Infections caused by the Delta variant (B.1.617.2) of SARS-CoV-2 are associated with increased viral loads compared to infections with the Alpha variant (B.1.1.7) or non-Variants of Concern

Christian von Wintersdorff Maastricht UMC+ Jozef Dingemans (  jozef.dingemans@mumc.nl ) Maastricht UMC+ https://orcid.org/0000-0001-8079-3087 Lieke van Alphen Department of Medical Microbiology, Care and Public Health Research Institute (CAPHRI), Maastricht University Medical Center+, Maastricht, The Netherlands. Petra Wolffs Maastricht University Medical Center Brian van der Veer Maastricht UMC+ Christian Hoebe South Limburg Public Health Service Paul Savelkoul Maastricht University Medical Centre


Introduction
During the ongoing COVID-19 pandemic, various variants of SARS-CoV-2 have emerged. Some of these variants have been labelled as variants of interest (VOI) or variants of concern (VOC) by the WHO due to their potentially increased transmissibility, disease severity or immune escape characteristics 1  has been much concern about the B.1.617.2 or Delta variant, which appears to be even more highly transmissible than the Alpha variant 3, 4, 5 and is also linked to possible immune escape 6,7,8 . In many countries, the Delta variant has either already replaced the Alpha variant as the dominant variant or is rapidly gaining ground and has causes major resurgences in reported cases.
As the Delta variant established itself as the new dominant SARS-CoV-2 variant in The Netherlands, a seemingly increase in detected viral loads was noticed during routine diagnostic testing at our department. Increased viral loads in infected persons could potentially contribute to the increased transmissibility of the Delta variant. In this study, we investigated the link between viral loads of the Delta and other SARS-CoV-2 variant in infected persons.

Viral load comparison based on variant-dominant time periods
In the rst comparison, three distinct time periods were determined in which certain SARS-CoV-2 variants were dominant in our testing population in South-Limburg, the Netherlands. These periods were labelled as the non-VOC period (December 2020 -February 2021), Alpha period (March 2021 -June 2021) and Delta period (July 2021). The prevalence of SARS-CoV-2 variants in these time periods was based on next-generation sequencing data of our genomic surveillance program (Figure 1), which is part of the Dutch national SARS-CoV-2 surveillance program. Figure 2A-B shows that the viral load was signi cantly higher in the time period in which infections were predominantly caused by the Delta variant (median CT 18 or 6.55 log 10 c/mL, Table 1), when compared to the non-VOC or Alpha periods (median CT 20 or 5.98 log 10 c/mL).

Viral load comparison of sequence-con rmed variants
Since the tested SARS-CoV-2 variants in the de ned time periods for non-VOC, Alpha and Delta are based on assumption and will also include a small portion of different variants, we aimed to con rm these ndings by comparing viral loads of only sequence-con rmed variants. To this end, viral loads of a subset of WGS-con rmed non-VOC (n=631), Alpha (n=1240) and Delta (n=87) variants were compared (table 1, gure 2C-D). These results con rmed that there was an overall higher viral load in the Delta variant group (median CT 16 or 7.11 log 10 c/mL, Table 1) when compared to the non-VOC (median CT 19 or 6.26 log 10 c/mL, Table 1) or Alpha variant (median CT 18 or 6.55 log 10 c/mL, Table 1). Additionally, the viral load of the Alpha variant group was increased compared to the non-VOC group.

Discussion
Recent studies have indicated that the delta variant is associated with increased viral loads, implying enhanced infectivity of this variant 3, 4, 5 . Nevertheless, the limited size of datasets used in these studies as well as the different diagnostic methods could lead to bias and preliminary conclusions with regards to the viral load and infectivity that is associated with the delta variant. In this study we have compared the CT-values and viral load obtained from samples that were tested positive during the time periods during which the non-VOC, alpha and delta variants were dominant, as evidenced by genomic surveillance of SARS-CoV-2 in the South Limburg region of The Netherlands. It was found that samples that were tested positive since the delta variant became dominant contained signi cantly higher loads of SARS-CoV-2 compared to samples that were tested during which the non-VOC or alpha variant were dominant. Based on the standard curve used for conversion of CT values to viral loads ( Figure S1A), it is estimated that samples harboring the delta variant contain about 4-fold higher loads of SARS-CoV-2 compared to samples harboring non-VOC or the alpha variant. These observations were con rmed using a subset of WGS-con rmed samples in which samples containing the delta variant harbored signi cantly higher loads of SARS-CoV-2 compared to their non-VOC (about 7-fold) or alpha variant (about 4-fold) counterparts. It has to be noted that the delta variant became dominant at the moment that > 60% of the population received at least one dose and > 35% of the total population was fully vaccinated in The Netherlands, leaving open the possibility that the observed differences in viral load might even be more pronounced in non-vaccinated individuals. Interestingly, when using WGS-con rmed data, which consists D614G mutation that enhances infectivity 9, 10, 11 and were much closer related to the original Wuhan strain than the non-VOC isolates in our study. One of the strengths of this study is the large dataset of samples of which the great majority was tested using the same or a highly comparable work ow ( Figure   S1). Furthermore, follow-up samples obtained from the same subject were ltered out of the dataset as much as possible, to avoid bias. Finally, extensive genomic surveillance in the region allowed us to accurately estimate the dominant period for each variant as WGS of a subset of samples con rmed the observed trends. Limitations of this study can be found in the fact that the available dataset of the delta variant is still limited in size compared to the other datasets and the fact that the non-VOC group consists of many different pangolin lineages.
In conclusion, our study shows that samples from individuals that are infected with the delta variant harbor about 4-fold higher loads of SARS-CoV-2 compared to individuals that are infected with non-VOC or the Alpha variant, which is signi cantly lower than previously reported 4 .

Clinical samples
In this study, nasopharyngeal samples that were tested positive for SARS-CoV-2 were included. Samples (n = 16,185) were collected from December 2020 until July 2021 by the South Limburg Public Health Service (n = 13,927), nursing homes (n = 1,932), general practitioners (n = 226) and commercial parties (n = 100). Samples from patients and personnel were excluded as they were regularly tested and could introduce bias in the dataset. All nasopharyngeal swabs were immediately deposited in 3 mL GLY viral transport medium (Mediaproducts BV) after collection and stored under the same conditions prior to testing.

Declarations
Ethical Statement The Medical Review Ethics Committee of the Maastricht UMC+ con rmed that the Medical Research Involving Human Subjects Act (WMO) does not apply to the above mentioned study and that an o cial approval of this study by the committee is not required (METC reference number 2021-2838).