ABSTRACT
Wastewater-based surveillance can be used as a complementary method to other SARS-CoV-2 surveillance systems. It allows the emergence and spread of infections and SARS-CoV-2 variants to be monitored in time and place. This study presents an RT-ddPCR method that targets the T19I amino acid mutation in the spike protein of the SARS-CoV-2 genomes, which is specific to the BA.2 variant (omicron). The T19I assay was evaluated both in silico and in vitro for its inclusivity, sensitivity, and specificity. Moreover, wastewater samples were used as a proof of concept to monitor and quantify the emergence of the BA.2 variant from January until May 2022 in the Brussels-Capital Region which covers a population of more than 1.2 million inhabitants. The in silico analysis showed that more than 99% of the BA.2 genomes could be characterized using the T19I assay. Subsequently, the sensitivity and specificity of the T19I assay were successfully experimentally evaluated. Thanks to our specific method design, the positive signal from the mutant probe and wild-type probe of the T19I assay was measured and the proportion of genomes with the T19I mutation, characteristic of the BA.2 mutant, compared to the entire SARS-CoV-2 population was calculated. The applicability of the proposed RT-ddPCR method was evaluated to monitor and quantify the emergence of the BA.2 variant over time. To validate this assay as a proof of concept, the measurement of the proportion of a specific circulating variant with genomes containing the T19I mutation in comparison to the total viral population was carried out in wastewater samples from wastewater treatment plants in the Brussels-Capital Region in the winter and spring of 2022. This emergence and proportional increase in BA.2 genomes correspond to what was observed in the surveillance using respiratory samples; however, the emergence was observed slightly earlier, which suggests that wastewater sampling could be an early warning system and could be an interesting alternative to extensive human testing.
ABSTRACT
Two adult female hippos in Zoo Antwerp who were naturally infected with SARS-CoV-2 showed nasal discharge for a few days. Virus was detected by immunocytochemistry and PCR in nasal swab samples and by PCR in faeces and pool water. Serology was also positive. No treatment was necessary.
ABSTRACT
Wastewater-based surveillance was conducted by the national public health authority to monitor SARS-CoV-2 circulation in the Belgian population. Over 5 million inhabitants representing 45% of the Belgian population were monitored throughout 42 wastewater treatment plants for 15 months comprising three major virus waves. During the entire period, a high correlation was observed between the daily new COVID-19 cases and the SARS-CoV-2 concentration in wastewater corrected for rain impact and covered population size. Three alerting indicators were included in the weekly epidemiological assessment: High Circulation, Fast Increase, and Increasing Trend. These indicators were computed on normalized concentrations per individual treatment plant to allow for a comparison with a reference period as well as between analyses performed by distinct laboratories. When the indicators were not corrected for rain impact, rainy events caused an underestimation of the indicators. Despite this negative impact, the indicators permitted us to effectively monitor the evolution of the fourth virus wave and were considered complementary and valuable information to conventional epidemiological indicators in the weekly wastewater reports communicated to the National Risk Assessment Group.
Subject(s)
COVID-19 , SARS-CoV-2 , Belgium/epidemiology , COVID-19/epidemiology , Humans , Public Health , RNA, Viral , WastewaterABSTRACT
The worldwide emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has highlighted the importance of rapid and reliable diagnostic testing to prevent and control the viral transmission. However, inaccurate results may occur due to false negatives (FN) caused by polymorphisms or point mutations related to the virus evolution and compromise the accuracy of the diagnostic tests. Therefore, PCR-based SARS-CoV-2 diagnostics should be evaluated and evolve together with the rapidly increasing number of new variants appearing around the world. However, even by using a large collection of samples, laboratories are not able to test a representative collection of samples that deals with the same level of diversity that is continuously evolving worldwide. In the present study, we proposed a methodology based on an in silico and in vitro analysis. First, we used all information offered by available whole-genome sequencing data for SARS-CoV-2 for the selection of the two PCR assays targeting two different regions in the genome, and to monitor the possible impact of virus evolution on the specificity of the primers and probes of the PCR assays during and after the development of the assays. Besides this first essential in silico evaluation, a minimal set of testing was proposed to generate experimental evidence on the method performance, such as specificity, sensitivity and applicability. Therefore, a duplex reverse-transcription droplet digital PCR (RT-ddPCR) method was evaluated in silico by using 154 489 whole-genome sequences of SARS-CoV-2 strains that were representative for the circulating strains around the world. The RT-ddPCR platform was selected as it presented several advantages to detect and quantify SARS-CoV-2 RNA in clinical samples and wastewater. Next, the assays were successfully experimentally evaluated for their sensitivity and specificity. A preliminary evaluation of the applicability of the developed method was performed using both clinical and wastewater samples.