Abstract
The emergence of new SARS-CoV-2 variants poses challenges to global surveillance efforts, necessitating swift actions in their detection, evaluation, and management. Among the most recent variants, Omicron BA.2.86 and its sub-lineages have gained attention due to their potential immune evasion properties. This study describes the development of a digital PCR assay for the rapid detection of BA.2.86 and its descendant lineages, in wastewater samples. By using this assay, we analyzed wastewater samples collected in Italy from September 2023 to January 2024. Our analysis revealed the presence of BA.2.86 lineages already in October 2023 with a minimal detection rate of 2% which then rapidly increased, becoming dominant by January 2024, accounting for a prevalence of 62%. The findings emphasize the significance of wastewater-based surveillance in tracking emerging variants and underscore the efficacy of targeted digital PCR assays for environmental monitoring.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
The emergence of new variants of SARS-CoV-2 remains a dynamic and ongoing phenomenon. It is important to keep abreast of new variants that may arise, through the development or targeted detection assays. Following the declaration of the end of the pandemic, surveillance efforts are limited due to a decline in clinical testing and sequencing (Attar Cohen et al., 2023). This presents a significant challenge in tracking the insurgence of new variants. By the end of 2023, the SARS-CoV-2 XBB lineages, mainly EG.5.1, predominated globally. However, in July 2023, a lineage very different from XBB emerged (World Health Organization (WHO), 2023), named BA.2.86. First detected in Denmark and Israel, it subsequently appeared in multiple regions worldwide. In Italy, BA.2.86 first appeared in September 2023 (Caccuri er al., 2023). At the time of writing, BA.2.86 consists of several different sub-lineages, namely BA.2.86.1 to BA.2.86.4, JQ.1, and JN.1- to JN.10 and is referred as BA.2.86* (BA.2.86X) lineage. By January 19, 2024, BA.2.86* lineage had become the dominant lineage in EU/EEA countries (European Centre for Disease Prevention and Control (ECDC), 2024a; European Centre for Disease Prevention and Control (ECDC), 2024b). Due to its high number of spike protein mutations, Omicron BA.2.86 was designated as a Variant Under Monitoring (VUM) on 17 August 2023, predicted to potentially evade vaccine-induced immunity against SARS-CoV-2 infection. Subsequently, in November 2023, the BA.2.86 variant was classified as a Variant Of Interest (VOI) (World Health Organization (WHO), 2023). A significant proportion of BA.2.86* sequences are attributed to the JN.1 sub-lineage, first detected in August 2023, which contains mutations of BA.2.86 + S:L455S. On December 19, 2023, the World Health Organization classified JN.1 as a separate VOI from the parent BA.2.86 lineage due to its rapidly increasing proportion (World Health Organization (WHO), 2023). As on 15 March 2024 JN.1, was the most reported variant of interest globally, accounting for 90.3% of sequences in week 9, while in the same week its parent lineage BA.2.86 was declining and accounted for 2.2% of sequences (World Health Organization (WHO), 2024). According to the Global Initiative on Sharing All Influenza Data (GISAID), 111,325 sequences with the BA.2.86* lineage have been detected in at least 110 countries and 54 US states since the lineage was identified (GISAID—Lineage Comparison). Of these, 93,523 (84%) were associated with JN.1.
The use of wastewater monitoring can provide a comprehensive approach to tracking the circulation and spread of different SARS-CoV-2 variants within a community or population (Bartel et al., 2024; Combe et al., 2024; Cutrupi et al., 2022; Espinosa-Gongora et al., 2023; Lipponen et al., 2024). This method provides a broader perspective beyond individual testing, offering insights into the overall viral landscape and aiding in the early detection of emerging variants (Sapoval et al., 2023).
Beginning in August 2023 and in the following months, BA.2.86* was detected in wastewater samples from several countries, including Sweden, France, Denmark, Germany and Thailand (Espinosa-Gongora et al., 2023; Wurtzer et al., 2024; Bartel et al., 2024; Rasmussen et al., 2023; Wannigama et al., 2023).
The aim of this study was to investigate the occurrence of BA.2.86* in wastewater throughout Italy in the period spanning from October 2023 to January 2024. Following the recommendation of the European Commission (Commission Recommendation (EU) 2021/472 of 17 March 2021 [https://eur-lex.europa.eu/eli/reco/2021/472]), regular environmental surveillance activities for SARS-CoV-2 variants have been carried out at the national level in the period October 2021–March 2023, through monthly monitoring (so called “flash surveys”). The reports are published regularly on the Istituto Superiore di Sanità (ISS) website dedicated to environmental monitoring of COVID-19 (https://www.iss.it/cov19-acque-reflue). Although the official surveillance programme ended in March 2023 due to the cessation of funding, voluntary testing continued. However, some regions reduced the frequency or number of sampling sites. In addition, due to cost considerations, there has been a shift from next-generation sequencing (NGS) of a long fragment of the spike protein to Sanger sequencing, which has limitations in identifying minority variants present in a sample with a mixture of variants. To address this issue, a digital RT-PCR assay (RT-dPCR) was developed to specifically detect BA.2.86*. By using this assay, we investigated when and where the BA.2.86* lineage was introduced in Italy and how it spread over time.
Material and Methods
Wastewater Sampling
A total of 507 samples collected between September 2023 and January 2024 were analysed in this study. The samples were part of the wastewater sample collection of the Italian SARI network for wastewater surveillance, implemented following to the Commission Recommendation 2021/472 and Legislative Decree (Decree of the Ministry of Health 30.10.2021, GU Serie Generale n.294 del 11-12-2021 [https://www.gazzettaufficiale.it/eli/gu/2021/12/11/294/sg/pdf]). Samples were collected as a 24-h composite and stored at 4 °C until analysis, which was performed within 48 h of collection. The list of samples is provided in Table S1. In particular, 111 samples were collected in September 2023 (in the week starting September, 4th), 110 samples in October 2023 (in the week starting October, 2nd), 97 in November 2023 (in the week starting November, 6th), 98 in December 2023 (in the week starting December, 1st) and 91 in January 2024 (in the week starting January, 8th). In total, 16 Regions and 2 Autonomous Provinces (A.P.) were involved in the study, with a total of 107 wastewater treatment plants monitored.
Viral Concentration, Nucleic Acids Extraction and SARS-CoV-2 Quantification by RT-qPCR
The SARI network laboratories managed the sampling process, viral concentration, and nucleic acid extraction using a standardized national protocol as previously described (La Rosa et al., 2021, 2022). Briefly, after heat inactivation at 56 °C for 30 min, 45 mL of the samples were concentrated using a polyethylene glycol (PEG)-based method (Wu et al., 2020). Centrifugation at 4500 × g for 30 min removed larger particles and debris. Subsequently the supernatant (40 mL) was mixed with 8% polyethylene glycol 8000 and 0.3 M NaCl. The mixture was then centrifuged at 12,000 × g for 2 h. The resulting pellet was resuspended in 2 mL of NucliSENS Lysis Buffer reagent for RNA extraction using magnetic silica beads. The eluted RNA (100 μL) was further purified with the OneStep PCR Inhibitor Removal Kit and stored at − 80 °C until molecular analysis. Purified RNAs were sent to the ISS for variant detection as part of the regular flash surveys undertaken since October 2021. The RT-qPCR assay for SARS-CoV-2 was also performed by each laboratory of the SARI network according to previously published protocols (La Rosa et al., 2021, 2022). Quality assurance controls (process control virus, inhibition control) were included to assess viral recovery and PCR inhibition, as previously described (La Rosa et al., 2021). After real-time PCR, the nucleic acids were stored at − 80 °C for further testing and shipped to ISS on dry ice for flash surveys on variants.
Assay for BA.2.86 Lineage Quantification by Digital RT-PCR
A specific assay was developed using Primer3Plus software (https://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) to selectively quantify the RNA levels of the BA.2.86 lineage of SARS-CoV-2. The assay targets the spike region, spanning from amino acid residue V445H to Y505H, with the probe binding to the region containing the characteristic Val deletion 483 (V483del), together with mutations N481K and E484K, which are specific to the BA.2.86 lineage. Primers and probes used in the present study are shown in Table 1.
To validate the BA.2.86 lineage assay for digital RT-PCR, the limit of detection (LOD) and the limit of quantification (LOQ)were calculated. To determine these parameters, four tenfold dilutions of a BA.2.86 control were tested in three independent runs as triplicates under the same conditions using RT-dPCR (Doğantürk et al., 2023). To evaluate the performance of the assay in wastewater samples, the BA.2.86 control was diluted in nucleic acids extracted from a wastewater sample collected in 2021, long before the circulation of the BA.2.86 variant. LOD95% was calculated according to Wilrich and Wilrich (2009), using the tools available at https://www.wiwiss.fu-berlin.de/fachbereich/vwl/iso/ehemalige/wilrich/index.html. LOQ was calculated as the lowest standard concentration that could be quantified with a CV value below 35% (Klymus et al., 2020).
Digital RT-PCR (RT-dPCR) analysis was conducted using the QIAcuity One 5-plex dPCR system (Qiagen, Hilden, Germany), along with the QIAcuity OneStep Advanced Probe Kit (Qiagen, Hilden, Germany). Each reaction consisted of 12 μL of reaction mixture per well containing 3 μL of Qiacuity 4 × OneStep Advanced Probe Master Mix, 0.12 μL of 100 × OneStep Advanced RT Mix (Reverse Transcription), 0.4 μM of primer forward (ID 2532, CAAGCTTGATTCTAAGCATAGTGG), 0.4 μM of primer reverse (ID 2534, CACCATAAGTGGGTCGGAAA), 0.2 μM of probe (ID 2533, HEX-CCGGTAACAAACCTTGTAAAGGTAA-BHQ-1), and 2.48 of RNase-Free water. Finally, 4 μL of extracted RNA was added as template. For each sample, two technical replicates were performed. Nucleic acids were amplified under the following conditions: reverse transcription for 40 min at 50 °C, enzyme activation for 2 min at 95 °C and 40 cycles of 5 s at 95 °C and 30 s at 60 °C. Partitions were imaged with 700 ms (HEX) exposure time, with gain set to 8. The QIAcuity Software Suite version 2.2.0.26 was used to determine sample thresholds using positive and no-template control wells (NTCs) with the manual global threshold approach. Samples were considered positive if they exhibited at least three positive partitions in one of the two replicates. The target concentration (copies/μl) in each sample was calculated using the instrument result and the formula:
Omicron BA.2.86* (g.c./L) = [dPCR result (copies/μl) × reaction volume/volume of tested RNA] × 100 × 25.
Where 100 is the total volume of extracted RNA and 25 is the ratio between the processed wastewater volume (40 mL) and the reference volume (1 L). Moreover, BA.2.86* viral loads were normalized for both the flow rate and the population equivalents of WWTP (genome copies/day*inhabitant).
Before testing environmental samples, the assay was tested on a clinical sample characterized as BA.2.86, used as a positive control, and, to asses specificity, on three clinical samples of different Omicron variants, namely XBB.1.5, XBB.1.16, and EG.5 (kindly provided by the Department of Infectious Diseases at the Istituto Superiore di Sanità in Rome). In parallel, the same controls were tested with the generic assay for SARS-CoV-2 (La Rosa et al., 2021), adapted for dPCR in this study.
A subset of wastewater samples (n = 28, including 5 samples from December 2023 and 23 from January 2024) was also amplified by conventional RT-PCR using the newly developed BA.2.86 primers and were subjected to Sanger sequencing to further confirm assay specificity and discriminate the JN.1 from the parental BA.2.86 variant. JN.1 differs from the parental variant for its L455S mutation, which falls within the fragment amplified by the newly designed assay.
Results
The generic SARS-CoV-2 test produced positive results on all clinical samples used as controls. Conversely, the specific test designed for BA.2.86 provided amplification on the BA.2.86 control, but did not detect the variants XBB.1.5, XBB.1.16, and EG.5, demonstrating its specificity for the BA.2.86 lineage. LOD95% calculated according to Wilrich and Wilrich (2009) was 1.82 copies/μL. The coefficient of determination (R2) value was calculated to be 0.9944, correlating with the expected concentrations. Therefore the lowest standard concentration that could be quantified with a CV value below 35% was dilution 3, corresponding to a LOQ of 9.16 copies/μL.
The generic assay detected SARS-CoV-2 in 447 out of 507 (88.2%) wastewater samples. The viral loads ranged from 2.5 × 102 to 8.4 × 106 (median value: 1.6 × 104) genome copies (g.c.)/L of wastewater (Supplementary Table 1). The concentration and extraction procedure had an average recovery of 25% (range 0.5–100%) as evaluated by seeding the samples with mengovirus or murine norovirus. The samples’ inhibition, assessed by RT-qPCR as ΔCq from a non-inhibited reference reaction, ranged from zero to 5.1 (median value: 0.17).
Out of 507 analysed samples, 116 (23%) tested positive for the BA.2.86 lineage, with viral concentrations ranging from 4.0 × 103 to 1.1 × 105 genome copies/L (g.c./L), as reported in Table 2.
The Omicron BA.2.86* was first detected in wastewater in Italy on 2 October 2023, in two samples collected from Pomezia (Lazio) and Trento (A.P. Trento). Figure 1 displays the number of positive and negative samples, as well as the increase in detection rate for the BA.2.86 lineage over time. During the analysed months, the variant rapidly spread: 2% and two Regions/A. P. in the week of 2–6 October 2023; 6% and 5 Regions/A. P. in the week of 6–10 November 2023; 53% and 14 Regions/A. P. in the week of 1–7 December 2023; and 62% and 13 Regions/A. P. in the week of 8–12 January 2024. The BA.2.86 variant was not detectable in any of the September samples analyses.The median BA.2.86 viral loads, normalized for flow rate and equivalent inhabitants, were slightly higher in December and January, as shown in the box plot in Fig. 2. Supplementary Table 1 summarises the BA.2.86* variant concentration in each sample.
Box plot illustrating the distribution of BA.2.86 lineage normalized concentrations in positive samples across months. Dots indicate individual measurements; the lines of the box represent the 25th, 50th and 75th percentile (bottom, middle, and top lines, respectively); whiskers show the range from minimum to maximum values, not including outliers
The occurrence of the Omicron BA.2.86* has progressively increased in most regions over the four months under monitoring, with some exceptions in regions with low sample numbers. Figure 3 provides a detailed geographic distribution and spread of the variant, including the increase in the number of regions where it was detected each month, and the detection rate in each region/A. P. over time.
Geographical distribution of BA.2.86 lineage prevalence over four months. The detection of the Omicron BA.2.86* is represented with increasing shades of blue based on its prevalence. Grey Regions/A.P. tested negative for the BA.2.86* variant. Regions/A.P. without any tested sample in a given month are shown in stripes
All 28 subset samples were successfully sequenced and characterised as a JN.1 variant, identified by the key mutation L455S.
Discussion
Numerous studies worldwide have demonstrated the feasibility of monitoring SARS-CoV-2 variants in wastewater (La Rosa et al., 2023; Rector et al., 2023) and more in general, its utility to monitor evolution and spread of endemic viruses (Yousif et al., 2023). This highlights the crucial role of wastewater surveillance as an alternative strategy for detecting viral variants circulating within communities (Bonanno Ferraro et al., 2022; Cutrupi et al., 2022; Heijnen et al., 2021; Hokajärvi et al., 2021; Islam et al., 2023; Karthikeyan, et al., 2022; La Rosa et al., 2022; Li et al., 2022; Reynolds et al., 2022; Róka et al., 2022; Vo et al., 2022; Wurtzer et al., 2022). Two approaches are commonly employed for variant detection: PCR-based methods such as RT-qPCR and RT-dPCR, and next-generation sequencing (NGS) followed by bioinformatics analysis. NGS provides high-throughput sequencing that can detect rare viral variants and provide detailed genomic characterization. However, implementing NGS requires significant expertise, infrastructure, and investment due to its high costs and complex bioinformatics analysis. On the other hand, the use of mutation RT-dPCR assays on SARS-CoV-2 RNA from wastewater can quickly, effectively and reliably monitor variants that are introduced and spread within a community (Tiwari et al., 2023). Due to these attributes, RT-dPCR was selected to track the BA.2.86 lineage that has surfaced in wastewater in recent months (Vigil et al., 2024).
In Italy, the BA.2.86 variant was initially reported in September 2023, appearing in two regions (Lombardia and Veneto), and accounting for 0.3% of the circulating variants according to clinical surveillance data (https://www.epicentro.iss.it/coronavirus/pdf/sars-cov-2-monitoraggio-varianti-indagini-rapide-settembre-2023.pdf). Subsequently, its prevalence escalated rapidly, reaching 1.3% in October, 11% in November, 51% in December and ultimately 85% in January (https://www.epicentro.iss.it/coronavirus/sars-cov-2-monitoraggio-varianti-indagini-rapide). The BA.2.86* showed a parallel evolutionary trend in wastewater samples. Its prevalence was very low in October, slightly increased in November, and had a significant surge in circulation in December and January. In particular, it is noteworthy that the sub-lineage JN.1 appeared in December 2023 and quickly replaced the parental BA.2.86 variant. In December, JN.1 had indeed a prevalence of 38.1% compared to 13.2% of BA.2.86. In January 2024, this trend intensified, with JN.1 dominating over BA.2.86 with 77.0% compared to only 6.1%.
During the four-month analysis, ISS conducted regular 'flash surveys' to sequence the long fragment (approximately 1400 bps) of the SARS-CoV-2 spike protein in wastewater samples using a previously established assay available on the ISS website (https://www.iss.it/cov19-acque-reflue). Sanger sequencing was employed, which is known to be less effective in identifying minor variants within complex matrices like wastewater, as it typically reveals prevalent ones. Indeed, while the BA.2.86* was detected in wastewater samples as early as October 2023 using the newly designed RT-dPCR method, it remained undetected by Sanger sequencing until December 2023, coinciding with the diffusion of the JN.1 sub-lineage across Italy. This suggests that the BA.2.86 lineage present in October and November, during periods of low variant circulation, escaped detection via Sanger sequencing due to its overshadowing by other prevalent variants (XBB.1.5*/XBB.1.9*, XBB.1.16*, XBB.2.3*, CH.1.1* and CM.7* variants). In December 2023, instead, 30% of sequences were identified as JN.1 by Sanger sequencing, a proportion that increased to 79% by January 2024. During December 2023 and January 2024, the JN.1 variant was detected at significantly higher proportion using both RT-dPCR and Sanger sequencing methodologies.
The here presented and used RT-dPCR assay accurately described the spread of the BA.2.86 lineage. However, unlike Sanger, this assay cannot differentiate sub-variants such as JN.1, but it only identifies the BA.2.86*. To confirm the specificity of the assay, and to attempt to discriminate JN.1, we sequenced by Sanger sequencing the short fragment amplified with the primers of the digital PCR, as JN.1 contains a specific mutation within this fragment, namely mutation L455S, not present in the other BA.2.86 sub-variants. All sequenced samples were identified as the JN.1 variant due to the presence of the L455S mutation. This confirms that the test can be used in combination with sequencing to differentiate between BA.2.86 and JN.1, which cannot be achieved using the RT-dPCR test alone.
Other studies monitored the spread of the BA.2.86 lineage through wastewater. In Sweden, the BA.2.86* was first detected in wastewater during the first week of August through NGS sequencing. It rapidly spread in the following weeks to most regions (Espinosa-Gongora et al., 2023). Genomic monitoring of SARS-CoV-2 in Berlin’s wastewater identified JN.1 as early as September 2023 using an Illumina COVIDSeq Test (Bartel et al., 2024). Results from wastewater surveillance sequencing in Denmark, indicated that the BA.2.86 variant was circulating in the country at a low level in mid-August (Rasmussen et al., 2023). The BA.2.86 variant was also detected in wastewater samples in Thailand as early as July 28, 2023 (Wannigama et al., 2023). Additionally, Wurtzer et al. used quantitative screening of BA.2.86 through RT-dPCR as part of wastewater monitoring program in France. Positive wastewater samples were first detected on 22 August 2023. Analysis of wastewater samples showed little circulation of this variant until 24 September. After that, a rapid acceleration was observed, leading to the detection of this variant in most wastewater in November (Wurtzer et al., 2024). NGS sequencing is usually preferred in most studies. However, a quantitative assay can also be a viable alternative, as it is an easily implemented, inexpensive, and highly informative approach to estimate the dynamics of emerging variants within populations.
The study has some limitations due to the low coverage of certain Italian regions, caused by the voluntary participation of SARI network laboratories in periodic flash surveys. This is reflected in the low number of samples analysed in certain areas and in the lack of consistency in sample collection, which could lead to bias in the representation of variant prevalence and circulation.
Overall, this study emphasises the importance of environmental surveillance as a tool for monitoring the emergence and spread of new variants, especially during periods of low virus circulation. Quantitative methods, such as RT-dPCR, can successfully detect single variant-specific mutations, increasing specificity and depth of detection in an environmental sample with multiple variants. The method presented provides a specific, sensitive and rapid approach to detect and monitor the presence of BA.2.86X variant in wastewater.
Data availability
No datasets were generated or analysed during the current study.
References
Attar Cohen, H., Mesfin, S., Ikejezie, J., Kassamali, Z., Campbell, F., Adele, S., Guinko, N., Idoko, F., Mirembe, B. B., Mitri, M. E., Nezu, I., Shimizu, K., Ngongheh, A. B., Sklenovska, N., Gumede, N., Mosha, F. S., Mohamed, B., Corpuz, A., Pebody, R., Pavlin, B. I. (2023). Surveillance for variants of SARS-CoV-2 to inform risk assessments. Bulletin of the World Health Organization, 101(11), 707–716. https://doi.org/10.2471/BLT.23.290093
Bartel, A., Grau, J. H., Bitzegeio, J., Werber, D., Linzner, N., Schumacher, V., Garske, S., Liere, K., Hackenbeck, T., Rupp, S. I., Sagebiel, D., Böckelmann, U., & Meixner, M. (2024). Timely monitoring of SARS-CoV-2 RNA fragments in wastewater shows the emergence of JN.1 (BA.2.86.1.1, Clade 23I) in Berlin, Germany. Viruses, 16(1), 102. https://doi.org/10.3390/v16010102
Bonanno Ferraro, G., Veneri, C., Mancini, P., Iaconelli, M., Suffredini, E., Bonadonna, L., Lucentini, L., Bowo-Ngandji, A., Kengne-Nde, C., Mbaga, D. S., Mahamat, G., Tazokong, H. R., Ebogo-Belobo, J. T., Njouom, R., Kenmoe, S., La Rosa, G. (2022). A State-of-the-Art scoping review on SARS-CoV-2 in sewage focusing on the potential of wastewater surveillance for the monitoring of the COVID-19 pandemic. Food and Environmental Virology, 14(4), 315–354. https://doi.org/10.1007/s12560-021-09498-6
Caccuri, F., Messali, S., Scarpa, F., Giovanetti, M., Ciccozzi, M., & Caruso, A. (2023). First detection of SARS-CoV-2 BA.2.86.1 in Italy. Journal of Medical Virology, 95(10), 29175. https://doi.org/10.1002/jmv.29175
Combe, M., Cherif, E., Deremarque, T., Rivera-Ingraham, G., Seck-Thiam, F., Justy, F., Doudou, J. C., Carod, J. F., Carage, T., Procureur, A., & Gozlan, R. E. (2024). Wastewater sequencing as a powerful tool to reveal SARS-CoV-2 variant introduction and spread in French Guiana, South America. The Science of the Total Environment, 924, 171645. https://doi.org/10.1016/j.scitotenv.2024.171645
Cutrupi, F., Cadonna, M., Manara, S., Postinghel, M., La Rosa, G., Suffredini, E., & Foladori, P. (2022). The wave of the SARS-CoV-2 Omicron variant resulted in a rapid spike and decline as highlighted by municipal wastewater surveillance. Environmental Technology & Innovation, 28, 102667. https://doi.org/10.1016/j.eti.2022.102667
Doğantürk, Y. E., Dağ-Güzel, A., & Kuşkucu, M. A. (2023). Development of a nanoplate-based digital PCR test method for quantitative detection of human adenovirus DNA. Infectious Diseases & Clinical Microbiology, 5(4), 353–366. https://doi.org/10.36519/idcm.2023.255
Espinosa-Gongora, C., Berg, C., Rehn, M., Varg, J. E., Dillner, L., Latorre-Margalef, N., Székely, A. J., Andersson, E., & Movert, E. (2023). Early detection of the emerging SARS-CoV-2 BA.2.86 lineage through integrated genomic surveillance of wastewater and COVID-19 cases in Sweden, weeks 31 to 38 2023. Euro Surveillance: Bulletin Europeen Sur Les Maladies Transmissibles = European Communicable Disease Bulletin, 28(46), 2300595. https://doi.org/10.2807/1560-7917.ES.2023.28.46.2300595
European Centre for Disease Prevention and Control (ECDC). (2024a). Communicable disease threats report, 14–20 January 2024, week 3. Available at: https://www.ecdc.europa.eu/en/publications-data/communicable-disease-threats-report-14-20-january-2024-week-3. Accessed May 3, 2024
European Centre for Disease Prevention and Control (ECDC). (2024b). Communicable disease threats report, 17–23 March 2024, week 12. Available at: https://www.ecdc.europa.eu/en/publications-data/communicable-disease-threats-report-17-23-march-2024-week-12. Accessed May 3, 2024
Heijnen, L., Elsinga, G., de Graaf, M., Molenkamp, R., Koopmans, M. P. G., & Medema, G. (2021). Droplet digital RT-PCR to detect SARS-CoV-2 signature mutations of variants of concern in wastewater. The Science of the Total Environment, 799, 149456. https://doi.org/10.1016/j.scitotenv.2021.149456
Hokajärvi, A. M., Rytkönen, A., Tiwari, A., Kauppinen, A., Oikarinen, S., Lehto, K. M., Kankaanpää, A., Gunnar, T., Al-Hello, H., Blomqvist, S., Miettinen, I. T., Savolainen-Kopra, C., & Pitkänen, T. (2021). The detection and stability of the SARS-CoV-2 RNA biomarkers in wastewater influent in Helsinki, Finland. The Science of the Total Environment, 770, 145274. https://doi.org/10.1016/j.scitotenv.2021.145274
Islam, M. A., Rahman, M. A., Jakariya, M., Bahadur, N. M., Hossen, F., Mukharjee, S. K., Hossain, M. S., Tasneem, A., Haque, M. A., Sera, F., Jahid, I. K., Ahmed, T., Hasan, M. N., Islam, M. T., Hossain, A., Amin, R., Tiwari, A., Didar-Ul-Alam, M., Dhama, K., Ahmed, F. (2023). A 30-day follow-up study on the prevalence of SARS-COV-2 genetic markers in wastewater from the residence of COVID-19 patient and comparison with clinical positivity. The Science of the Total Environment, 858(Pt 3), 159350. https://doi.org/10.1016/j.scitotenv.2022.159350
Karthikeyan, S., Levy, J. I., De Hoff, P., Humphrey, G., Birmingham, A., Jepsen, K., Farmer, S., Tubb, H. M., Valles, T., Tribelhorn, C. E., Tsai, R., Aigner, S., Sathe, S., Moshiri, N., Henson, B., Mark, A. M., Hakim, A., Baer, N. A., Barber, T., Knight, R. (2022). Wastewater sequencing reveals early cryptic SARS-CoV-2 variant transmission. Nature, 609(7925), 101–108. https://doi.org/10.1038/s41586-022-05049-6
Klymus, K. E., Merkes, C. M., Allison, M. J., et al. (2020). Reporting the limits of detection and quantification for environmental DNA assays. Environmental DNA, 2, 271–282. https://doi.org/10.1002/edn3.29
La Rosa, G., Bonadonna, L., & Suffredini, E. (2021). Protocollo della Sorveglianza di SARS-CoV-2 in reflui urbani (SARI) - rev. 3 (Rev. 3). Zenodo. https://doi.org/10.5281/zenodo.5758725. Accessed May 3, 2024
La Rosa, G., Brandtner, D., Bonanno Ferraro, G., Veneri, C., Mancini, P., Iaconelli, M., Lucentini, L., Del Giudice, C., Orlandi, L., SARI Network, & Suffredini, E. (2023). Wastewater surveillance of SARS-CoV-2 variants in October-November 2022 in Italy: detection of XBB.1, BA.2.75 and rapid spread of the BQ.1 lineage. The Science of the Total Environment, 873, 162339. https://doi.org/10.1016/j.scitotenv.2023.162339
La Rosa, G., Iaconelli, M., Veneri, C., Mancini, P., Bonanno Ferraro, G., Brandtner, D., Lucentini, L., Bonadonna, L., Rossi, M., Grigioni, M., SARI Network, & Suffredini, E. (2022). The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance. The Science of the Total Environment, 837, 155767. https://doi.org/10.1016/j.scitotenv.2022.155767
Li, L., Uppal, T., Hartley, P. D., Gorzalski, A., Pandori, M., Picker, M. A., Verma, S. C., & Pagilla, K. (2022). Detecting SARS-CoV-2 variants in wastewater and their correlation with circulating variants in the communities. Scientific Reports, 12(1), 16141. https://doi.org/10.1038/s41598-022-20219-2
Lipponen, A., Kolehmainen, A., Oikarinen, S., Hokajärvi, A. M., Lehto, K. M., Heikinheimo, A., Halkilahti, J., Juutinen, A., Luomala, O., Smura, T., Liitsola, K., Blomqvist, S., Savolainen-Kopra, C., Pitkänen, T., WastPan Study Group. (2024). Detection of SARS-COV-2 variants and their proportions in wastewater samples using next-generation sequencing in Finland. Scientific Reports, 14(1), 7751. https://doi.org/10.1038/s41598-024-58113-8
Rasmussen, M., Møller, F. T., Gunalan, V., Baig, S., Bennedbæk, M., Christiansen, L. E., Cohen, A. S., Ellegaard, K., Fomsgaard, A., Franck, K. T., Larsen, N. B., Larsen, T. G., Lassaunière, R., Polacek, C., Qvesel, A. G., Sieber, R. N., Rasmussen, L. D., Stegger, M., Spiess, K., Jokelainen, P. (2023). First cases of SARS-CoV-2 BA.2.86 in Denmark, 2023. Euro Surveillance: Bulletin Europeen Sur Les Maladies Transmissibles = European Communicable Disease Bulletin, 28(36), 2300460. https://doi.org/10.2807/1560-7917.ES.2023.28.36.2300460
Rector, A., Bloemen, M., Thijssen, M., Delang, L., Raymenants, J., Thibaut, J., Pussig, B., Fondu, L., Aertgeerts, B., Van Ranst, M., Van Geet, C., Arnout, J., & Wollants, E. (2023). Monitoring of SARS-CoV-2 concentration and circulation of variants of concern in wastewater of Leuven, Belgium. Journal of Medical Virology, 95(2), e28587. https://doi.org/10.1002/jmv.28587
Reynolds, L. J., Gonzalez, G., Sala-Comorera, L., Martin, N. A., Byrne, A., Fennema, S., Holohan, N., Kuntamukkula, S. R., Sarwar, N., Nolan, T. M., Stephens, J. H., Whitty, M., Bennett, C., Luu, Q., Morley, U., Yandle, Z., Dean, J., Joyce, E., O’Sullivan, J. J., Meijer, W. G. (2022). SARS-CoV-2 variant trends in Ireland: Wastewater-based epidemiology and clinical surveillance. The Science of the Total Environment, 838(Pt 2), 155828. https://doi.org/10.1016/j.scitotenv.2022.155828
Róka, E., Déri, D., Khayer, B., Kis, Z., Schuler, E., Magyar, N., Pályi, B., Pándics, T., & Vargha, M. (2022). SARS-CoV-2 variant detection from wastewater: Rapid spread of B.1.1.7 lineage in Hungary. Journal of Water and Health, 20(2), 277–286. https://doi.org/10.2166/wh.2022.179
Sapoval, N., Liu, Y., Lou, E. G., Hopkins, L., Ensor, K. B., Schneider, R., Stadler, L. B., & Treangen, T. J. (2023). Enabling accurate and early detection of recently emerged SARS-CoV-2 variants of concern in wastewater. Nature Communications, 14(1), 2834. https://doi.org/10.1038/s41467-023-38184-3
Tiwari, A., Adhikari, S., Zhang, S., Solomon, T. B., Lipponen, A., Islam, M. A., Thakali, O., Sangkham, S., Shaheen, M. N. F., Jiang, G., et al. (2023). Tracing COVID-19 trails in wastewater: a systematic review of SARS-CoV-2 surveillance with viral variants. Water, 15(6), 1018. https://doi.org/10.3390/w15061018
Vigil, K., D’Souza, N., Bazner, J., Cedraz, F. M., Fisch, S., Rose, J. B., & Aw, T. G. (2024). Long-term monitoring of SARS-CoV-2 variants in wastewater using a coordinated workflow of droplet digital PCR and nanopore sequencing. Water Research, 254, 121338. https://doi.org/10.1016/j.watres.2024.121338
Vo, V., Tillett, R. L., Papp, K., Shen, S., Gu, R., Gorzalski, A., Siao, D., Markland, R., Chang, C.-L., Baker, H., et al. (2022). Use of wastewater surveillance for early detection of alpha and epsilon SARS-CoV-2 variants of concern and estimation of overall COVID-19 infection burden. Science of the Total Environment, 835, 155410. https://doi.org/10.1016/j.scitotenv.2022
Wannigama, D. L., Amarasiri, M., Phattharapornjaroen, P., Hurst, C., Modchang, C., Chadsuthi, S., Anupong, S., Miyanaga, K., Cui, L., Fernandez, S., Huang, A. T., Ounjai, P., Tacharoenmuang, R., Ragupathi, N. K. D., Sano, D., Furukawa, T., Sei, K., Leelahavanichkul, A., Kanjanabuch, T., Pathogen Hunters Research Team. (2023). Tracing the new SARS-CoV-2 variant BA.2.86 in the community through wastewater surveillance in Bangkok, Thailand. The Lancet Infectious Diseases, 23(11), e464–e466. https://doi.org/10.1016/S1473-3099(23)00620-5
Wilrich, C., & Wilrich, P.-T.H. (2009). EXCEL program for the estimation of the POD function and the LOD of a qualitative microbiological measurement method. Journal of AOAC International, 92, 1763–1772.
World Health Organization (WHO). (2023). Available at: https://www.who.int/docs/default-source/coronaviruse/21112023_ba.2.86_ire.pdf?sfvrsn=8876def1_3. Accessed 21 Nov 2023.
World Health Organization (WHO). (2024). COVID-19 epidemiological update–15 March 2024. Available at: https://www.who.int/publications/m/item/covid-19-epidemiological-update-15-march-2024. Accessed 15 Mar 2024.
Wu, F., Zhang, J., Xiao, A., Gu, X., Lee, W. L., Armas, F., Kauffman, K., Hanage, W., Matus, M., Ghaeli, N., Endo, N., Duvallet, C., Poyet, M., Moniz, K., Washburne, A. D., Erickson, T. B., Chai, P. R., Thompson, J., & Alm, E. J. (2020). SARS-CoV-2 titers in wastewater are higher than expected from clinically confirmed cases. mSystems, 5(4), e00614-e620. https://doi.org/10.1128/mSystems.00614-20
Wurtzer, S., Guilbaud, R., Levert, M., Fagour, N., Le Hingrat, Q., Descamps, D., Tarantola, A., Grellet, S., Londinsky, N., Moskovoy, J. M., Mouchel, J. M., Charpentier, C., & Moulin, L. (2024). BA.2.86 variant emergence and spread dynamics through wastewater monitoring in Paris, France. The Science of the Total Environment, 917, 170355. https://doi.org/10.1016/j.scitotenv.2024.170355
Wurtzer, S., Waldman, P., Levert, M., Cluzel, N., Almayrac, J. L., Charpentier, C., Masnada, S., Gillon-Ritz, M., Mouchel, J. M., Maday, Y., Boni, M., OBEPINE Consortium, AP-HP Virologist Group, Marechal, V., & Moulin, L. (2022). SARS-CoV-2 genome quantification in wastewaters at regional and city scale allows precise monitoring of the whole outbreaks dynamics and variants spreading in the population. The Science of the Total Environment, 810, 152213. https://doi.org/10.1016/j.scitotenv.2021.152213
Yousif, M., Rachida, S., Taukobong, S., Ndlovu, N., Iwu-Jaja, C., Howard, W., Moonsamy, S., Mhlambi, N., Gwala, S., Levy, J. I., Andersen, K. G., Scheepers, C., von Gottberg, A., Wolter, N., Bhiman, J. N., Amoako, D. G., Ismail, A., Suchard, M., & McCarthy, K. (2023). SARS-CoV-2 genomic surveillance in wastewater as a model for monitoring evolution of endemic viruses. Nature Communications, 14(1), 6325. https://doi.org/10.1038/s41467-023-41369-5
Acknowledgements
We wish to thank Dr. Paola Stefanelli (Istituto Superiore di Sanità, Department of Infectious Diseases) for sharing clinical omicron samples used as controls for specificity. This research was supported by EU funding within the NextGeneration EU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, PE13 INF-ACT). We also would like to thank Monica Monfrinotti and Giorgio Giorgi (ARPA Lazio), Giulia Palumbi (ARPA Liguria), Elena Ballarini (ARPA Marche), Andrea Franzetti (Università di Milano-Bicocca).
The SARI network (“Sorveglianza Ambientale di SARS-CoV-2 attraverso i Reflui urbani in Italia”). Abruzzo: Paolo Torlontano (Regione Abruzzo); Giuseppe Aprea, Silvia Scattolini, Vicdalia Aniela Acciari (Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise “G. Caporale”). Basilicata: Michele La Bianca (Regione Basilicata); Rosa Anna Cifarelli, Achille Palma, Giuseppe Lauria, Giovanna La Vecchia (Agenzia Regionale per la Protezione dell'Ambiente Basilicata – ARPAB). Campania: Vincenzo Giordano (Regione Campania); Luigi Cossentino (Arpac - Agenzia Regionale per la Protezione Ambientale in Campania); Francesca Pennino, Annalisa Lombardi (Università degli Studi di Napoli “Federico II”). Emilia-Romagna: Lisa Gentili, Paola Angelini (Regione Emilia-Romagna); Daniele Nasci (HERATech); Giovanni Alborali, Nicoletta Formenti, Flavia Guarneri (Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna); Nadia Fontani, Marco Guercio (IREN). Friuli-Venezia Giulia: Marika Mariuz, Gabriella Trani, (Direzione Centrale Salute FVG); Anna Pariani, Laura De Lellis (LABORATORIO HERAtech di Sasso Marconi –BO). Lazio: Carla Ancona (DEPLAZIO - Dipartimento di Epidemiologia del Servizio Sanitario Regionale - Regione Lazio), Alessandra Barca, Flavia Serio (Regione Lazio), Doriana Antonella Giorgi, Irene Ferrante, Valeria Capparuccini (ARPA Lazio - Agenzia Regionale per la Protezione Ambientale del Lazio), Maria Teresa Scicluna, Antonella Cersini, Gabriele Pietrella (IZSLT - Istituto Zooprofilattico Sperimentale del Lazio e della Toscana). Liguria: Elena Nicosia (Regione Liguria settore tutela della salute negli ambienti di vita e di lavoro); Nadia Fontani, Marco Guercio (Iren); Elena Grasselli (UNIGE - DISTAV); Alberto Izzotti (UNIGE – DIMES); Irene Tomesani (UNIGE); Marta Bellisomi, Stefano Rosatto (ARPAL). Lombardia: Emanuela Ammoni, Danilo Cereda (Regione Lombardia); Barbara Bertasi, Marina Nadia Losio (IZSLER - Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia); Desdemona Oliva (CAP Holding); Sara Castiglioni, Silvia Schiarea (Istituto Mario Negri IRCCS); Sandro Binda, Valeria Primache, Laura Pellegrinelli (Università degli Studi di Milano, Dipartimento di Scienze Biomediche per la Salute); Clementina Cocuzza, Rosario Musumeci (Università di Milano-Bicocca). Marche: Luigi Bolognini, Fabio Filippetti (Regione Marche); Marta Paniccia’, Sara Briscolini (IZSUM - Istituto Zooprofilattico Sperimentale Umbria Marche); Silvia Magi, Annalisa Grucci (ARPAM). Molise: Michele Colitti, Angela Ciccaglione (Regione Molise); Carmen Montanaro (ASReM); Piemonte: Bartolomeo Griglio; Angela Costa (Regione Piemonte); Lucia Decastelli, Angelo Romano, Manila Bianchi (IZSTO - Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d'Aosta SC Sicurezza e Qualità degli Alimenti); Elisabetta Carraro, Cristina Pignata (Dipartimento di Scienze della Sanità Pubblica e Pediatriche, Università di Torino), Manuela Macrì, Silvia Bonetta (Dipartimento di Scienze della Vita e Biologia dei Sistemi, Università di Torino). Puglia: Nehludoff Albano, Giuseppe Di Vittorio, Onofrio Mongelli (Regione Puglia); Francesca Apollonio, Francesco Triggiano, Osvalda De Giglio, Maria Teresa Montagna (Laboratorio di Igiene dell'Ambiente e degli Alimenti, Dipartimento Interdisciplinare di Medicina, Università degli Studi di Bari Aldo Moro). Sicilia: Mario Palermo (Regione Sicilia); Carmelo Massimo Maida, Walter Mazzucco, Fabio Tramuto (Università degli Studi di Palermo-Dipartimento PROMISE - sezione di Igiene); Simona De Grazia, Giovanni Maurizio Giammanco, Chiara Filizzolo (Centro di Riferimento Regionale per la Sorveglianza delle Paralisi Flaccide Acute (PFA) e ambientale della circolazione di poliovirus in Sicilia - AOUP Palermo); Giuseppa Purpari, Francesca Gucciardi (IZS - Istituto Zooprofilattico Sperimentale della Sicilia), Margherita Ferrante, Antonella Agodi, Martina Barchitta (Università degli Studi di Catania - Dipartimento “G. F. Ingrassia”). Toscana: Piergiuseppe Cala’ (Az. USL Toscana Centro); Annalaura Carducci, Marco Verani, Ileana Federigi (Laboratorio di Igiene e Virologia Ambientale - Dipartimento di Biologia Università di Pisa). Umbria: Salvatore Macrì (Regione Umbria); Ermanno Federici, Maya Petricciuolo, Agnese Carnevali (Laboratorio Microbiologia Applicata e Ambientale, DCBB Università di Perugia). Veneto: Francesca Russo, Gisella Pitter, Vanessa Groppi (Regione Veneto); Franco Rigoli, Marco Zampini (ARPAV - Agenzia Regionale per la Prevenzione e Protezione Ambientale del Veneto); Tatjana Baldovin, Irene Amoruso (Universita’ di Padova). AP Trento: Maria Cadonna, Mattia Postinghel (ADEP SGI PAT); Paola Foladori (Università di Trento).AP Bolzano: Lorella Zago (P.A. Bolzano), Alberta Stenico, Morelli Marco, Dossena Matteo (Laboratorio biologico - Agenzia provinciale per l'ambiente e la tutela del clima (APPA).
Funding
This article is funded by EU funding within the NextGeneration EU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases, Project no. PE00000007, PE13 INF-ACT.
Author information
Authors and Affiliations
Consortia
Contributions
Conceptualization and Writing—Original Draft CV, ES, GLR Investigation CV, DB, PM, GBF, MI, the SARI network Methodology ad validation CV, DB, PM, GBF, MI, ES, MP, GL, VP, BMG, AM, GLR Writing—Review & Editing CV, DB, PM, GBF, MI, ES, MP, GL, VP, BMG, AM, the SARI network, GLR.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Group members of the SARI Network is listed in acknowledgements section.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Veneri, C., Brandtner, D., Mancini, P. et al. Tracking the Spread of the BA.2.86 Lineage in Italy Through Wastewater Analysis. Food Environ Virol (2024). https://doi.org/10.1007/s12560-024-09607-1
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s12560-024-09607-1