Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma

Research

JAMA | PreliminaryCommunication

Chenguang Shen, PhD; Zhaoqin Wang, PhD; Fang Zhao, PhD; Yang Yang, MD; Jinxiu Li, MD; Jing Yuan, MD; Fuxiang Wang, MD; Delin Li, PhD; Minghui Yang, PhD; Li Xing, MM; Jinli Wei, MM; Haixia Xiao, PhD; Yan Yang, MM; Jiuxin Qu, MD; Ling Qing, MM; Li Chen, MD; Zhixiang Xu, MM; Ling Peng, MM; Yanjie Li, MM; Haixia Zheng, MM; Feng Chen, MM; Kun Huang, MM; Yujing Jiang, MM; Dongjing Liu, MD; Zheng Zhang, MD; Yingxia Liu, MD; Lei Liu, MD

Editorial

Audio and Video and Supplemental content

Author Affiliations: Author affiliations are listed at the end of this article.

Corresponding Authors: Yingxia
Liu, MD (yingxialiu@hotmail.com), Zheng Zhang, MD (zhangzheng1975 @aliyun.com), and Lei Liu, MD (liulei3322@aliyun.com), Shenzhen Third People’s Hospital, Second Hospital Affiliated to Southern University of Science and Technology, No. 29, Bulan Road, Longgang District, Shenzhen 518112, China.

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IMPORTANCE Coronavirus disease 2019 (COVID-19) is a pandemic with no specific therapeutic agents and substantial mortality. It is critical to find new treatments.

OBJECTIVE To determine whether convalescent plasma transfusion may be beneficial in the treatment of critically ill patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

DESIGN, SETTING, AND PARTICIPANTS Case series of 5 critically ill patients with laboratory-confirmed COVID-19 and acute respiratory distress syndrome (ARDS) who met the following criteria: severe pneumonia with rapid progression and continuously high viral load despite antiviral treatment; PAO2/FIO2 <300; and mechanical ventilation. All 5 were treated with convalescent plasma transfusion. The study was conducted at the infectious disease department, Shenzhen Third People’s Hospital in Shenzhen, China, from January 20, 2020, to March 25, 2020; final date of follow-up was March 25, 2020. Clinical outcomes were compared before and after convalescent plasma transfusion.

EXPOSURES Patients received transfusion with convalescent plasma with a SARS-CoV-2– specific antibody (IgG) binding titer greater than 1:1000 (end point dilution titer, by enzyme-linked immunosorbent assay [ELISA]) and a neutralization titer greater than 40 (end point dilution titer) that had been obtained from 5 patients who recovered from COVID-19. Convalescent plasma was administered between 10 and 22 days after admission.

MAIN OUTCOMES AND MEASURES Changes of body temperature, Sequential Organ Failure Assessment (SOFA) score (range 0-24, with higher scores indicating more severe illness), PAO2/FIO2, viral load, serum antibody titer, routine blood biochemical index, ARDS, and ventilatory and extracorporeal membrane oxygenation (ECMO) supports before and after convalescent plasma transfusion.

RESULTS All 5 patients (age range, 36-65 years; 2 women) were receiving mechanical ventilation at the time of treatment and all had received antiviral agents and methylprednisolone. Following plasma transfusion, body temperature normalized within 3 days in 4 of 5 patients, the SOFA score decreased, and PAO2/FIO2 increased within 12 days (range, 172-276 before and 284-366 after). Viral loads also decreased and became negative within 12 days after the transfusion, and SARS-CoV-2–specific ELISA and neutralizing antibody titers increased following the transfusion (range, 40-60 before and 80-320 on day 7). ARDS resolved in 4 patients at 12 days after transfusion, and 3 patients were weaned from mechanical ventilation within 2 weeks of treatment. Of the 5 patients, 3 have been discharged from the hospital (length of stay: 53, 51, and 55 days), and 2 are in stable condition at 37 days after transfusion.

CONCLUSIONS AND RELEVANCE In this preliminary uncontrolled case series of 5 critically ill patients with COVID-19 and ARDS, administration of convalescent plasma containing neutralizing antibody was followed by improvement in their clinical status. The limited sample size and study design preclude a definitive statement about the potential effectiveness of this treatment, and these observations require evaluation in clinical trials.

JAMA. doi:10.1001/jama.2020.4783 Published online March 27, 2020.

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Research Preliminary Communication

Treatment of Critically Ill Patients With COVID-19 With Convalescent Plasma

The epidemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originating in Wuhan,

Key Points

Question Couldadministrationofconvalescentplasma transfusion be beneficial in the treatment of critically ill patients with coronavirus disease 2019 (COVID-19)?

Findings Inthisuncontrolledcaseseriesof5criticallyillpatients with COVID-19 and acute respiratory distress syndrome (ARDS), administration of convalescent plasma containing neutralizing antibody was followed by an improvement in clinical status.

Meaning Thesepreliminaryfindingsraisethepossibilitythat convalescent plasma transfusion may be helpful in the treatment of critically ill patients with COVID-19 and ARDS, but this approach requires evaluation in randomized clinical trials.

1
China, has rapidly spread worldwide. As of March 24,

2020, China had reported 81 767 cases with 3281 deaths, and the World Health Organization declared coronavirus disease 2019 (COVID-19) a pandemic. As of March 18, 2020, cases were reported in approximately 195 countries.2

No specific therapeutic agents or vaccines for COVID-19 are available.3 Several therapies, such as remdesivir and favipira- vir, are under investigation,3,4 but the antiviral efficacy of these drugs is not yet known. The use of convalescent plasma was recommended as an empirical treatment during outbreaks of Ebola virus in 2014, and a protocol for treatment of Middle East respiratory syndrome coronavirus with convalescent plasma was established in 2015.5 This approach with other viral infec- tions such as SARS-CoV, H5N1 avian influenza, and H1N1 in- fluenza also suggested that transfusion of convalescent plasma was effective.6-10 In previous reports, most of the patients re- ceived the convalescent plasma by single transfusion.9-11 In a study involving patients with pandemic influenza A(H1N1) 2009 virus infection, treatment of severe infection with convales- cent plasma (n = 20 patients) was associated with reduced re- spiratory tract viral load, serum cytokine response, and mortality.10 In another study involving 80 patients with SARS, administration of convalescent plasma was associated with a higher rate of hospital bxdischarge at day 22 from symptom on- set compared with patients who did not receive convalescent plasma.12 Accordingly, these findings raise the hypothesis that use of convalescent plasma transfusion could be beneficial in patients infected with SARS-CoV-2.

The purpose of this study was to describe the initial clini- cal experience with convalescent plasma transfusion admin- istered to critically ill patients with COVID-19.

Methods

This study was conducted at the infectious disease depart- ment, Shenzhen Third People’s Hospital, Shenzhen, China, from January 20, 2020, to March 25, 2020, and the final date of follow-up was March 25, 2020. The study was approved by the ethics committees from Shenzhen Third People’s Hospi- tal, and each patient gave written informed consent.

Patients

Patients with laboratory confirmed COVID-19, diagnosed using quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) (GeneoDX Co, Ltd)13 were eligible to receive convalescent plasma treatment if they fulfilled the following criteria: (1) had severe pneumonia with rapid pro- gression and continuously high viral load despite antiviral treatment; (2) PAO2/FIO2 of <300 (PAO2 measured in mm Hg and FIO2 measured as fraction of inspired oxygen)14; and (3) were currently or had been supported with mechanical ventilation. The serum of each recipient was obtained and enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody titers were tested one day prior to the convalescent plasma transfu- sion. The ABO blood types of the patients were determined for

potential compatibility with the convalescent plasma donor, and each received 2 consecutive transfusions of 200 to 250 mL of ABO-compatible convalescent plasma (400 mL of convales- cent plasma in total) on the same day it was obtained from the donor. The patients received antiviral agents continuously un- til the SARS-CoV-2 viral loads became negative.

Disease Severity Classification

Patients with laboratory-confirmed COVID-19 infection who had any of the following were considered in critical condi- tion: (1) respiratory failure requiring mechanical ventilation, (2) shock, identified by the use of vasopressor therapy and el- evated lactate levels (>2 mmol/L) despite adequate fluid re- suscitation, or (3) failure of other organs requiring admission to the intensive care unit (ICU).

Donors

The 5 donors of convalescent plasma were between the ages of 18 and 60 years. The donors had recovered from SARS- CoV-2 infection and were invited to donate their convales- cent plasma after written informed consent was obtained. All donors had been previously diagnosed with laboratory- confirmed COVID-19 and subsequently tested negative for SARS-CoV-2 and other respiratory viruses, as well as for hepatitis B virus, hepatitis C virus, HIV, and syphilis at the time of blood donation. The donors had been well (asymp- tomatic) for at least 10 days, with a serum SARS-CoV-2– specific ELISA antibody titer higher than 1:1000 and a neu- tralizing antibody titer greater than 40. Following donation, 400 mL of convalescent plasma was obtained from each donor by apheresis, and the plasma was immediately trans- fused to the recipients on the same day it was obtained.

Clinical Information

Clinical information for the 5 patients before and after conva- lescent plasma transfusion was obtained from a review of the hospital computer medical system and included the follow- ing: demographic data, days of admission from symptom on- set, and presenting symptoms; data about various treat- ments, including mechanical ventilation, antiviral therapies, and steroids; clinical data, including body temperature, PAO2/FIO2, and Sequential Organ Failure Assessment (SOFA) score (range 0-24, with higher scores indicating more severe

E2 JAMA Published online March 27, 2020 (Reprinted)
© 2020 American Medical Association. All rights reserved.

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Treatment of Critically Ill Patients With COVID-19 With Convalescent Plasma

Preliminary Communication Research

illness); laboratory data, including white blood cell count, lym- phocyte count, chemistry panels assessing liver and kidney function, cycle threshold value (Ct), inflammatory factors C-reactive protein (CRP), procalcitonin, and IL-6, and serum antibody titer (IgG, IgM, and neutralizing antibodies); data from chest imaging studies; and information on complications, such as acute respiratory distress syndrome (ARDS), bacterial pneu- monia, and multiple organ dysfunction syndrome.

Quantitative RT-PCR

The qRT-PCR for SARS-CoV-2 was assessed as described previously.13 Nasopharyngeal specimens collected during hos- pitalization were sent to the laboratory in a viral transport case. Total nucleic acid extraction from the samples was per- formed using the QIAamp RNA Viral Kit (Qiagen), and qRT-PCR was performed using a commercial kit specific for 2019-nCoV detection (GeneoDX Co) approved by the China Food and Drug Administration. Each RT-PCR assay provided a Ct value, which is the number of cycles required for the fluorescent signal to cross the threshold for a positive test: a higher Ct value is cor- related with a lower viral load. The specimens were consid- ered positive if the Ct value was 37.0 or lower and negative if the results were undetermined. Specimens with a Ct value higher than 37 were repeated. The specimen was considered positive if the repeated results were the same as the initial re- sult and between 37 and 40. If the repeated Ct was undetect- able, the specimen was considered negative. All procedures involving clinical specimens and SARS-CoV-2 were per- formed in a biosafety level 3 laboratory. The Ct values of the 5

recipients were obtained on day −1, day 1, day 3, day 7, and day 12 after the transfusion.

ELISA

Microtiter plates (Sangon Biotech) were coated overnight at 4 °C with 4 μg/mL recombinant SARS-CoV-2 RBD (receptor bind- ing domain) proteins (50 μL per well) expressed by our labo- ratory through 293-T cells. The plates were washed 3 times with phosphate-buffered saline (PBS) containing 0.1% vol/vol Tween-20 (PBST) and blocked with blocking solution (PBS con- taining 2% wt/vol nonfat dry milk) for 2 hours at 37 °C. The plates were then washed with PBST. The serum samples were diluted to 200-fold into PBS as initial concentration, and se- rial 3-fold dilutions of serum was added to the wells and in- cubated at 37 °C for 60 minutes. After 3 washes, 100 μL of horseradish peroxidase–conjugated goat anti–human IgG (for IgG antibody titer detection) and IgM (for IgM antibody titer detection) antibodies solution (Sangon Biotech) were added to each plate, respectively, and incubated at 37 °C for 60 min- utes. After 5 washes, 100 μL of tetramethylbenzidine sub- strate (Sangon Biotech) was added at room temperature in the dark. After 15 minutes, the reaction was stopped with a 2 M H2SO4 solution (sulfuric acid). The absorbance was mea- sured at 450 nm. All samples were run in triplicate. The ELISA titers were determined by end point dilution.

Serum Neutralization Assay

Vero cells (104) were seeded 24 hours before the infection in a 96-well plate (Costar). On the day of infection, the cells were

Table 1. Clinical Characteristics of SARS-CoV-2-Infected Patients Who Received Convalescent Plasma

Patient 12345

Sex
Age, y
Weight, kg
Smoking
Bloodtype
Coexisting chronic diseases

Disease presentation and course

Estimated incubation period, da

Interval between symptom onset and admission, d

Interval between admission and plasma transfusion, d

Complications prior to plasma transfusion

Most severe disease classification Treatments

Steroids Antivirals

Male 70s 55 No

B None

1 2

Bacterial pneumonia; severe ARDS; MODS

Critical

Methylprednisolone

Lopinavir/ritonavir; interferon alfa-1b; favipiravir

Male 60s 85 No

Hypertension; mitral insufficiency

7 4

Bacterial pneumonia; fungal pneumonia; severe ARDS; myocardial damage

Critical

Methylprednisolone

Lopinavir/ritonavir; arbidol; darunavir

Female 50s
60
No

B None

3 2

20
Severe ARDS

Critical

Methylprednisolone

Lopinavir/ritonavir; interferon alfa-1b;

Female 30s 41.5 No

A None

7 2

19
Severe ARDS

Critical

Methylprednisolone

Interferon alfa-1b; favipiravir

Male 60s 87 No

B None

15 3

20
Severe ARDS

Critical

Methylprednisolone

Lopinavir/ritonavir; interferon alfa-1b

Abreviations: ARDS, acute respiratory distress syndrome; MODS, multiple organ dysfunction syndrome; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

a Estimated incubation period defined as interval between estimated exposure to SARS-CoV-2 and symptom onset.

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Research Preliminary Communication Treatment of Critically Ill Patients With COVID-19 With Convalescent Plasma

Table 2. Comparison of Viral Load, Clinical Indexes, and Laboratory Results Before and After Convalescent Plasma Transfusion

Patient 12345

Clinical characteristics

Body temperature, °C

Just before transfusion Day 1 posttransfusion Day 3 posttransfusion Day 7 posttransfusion Day 12 posttransfusion

SOFA scorea
Just before transfusion Day 1 posttransfusion Day 3 posttransfusion Day 5 posttransfusion Day 7 posttransfusion Day 12 posttransfusion

PAO2 /FIO2 b
Just before transfusion Day 1 posttransfusion Day 3 posttransfusion Day 7 posttransfusion Day 12 posttransfusion

Ct valuec (viral load proxy) On admission to hospital

Lowest value during hospitalizationd (highest viral load)

Just before plasma transfusion Day 1 posttransfusion
Day 3 posttransfusion
Day 7 posttransfusion

Day 12 posttransfusion Mechanical ventilation

Onset, days before transfusion

Extubated, days posttransfusion ECMO

Onset, days before transfusion

Removal, days posttransfusion

Laboratory findings

38.6 39.0 38.5 36.8 38.1 36.6 37.8 37.2 37.0 36.8

5 10 4 12 6 10 5 11 3 7 2 4

276 209 300 134 220 230 245 206 284 316

23.0 19.7 19.2 19.7

28.5 22.0 30.0 23.7 34.4 25.0 38.0 32.0 Negative Negative

11 2 Intubated Intubated

Not received 1 NA 5

37.6 37.7 37.0 36.5 36.6

3 4 3 2 2 2

172 184 164 220 342

18.9 18.9

33.0 38.5 Negative Negative Negative

12 2

Not received NA

65. 108.3 78.7 74.7 6.2 NT

0.1 0.1 0.09 0.08 0.04 NT

38.3 39.0 37.9 39.0 36.6 36.8 37.9 36.8 36.8 37.9

3 2 3 2 2 2 2 2 2 1 1 1

188 205 242 292 233 304 290 230 322 366

38.0 28.0 26.6 26.5

26.6 35.9 28.0 Negative Negative Negative Negative Negative Negative Negative

9 2 9 9

Not received
NA NA

156.0 173.1 NT 186.8 160.8 233.7 NT 260.4 9.6 5.5 5.8 3.2

0.2 0.2 0.08 0.4 0.07 1.5 NT 0.9 0.04 0.09 0.04 0.07

(continued)

Not received

C-reactive protein, mg/L (normal range, <8)

Before transfusion
Day 1 posttransfusion Day 3 posttransfusion Day 5 posttransfusion Day 7 posttransfusion Day 12 posttransfusion

Procalcitonin, ng/mL (normal range, <0.1) Before transfusion
Day 1 posttransfusion
Day 3 posttransfusion

Day 5 posttransfusion Day 7 posttransfusion Day 12 posttransfusion

E4 JAMA Published online March 27, 2020

163.4 242.8 146.2 223.0 115.1 75.2 31.3 10.4 31.2 13.9 5.3 33.1

1.2 7.3 1.3 19.7 1.6 13.9 0.9 1.8 1.1 0.1 0.4 0.2

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Treatment of Critically Ill Patients With COVID-19 With Convalescent Plasma

Preliminary Communication

Research

Table 2. Comparison of Viral Load, Clinical Indexes, and Laboratory Results Before and After Convalescent Plasma Transfusion (continued)

Patient 12345

IL-6, pg/mL (normal range, 0-7)

Before transfusion
Day 1 posttransfusion Day 3 posttransfusion Day 5 posttransfusion Day 7 posttransfusion Day 12 posttransfusion

Length of hospital stay, d
Current status as of March 25, 2020

70.5

74.9

34.5

24.1

30.8

6.1

Remains hospitalized

Stable, still receiving mechanical ventilation

438.2

1045.0

334.1

29.8

31.8

Remains hospitalized

Stable, still receiving mechanical ventilation

63.9 79.1 87.8 118.5 39.3 NT 67.0 25.8 797.9 590.5 NT NT 174.3 34.0 69.9 NT 2.7 54.9 53 51 55

Discharged Discharged Discharged home home home

Abbreviations: Ct, cycle threshold; ECMO, extracorporeal membrane oxygenation; NT, not tested.

. a The SOFA score is calculated using 6 systems: respiratory, coagulation, hepatic, cardiovascular, central nervous system, and kidney. A score of 0 is given for normal function through to 4 for most abnormal for each system. The worst values on each day are recorded, and the final SOFA score is the sum of the scores of each system.

. b PAO2/FIO2 ratio was defined as the ratio of the partial pressure of arterial oxygen to the percentage of inspired oxygen.

. c Cycle threshold is the number of polymerase chain reaction cycles required for gene amplification. A higher Ct value is correlated with a lower viral load.

. d Lowest value (highest viral load) between hospital admission and plasma transfusion.

washed twice. Serum samples from patients were incubated at 56 °C for 30 minutes and then diluted 2-fold in cell culture medium (modified eagle medium). Aliquots (40 μL) of di- luted serum samples (from 2-fold to 2056-fold) were added to 50 μL of cell culture medium containing 50 times the tissue culture infective dose (TCID50) of the BetaCoV/Shenzhen/ SZTH-003/2020 strain virus (isolated from this hospital, GI- SAID access number: EPI_ISL_406594)15 on a 96-well plate and incubated at 37 °C for 2 hours in CO2 5% vol/vol. Virus anti- body mix was then added to cells in 96-well plates and plates were incubated at 37 °C with microscopic examination for cy- topathic effect after a 5-day incubation. The highest dilution of serum that showed inhibition activity of SARS-CoV-2 was recorded as the neutralizing antibody titer. Assays were per- formed in triplicate with negative control samples from healthy volunteers.

Results

Five patients (age range, 36-73 years; 2 women) were treated with convalescent serum. None were smokers, and 4 of 5 had no preexisting medical conditions. All 5 had received various antiviral agents and steroids (Table 1). Convalescent plasma was administered between 10 and 22 days after admission.

The Ct value at the time of admission ranged from 18.9 to 38.0, and on the day of plasma transfusion from 22.0 to 35.9 (Table 2 and Figure 1A). It increased (improved) within 1 day after transfusion. The Ct value of patient 5 became negative on posttransfusion day 1, patient 3 and patient 4 became nega- tive on day 3, and patient 1 and patient 2 became negative on day 12 after the transfusion (Table 2).

The SOFA score ranged from 2 to 10 prior to plasma trans- fusion, and decreased to a range of 1 to 4 at 12 days following transfusion (Table 2 and Figure 1B). The PAO2/FIO2 ranged from 172 to 276 prior to transfusion, and increased (improved) for 4 of 5 patients within 7 days after transfusion (overall range, 206-290), and increased substantially (range, 284-366) on the 12th day after the plasma treatment (Table 2 and Figure 1C). Body temperature ranged from 37.6 to 39.0 °C before plasma transfusion and declined to the normal range on the third day after the transfusion (Table 2 and Figure 1D).

After the treatment, the values of the inflammatory bio- markers CRP, procalcitonin, and IL-6 of patients 1, 2, 4, and 5 decreased; the values of CRP and procalcitonin of patient 3 de- creased (Table 2).

The computed tomography scans of the lungs of these pa- tients all demonstrated severe pneumonia prior to plasma transfusion and showed improvement of the pulmonary le- sion of patient 1 on the third day after the plasma transfusion (eFigure 1 in the Supplement) and gradual resolution of pul- monary lesions of other patients at 3 days after the plasma treat- ment (eFigures 2, 3, 4, and 5 in the Supplement).

One day prior to convalescent plasma administration, the RBD-specific IgG and IgM ELISA titers of the donors ranged be- tween 1800 and 16 200 (ELISA end point dilution titers) (Table 3). The neutralization titers against SARS-CoV-2 ranged between 80 and 480 (neutralizing end point dilution titers). The RBD-specific IgG ELISA titers of 5 recipients ranged be- tween 1800 and 48 600 and the IgM titers between 5400 and 145 800 a day prior to the convalescent transfusion (eTable in the Supplement). After the transfusion of convalescent plasma, the titers of IgG and IgM in the sera of these patients in- creased in a time-dependent manner. The IgG titers of the

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Research Preliminary Communication Treatment of Critically Ill Patients With COVID-19 With Convalescent Plasma

Figure 1. Temporal Changes of Cycle Threshold Value, PAO2/FIO2, SOFA Score, and Body Temperature in Patients Receiving Convalescent Plasma Transfusion

A Cycle threshold 45

SOFA score 15

0
013 7 12 013 7 12

Days posttransfusion

C PAO2/FIO2 400

300

200

100

Body temperature 40

36
013 7 12 013 7 12

Days posttransfusion

(range 0-24, with higher scores indicating more severe illness; see footnote to Table 2 for more complete definition). C, Change in PAO2/FIO2 ratio of the treated patients from day 0 to day 12 after treatment. D, Change in body temperature of the 5 patients following plasma transfusion.

Donorsa 12345

A, Change in cycle threshold (Ct) value in nasopharyngeal swabs of infected patients at day 0, day 3, day 7, and day 12 after the plasma transfusion. A Ct value of 40 was defined as undetectable. B, Change in Sequential Organ Failure Assessment (SOFA) score of the patients with convalescent plasma treatment

Table 3. Characteristics and Antibody Titer of Convalescent Plasma Donors

Blood type
Donated plasma volume, mL
Interval between symptom onset and discharge, d Interval between discharge and plasma donation, d RBD-specific IgG ELISA titerb
RBD-specific IgM ELISA titerc
Neutralizing antibody titerd

Abbreviation: RBD, receptor binding domain.

a Donors-patients were matched by number (donor 1 gave plasma to patient 1, etc).

b ELISA end point dilution titers (IgG antibody). The expected titer of negative control from a healthy person is 200.

treated patients increased to 145 800, 5400, 5400, 145 800 and 145 800, and the IgM titers increased to 145 800, 5400, 5400, 437 400 and 145 800, respectively, at 3 days after transfu-

B
400 11
11
16 200 16 200 240

B B A 400 400 400 11 13 13 11 13 11 1800 1800 5400 1800 5400 5400 80 120 240

B
400 11
12
16 200 5400 480

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c ELISA end point dilution titers (IgM antibody). The expected titer of negative control from a healthy person is 200.

d Neutralization end point dilution titers. The expected titer of negative control from a healthy person is 10.

sion. These IgG and IgM titers maintained a high level at 7 days after transfusion (Figure 2A and 2B; eTable in the Supple- ment). The neutralizing antibody titers of the 5 recipients

Patients

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PAO2/FIO2 Ct value

Body temperature, °C SOFA score

Treatment of Critically Ill Patients With COVID-19 With Convalescent Plasma Preliminary Communication Research

Figure 2. Changes of Receptor Binding Domain–Specific IgG and IgM ELISA and Neutralizing Antibody Titers Before and After Convalescent Plasma Transfusion in Patients

RBD-specific IgG ELISA titer 150

100

RBD-specific IgM ELISA titer 150

100

Neutralizing antibody titer 1000

100

1 1 10
013 7013 7013 7

Days posttransfusion Days posttransfusion Days posttransfusion

Higher titer values indicate greater protection. A, Variation of RBD-specific IgG ELISA titer. B, Variation of RBD-specific IgM ELISA titer. C, Variation of neutralizing antibody titer against SARS-CoV-2 in recipients in day 0, day 1,

ranged between 40 and 160 before transfusion; one day after transfusion, the titers increased to 320, 80, 80, 160, and 240; on day 7, they were 320, 160, 160, 240, and 480, respectively (Figure 2C; eTable in the Supplement).

All 5 patients were receiving mechanical ventilation at the time of transfusion, and 3 patients (patients 3, 4, and 5) were weaned from mechanical ventilation (Table 2). Patient 2 was receiving ECMO at the time of plasma treatment but did not require ECMO on day 5 after transfusion (Table 2). Patients 3, 4, and 5 were discharged from the hospital (length of stay: 53, 51, and 55 days, respectively). As of March 25, 2020, patients 1 and 2 remained hospitalized, with lengths of stay of 37 days each.

Discussion

In this case series, 5 patients who were critically ill with COVID-19 were treated with convalescent plasma. As assessed by Ct, viral load declined within days of treatment with convalescent plasma, and the clinical conditions of these patients improved, as indicated by body temperature reduction, improved PAO2 /FIO2 , and chest imaging. Four patients who had been receiving mechanical ventilation and ECMO no longer required respiratory support by 9 days after plasma transfusion.

Previous studies have reported the use of convalescent plasma transfusion in the treatment of various infections.6,10,16 For example, patients (n = 50) with SARS had a significantly higher discharge rate by day 22 following onset of illness (73.4% vs 19.0%; P<.001) and lower case-fatality rate (0% vs 23.8%; P = .049) in the convalescent plasma treatment group (n = 19 patients) when compared with steroid treatment group (n = 21).17 In another study of 93 patients with influenza A(H1N1), patients who received convalescent plasma treat- ment (n = 20) compared with those in the control group (n = 73)

day 3, and day 7 following transfusion. The identical line segments were adjusted slightly to avoid superimposition. RBD indicates receptor binding domain.

had significantly fewer deaths (20% vs 54.8%; P = .01) and a lower median lymphocyte count on ICU admission.10

In this study, collection and transfusion of the plasma were done as previously reported.10 In addition, plasma was obtained from the donors and transfused in the recipients on the same day, which helps preserve the natural activity of the plasma.

Studies have shown that viral loads are highly correlated with disease severity and progression.18 Fatal outcome of hu- man influenza A(H5N1) has been associated with high viral load and hypercytokinemia.19 Apart from antiviral treatment, virus- specific neutralizing antibody, which could accelerate virus clearance and prevent entry into target cells, serves as the main mechanism for the restriction and clearance of the viruses by the host.20-22 In the current study, SARS-CoV-2 was still detect- able in all 5 patents even though antiviral treatment had been given for at least 10 days, although viral load decreased and be- came undetectable soon after convalescent plasma treatment. As determined by ELISA, all plasma from the donors had high virus-specific IgG and IgM ELISA titers. Moreover, the neutral- izing antibody titers, vital for the restriction of viral infection of the 5 recipients, significantly increased after plasma trans- fusion. The results highlight the possibility that antibodies from convalescent plasma may have contributed to the clearance of the virus and also the improvement of symptoms. In addition to viral neutralizing antibodies, acceleration of infected cell clearance by antibodies has also been found in an in vivo study of HIV-1 virus.23 In the current study, all patients received an- tiviral agents, including interferon and lopinavir/ritonavir, dur- ing and following convalescent plasma treatment, which also may have contributed to the viral clearance observed.

Limitations

This study has several limitations. First, this was a small case series that included no controls. Second, it is unclear if these patients would have improved without transfusion of

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Patients

IgG ELISA titer, in thousands

IgM ELISA titer, in thousands

Neutralizing antibody titer

Research Preliminary Communication

Treatment of Critically Ill Patients With COVID-19 With Convalescent Plasma

convalescent plasma, although the change in Ct and PAO2/ FIO2 represent encouraging findings. Third, all patients were treated with multiple other agents (including antiviral medi- cations), and it is not possible to determine whether the im- provement observed could have been related to therapies other than convalescent plasma. Fourth, plasma transfusion was ad- ministered 10 to 22 days after admission; whether a different timing of administration would have been associated with dif- ferent outcomes cannot be determined. Fifth, whether this ap- proach would reduce case-fatality rates is unknown.

Conclusions

In this preliminary uncontrolled case series of 5 critically ill pa- tients with COVID-19 and ARDS, administration of convales- cent plasma containing neutralizing antibody was followed by improvement in the patients’ clinical status. The limited sample size and study design preclude a definitive statement about the potential effectiveness of this treatment, and these obser- vations require evaluation in clinical trials.

ARTICLE INFORMATION

Accepted for Publication: March 20, 2020. Published Online: March 27, 2020.

doi:10.1001/jama.2020.4783

Author Affiliations: Shenzhen Key Laboratory of Pathogen and Immunity, National Clinical Research Center for Infectious Disease, State Key Discipline of Infectious Disease, Shenzhen Third People’s Hospital, Second Hospital Affiliated to Southern University of Science and Technology, Shenzhen, China (Shen, Z. Wang, Zhao, Y. Yang, J. Li, Yuan,

F. Wang, D. Li, M. Yang, Xing, Wei, Xiao, Y. Yang, Qu, Qing, L. Chen, Xu, Peng, Y. Li, Zheng, F. Chen, Huang, Jiang, D. Liu, Zhang, Y. Liu, L. Liu); Laboratory of Protein Engineering and Vaccines, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences (CAS), Tianjin, China (D. Li, Xiao).

Author Contributions: Dr L. Liu had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Drs Shen, Z. Wang, Zhao, and Y. Yang contributed equally.

Concept and design: Shen, Z. Wang, Yuan, F. Wang, D. Liu, Zhang, Y. Liu, L. Liu.
Acquisition, analysis, or interpretation of data: Shen, Yang Yang, J. Li, Yuan, D. Li, M. Yang, Xing, Wei, Xiao, Yan Yang, Qu, Qing, L. Chen, Xu, Peng, Y. Li, Zheng, F. Chen, Huang, Jiang, Y. Liu, L. Liu.

Drafting of the manuscript: Shen, Zhao, Yang Yang, J. Li, Yuan, F. Wang, M. Yang, Xing, Wei, Xiao, Yan Yang, Qu, Qing, L. Chen, Xu, Zheng, Huang, Jiang, D. Liu, Y. Liu, L. Liu.

Critical revision of the manuscript for important intellectual content: Shen, Z. Wang, Yang Yang, Yuan, D. Li, Peng, Y. Li, F. Chen, Zhang, Y. Liu, L. Liu. Statistical analysis: Yuan.

Obtained funding: Yuan, Zhang, Y. Liu, L. Liu. Administrative, technical, or material support: Shen, Zhao, J. Li, Yuan, F. Wang, D. Li, M. Yang, Yan Yang, Qu, Qing, L. Chen, Zhang.
Supervision: Z. Wang, Yuan, Zhang, Y. Liu, L. Liu.

Conflict of Interest Disclosures: None reported.

Funding/Support: This work was supported by the National Science and Technology Major Project (2018ZX10711001, 2017ZX10103011, 2017ZX10204401), Sanming Project of Medicine in Shenzhen (SZSM201412003, SZSM201512005), China Postdoctoral Science Foundation (2019T120147, 2018M641508), Shenzhen Science and Technology Research and Development Project (202002073000001), National Natural Science Foundation of China (81902058), Shenzhen Science and Technology Research and Development Project (202002073000002), and The Key Technology R&D Program of Tianjin (17YFZCSY01090).

Role of the Funder/Sponsor: The funding agencies had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

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