Successful Treatment of Convalescent Plasma Therapy in Three Patients With Severe SARS-CoV-2 Infection in Fuzhou, China

Background Up to now, there is no specic treatment for coronavirus disease 2019 (COVID-19) yet except for general supportive care. Hence, it will be critical to nd a new strategy for COVID-19. The study is to explore whether convalescent plasma transfusion may be benecial in the treatment of severe patients with COVID-19. Methods This is a retrospective analysis of three severe patients with laboratory-conrmed COVID-19 and admitted in Fuzhou pulmonary hospital of Fujian province from February 18th, to May 15th,who met the following criteria: (1) within 3 weeks of symptom onset (cid:0) laboratory conrmed cases or who had viremia conformed by clinical experts (cid:0)(cid:0) 2 (cid:0) Severe patients with rapidly progress or the early stage of critically ill patients or who required plasma therapy were comprehensively evaluated by clinical experts. The data of clinical manifestations and the progresses of disease monitored by blood-gas analysis, biochemical tests, routine examine, radiological exam were abstracted and then analysis the changes before and after convalescent plasma transfusion. All three (one male and two females; age range, 57-65years) were treated with plasma Two patients the novel coronavirus within 3 days after the transfusion. No adverse effects and severe complications were observed. Conclusions In this preliminary uncontrolled case series of 3severe patients with COVID-19, convalescent plasma could be as a promising therapy for COVID-19 without corticosteroids and no serious adverse reactions associated with the transfusion of convalescent plasma were observed, which would improve the clinical outcomes following by improvement in their clinical status. Using the convalescent plasma at the early stage(less than 10 days) of disease could be more effective. Anticoagulation is necessary for severe patients with COVID-19 given the state of hypercoagulability. However, given the small sample size and limited study design, naturally these results should be taken with a grain of salt until replicated by other further investigation in larger well-controlled trials.


Introduction
Since late December 2019, an outbreak of severe acute respiratory syndrome coronavirus-2(SARS-CoV-2) infection which was named by World Health Organization (WHO) as coronavirus disease 2019(COVID-2019), was initially emerged in Wuhan, China [1][2][3].The epidemic spread rapidly worldwide and become an unprecedented global public health crisis [4,5].As of May 16, 2020, COVID-19 had spread up to more than 200 countries and regions, that had been reported by WHO, with a total number of 4,425485 cases, including 302056 cases (6.83%) [6]. At the moment, there are still no speci c therapies approved by the China Food and Drug Administration (CFDA) or U.S. Food and Drug Administration (FDA) for SARS-CoV-2, the virus that causes COVID-19, therapeutic strategy is being studied in addition to supportive care [7], while some drugs are still under investigation. Several agents are being used under clinical trial and compassionate use protocols based on in vitro activity (against SARS-CoV-2 or related viruses) and on limited clinical experience [8]. E cacy has not been established for any drug therapy. Several agents seem to be clinical bene cial, but their e cacy is far from satisfactory [9].A recent trial showed lopinavirritonavir has no treatment bene t for severe illness caused by SARS-CoV-2 [10].A randomized, doubleblind, placebo-controlled, multicentre trial [11] revealed remdesivir was not associated with a difference in time to clinical improvement (hazard ratio 1.23 [95% CI 0.87-1.75]),which was not statistically signi cant. Although patients receiving remdesivir had a numerically faster time to clinical improvement than those receiving placebo among patients with symptom duration of 10 days or less (hazard ratio 1.52 [0.95-2.43]).To the end, the effective vaccine and speci c antiviral medicines are unavailable, therefore there are urgent needs to look for an COVID-19-speci c treatment, especially among the severe patients. Previous reports [12] have shown treatment with convalescent plasma collated from recovered patients would have a shorter hospital stay and lower mortality than the comparator group(high-dose steroids pulse therapy), and no immediate adverse effect noted after plasma infusion. A meta-analysis involving 1703 in uenza pneumonia patients who received in uenza-convalescent human blood products, showed treatment with convalescent plasma could reduce virus load and pool absolute reduction of 21% in mortality [13]. However, the e cacy of convalescent plasma in critically ill patients with SARSCoV-2 infection remains unclear. Here, we performed this timely pilot study in three participating patients to describe whether it is also effective to use convalescent plasma therapy in the COVID-19 setting and discuss the preliminary experience of convalescent plasma transfusion administered to critically ill patients with COVID-19.

Methods
We did this timely descriptive study in 3 patients with COVID-19 in Fuzhou Pulmonary Hospital of Fujian province, which was the designated hospital in Fuzhou, China, from February 18 th , to March 30 th , 2020, and the nal date of follow-up was April 30 th , 2020. The study was reviewed and approved by the medical ethics committees of Fuzhou Pulmonary Hospital of Fujian province, and each participant gave written informed consent.

Patients
Patients with COVID-19 were laboratory con rmed by using throat swab SARS-CoV-2 real-time PCR(Detection Kit for 2019 Novel Coronavirus RNA PCR-Fluorescence Probing) (DAAN GENE Co, Ltd),who had been empirically treated with anti-viral drug arbidol or lopinavir-ritonavir, which are also recommended by the New Coronavirus Pneumonia Diagnosis and Treatment Program (6th edition) published by the National Health Commission of China a Patients, who were in accordance with the New Coronavirus Pneumonia Convalescent Plasma Therapy Guidance of China (2nd edition) b , were given the convalescent plasma treatment if they ful lled the following criteria: (1) within 3 weeks of symptom onset laboratory con rmed cases or who had viremia conformed by clinical experts 2 Severe patients with rapidly progress or the early stage of critically ill patients or who required plasma therapy were comprehensively evaluated by clinical experts.
Exclusion criteria were b : (1) patients allergic to plasma content or citrate; (2) The terminal stage of critical illness ;( 3) patients who developed multiple organ dysfunction syndrome ;( 4) patients who were comprehensive assessment by clinician unsuitable to use convalescent plasma.

Donors and convalescent plasma transfusion 2.21 Donors
The three donors of convalescent plasma were invited to donate their convalescent plasma after written informed consent was obtained. The convalescent plasma donors must ful lled the following criteria b : (1) at least 3 weeks following symptom onset (2)comply with the latest version of the COVID-19 diagnosis and treatment protocol to remove isolation and discharge standards; (3) between the ages of 18 and 55 years; (4) No less than 50 kg for males and 45 kg for females; (5) No history of menstrual blood disease; (6)patients who were comprehensive assessment by clinician unsuitable to donate plasma.

Test items
All donors had been tested negative SARS-CoV-2, as well as for hepatitis B virus, hepatitis C virus, HIV, and syphilis at the time of blood donation. The novel coronavirus serum/plasma IgG antibody was found to be active and still positive after 160-fold dilution according to the reagent speci cation; or novel coronavirus serum/plasma total antibody was found to be reactive by qualitative test and was still positive after 320-fold dilution according to the reagent speci cation. Enzyme-linked immunosorbent assay (ELISA) should be used for the detection, and adequate evaluation or reference to the evaluation data shall be made to ensure the quality of the detection.

Convalescent plasma transfusion
According to the principle of cross compatibility, the eligible patients received the transfusion of ABOcompatible convalescent plasma. The rst 15 minutes of infusion should be given at a slow speed, and the occurrence of adverse reactions to blood transfusion should be closely monitored. If there is no adverse reaction, the clinician will adjust the infusion speed according to the patient's condition. Infusion dose was 200ml for each cycle.

Data sources and Statistical Analysis
Clinical information of the 3 cases was reviewed of the hospital compute system and included the following: epidemiological and demographic data, exposure history, clinical and laboratory examination, radiological characteristics and outcome disease. Continuous variables were presented as the median and IQR. Graphs were plotted using GraphPad Prism 7.0. Statistical software used included SPSS 22.0.

Results
A total number of three patients (one male and two females; age range, 57-65years) were treated with convalescent plasma during the study. Two of three had preexisting medical conditions. None of the patients was affected by cluster infection and exposure to Huanan Seafood Wholesale Market. General characteristics of patients in the study were listed in Table 1. The most common symptoms were fever (three cases, 3/3) and cough (two cases, 2/3). All the three patients were without dyspnea or hypoxemia.
Two patients had underlying chronic diseases, including cerebrovascular diseases, diabetes and hypertension. All patients were treated with a combination of two antiviral drugs (lopinaviritonavir or arbidol combined with IFN-), whereas none of the patients were given glucocorticoids ( Table 2).
Antibacterial treatment was used when bacterial infections had been con rmed, and then only one patient received ceftriaxone sodium (i.v.) (2.0g qd). Hydroxychloroquine or chloroquine had been administered to two patients. Throat swab nucleic acid test of most patients (2/3) became negative for the novel coronavirus within 3 days after the transfusion (Table 3).

Discussion
The study of the passive immunizations for the prevention and treatment of human infectious diseases began in the 20 th century [14].To date, there is no speci c therapeutics for SARS-CoV-2 infection, and therefore administration of convalescent plasma can be still an important treatment [15].Previous evidence has proven that the use of convalescent plasma transfusion in the treatment of various infectious, especially in cororavirus infectious [12,16].
There were such three cases in our study all of which were severe patients with COVID-19 and treated with convalescent plasma. Case One had many underlying diseases heavy, and the other two cases rapidly progressed after being admitted to hospital. Especially in case 3 that pulmonary lesion progression exceeded 50% in a short period of time. However, after the treatment of convalescent plasma, the conditions of the 3 patients were well controlled in 3 days. The in ammatory indicators, including IL-6, CRP and blood routine examination were monitored regularly. Both clinical assessment and laboratory variables, the symptoms were signi cantly improved and lung lesions were signi cantly absorbed within 2 weeks.
However, to date, there are no proven options for prophylaxis for those who have been exposed to SARS-CoV-2, nor therapy for those who develop COVID-19 [17].Previous studies have reported the use of convalescent plasma transfusion as the treatment for COVID-19 Patients [18][19][20][21][22].Similar to those, our study suggests that convalescent plasma from patients who have recovered from COVID-19 infection might be a promising treatment to treat patients without causing any severe adverse effects. The possible explanations for the e cacy of convalescent plasma therapy is that the antibodies from convalescent plasma might not only limited to free viral clearance and blocking new infection, but also included acceleration of infected cell clearance [23,24].The patient usually develops a primary immune response by days 10-14, so using the convalescent plasma at the early stage of disease could be more effective. The time from onset of illness to convalescent plasma transfusion was within 10 days, and this might be one of the contributions to the good prognosis.
The current guidelines emphasizes that systematic corticosteroids should not be given routinely for the treatment of COVID-19 [25], however, whether to use the corticosteroids for COVID-19 patients remains a controversial topic [26].The nonuse of corticosteroids is the major different from the current study [18-20, 22, 27].
Although it was reported that dexamethasone could cut deaths by one-third among patients critically ill with COVID-19 [28], routinely use for the treatment of COVID-19 should need much more evidence. From our results, systematic corticosteroids for non-critical cases are not a must. Considering that we observed differing degrees of increase in D-dimer levels in all patients therefore anticoagulation is necessary for severe patients with COVID-19 given the state of hypercoagulability.

Conclusions
Convalescent plasma shows a promising therapeutic effect without corticosteroids and low risk in the treatment of severe COVID-19 patients. Using the convalescent plasma at the early stage of disease could be more effective.Anticoagulation is necessary for severe patients with COVID-19 given the state of hypercoagulability. However, given the small sample size and limited study design, naturally these results should be taken with a grain of salt until replicated by other further investigation in larger well-controlled trials.

Limitations
There are several limitations in this study. First, this was a retrospective analysis that had small case series and included no controls. Second, similar to most Chinese hospital, we lack the ability to detect the viral loads that may correlate with disease severity and progression. Third, in the current study, all patients received antiviral agents, including interferon and lopinavir/ritonavir, during and following convalescent plasma treatment, which also may have contributed to the viral clearance observed. Fifth, evidentially should need to further study in larger well-controlled trials. The datasets used or analyzed in the study are available from the corresponding author on reasonable request.

Con ict of interests
We declare no competing interests.