key: cord-0799874-pauiniri
authors: Park, Sunghee; Lim, So Yun; Kim, Ji Yeun; Park, Heedo; Lim, Joon Seo; Bae, Seongman; Kim, Jeonghun; Jung, Jiwon; Kim, Min Jae; Chong, Yong Pil; Choi, Sang-Ho; Lee, Sang-Oh; Kim, Yang Soo; Park, Man-Seong; Kim, Sung-Han
title: Clinical and virological characteristics of SARS-CoV-2 B.1.617.2 (Delta) variant: a prospective cohort study
date: 2022-04-01
journal: Clin Infect Dis
DOI: 10.1093/cid/ciac239
sha: 43c4f5793d4471b1dd9733db15fd90b2356d6521
doc_id: 799874
cord_uid: pauiniri

BACKGROUND: Data on the clinical and virological characteristics of the delta variant of SARS-CoV-2 are limited. This prospective cohort study compared the characteristics of the delta variant to other variants. METHODS: Adult patients with mild COVID-19 who agreed to daily saliva sampling at a community isolation facility in South Korea between July and August 2021 were enrolled. Scores of 28 COVID-19-related symptoms were recorded daily. The genomic RNA and subgenomic RNA from saliva samples were measured by real-time reverse-transcriptase-PCR. Cell cultures were performed on saliva samples with positive genomic RNA results. RESULTS: A total of 141 patients (delta group, n = 108 [77%]; non-delta group, n = 33 [23%]) were enrolled. Myalgia was more common in the delta group than in the non-delta group (52% vs. 27%, P = .03). Total symptom scores were significantly higher in the delta group between days 3 to 10 after symptom onset. Initial genomic RNA titers were similar between the two groups; however, during the late course of disease, genomic RNA titers were higher in the delta group. Negative conversion of subgenomic RNA was slower in the delta group (median 9 vs. 5 days; P < .001). The duration of viral shedding in terms of positive viral culture was also longer in the delta group (median 5 vs. 3 days; P = .002). CONCLUSIONS: COVID-19 patients infected with the delta variant exhibited prolonged viable viral shedding with more severe symptoms than those infected with non-delta variants.

The coronavirus disease (COVID-19) outbreak due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected more than 240 million patients and caused almost 5 million deaths worldwide [1] . While the development of several vaccines resulted in a temporary decline in the number of cases, hospitalizations, and deaths due to COVID-19 [2, 3] , the emergence of variants including the SARS-CoV-2 Pango lineage B.1.617.2, also known as the delta variant, has caused a new wave of the pandemic [4, 5] .

The delta variant, first identified in India in October 2020, has since become the predominant SARS-CoV-2 variant and currently accounts for more than 99% of the cases in South Korea [6] as well as in the United States [7] . The delta variant is highly transmissible, with a transmission advantage of 66% over the alpha variant in England [8] and over 79% in certain regions of France [9] . A previous study reported that compared with other variants, the delta variant exhibits increased disease severity, higher viral loads, and a longer duration of positive PCR results [10] . However, comparative data between the delta variant with other variants in terms of symptom presentation and the duration of viable virus shedding are limited. The period between July 2021 and August 2021 in South Korea was the transitional time before the delta era when non-delta variants and the delta variant were co-circulating.

The proportion of the delta variant in South Korea was 30% in early July, 62% in early August, and 97% in late August [6] . In this study, we compared the clinical and virological characteristics of the delta variant to non-delta variants in adult patients with mild COVID-

A c c e p t e d M a n u s c r i p t

At the time of the study, confirmed asymptomatic or mildly symptomatic COVID-19 patients in South Korea were isolated at non-hospital community facilities. The patients were monitored for at least 10 days after the diagnosis, and those who needed further evaluation or medical care were transferred to nearby hospitals. Following government health policies, patients whose symptoms improved or no longer worsened after 10 days since the diagnosis were discharged from the facility. Between July 20 and August 20, 2021, patients with mild COVID-19 above the age of 18 were recruited at a designated non-hospital community facility in Seoul, South Korea. All participants provided written informed consent. This study was approved by the institutional review board of Asan Medical Center (Seoul, South Korea).

Patients were instructed to record their symptom scores twice daily (8 AM and 5 PM) in an electronic diary that was accessible to the medical staff. The severity of each of the 28 COVID-19-related symptoms was scored from 0 to 3 points as follows: i) score of 0 (no symptoms), ii) score of 1 (transient or mild discomfort, no interference with daily activities, and no requirement of medical intervention or therapy), iii) score of 2 (mild-to-moderate limitations in daily activities, and symptoms are controlled by medical intervention or therapy), and iv) score of 3 (substantial limitations in daily activities, and symptoms are not well-controlled even with medical intervention or therapy). The questionnaire sheets for the COVID-19-related symptoms are provided in the Supplementary Materials. The mean of the sum of all 28 COVID-19-related symptom scores ("total symptom scores") per participant per day was used to compare the two groups, based on the method used to evaluate the dynamics of symptoms in a previous meta-analysis of human influenza patients [11] . The A c c e p t e d M a n u s c r i p t dynamics of the mean of total symptom scores according to time was drawn for each group, and the mean total symptom scores for each day after symptom onset were compared between the delta and non-delta groups.

Self-collected saliva samples were obtained from the patients starting from the day of study enrollment until the day of discharge. Each day, 2 mL of saliva was collected into an airtight container that was provided after the patient agreed to participate in the study.

Patients were instructed to avoid food, water, and teeth brushing for at least 30 minutes prior to sample collection. Saliva samples were picked up within an hour by the medical staff and transported to a designated laboratory where they were aliquoted and stored at -80 °C until use. For the viral kinetic study, patients infected with the delta variant were randomly selected after matching age and sex with the patients infected with a non-delta variant.

The collected saliva samples were inactivated at 65℃ for 30 min in a special negative pressure laboratory. Genomic viral RNA was extracted from the specimens using a QIAamp Supplementary Table 1 .

Subgenomic RNAs of the SARS-CoV-2 N and S genes were detected by multiplex realtime RT-PCR assays as described in our previous study [12] . The shared forward primer was designed in the 5' leader sequence, and reverse primers and probes were located in the gene sequences coding the N and S proteins (Supplementary Table 2 

Categorical variables were compared using chi-squared or Fisher's exact test, and continuous variables were compared with the Mann-Whitney U test or Kruskal-Wallis test, as appropriate. Time-wise differences in the dynamics of viral shedding and symptom score between patients infected with the delta variant and those infected with a non-delta variant were compared using a generalized linear mixed model. Interactions between time and variant groups (delta vs. non-delta) were also evaluated. Viral shedding values less than the lower limit of quantification (LoQ; 2.6 log 10 copies/mL) of the RT-PCR assay but with positive qualitative results were imputed with half of the lower LoQ. Negative RNA values were imputed with 0 log 10 copies/mL. In addition, we performed survival analysis to estimate the negative conversion rate of PCR using the Kaplan-Meier plot and log-rank test.

Nonparametric maximum-likelihood estimation (NPMLE) was used to estimate the A c c e p t e d M a n u s c r i p t proportion of the viral shedding in terms of subgenomic RNA and viral culture due to interval censoring data. All tests of significance were two-tailed and P values < .05 were considered significant. Data were analyzed using SPSS Statistics for Windows, version 23.0 (IBM Corp., Armonk, NY, USA) or R version 4.0.4 (R Project for Statistical Computing, Vienna, Austria).

The study patient flow chart is shown in Figure 1 Table 1 . There were no significant differences in terms of age, gender, and the duration from symptom onset to isolation facility admission between the delta group and the non-delta group. In addition, there were no significant differences between the delta group and the non-delta group in terms of underlying diseases or the proportion of patients with obesity or smoking habits. Nine (8.3%) patients in the delta group and 7 (21.2%) patients in the non-delta group were presymptomatic (P = .06). In both groups, the most common M a n u s c r i p t symptoms were fever, cough, sore throat, myalgia, and headache, and there were no significant differences in the prevalence of each symptom between the two groups (Table 1) , with the exception of a higher proportion of myalgia in the delta group (51.5% vs. 26.9%, P = .03) and a higher proportion of loss of taste in the non-delta group (2.0% vs. 26.9%, P < .001). Abnormal infiltrations on chest imaging were seen in approximately 9% of patients in each group (9.3% vs. 9.1%, P > .99).

A total of 9 patients were transferred to a hospital facility due to symptom aggravation, among whom 8 were infected with the delta variant (7.4% vs. 3.0%, P = .69). The remaining 132 patients showed stable or reduced symptoms and were discharged from the facility. The Table 3 . In both the delta group and the non-delta group, the mean of total symptom scores peaked around 2 to 4 days after symptom onset; the peak of the mean of total symptom scores was significantly higher in the delta group compared with the non-delta group (13 vs. 8, P = .03). The mean of the total symptom scores were significantly different between the two groups on days 3 to 10 after symptom onset ( Figure 2 ).

Of the 108 patients infected with the delta variant, 33 were matched with those in the non-delta group according to age and sex (Figure 1 ). Baseline clinical characteristics of these matched patients are shown in Supplementary Table 4 . There were no significant differences in terms of age, gender, underlying diseases, and the duration from symptom onset to A c c e p t e d M a n u s c r i p t isolation facility admission between the delta group and the non-delta group. A total of 366 samples from the 66 patients were analyzed using genomic RNA assay and their viral shedding kinetics are shown in Figure 3 . The initial viral loads (log 10 copies/mL) were not significantly different between the delta group and the non-delta group; however, during the late course of disease (day 3 to day 10 after symptom onset), the genomic RNA titers were significantly higher in the delta group than in the non-delta group (Figure 3) . In all patients, viral loads decreased according to time (P for time effect < .001), and the viral loads were significantly different between the two groups over time (P for group effect = .046). No significant group-by-time interaction effect was observed (P for interaction = .41). However, the degree of declines in the viral loads from the peak values was more rapid in the non-delta group than in the delta group, as the median number of days from symptom onset to negative conversion of genomic RNA detection was 9 days (95% CI, 8-11) in the non-delta group compared with 11 days (lower limit of the 95% CI: 9 days; upper limit of the 95% CI: not available in our data) in the delta group (P by log-rank test = .02, Figure 4A ).

We further analyzed subgenomic RNA from all saliva samples to infer the duration and prevalence of viable virus shedding. As shown in Figures 3B-3D , subgenomic RNAs were detected for a considerably longer duration in the delta group than in the non-delta group. In the delta group, subgenomic RNA was positive up to 12 days after symptom onset, with the rate of subgenomic RNA positivity gradually decreasing over time ( Figure 3C and Supplementary Table 5 ). In the non-delta group, subgenomic RNA was positive up to 9 days after diagnosis and not thereafter ( Figure 3D and Supplementary Table 5 ).

In addition, we performed cell cultures by using 231 saliva samples with positive genomic RNA results. As shown in Figures 3C and 3D , viable viruses were detected for a A c c e p t e d M a n u s c r i p t considerably longer duration in the delta group than in the non-delta group. In the delta group, cell culture revealed positive results up to 11 days after symptom onset, with the rate of cell culture positivity gradually decreasing over time ( Figure 3C and Supplementary Table   6 ). In the non-delta group, cell culture exhibited positive results only until day 4 after diagnosis and not thereafter ( Figure 3D and Supplementary Table 6 ). Moreover, the negative conversion of subgenomic RNA was slower in the delta group at a median of 9 days from symptom onset (lower limit of the 95% CI: 6 days; upper limit of the 95% CI: not available in our data) compared with the non-delta group at a median of 5 days (95% CI, 1-6; P < .001; Figure 4B and 4C). The time to negative conversion of viral culture was also longer in the delta group (median, 5 days; 95% CI, 3-6) compared with the non-delta group (median, 3 days; 95% CI, 1-3; P = .002; Figure 4D and 4E).

A previous study reported that the delta variant exhibited increased disease severity and had higher and longer viral loads than other variants [10] , suggesting a higher transmission potential. However, comparative data on the kinetics of viable viral shedding between the delta variant and non-delta variants are limited. Our study clearly demonstrated that patients with the delta variant had prolonged viable viral shedding by subgenomic RNA assay and cell culture when compared with other variants. Previous studies reported that in most patients with mild-to-moderate COVID-19, viable virus was not isolated after 10 days following symptom onset [13, 14] . In contrast, in some patients with severe COVID-19, viable virus was isolated between 10 and 20 days after symptom onset [15, 16] . Our previous study on asymptomatic or mild COVID-19 patients before the emergence of the delta variant also demonstrated that the duration of viable virus shedding of symptomatic COVID-19 patients was longer than that of asymptomatic patients with SARS-CoV-2 infection, and that the timewise changes in viral shedding kinetics after symptom onset overlapped the changes in A c c e p t e d M a n u s c r i p t symptom scores [17] . In this context, our findings on the higher symptom scores and higher viral load with more prolonged viable viral shedding in the delta group than in the non-delta group are consistent with the previous studies [13] [14] [15] [16] [17] .

Currently, the CDC recommends that isolation and precautions may be discontinued This study has several limitations. First, there may be unmeasured confounding variables between the delta variant and non-delta variant groups. However, as randomized trials are neither feasible nor ethical in this setting, alternative study designs are needed to answer important policy questions. Our study has strengths in terms of the similar distribution of baseline characteristics between the two groups. In addition, we had a unique chance to compare patients who were infected with the delta strain and those who were infected with non-delta strains during the same period, which may reduce the biases that may entail comparisons between contemporary and historical cohorts. Second, Lechien et al. developed the COVID-19 Symptom Index (CSI) [18] , and demonstrated the internal consistency of the CSI by assessing the test-retest reliability and the external validity by a correlation analysis between the CSI and other scoring systems such as SNOT-22. The weakness of our study is the lack of internal and external validation of the symptom scoring system used in our study.

However, our previous study conducted in the same isolation facility between January 10 and M a n u s c r i p t A c c e p t e d M a n u s c r i p t group. Summary curves for the delta group (n=108) and non-delta group (n=33) were compared using mean ± standard error of the mean. *P < .05; **P < .01. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 

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A c c e p t e d M a n u s c r i p t February 22, 2021, when the non-delta variant was dominant in South Korea [17] showed similar symptom scores compared to those in patients with the non-delta variant between July 20 and August 20, 2021 in this study (Supplementary Figure 2) . Finally, during the study period, only a limited number of fully vaccinated patients with breakthrough delta variant infection were included. Previous studies revealed that vaccination reduced the risk of onward transmission by accelerating viral clearance [19, 20] . Therefore, our findings may not be generalized in the setting of a highly vaccinated population.In conclusion, COVID-19 patients infected with the delta variant had more systemic symptoms with more prolonged symptom duration than those infected with non-delta variants. In addition, patients with the delta variant exhibited higher viral loads and more prolonged viable viral shedding than those with non-delta variants. 

There are no conflicts of interest for any of the authors.