Does PCR Cycle Threshold for SARS-CoV-2 have Association with ABO Blood Group Types?

A cluster of cases with pneumonia with unknown cause were noticed in Wuhan, China in December 2019. In January 2020, World Health Organisation (WHO) officially announced a new virus initially named as the 2019-novel coronavirus (2019-nCoV) and later changed to SARS-CoV-2 as the cause of disease. Later on, in February 2020, WHO officially named the disease as COVID-19. Soon the disease spread globally and in March 2020, WHO declared the coronavirus outbreak a pandemic [1]. As of 14 August 2020, globally a total of 20,687,815 confirmed cases including 750,400 deaths has been reported with 2,461,190 cases including 48,040 deaths alone in India [2]. This has led to the shift in focus of attention of the healthcare workers, scientists and researchers worldwide towards this disease.

RT-qPCR test from respiratory samples (nasopharynx/oropharynx) is currently the standard method for the diagnosis of SARS-CoV-2 infection. This test is semiquantitative in nature and the Ct value is a surrogate marker for a measure of the viral load in the sample [3]. Ct values denote the number of amplification cycles required for the target gene to achieve or exceed a specified threshold level. Ct value is inversely proportional to the viral load and several studies have reported association of lower Ct value with increased probability of progression to severe disease, infectivity and greater transmissibility [4-7].

The antigenic determinants of ABO blood groups have been reported in past to be associated with certain viral infections such as rotavirus, noroviruses (NoVs), dengue virus, Norwalk virus and hepatitis B virus [8-11]. During this ongoing pandemic, researchers have demonstrated linkage of ABO blood group type with COVID-19 infection. Cheng Y et al., showed that individuals with O blood group as compared to non-O blood group ones were less susceptible to infection with SARS-CoV-2 [12]. Zhao J et al., based on the comparison of ABO blood groups of controls from the general population with SARS-CoV-2 infected patients reported a significantly higher risk of the infection in individuals with blood group A and lower risk in blood group O individuals [13]. Similar finding has been reported by Zietz M and Tatonetti NP in a cohort of 7770 SARS-CoV-2 positive cases [14].

Keeping in view the association of susceptibility of SARS-CoV-2 infection with ABO blood group types and Ct value with that of disease severity and infectivity rate, we aimed to assess the relationship between RT-qPCR Ct value for SARS-CoV-2 and ABO blood group type and to determine any blood type dependent difference in viral load. To the best of our knowledge, this is the first study to focus on such an association.

Materials and Methods

A prospective and observational study was carried out at ICMR approved COVID-19 diagnostic facility of Western Maharashtra, India. Convenient sampling was used wherein all consecutive patients admitted to our tertiary care centre between mid-July to mid-August 2020 were recruited for COVID-19 testing and blood grouping. Only patients who were found to be positive for SARS-CoV-2 by RT-qPCR were included in the study. Asymptomatic, non-admitted COVID-19 positive patients were excluded from the study. Samples obtained from all the consecutive admitted patients with COVID-19 helped us to prevent selection bias. This was a pilot study as there is no data on prior studies in Indian settings. The study was approved by the ethical Committee of the institution (IEC/2020/289) and written informed consent was obtained from all the patients.

Nasopharyngeal/Oropharyngeal swabs were collected in Viral Transport Medium (VTM) and were tested for SARS-CoV-2 within 24 hours of collection using commercially available TRUPCR SARS-CoV-2 RT qPCR kit version 2.0 (Cat No. 3B304, Kilpest India Ltd, Bhopal, India) on Light Cycler 480 II (Roche) platform (96 well plates). The samples were reported positive based on the detection of pan-Sarbecovirus E-gene sequence and SARS-CoV-2 specific RdRP gene sequence in accordance with the manufacturer instructions. Ct values for both E and RdRP genes obtained at the time of admission including age and gender of all the patients tested positive for SARS-CoV-2 were recorded. In addition, whole blood venous sample in EDTA vacutainer of all admitted 116 patients were obtained for ABO blood group typing. Records of blood groups of voluntary blood donors aged between 18 to 60 years at our hospital were obtained as control group to reflect the distribution of blood group types in the general population. These records were obtained from September 2019 to November 2019 to avoid inclusion of asymptomatic SARS-CoV-2 positivity. This control group was necessary to study any significant difference in the distribution of blood groups between SARS-CoV-2 positive and negative groups.

Statistical Analysis

The data were expressed as mean and Standard Deviation (SD) of Ct values and were subjected to Welch’s t-test for evaluation of statistical significance of association of Ct value of E and RdRP gene with different blood group types, age groups and gender. Fisher’s-exact test was used for comparison of ABO blood group distribution in SARS-CoV-2 positive patients and Control group. The statistical significance level was set at p<0.05. All statistical computations were performed using GraphPad Prism version 5.00 for Windows, (GraphPad Software, San Diego California USA).

Results

Distribution of ABO Blood Group in SARS-Cov-2 Positive Patients

Of the 116 patients positive for SARS-CoV-2 enrolled in the study, ABO blood group typing revealed that 33 (28.45%) had type A, 34 (29.31%) had type B, 41 (35.34%) were of type O and remaining 08 (6.90%) had AB blood group type. This distribution in COVID-19 patients was compared to control group comprising of 882 non-COVID-19 healthy blood donors. In the control group, type B was the most common followed by type O while type AB was the least common [Table/Fig-1]. The SARS-CoV-2 positive patient group comprised of higher percentages of individuals with type O and was the most common type followed by type B blood group. However, no significant difference was noted in the distribution of ABO blood types between the two groups i.e., SARS-CoV-2 positive and control [Table/Fig-1].

[Table/Fig-1]:

Comparison of ABO blood group distribution in SARS-CoV-2 positive patients and Control group.

	SARS-CoV-2 +ve	Control group	p-value
Total (n)	116	882
ABO blood group
A	33 (28.45%)	256 (29.02%)	0.9841
B	34 (29.31%)	266 (30.16%)	0.9365
AB	08 (6.90%)	98 (11.11%)	0.2206
O	41 (35.34%)	262 (29.70%)	0.2566

Fisher’s-exact test was used to calculate p-value (significant level p<0.05)

Cycle Threshold (Ct) Values with Respect to Age, Gender and ABO Blood Group Types in SARS-CoV-2 Positive Patients

The study cohort comprised of 82 males and 34 females and was categorised into different age groups as shown in [Table/Fig-2]. The mean and SD of Ct values for E and RdRP gene in different age group, gender and ABO blood group types are shown in [Table/Fig-2]. Scatter dot plot of Ct values for both E gene and RdRP gene in different age group are shown in [Table/Fig-3,4], respectively while for that in different blood group types are plotted in [Table/Fig-5,6]. No statistical significance was noted in the Ct values with respect to age, gender and ABO blood group types either within or between the groups.

[Table/Fig-2]:

Cycle threshold (Ct) values of E gene and RdRP gene in SARS-CoV-2 positive patients and its correlation in different age group, gender and ABO blood group types.

Characteristic	No. of samples	E gene		RdRP gene
		Mean	SD	p-value	Mean	SD	p-value
All samples	116	26.29	5.65	-	28.20	5.51	-
Age (years)
≤20	06	24.95	4.77	0.7702	26.11	4.29	0.6076
21-40	47	25.34	6.15	0.4641	27.44	5.96	0.4952
41-60	26	27.06	6.03	0.6290	29.08	5.67	0.8061
61-80	33	27.11	4.83	0.3081	28.72	4.69	0.2961
>80	04	27.59	4.64	0.8608	30.39	7.19	0.3398
Gender
Males	82	26.05	5.74	0.8499	27.84	5.46	0.9446
Females	34	26.85	5.49	0.9066	29.09	5.60	0.8661
ABO blood group
A	33	25.36	5.67	0.9373	27.54	5.13	0.6582
B	34	25.59	5.36	0.7508	27.40	5.02	0.5449
AB	08	28.68	3.90	0.2980	30.06	4.60	0.6489
O	41	27.15	6.06	0.5630	29.04	6.28	0.2861

SD: Standard deviation

p-values are based on a comparison of mean CT values of each age group, gender and ABO blood group type vs the mean of all Ct values for E gene and RdRP gene using Welch’s t-test

[Table/Fig-3]:

Scatter dot plot with line at mean and range showing comparison of Cycle Threshold (Ct) of E gene by age group.

[Table/Fig-4]:

Scatter dot plot with line at mean and range showing comparison of Cycle Threshold (Ct) of RdRP gene by age group.

[Table/Fig-5]:

Scatter dot plot with line at mean and range showing comparison of Cycle Threshold (Ct) of E gene by ABO blood group type.

[Table/Fig-6]:

Scatter dot plot with line at mean and range showing comparison of Cycle Threshold (Ct) of RdRP gene by ABO blood group type.

Discussion

Blood group antigens are well known to modify the immune response to infection and based on the differences in blood group antigen expression, individuals may be susceptible or protected against certain infections [15]. Few studies mostly originating from China, have demonstrated that patients with blood group A were more susceptible to SARS-CoV-2 infection, whereas patients with blood group O relatively had a lower risk [13,14,16]. Coronavirus is an enveloped virus that targets host cells via viral adhesion glycoproteins. The SARS-CoV spike (S) protein is such a viral adhesion glycoprotein. Guillon P et al., have demonstrated that the natural human anti-A antibodies might block the interaction between SARS-CoV spike protein and its receptor angiotensin-converting enzyme 2, thus providing protection. This could well be explained hy individuals with blood group A with absence of naturally occurring anti-A antibodies tend to be more susceptible to SARS-CoV infection [17].

Several studies on COVID-19 have demonstrated that lower Ct values were associated with more severe disease and infectivity [4-7]. This is in conformity with earlier studies published on Ct values in other respiratory infections such as Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Respiratory Syncytial virus [18,19]. The association of susceptibility of SARS-CoV-2 infection with ABO blood group types and Ct value with that of disease severity and infectivity rate led us to assess the relationship between RT-qPCR Ct value for SARS-CoV-2 and ABO blood group type and to determine any blood type dependent difference in viral load.

Present study did not showed any association of ABO blood group type with susceptibility to infection by SARS-CoV-2. It also did not reveal any significant difference in Ct values for pan-Sarbecovirus E and SARS-CoV-2 specific RdRP gene in different blood group types. These results were in variance with those reported earlier [4-7,13,14,16]. This variance in the results may be explained based on the differences in the ethnicity of the population studied. There is a definite racial element to ABO blood typing [20,21]. Present study certainly raises a pertinent question on the effects of ethnicity on COVID-19 susceptibility and severity. Moreover, the time from onset of symptoms to sampling varied between patients which could have affected the Ct values.

Unlike the study by Buchan BW et al., where they showed significant correlation of Ct value with age between 80-89 years, present study did not show any significance with different age group. This difference may be attributed to very small number of patients in this group i.e., 4 patients comprising just 3.45% of the total cohort. However, present study result of Ct values showing no statistical significance with gender difference corroborated with that of Buchan BW et al., [22].

Limitation(s)

The main limitation in this study was the small sample size. Moreover, there was a lead time bias as the time from onset of symptoms to sampling differed between patients. The Ct value of SARS-CoV-2 is known to vary during the course of infection and tends to be higher in early symptomatic stage. Additionally, Berksonian bias cannot be ruled out as our study population includes hospitalised patients. However, present centre being a multi-speciality government hospital, people from different strata and socio-economic status were screened for SARS-CoV-2 infection before admission, during current pandemic.

Conclusion(s)

Present study did not find any association of Ct values for SARS-CoV-2 with ABO blood group types. However, the knowledge on COVID-19 is constantly evolving and a wide range in response and course of COVID-19 disease has been noted in different ethnic population. Since there is no published data from India on association of ABO blood group types with susceptibility to SARS-CoV-2 infection and its correlation with Ct values. Therefore, further large-scale studies based on Indian population are needed for more definitive results.

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