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Convalescent plasma: A possible treatment protocol for COVID- 19 patients suffering from diabetes or underlying liver diseases

Background & Aim As on date, no specific treatment is available for devastating COVID-19 (SARS-CoV-2) infection. This pandemic viral infection has affected over 200 countries within a very short time and created a calamitous situation across the globe. As per WHO guidelines, the treatment is mainly symptomatic and supportive. This clinical protocol has not proven sufficient to save the lives of COVID-19 patients suffering from diabetes or having underlying liver diseases; hence there is utmost need to tackle this situation by other means such as Convalescent Plasma (CP) therapy. Methods A comprehensive literature survey was carriedout using Elsevier, PubMed, Taylor & Francis, Springer, Nature and Google search engines. Results The patients suffering from diabetes or liver dysfunction or any other underlying diseases are at greatest risk of SARS-CoV-2 infection. From the study, it is proved that plasma collected from the recovered patients of viral infection has considerable potential to treat the viral disease without the occurrence of adverse effects. Conclusion The CP therapy can be a possible life saving alternative to treat critical COVID-19 patients having diabetes or underlying liver dysfunction. Hence, randomised clinical trials are recommended at the earliest to save the lives of infected individuals of COVID-19.


Introduction
A novel Coronavirus disease 2019  is the infection of the respiratory tract caused by Severe acute respiratory syndrome coronavirus À2 (SARS-CoV-2) which has created a disastrous situation in most of the countries. The first case of COVID-19 was reported in China's Wuhan State (capital of Hubei province) in December 2019 [1].
It is a highly infectious disease and almost reached every country across the globe (Over 200 counties) within a very short span. By April 20, 2020 over 24 lakh people were infected with COVID-19 and caused over 1.7 lakh deaths worldwide [2]. The mortality rate based on the cases which had an outcome is on higher side, i.e. 20% [2]. Most of COVID-19 patients were asymptomatic (or with very mild symptoms) and recovered themselves, which were very difficult to be detected; otherwise the total number of COVID-19 cases reported so far would be on the higher side. As on date, no effective therapy is available to treat devastating SARS-CoV-2, the treatment is mainly experimental or empirical.
According to the figures of the International Diabetes Federation, more than 463 million peoples were suffering from diabetes globally [3]. Very limited data is available regarding COVID-19 patients with diabetes, but it is reported that the diabetic patients are at the utmost risk of SARS-CoV-2 infection. The recent report published by Chinese Centre for Disease Control and Prevention of 72,314 cases of COVID-19 shown that, the mortality rate is on the higher side if the virus infected patient is suffering from diabetes. (7.3% in patients with diabetes while only 2.3% in other patients) [4,5]. Similarly, those suffering from advanced chronic liver diseases are also at greatest risk of infection owing to autoimmune liver diseases and cirrhosis-associated immune dysfunction [6]. It has been confirmed that, the liver cells get infected with SARS-CoV-2 infection as the receptor of the virus is ACE2, which has been revealed to be expressed on cholangiocytes [7,8]. The study confirmed that, 43 out of 99 patients infected with SARS-CoV-2 had some sort of liver function abnormality.
The drugs like Chloroquine, Hydroxychloroquine and Remdesivir [9] were reported to be effective in some studies, but not efficacious in critically ill and aged patients of COVID-19. The patients with diabetes or any other severe underlying condition may develop serious respiratory diseases if infected with the COVID-19 infection. To combat such a devastating situation, this article highlights research in the fields of neutralizing antibodies (nAbs) present in convalescent plasma for the treatment of SARS-CoV-2.

Search methods
A comprehensive literature survey was carried out using Elsevier, PubMed, Taylor & Francis, Springer, Nature and Google search engines to prepare a descriptive review on this topic. The Professors from University of Oxford, UK and SP Pune University, India as well as the Physicians of COVID 19 hospitals of India were consulted to discuss the use of the CP therapy and its potential to combat COVID-19 infection. The search terms, "SARS-CoV-2", "COVID-19" and "Neutralizing Antibodies" along with "Convalescent plasma", "Diabetes" and "Liver diseases" were thoroughly investigated using search engines.

Structure of SARS-CoV-2
The SARS-CoV-2 is an enveloped, single-stranded and positive (þ) sense RNA virus. It belongs to the beta-CoV genera of Coronaviridae family. Structural proteins like membrane (M), nucleocapsid (N), spike protein (S) and envelope (E) protein play a vital role in the entry of virus and its replication in the host cell [10].

Neutralizing antibodies from CP
The recovered patients of COVID-19 have high neutralizing antibody titre, which can be an important donor source of Convalescent Plasma. The neutralizing antibodies can be efficacious in virus clearance and can act as a key immune product to treat viral diseases [11]. If nAbs specific to particular virus are administered in infected individual, it may reduce viral replication and disease severity. It may be possible means to limit the COVID-19 pandemic infection.
The S protein of SARS-CoV-2 is one of the major targets for developing neutralizing antibodies to inhibit the binding and fusion of SARS-CoV-2. Schematic mechanism of neutralizing antibodies is highlighted in Fig. 1. Neutralizing antibodies binds with Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein as shown in the aforesaid figure. The protruding portion (blue colour) highlights the antibody epitope [12]. It has been reported that the ACE2 is the cell entry receptor for SARS-CoV-2 as like SARS-CoV because ACE2 shows binding to the receptor binding domain of both SARS-CoV and SARS-CoV-2 [13]. In the present scenario the research in the globe is focused on identifying neutralizing antibodies which can target the spike protein responsible for viral entry into the host cell, producing protective results in affected individuals.

Research in the field of convalescent plasma therapy
It has been reported that; the antibodies isolated from the recovered individuals of viral diseases were administered to an infected individual at an early stage, may magnificently reduce the viral load and disease mortality associated with SARS viral infections [11]. Suppression of the viraemia was also reported because of antibodies present in convalescent plasma.
Hung and his associates highlighted the effective use of convalescent plasma in H1N1 viral infection. The dramatic reduction in viral load was observed within 5e7 days of symptom onset [14]. Besides, significant reduction in mortality was also observed in patients treated with convalescent plasma.
The research conducted by Hung IF et al. revealed successful treatment over 20 patients suffered with pandemic influenza A (H1N1)2009 viral infection [15]. The serum cytokine response, viral load of the respiratory tract and the mortality rate were greatly reduced with the treatment of convalescent plasma.
In a study in Hong Kong, 80 patients who were suffering from severe acute respiratory syndrome (SARS) infection were administered with convalescent plasma [16]. Some patients received convalescent plasma after day 14 of infection, while some received immediately after the infection. It was observed that, patients treated earlier successfully recovered from the clinical symptoms of infection than those who received plasma after day 14. It signifies the efficacy of CP therapy, which depends on how early you start the treatment of affected individuals after confirmed identification of infection.
A clinical study conducted on three patients of SARS infection in Taiwan also highlights the effective use of convalescent plasma [17]. All these three patients were critical and did not respond to available therapy. They were administered with convalescent plasma and the therapy was found to be successful within a span of 24 h of administration of CP. The viral load decreased from day 2, the hyperthermia dropped dramatically and all patients survived. The major limitation of the study was a very small sample size.
Effectiveness of CP therapy along with brincidofovir was also reported against the devastating Ebola virus disease in the year 2015 in Africa [18]. The viral load decreased immediately after starting investigational therapy with CP in most of the infected persons.
Zhou B. highlighted promising use of passive immunotherapy for the treatment of influenza A (H5N1) infection in China [19]. The critical patients were not responding to Oseltamivir alone, but recovered by combinational approach using Oseltamivir with CP therapy. Immediately after transfusion (within 8 h) of CP therapy, the viral load was found to be reduced by a factor of 12 (from 1.68 Â 105 to 1.42 Â 104 copies per millilitre).
Arabi Y. has also demonstrated the use of CP at early stages of MERS-CoV [20] which has considerable potential for clinical improvements in affected individuals.
Chenguang S. efficaciously carried out treatment of five critical patients of SARS eCoV-2 (COVID-19) with convalescent plasma [21]. The significant improvements in clinical status of four infected individuals out of five were reported within ten days of transfer of convalescent plasma. The body temperature normalised within three days of initiation of treatment. It was observed that, the viral loads were also decreased and it seems to be negative after 12 days of transfer of CP.
Chen L. has clearly stated the use of convalescent plasma for COVID-19 as a promising approach [22]. It has emphasised on the successful use of CP against the SARS -CoV, Ebola, MERS and H1N1 viral species.
One of the promising clinical studies carried out against the devastating SARSeCoV-2 (COVID-19) successfully demonstrated the operational use of CP therapy [23]. The therapy was adopted to treat 10 severe adult cases from China who was intolerant to available therapy. The single dose of 200 mL of CP showed the remarkable results in affected serious patients. The dose was well tolerated by all individuals with minimal side effects. The clinical symptoms were improved within 3 days of CP therapy and the viraemia was also disappeared on day 7. The increase in oxyhaemoglobin saturation on day 7 along with lymphocyte counts was also observed.
The recent useful study carried out in China over 175 patients recovered from COVID-19 viral infection clearly highlights the generation of SARS-CoV-2-specific neutralizing antibodies (spike binding) from day 10e15 after infection [24]. From day 10 the peak levels of neutralizing antibodies were detected in all patients and these antibodies were remained stable thereafter.
The clinical trials of Convalescent plasma therapy conducted/ initiated against COVID-19 pandemic across the globe are summarised in Table 1. The said table highlights the doses, participants and findings of different studies of CP therapy.

India's perspectives for convalescent plasma therapy
In India, Drug Controller General of India (DCGI) has given permission to the Indian Council of Medical Research (ICMR) as well as other research agencies/institutes of medical sciences to conduct the clinical trials using Convalescent Plasma [28,29]. The DCGI has given a protocol for carrying out a phase II, open label, randomised controlled trials for assessing the safety and efficacy of convalescent plasma [30]. The given document highlights the policies to be pursued while adopting CP therapy in India. The Delhi, Kerala and the Maharashtra were states of India where the trials were initiated to combat devastating COVID-19 infection in a large number of patients.
The first patient in India (aged 49) who was treated with convalescent plasma therapy in Delhi has now recovered from COVID-19 and was discharged from hospital on April 24, 2020 [31]. This patient was admitted in hospital with history of respiratory diseases and moderate symptoms. In the next few days his condition worsened and he developed pneumonia with Type I respiratory failure. He did not respond to the available therapy, later given ventilator support, but the condition deteriorated with time, therefore the CP administration started as per the established protocol from day 10 of infection. The progressive improvement was observed in the patient from day two and on the fourth day of transfusion of CP the ventilator support was weaned off. The patient was successfully recovered from infection within 10 days of transfusion of convalescent plasma.
In India, many of the recovered patients of COVID -19 are ready to donate their plasma to save the lives of other infected individuals. The plasma collected from one recovered patient can be sufficient to treat two to three COVID -19 patients.

Protocol of convalescent plasma transfer
In the present outbreak of COVID-19, whole blood consisting of plasma collected from SARSeCoV-2 patients, which may be administered to infected persons and can be investigated as one of the possible means of treatment. The schematic representation of passive antibody therapy for COVID -19 patients, highlighting the major steps is shown in Fig. 2.
The foremost steps of CP therapy can be summarised as below [28,30]:

To identify the blood donors who have been recovered from
SARSeCoV-2 viral infections. 2. Pre-donation screening should be carried out. The donor should be free from any signs of SARS-CoV-2 infection and two negative results should be obtained for SARS-CoV-2 by molecular testing of blood or nasopharyngeal swab testing. 3. The informed consent of the donors should be collected. 4. The donor's blood grouping and screening for transfusion transmissible infections should be carried out. 5. The blood is to be withdrawn from the recovered person of COVID-19 infection, as per the standard parameters/established protocol. 6. The plasma has to be separated from whole blood and investigated for presence of virus-neutralizing antibodies by the experts. 7. The storage and transportation of CP to the respective hospitals for the transfusions. 8. Compatibility testing of critically ill and aged patients of COVID-19 should be carried out. 9. The informed consent from a patient or his legal heirs (if unconscious) should be taken. 10. The respiratory rate of the patient should be > 24/min and SaO2 < 93% on room air or PaO2/FiO2 <300. 11. The clinical transfusion process should be carried out as per the existing protocol. 12. The continuous assessment of the success of this empirical therapy should be carried out for 7e10 days. The viral load, antibody levels and other clinical tests should be monitored. 13. The adverse effects (if any) after the administration of CP should be closely monitored.

Adverse effects of CP therapy
No serious adverse effects were reported in the CP therapy previously carried out in SARS, MERS, H1N1 and Ebola infection outbreak [15,16,20]. The therapy was well tolerated in most of the patients while some reported non-lethal adverse effects. The adverse effects like increase in body temperature, nausea, itching, chills and skin rashes were reported in some studies [32]. These events were resolved spontaneously by symptomatic treatment or by reducing the rate of transfusion. The transfusion related adverse effects like circulatory overload, anaphylactic reactions and transfusion related acute lung injuries (TRALI) were also reported in few patients [33]. The incidence of TRALI is very rare (affecting one in every 5000 patients) and observed in severe patients only.

Cautions of CP therapy
The risk of Hepatitis B virus, Hepatitis C virus and HIV disease transmission through the donated plasma should be thoroughly investigated. Very limited information is available on the safety of -The close contacts of those patients and health care workers were also allowed to enrol for the study.
[27] CP therapy in pregnant patients of viral diseases; hence sufficient results are warranted prior to use of CP therapy in pregnancy.

Conclusion
From the evidences highlighted in the aforesaid study, convalescent plasma from the recovered patients of COVID-19 can be effectively used as a treatment protocol for SARSeCoV-2 viral infected patients having diabetes or underlying liver diseases. The CP therapy has greatest potential to treat the devastating COVID -19 viral infections. Most of the earlier studies were carried out with fewer numbers of patients having diabetes or liver diseases; hence further in-vivo studies on a large scale will be required to generate statistically significant data in terms of its efficacy and to investigate possible adverse effects associated with it. The optimal dose, time of administration and clinical aspects of CP therapy should be investigated. There is utmost need to assess available in vitro-in vivo data and to carry out randomised clinical trials on COVID -19 patients having diabetes or any other comorbidity to save the valuable lives of infected individuals across the globe.

Ethical approval
Not required.

Financial support
The work is not funded by any government or private agencies.