflammation, endothelial dysfunction, pathologic coagulation and finally on outcome (6). Targeting CRS has been recently proposed in the treatment of the most severe Covid-19 patients by using monoclonal antibodies directed against IL-6 or IL-1-Beta (7). Unfortunately, the best target is still unknown and these treatments are associated to a long- lasting immunosuppression.

Plasma exchange or plasma exchange therapy (PET or TPE) offers a unique opportunity to rapidly reduce several circulating cytokines present in severe cases of Covid-19. Indeed, TPE was found able to reduce pro-inflammatory cytokines in sepsis/septic shock even if, until now, no effect on outcome could be demonstrated (8-10). Recently, some authors reported the use of TPE in Covid-19 with however unconclusive results, using fresh frozen plasma (FFP) as replacement fluid (16, 17) Kesici et al (18) suggest that TPE applied with convalescent plasma (CP) should be considered as a therapeutic option in severe Covid-19 patients within the first week of symptom onset. Lin et al (19) reported TPE associated with continuous venovenous hemofiltration (CVVH). Ma et al (20) reported using blood purification therapies, including TPE and adsorption for treating Covid-19 patients.

Ma et al (Clinical Immunology, vol. 214 2020, 108408, DOI:

10.1016/j. dim.2020.108408) do not disclose a composition comprising 4-6% albumin or its use in plasma exchange.

Lerzan et al (Brain, Behavior and Immunity, vol. 87, 2020, 155-158, DOI: 10.1016/J. BBI.2020.05.022) discloses the treatment of autoimmune meningoencephalitis caused by Covid-19 in patients using plasmapheresis with albumin. This document does not disclose a composition comprising 4-6% albumin or its use in plasma exchange. Keith et al (Critical Care, 24 (1), 2020, DOI: 10.1186/s 13054-020-2836-4) proposes the use of therapeutic plasma exchange for the treatment of fulminant Covid-19 infection. This document does not disclose any treatment.

Rimmer et al (Critical Care, 18(6), 2014, 699, DOI: 10.1186/S 13054-014-0699- 2) is a review and meta-analysis of the use of plasma exchange for the treatment of sepsis and septic shock (abstract). Plasma exchange or plasma filtration involves the removal of plasma and replacement with normal saline, albumin or fresh frozen plasma (page 2, left column, paragraph 2). This document does not disclose Covid-19 patients.

Yong et al (Frontiers of Medicine, 14(2), 2020, 232-248, DOI: 10.1007/S 11684- 020-0774-9) reviews the treatments for patients coronavirus disease (abstract), including the use of plasmapheresis and filtration, plasma filtration absorption and protein A immune absorption for removing inflammatory cytokines and protecting organs. This document does not disclose a solution comprising 4-6% albumin.

Fang et al (Current Medical Science, 40(2), 2020, 275-280, DOI: 10.1007/S11596-020-2172-6) discloses the clinical characteristics of children with coronavirus, in which a child 1 year old with Covid-19 was subjected to plasma exchange. This document does not disclose a solution comprising 4-6% albumin.

US 6627 151 discloses a method of treating small vessel disease of the heart in a subject comprising the step of withdrawing blood, and treating the blood with plasmapheresis, and reinfusing the blood into the subject. This document does not disclose Covid-19 patients

Bobati et al (Journal of Clinical and Diagnostic Research, 11(8), 2017, EC35- EC37, DOI: 10.7860MCDR/2017/27073.10480) discloses the use of therapeutic plasma exchange in the treatment patients with neurological and non-neurological diseases, such as Guillain-Barre Syndrome. This document does not disclose Covid-19 patients.

Hua et al (International Journal of Antimicrobial Agents, 56(2), 2020, DOI:

10.1016/J.IJANTIMICAG.2020.105974) discloses the treatment of severe Covid-19 infection in a patient by plasma exchange (abstract) with frozen plasma used as the replacement solution. This document does not disclose a solution comprising 4-6% albumin or its use.

Clinical Trial NCT04374539 (clinicaltrials.gov) discloses the protocol of an ongoing clinical trial of plasma exchange for patients with Covid-19. Zhang et al (British Journal of Haematology, 190(4), 2020, DOI: 10.1111/bjh.16890) discloses the use of plasma exchange for treating severe Covid-19 patients and thereby reduce the cytokine storm. This document does not disclose the use of a solution comprising albumin.

Plasma exchange is the removal and retention of plasma, with return of the cellular components to the patients, in order to remove antibodies, toxins or abnormal proteins that are causing the clinical symptoms. The patient’s blood goes through an apheresis machine, where the blood is centrifuged or filtered so as to isolate the plasma fraction, such plasma fraction is removed, and the red blood cells, white blood cells and platelets are reinfused to the patient, along with replacement fluid.

Typically, 30-40 mL/kg of plasma (1-1.5 plasma volumes) are removed at each procedure and replaced with Frozen Fresh Plasma, isotonic 4.5 or 5.0% human albumin solution or mixture of such. Frozen Fresh Plasma refers to the plasma portion of whole blood, centrifuged, separated, and frozen solid at -18 °C or colder within eight hours of collection, but may also be used to designate any transfused plasma product.

Blood plasma makes up about 55% of the body's total blood volume and its volume can be estimated by the formula: Estimated plasma volume (in liters) = 0.07 x weight (kg) x (1 - hematocrit) .

A one plasma volume (PV) exchange removes about 63% of intravascular constituents, 1.2 PV approximately around 70%, 1.5 PV approximately around 75%, and 2 PV remove around 85% of the constituents.

The inventors have determined that it is possible to treat Covid-19 patients, including those under mechanical ventilation by performing plasma exchange, and replacing the removed and discarded plasma by an isotonic solution containing from 4-6% albumin (preferably about 5% albumin, or 5% albumin).

The term “around” or “about” is meant to encompass variations of ±3%, or in preferred instances ±1%, as such variations are appropriate to perform the disclosed methods.

The invention thus relates to an isotonic solution containing from 4-6% albumin for use thereof for the treatment of a Covid-19 patients, as a plasma replacement in plasma exchange therapy. In the preferred embodiment, the isotonic solution contains 5% albumin. Such isotonic solution is known in the art and is commercialized for instance under the name of Viabelex by the Laboratoire Frangais des Biotechnologies (LFB, Les Ulis, France). It can be dosed at 4% (40 mg/ml_) or at 20% (200 mg/ml_). When the solution is highly dosed, it is diluted in saline to obtain the appropriate concentration. Such solution also contains sodium chloride and/or sodium caprylate and water so that it can be injected. The solution is isotonic for plasma, meaning that it has the same osmolarity or osmolality than plasma (280 to 295 mOsm/L).

In the context of the invention, it is preferred when the isotonic solution is the only solution reinjected to the patient as plasma replacement. In other words, the solution is the sole replacement fluid used in the plasma exchange therapy. This means that no frozen fresh plasma, or convalescent plasma is used.

The isotonic solution replaces plasma which has been discarded, so as to remove any factor that may be responsible for the cytokine storm observed in critically ill Covid-19 patients, in particular IL-6. A plasma volume exchange of 1 to 1.5, in particular of 1.2 is appropriate for using the isotonic solution.

The “cleaning” of the patient’s plasma, leading to clinical improvement is generally not observed after one plasma exchange session. It is preferred to repeat such session, and when the isotonic solution is used multiple times. In particular, it is used from 3 to 5 times, once every 48 hours, particularly 4 times, once every 48 hours. However, the physician may decide to increase or decrease the number of uses or the duration between two uses, depending on the patient’s clinical condition.

The isotonic solution can be used for any patient with Covid-19. However, since the plasma exchange procedure is a heavy clinical procedure, it is preferred when it is used for patients with severe Covid-19, in particular for patients in need for oxygen assistance, in particular patients with acute respiratory distress syndrome and/or multi-organ failure. The example shows that it can be used when the patient is under mechanical ventilation.

The invention also relates to a method for treating a patient with Covid-19, comprising performing plasma exchange for the patient, wherein an isotonic solution containing from 4 to 6% albumin is used as the sole replacement fluid for plasma before reinjection to the patient. The embodiments disclosed above are also applicable to this method.

The invention also relates to a method for preparing a composition for treating a Covid-19 patient, replacing removed plasma from a patient with Covid-19 with an isotonic solution containing from 4 to 6% albumin as the sole replacement fluid, wherein from 1 to 1.5 volumes of patient has been removed. Such composition is a medicament used for the treatment of the Covid-19 patient. The medicament thus consists in a composition which contains from 13.75% to 22% of plasma from the patient, from 33% to 41.25% of an isotonic solution containing from 4 to 6 % of albumin, about 4% of white cells, about 41% of blood cells and about 0.01% of platelets, such cells and platelets originating from the patient. This method is performed ex vivo and doesn’t include the step of obtaining the patient’s plasma or of reinjecting the composition to the patient.

These percentages correspond to the replacement of 1 to 1.5 plasma volumes by the isotonic solution, and considering that the blood plasma is about 55% of the blood volume, with is complemented by about 4% of white cells, about 41% of blood cells and traces of platelets. When a plasma volume of 1.2 is removed (corresponding to the removal of around 70% of the plasma content) and replaced with the isotonic solution, the composition contains 16.5% of plasma from the patient, 39.5% of the isotonic solution containing from 4 to 6 % of albumin, about 4% of white cells, about 41% of blood cells and about 0.01% of platelets.

Due to the fact that there is some patient’s plasma remaining in the composition, even after addition of the isotonic solution, such composition contains antibodies or T lymphocytes against an SARS-CoV-2 antigen. Indeed, since the patient was infected with the SARS-CoV-2 virus, some immune response (humoral and cellular) is observable for the patient, thus leading to the presence of such antibodies or T lymphocytes.

The composition herein disclosed is obviously susceptible to be obtained by the method disclosed above. It is to be noted that the composition is generally administered to the patient as soon as it is produced in the apparatus performing the plasma removal. Indeed, the plasma exchange therapy requires some kind of extracorporal circulation, and the composition is not intended to be stored.

The invention also relates to the composition herein disclosed, for use thereof for the treatment of a Covid-19 patient. In particular, the plasma was isolated from the patient receiving the treatment. The composition is thus prepared from a patient’s plasma and reinjected to this patient.

All of the embodiments mentioned below can also be used for treatment of patients with COVID-19 related disease (diseases which appear as a consequence of SARS-CoV-2 infection or following such infection), in particular diseases which imply neurologic impairment, such as the ones disclosed in the examples (encephalitis).

Such treatment is of particular interest when the encephalitis is characterized by presence of small deep white matter lesions rather than diffuse confluent lesions of the deep white matter as seen by brain IRM.

The isotonic solution may be used in combination with corticosteroids.

In summary, using the isotonic solution as disclosed as the sole replacement fluid in TPE offers a lots of benefits:

Such solutions are widely available and more easily than fresh frozen plasma

These solutions are more safe than fresh frozen plasma, in view of their manufacture processes that lower the risk of contamination, where fresh frozen plasma or convalescent plasma that may contain pathogens These solutions reduce the anaphylactic risk (or other risks) that is sometimes observed with plasma transfusion (15).

FIGURES

Figure 1: evolution of the Pa02/Fi02 (A), IL-6 (B), D-dimers (C), fibrinogene (D), and CRP € values over time following TEP (represented by the four top arrows with dotted lines). A Pseudomonas aeruginosa ventilator-associated pneumoniae is represented by the arrow on the left (B and D panels). The mechanical ventilation duration is represented by the lung icon, the administration of sedative is represented by the syringe icon and the administration of neuromuscular blockers is represented by the biceps icon.

Figure 2: Significant MRI findings in Case #1: bilateral hyperintense lesions in fluid attenuated inversion recovery (T2/FLAIR) with gadolinium enhancement; Case #2: hyperintense lesions in T2/FLAIR with microhemorrhages; Case#3: multiple microhemorrhages in T2/FLAIR and diffusion sequences without gadolinium enhancement. Case #4: confluent hyperintense lesions in T2/FLAIR with gadolinium enhancement; Case #5: confluent hyperintense lesions in T2/FLAIR sequence with slight gadolinium enhancement.

Figure 3: Clinical presentation and time course of the disease. Abbreviations: COVID-19, coronavirus disease 2019; ICU, intensive care unit; NP, not performed; PLEX, plasma exchange; SAPS 2, simplified acute physiology score 2; SARS- CoV-2, severe acute respiratory syndrome coronavirus. * epinephrine > 0.1 pg/kg/min OR norepinephrine > 0.1 pg/kg/min

Figure 4: Complementary explorations findings. Abbreviations: COVID-19, coronavirus disease 2019; CSF, cerebrospinal fluid; EEG, electroencephalogram; IL-6, interleukin-6; MRI, magnetic resonance imaging; FLAIR, fluid-attenuated inversion recovery; SWAN, susceptibility weighted magnetic resonance sequences; NP, not performed; RT-PCR, Reverse transcription-polymerase chain- reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2

EXAMPLES

Example 1. Rapid resolution of ARDS secondary to SARS-CoV-2 infection by TPE

A 74-year-old man was admitted to ICU for acute respiratory distress that rapidly worsened over 3 days after having been in close contact with his son in-law presenting with signs highly suggestive of Covid-19. His past medical history was remarkable for hypertension treated with an association of calcium-channel and beta-blockers, a recent diagnosis of diabetes mellitus needing only dietary measures, and overweight (body mass index of 28,8 kg/m²). Upon ICU admission, respiratory rate was at 32 per minute and oxygen blood saturation (Sat0₂) was at 81% in room air. Despite 15 liters per minute of oxygen through a facemask, Sat0₂ was only at 91%. Orotracheal intubation to put the patient on mechanical ventilation was decided. Sedatives and neuromuscular blockers (NMB) were started simultaneously with lung protective ventilation. Biological values on admission are shown in Table 1.

Variable Patient Reference Range results

Tracheal Aspirate RT-PCR for SARS-CoV-2 Positive

Blood

Hemoglobin (g/dl) 15.0 13-17.5

Platelets (per mm³) 180,000 150,000-400,000

White-cell count (per mm³) 9290 4000-10,000

Differential count (per mm³)

Total neutrophils 7170 2000-7500 Total lymphocytes 1680 1500-4000 Total monocytes 400 150-1000 Procalcitonine (pg/l) 0.71 < 0.1

High-sensitivity C-reactive protein (mg/liter) 75 < 5 Fibrinogen (g/liter) 5.1 2-4

D-Dimers (ng/ml) 890 < 500

NT-pro-BNP (ng/l) 72.6 < 300 lnterleukine-6 (pg/ml) 114.2 0-6.5

Serologic testing for human Negative immunodeficiency virus Serologic testing for hepatitis b & c Negative

Table 1. Biological values on patient’s admission Reference ranges are affected by many variables, including the patient population and the laboratory methods used. The ranges used at Pitie-Salpetriere Hospital are for adults who are not pregnant and do not have medical conditions that could affect the results. They may therefore not be appropriate for all patients.

RT-PCR on nasopharyngeal swab confirmed SARS-CoV-2 infection. Chest X- ray revealed bilateral interstitial infiltrates suggestive of Covid-19 related ARDS. Empiric antibiotic therapy with IV cefotaxime was introduced on arrival, switched to piperacillin and tazobactam for seven days because of distal protected aspirate positive for oral flora.

Despite this treatment, the patient respiratory status worsened over the 7 first days with a Pa0₂/Fi0₂ ratio at 79 under PEEP at 14 cmH₂0 (pulmonary dynamic compliance at 27,6 ml_/cmH₂0) at day 7. The levels of CRP (110 mg/L, normal range < 5), fibrinogen (5.0 g/L, normal range 2-4) and IL-6 (306 pg/mL, normal range < 6,5) were increased. TPE indication was retained and the patient underwent 4 consecutive sessions every 48 hours (Spectra Optia, TerumoBCT, Lakewood, CO, USA), with 5% albumin as the sole replacement solution, exchanging 1.2 plasma volumes with a blood flow of 60 (40-70) mL/min for a duration of 110 (93-120) minutes. Anticoagulation was achieved by regional citrate infusion (1/12). Evolution of Pa0₂/Fi0₂ ratio, CRP, fibrinogen and IL-6 levels are shown in Figure 1. The last TPE session occurred on day 13.

Results

The patient respiratory status rapidly improved. He could be weaned from NMB at day 12, from sedatives on day 20 and finally from mechanical ventilation on day 21. Whereas Pa0₂/Fi0₂ ratio increased, IL-6 serum levels decreased under TPE treatment after initial rebounds, as expected with a protein of low molecular weight (24 kDa), short half-life, and broad distribution space (11). The patient’s status remained stable as the IL-6 levels (Figure 1).

In this patient, TPE was used as a unique strategy to mitigate CRS associated to Covid-19 without any a priori of the most important cytokine implicated in the syndrome. Our patient spectacularly improved under treatment, as compared to the dramatical outcomes described in Covid-19 patients admitted to ICU (13), and had no relapse after TPE discontinuation of both his respiratory status and his plasmatic IL-6 levels (except for a peak due to Pseudomonas aeruginosa ventilator-associated penumoniae). TPE has the advantage over monoclonal antibodies targeting pro-inflammatory cytokines of his transient effect on immune system precluding any long-lasting immunosuppression.

In addition, the protocol that was used here, with 5% albumin as sole replacement fluid, offers major benefits in terms of availability and security as compared to replacement with fresh frozen plasma or convalescent plasma that has been proposed for treatment of critically ill Covid-19 patients (14), with inherent anaphylactic risk.

Example 2. Severe COVID-19-related encephalitis can respond to immunotherapy

Background: Neurological manifestations have been reported during coronavirus disease-2019 (COVID-19) but encephalitis has seldom been reported. The role immunotherapy is unclear. Methods: We report five consecutive patients with severe COVID-19-related encephalitis treated with plasma exchange (PLEX) and corticosteroids in a 16-bed French Neuro-intensive care unit. Encephalitis was diagnosed after sedation withdrawal in patients initially admitted for a COVID-19 related acute respiratory distress syndrome. PLEX and corticosteroids were proposed for these brain-injured patients, as an immune-related mechanism was suspected.

Results: Patients (4 males, one female) were aged 37 to 77 years and presented with various central neurological manifestations including severe consciousness impairment, movement disorders, and brainstem dysfunction. In addition to brain involvement, one patient had a COVID-19 related acute inflammatory polyradiculoneuropathy. All patients had pathological brain magnetic resonance imaging findings, whereas cerebrospinal fluid (CSF) cytological and biochemical results were unremarkable. Reverse transcription-polymerase chain- reaction (RT-PCR) assay of the CSF was negative for SARS-CoV-2 for all patients. The interval between COVID-19 symptoms onset and diagnosis of neurological impairment ranged from 17 to 36 days. Three patients showed a rapid neurological improvement with consciousness recovery within 7 days after immunotherapy initiation.

Conclusions: Despite potentially severe clinical presentations, some COVID- 19 related encephalitis patients can respond to immunotherapy (PLEX and corticosteroids) supporting the hypothesis of a host-immune response to SARS- CoV-2.

Since December 2019, an outbreak of infections due to severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide and various coronavirus disease-2019 (COVID-19)-related neurological symptoms have been reported (Filatov et al., 2020, Helms et ai, 2020b; Mao et ai, 2020; Moriguchi et ai, 2020; Oxley et ai, 2020; Poyiadji et al., 2020; Khoo et ai, 2020; Paterson et al., 2020).

Although a neuro-invasive potential of SARS-CoV-2 is suspected - as for others coronaviruses -, there are few reports of COVID-19-associated encephalitis (Huang et al., 2020; Le Guennec et al., 2020; Moriguchi et al., 2020; Paterson et al., 2020). An immune-mediated mechanism has been proposed to explain coronaviruses associated encephalitis (Weyhern et al., 2020), and immunotherapy has shown interesting results in a recent case series of COVID-19 mild meningoencephalitis (PLEX; Dogan et ai, 2020) and one out of two patients with encephalitis in a more recent series (immunoglobulins; Paterson eta!., 2020).

Herein, we report a case series of five patients treated by PLEX and corticosteroids for severe COVID-19 related encephalitis.

MATERIALS AND METHODS

We report a consecutive series of patients diagnosed with severe COVID-19- related encephalitis treated with plasma exchange (PLEX) and corticosteroids. SARS-CoV-2 was detected by reverse transcription-polymerase chain-reaction (RT-PCR) in nasopharyngeal swab or tracheal aspirate. In the absence of other causes of consciousness impairment (i.e. toxic or metabolic), COVID-19 related neurological disorder was suspected to be responsible for delayed awakening after sedation withdrawal.

All patients had routine blood tests, cerebrospinal fluid (CSF) analysis, electroencephalogram (EEG), and brain magnetic resonance imaging (MRI). A comprehensive workup was performed in all patients to rule out an alternative diagnosis, including PCR detection of Herpesviridae in CSF, and testing for neuronal antibodies in the blood and the CSF (see Supplementary material for details). 5

Because of the suspicion of post-infectious inflammatory or immune-mediated encephalitis, PLEX sessions (5 to 10 with albumin as a replacement solution; see Supplementary material for details) and corticosteroids infusions (methylprednisolone 1g/day - 5 to 10 days) were proposed as a therapeutic strategy. Consent was obtained from patient's legal surrogate in accordance with French regulations. The study received approval from the Sorbonne University Ethic Committee (CER-202028 on 24/04/2020).

DETAILED DESCRIPTION OF THE 5 CASES

Case #1 is a 40-50 year-old male hospitalized in ICU, intubated and mechanically ventilated for a severe ARDS 10 days after COVID-19 symptoms onset. He received no specific treatment for COVID-19. Sedatives were stopped at day 28 after symptoms onset, and neurological examination revealed an alteration of consciousness (spontaneous eye opening, no response to simple orders), oculomotor disturbances (skew deviation and internuclear ophthalmoplegia), a bilateral upper motor neuron syndrome (bilateral ankle clonus and diffuse brisk deep tendon reflexes) and axial and lower limbs myorhythmia. He was clinically in an unresponsive wakefulness syndrome. EEGs performed at days 34 and 41 showed a nonspecific bilateral frontotemporal slow activity. Brain MRI at day 43 was remarkable for several bilateral hyperintense lesions in fluid attenuated inversion recovery (T2/FLAIR) and diffusion sequences, without reduction in apparent diffusion coefficient (ADC) in deep hemispheric white matter lesions with gadolinium enhancement. Lumbar puncture (LP) at day 43 was unremarkable. Brain MRI at day 50 showed similar findings, with a new lesion within the posterolateral left part of the pons on T2/FLAIR in accordance with neurological symptoms. The patient was still unresponsive at day 51 (FOUR-score = 11 [E3 M3 B3 R1], 23 days after sedation withdrawal). Corticosteroid infusions (1g/day IV methylprednisolone for 10 days) and PLEX (5 sessions) were respectively started at day 52 and day 57 with a rapid neurological improvement starting by a decrease in myoclonus and consciousness improvement allowing functional communication at day 63. Improvement of swallowing allowed withdrawal of mechanical ventilation at day 69, and a walking into the ward at day 70. Only a mild dysexecutive syndrome was observed at ICU discharge (on day 85).

Case #2 is a 50-60 year-old male hospitalized in the ICU, intubated and mechanically ventilated for severe ARDS 6 days after COVID-19 symptoms onset. He received no specific treatment for COVID-19. Sedatives were stopped at day 36 after symptoms onset, and neurological examination revealed a coma (no eye opening nor response to painful stimulation), and oculomotor disturbances (skew deviation and internuclear ophthalmoplegia). Brain MRI at day 41 showed a small hemorrhagic lesion of the left insula, a posterior hyperintensity of the pons, multiple microhemorrhages especially in the pontine tegmentum, and multiple left parieto occipital punctiform T2 hyperintense lesions without gadolinium enhancement. CSF examination at day 43 showed 1 cell/mm3, with normal levels of protein and glucose. Brain MRI at day 55 showed a regression of the posterior hyperintensity of the pons, and a stability of the other lesions. Neurophysiological exploration at day 55 included somatosensory evoked potential showing bilateral presence of N20 but with an increased P14- N20 inter-latency and, brainstem auditory evoked potentials showing medulo-pontic involvement (low III and V wave amplitudes, increased l-V inter-latency) in relation with a brainstem dysfunction. EEGs at days 57 and 63 showed nonspecific slow-wave activity, poorly reactive, without any epileptic patterns. Electroneuromyography at day 65 showed signs of mild polyneuropathy compatible with critical illness polyneuropathy. The patient was still unresponsive at day 65 (FOUR-score = 7 [E2 MO B4 R1], 29 days after sedation withdrawal). Corticosteroid infusions (1g day IV methylprednisolone for 5 days) and PLEX (5 sessions) were respectively started at day 66 and day 69, associated with a rapid consciousness improvement allowing functional communication at day 71, withdrawal of mechanical ventilation at day 88. and a transfer to rehabilitation center at 116 days. The patient was then fully conscious, with a mild dysexecutive syndrome.

Case #3 is a 60-70 year-old male hospitalized in the ICU, intubated and mechanically ventilated for severe ARDS the day of COVID-19 symptoms onset. He received no specific treatment for COVID-19. Sedatives were stopped at day 17, and neurological examination showed an alteration of consciousness (spontaneous eye opening, no response to simple orders), oculomotor disturbances (brisk horizontal oculocephalogyric reflex, ocular bobbing), myorhythmias of the tongue and flaccid quadriplegia with facial diplegia, areflexia and dysautonomia. He was clinically in an unresponsive wakefulness syndrome. EEG at day 25 showed nonspecific slow-wave activity, reactive to auditory stimuli, without any epileptic patterns. Brain MRI at day 30 was notable for multiple microhemorrhages within the corpus callosum and bilateral diffuse slightly hyperintense lesions of the deep subcortical white matter in T2/FLAIR and diffusion sequences. There were also two small ischemic lesions, of the right pallidum and the hypothalamus. CSF examination showed albuminocytologic dissociation with 4 cells/mm3, and high level of protein at 1.54g/L. Somatosensory evoked potential at day 45 showed slight bilateral N20 (inter - latencies were not reliable due to peripheral involvement). Brain MRI at day 48 showed new multiple microhemorrhages involving the corpus callosum, and a stability of the other lesions. Electroneuromyography at day 49 showed complete abolition of sensory and motor potential in four limbs, suggesting a COVID-19-related Guillain-Barre syndrome in addition to the encephalitis. The patient was still unresponsive at day 47 (FOUR-score = 8 [E3 M0 B4 R1], 30 days after sedation withdrawal). Corticosteroid infusions (1g day IV methylprednisolone for 5 days) and PLEX (10 sessions) were respectively started at day 49 and day 48, with a consciousness improvement and a regression of ocular bobbing, allowing functional communication (following verbal commands with eye tracking) at day 55, and a partial trapezius muscles strength recovery. Severe motor deficit of the 4 limbs with facial diplegia was still observed.

Case #4 is a 30-40 year-old male hospitalized in the ICU, intubated and mechanically ventilated for severe ARDS 10 days after COVID-19 symptoms onset. He initially received hydroxychloroquine plus spiramycin for 2 days (QT prolongation). Sedatives were stopped at day 33 after symptoms onset, and neurological examination revealed an alteration of consciousness (spontaneous non-goal directed movement, spontaneous eye opening, no eye fixation or following, no response to verbal commands). He was clinically in an unresponsive wakefulness syndrome. CSF examination at day 34 revealed a mild pleocytosis with 10 cells/mm3, with normal levels of protein and glucose. Brain MRI at day 39 was remarkable for several confluent hyperintense lesions in T2/FLAIR sequence, located within the periventricular and deep supratentorial white matter. There were also multiple symmetrical bilateral focal lesions of centrum semiovale, pallidum and periventricular white matter, hyperintense in T2/FLAIR and diffusion sequences with gadolinium enhancement. EEGs at days 34, 52, 58 and 65 showed nonspecific diffuse slow-wave activity, unreactive, without any epileptic patterns. Somatosensory evoked potential at day 60 showed bilateral presence of N20. The patient was still unresponsive at day 41 (FOUR-score = 8 [E3 M0 B4 R1], 8 days after sedation withdrawal). Corticosteroid infusions (1g day IV methylprednisolone for 10 days) and PLEX (10 sessions) were respectively started at day 42 and day 45 without any consciousness improvement leading to withdrawal of care and death at day 81.

Case #5 is a 70-80 year-old female hospitalized in the ICU, intubated and mechanically ventilated for severe ARDS 10 days after COVID-19 symptoms onset. She received no specific treatment for COVID-19 but initial cefotaxime plus spiramycine for a suspected community-acquired pneumonia. Sedatives were stopped at day 22 after symptoms onset, and neurological examination showed an alteration of consciousness (spontaneous eye opening, no response to simple orders). She was clinically in an unresponsive wakefulness syndrome. Brain MRI at day 35 was notable for several confluent hyperintense lesions in T2/FLAIR sequence, located within the periventricular and deep supratentorial white matter. They mostly had necrotic centers, with slight peripheral gadolinium enhancement. CSF examination at day 39 was normal. EEG at day 48 showed nonspecific diffuse slow-wave activity, inconstantly reactive, without any epileptic patterns. The patient was still unresponsive at day 39 (FOUR-score = 6 [E1 MO B4 R1], 17 days after sedation withdrawal). Corticosteroid infusions (1g day IV methylprednisolone for 5 days) and PLEX (5 sessions) were respectively started at day 40 and day 50 without consciousness improvement, leading to withdrawal of care and death at day 75.

For all patients, routine etiological workup included:

1/Blood:

• Serologic testing for human immunodeficiency virus

• Serologic testing for hepatitis b & c

• Antiphospholipid antibodies

• Autoantibodies to neutrophil cytoplasmic antigens

• Anti-ENA antibodies

• Anti-DNA antibodies

• Antinuclear antibodies

• Anti-aquaporin 4 antibodies

• Onconeural antibody testing including anti-NMDAr, -VGKC (LGI1, CASPR2), AMPA1/2, -DPPX, -GABAb, -Hu, -Ri, -Yo, -Tr, -CV2, -GAD antibodies

2/Cerebrospinal fluid:

• Gram stain and culture

• Auramine O staining for mycobacteria

• PCR for Herpes Simplex Virus 1 & 2

• PCR for Varicella-zooster-virus

• PCR for Cytomegalovirus

• PCR for Epstein-Barr-Virus

• PCR for Enterovirus

• PCR for Adenovirus

• Interferon-alpha level (were all undetectable)

• PCR for Toxoplasma gondii

• PCR for Aspergillus fumigatus

• Detection of Mucorales DNA

• Oligoclonal bands research

• Onconeural antibody testing including anti-NMDAr, -VGKC (LGI1, CASPR2), AMPA1/2, -DPPX, -GABAb, -Hu, -Ri, -Yo, -Tr, -CV2, -GAD antibodies

• Anti-aquaporin 4 antibodies PLASMA EXCHANGE TECHNIQUE

Plasma exchanges were performed with a centrifuge-driven cell separator Spectra Optia (TerumoBCT®, Lakewood, CO, USA), with 5% albumin as the sole replacement solution exchanging 1.2 plasma volumes with a blood flow of 60 (40- 70) mL/min for a duration of 110 (93-120) minutes. Anticoagulation was achieved by regional citrate infusion. Vascular access was established by venous insertion of double-lumen hemodialysis catheter.

RESULTS

Five patients aged between 37 and 77 years with suspected COVID-19 related encephalitis presenting with altered consciousness were treated by PLEX and corticosteroids. They all fulfilled diagnosis criteria for possible immune encephalitis according to Graus et al., 2016. Three had a rapid and marked improvement (cases #1 , #2 and #3) whereas two did not respond and eventually died (cases #4 and #5).

The clinical presentation and the time course of the disease are summarized in Figure 3, complementary explorations findings are summarized in Figure 4 (a detailed history is available in for each patient below). Patients had no prior history of neurological disease. They were intubated and mechanically ventilated for COVID-19 related ARDS. After sedation withdrawal (day 12 to 30 from initiation), they presented severe and persistent consciousness disorder (comatose state or unresponsive wakefulness syndrome), three had oculomotor disturbances (cases #1, #2 and #3) and one had peripheral symptoms attributed to Guillain-Barre syndrome (case #3). CSF examinations were unremarkable except in one patient with albuminocytologic dissociation (case #3), and one with mild pleocytosis (case #4). RT-PCR assays of the CSF were negative for SARS-CoV-2, as common viruses for all patients (see Supplementary material for details). Neuronal antibodies were negative in serum and CSF. None of the patients had signs of thrombotic microangiopathy (no hemolysis, normal levels of ADAMTS13 activity and antigen). EEGs showed unspecific slow-wave activity. Brain MRIs mostly showed bilateral hyperintense lesions in the deep and periventricular supratentorial white matter, either punctiform or slightly diffuse (cases #1, #2 and #3) or diffuse and confluent (cases #4 and #5), associated with lesions in the pons for 2 patients (cases #1 and #2; Figure 2) and gadolinium contrast enhancement for 3 patients (cases #1, #4 and #5).

All patients received immunotherapy combining corticosteroids infusions (IV methylprednisolone 1g/day for 5 to 10 days) and PLEX with albumin (5 to 10 sessions). It is worth noting that neurological impairment remained unchanged in for all patients with severe consciousness disorder despite cessation of sedation for 9-33 days. Three patients (cases #1, #2 and #3) showed neurological improvement few days after immunotherapy initiation (6, 2 and 7 days respectively), with consciousness recovery allowing ICU discharge and transfer to rehabilitation. Two patients (cases #4 and #5) showed no signs of consciousness improvement and died after discontinuation of life-sustaining therapies.

DISCUSSION

We report the first case series of patients with severe COVID-19 related encephalitis including consciousness impairment treated by PLEX and corticosteroids with an excellent response in 3 out of 5 patients.

Relation Between Immunotherapy and Clinical Improvement

Reports on patients with positive SARS-CoV-2 RT-PCR assay in the CSF are scarce (Huang et al., 2020; Moriguchi et al., 2020) and, most patients had moderate acute cognitive impairment without pleocytosi (Helms et al., 2020a) or, mildly elevated CSF cell counts (Bernard-Valnet et al., 2020). Likewise, Guillain- Barre and Miller Fisher syndromes, acute necrotizing hemorrhagic encephalopathy, and acute disseminated encephalomyelitis have also been described in COVID-19 patients, suggesting a host-immune response mechanism rather than a direct neuro-invasion of the SARS-Cov-2 (Toscano et al., 2020; Novi et al. , 2020; Gutierrez-Ortiz etal., 2020).

In the present cases, the secondary neurological involvement (no prior neurological initial symptoms), and the absence of SARS-CoV-2 in the CSFs point towards a post-infectious mechanism rather than a direct viral neuro-invasion as suggested by Weyhern et al, although no oligoclonal bands and low lnterleukine-6 were found in the CSFs.

The rapid clinical improvement (i.e 6, 2 and 7 days for cases #1, #2 and #3 respectively) after immunotherapy initiation was at striking contrast with the protracted neurological impairment (respectively 24, 30 and 31 days after sedation withdrawal) before treatment initiation. Such feature supports an inflammatory or immune process. In the instance of critical illness, delayed awakening and cognitive impairment such as delirium may result from many factors such as hypoxic encephalopathy, metabolic disturbances, side effects of sedation in the case of ICU patients (Mazeraud et a!., 2018). However, ICU related brain injuries had never been reported 11 to be responsive to immunotherapy. Although we cannot rule out a spontaneous recovery (Fischer et al., 2020), the rapid improvement after immunotherapy initiation seems to point towards a therapeutic effect.

Differences Between Responders and Non-Responders

PLEX and corticosteroids’ responders (cases #1, #2 and #3) and non responders (cases #4 and #5) shared similar disease courses (severe COVID-19 related ARDS, mechanical ventilation and sedation for several weeks, severe consciousness impairment which persisted several weeks after sedation withdrawal, unremarkable CSF findings).

Differences in treatment response may be related to lesion severity observed on MRI between the 2 groups. The responders mainly had small deep white matter lesions while non-responders had more diffuse confluent lesions of the deep white matter. Time of treatment since diagnosis does not seem to be a relevant factor since non-responders had earlier immunotherapy compared to responders (40 and 42 days after COVID-19 symptoms onset for the non-responders, versus 48, 52 and 66 days for the responders). Another cause of treatment failure can also be related to the underlying mechanism: non-responders may have had irreversible necrotic lesions related to vasculopathy and coagulopathy as often seen after COVID-19 infection especially in the lungs (Helms etai, 2020b).

CONCLUSION

Immunotherapy combining PLEX and corticosteroids can be effective in the treatment of severe COVID-19 related encephalitis. The exact pathophysiological mechanism underlying brain injury in these patients has yet to be clarified but a host-immune response to SARS-CoV-2 appears to be a plausible hypothesis. Further studies are necessary to confirm the therapeutic effect of combined PLEX and corticosteroids, and to determine the optimal treatment approach of these patients.

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4/abstract

Claims

1. Isotonic solution containing from 4-6% albumin for use thereof for the treatment of a Covid-19 patient or a COVID-19-related disease, as the sole replacement fluid for plasma replacement in plasma exchange therapy.

2. Isotonic solution for use according to claim 1, wherein the COVID-19-related disease is a neurologic disease, in particular an encephalitis.

3. Isotonic solution for use according to claim 2, wherein the COVID-19-related disease is a encephalitis and wherein the patient presents small deep white matter lesions rather than diffuse confluent lesions of the deep white matter.

4. Isotonic solution for use according to any one of claims 1 to 3, which contains 5% albumin.

5. Isotonic solution for use according to any one of claims 1 to 4, wherein from 1 to 1.5 volumes of plasma is exchanged.

6. Isotonic solution for use according to any one of claims 1 to 5, wherein it is used from 3 to 5 times every 48 hours.

7. Isotonic solution for use according to any one of claims 1 to 6, wherein the patient is under mechanical ventilation.

8. Isotonic solution for use according to any one of claims 1 to 7, wherein it is used in combination with corticosteroids.

9. A method for preparing a composition for treating a COVID-19 patient, replacing removed plasma from a patient with COVID-19 with an isotonic solution containing from 4 to 6% albumin as the sole replacement fluid, wherein from 1 to 1.5 volumes of patient has been removed.

10. An composition for treating COVID-19, containing from 13.75 to 22% plasma, from 33 to 41.25% of an isotonic solution containing from 4 to 6 % of albumin, about 4% of white cells, about 41 % of blood cells and about 0.01 % of platelets,

11. The composition of claim 10, which contains antibodies or T lymphocytes against an SARS-CoV-2 antigen.

12. The composition of claim 10 or 11, which is susceptible to be obtained by the method of claim 6.

13. The composition of any one of claims 10 to 12 for use thereof for the treatment of a COVID-19 patient or of a patient with a COVID-19 related disease.

14. The composition for use of claim 13, wherein the COVID-19 related disease is a neurologic disease.

15. The composition for use of claim 13 or 14, wherein the plasma was isolated from the patient receiving the treatment.