key: cord-1029193-ftm2h2hr
authors: Yaniv, Karin; Ozer, Eden; Shagan, Marilou; Paitan, Yossi; Granek, Rony; Kushmaro, Ariel
title: Managing an evolving pandemic: Cryptic circulation of the Delta variant during the Omicron rise
date: 2022-04-30
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2022.155599
sha: f3c48d96670abbd255f77a1889e241bbcc2265f9
doc_id: 1029193
cord_uid: ftm2h2hr

SARS-CoV-2 continued circulation results in mutations and the emergence of various variants. Until now, whenever a new, dominant, variant appeared, it overpowered its predecessor after a short parallel period. The latest variant of concern, Omicron, is spreading swiftly around the world with record morbidity reports. Unlike the Delta variant, previously considered to be the main variant of concern in most countries, including Israel, the dynamics of the Omicron variant showed different characteristics. To enable quick assessment of the spread of this variant we developed an RT-qPCR primers-probe set for the direct detection of Omicron variant. Characterized as highly specific and sensitive, the new Omicron detection set was deployed on clinical and wastewater samples. In contrast to the expected dynamics whereupon the Delta variant diminishes as Omicron variant increases, representative results received from wastewater detection indicated a cryptic circulation of the Delta variant even with the increased levels of Omicron variant. Resulting wastewater data illustrated the very initial Delta-Omicron dynamics occurring in real time. Despite this, the future development and dynamics of the two variants side-by-side is still mainly unknown. Based on the initial results, a double susceptible-infected-recovered model was developed for the Delta and Omicron variants. According to the developed model, it can be expected that the Omicron levels will decrease until eliminated, while Delta variant will maintain its cryptic circulation. If this comes to pass, the mentioned cryptic circulation may result in the reemergence of a Delta morbidity wave or in the possible generation of a new threatening variant. In conclusion, the deployment of wastewater-based epidemiology is recommended as a convenient and representative tool for pandemic containment.

SARS-CoV-2 virus is circulating worldwide and is undergoing mutations resulting in the continuous emergence of new variants (Tao et al., 2021) . Different variants could differ in various clinical parameters, including symptom severity, infection rate and immune response (Hoffmann et al., 2021a (Hoffmann et al., , 2021b Jewell, 2021) . Therefore, when a new variant emerges, even in highly vaccinated populations, immunity might not be absolute. This may occur through vaccination deterioration over time, or through mutated targeting regions for neutralizing antibodies (generated by vaccine or previous variant recovery). One of the latest variants to materialize is the Omicron variant of concern (B.1.1.529) (Karim and Karim, 2021) . This variant demonstrates numerous mutations especially in the spike protein gene (S gene) when compared to the original SARS-CoV-2 or other variants of concern (Venkatakrishnan et al., 2021) .

Therefore, despite vaccination efforts in Israel, with a large portion of the population being vaccinated between the 1 st to 4 th dose of vaccine and despite high infection rates by previous variants, the Omicron variant had now rooted itself in Israel ("Israeli Ministry of Health Dashboard," n.d.; Leshem et al., 2022) .

One methodology for assessment of viral infection is through wastewater-based epidemiology, a convenient tool that can provide one of the most accurate representation of the extent of virus' prevalence in a population (Collivignarelli et al., 2020; La Rosa et al., 2020a , 2020b Martin et al., 2020; Westhaus et al., 2021; Yaniv et al., 2021a) . Using such a tool can offer an early warning system that can provide the impetus for developing a vast clinical J o u r n a l P r e -p r o o f Journal Pre-proof examination infrastructure. Using wastewater surveillance, it is possible to receive significant amount of information without having to depend on community cooperation and clinical testing resources. Previous reports from our group (Yaniv et al., 2021b (Yaniv et al., , 2021a , continuously monitored SARS-CoV-2 prevalence in wastewater of a single city in Israel (the fourth in size) utilizing reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Targeting variants of concern, we detected the shifts between the original SARS-CoV-2 and the Alpha variant (Yaniv et al., 2021b) , and between the Alpha and Delta variants (Yaniv et al., 2021a) . Considering clinical reports regarding the Omicron variant infection rate, it was therefore expected that as occurred in previous infections, this variant would appear in the wastewater and "overpower" its predecessor, the Delta variant.

The variant-population relations are dependent on many factors, some are circumstantial and others are specific to each variant. Infections caused by Delta or Omicron in the current morbidity wave might be prevented due to various levels of vaccination or through crossimmunization by the two variants. The cross-immunization may be symmetric, i.e., a recovered individual from a Delta infection is protected against infection by Omicron at the same level that a recovered individual from an Omicron infection is protected against Delta, or may be asymmetric (i.e., non-equal protections). In fact, recent studies (Laurie et al., 2022; Suryawanshi et al., 2022) suggest that the asymmetric case is more likely, but the extent of this asymmetry has not yet been quantified. until processed. Samples underwent a duplicate RNA extraction directly from the wastewater sample, according to Zymo Environmental Water RNA (Zymo Research R2042) manufacture protocol, including enrichment step starting from 5 mL sample. MS2 phage exist in nondetectable levels in Israel's wastewater, therefore spiking wastewater samples with high concentration of the phage allows its usage as external control. We added 10 5 copies of MS2 phage to 5 mL of each sample prior to the initial lysis step in the RNA extraction protocol. The final elution volume was 35 μL of RNase free water. RNA samples were kept at -80 o C.

Wastewater samples resulted in negative detection of a variant, underwent a concentration protocol using MCE electronegative membrane procedure as was previously described (Yaniv et al., 2021a) . However, none of the concentrated samples manifested a new signal, meaning there was a full correlation between the Zymo kit results and the concentrated results.

The Wastewater's RNA were subjected to an RT-qPCRs assays as previously described (Yaniv et al., 2021a (Yaniv et al., , 2021b . Briefly, reaction final volume is 20 µL with primers and probes concentration of 0.5 µM and 0.2 µM respectively. Each reaction mixture was added with 5 µL of RNA sample. Assay steps were preformed according to manufacture recommended protocol (One Step PrimeScript III RT-qPCR mix RR600 TAKARA, Japan) using Applied Biosystems Thermocycler (Thermo Scientific). RNA copy number per Liter of wastewater for raw sample was calculated using an equation available in the SI file (page 17). Each RT-qPCR assay run included relevant quality controls (such as: Non template control (NTC) and MS2 phage detection for wastewater RNA sample (Dreier et al., 2005) ). (Table S2 ). Negative controls were carried out in two ways. First a negative control was by a DNA gene block matching the original SARS-CoV-2 sequence relevant for the tested primersprobe set (Table S2 ). Further negative controls were performed using Wastewater samples confirmed as positive for the original variant, Alpha variant of concern or Delta variant of concern. Serial dilutions for the relevant gene block were prepared based on copy number calculations. The resulting Ct values (Y axis) were plotted against the log copy number of the DNA gene block template (X axis). While not being able to represent the cDNA synthesis step, DNA standards are acceptable and of high use (Ahmed et al., 2021 (Ahmed et al., , 2020 Bivins et al., 2021; Gerrity et al., 2021; Medema et al., 2020; Philo et al., 2021) . In accordance, copy number calculations were accounted for the use of a double stranded DNA instead of single strand RNA.

Each concentration was examined by ten repetitions and a standard deviation was calculated. In addition, linear regression was employed between the log copy number and the Ct values from the RT-qPCR results.

In order to assess the designed Omicron RT-qPCR assay's sensitivity in a wastewater matrix, we used RNA extracted from pre-determined SARS-CoV-2-negative wastewater sample.

The predetermined negative RNA sample was examined using our Omicron detection set. We added known concentration of the gene block used as positive control for calibration (0/10 0 /10 1 /10 2 ) to the negative RNA sample (1:10 v/v). Each spiking condition was measured by J o u r n a l P r e -p r o o f Journal Pre-proof eight repetitions. Ct values were plotted to correspond to the new probes limit of detection in a complex wastewater environment.

We present here a double susceptible-infected-recovered (SIR) model with cross-variant immunization and time-dependent waning immunity. We define the following time-dependent variables:

and are the fractions of actively infected populations in Delta and Omicron, respectively.

(ii) and are the effective fractions of susceptible populations to Delta and Omicron infections, respectively, henceforth "susceptibilities". These variables present an average over the diverse immunity presented in the population, although in the original SIR model they simply present the fraction of population that is neither actively infected nor recovered.

(iii) and are the fractions of recovered population from Delta and Omicron infections, respectively, in the present outbreak of infection. The contribution of recovered individuals from previous outbreaks is accounted for in the initial conditions for and .

In addition, we use the following (time-independent) parameters:

(i) and are the infection time-period of Delta and Omicron, respectively.

(ii) and are the basic reproduction numbers of Delta and Omicron, respectively.

J o u r n a l P r e -p r o o f

We note in passing that the actual (time-dependent) reproduction numbers of Delta and Omicron (respectively) are given by and , making the actual reproduction numbers much below the basic ones.

The model equations are as follows:

where and take the following expressions, accounting for both cross-variant immunization and waning immunity that is linear in time,

where and are the initial susceptibilities ( ) that were gained from past pandemic waves and vaccination (using the relation ) ), is the rate at which the relative immunity decreases, represents the relative mean protection against Delta infection that a newly recovered individual from Omicron gained, and similarly represents the relative mean protection against Omicron infection that a newly recovered individual from Delta gained. between 1.4*10 6 and 1.4*10 0 copies per µL reaction (Fig. 1b) . LOD was determined as the lowest dilution tested in which a Ct value of <40 was received for more than 95% of the measurements and was found to be 1.4*10 0 copies per µL reaction. When translated to wastewater monitoring and detection using the equation appearing in the SI file (page 17), the new set LOD is 4.7*10 2 RNA copies per L wastewater (Ct=40). Apart from being found as sensitive, the newly designed Omicron set did not have cross-reactivity with any of the tested negative controls. Thus, if original variant gene block showed positive signals in wastewater samples (to the original/Alpha/Delta variants), a signal from the Omicron set did not manifest, proving this set to be highly specific.

We further evaluated the sensitivity and the stability of the designed Omicron set in wastewater matrix. Wastewater contains many factors that may inhibit the RT-qPCR assay due to the complexity of the environment, therefore it was crucial to evaluate the designed set functionality in such an environment if it is aimed for wastewater detection. Predetermined negative for SARS-CoV-2 wastewater sample, were spiked with Omicron positive gene block at different concentrations (Fig. 1c) . As can be seen in Figure 1c , despite the wastewater's difficult environment, the sensitivity of the Omicron set was not compromised and the LOD remained as before (1.4*10 0 copies per µL reaction and 4.7*10 2 RNA copies per L wastewater). Table S3 ). In all samples, a signal manifested for one of the sets, meaning either Delta or Omicron, apart from a single sample positive for both. Such a case could be explained by double variants infection or by a new variant possessing the two sets targets, regardless this is very rare (<0.5%).

In samples are therefore high-risk targets worth of sequencing, more than random survey sequencing and can help refine sequencing efforts. It is therefore recommended to embrace an approach of RT-qPCR assays development for circulating variants and utilizing these assays on routine testing, thus discovering infections suspected as new variants that should be sequenced.

Our group continuously monitors Israel's 4 th largest city's (Beer-Sheva) wastewater since the beginning of the COVID-19 pandemic. We monitor general SARS-CoV-2 levels using N gene detection by CDC's N2 set together with our previously published set (Yaniv et al., 2021b) .

In addition, a more specific variant detection is being employed in accordance with the most relevant variants of concern distribution in the world and specifically in Israel. Utilizing such a It is important to note that N gene detection levels are higher than S gene detection levels, most likely due to transcriptional level differences during the SARS-CoV-2 "lifespan" (Emanuel et al., 2021; Kim et al., 2020) . columns) and Omicron variant of concern detected by Ins214 S set (orange columns Usually, wastewater detection precedes virus' presence in the population between a few days to several weeks prior to clinical reports (Chavarria-Miró et al., 2021; La Rosa et al., 2021; Róka et al., 2021; Wu et al., 2022) . In the case of Omicron detection in the wastewater, variant's presence preceded population's morbidity increase by approximately a week prior to clinical reports (Fig. 3a) . This was somewhat expected in the case of the Omicron and reflected the J o u r n a l P r e -p r o o f with cross-immunization after recovery and time-dependent waning immunity. Adapting possible model parameters based on initial data received from wastewater, together with information from the Ministry of Health database, and a variety of other sources (see SI), it possible to shed light on the recent parallel spread of Omicron and Delta and to predict its future trends.

(and non-infecting) stage is relatively short, about two days for the parent virus and most of its variants, and probably down to one day for Omicron, it was neglected here. The presymptomatic-infecting stage is about 3 days long for most variants (between days 3-5 from infection) (He et al., 2020; Lauer et al., 2020) , and is likely to be around 1-2 days long for Omicron (between days 3-4 from infection) (European Centre for Disease Prevention and Control, 2022), and the difference in the level of infectiousness during the symptomatic stage is obviously present. For simplicity, we used here only the SIR model for both variants, introducing the differences between them in the choice of model parameters. In addition, we do not include here the shedding processes of Delta and Omicron virions to wastewater (Proverbio et al., 2022) , which could allow better comparison with wastewater data, as this will require the use of yet unknown parameters.

We relied on an asymmetric cross-immunization, where on the average, considering the status of vaccination and past infections of the population, a person recovering from Omicron gained 4 times less protection against a Delta infection, than a person recovering from Delta gained protection against an Omicron infection (further explained in the SI). Moreover, to receive a better correlation with the steady, cryptic circulation of the Delta observed in wastewater, a linear in time waning of immunity was considered. The resulting model predicts a significant decrease in Omicron levels within the near future, diminishing until fully

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