Association of ABO blood groups with presentation and outcomes of confirmed SARS CoV-2 infection: A prospective study in the largest COVID-19 dedicated hospital in Bangladesh.

BACKGROUND: Globally, studies have shown conflicting results regarding the association of blood groups with SARS CoV-2 infection.
OBJECTIVE: To observe the association between ABO blood groups and the presentation and outcomes of confirmed COVID-19 cases. DESIGN, SETTING, AND PARTICIPANTS: This was a prospective cohort study of patients with mild-to-moderately severe COVID-19 infections who presented in the COVID-19 unit of Dhaka Medical College Hospital and were enrolled between 01 June and 25 August, 2020. Patients were followed up for at least 30 days after disease onset. We grouped participants with A-positive and A-negative blood groups into group I and participants with other blood groups into group II.
RESULTS: The cohort included 438 patients; 52 patients were lost to follow-up, five died, and 381 completed the study. The prevalence of blood group A [144 (32.9%)] was significantly higher among COVID-19 patients than in the general population (p < 0.001). The presenting age [mean (SD)] of group I [42.1 (14.5)] was higher than that of group II [38.8 (12.4), p = 0.014]. Sex (p = 0.23) and co-morbidity (hypertension, p = 0.34; diabetes, p = 0.13) did not differ between the patients in groups I and II. No differences were observed regarding important presenting symptoms, including fever (p = 0.72), cough (p = 0.69), and respiratory distress (p = 0.09). There was no significant difference in the median duration of symptoms in the two group (12 days), and conversion to the next level of severity was observed in 26 (20.6%) and 36 patients (13.8%) in group I and II, respectively. However, persistent positivity of RT-PCR at 14 days of initial positivity was more frequent among the patients in group I [24 (19%)] than among those in group II [29 (11.1%)].
CONCLUSIONS: The prevalence of blood group A was higher among COVID-19 patients. Although ABO blood groups were not associated with the presentation or recovery period of COVID-19, patients with blood group A had delayed seroconversion.

Introduction

The World Health Organization (WHO) first reported the emergence of COVID-19 infection in Wuhan City, China in late December [1]. Subsequently, the disease caused by this novel virus was declared to be a pandemic on March 12, 2020 [2]. As of September 19, 2020, there have been 30,369,778 confirmed cases of COVID-19, including 948,795 deaths reported to the WHO [3]. The presentation of COVID-19 varies widely. The most common symptoms of COVID-19 include fatigue, fever, dry cough, respiratory distress, and anosmia. Moreover, a large proportion of patients remain asymptomatic [4]. Its behavior also shows regional variation. A genome-wide association study on severe COVID-19 with respiratory failure detected cross-replicating associations with rs11385942 at locus 3p21.31 and with rs657152 at locus 9q34.2. The association signal at locus 9q34.2 coincided with the ABO blood group locus. They found a higher risk in individuals with blood group A than in those with other blood groups [5]. Other viruses, such as HBV [6], SARS-COV [7], and MERS-COV [8], are susceptible to ABO blood groups. The mechanisms underlying the association between the blood groups are still unclear. Histo-blood group antigens are expressed on endothelial cells (ECs) and platelets [9]. The SARS-CoV-2 enters the human body through replication in epithelial cells of the respiratory and digestive tracts. They have the ability to synthesize ABH carbohydrate epitopes. It has been hypothesized that the S protein of the virions produced by either A or B individuals can be decorated with A or B carbohydrate epitopes, respectively. Guillon et al. [10] reported that the interaction between the S protein and angiotensin-converting enzyme 2 (ACE-2) is specifically inhibited by human natural anti-A antibodies. SARS-CoV and SARS-CoV-2 have similar nucleic acid sequences and similar receptor combinations with ACE-2 [11]. It was also found that the non-A type is a risk factor for venous thromboembolism, which is one of the causes of COVID-19 deaths [12]. However, different studies have shown contradictory results regarding the influence of blood groups on the susceptibility and outcome of COVID-19 infections [13-16]. Therefore, this study was conducted to observe the association of ABO blood groups with the presentation and outcome of confirmed COVID-19 infections in the largest COVID-19 dedicated hospital in Bangladesh.

Materials and methods

This single-center prospective cohort study was conducted to evaluate the association of the ABO blood groups with the presentation and outcomes of confirmed COVID -19 infections in hospitalized and outdoor patients with COVID-19. The outcomes in this research included: A. Duration required for clinical improvement, as defined below; B. Proportion of patients converted to the next level of severity; C. Proportion of the patients remaining positive for RT-PCR of COVID-19 on day 14 after the initial positivity; D. Development of post-COVID syndrome as defined below. The participants were enrolled from 01 June to 25 August, 2020. The study was conducted in the Department of Medicine, Dhaka Medical College Hospital. The recruitment was limited to patients who were more than 18 years of age with confirmed COVID-19 (RT-PCR positive) infection. Patients with hemoglobinopathies or other blood disorders were excluded from the study. Written informed consent was obtained from all the patients. Ethical approval was obtained from the ethical review committee of the institute. The capacity to provide consent was determined by the investigators in the presence of an attendant, a testimony of the attendant was obtained in the consent form, and the ethical review committee approved this consent procedure during the approval of the protocol. Those who were minors or unable to provide consent were excluded from the study. For the estimation, we grouped A positive and A negative blood groups into group I and other blood groups, such as B, AB, and O, irrespective of their Rh status, into group II. An assumption that the expected proportions to be cured from COVID-19 by day 12 in group I (blood group A) and group II (blood groups B, O, AB) are 0.70 and 0.90, respectively. Thus, we required a total of 378 samples at a 1:2 ratio, which would provide a power of at least 90% in two-tailed tests and a p value less than 0.05, to detect significant differences between the groups. Therefore, considering a 10% dropout rate, we needed 416 samples in total.

Procedure

A case record form was constructed to collect baseline information, such as demographics, blood groups, clinical signs and symptoms, comorbidities, and oxygen saturation. Routine investigations, including CBC, ESR, CRP, creatinine, RBS, SGPT, chest X-ray, and D-dimer were advised on enrollment. Real-time-polymerase chain reaction testing for COVID-19 was performed 14 days after the initial positive test in all patients. The patients were followed up directly or over the telephone with at least three-day intervals up to 30 days from the onset of the disease. Clinical improvement in patients was assessed according to the improvement criteria of the WHO and Bangladesh guidelines [17,18], which required that the body temperature remained normal for at least 3 days, respiratory symptoms were significantly improved (respiratory rate < 25 and no dyspnea), and SpO2 >93% was achieved without assisted oxygen inhalation. Mild disease was defined as the symptoms of an upper respiratory tract viral infection, including mild fever, cough (dry), sore throat, nasal congestion, malaise, headache, muscle pain, anosmia, or malaise. Moderate disease, including respiratory symptoms, such as cough and shortness of breath are present without signs of severe pneumonia. Severe disease included severe dyspnea, tachypnea (> 30 breaths/min), and hypoxia (SpO2 < 90% in room air). These classifications were made according to the World Health Organization and national guidelines of Bangladesh [17,18]. In this study, we assessed the proportion of patients with early recovery (clinical improvement within 7 days of symptom onset), late recovery (clinical improvement required ≥12 days), severity conversion (patients progress to more serious disease), persistently positive for RT-PCR of COVID-19 (positive RT-PCR on a 14 day test), and post-COVID syndrome (in the absence of any definition, we defined it as 1. Persistence of illness with signs and symptoms beyond virologic clearance 2. Development of new symptoms within 1 month after the initial clinical and virologic cure, the etiology of which is postulated to be viral infection.

Statistical analysis

The data were analyzed using STATA version-15.1, StataCorp, 4905 Lakeway Drive, College Station, Texas 77845 USA. To compare the variables between group I (blood group A) and group II (other blood groups), an independent sample t-test was used. The chi-square test was used to analyze the categorical variables. For the outcome assessment, relative risk (RR) with 95% CI was measured for qualitative variables categorizing groups I and II. The difference in the median time-to-recovery between group I and group II was determined with cumulative incidence by blood group from the Fine-Gray Model. The hazard ratios were calculated using cause-specific hazard models. Categorical variables are presented as n (%), normally distributed continuously as mean (SD), and skewed continuous variables as median (IQR). The statistical significance was set at P <0.05.

Results

Of the 554 patients who were screened and assessed for eligibility, a total of 438 patients were enrolled in the study. A total of 52 patients were lost to follow-up, 5 patients died, and 381 patients completed the follow-up. The analysis was performed on 438 patients (Fig 1).

Fig 1

Sample selection in this prospective cohort study in adults with COVID-19 infection.

Among them, 188 patients were admitted to the hospital. The mean age of the patients was 39.8 (13). The majority of the participants were younger than 40 years [266 participants (60.6%)] and men were predominantly affected [258 (58.9%)]. Most of the patients presented with fever (333 patients, 76%) and cough (274 patients, 62.6%). Respiratory distress developed in 191 patients (43.6%) during the disease course. The majority of the patients [304 patients (69.4%)] exhibited mild severity. Overall, 113 (25.7%) patients presented with co-morbidities. Among them, hypertension was present in 63 (14.4%) and diabetes in 55 (12.6%) patients (Table 1).

Table 1

Baseline characteristics of COVID-19 patients with different blood groups (N = 438).

Variables	Total (N = 438)	Group Ic (N = 144)	Group IId (N = 294)	p-value
Age (years) Mean (SD)	39.8(13.2)	42.1(14.5)	38.8(12.4)	0.014
Sex (Male), n (%)	258(58.9)	79(54.9)	179(60.9)	0.23
Symptoms
Fever, n (%)	333(76.0)	111(77.1)	222(75.5)	0.72
Cough, n (%)	274(62.6)	92(63.9)	182(61.9)	0.69
Running Nose, n (%)	39(8.8)	13(9.0)	26(8.8)	0.95
Respiratory Distressa, n (%)	191(43.6)	71(49.3)	120(40.8)	0.09
Sore throat, n (%)	101(23.1)	32(22.2)	69(23.5)	0.77
Hoarseness of voice, n (%)	16(3.7)	2(1.4)	14(4.8)	0.08
Diarrhea, n (%)	37(8.4)	9(6.2)	28(9.5)	0.25
Vomiting, n (%)	27(6.2)	8(5.6)	19(6.5)	0.71
Anorexia, n (%)	122(27.9)	49(34.0)	73(24.8)	0.04
Anosmia, n (%)	155(35.4)	42(29.2)	113(38.4)	0.06
Headache, n (%)	95(21.7)	29(20.1)	66(22.4)	0.58
Lethargy, n (%)	112(25.6)	32(22.2)	80(27.2)	0.26
Body ache, n (%)	73(16.7)	25(17.4)	48(16.3)	0.79
Hypertension, n (%)	63(14.4)	24(16.7)	39(13.3)	0.34
Diabetes, n (%)	55(12.6)	23(16.0)	32(10.9)	0.13
Severity gradeb at presentation
Mild, n (%)	304(69.4)	94(65.3)	210(71.4)	0.19
Moderate, n (%)	134(30.6)	50(34.7)	84(28.6)

a Shortness of breath, respiratory rate >25/min, or oxygen saturation <93%.

b Disease severity at presentation: mild symptoms of an upper respiratory tract viral infection, including mild fever, cough (dry), sore throat, nasal congestion, malaise, headache, muscle pain, anosmia, or malaise.

Moderate respiratory symptoms, such as cough and shortness of breath are present without signs of severe pneumonia (tachypnea > 30 breaths/min, and hypoxia: SpO2 < 90% on room air).

C Blood group A positive and negative.

d Other blood groups-B, AB, and O including Rh status.

The average median (IQR) duration of illness was 12 (range, 8–16) days. In Group I, the IQR was 12 days (9–16 days) and Group II it was 12 days (8–15 days) (HR, 95% CI, 1.14, 0.91–1.41, p = 0.25) (Table 2).

Table 2

Duration of illness of COVID-19 patients among Group I and Group II.

Attribute	Total (N = 386)	Group I (N = 126)	Group II (N = 260)	Cause-Specific Hazard Ratio 95% CI	p-value
Total duration of illness, Median (IQR)	12 (8–16)	12 (9–16)	12 (8–15)	1.14 (0.91–1.41)	0.25

The recovery within 7 days was 202 (52.3%) for the total population, 60 (47.6%) in Group II, and 142 (54.6%) in Group II (RR, 1.15; 95% CI, 0.93–1.43; p = 0.20). In 111 (28.8%) patients, the symptoms persisted for more than 12 days. In group I and group II it was 39 (31.0%) and 72(27.7%), respectively (RR, 1.12; 95% CI, 0.81–1.55; p = 0.51). A total of 62 (16.1%) patients progressed to the next level of severity, including 26 patients (20.6%) in group I and 36 (13.9%) in group II (RR, 1.49; 95% CI, 0.94–2.35; p = 0.09). The number of patients who remained positive even 14 days after initial positivity was 53 (13.7%); among them, 24 patients (19.0%) were in group I and 29 (11.1%) were in group II (RR, 1.71; 95% CI, 1.04–2.81; p = 0.04). After improvement of the initial symptoms, 172 (44.6%) patients developed post-COVID syndrome, including 65 patients (51.6%) in group I and 107 (41.1%) in group II (RR, 1.25; 95% CI, 1.00–1.57; p = 0.05; Table 3).

Table 3

Outcome of COVID-19 patients among Group I and Group II.

Attribute	Total (N = 386) n (%)	Group I (N = 126) n (%)	Group II (N = 260) n (%)	Relative Risk 95% CI	p-value
Recoverya,b within 7§ days	202 (52.3)	60 (47.6)	142 (54.6)	1.15 (0.93–1.42)	0.20
Persistence of symptoms 12 days or more	111 (28.8)	39 (31.0)	72 (27.7)	1.12 (0.81–1.55)	0.51
Conversion to next level of severityc	62 (16.1)	26 (20.6)	36 (13.9)	1.49 (0.94–2.35)	0.09
Persistent positivityd	53 (13.7)	24 (19.0)	29 (11.1)	1.71 (1.04–2.81)	0.04
Post COVID syndromee	172 (44.6)	65 (51.6)	107 (41.1)	1.25 (1.00–1.57)	0.05

a Clinical recovery was defined as a normal body temperature for at least 3 days, improved respiratory symptoms defined as no shortness of breath and respiratory rate <25/min, and an oxygen saturation greater than 93% without supplemental oxygen.

b Response criteria was that the patient had recovered clinically as defined above. The day at which clinical recovery started was considered as the response day.

c Disease stages were defined as mild symptoms of an upper respiratory tract viral infection, including mild fever, cough (dry), sore throat, nasal congestion, malaise, headache, muscle pain, anosmia, or malaise. Moderate respiratory symptoms, such as cough and shortness of breath are present without signs of severe pneumonia (tachypnea > 30 breaths/min, and hypoxia: SpO2 < 90% on room air. Severe tachypnea > 30 breaths/min, and hypoxia: SpO2 < 90% on room air.

d Persistent RT-PCR positive patient remained positive for RT-PCR at 14 days test.

eIn the absence of any definition, we defined it as 1. Persistence of illness signs and symptoms beyond virologic clearance 2. New development of symptoms within 1 month after initial clinical and virologic cure, the etiology of which is postulated to be viral infection.

§Days calculated from the onset of symptoms to the day of clinical recovery.

Group I: Blood group A, irrespective of Rh phenotype.

Group II: Blood groups B, AB, and O, irrespective of Rh phenotype.

There were no significant differences in the time to recovery from illness between groups I and II in terms of the cumulative incidence by blood group from the Fine-Gray Model (Fig 2).

Fig 2

Cumulative incidence by blood group from Fine-Gray Model.

Discussion

In the current study, the main aim was to observe the association of the different blood groups on the presenting features and outcomes of mild-to-moderate COVID-19 infections. No differences were observed with respect to the presentation and duration of recovery among the different blood groups. In this study, the majority of the patients were young. It was observed that 60.4% of the affected patients were aged < 40 years, and 8% of the patients were aged > 60 years. Among the affected individuals in this study, 144 patients had blood group A (32.8%), 148 had blood group B (33.7%), 52 had blood group AB (11.9%), and 94 had blood group O (21.5%). In a study [19] conducted by the Blood Bank of Dhaka Medical College, the proportions of different blood groups in the community were as follows: A, 21.8%; B, 37.5%; AB, 8.9%; and O, 31.8%. A higher prevalence of blood group A was observed among the affected individuals. A one-sample t-test revealed a p value of < 0.001. A Turkish study [20] found similar results. Most patients presented with fever, cough, anosmia, anorexia, and lethargy. We did not find any significant differences in symptoms between group I (blood group A) and group II (other blood groups); similar results were found in another study [13]. In this study, comorbidities were present in approximately 26% of the cases. The most important factors were hypertension and diabetes. We observed no statistically significant differences in the presenting features and comorbidities among the patients with different blood groups. Globally, approximately 80% of adults present with mild (40%) to moderate illness (40%), 15% with severe illness, and 5% with critical illness [21]. In this study, 69.4% of patients showed mild disease, and 30.6% had moderate severity. It was observed that the patient’s blood group did not have any effect on their presenting severity (p = 0.62); a similar observation was reported by Latz et al. [13]. Although a majority of the patients in this study recovered, five patients died. As the number of patients was low (1.3%), no analysis was performed regarding the death of the patients. Among them, one patient was of blood group A, two were of blood group B, and two were of blood group O. Four patients died in the hospital due to respiratory failure resulting from COVID pneumonia; one died at home, and the etiology was not determined. The median duration of the symptoms was 12 days. The blood group did not have any influence on the symptom duration. However, Zietz [15] described that the blood group A had less severity of illness, less intubation, and less mortality. More than 50% of the patients recovered within seven days. In the present study, 28% of the patients’ symptoms persisted for more than 12 days. It was determined that the blood group did not have any influence on patient recovery. Approximately 16% of the patients converted to the next level of severity, that is, from mild-to-moderate, severe, and death. The blood group did not show any association with the conversion. Approximately 13.7% of the patients remained positive for COVID-19 in RT-PCR tests even after 14 days of initial positivity. The blood group A had a higher relative risk of delayed seroconversion. Latz et al [13], Zeng X et al [16] found no differences in their study. Approximately 44% developed different post-COVID symptoms. It was observed that post-COVID fatigue was the most prominent symptom. We did not find any significant relationship between the blood groups and post-COVID syndrome. This study was performed on patients > 18 years of age with mild-to-moderate severity of illness. Therefore, our findings cannot be generalized. Therefore, this should be interpreted with caution.

This study has a few limitations. This was a single-center study, and the sample size was limited. We did not include the severe cases, and most of the follow-up was conducted through virtual interviews, which has some inherent drawbacks for the study.

Conclusions

The prevalence of blood group A was higher among COVID-19 patients. The ABO blood group was not associated with the presentation and recovery period of COVID-19. However, the patients of blood group A had a higher risk of persistent positivity. To detect the susceptibility of the infection, further studies on the individuals with confirmed exposure to COVID-19 infection should be conducted.

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4 Feb 2021

PONE-D-20-36898

Impact of ABO Blood Groups on Presentation and Outcomes of Confirmed SARS Cov-2 infection: A prospective study in a largest COVID-19 Dedicated Hospital in Bangladesh.

PLOS ONE

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Reviewer #1: Good clinical report. Noticed patients were young! What were the causes of death of 5 patients? Do you have any long term follow up?

Reviewer #2: Here is a list of specific comments. Note: without line and page numbering, it was difficult to reference the reviews.

1. Because “impact” inferred causality, I suggest replacing “impact” with ‘association’ throughout the manuscript.

2. Abstract, Results, “the Presenting age . . . did not differ among the groups”: “The groups” were not clear in Abstract. Did it refer to the blood groups?

3. Materials and Methods, “this single centered ...outcome of Confirmed COVID-19 ...”: I suggest explicitly defining the outcome.

4. Materials and Methods, “for the estimation we grouped A . . . ”: I suggest including brief information about Groups I and II in Abstract.

5. Materials and Methods, “the estimated required sample size . . . ”: For the calculation of the required sample size, I suggest including the proportions of Groups I and II, the reference risk (i.e., risk in Group II), and the test statistic. Also, please specify the risk referred to the risk of recovery.

6. Statistical Analysis, “sample size 377 ...”: The sentence regarding the required sample size had been previously mentioned. I suggest excluding it.

7. Statistical Analysis, “to compare the groups A, B, AB and O . . . ”: The groups of A, B, AB and O contradicted the previously defined Groups I and II in the Materials and Methods section. I suggest using the same definitions of blood groups consistently throughout the manuscript.

8. Statistical Analysis, “the difference in median time-to-recovery . . . ”: For recovery, death was a competing event. Instead of stating “cox regression analysis”, I suggest stating ‘cause-specific hazards models’. No change to the models; it was just terminology. However, the use of Kaplan-Meier method and log-rank test were different. I suggest replacing them with ‘cumulative incidence functions method’ and ’Gray’s test’.

9. Results, “438 patients were enrolled . . . ”: Because survival analysis was used, the 52 lost-to-follow-up patients could be included in the analyses.

10. Results, “maximum follow up days was 45”: Weren’t the patients followed up to 30 days?

11. Table 1:

(11a) I suggest adding a column including Group II (i.e., blood groups B, AB and O) and a column of p-values comparing Groups I and II.

(11b) I suggest revising the column header “significance” as ‘p-values’.

12. Table 2: Please clarify in the table what the “Risk ratio/Hazard ratio” column referred to. Per the interpretation in the footnote, it seemed to refer the interactions between blood groups and attributes. If so, I suggest reporting hazard ratios for attributes in Group I and Group II because hazard ratios for interactions were not interpretable directly.

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10 Mar 2021

A rebuttal letter

To

Editor in chief,

PLOS ONE

Subject: In response to review of the manuscript entitled “Association of ABO blood groups with presentation and outcomes of confirmed SARS CoV-2 infection: A prospective study in the largest COVID-19 dedicated hospital in Bangladesh”.

Dear Sir,

Thank you for reviewing my manuscript. I have tried to address each point raised by the academic editor and the reviewers.

Response to Academic editor

PLOS ONE style-I have revised the manuscript according to PLOS ONE style.

The manuscript was edited by Editage. A copy was uploaded as supporting information. A clean copy of edited manuscript was uploaded as manuscript file.

P value =0.00 was mentioned in 2 occasion in the manuscript. I have corrected it to p <0.001 in the result section of the abstract and the discussion part of the manuscript.

Discussion- A one-sample t-test revealed a p value of < 0.001. (Page-11, line 205)

Abstract- The prevalence of blood group A [144 (32.9%)] was significantly higher among COVID-19 patients than in the general population (p < 0.001). (Page 2, line 36, 37)

Description about How capacity to give consent was determined was added in the methodology section in the following way-

The capacity to provide consent was determined by the investigators in the presence of an attendant, a testimony of the attendant was obtained in the consent form, and the ethical review committee approved this consent procedure during the approval of the protocol. Those who were minors or unable to provide consent were excluded from the study. (Page-5, line 87-91)

The title was amended as per reviewer’s advice both in the online submission form and manuscript as- “Association of ABO blood groups with presentation and outcomes of confirmed SARS CoV-2 infection: A prospective study in the largest COVID-19 dedicated hospital in Bangladesh”

Caption of the supporting information file was added at the end of the manuscript.

Response to Reviewer’s:

Is the manuscript technically sound, and do the data support the conclusions?

In response to Reviewer-2: I have tried to revise the manuscript according to your advice. In the subsequent section I shall explain it.

Has the statistical analysis been performed appropriately and rigorously?

In response to Reviewer 2: Initially the statistical analysis was done with SPSS version 20. Some analysis you advised in the comment section like cumulative incidence function method was not possible in SPSS. So I revised the statistical analysis and did it with STATA version-15.1, StataCorp, 4905 Lakeway Drive, College Station, Texas 77845 USA. The change was made according to your advice which will be discussed in the subsequent sections. (Page-6, line 120-128)

Have the authors made all data underlying the findings in their manuscript fully available?

In response to Reviewer 2: Data was made available to PLOS ONE and Dryad data repository, will be available to third party after the publication of the manuscript.

DOI https://doi.org/10.5061/dryad.dv41ns1xk

Is the manuscript presented in an intelligible fashion and written in Standard English?

Both reviewers response was yes.

Review comments to author:

Reviewer 1: Thanks for the appraisal.

Most of the patients were young: In this study the most of the study population were less than 40 years (about 60%).This is different from the Europe and USA. In USA older patients (ages ≥65 years) accounted for about 31% of all cases, in this study it was only 8%.This is probably due sociocultural background of Bangladesh. Here proportion of elderly population (5%) is less the western world (North America 16%, Europe 21%). More over many of our participants were health professionals.

Cause of the death: total 4 of our patient died in Hospital due to respiratory failure due to COVID pneumonia. One of our patient died at home, the etiology was undetermined. ( Page-12, line 217-218)

Long term follow up: No we don’t have long term follow up of all the patients.

In response to Reviewer 2: Sorry for your discomfort. This was my second only writing to an international journal, I failed to understand the instruction. In the revised manuscript I have added the line and page numbering. Thank you for your suggestion, it helped to enrich my knowledge and views.

Because “impact” inferred causality, I suggest replacing “impact” with ‘association’ throughout the manuscript.

Response: I have replaced the impact with association throughout the manuscript and renamed the title as Association of ABO blood groups with presentation and outcomes of confirmed SARS CoV-2 infection: A prospective study in the largest COVID-19 dedicated hospital in Bangladesh

Abstract, Results, “the Presenting age . . . did not differ among the groups”: “The groups” were not clear in Abstract. Did it refer to the blood groups?

Response: I have clarified it in the revised manuscript and rewrite it as “The presenting age [mean (SD)] of group I [42.1 (14.5)] was higher than that of group II [38.8 (12.4), p=0.014]. Sex (p=0.23) and co-morbidity (hypertension, p=0.34; diabetes, p=0.13) did not differ between the patients in groups I and II”. ( Page 2, line 37-39)

Note: Previously we have compared A, B, AB, O and found no difference in the age of presentation. But this time when we compared between blood group A (group I) and other blood groups (group II) according to your suggestion, we found a significant difference. More over this time analysis was done with 438 patient including the lost to follow up patients.

Materials and Methods, “this single centered ...outcome of Confirmed COVID-19 ...”: I suggest explicitly defining the outcome.

Response: In the revised manuscript the following lines are added to define the outcome explicitly- The outcomes in this research included: A. Duration required for clinical improvement, as defined below; B. Proportion of patients converted to the next level of severity; C. Proportion of the patients remaining positive for RT-PCR of COVID-19 on day 14 after the initial positivity; D. Development of post-COVID syndrome as defined below.(Page 4, line 79-82)

Materials and Methods, “for the estimation we grouped A . . . ”: I suggest including brief information about Groups I and II in Abstract.

Response: According to your suggestion the following line was added in the abstract in the designs, settings part- We grouped participants with A-positive and A-negative blood groups into group I and participants with other blood groups into group II. (Page 2, line-33, 34)

Materials and Methods, “the estimated required sample size . . . ”: For the calculation of the required sample size, I suggest including the proportions of Groups I and II, the reference risk (i.e., risk in Group II), and the test statistic. Also, please specify the risk referred to the risk of recovery.

Response: According to your suggestion we estimated our sample size as follows- An assumption that the expected proportions to be cured from COVID-19 by day 12 in group I (blood group A) and group II (blood groups B, O, AB) are 0.70 and 0.90, respectively. Thus, we required a total of 378 samples at a 1:2 ratio, which would provide a power of at least 90% in two-tailed tests and a p value less than 0.05, to detect significant differences between the groups. Therefore, considering a 10% dropout rate, we needed 416 samples in total. (Page 5, Line-93-96)

Note: formula used

n=(r+1)/r (p^* (1-p^* ) 〖(Z_β+Z_(α/2))〗^2)/〖〖(p〗_1- p_2)〗^2

r = ratio of group-1 and group - 2

p*= Average of proportion

Zα/2 = Level of significance

Zβ = Power of the test

p1 - p2= Effect size

p1 = Proportion in group - 1

p2 = Proportion in group - 2

Note: we were forced to assume the proportion as we found no suitable study to make a reference regarding the proportion of cure by day 12.

Statistical Analysis, “sample size 377 ...”: The sentence regarding the required sample size had been previously mentioned. I suggest excluding it.

Response: It has been excluded

Statistical Analysis, “to compare the groups A, B, AB and O . . . ”: The groups of A, B, AB and O contradicted the previously defined Groups I and II in the Materials and Methods section. I suggest using the same definitions of blood groups consistently throughout the manuscript.

Response: To compare the variables between group I (blood group A) and group II (other blood groups), an independent sample t-test was used. (page-6, line 121-122)

Statistical Analysis, “the difference in median time-to-recovery . . . ”: For recovery, death was a competing event. Instead of stating “cox regression analysis”, I suggest stating ‘cause-specific hazards models’. No change to the models; it was just terminology. However, the use of Kaplan-Meier method and log-rank test were different. I suggest replacing them with ‘cumulative incidence functions method’ and ’Gray’s test’.

Response: I have re do the analysis according to your suggestion and rewritten the section as- The difference in the median time-to-recovery between group I and group II was determined with cumulative incidence by blood group from the Fine-Gray Model. The hazard ratios were calculated using cause-specific hazard models. Categorical variables are presented as n (%), normally distributed continuously as mean (SD), and skewed continuous variables as median (IQR). The statistical significance was set at P <0.05. (Page-6, line 124-128)

Results, “438 patients were enrolled . . . ”: Because survival analysis was used, the 52 lost-to-follow-up patients could be included in the analyses.

Response: In the revised manuscript we analyze all 438 patients

. Results, “maximum follow up days was 45”: Weren’t the patients followed up to 30 days?

Response: It was a mistake, omitted.

Table 1:

(11a) I suggest adding a column including Group II (i.e., blood groups B, AB and O) and a column of p-values comparing Groups I and II.

(11b) I suggest revising the column header “significance” as ‘p-values’.

Response: Table I is reformed as-

Table 1: Baseline characteristics of 438 COVID19 patients with different Blood Group

12. Table 2: Please clarify in the table what the “Risk ratio/Hazard ratio” column referred to. Per the interpretation in the footnote, it seemed to refer the interactions between blood groups and attributes. If so, I suggest reporting hazard ratios for attributes in Group I and Group II because hazard ratios for interactions were not interpretable directly

Response: we split table 2 to table 2 and table 3 as follows. Table 3 variable were qualitative variable, so we could compute hazard ratio in this instances.

Table 2: Duration of illness of COVID-19 patients among Group-I and Group-II

Table 3: Outcome of COVID-19 patients among Group-I and Group-II

I hope I have tried my level best to address all of your point raised during the review process. Please consider my manuscript for publication in PLOS ONE.

Thanks

Dr. Reaz Mahmud

Assistant professor Neurology

Dhaka Medical College

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

15 Mar 2021

Association of ABO blood groups with presentation and outcomes of confirmed SARS CoV-2 infection: A prospective study in the largest COVID-19 dedicated hospital in Bangladesh

PONE-D-20-36898R1

Dear Dr. Mahmud,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Prof. Raffaele Serra, M.D., Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

amended manuscript is acceptable

Reviewers' comments:

22 Mar 2021

PONE-D-20-36898R1

Association of ABO blood groups with presentation and outcomes of confirmed SARS CoV-2 infection: A prospective study in the largest COVID-19 dedicated hospital in Bangladesh

Dear Dr. Mahmud:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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