Asian Journal of Transfusion Science
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ORIGINAL ARTICLE  
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A study to identify severe acute respiratory syndrome coronavirus 2 in erythrocytes of patients suffering from coronavirus disease-19 at an Apex tertiary care institute in Andhra Pradesh, South India


1 Department of Transfusion Medicine and Hemotherapy, All India Institute of Medical Sciences, Mangalagiri, Andhra Pradesh, India
2 Department of Microbiology, All India Institute of Medical Sciences, Mangalagiri, Andhra Pradesh, India
3 Department of Anaesthesiology, All India Institute of Medical Sciences, Mangalagiri, Andhra Pradesh, India

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Date of Submission12-Jul-2021
Date of Decision21-Feb-2022
Date of Acceptance06-Mar-2022
Date of Web Publication26-Sep-2022
 

   Abstract 

CONTEXT: Coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) in the latest coronavirus disease (COVID-19) pandemic has been shown to cause a rise of ferritin levels through a possible viremia. Hemoglobin in the erythrocyte (red blood cell [RBC]) is a potential source of ferritin and we hypothesized that erythrocyte might be infected by this virus. Any infected RBC in asymptomatic blood donors might become a huge threat to the blood.
AIM: The aim of the study is to identify the presence of SARS-CoV-2 ribonucleic Acid (RNA) in RBC of patients suffering from COVID-19 disease.
SETTING AND DESIGN: A prospective study was performed to identify the presence of SARS-CoV-2 RNA in erythrocytes of patients with COVID-19.
SUBJECTS AND METHODS: Ten samples were collected from patients with COVID-19 at different times during their admission. After sampling, these were processed and stored like Packed red blood cells (PRBCs). These units were further processed by washing or leukoreduction with further sampling. All these samples were tested for the detection of SARS-CoV-2 RNA by real-time-polymerase chain reaction.
STATISTICAL ANALYSIS: Descriptive statistics with Microsoft Excel were performed.
RESULTS: The study could not identify the SARS-CoV-2 virus in the erythrocytes or plasma of infected patients.
CONCLUSION: SARS-CoV-2 virus may not be a transfusion transmissible infection, however, this needs to be confirmed with a larger study. It is recommended to have a lookout hemovigilance policy given newer variants of SARS-CoV-2 and for possible new viruses that may emerge in the future.

Keywords: Coronavirus disease-19, erythrocytes, leukoreduction, real-time polymerase chain reaction, severe acute respiratory syndrome coronavirus 2, washing


How to cite this URL:
Kumar IS, Babu MV, Tripathi M. A study to identify severe acute respiratory syndrome coronavirus 2 in erythrocytes of patients suffering from coronavirus disease-19 at an Apex tertiary care institute in Andhra Pradesh, South India. Asian J Transfus Sci [Epub ahead of print] [cited 2022 Dec 4]. Available from: https://www.ajts.org/preprintarticle.asp?id=356902



   Introduction Top


The novel coronavirus 2019, a positive-sense single-stranded ribonucleic Acid (RNA) virus, which started as an epidemic in Wuhan, China, has become a pandemic affecting most countries in the world and infected a total of 274,628,461 people, has caused deaths of 5,358,978 people across the globe as per the World Health Organization.[1] India, a developing country with the second-largest population, has reported a total of 34,752,164 people being infected and a total of 478,007 deaths due to coronavirus disease (COVID-19).[2]

The infection spreads by aerosols. Blood transmission has not been reported so far, but COVID-19 is reported to attack the 1-beta chain of hemoglobin and capture porphyrin to inhibit human heme metabolism.[3] Its metabolite ferritin has been used as a prognostic marker too in blood. Angiotensin-converting enzyme 2 has been identified as a functional receptor for severe acute respiratory syndrome coronavirus (SARS-CoV) for entering cells and multiplication intracellularly.[4]

Currently, there are no studies relating the presence of SARS-CoV-2 RNA in erythrocytes of blood. With the second and third waves of COVID-19 in India, more and more asymptomatic infections are being found among COVID-19-positive cases. If such asymptomatic patients become blood donors and if COVID-19 is transmissible through blood, it becomes a huge threat. Thus, we planned this project to detect the SARS-CoV-2 RNA in erythrocytes, plasma, leukoreduced, and/or 3–6 times normal saline washed red blood cells (RBCs) and corresponding eluates. Secondary objectives were to analyze the data to ascertain the effect of presence or absence of fever in patients, storage of red cells and plasma on the virus, the effect of washing and leukoreduction on viral levels and to possibly formulate strategies to prevent infection through blood transfusion and improve blood safety program.


   Subjects and Methods Top


This is a prospective observational study performed after getting requisite approval from the institutional ethics body and registered in the Clinical Trial Registry of India bearing registration number CTRI/2021/01/030630. The personnel involved were provided with personnel protective equipment and the biomedical waste generated was discarded appropriately.

Sample size calculation

Given the lack of prevalence statistics about SARS-CoV-2, RBC infectivity rate, and transfusion transmission rate at the time of inception of the study, Formal sample size and power could not be calculated for the present study. However, keeping in view the feasibility and available resources, it was justifiably proposed and the study was performed by collecting 10 samples from patients with COVID-19 after getting necessary approvals.

Selection and description of participants

It was assumed that SARS-CoV-2 will become an endemic infecting a large group of population. Hence, it was decided to include both patients with a confirmed diagnosis and any healthy asymptomatic donors whose samples test positive for COVID-19. However, during the study, blood was collected only from patients, and donors were not sampled as none were found positive for the SARS-CoV-2 virus.

Inclusion criteria

The samples were collected from consented COVID-19 patients with confirmed illness by real-time-polymerase chain reaction (RT-PCR).

Details of participants

Three patients consented to participate in the study.

Sample collection algorithm

As the presence of SARS-CoV-2 RNA in the blood sample is unknown, and at what period during disease the highest titers of viral RNA can be found in the blood is unknown, sample collection algorithm was devised and followed as depicted in [Figure 1]. This was done assuming that the viremia can be present at some point in the clinical course of the disease from the three consented patients [Table 1].
Figure 1: Sample collection algorithm

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Table 1: Demographic characteristics of patients in the study

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The sample collection algorithm was also planned to give equal or almost equal power to analyze the possible unknown effect of fever, washing, leukoreduction, 35 days storage with citrate phosphate dextrose adenine anticoagulant (CPDA) preservative, 42 days storage with saline adenine glucose mannitol additive solution (SAGM) [Table 2].
Table 2: Sample collection, processing, and storage algorithm used in study

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Sample collection

One blood sample of 20 mL each was collected into quadruple blood bags under aseptic conditions from each of the three patients between 1 and 3, 4 and 6 days, 7 and 9 days after symptom onset, respectively. One blood sample was collected after 9 days in one patient [Table 2]. All samples thus were collected into individual primary blood bags of the quadruple bags with 2.8 mL CPDA (Polymed Medical Devices, Faridabad, Haryana, India). The anticoagulant volume was reduced in a closed manner using a satellite bag to compensate for 20 mL sample collection emulating normal blood collection procedures.

Sample processing and storage

The bags were labeled with unique 4-digit unit numbers, specific for study thereby blinding the nature and type of samples before processing. The entire processing and storage were done as per the departmental standard operating procedures for preparation of packed red cells, fresh frozen plasma, and storage of same. Similarly, departmental standard operating procedures were followed for washing and leukoreduction.

Technical information

Component preparation, storage, sample aliquoting

The primary blood sample was centrifuged at 2500 RPM (heavy spin) for 10 min at 4°C in Heraeus Cryofuge 16 Blood Bag centrifuge (Thermo Fisher Scientific, Am Kalkberg, Germany). Plasma and packed red cells (PRBC) were prepared using a manual plasma expressor. Aliquots of packed red cells and plasma were collected under a closed system and freeze preserved as secondary samples with unique 4-digit code for RT-PCR testing. Thus, 10 red cell samples and 10 plasma samples could be prepared for testing by RT-PCR from 10 blood bags. Platelet concentrates were not prepared in the study, hence, platelet-rich plasma or buffy coat method cannot be commented upon. In 6 packed red cells, supernatant plasma was removed completely and 4.4 mL SAGM was added. Four packed red cells without SAGM have been stored for 35 days at 2°C–6°C in a separate blood bag refrigerator assigned for the study. Six packed red cells with SAGM were stored for 42 days at 2°C–6°C in the assigned blood bag refrigerator.

On the last day of the shelf life of these PRBC, leukoreduction using a red cell filter with an attached transfer bag (Terumo Penpol, Trivandrum, India) was performed for five packed red cells [Table 2].

This was followed by washing of all ten PRBCs using normal saline, six times manually. The washing step was performed in a closed system using laminar airflow, sterile connecting device, and refrigerated centrifuge. Heavy spin was used for centrifuging packed red cells, followed by removal of the supernatant. Normal saline was added subsequently and mixed. The same was followed 5 more times for preparing 6 times washed PRBC.

Samples of residual red cells at the end of 3rd wash (10 samples) and supernatant saline (10 samples) were aliquoted. Similarly, residual red cells (10 samples) and supernatant saline (10 samples) were aliquoted at the end of the 6th wash. These were labeled as tertiary samples with unique 4-digit codes. Thus, finally, 20 secondary and 40 tertiary samples freeze preserved were collected for further testing for SARS-CoV-2 RNA by RT-PCR.

Severe acute respiratory syndrome coronavirus 2 ribonucleic acid detection by real-time-polymerase chain reaction

All sixty samples (secondary and tertiary) were subjected to COVID-19 testing appropriately at an institutional ICMR approved laboratory.

The samples were tested by RT-PCR to detect SARS-CoV-2 Nucleocapsid (N) gene and confirmatory genes. QIAamp® Viral RNA Mini Kit (QIAGEN) was used to purify viral RNA from blood. The isolated RNA was processed and subjected to multiplex real-time RT-PCR using Q-Line® molecular novel coronavirus (COVID-19) RT-PCR detection kit (POCT Services Pvt. Ltd) that detects two targets: N-gene and ORF1ab gene of SARS CoV-2. The RT-PCR was performed in QuantStudio™ 5 Real-Time PCR System (Applied BiosystemsTM, ThermoFisher Scientific).

Result interpretation was made based on the cycle threshold (Ct) reference value of the target gene as <40 and validated with the detection of internal control (housekeeping gene) in each sample. A positive result was given when there was an amplification of both the N gene and ORF1ab gene [Figure 2]. The sample was retested if there was an amplification of a single target gene. A negative result was given when there was no amplification of either of the target gene.
Figure 2: Real-time polymerase chain reaction text plot of a patient

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Ethics

The study was performed after obtaining ethics committee permission and the study has been performed in convention with international standards.

Statistics

Descriptive statistics have been performed using Microsoft Excel 2007. Analysis of the obtained data was planned considering a two sided, P < 0.05 to be statistically significant, and multivariate regression analysis was to be used in the study subject to the viral levels for assessing the effect of patient-related factors such as fever and other symptoms, processing related factors such as washing, leukoreduction, storage on COVID-19 levels in blood samples.


   Results Top


Patient demographics

The demographic and clinical features of the three patients enrolled in the study are as tabulated in [Table 1]. All patients had mild COVID-19 illness; they were all in the age group of 25–30 years. The source of infection was either hospital-acquired (n = 2) or because of travel from an epidemic area (n = 1).

Ten samples were collected from these 3 patients as per the study design and these were further processed to result in 20 secondary and 40 tertiary samples.

Severe acute respiratory syndrome coronavirus 2 ribonucleic acid testing

As per the study design, twenty secondary and forty tertiary samples were tested for the detection of SARS-CoV-2 RNA by RT-PCR.

Sample positivity

During the present study, all the 60 samples tested by RT-PCR were found to be negative for SARS-CoV-2 RNA.

The analysis could not be performed as to the viral levels and symptoms in patients, the effects of washing, storage, leukoreduction as all the samples were negative for the viral RNA


   Discussion Top


The present study shows that SARS-CoV-2 RNA may not be a transfusion transmissible infection. This is postulated because of the lack of SARS-CoV-2 RNA in the red cells or plasma of patients with mild symptoms and with considerable viral load in routine testing. It is also being postulated that any pathogenesis pertaining to red cell abnormalities and coagulation abnormalities in COVID-19 may not be because of the actual viral infection. This may be because of some other mechanisms cascading through multiple known or unknown chemicals/proteins.

In early 2000, during the first epidemic of SARS-CoV, there have been reports of detection of very low levels of viral RNA in plasma of patients who had a considerable viral load in sputum samples. During their studies, techniques such as ultracentrifugation and in-house nested PCR were used. No detectable levels of the virus could be found in two contacts of the primary case who also had a high viral load in sputum samples.[5] Later on, some studies have shown that SARS-CoV can infect mononuclear cells and monocyte-derived dendritic cells and replicate in a self-limiting manner.[6],[7] Despite all these, there were never any reports of SARS-CoV transmission through a blood transfusion during the first outbreak in early 2000.

Similarly, a large study comprising of 31,151 donors was tested for SARS-CoV and none of them had the virus in detectable levels. They opined that testing can be helpful in preventing possible transfusion transmission of SARS-CoV.[8]

Similar to other numerous infections that can spread through blood transfusion such as HIV, hepatitis, malaria, syphilis; COVID-19 is also assumed to be transmissible through blood transfusion with numerous guidelines suggesting deferral of blood donors post COVID-19 for up to 28 days or till they turn negative by RT-PCR for COVID. However, there is an inherent risk that the donor might be exposed without himself knowing, be in a window period, or as an asymptomatic carrier. Such donors can transmit the virus, especially if they are asymptomatic. However, there are no case reports of COVID-19 transmission through blood despite the pandemic of more than 1 year.

There is one report from the Republic of Korea, which states of an aplastic anemia patient who received an apheresis platelet unit from an asymptomatic donor who subsequently turned to be COVID positive after 1 day. The recipient in this case report was tested four times subsequently in a span of around 15 days and found to be negative and asymptomatic.[9] In a similar report from Madrid, Spain wherein a stem cell transplant was performed from a donor in the incubation phase of COVID-19 and the patient outcome was uneventful without any infection of COVID-19, till day-100 arising in the recipient. The authors opined that the possible low levels of COVID-19 viral RNA in plasma of patients with COVID-19 could support the safety of blood products including hematopoietic stem cells.[10] Given all the above, it is being postulated that COVID-19 might not be transmitted through blood transfusion. A possible reason for the lack of viremia in patients might be viral particle homing onto endothelial cells/unknown susceptible tissue or fast clearance from circulation. Another possibility is the presence of viral subcomponents akin to Middle East Respiratory Syndrome wherein shedding occurs of virus in a small number of patients, but it cannot be isolated from blood thereby noninfectious even in severe patients.[11]

In a publication from France, wherein the French National Blood Service has performed a retrospective analysis-trace back and has shown similar results wherein <1% (extremely rare) of individuals develop viremia and the virus could be detected by concentration techniques, however, the virus could not be isolated and possibly is shed viral RNA as the corresponding plasma was found to be noninfectious. This study emphasized the role of a strong hemovigilance program as a major cornerstone in blood safety.[12] There are no case reports available regarding the transmission of SARS-CoV-2/SARS-CoV by blood transfusion to date.

An Australian publication stresses that the transfusion-transmitted risk of SARS-CoV-2 appears to be low and to maintain a sufficient stock of blood supply for the needy with minimizing transmission of SARS-CoV-2 to donors and staff is the biggest challenge.[13] Another systematic review also stated the same regarding the possible risk of blood transmission of SARS-CoV-2.[14]

The latest recommendation in India by the National AIDS Control Organization is that a person can donate blood after 14 days after recovery from a COVID-19 infection and RT-PCR negative or after vaccination[15],[16] and came down from 28 days.[17] This period may be shortened in the future when due to pandemics; a suitable eligible population might not be available to donate blood.

The effects of washing, leukoreduction, anticoagulants, duration of red cell preservation could not be analyzed in the present study as the virus could not be detected in the primary blood samples of the patients suffering from COVID-19.

Subject to the rise of newer variants in the future that may spread through blood transfusion, the hemovigilance program needs to be strengthened to identify such novel transfusion transmissible infections. This is to detect early an epidemic situation and give corresponding guidelines promptly.

If all other measures fail, the development of new pathogen reduction technologies, utilization of existing pathogen reduction technologies is recommended. However, patient affordability and specialized component availability might become constraints. All the blood transfusion recipients have the right to know the risks of blood transfusion, the alternatives available, and the right to make their own choice, especially when it involves an irreversible situation like blood transfusion in their life.

Limitations

  1. The present study may not be generalizable to all the variants especially given the high degree of diversity and mutation rate of the virus
  2. The sample size of the present study is small. Although the present study has been designed to include asymptomatic carriers, symptomatic patients at different stages of infection, due to the paucity of data of actual COVID-19 viral RNA positivity in erythrocytes of patients suffering from COVID-19 disease, the sample size could not be calculated.



   Conclusion Top


SARS-CoV-2 could not be detected in blood samples of patients with COVID-19 disease in the present study, raising the possibility that it may not be transmissible through blood. This may drastically affect the present restrictive strategy to defer possible donors for long periods from 28 days[16] to a more liberal strategy thereby preventing blood shortage. The effect of leukoreduction, washing on COVID-19 viral RNA content in blood components could not be ascertained in the present study. A “lookout” hemovigilance strategy is needed to detect any mutations leading to transmission of SARS-CoV-2 through blood.

Acknowledgments

I acknowledge the help and relentless support of the Faculty and staff of the Departments of Anaesthesiology, Transfusion Medicine and Hemotherapy and Microbiology, All India Institute of Medical Sciences, Mangalagiri at all times.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
World Health Organization. COVID-19 Dashboard. Available from: https://covid19.who.int/. [Last accessed on 2021 Dec 22].  Back to cited text no. 1
    
2.
World Health Organization. COVID-19 Dashboard. Available from: https://covid19.who.int/region/searo/country/in. [Last accessed on 2021 Dec 22].  Back to cited text no. 2
    
3.
Liu W, Li H. COVID-19: Attacks the 1-Beta Chain of Hemoglobin and Captures the Porphyrin to Inhibit Human Heme Metabolism. ChemRxiv 2020;1:31. Available from: https://doi.org/10.26434/chemrxiv.11938173.v6. [Last accessed on 2021 Dec 22].  Back to cited text no. 3
    
4.
Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne MA, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 2003;426:450-4.  Back to cited text no. 4
    
5.
Drosten C, Günther S, Preiser W, van der Werf S, Brodt HR, Becker S, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 2003;348:1967-76.  Back to cited text no. 5
    
6.
Yilla M, Harcourt BH, Hickman CJ, McGrew M, Tamin A, Goldsmith CS, et al. SARS-coronavirus replication in human peripheral monocytes/macrophages. Virus Res 2005;107:93-101.  Back to cited text no. 6
    
7.
Law HK, Cheung CY, Ng HY, Sia SF, Chan YO, Luk W, et al. Chemokine up-regulation in SARS-coronavirus-infected, monocyte-derived human dendritic cells. Blood 2005;106:2366-74.  Back to cited text no. 7
    
8.
Schmidt M, Brixner V, Ruster B, Hourfar MK, Drosten C, Preiser W, et al. NAT screening of blood donors for severe acute respiratory syndrome coronavirus can potentially prevent transfusion associated transmissions. Transfusion 2004;44:470-5.  Back to cited text no. 8
    
9.
Cho HJ, Koo JW, Roh SK, Kim YK, Suh JS, Moon JH, et al. COVID-19 transmission and blood transfusion: A case report. J Infect Public Health 2020;13:1678-9.  Back to cited text no. 9
    
10.
Lázaro Del Campo P, de Paz Arias R, Ramírez López A, de la Cruz Benito B, Humala Barbier K, Sánchez Vadillo I, et al. No transmission of SARS-CoV-2 in a patient undergoing allogeneic hematopoietic cell transplantation from a matched-related donor with unknown COVID-19. Transfus Apher Sci 2020;59:102921.  Back to cited text no. 10
    
11.
Corman VM, Albarrak AM, Omrani AS, Albarrak MM, Farah ME, Almasri M, et al. Viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection. Clin Infect Dis 2016;62:477-83.  Back to cited text no. 11
    
12.
Cappy P, Candotti D, Sauvage V, Lucas Q, Boizeau L, Gomez J, et al. No evidence of SARS-CoV-2 transfusion transmission despite RNA detection in blood donors showing symptoms after donation. Blood 2020;136:1888-91.  Back to cited text no. 12
    
13.
Kiely P, Hoad VC, Seed CR, Gosbell IB. Severe acute respiratory syndrome coronavirus-2: Implications for blood safety and sufficiency. Vox Sang 2021;116:155-66.  Back to cited text no. 13
    
14.
Leblanc JF, Germain M, Delage G, O'Brien S, Drews SJ, Lewin A. Risk of transmission of severe acute respiratory syndrome coronavirus 2 by transfusion: A literature review. Transfusion 2020;60:3046-54.  Back to cited text no. 14
    
15.
DO.No.1940407/2020/Imm (2021 May 19), from the Ministry of Health and family Welfare, Department of Health and Family Welfare, Government of India.  Back to cited text no. 15
    
16.
TNN. Days between vaccination and blood donation cut from 28 to 14. The Times of India; 2020. Available from: https://timesofindia.indiatimes.com/city/mumbai/mumbai-days-between-vax-and-blood-donation-cut-from-28-to-14/articleshow/82418617.cms?msclkid=9a16186bc52d11eca0704db940c39e1c.html. [Last accessed on 2022 Apr 26].  Back to cited text no. 16
    
17.
National Guidance to Blood Transfusion Services in India in light of Covid-19 Pandemic. 2020. http://naco.gov.in/blood-transfusion-services-publications [Last accessed on 2021 Dec 22].  Back to cited text no. 17
    

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Correspondence Address:
I. S. Chaitanya Kumar,
Department of Transfusion Medicine and Hemotherapy, All India Institute of Medical Sciences, Mangalagiri, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ajts.ajts_98_21



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