Besides nasopharyngeal swabs, monkeypox virus (MPXV) DNA has been detected in a variety of samples such as saliva, semen, urine and fecal samples. Using the environmental surveillance network previously developed in Spain for the routine wastewater surveillance of SARS-CoV-2 (VATar COVID-19), we have analyzed the presence of MPXV DNA in wastewater from different areas of Spain. Samples (n = 312) from 24 different wastewater treatment plants were obtained between May 9 (week 19 of 2022) and August 4 (week 31 of 2022). Following concentration of viral particles by a validated aluminum adsorption-precipitation method, a qPCR procedure allowed us to detect MPXV DNA in 56 wastewater samples collected from May 16 to August 4, 2022, with values ranging between 2.2 × 10³ to 8.7 × 10⁴ genome copies (gc)/L. This study shows that MPXV DNA can be reproducibly detected by qPCR in longitudinal samples collected from different Spanish wastewater treatment plants. According to data from the National Epidemiological Surveillance Network (RENAVE) in Spain a total of 6,119 cases have been confirmed as of August 19, 2022. However, and based on the wastewater data, the reported clinical cases seem to be underestimated and asymptomatic infections may be more frequent than expected.

Wastewater based epidemiology (WBE) offers an overview of the SARS-CoV-2 variants circulating among the population thereby serving as a proper surveillance method.

Shortly after the beginning of the SARS-CoV-2 pandemic, many countries implemented sewage sentinel systems to monitor the circulation of the virus in the population. A fundamental part of these surveillance programs is the variant tracking through sequencing approaches to monitor and identify new variants or mutations that may be of importance. Two of the main sequencing platforms are Illumina and Oxford Nanopore Technologies. Here, we compare the performance of MiSeq (Illumina) and MinION (Oxford Nanopore Technologies), as well as two different data processing pipelines, to determine the effect they may have on the results. MiSeq showed higher sequencing coverage, lower error rate, and better capacity to detect and accurately estimate variant abundances than MinION R9.4.1 flow cell data. The use of different variant callers (LoFreq and iVar) and approaches to calculate the variant proportions had a remarkable impact on the results generated from wastewater samples. Freyja, coupled with iVar, may be more sensitive and accurate than LoFreq, especially with MinION data, but it comes at the cost of having a higher error rate. The analysis of MinION R10.4.1 flow cell data using Freyja combined with iVar narrows the gap with MiSeq performance in terms of read quality, accuracy, sensitivity, and number of detected mutations. Although MiSeq should still be considered as the standard method for SARS-CoV-2 variant tracking, MinION’s versatility and rapid turnaround time may represent a clear advantage during the ongoing pandemic.

Wastewater analysis serves as a complementary tool to clinical data in monitoring both the quantity and diversity of circulating enteroviruses among the population. While enterovirus infections typically result in asymptomatic or mild disease, certain types can lead to severe symptoms, highlighting the importance of tracking their prevalence and distribution. Here we present both a RTqPCR assay targeting a conserved region in the 5’ end of the enterovirus genome to detect and quantify its presence in sewage along with a sequencing pipeline based in MinION platform (Oxford Nanopore Technologies) to characterize circulating enterovirus types in samples with sufficient virus load (Cq≤33). Two sequencing approaches are possible: one targeting all enterovirus types (1), and the other specifically focusing on Cluster C enterovirus (2), including poliovirus. The obtained data are processed using a designed pipeline to quantify the proportions of different enterovirus types, providing valuable insights into the spread of potentially severe types.

During the evolution of the SARS-CoV-2 pandemic, new variants of the virus have emerged and spread worldwide. The increased transmissibility and proclivity of some of these variants to cause more serious disease threatens public health responses against the virus, and they are classified as variants of concern (Variants of Concern, VOCs). While Next-Generation-Sequencing (NGS) is the gold standard to identify VOC, it cannot always be rapidly implemented in some settings to provide information as an early warning tool. Duplex quantitative real time RTqPCR assays offer a sensitive and easy-to-use tool to detect, discriminate, and estimate relative proportions of SARS-CoV-2 variants containing VOC-specific signature mutations from variants lacking it, using allelic discrimination probes. We developed three multiplexed RTqPCR assays that can detect Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) VOCs by targeting 22281_22289DelCTTTACTTG, 28262_28263InsAACA and 22029_22034DelAGTTCA deletions/insertions in their genomes, respectively. Beta and Delta deletions are mapped to the S gene (residues 241/243 and 157/158, respectively), while Gamma insertion is located between the end of ORF8 and the beginning of N gene. Our specific duplex RTqPCR assays have been adapted from a previously designed duplex RTqPCR assay used to estimate the relative proportion of genomes containing 21765-21770DelTACATG mutation affecting residues HV69/70, a signature mutation of Alpha VOC (Carcereny et al., 2021). All duplex RTqPCR assays targeting signature mutations of VOCs may be used as a complementary tool to NGS for rapid variant tracking and surveillance in wastewater-based epidemiology.

During the evolution of the SARS-CoV-2 pandemic, new variants of the virus have emerged and spread worldwide. The increased transmissibility and proclivity of some of these variants to cause more serious disease threatens public health responses against the virus, thus being its monitoring relevant. While Next-Generation-Sequencing (NGS) is the gold standard to identify variants, it cannot always be rapidly implemented in some settings to provide information as an early warning tool. Duplex quantitative real time RTqPCR assays offer a sensitive and easy-to-use tool to detect, discriminate, and estimate relative proportions of SARS-CoV-2 variants containing signature mutations from variants lacking it, using allelic discrimination probes. We developed ten multiplexed RTqPCR assays that can detect Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron (B.1.1.529), Omicron BA.1 (B.1.1.529.1), Omicron BA.2 (B.1.1.529.2), Omicron FLip (XBB.1.5-like + L455F + F456L), Omicron BA.2.86 (B.1.1.529.2.86) and Omicron DeFLiRT (JN.1 + delS31) variants by targeting 21765_21770DelTACATG, 22281_22289DelCTTTACTTG, 28262_28263InsAACA, 22029_22034DelAGTTCA, 28362_28370DelGAGAACGCA, 22121InsGAGCCAGAA, 21633_21641DelTACCCCCTG, G22927T, T22928C and T22930A (L455F and F456L SNVs), ins21610ATGCCGCTGTTT and 21654_21656DelCTT deletions, insertions and SNVs in their genomes. Alpha, Beta, Delta, Omicron BA.1, Omicron BA.2, Omicron Flip, Omicron BA.2.86 and Omicron DeFLiRT markers are mapped to the S gene (residues 69/70, 241/243, 157/158, 214, 25/27, 455/456, 16 and 31 respectively), Gamma insertion is located between the end of ORF8 and the beginning of N gene, and Omicron (B.1.1.529) deletion is mapped to the N gene (residues 31/33). The duplex RTqPCR assay targeting 21765-21770DelTACATG mutation affecting residues HV69/70 has been previously used to estimate the relative proportion of Alpha VOC (Carcereny et al., 2021), and is also suitable to estimate the proportions of Omicron BA.4 and BA.5. All duplex RTqPCR assays targeting signature mutations may be used as a complementary tool to NGS for rapid variant tracking and surveillance in wastewater-based epidemiology.