A retrospective analysis of clinical features of patients hospitalized with SARS-CoV-2 Omicron variants BA.1 and BA.2

The causative agent of the ongoing Corona virus disease 2019 (COVID-19) pandemic, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has acquired a considerable amount of mutations, leading to changes in clinical manifestations and increased transmission. Recent studies based on animal disease models and data from the general population were reporting a higher pathogenicity of the BA.2 sublineage compared to BA.1. The aim of this study was to provide real world data on patients with the SARS-CoV-2 Omicron BA.1 and BA.2 subvariants treated at our center, highlighting similarities and differences in the clinical disease course. We retrospectively collected and analyzed the data of adult patients admitted with confirmed SARS-CoV-2 infection at the Department for Infectious Diseases and Tropical Medicine, Klinik Favoriten, Vienna, Austria. Patient characteristics including age, underlying diseases, vaccination status and outcome were compared between patients with the BA.1 and BA.2 subvariants. Between January 2022 and May 2022 we included 168 patients infected with Omicron BA.1 and 100 patients with BA.2. Patients admitted with BA.2 were significantly older, more often fully immunized and required less dexamethasone than patients with BA.1. No substantial differences were identified between patients infected with BA.1 and BA.2 regarding BMI, laboratory findings, need for supplemental oxygen, mortality and other evaluated comorbidities excepting active malignancies. The significantly larger percentage of fully immunized patients admitted with BA.2 is pointing to an increased transmissibility of this subvariant, while the comparable outcome of a somewhat older and sicker patient population might be indicative of reduced virulence.


Methods
We retrospectively collected and analyzed the data of adult subsequent patients admitted with confirmed SARS-CoV-2 infection at the general ward and intensive care unit of the Department for Infectious Diseases and Tropical Medicine, Klinik Favoriten, Vienna, Austria.
We included data of all consecutive patients which were RTPCR positive for BA.1 and admitted to our department between 01.01.2022 and 28.02.2022 and all consecutive patients which were RTPCR positive for BA.2 and admitted to our department between 02.02.2022 and 04.05.2022, except patients who were not discharged or deceased until the period of data collection. Data was collected between 03.03.2022 and 14.07.2022.
The presence of SARS-CoV-2 was analyzed on a Cobas 6800 system (Roche, Mannheim, Germany) using real-time polymerase chain reaction Cobas SARS-CoV-2 Qualitative assay (Roche, Mannheim, Germany) for qualitative analyses.
The PCR assay represents a dual target test, designed for the detection of SARS-CoV-2 RNA in nasopharyngeal swab samples of infected patients. Conserved regions within the ORF 1a/b and E genes are targeted to ensure high accuracy.
Routine application of test revealed high sensitivity for samples with Ct < 30. Variant identification was based on melting characteristics specific for the mutations BA.1 and BA.2 according to the manufacturer's instructions.
This immunoassay allows in vitro quantitative determination of total antibodies to the SARS-CoV-2 S protein RBD in human serum.
The results are displayed in Roche units (U)/ml, which correlate to the WHO standard (binding antibody units (BAU)/ml). According to the manufacturer's method sheet, samples with anti-SARS-CoV-2 S concentrations above the measuring range (0.4-250 U/mL) were diluted up to 1:10. Higher results were reported as > 2500 U/mL. The overall specificity in the package insert of the manufacturer was 99.98%, sensitivity 98,8%, respectively. The data regarding patient vaccination status was obtained from the Austrian national registry for COVID-19 vaccinations. Fully vaccinated patients were considered those with completed primary vaccination scheme.
Our department applies the criteria of the WHO clinical progression scale regarding to mild, moderate and severe COVID-19 cases 36 . Mild cases are considered those which are either asymptomatic or show SARS-CoV-2 typical symptoms, but do not require hospitalisation.
Moderate disease is defined as COVID-19 associated hospitalisation with, at most, the need for low-flow oxygen supplementation.
Severe cases are defined as COVID-19 associated hospitalisation with the need of high-flow oxygen suplementation, non-invasive ventilation or mechanical ventilation. Statistical analysis. The statistical analyses were carried out in GraphPad Prism Version 9. Categorical data was tested for significance with the Fisher's exact test. Non-categorical data was first tested for normality (D' Agostino & Pearson test, Anderson-Darling test, Shapiro-Wilk test and Kolmogorov-Smirnov test). The passing of the normality test led to a t-test, while data that wasn't normally distributed was tested with the Mann-Whitney test or, when comparing antibody levels after 0, 1, 2 and 3 vaccines, the Kruskal-Wallis Test.
Ethical approval and consent to participate. Ethics approval from "Ethikkommission der Stadt Wien" was obtained, informed consent has been waived by the approving ethics committee. All methods were performed in accordance with relevant guidelines and regulations.

Results
The characteristics of our patient population are summarized in Table 1.
We Underlying diseases. Patients admitted with BA.2 were burdened by active malignancies in a larger quota.
The rest of the reviewed comorbidities did not show significant differences but there was a tendency of more underlying diseases in BA.2 patients (eg. chronic kidney disease 22% in BA.1 vs 33% in BA.2). The most prevalent underlying disease present in patients infected with BA.1 and BA.2 was arterial hypertension with 58,9% and 58%, (p = 0,8986) respectively. Table 1. Patient characteristics. Age, body mass index, Ct-value at admission, fraction of inspired oxygen (FiO 2 ) at admission, Respiratory rate at admission, Days since onset of symptoms, CRP, Leucocytes, BUN, Ferritin are shown as mean values with standard deviation. U = Roche units = binding antibody units (BAU); Ct = cycle treshold of the polymerase chain reaction test; F i O 2 = fraction of inspired oxygen; mg = milligram; L = liter; G. = Giga; dL = deciliter; μg = microgram; ICU = intensive care unit; fully vaccinated = 3 or more vaccines against SARS-CoV-2; active malignancy = diagnosed malignancy currently receiving anticancertherapy and/or under current surveillance; days since onset = days since onset of symptoms or first positive PCR-Test in case of asymptomatic patients. www.nature.com/scientificreports/ COVID-19 admission. The Ct-value, F i O 2 , respiratory rate and laboratory findings on admission were comparable in both groups-indicating no significant difference in CRP, BUN, leucocytes or ferritin.

COVID-19 disease course.
Among all patients infected with BA.1, 36% were administered dexamethasone, while only 23% BA.2 patients seemed to be needing a glucocorticoid therapy (p = 0,0397). Our Standardof-Practice (SOP) recommends dexamethasone treatment in patients with need for supplemental oxygen caused by COVID-19 disease. We consider a dexamethasone treatment to be a better indicator for a moderate/severe COVID-19 disease course than the need for oxygen supplementation, as the latter had more confounding variables.

Need for supplemental oxygen and Ct-values.
There were no significant differences in Ct-value upon admission and during the in-hospital stay (Fig. 1), FiO 2 and respiratory rate on admission, but significant difference in days between onset and hospital admission, highlighting a shorter duration for patients infected with BA.2.
The proportions of patients needing supplemental oxygen in the form of low-flow, high-flow oxygen or mechanical ventilation (Fig. 2), as well as an ICU stay were similar in both groups. The duration of hospital stay and elapsed days until Ct-values surpassed 30 were also comparable (Fig. 1). Unsurprisingly, the mortality of the two groups was also alike: 13 (8%) BA.1 and 11 (11%) BA.2 patients died during their hospital stay.
The antibody concentration did have an effect on the need for supplemental oxygen in both sublineages: lower antibody levels were associated with more need for supplemental oxygen. BA.1 patients needing supplemental oxygen had a median antibody value of 157,5 U/mL, while those who did not require it had a median antibody value of 1128 U/mL (p = 0,0123). BA.2 patients had similar differences, with 539 U/mL in the case of those who  www.nature.com/scientificreports/ required supplemental oxygen and 1992 U/mL in the case of those who did not (p = 0,0489). The number of vaccines was directly proportional with the concentration of antibodies in our patients (p < 0,0001).

Discussion
Transmissibility. At our department, BA.1 was most prevalent during the months of January 2022-late February 2022, coinciding with a spike of COVID-19 cases in the general population of Austria with a maximum of 40.419 new cases on the 10.02.2022. Immediately followed by this wave came another with even higher numbers of daily new infections, peaking at 64.038 new cases on the 17.03.2022, and coinciding with the predominance of BA.2 at our department 13,35 . This epidemiological data is consistent with the predictions and findings of other studies which mention a higher transmissibility of BA.2 in comparison to BA.1 21,29,30 . Virulence. It is crucial to distinguish between patients who were hospitalized primarily because of COVID-19 and received treatment for it, and those who were hospitalized for a different reason and transferred to our department solely for isolation purposes. This study evaluated the need for supplemental oxygen and COVID-specific treatment according to our SOP. As less than half of our patients (43% of BA.1 patients and 41% of BA.2 patients) needed supplemental oxygen (Fig. 2), it could be assumed that only a fraction of the admissions were primary COVID-19 cases. Likewise, one previous study, which was conducted in a medical center in the Netherlands between December 2021 and February 2022, found that only 45% of the adult patients admitted with the SARS-CoV-2 Omicron variant were primary COVID-19 cases 37 .
Interestingly, a significantly higher proportion of BA.1 patients received dexamethasone. This presumably indicated that a significantly higher percentage of BA.1 patients needed glucocorticoids for a COVID-19-associated hyperinflammatory syndrome as compared to patients with BA.2.
There was no significant difference between those infected with BA.1 and BA.2 with regard to the degree of oxygen supplementation. This could be indicative of a generally mild clinical manifestation considering the high age of BA.2 patients. However, our small sample size might have diluted a difference between groups, as the average FiO 2 was numerically lower in BA.2 patients. In addition, higher antibody titers had a protective character against fulminant disease with a need for oxygen therapy in both sublineages. As the number of vaccines correlated with the concentration of antibodies in our patients, this finding was consistent with previous data which points to booster vaccines positively affecting the clinical course, despite infection with newer variants which have a notable ability for immune escape and for causing breakthrough infections 38,39 .
There was no statistically significant difference in BMIs of patients admitted with BA.1 and BA.2. This notwithstanding, it is important to mention that those in need of mechanical ventilation had a significantly higher BMI (p = 0,0347), supporting existing evidence 40,41 and confirming that this remains a risk factor in the case of infections with BA.1 and BA.2 as well.
The results of our study confirm previous research which indicates a higher transmissibility of BA.2 29 , as it can escape vaccine-induced antibodies and can lead to hospitalization in fully immunized patients in a larger proportion than BA.1 (p = 0,001). Contrary to previous evidence based on infections in the general population 10 and predictions derived from animal models 31 which report a stronger symptomatology and increased disease severity of BA.2, our findings present a change in the demographics of patients hospitalized with BA.2, such as older age and a larger proportion of patients with active malignancies, but a similar outcome to BA.1 (need for oxygen, ICU stay, respiratory rate on admission, laboratory findings), suggesting a somewhat lower virulence of BA.2. The higher age at admission in the case of BA.2 may also be indicative of reduced need for hospitalization in younger patients, or of increased community spread in older patients.
This study has a number of limitations. As a retrospective single center study with a rather low sample size, there is a high susceptibility to bias. Furthermore, we included the data of only 100 BA.2 patients, which might have been too little to unveil greater differences between the two subvariants.
Another limitation is the early discharge of patients with mild disease or rapid improvement of their clinical status-these patients were administered antiviral therapy and antibodies and sometimes discharged while still infectious. As a consequence, we lack information about the necessary time to clear the virus in patients with mild clinical manifestations.
Exact FiO 2 values needed during the first 15 days of stay at our department were thoroughly documented. Nevertheless, some values are missing due to patients being discharged, transferred, moribund patients not tolerating high-flow oxygen supplementation or in case of death. This could have led to a bias regarding the cumulative FiO 2 values of the later days.
To summarize our evidence, the behavior of SARS-CoV-2 might take a natural evolutionary course. Similar to many other viruses, the mutations that provide a growth advantage and increased fitness prevail. As the newer variants of SARS-CoV-2 show higher transmissibility by escaping neutralizing antibodies, they might also cause less severe illness, hopefully leading in a more distant future to the possibility of a normalized coexistence with this pathogen.

Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.