We targeted the wastewater treatment plant (WTP) of Italy’s largest airport, Fiumicino Airport, in Rome, which had ≈3,000,000 passengers/month during May–July 2022 (https://fiumicinoairport.com/statistics). This WTP has a global capacity of 4,000 m³ per day. We collected 24-hour composite wastewater samples twice a week during May 30–August 3, 2022, for a total of 20 samples.

Before viral concentration, we pretreated samples in a water bath at 56°C for 30 min to inactivate the virus and protect laboratory technicians, as per a previous study (9). We used a polyethylene glycol/sodium chloride precipitation protocol originally developed for SARS-CoV-2 environmental surveillance (10,11) but modified the protocol by increasing the initial wastewater volume to 90 mL (2 tubes of 45 mL) and eluting all the extracted nucleic acids in 50 µL of elution buffer supplied with the kit. We used NucliSens miniMAG (bioMérieux, https://www.biomerieux.com) semi-automatic extraction platform to extract nucleic acids. We used OneStep PCR Inhibitor Removal Kit (Zymo Research, https://www.zymoresearch.com) to purify DNA.

We used 3 different real-time PCR assays: 2 published in 2004 that target the N3R and F3L genes (12), and 1 developed in 2010 by the US Centers for Disease Control and Prevention, G2R_G generic real-time PCR assay (13), which targets the G2R region of the tumor necrosis factor receptor gene. After comparing primers and probes with sequences of the current outbreak, we noted mismatches in primers, probes, or both. Therefore, we designed and tested novel primers and probes that had 100% nucleotide identity with current outbreak sequences, then compared these with the original primers and probes (Table 1). We used MPXV (Slovenia ex Gran Canaria) DNA (European Virus Archive Global [EVAg]; https://www.european-virus-archive.com) as a control for testing primers and probes (Appendix). We further optimized the assays by evaluating different real-time PCR reagents and primer/probe concentrations (Appendix). We prepared reaction mixes in 25 μL by using TaqPath BactoPure Microbial Detection Master Mix (Thermo Fisher Scientific, https://www.thermofisher.com), 800 mmol of each primer, 500 nmol of the probe, and 5 μL of sample. Amplification conditions included an initial activation step at 95°C for 2 min and 50 cycles of 10 s at 95°C and 30 s at 60°C. We included 10-fold dilutions of the standardized EVAg MPXV DNA (range 740–0.74 copies/μL) in the runs as positive controls and for the rough estimation of viral loads. For each assay, we assessed the limit of detection at 50% (LOD₅₀) on a pure target (i.e., EVAg MPXV DNA) and on MPXV DNA diluted in nucleic acids previously extracted from wastewater samples collected in Europe before monkeypox emerged.

We designed nested PCR assays targeting the same regions as the real-time PCR assays to confirm results by amplicon sequencing using Primer3Plus software (https://www.primer3plus.com) (Table 1). We performed reactions by using 1 μL of 10 μmol primer and 2 μL of sample, and Platinum SuperFi II Green PCR Master Mix (Thermo Fisher Scientific) in a final volume of 25 μL. PCR amplicons on both strands were sequenced by Bio-Fab Research (https://www.biofabresearch.com).

All real-time PCR assays successfully amplified the EVAg MPXV DNA. Compared with the original assay, the modified G2R_G assay showed a decrease in the average quantification cycle (Cq) values of 1.34 cycles (21.93 vs. 23.28), demonstrating a better performance. Therefore, we performed subsequent optimization activities and screening of wastewater samples by using the F3L and N3R assays as originally designed but modified the G2R_G assay for our study.

On pure MPXV DNA, the real-time F3L assay had an LOD₅₀ of 0.21 copies/μL, the N3L assay had an LOD₅₀ of 0.31 copies/μL, and G2R_G had an LOD₅₀ of 0.21 copies/μL. For nucleic acids extracted from sewage samples spiked with MPXV, F3L had an LOD₅₀ of 0.43 copies/μL and 2.16 copies/reaction, N3L had an LOD50 of 0.33 copies/μL and 1.65 copies/reaction, and G2R_G had an LOD₅₀ of 0.31 copies/μL and 1.55 copies/reaction (Appendix).

Cq values ranged from 38.37–40.18 for 2 wastewater samples that tested positive by real-time PCR (Table 2), indicating relatively low DNA concentrations in the tested samples. Consensus sequences found 100% similarity by BLAST analysis between study sequences and MPXV strains available in GenBank (accession no. OX248696), thus confirming the presence of MPXV DNA.

A crucial aim of infectious disease surveillance is early detection of cases, outbreaks, and clusters, which is essential for disease control. We explored possible methods for monitoring MPXV through wastewater surveillance, a well-established complementary epidemiologic tool used successfully for viral infectious diseases, including SARS-CoV-2 and polio.

Monkeypox prevalence in the general population was low at the time of sample collection, only 20 cases had been detected in Italy as of May 30, 2022. Thus, to maximize the probability of positive samples among those collected, we tested wastewater samples from a large transportation hub, through which millions of persons travel to and from numerous countries. Because harmonized methods for detecting MPXV in wastewater are not yet available, we tested 3 different real-time PCR assays previously designed for clinical samples. We modified the assays by introducing changes in the primer and probe sequences to mitigate the effect of nucleotide mismatches. Among 20 samples, 3 tested positive for MPXV by real-time or nested PCR and sequencing.

In the next stage, we will test wastewater samples from WTPs enrolled in official SARS-CoV-2 environmental surveillance throughout Italy, to map the geographic distribution of MPXV in the country. Further research efforts should focus on elucidating how detection of viral DNA in sewage can be related to reported and confirmed cases. Factors affecting MPXV detection in wastewater also should be studied, including routes and duration of virus shedding by infected persons, environmental persistence, and analytical sensitivity of the methods used (14).

In conclusion, we adapted SARS-CoV-2 wastewater surveillance for MPXV detection in a large airport WTP. Our methods can be applied to wastewater-based epidemiology for monkeypox outbreaks and provides basic tools, including analytic methods. Wastewater surveillance can be rapidly adapted to detect emerging threats, including monkeypox.