Dear Editor
In this journal, our previous study commented on the high amounts of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in aerosols exhaled by patients with Omicron variant infection and the increased transmissibility of Omicron variants1. Previous study by Salvagno GL, et al. announced that aerosols released by patients infected with SARS-CoV-2 Omicron variant may contain higher viral particles than those released by subjects infected with previous SARS-CoV-2 strains, only based on the comparison of the viral load in nasopharyngeal specimens2. However, no direct evidence showed that the viral load in nasopharyngeal specimens and exhaled viral aerosols had the same trends. In this study, we not only gave the data support for the relationship between viral load in nasopharyngeal specimens and viral aerosols exhaled from corona virus disease 2019 (COVID-19) patients, but also further explored the variety of virus release from patients with Omicron BA.1 or BA.2 subvariant infection.
Since 26 November 2021, when the SARS-CoV-2 variant B.1.1.529 was classified by the World Health Organization (WHO) as one of the variants of concern (VOC) and named Omicron, the Omicron subvariant has been spreading around the world3. According to WHO's weekly report on the epidemiology of COVID-19 on 22 March, 99.8% of gene sequences submitted to the Global Initiative of Sharing All Influenza Data (GISAID) in the past 30 days are Omicron, and 85.96% of them are BA.2 subvariants. An updated analysis published on 11 March 2022 by the United Kingdom4, which used data on samples collected between 01 December 2021 and 01 March 2022, confirmed that BA.2 had a higher growth rate compared to BA.1 and higher secondary attack rates for household and non-household contacts. The Omicron BA.2 subvariant is known as "invisible Omicron" because it no longer has the detection characteristics of the Omicron variant due to its mutation at the S protein gene, which may be one of the reasons for its higher spread advantage. Recent studies observed that the Omicron BA.2 subvariant of SARS-CoV-2 has similar infectivity and pathogenicity as the Omicron BA.1 subvariant in rodent models5. However, the degree of difference in airborne transmissibility between Omicron BA.2 and BA.1 subvariants remains unclear.
In this study, we monitored the aerosol emission characteristics of patients infected with Omicron BA.2 or BA.1 subvariant and virus growth rates in human cells, and we also analyzed the relationship between viral load in upper respiratory tract and the amount of exhaled viral aerosols. Those explorations may provide the evidence for the higher transmissibility of Omicron BA.2 subvariant.
51 patients diagnosed with Omicron BA.2 infection and 34 patients diagnosed with Omicron BA.1 infection were recruited in this study (Supplementary Table 1). The viral RNA tests of throat swab after admission were performed by using a SARS-CoV-2 test kit (Liferiver, Shanghai ZJ Bio-Tech Co., Ltd, Shanghai, China). 34 patients infected with Omicron BA.1 subvariant were selected from the group of Omicron patients in our previous study and the previous data of throat swab tests after admission were also discussed in this study1. Exhaled breath condensate (EBC) samples were collected for 5 min from all the patients by using a BioScreen device (Dingblue Technology Co., LTD, Beijing, China) and 800 µL EBC samples was collected form each patient. All collected EBC samples were examined for SARS-CoV-2 using the same SARS-CoV-2 test kit, targeting both the ORF1ab and N genes. The viral load in all EBC samples were confirmed by using standard curve based on SARS-CoV-2 RNA reference material containing ORF1ab, N and E genes (provided by China National Institute of Metrology and calculation details are provided in Supplementary Information). The virus breath emission rate (BER) and virus concentration in exhaled air of each patient were calculated by the equation that had been published in previous studies1 , 6. Growth rates of seven SARS-CoV-2 strains including BA.1.1, BA.1.17, BA.2.2, BA.2.3, BA.2.10, Delta and WT in Caco2 cells were obtained (experiment details are provided in supplementary information).
As shown in Fig. 1 A, methods involved in this study are throat swabs sampling, exhaled breath condensate (EBC) collection and SARS-CoV-2 RNA detection. The total positive rate of EBC samples from Omicron BA.1 subvariant patients were 17.65% (Fig. 1 B). The total positive rate of EBC samples from Omicron BA.2 subvariant patients were 29.41% (Fig. 1 B). The throat swab COVID-19 tests targeting the ORF1ab and N genes after admission of all 85 patients were positive, and significant differences of Ct value were found among the BA.1 and BA.2 groups (for N gene, *P = 0.027; for ORF1ab gene, **P = 0.009) (Fig. 1 C).
Fig. 1.
Aerosol emission characteristics of SARS-CoV-2 Omicron BA.1 and BA.2 subvariants, virus growth rates in Caco2 cells, and the relationship between viral load in upper respiratory tract and breath emission rate (BER) of COVID-19 patients. (A) Methods involved in this study, including throat swabs sampling, exhaled breath condensate (EBC) collection and SARS-CoV-2 RNA detection; (B) The proportion of single and double positive EBCs. These data were obtained from 34 patients infected with Omicron BA.1 and 51 patients infected with Omicron BA.2. Single positive means that one of the ORF1ab, N and E genes tests positive and double positive means that any two of the three genes test positive. Light red represents single positive EBCs, dark red represents double positive EBCs and light blue represents negative EBCs; (C) Ct values of throat swabs at admission. The data in the figure are RNA test data of throat swabs from the 85 patients at admission. Blue and red represent ORF1ab gene and N gene of SARS-CoV-2, respectively. Statistical significance between two groups was calculated by unpaired Student's t-test. Values are expressed as the mean ± standard error of the mean (SEM). *P < 0.05, **P <0.01; (D) BER of patients with Omicron BA.1 or BA.2 subvariant infection in early stage (<7days). Orange dots represent each data, the horizontal lines represent maximum value, 25% percentile, 50% percentile (median), 75% percentile and minimum value (from bottom to top), respectively; (E) Growth rates of different SARS-CoV-2 strains in Caco2 cells including BA.1.1, BA.1.17, BA.2.2, BA.2.3, BA.2.10, Delta and WT. The broken lines in different colors represent different SARS-CoV-2 strains; (F) Variation trends of BER and Ct value of throat swabs. Data were obtained from absolute quantitative data of N gene sourced from positive patients. The patients with positive EBCs were divided into two groups, including group high and group low, according to their Ct value of throat swabs. Yellow represents the average Ct value of throat swabs and the blue represents average BER.
The average BER of BA.1 (1.60 × 105 copies/hour) and BA.2 (5.03 × 105 copies/hour) subvariant patients were similar in early stage (<7days) ( Fig. 1 D). The overall BER from Omicron BA.1 subvariant patients was from 1.48 × 104 to 4.04 × 105 copies/hour (Supplementary Table 1). The overall BER from Omicron BA.2 subvariant patients was from 2.06 × 104 to 2.93 × 106 copies/hour (Supplementary Table 1). In order to further explain the similar virus emission characteristics of BA.1 and BA.2 patients, growth rates of WT, Delta, BA.1 and BA.2 in Caco2 cells were measured, and we found that BA.1 and BA.2 had similar growth rate at each time point (Fig. 1 E).
Furthermore, we explored the relationship between the viral loads in upper respiratory tract and BER. As shown in Fig. 1 F, patients were divided into high group and low group based on upper respiratory viral loads. We found that the viral load in upper respiratory tract had the similar variation trend with the BER, which was identified by both the BA.1 and BA.2 patients (Fig. 1 F).
In this study, we compared the SARS-CoV-2 aerosol emission from patients with Omicron BA.1 or BA.2 subvariant infection. The results showed that BA.2 subvariant patients had higher viral load in upper respiratory and EBC positive rate, compared with BA.1 subvariant patients. However, in previous study, it was found that BA.2 is ∼1.4 times more transmissible than BA.17, which cannot be well explained by their similar SARS-CoV-2 aerosol emission characteristics and same growth rates in human cells. Although the upper limit of exhaled virus concentrations from BA.2 patients was higher than that from BA.1 patients and might add extra contribution of super-spreaders to the epidemic, other explorations were still urgent to find multiple factors affecting the wide spread of Omicron subvariants, such as the environmental tolerance, immune escape ability, diversified modes of transmission, et al. Finally, we found that the viral load in upper respiratory tract and the amount of exhaled viral aerosols had the same variation trends, which may provide evidence for the increasing transmissibility of different SARS-CoV-2 variants when the viral loads in upper respiratory tract also increased.
Ethics statement
Sample collection and all experiments in the present study were performed with Ethical Approval given by Ethics Committee of the Center for Disease Control and Prevention of Xiamen.
Funding
This work was financially supported by the Beijing Science and Technology New Star Program (Z211100002121064) and Fujian Province Health Science and Technology Project (2020CXB050).
Declaration of Competing Interest
The authors declare no competing interests.
Acknowledgement
The authors thank the patients who took part in this study.
Footnotes
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jinf.2022.05.035.
Appendix. Supplementary materials
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