Role of Convalescent Plasma Therapy in Patients with Moderate to Severe COVID 19

Significance: Given the lack of evidence of effective treatments and vaccines for Corona virus disease-19(COVID-19), several treatment options are under investigation, including Convalescent Plasma. The FDA granted emergency use authorization (EUA) on August 23, 2020 for use of convalescent plasma in hospitalized patients with COVID-19 but further data from randomized clinical trials are needed. Objective: The objective of the study was to find evidence of efficacy of Convalescent Plasma therapy in patients with moderate to severe COVID-19. Study Design, Setting, and Sample size: Open label, observational study, where participants were allocated using predefined assessment criteria. The study included 42 patients admitted in Fatima Memorial Hospital, Lahore with moderate to severe COVID-19. The study was terminated early after 42 patients of a planned sample size of 50 patients due to no further admissions of COVID-19 in the hospital. Groups: Standard treatment for COVID-19 disease (antibiotics, steroids, Tocilizumab, Remdesivir) according to disease severity was given in 15 patients (control group), while Convalescent plasma was administered in addition to standard treatment in 27 patients (cases). Primary and Secondary Outcomes: Primary outcome was to see clinical improvement in patients with moderate to severe COVID-19, defined as patient recovered and discharged home. Secondary outcomes looked at oxygen requirement in both cases and controls after administration of treatment and any improvement in biochemical markers. Results: 42 patients were observed in the study. 27 patients assigned to the cases group received standard treatment and convalescent plasma therapy for moderate to severe Covid-19 disease whilst 15 patients received standard treatment only, assigned as control group. The median age of patients in the convalescent plasma group was 59.81±10.14 and 54.37 ±12.93 in the control group (p-value = 0.086). There was no statistically significant difference in baseline characteristics between the cases and control group. Primary outcome measured as patient recovered and discharged home was achieved in 64.3% of patients in the cases group and 35.7% in the control group (p value = 1). Secondary outcome compared oxygen requirements in cases (median 20 l/min) and controls (median 8 l/min) after treatment. There was no statistically significant difference in oxygen requirements between cases and controls after treatment (p=0.059). Biochemical markers of inflammatory response such as Total leukocyte count, D-dimer, Lactate dehydrogenase (LDH) did not show any statistically significant difference in the two groups. CRP however decreased markedly in those patients treated with Convalescent plasma in addition to standard treatment when compared with control group (p value= 0.002). Also, serum Ferritin was also found to be much lower in cases as compared to controls (p value = 0.001). No adverse effects were seen in the cases after administration of convalescent plasma. Conclusion: Convalescent Plasma therapy administered in addition to standard treatment did not result in a statically significant improvement in patient survival in patients with moderate to severe COVID-19 when compared with standard treatment alone. There was however, a significant reduction in two inflammatory markers associated with severe disease i.e. C-reactive protein (CRP) and serum Ferritin in the cases group. The study results may have failed to detect a clinically significant difference because of early termination of the study resulting in small sample size.

Several observational studies, and randomized control trials are underway evaluating the efficacy of Tocilizumab, an interleukin-6(IL-6) receptor inhibitor. This immunomodulatory drug already used for rheumatic diseases and cytokine release syndrome is being investigated for treatment of severe COVID-19.
Currently, there is little evidence for effective treatments of COVID-19 and vaccines are under trial. Several treatments are under investigation which have previously been used in pandemics, including the Spanish flu, SARS-Co V, West Nile virus, and more recently, Ebola virus. 3,4,5,6 Passive immunization has been used in prevention and management of infectious diseases since early 20th century 3 . This includes Convalescent Plasma, a passive immune therapy which requires administration of specific antibodies against the agent for treating an infectious disease caused by that agent. Convalescent plasma is obtained using apheresis from patients recovered from the disease. Mechanism of action is viral neutralisation 2 . Most observational studies to date suggest administration of convalescent plasma(CP), preferably with high antibody titer, in early disease, prior to development of life-threatening inflammatory disease.
During apheresis, in addition to neutralizing antibodies (NAbs), other proteins such as antiinflammatory cytokines, clotting factors, natural antibodies, defensins, pentraxins and other undefined proteins are obtained from donors 7 .In this sense, transfusion of CP to infected patients may provide further benefits such as immunomodulation via amelioration of severe inflammatory response 8 . The latter could be the case of COVID-19 in which an over-activation of the immune system may come with systemic hyperinflammation or "cytokine storm" driven by IL-1β, IL-2, IL-6, IL-17, IL-8er, TNFα and CCL2. This inflammatory reaction may perpetuate pulmonary damage entailing fibrosis and reduction of pulmonary capacity 9,10 .
In the United States, the Food and drug administration (FDA) is accepting investigational new drug applications for use of convalescent plasma for patients with severe or life-threatening COVID-19 .One case series described administration of plasma from donors who had completely recovered from COVID-19 to five patients with severe COVID-19 on mechanical ventilation and persistently high viral titers despite investigational antiviral treatment 11 .
The patients had decreased nasopharyngeal viral load, decreased disease severity score, and improved oxygenation by 12 days after transfusion 11 . However, these findings do not establish a causal effect, and the efficacy of convalescent plasma remains unknown.
The convalescent donors must undergo standard predonation assessment to ensure compliance with current regulations regarding plasma donation 12 . Currently, convalescent donors between 18 and 65 are considered as subjects without infectious symptomatology and a negative test for COVID-19 after 14 days of recovery. These tests must be repeated 48 h later and at the moment of donation 12 . Apheresis is the recommended procedure to obtain plasma. This procedure is based on a continuous centrifugation of blood from donor to allow selective collection plasma. The efficiency of this technique is around 400-800 mL from a single apheresis donation. This amount of plasma could be storage in units of 200 or 250 mL, and frozen within 24 h of collection to be used in further transfusions 13 .
We want to know whether plasma from people who have recovered from COVID-19 is an effective treatment for people with COVID-19 in our geography, and whether this treatment causes any unwanted effects.

Methods: Study design:
It was an open label, observational study, where participants were allocated using predefined protocol. Those patients who met the inclusion criteria of study after signing an informed consent either by the patient or the person legally authorized to sign were included. Patients not receiving convalescent plasma therapy were considered as control and were given standard available treatment as approved by FDA and local authorities. Standard treatment included giving antibiotics, steroids (as recommended by the Recovery Trial, United Kingdom,UK), Tocilizumab (for patients in cytokine storm) and Remdesivir (antiviral medication).

Sample size:
Our sample size was aimed at 50 patients calculated by the results from previous study which showed improvement in 80% of patients. However due to diminished cases of COVID-19 in the region, no further admissions were done and the study was terminated.

Sampling technique:
Our sampling technique was probability sampling according to inclusion and exclusion criteria.

Duration of study:
The study was conducted over a three month period, from May 2020 till July 2020.

CP infusion protocols:
Patients will receive an initial dose of 200 mL, followed by one or two additional doses of 200mL according to disease severity and tolerance of the infusion. Predefined Parameters of disease severity will be assessed 12 to 24 hourly.

Standard Treatment:
Standard treatment was given to all patients and included anti-bacterial medication (Azithromycin for all patients and Meropenem for secondary infections), steroids (dexamethasone, methylprednisolone), anti-coagulants (enoxaparin, heparin), Tocilizumab (for patients in cytokine storm) and anti-viral medication (Remdesivir). This was provided based on latest national guidelines and hospital protocols.

Outcome Measures:
The primary endpoint of the study was patient recovery and discharge home. Secondary outcomes assessed the response in patients who received convalescent plasma compared with patients receiving standard treatment.
Assessment of response in patient was measured using following parameters: 1 .Clinical parameters: a. Improvement in oxygen saturation b.
Decrease oxygen requirements c.
Weaning of from ventilator support d. Normalization

Results:
A total of 42 patients were observed in the study. 27 of these patients received standard treatment and convalescent plasma therapy (cases) for moderate to severe Covid-19 disease whilst 15 received standard treatment, assigned as control group.
The median age of patients in the convalescent plasma group was 59.81±10.14 and 54.37 ±12.93 in the control group (P value = 0.086). There was no statistically significant gender difference between the convalescent plasma group and the control group (p value = 0.734).   Hypertension was the most prevalent co-morbidity with 74.1% (20) patients in the convalescent plasma group and 33.3 % (5) patients in the control group known to have the disease. Diabetes, the second most prevalent condition was found in 63 % (17) patients), while 46.7% (7) patients suffered from diabetes in the control group, p value =0.347. A total of 6 patients (22.2%) had a history of cardiovascular disease in the convalescent plasma group while 1 patient (6.7%) was known to have cardiovascular disease in the control group. Chronic kidney disease was found in 7.4% of patients in convalescent plasma group and 6.7% in the controls. Although, 7 patients in the convalescent plasma group were obese, none in the control were obese. However, this difference was not found to be statistically significant as p value was 0.038. The most common lung disease was Asthma.

Figure: 2: Presenting symptoms in convalescent plasma group (case) and standard treat group(control)
Patients with known history of lung disease were equally distributed in both groups. Table-

Case Control
No statistically significant differences were found in baseline demographics of the cases and controls group.
Secondary outcome included reviewing patient's oxygen requirement and oxyhaemoglobin saturation, SaO2. Oxygen requirement was defined as the minimum amount of oxygen required to keep SaO2>94%. Patient's oxygen requirements were measured on a daily basis before and after treatment. All patients required oxygen on admission (minimum oxygen requirement was 2L and maximum was 15L). The oxygen requirement increased with disease progression. However, there was no significant difference in reduction of oxygen requirements between cases and controls. Graph-1.

Graph:1: Representing oxygen requirement IQR in patients before and after treatment in cases and controls.
The assessment of response in patient was also measured using laboratory parameters mainly inflammatory markers such as D-Dimers, LDH, CRP & serum Ferritin levels. Total leukocyte count was measured for any secondary bacterial infections. There was no statistical difference in the laboratory parameters such as TLC, d-dimers, CRP, LDH, Creatinine and ALT between the cases and controls. Oxygen requirement (pre treatment) Oxygen requirement (post treatment)

Controls
Graph:2-Depicts the trend in inflammatory markers before and after treatment with convalescent plasma along with standard treatment.

Graph: 3-Depicts the trend in inflammatory markers in patients who received standard treatment.
The primary end point was patient recovery and discharge home. The discharge criteria was based on normalization of body temperature, resolution of presenting symptoms, maintaining oxygen saturation >94% at room air at rest and a repeat PCR negative and chest x ray showing decreasing infiltrates more than 50% improvement from base line.
The rate of recovery in the two groups was 64.3% versus 35.7% (p-value= 1) signifying that convalescent plasma did not improve patient survival. No adverse events were reported after the transfusion of convalescent plasma. Figure  Both trials did not indicate any reduction in mortality. Our research data also suggested that convalescent plasma therapy did not add any survival benefit in this patient cohort when compared with current standard treatment. This is contrary to the some of the results of the secondary end point, where a clear improvement in some of the biochemical markers such as CRP, Serum Ferritin is seen. The remaining biochemical markers suggest a decline in the inflammatory process and improvement in patient oxyhaemoglobin Saturation,SaO2 irrespective of the administration of convalescent plasma.
Our study had its limitations as it was only a single centre study restricted to a specific group of people. We were also unable to attain our sample size of 50 participants due to a reduction in number of patients admitted with severe SARS COV2. There were also confounding variables such as administration of other immunomodulatory drugs such as Tocilizumab for cytokine storm and use of steroids which are known to be effective in moderate to severe COVID 19 disease (Recovery Trial)*. The anti-viral drug, Remdesivir was given to ten patients. Remdesivir has been found to improve recovery rate in patients when compared with placebo*.
Our study failed to demonstrate any improvement in survival when compared with standard treatment. Our recommendations based on the study are that Convalescent plasma is safe therapeutic option and may have a role when administered early in the viral replication stage (first 14 days) especially when other treatments may be contraindicated, as it demonstrated reduction in inflammatory markers.