key: cord-1004684-hbt4tnpc
authors: Ye, Mingxiang; Fu, Dian; Ren, Yi; Wang, Faxiang; Wang, Dong; Zhang, Fang; Xia, Xinyi; Lv, Tangfeng
title: Treatment with convalescent plasma for COVID‐19 patients in Wuhan, China
date: 2020-04-15
journal: J Med Virol
DOI: 10.1002/jmv.25882
sha: 879415152d253eb38de4999ae7efd745230c15d8
doc_id: 1004684
cord_uid: hbt4tnpc

The discovery of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and the outbreak of coronavirus disease 2019 (COVID‐19) are causing public health emergency. A handful of literatures have summarized its clinical and radiologic features, whereas therapies for COVID‐19 are rather limited. In order to evaluate the efficacy of convalescent plasma therapy in COVID‐19 patients, we did this timely descriptive study. 6 laboratory confirmed COVID‐19 patients were enrolled and received the transfusion of ABO‐compatible convalescent plasma. The efficacy of this intervention was determined by the alleviation of symptoms, changes in radiologic abnormalities and laboratory tests. No obvious adverse effect observed during the treatment. Transfusion of convalescent plasma led to a resolution of ground glass opacities (GGOs) and consolidation in patient #1, #2, #3, #4 and #6. In patient #1 and #5 who presented with SARS‐CoV‐2 in throat swab, convalescent plasma therapy elicited an elimination of virus. Serologic analysis indicated an immediate increase in anti‐SARS‐CoV‐2 antibody titers in patient #2 and #3, but not in patient #1. This study indicates that convalescent plasma therapy is effective and specific for COVID‐19. This intervention has a special significance for eliminating SARS‐CoV‐2 and is believed to be a promising state‐of‐art therapy during COVID‐19 pandemic crisis. This article is protected by copyright. All rights reserved.

The global outbreak of a novel human coronavirus, newly named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the international committee on taxonomy of viruses, has attracted increasing attentions and public emergency 1,2 . This virus was initially detected in Wuhan, China, in December 2019. A cluster of pneumonia patients manifesting as fever, cough, and dyspnea with unknown etiology emerged at that time [3] [4] [5] . The virus was presumed to be zoonotic because preliminary investigation demonstrated that the first generation patients in Wuhan geographically linked to Huanan seafood whole sale market where live animals were sold. While patients outside of Wuhan usually had traveled to the city, or had contact with city residents 6 . These epidemiologic findings strongly suggest that SARS-CoV-2 transmits from human-to-human, and causes the disease now named coronavirus disease 2019 (COVID- 19) 7 . By the end of March, 2020, COVID-19 has spread up to 199 countries and causing more than 27000 deaths 8 .

SARS-CoV-2 belongs to the β-coronavirus family. Its genome is a single-stranded RNA composed of about 30 kb nucleotides, which encodes four This article is protected by copyright. All rights reserved. Accepted Article major structural proteins: spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N). Among these proteins, the S protein is of special interest because this club-shaped glycoprotein spikes give the virus a crown-like appearance 9 . Translational studies have demonstrated that the interaction between the receptor binding motif of S protein and the angiotensin-converting enzyme 2 (ACE2) mediates the recognition and entry of SARS-CoV-2 into the host cells, and ACE2 is defined as a putative receptor for SARS-CoV-2 10, 11 . The homogeneity in the receptor binding domain between SARS-CoV-2 and SARS-CoV underlies their overlapping pathogenicity and biological properties. Indeed, the clinical manifestations and radiologic features of COVID-19 and those of SARS are quite similar 12, 13 . For example, both diseases are highly infectious, and the incubation period ranges from several days to two weeks. Common symptoms at the onset of disease include fever, cough, myalgia and shortness of breath. Laboratory test may indicate white blood cell count below normal range, lymphopenia, hypoxaemia, deranged liver and renal function 3, 5 .

The typical radiologic abnormalities include multifocal ground glass opacities (GGOs) and subsegmental areas of consolidation [14] [15] [16] .

At the moment, therapeutic strategy for COVID-19 is largely supportive 17 .

Several off-label anti-viral and anti-HIV agents seem to be clinical beneficial, but their efficacy is far from satisfactory 18 

plasma group had a shorter hospital stay and lower mortality than the comparator group, and no immediate adverse effect noted after plasma infusion 20 . A systemic meta-analysis involving 1703 influenza pneumonia patients who received influenza-convalescent human blood products, showed reduced virus load and pooled absolute reduction of 21% in mortality 21 . Since the number of COVID-19 cases and disease-related death is increasing at an incredible speed, an urgent question that needs to be addressed promptly is whether it is also effective to use convalescent plasma therapy in the COVID-19 setting. One going clinical trial is recruiting patient for anti-SARS-CoV-2 convalescent plasma therapy in Shanghai,

China, but no relevant data has been announced yet (NCT04292340). The outcomes of this trial are definitely essential for formulating the principles of therapeutic strategy.

In this study, we provided preliminary data showing the efficacy of convalescent plasma therapy in COVID-19 patients. We found this intervention was effective in improving patient's symptoms and ameliorating radiologic abnormalities. More timely multi-center randomized clinical trials are warranted to determine the safety and efficacy of convalescent plasma therapy for COVID-19.

During the outbreak of SARS-CoV-2 infection in Wuhan, the number of COVID-19 patients far exceeded the capacity of local hospital. Therefore, the government built two designated hospital in Wuhan, one of which was named Huoshenshan. We did this study in 6 COVID-19 patients admitted to Wuhan Huoshenshan Hospital from February 11 th to March 12 th , 2020. All the patients This article is protected by copyright. All rights reserved. Table 1 . This study was reviewed and approved by the Medical Ethical Committee of Wuhan Huoshenshan Hospital. Written informed consent was obtained from each participant.

Convalescent plasma was collected from patients who had recovered from COVID-19. Recovery was defined as an afebrile status for at least 3 days, alleviation of respiratory symptoms, negative for SARS-CoV-2 nucleic acid for consecutive two RT-PCR tests, and at least 3 weeks following disease onset. The donors need to be seronegative for anti-HBV, HCV and HIV, and seropositive for Accepted Article anti-SARS-CoV-2. As a routine check with plasma donation, the convalescent plasma was also confirmed free of residual SARS-CoV-2 by real time PCR.

Eligible patients received the transfusion of ABO-compatible convalescent plasma as soon as the plasma was available to the institution. In accordance with the New Coronavirus Pneumonia Convalescent Plasma Therapy Guidance of China (2 nd edition) 2 , patients received at least one cycle of ABO-compatible convalescent plasma transfusion (200ml for each cycle). Each transfusion was administered over a 30-minute period.

Throat swabs were taken and immediately put into transport tube. All the tested samples were processed under airborne precaution. The SARS-CoV-2 nucleic acid was detected by reverse transcription and real-time PCR assays using commercial detection kit (Changsha Sansure Biotech). Two independent primers that match the open reading frame1ab (ORF1ab) and the nucleocapsid protein (N) fragments were used. RNase P was used as equal loading control. Reverse transcription and real-time PCR were performed according to the manufacturer's recommendations.

Each transcript provided a cycle threshold value (Ct value), which is the number of cycles required for the fluorescent signal. A Ct value less than 40 was defined as a positive result, and a Ct value exceeds 40 was defined as a negative test.

Serum anti-SARS-CoV-2 IgM and IgG were measured by chemiluminescence using commercially available kits (Shenzhen YHLO Biotech). Briefly, blood sample was centrifugated at room temperature and the supernatant was removed and incubated with the SARS-CoV-2 antigen-coated magnetic beads. The antigen-antibody complex captured by the beads slurry was gently precipitated by a magnetic separation rack. The beads were then incubated with acridinium ester-labeled mouse anti-human IgM or IgG antibody and reacted with hydrogen peroxide in excitation buffer. Relative luminescence intensity was recorded in an iFlash-3000 chemiluminescence system.

Adverse events and serious adverse events associated with convalescent plasma transfusion were assessed by the treating clinician. During transfusion, patients were under continuous supervision, with vital signs checked every 15 minutes and at 4 hours after the end of the intervention.

The primary outcome was the improvement in symptoms and chest CT in the following days after indicated intervention. Blood and swab samples were obtained to measure serum anti-SARS-CoV-2 IgM and IgG titers and throat SARS-CoV-2 nucleic acid, respectively.

A total number of 6 patients were assessed during the study period. This study was initially designed in the middle of February, however, the ABO-compatible convalescent plasma was not available until the beginning of March. We actually recruited the first participant on March 5 th and the last participant on March 18 th , This article is protected by copyright. All rights reserved.

respectively. The other issue needed to be addressed was that Wuhan Huoshenshan Hospital did not receive COVID-19 patients directly from the community, instead, our patients were transferred from other non-designated hospital. Therefore, our patients had been treated elsewhere and were at a relatively late course of the disease when admitted to the institution. Most patients manifested as fever, shortness of breath and non-productive cough at the onset of the disease. Other common clinical manifestations included fatigue and myalgia ( Table 1) . As the patients have been treated previously, blood cell count, coagulation test and biochemical analysis upon admission was mostly normal. In terms of inflammation indicators, patient #3 had a slight increase in C-reactive protein (6.73mg/L) and procalcitonin (0.17μg/L). No adverse reactions were observed in the six patients during plasma transfusion and in the following three days. March 11 th and March 14 th were comparable to those before plasma therapy. After carefully evaluation, the patient was considered as cured and was ready to discharge from hospital.

A 75-year-old woman had fatigue and shortness of breath on February 2 nd . She did not have fever and myalgia. She was positive for SARS-CoV-2 throat test and admitted to our hospital on February 12 th . At admission, her saturation was 75% and she received oxygen therapy through nasal catheter. Chest CT scan on February 22 nd showed multiple subpleural consolidation in bilateral lungs ( Figure   2 ). Her throat swab was negative for SARS-CoV-2, but she still felt respiratory distress and need oxygen supplement. On March 5 th , repeated chest CT examination suggested a partial resolution of the consolidation in the left down lobe, whereas the majority of lesions in the right lung were not resolved ( Figure   2 ). Therefore, the convalescent plasma was given on March 5 th . Before the treatment, serum anti-SARS-CoV-2 antibodies titers for IgM and IgG were 47 and 106, respectively. The patient reported that she felt an alleviation of respiratory distress after the treatment and the second cycle of convalescent plasma was given on March 9 th . Re-evaluation of serum anti-SARS-CoV-2 antibodies on March 11 th suggested a two-fold increase in IgM and IgG titers. Although chest CT on March 11 th did not indicated a radiologic improvement, the patient did not require oxygen therapy and the saturation was 99% on ambient air. In order to track the dynamic This article is protected by copyright. All rights reserved.

changes of consolidation, repeated chest CT scan was done on March 18 th . Figure   2 showed representative images illustrating the evolution of consolidation. After two cycles of convalescent plasma intervention, the density of consolidation was gradually reduced and turned into scattered GGOs with subpleural line. Three independent throat swab tests were all negative for SARS-CoV-2. The patient was considered as cured and was under further clinical monitoring. 

showed a complete resolution of the consolidation and gradually resolution of GGOs around the reticular vacuole. Of noted, a septal line appeared in left down lobe ( Figure 3) . Arterial blood gas analysis on March 15 th indicated a PO2/FiO2 ratio of 330. The patient was cured and has discharged from hospital.

A 63-year-old woman with ten years history of Sjögren syndrome had fever This article is protected by copyright. All rights reserved.

A 28-year-old woman experienced fatigue and myalgia on February 10 th . Her throat swab was positive for SARS-CoV-2 but her chest CT was otherwise normal. Before discharge from a local hospital, two consecutive throat swab tests were negative. She was asymptomatic and sent to our hospital for further clinical evaluation on March 5 th . At admission, serologic anti-SARS-CoV-2 antibody test was positive. The first throat swab test in our institution was negative, whereas it turned positive on the following days. Chest CT examination on March 8 th did not showed lung lesions ( Figure 5 ). This asymptomatic patient without radiologic abnormalities was recognized as a post-discharge SARS-CoV-2-positive COVID-19 patient, and could be a potential source of infection. Thus, convalescent plasma therapy was used on March 13 th . Several consecutive SARS-CoV-2 throat swab tests after the intervention were negative. The patient was discharged from hospital on March 19 th .

A 57-year-old man had fever, cough, shortness of breath and myalgia since January 29 th . He was tested for throat swab SARS-CoV-2, and the result was positive. He was sent to our hospital on March 12 th . Laboratory blood test indicated titers of anti-SARS-CoV-2 IgM and IgG were 16 and 217, respectively.

Chest CT examination on March 14 th showed GGOs without clear boundary in the peripheral region and down lobe of bilateral lungs ( Figure 6 ). Although repeated throat swab test after admission was negative, the patient complained that he still had respiratory distress. The convalescent plasma was given on March 18 th , and the patient reported a markedly relief of symptom. Figure 6 showed the radiologic changes 3 days after indicated intervention, in which GGOs were generally resolved after convalescent plasma transfusion. The patient was discharged from hospital on March 22 nd .

This descriptive study highlights convalescent plasma therapy as an effective and specific treatment for COVID-19. According to the experience of SARS and severe influenza, convalescent plasma is recommended to use as early as possible because the production of endogenous IgM and IgG antibodies peaks at two weeks and four weeks after infection, respectively 20, 21 . However, patients admitted to Wuhan Huoshenshan Hospital have already been treated elsewhere and the duration from the onset of disease to admission usually exceeds four weeks. Fortunately, convalescent plasma therapy is still functional in the 6 patients, and all patients did not admit to ICU during treatment. To the best of our knowledge, this is a timely study evaluating the efficacy of convalescent plasma therapy in COVID-19 patients with distinct radiologic, laboratory and clinical features.

In compliance with the New Coronavirus Pneumonia Diagnosis and Treatment Program (6 th edition), the principle of discharge was based on a relief of symptoms, obvious absorption of abnormalities in chest CT, abatement of fever, and viral clearance with throat swab for two consecutive tests. In our study, we found detectable SARS-CoV-2 in patient #5 who has already discharged from a local hospital. This is in agreement with a previous report that the virus RNA persists for a median of 20 days in survivors. 3 Strikingly, patient #5 represents a group of post-discharge asymptomatic "walking COVID-19" cases that might serve as a possible source to propagate the outbreak 22 . Indeed, a recent autopsy study of COVID-19 patient's lung indicates the presence of SARS-CoV-2 particles in bronchial mucosal epithelial and type II alveolar epithelial cells 23 .

This has important implications for reconsidering the period of patient isolation, the principle of discharge, and warrants highly efficient that can eliminate SARS-CoV-2. Yeh and colleagues tested the efficacy of convalescent plasma therapy in three SARS patients, and they found viral load dropped from 495×10 3 , 76×10 3 or 650×10 3 copies/mL to zero or 1 copy/mL one day after transfusion.

Anti-SARS-CoV IgM and IgG also increased in a time-dependent manner following transfusion 24 . This treatment is also effective for influenza A (H5N1) infection, in which convalescent plasma therapy led to a 12-fold decrease in blood virus load during the first 8 hours after transfusion, and the virus was undetectable within 32 hours 25 Another important finding in our study is the dynamic change of radiologic abnormalities. We found a rapid and dramatic radiologic improvement in patient #6, who manifested as extensive pure GGOs. This effect could be recaptured in patient #2 and patient #3, while the absorption of consolidation takes a relatively longer time. Intriguingly, our initial chest CT evaluation of patient #3 showed the patient's lung was in bad condition and respiratory distress did not relieve after standard supportive treatment. Although he was negative for throat swab test and This article is protected by copyright. All rights reserved.

the anti-SARS-CoV-2 IgM and IgG were detectable, it is reasonable to believe a clearance of virus in the upper respiratory tract, while SARS-CoV-2 may still exist in the lower respiratory tract and lung. Bronchoalveolar lavage fluid SARS-CoV-2 test would be informative, but bronchoscopy examination was not applicable in case of air-borne droplets transmission. We treated patient #3 with the transfusion of convalescent plasma. As a consequence, the patient had a significant improvement of his symptom, as well as a gradually resolution of consolidation.

Our study is different from previous SARS and influenza cases because convalescent plasma was used in a relatively late course of disease. We found it is still clinically beneficial in all the 6 cases. The mechanism of action in this setting was not fully understood. We speculated that the anti-SARS-CoV-2 IgM and IgG directly neutralizes the virus, and the anti-inflammatory contents may prevent cytokine storms. For the latter hypothesis, there is great debate of using corticosteroids. Evidence in SARS suggests that corticosteroids do not reduce mortality, but rather delayed viral clearance 26 . Therefore, corticosteroids should not be routinely given, like what we have done in patient #4 with Sjögren syndrome.

This study is limited by the small sample size. However, by including representative patients with distinct radiologic, laboratory and clinical features, we believe our study population is representative of COVID-19 patients in Wuhan.

Since more and more patients have recovered from the infection of SARS-CoV-2, voluntary donation of convalescent plasma would be definitely encouraged and appreciated. Taken together, COVID-19 is becoming a global health threat, reliable treatment is crucial for reducing mortality and preventing disease outbreak. SARS-CoV-2-specific therapies, including convalescent plasma from 

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All authors are voluntary front-line clinicians in Wuhan Huoshenshan Hospital.We express gratitude to all the participants and their families for the invaluable support to the study.

This study was partially sponsored by grants National Natural Science Foundation of China (#81802301 to Mingxiang Ye, #81772500 to Tangfeng Lv), and JiangsuProvincial Key Research and Development Program (BE2018713 to Xinyi Xia).The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.

We declare no competing interests.