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Published in final edited form as: Sci Total Environ. 2023 Aug 11;903:166230. doi: 10.1016/j.scitotenv.2023.166230

Enhanced detection of mpox virus in wastewater using a pre-amplification approach: A pilot study informing population-level monitoring of low-titer pathogens

Devin A Bowes 1,*, Katherine B Henke 1,*, Erin M Driver 1, Melanie Engstrom Newell 1, Izabella Block 1, Gray Shaffer 1, Arvind Varsani 2,3,4, Matthew Scotch 1,5, Rolf U Halden 1,6,7,8,**
PMCID: PMC10592092  NIHMSID: NIHMS1926972  PMID: 37574063

Abstract

A recent outbreak of the mpox virus (MPXV) occurred in non-endemic regions of the world beginning in May 2022. Pathogen surveillance systems faced pressure to quickly establish response protocols, offering an opportunity to employ wastewater-based epidemiology (WBE) for population-level monitoring. The pilot study reported herein aimed to: (i) develop a reliable protocol for MPXV DNA detection in wastewater which would reduce false negative reporting, (ii) test this protocol on wastewater from various regions across the United States, and (iii) conduct a state of the science review of the current literature reporting on experimental methods for MPXV detection using WBE. Twenty-four-hour composite samples of untreated municipal wastewater were collected from the states of New Jersey, Georgia, Illinois, Texas, Arizona, and Washington beginning July 3rd, 2022 through October 16th, 2022 (n = 60). Samples underwent vacuum filtration, DNA extraction from captured solids, MPXV DNA pre-amplification, and qPCR analysis. Of the 60 samples analyzed, a total of eight (13%) tested positive for MPXV in the states of Washington, Texas, New Jersey, and Illinois. The presence of clade IIb MPXV DNA in these samples was confirmed via Sanger sequencing and integration of pre-amplification prior to qPCR decreased the rate of false negative detections by 87% as compared to qPCR analysis alone. Wastewater-derived detections of MPXV were compared to clinical datasets, with 50% of detections occurring as clinical cases were increasing/peaking and 50% occurring as clinical cases waned. Results from the literature review (n = 9 studies) revealed successful strategies for the detection of MPXV DNA in wastewater, however also emphasized a need for further method optimization and standardization. Overall, this work highlights the use of pre-amplification prior to qPCR detection as a means to capture the presence of MPXV DNA in community wastewater and offers guidance for monitoring low-titer pathogens via WBE.

Keywords: Wastewater-based epidemiology, monkeypox virus, wastewater surveillance, wastewater solids, emerging pathogens, public health

Graphical Abstract

graphic file with name nihms-1926972-f0001.jpg

1. Introduction

Between May 13th and May 21st, 2022, the World Health Organization (WHO) received 92 reports of laboratory-confirmed cases of mpox (formerly known as monkeypox (World Health Organization, 2022a)) in 12 non-endemic countries (World Health Organization, 2022b). The first reported case in the United States (U.S.) occurred in Boston, Massachusetts on May 18th in an individual who had recently traveled outside the country (Massachusetts Department of Public Health, 2022). The multi-country outbreak continued to progress throughout the summer with more than 16,000 confirmed cases and five deaths reported across 75 countries/areas/territories by July 22nd, including more than 2,300 cases in the United States (World Health Organization, 2022c). At that time, the WHO director announced that the mpox outbreak should be considered a public health emergency of international concern, with the U.S. Department of Health and Human Services officially declaring a public health emergency by early August (U.S. Department of Health and Human Services, 2022; World Health Organization, 2022d). As of March 15, 2023, the U.S. states with the most reported mpox cases included California (5,746 cases), New York (4,238), Texas (2,923), Florida (2,885), Georgia (1,993), and Illinois (1,436) (Centers for Disease Control and Prevention, 2023a).

Mpox is a zoonotic disease caused by the mpox virus (MPXV), an enveloped, double-stranded DNA virus belonging to the Orthopoxvirus genus of the Poxviridae family. Mpox is typically considered a rare infectious illness endemic in regions of Western and Central Africa and can be classified into three clades (I, IIa, and IIb) (Happi et al., 2022). The mpox variants circulating during the 2022 outbreak can largely be traced to clade IIb (Luna et al., 2022). The incubation period for mpox can last from 4–21 days, though it typically ranges between 7–14 days (Altindis et al., 2022; Mitjà et al., 2023). Disease symptoms may include fever, chills, headache, body aches, fatigue, and swollen lymph nodes, along with a characteristic rash that may develop before or after other symptoms and may present on different parts of the body (Altindis et al., 2022; Mitjà et al., 2023; Titanji et al., 2022). Although information on MPXV shedding rates is currently limited, MPXV DNA has been identified from skin lesions, saliva, urine, feces, semen, and blood of infected individuals, as well as from throat/nasopharyngeal and anal/rectal swabs (Antinori et al., 2022; Palich et al., 2023; Peiró-Mestres et al., 2022).

At the onset of the 2022 mpox outbreak, clinical surveillance systems experienced several challenges that hampered timely testing and reporting, including disease stigma (mpox burden was particularly high amongst men who have sex with men (Mitjà et al., 2023)) and surveillance fatigue from the ongoing COVID-19 pandemic (Zaheer et al., 2022). These challenges, combined with clinical observations revealing that infected individuals shed the virus from skin lesions as well as in bodily fluids and feces, raised the question of whether wastewater-based epidemiology (WBE) could be employed to monitor the spread of mpox in communities. Given the demonstrated success of WBE as a useful and cost-effective tool for population-level monitoring of SARS-CoV-2 throughout the COVID-19 pandemic (Bibby et al., 2021; Bivins et al., 2020; Bowes et al., 2023) and the recent wide-scale adoption of WBE around the globe, multiple research teams had access to the wastewater samples and basic laboratory infrastructure and protocols necessary to test for this emergent virus. To date, studies have reported the detection of MPXV DNA in wastewater samples from the Netherlands (de Jonge et al., 2022), Spain (Girón-Guzmán et al., 2023), Italy (La Rosa et al., 2023), France (Wurtzer et al., 2022), Thailand (Wannigama et al., 2023), the United States (Florida (Sharkey et al., 2022) and California (Wolfe et al., 2023)), Canada (Mejia et al., 2022), and Poland (Gazecka et al., 2023).

While the aforementioned studies report successful implementation of this novel application of WBE, some limitations were noted that prompted further investigation. In particular, the relatively low concentrations of MPXV DNA in wastewater resulted in high (i.e., >35) and oftentimes irreproducible Ct values in qPCR analyses (de Jonge et al., 2022; Girón-Guzmán et al., 2023; La Rosa et al., 2023; Mejia et al., 2022; Wurtzer et al., 2022), thereby increasing the potential for erroneous underreporting (i.e., false negative results). In an effort to address this challenge, we conducted a multifaceted study to: (i) develop a method for MPXV DNA detection in wastewater that would reduce false negative reporting, (ii) test this method on wastewater samples from various regions across the U.S., and (iii) conduct a state of the science review of the literature reporting on MPXV detection in wastewater to understand the current landscape and inform future population-level monitoring of low-titer pathogens to support public health decision-making.

2. Methods

2.1. Wastewater sample collection

Twenty-four-hour time- and/or flow-weighted composite samples of raw, untreated wastewater were collected every other week at either the wastewater treatment plant level or from within the sewer infrastructure using refrigerated automated samplers (Teledyne ISCO, Lincoln, NE) from the week of July 3rd, 2022 through the week of October 9th, 2022. Samples collected the week of February 27th, 2022 were also processed using the developed method to demonstrate absence of MPXV in U.S. wastewater prior to the 2022 outbreak. In total, 60 samples were collected from six states across the U.S. including New York, Georgia, Illinois, Texas, Arizona, and Washington. After collection, samples were stored in high-density polyethylene bottles, packed in Styrofoam coolers with wet ice and/or reusable ice packs, and sent via overnight shipping to the laboratory at Arizona State University for analysis. Upon arrival, sample metadata were documented where applicable (e.g., flow measurements, estimated population served, etc.) (Table S1) and samples were either immediately processed or temporarily stored at −20 °C.

2.2. Sample processing and DNA extraction

Each raw wastewater sample was thoroughly mixed and subsequently filtered (~150 mL) using a disposable vacuum filtration unit (Fisher Scientific, Chino, CA) fitted with a 0.22 μm polycarbonate membrane filter (47 mm) (EMD Millipore, Burlington, MA). The filtrate was subsequently discarded and the 0.22 μm filter with captured solids was removed from the filter unit, segmented into quarters, and transferred to an individual bead beating tube supplied within the QIAGEN AllPrep PowerViral DNA/RNA extraction kit (QIAGEN, Germantown, MD). DNA was then extracted from the wastewater solids according to the manufacturer’s recommendations, including inhibitor removal, with a few modifications to maximize recovery and increase concentrations of DNA extracts: 1) bead beating was performed for 20 minutes instead of the recommended 10 minutes, 2) the final incubation step with nuclease-free water was increased from 1 minute to 5 minutes, and 3) the final elution volume was 50 μL instead of the recommended 100 μL. DNA extracts were temporarily stored at −20 °C prior to analysis.

Whole-process and DNA extraction controls (deionized water; n = 3 for each control) were performed to assess sample contamination throughout the filtration and/or extraction processes. No MPXV DNA contamination was identified in these controls. To assess recovery, a subset of raw wastewater samples (n = 5) were spiked with murine gammaherpesvirus (MHV68), an enveloped, double-stranded DNA virus in the Herpesviridae family (~150–200 nm in diameter) (Bortz et al., 2003; Stewart et al., 1996), as a MPXV surrogate (~200–250 nm diameter) (Cho and Wenner, 1973). Here, 1.55 × 105 copies of MHV68 were spiked into raw wastewater samples (~150 mL) prior to the same sample filtration and DNA extraction protocol described above. Details for qPCR analysis of these recovery experiments can be found in the Supplementary Information (Section S1 and Table S2).

2.3. Pre-amplification and qPCR

DNA extracts were analyzed for MPXV DNA using consecutive steps of pre-amplification and qPCR. Specifically, the G2R region of the MPXV genome was targeted using the G2R_G primers and probe originally described by Li et al. (2010) and modified by de Jonge et al. (2022) (Table S2) to better reflect the MPXV variants circulating during the 2022 outbreak. Pre-amplification was performed using the BioRad iScript Explore One-Step Reverse Transcriptase and PreAMP Kit (BioRad, Hercules, CA), excluding the provided DNase and reverse transcriptase reagents, and carried out with a Veriti Dx thermal cycler (Applied Biosystems, Waltham, MA). Each 30 μL reaction consisted of the following components: 0.3 μL each of forward and reverse primer (final concentration of 100 nM each) (Integrated DNA Technologies, Coralville, IA), 15 μL of SSO Advanced Preamp Supermix, 0.6 μL of iScript Explore Reaction Booster, 5 μL of DNA template, and the remaining volume of nuclease-free water. Thermal cycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 35 cycles of 95 °C for 15 s and 59 °C for 1 min. No-template controls (PCR-grade water) were included to ensure that no contamination was introduced in the laboratory. Pre-amplified samples were stored at −20 °C prior to qPCR analysis.

qPCR was performed using an Applied Biosystems QuantStudio™ 5 Real-Time PCR system with the aforementioned G2R_G primers and probe. Each 20 μL qPCR reaction contained the following components: 0.4 μL each of forward and reverse primer (final concentration of 400 nM each), 0.4 μL of fluorescently-labeled hydrolysis probe (final concentration of 200 nM) (Integrated DNA Technologies, Coralville, IA), 10 μL of TaqMan Universal PCR Mastermix (Applied Biosystems, Waltham, MA), 2 μL of DNA template (either from the pre-amplification step, if performed, or from the original DNA extract), and the remaining volume of nuclease-free water. Thermal cycling conditions were as follows: 95 °C for 10 min followed by 50 cycles of 95 °C for 15 s and 59 °C for 1 min. All samples and standards for qPCR were analyzed in triplicate with a standard curve and no-template controls (PCR-grade water) on every plate. The positive control was provided by the National Institute of Standards and Technology (NIST) (Research Grade Test Material 10223) and consisted of linearized plasmid DNA (3,376 bp) containing nine PCR target regions of the MPXV genome at a concentration of ~1.1 × 105 plasmid copies/μL. qPCR standard curves consisted of 10-fold serial dilutions of the NIST standard ranging from 105 to 100 plasmid copies/μL using PCR-grade water. Standard curves were linear and Ct values between replicates were reproducible in the range of 105 to 102 plasmid copies/μL, with an average PCR efficiency of 100% (slope = −3.32, y-intercept = 38.47, R2 = 1.00). qPCR products from wastewater samples with positive MPXV DNA detections were analyzed by gel electrophoresis on a 3% agarose/TBE gel, revealing amplicons of the expected length.

2.4. Sanger sequencing

DNA extracts which tested positive for MPXV DNA via pre-amplification/qPCR were analyzed further via Sanger sequencing. Again, the G2R region of the MPXV genome was targeted, though a different reverse primer was used in order to generate a longer amplicon (475 bp) more amenable to sequencing (Table S2). Pre-amplification was performed as described above, with each 30 μL reaction consisting of the following components: 0.3 μL each of forward and reverse primer (final concentration of 100 nM each) (Integrated DNA Technologies, Coralville, IA), 15 μL of SSO Advanced Preamp Supermix, 0.6 μL of iScript Explore Reaction Booster, 5 μL of DNA template, and the remaining volume of nuclease-free water. Thermal cycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 35 cycles of 95 °C for 15 s and 58 °C for 2 min. A second round of PCR was then performed on these pre-amplified samples, with each 20 μL reaction containing the following components: 0.4 μL each of forward and reverse primer (final concentration of 400 nM each), 10 μL of TaqMan Universal PCR Mastermix (Applied Biosystems, Waltham, MA), 2 μL of pre-amplified DNA template, and the remaining volume of nuclease-free water. Thermal cycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 50 cycles of 95 °C for 15 s and 58 °C for 2 min. Second-round PCR products were submitted to the Arizona State University Genomics Facility for Sanger sequencing of both strands.

Sequencing data were analyzed using ApE version 3.1.3 (Davis and Jorgensen, 2022). Where quality sequencing data were obtained for both strands, sequences were aligned using the EMBOSS merger tool (Rice et al., 2000) and consensus sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990). Clade/lineage assignment was performed using Nextclade (Aksamentov et al., 2021).

2.5. Data analysis

Analysis of qPCR data was performed using the QuantStudio™ Data and Analysis Software (version 1.2) from Thermo Scientific (Waltham, MA). Clinical mpox case counts were retrieved from publicly available datasets from the counties where wastewater samples were collected and de-identified by reporting at the state level. In states with multiple collection sites across different counties, county-level data were aggregated. Each state’s reported daily mpox clinical cases were then aggregated by week to assess alongside wastewater-derived detections of MPXV DNA. All data were organized and analyzed using Microsoft Excel 2019 (version 16.7).

2.6. State of the science literature review

To understand the current status of MPXV DNA detection in wastewater, a state of the science review of the literature was conducted focusing solely on studies reporting experimental investigation. Complete details of this review process are documented in the Supplementary Information (Section S2 and Figure S1).

3. Results

Here, we present a method for extracting MPXV DNA from captured wastewater solids coupled with a two-step detection protocol consisting of pre-amplification followed by qPCR to reduce false negative reporting. We applied this method to raw municipal wastewater collected from within six states across the United States over a period of four months. Resultant wastewater-derived data were then compared to clinical reports of mpox cases from the corresponding regions. Finally, a state of the science review was conducted to understand the current status of MPXV detection in community wastewater and inform future work.

3.1. Sample preparation and DNA extraction method performance

To evaluate recovery of MPXV DNA with the extraction method reported herein, a subset (n = 5) of raw wastewater samples from the sample set were spiked with a surrogate virus, MHV68. Spiking of MHV68 into raw wastewater occurred prior to filtration and the samples were subjected to the same extraction steps as all other samples. All samples were analyzed via qPCR in triplicate (no pre-amplification) and average calculated recovery ranged from 12–35% (average 22 ± 10%) (Table S3). Whole-process blanks and DNA extraction blanks were also performed using deionized water (n = 3 each) and confirmed that no MPXV contamination was introduced in the laboratory.

3.2. Mpox viral DNA detections in community wastewater

In total, 60 raw wastewater samples were processed and analyzed for MPXV DNA. Prior work from others as well as initial results from this study indicated that MPXV DNA would be present at relatively low concentrations in wastewater (de Jonge et al., 2022; Girón-Guzmán et al., 2023; La Rosa et al., 2023; Mejia et al., 2022; Sharkey et al., 2022; Wannigama et al., 2023; Wolfe et al., 2023; Wurtzer et al., 2022). Thus, pre-amplification was employed to increase the concentration of the target MPXV sequence in the DNA extracts, thereby decreasing the potential for false negative detections in subsequent qPCR analyses. The incorporation of this pre-amplification step was motivated by prior studies which employed pre-amplification prior to qPCR for sensitive detection of SARS-CoV-2 RNA in wastewater samples (Ando et al., 2022; Katayama et al., 2023). In total, eight samples (13%) resulted in positive detections: one in Illinois, one in New Jersey, two in Texas, and four in Washington (Figure 1). Positive wastewater samples ranged in date from the week of July 3rd, 2022 through the week of October 9th, 2022 (Figure 1), coinciding with the general period of the 2022 mpox outbreak in the United States (based on clinical reports) (Centers for Disease Control and Prevention, 2023b). Cycle threshold (Ct) values for positive wastewater samples are provided in Table S4.

Figure 1.

Figure 1.

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MPXV DNA detections in wastewater. Left panel shows a map of the United States indicating states included in this study and states where MPXV DNA was detected in wastewater. For each state, the percentage of samples that tested positive at any point during the study period is indicated. Right panel shows detections of MPXV DNA by site over the period of February 27th through October 16th, 2022. Dates are provided in month/day format. State abbreviations with subscripts indicate multiple sampling sites within the same state (TX = 3 sites; WA = 2 sites). Note that the absence of MPXV DNA in any given wastewater sample does not preclude the presence of mpox cases within the corresponding community/state at the time of sampling.

Importantly, the presence of MPXV DNA in positive wastewater samples was validated via Sanger sequencing of a 475 bp amplicon. Quality sequencing data were obtained for seven of the eight samples and consensus sequences were analyzed via BLAST. In every case, at least the top 500 hits were to the mpox genome (100% query cover, 100% identity). Sequences and BLAST hits are summarized in Table S5. Consensus sequences were also analyzed using Nextclade for clade/lineage assignment, and all seven sequences were assigned to clade IIb, lineage A.1.1. This clade assignment is in agreement with existing knowledge that variants circulating during the 2022 mpox outbreak can largely be traced to clade IIb (Luna et al., 2022). We did not obtain quality sequencing data for one of the eight qPCR-positive samples (TX1, week of September 25th). This could be due to an insufficient amount of MPXV DNA target in the sample prepared for sequencing and/or the inherent complexity of DNA extracts derived from wastewater samples (Mejia et al., 2022).

Reproducibility of the pre-amplification plus qPCR detection method reported herein was evaluated by performing multiple pre-amplification experiments for most MPXV DNA-positive samples. Each pre-amplification experiment was analyzed in triplicate via qPCR and all Ct values are provided in Table S4. For comparison, DNA from the positive samples was also analyzed via qPCR without pre-amplification. Overall, the qPCR-only detection method yielded high (38–47), often irreproducible Ct values (Figure S2), with many replicates providing no detection even for samples where the presence of MPXV DNA was confirmed via Sanger sequencing, resulting in a false negative rate of 39% (i.e., 17 non-detects out of 44 total qPCR replicates of positive samples) (Table S4). Conversely, the pre-amplification plus qPCR detection method yielded low (6–10), reproducible Ct values (Figure S2) with only one false negative amongst 20 total pre-amplification experiments (5%) (Table S4). This represents an 87% reduction in the false negative rate as compared to detection using qPCR alone.

3.3. Comparison to clinically reported cases

The wastewater detections of MPXV in the states of New Jersey, Illinois, Washington, and Texas were compared with clinical reports of mpox cases in the counties where the samples were collected. County-level mpox case counts were retrieved from publicly available datasets and are shown here by state for de-identification purposes. Note that due to this de-identification process, the number of clinical cases displayed here should not be interpreted as the total case count per state. Each state’s clinical case data were aggregated by week and assessed alongside wastewater-derived detections of MPXV DNA (Figure 2). Clinical reports of mpox began to increase in mid-June in the states of Texas, Washington, and New Jersey, going from five (combined) reported cases the week of June 12th to 70 cases the week of June 26th. The first wastewater-derived detection in this study occurred in Washington the following week of July 3rd. MPXV DNA was then detected in three states (Washington, New Jersey, and Illinois) the week of July 31st, near the peak of clinical case reports (242 total cases across all four states). By mid-August, clinical case reports had begun to decline in all four states, though MPXV DNA was still detected in wastewater during this period. Four more wastewater detections occurred the weeks of August 28th (Washington), September 25th (Texas and Washington), and October 9th (Texas), resulting in 50% of the eight total wastewater detections occurring during the initial increase in reported mpox cases and 50% occurring as clinical cases waned (Figure 2).

Figure 2.

Figure 2.

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Comparison of mpox clinical cases reported from the week of May 8th, 2022 through the week of October 23rd, 2022 to wastewater detections of MPXV DNA in this study for the states of Texas, Washington, New Jersey, and Illinois. Mpox clinical case counts were retrieved from the counties where positive wastewater samples were collected and de-identified by reporting here at the state level. Dates are provided in month/day format. Wastewater results across the indicated time period are represented by the bars above each graph. A red square indicates that MPXV DNA was detected in at least one sample from the corresponding state that week. A green square indicates that wastewater sample(s) from that week were analyzed but MPXV DNA was not detected in any sample. Grey indicates that no wastewater sample was analyzed that week.

4. Discussion

The rapid emergence of mpox in non-endemic regions of the world in May of 2022 prompted the investigation of wastewater-based epidemiology as a tool to assist in public health monitoring and decision-making. Multiple groups sought to rapidly accommodate this need, some by leveraging existing SARS-CoV-2/COVID-19 monitoring campaigns and/or adapting their protocols for the detection of MPXV DNA (de Jonge et al., 2022; Girón-Guzmán et al., 2023; La Rosa et al., 2023; Sharkey et al., 2022; Wannigama et al., 2023; Wolfe et al., 2023; Wurtzer et al., 2022). Here, we present a method for MPXV DNA extraction from the captured solids of raw wastewater coupled with a PCR detection protocol that reduces the chance of false negatives when reporting on low-titer pathogens, such as MPXV. Samples were collected from within six states across the United States (New Jersey, Illinois, Washington, Texas, Arizona, and Georgia) throughout the 2022 mpox outbreak (between July 3rd and October 16th, 2022), as well as prior to the outbreak (February-March, 2022) to confirm the absence of MPXV DNA in U.S. wastewater at that time.

Although multiple groups sought to adapt existing SARS-CoV-2 monitoring campaigns for the detection of MPXV in wastewater, there are noteworthy differences between these two viruses/outbreaks. For example, SARS-CoV-2 is an RNA virus while mpox is a DNA virus, and the scale of the 2022 mpox outbreak in the U.S. was significantly smaller than the scale of the COVID-19 pandemic (26,820 reported U.S. mpox cases between July 3rd and October 15th, 2022 versus 9,359,712 reported U.S. COVID-19 cases between July 6th and October 12th, 2022) (Centers for Disease Control and Prevention, 2023b, 2023c). The relatively low mpox case counts meant that lower quantities of MPXV DNA would be expected in wastewater samples as compared to SARS-CoV-2 RNA, which has been detected at quantifiable levels in both liquid and solid matrices (Graham et al., 2021; Kim et al., 2022; Li et al., 2021). Early on in this study, an established SARS-CoV-2 processing pipeline was tested to determine its feasibility for MPXV DNA detection (Bowes et al., 2023). In short, raw wastewater was vacuum filtered (0.45 μm polyethersulfone membrane) and concentrates were prepared using 10 kDa molecular weight cutoff centrifugal filters. However, DNA yield using this approach was low and MPXV DNA was not detected in any of these samples via qPCR.

Since MPXV is known to be shed in feces (Antinori et al., 2022; Peiró-Mestres et al., 2022), it was decided in this study, as in others (de Jonge et al., 2022; Mejia et al., 2022; Sharkey et al., 2022; Wolfe et al., 2023; Wurtzer et al., 2022), to transition to the use of captured (and/or settled) solids as a better matrix for this application. However, detection by qPCR alone was not always reliable, as shown in Table S4 where qPCR technical replicates were not reproducible or, in some cases, no detections were achieved at all. A review of the literature revealed this irreproducibility phenomenon in other reports of mpox monitoring in wastewater as well (Girón-Guzmán et al., 2023; La Rosa et al., 2023; Wurtzer et al., 2022). Furthermore, the Ct values of the MPXV DNA-positive samples (38–47) were outside the range of the standard curve used in this study. This observation, in addition to irreproducible qPCR replicates, raised the question of whether accurate quantification could be achieved. This led to the integration of pre-amplification of MPXV DNA prior to qPCR analysis in order to reduce the potential for false negatives, thereby increasing confidence in the reported results. Incorporation of this pre-amplification step revealed that MPXV DNA in one sample (TX1 on October 10th) would have otherwise gone entirely undetected if only qPCR methods were implemented (Table S4, Figure S2). Five other samples consistently tested positive for MPXV DNA using the combined pre-amplification plus qPCR approach, but yielded a mixture of detects and non-detects when qPCR was employed without pre-amplification (Ct values ranging between 38–46). Only two samples resulted in 100% reproducible detections between all replicates using qPCR alone (Table S4). Both of these samples were from the same site in Washington during the weeks of July 31st and August 28th; the former near the height of the outbreak in that state according to clinical case reports (57 cases) and the latter as cases waned (25 cases) (Figure 2). In summary, incorporating pre-amplification prior to qPCR minimized doubt in reporting the presence or absence of MPXV DNA in community wastewater, thereby decreasing the false negative rate from 39% to 5% (87% reduction) (Table S4) while still yielding sound qualitative data, as demonstrated by the validation of positive samples via Sanger sequencing.

After performing multiple experiments to validate detections, a total of eight wastewater samples from four states tested positive for MPXV DNA throughout the duration of this study: Washington (four detections), Texas (two), New Jersey (one), and Illinois (one). These eight detections were overlapped with clinical case reports from the same regions to compare trends (Figure 2). Four out of the eight wastewater detections (50%) occurred as clinical cases were increasing/peaking throughout the month of July. Three of these four detections occurred near the height of clinical case reports across all four states (week of July 31st), which seems reasonable given the number of individuals who fell ill during this period and were actively shedding the virus into wastewater. The remaining four wastewater detections (50%) occurred as clinical case reports were decreasing in late summer and early fall (autumn), demonstrating the robust ability of WBE to continue to capture the signal of viral presence in communities. Notably, the wastewater detection from a site in Texas during the week of October 9th occurred when only seven new clinical cases were reported. This ability of WBE to detect pathogens even during periods of low clinical case counts has been demonstrated repeatedly when monitoring for SARS-CoV-2 throughout the various waves of the global COVID-19 pandemic (Acosta et al., 2022; Gonçalves et al., 2021).

There were some observed disparities between the clinical and wastewater datasets, including wastewater non-detections at times of relatively high clinical case counts and vice versa. It is important to note that there are intricacies associated with each dataset that should be considered. For instance, clinical case count data can be impacted by factors such as access to testing, willingness to seek testing, and testing fatigue, as was demonstrated throughout the COVID-19 pandemic (Bowes et al., 2023; Embrett et al., 2022; Olesen et al., 2021; Safford et al., 2022). Additionally, the clinical case counts presented in Figure 2 were obtained at the county level, then aggregated, where applicable, by state. The wastewater samples were largely collected from wastewater treatment plants (WWTPs), which may serve a subset of the county’s residents. Thus, if mpox cases were clustered in an area of a county not served by the sampled WWTP, these cases likely would not be reflected in the wastewater samples analyzed. Furthermore, while WBE has proven to be an effective, low-cost, and anonymized technique for pathogen surveillance, WBE has its own set of challenges that must be considered, not least of which are in-sewer target dilution and degradation (Hart and Halden, 2020). These factors can severely impact the ability to detect a pathogen sequence of interest at the WWTP level, even if there are infected individuals in the catchment area. Additionally, sampling more often (e.g., weekly or daily instead of every other week) likely would provide a more granular, continuous picture of mpox spread within a community, and further optimizing the DNA extraction protocol described herein may enable more sensitive detection of MPXV DNA in the future. Overall, we suggest engaging with both clinical and wastewater datasets for the most complete picture of mpox spread within a community of interest.

Since the start of the 2022 mpox outbreak, MPXV DNA detection in wastewater has been reported from multiple countries and regions throughout the world, including the Netherlands (de Jonge et al., 2022), Spain (Girón-Guzmán et al., 2023), Italy (La Rosa et al., 2023), France (Wurtzer et al., 2022), Canada (Mejia et al., 2022), Thailand (Wannigama et al., 2023), Poland (Gazecka et al., 2023), and the United States (Sharkey et al., 2022; Wolfe et al., 2023), highlighting the scale of the outbreak and the utility of WBE for supporting public health strategies. While the study reported herein offers a reliable method for detecting MPXV in wastewater that minimizes the chances of reporting false negatives, certain challenges prompted an in-depth investigation of the current literature to understand how other groups approached detecting, monitoring, and quantifying MPXV DNA in wastewater. In total, nine experimental studies reporting on MPXV DNA detection in wastewater were analyzed, consisting of both peer-reviewed (seven) and non-peer-reviewed (i.e., preprint; two) reports (Table 1). Key features of each study were noted, including study date range, study location, source(s) of wastewater (WWTP, in-sewer, etc.), type and frequency of sample collection (grab versus composite, daily versus weekly, etc.), total number of samples collected and analyzed, sample preparation protocol, nucleic acid extraction method, qPCR assay(s) employed, number of MPXV detections, quantification and Ct value range, reported controls, and method validation. This information is summarized in Table 1 along with corresponding information for the method reported in this work. Most often, samples were collected from wastewater treatment plants (WWTPs). While MPXV DNA could be detected at the WWTP level, it is noted that higher concentrations may be measured and enhanced spatial resolution achieved if sampling occurred further upstream, such as at neighborhood- or building-level. The number of samples analyzed ranged greatly amongst all studies (approximately 20 to >600) and consisted of a mix of either grab or 24-hour composite samples. Sample collection frequencies also varied (e.g., daily, weekly, twice weekly, or every other week).

Table 1.

Results from state of the science literature review of experimental studies reporting on mpox virus detection in wastewater.

Study date range Location Source(s) of wastewater Type(s) of samples and frequency of collectiona Number of samples reported in studya
May 16th – July 3rd, 2022b Amsterdam, Netherlands 2 city WWTPs; 1 airport WWTP; 5 city districts City WWTPs: 24-h composite samples; Airport WWTP: unspecified sample type; City districts: composite samples 108 total samples; City WWTPs: 50 samples, airport WWTP: 23 samples, city districts: 35 samples
May 9th – August 4th, 2022 Spain 24 WWTPs Grab samples 312 total samples
May 30th – August 3rd, 2022 Rome, Italy Airport WWTP 24-h composite samples, 2×/week 20 total samples
Unspecified, at least July 11th – July 20th, 2022 Miami-Dade county, Florida, USA WWTP and hospital Grab and composite samples; DNA: weekly from both locations; RNA: daily from WWTP and weekly from hospital NR
April 11th – July 11th, 2022b Paris, France 16 sewersheds 24-h composite samples, 1×/week 321 total samples
June 28th – September 30th, 2022 10 anonymized cities in Canada 22 WWTPs 24-h composite samples, 2×/week ~616 total samples
May – August, 2022b Bangkok, Thailand 63 sewered and non-sewered locations Samples collected every two weeks 378 total samples
June 19th – August 1st, 2022 Greater San Francisco Bay and Sacramento areas, California, USA 9 WWTPs Solids: daily; liquid: 24-h composite, daily from two WWTPs 407 total solids samples; 15 total liquid samples
July to December, 2022 Poznan, Poland 2 WWTPs 24-h composite samples, 1×/week 44 total samples
July 4th – October 16th, 2022b New Jersey, Georgia, Illinois, Texas, Arizona, and Washington, USA 8 WWTPs; 1 in-sewer (pre-WWTP) 24-h composite samples every other week 60 total samples
Sample preparation protocol Nucleic acid extraction/purification protocol qPCR assay(s) Quantification
Centrifugation to isolate solids QIAGEN DNeasy blood and tissue kit followed by Zymo Research OneStep PCR inhibitor removal kit Multiplexed using modified generic and West African MPXV assays from Li et al. (2010) No
Aluminum-based adsorption precipitation Promega Maxwell RSC PureFood GMO and authentication kit using the Promega Maxwell RSC instrument Generic and West African MPXV assays from Li et al. (2010) Yes (2.2 × 103 – 8.7 × 104 gc/L)
PEG/NaCl precipitation bioMerieux NucliSens miniMAG semi-automatic extraction platform followed by Zymo Research OneStep PCR inhibitor removal kit Modified generic MPXV assay from Li et al. (2010) and N3R and F3L assays from Kulesh et al. (2004) No
DNA: wastewater solids prepared via vacuum filtration; RNA: wastewater solids prepared via electronegative vacuum filtration DNA: Solids homogenized prior to using ZymoBIOMICS DNA Miniprep kit; RNA work: Zymo Research Quick-RNA Viral kit modified for the reduction of PCR inhibitors Assay targeting the CrmB region of the MPXV genome Yes; DNA prep: 4.7 × 103 – 6.8 × 103 gc/L; RNA prep: ~7.5 × 102 – 6.2 × 103 gc/L
Centrifugation to isolate solids QIAGEN PowerFecal Pro kit using the QIAsymphony automated extractor followed by Zymo Research OneStep PCR inhibitor removal kit Thermo Fisher assay #Vi07922155_s1 Yes (~2 × 103 – 4 × 104 gc/L)
Centrifugation to isolate solids Solids subjected to bead beating prior to DNA extraction using QIAGEN MagAttract PowerMicrobiome kit using Thermo Fisher KingFisher Flex instrument Generic and West African MPXV assays from Li et al. (2010) and G2R_NML assay (developed in-house) Yes (below sample limit of quantification (~3 × 103) – ~2 × 104 gc/L)
Centrifugation of wastewater and concentration of supernatant (100 kDa MWCO filters) QIAGEN DNeasy PowerSoil Pro kit Generic MPXV assay from CDC (Centers for Disease Control and Prevention Poxvirus and Rabies Branch, 2022) Yes (1.1 × 104 – 9.3 × 104 gc/L)
Solids: Settled wastewater solids collected from WWTPs, centrifuged and homogenized; Liquids: Ceres Nanotrap particles with Thermo Scientific KingFisher Flex system Solids: PerkinElmer chemagic Viral DNA/RNA 300 kit H96 using the chemagic 360 instrument, followed by Zymo Research OneStep-96 PCR inhibitor removal kit; Liquids: Applied Biosystems MagMAX Viral/Pathogen nucleic acid isolation kit using Thermo Fisher KingFisher Flex system followed by Zymo Research OneStep-96 PCR inhibitor removal kit Generic and West African MPXV assays from Li et al. (2010) (used digital droplet PCR) Yes (~9 × 102 – 2.4 × 104 gc/g dry weight of wastewater solids)
Samples concentrated (specifics not provided) Promega Wizard Enviro TNA kit Thermo Fisher assay #Vi07922155_s1 No
Wastewater solids prepared via vacuum filtration QIAGEN AllPrep PowerViral DNA/RNA extraction kit including PCR inhibitor removal Generic MPXV assay originally developed by Li et al. (2010) and modified by de Jonge et al. (2022) No
Recovery Number of detections a Range of Ct values Controls/validation reported
NR 45 35.7 – 42.9 Pre-outbreak samples as negative controls; Positive and negative qPCR controls; Positive samples required detection using two MPXV qPCR assays; Seminested PCR followed by gel electrophoresis and Sanger sequencing of a selection of positive samples with Ct values < 40
MPXV spike-in into negative WW samples (31.5 ± 15.9% undiluted samples; 45.5 ± 25.7% recovery 10-fold diluted samples) 56 34.3 – 44.3 Negative DNA extraction controls; Positive and negative qPCR controls; Subset of samples tested positive using both MPXV qPCR assays
NR 2 38.4 – 40.2 Positive qPCR controls; One sample positive using two MPXV qPCR assays; Nested PCR followed by gel electrophoresis and Sanger sequencing (one sample that tested positive via qPCR could not be confirmed via sequencing)
NR DNA: 2; RNA: 6 NR RNA: Sanger sequencing of two positive samples
Bovine coronavirus (75% recovery (CV of 12%)) 34 31.3 – 39.0 Pre-outbreak samples as negative controls; Extraction blanks; qPCR reproducibility assessed using pepper mild mottle virus (CV of 15%); Positive controls included for qPCR; Digital PCR performed on positive samples for confirmation and quantification (91% of qPCR detections confirmed via dPCR); Negative controls included for dPCR
NR Not specified, at least 77 ~31 – 40 Positive and negative qPCR controls; Subset of samples tested positive using >1 MPXV qPCR assay; Sanger sequencing of amplicons from qPCR-positive samples (validation rates of 16 – 76%, depending on amplicon)
NR 22 NR Samples prior to outbreak included as negative controls; Positive qPCR controls; Positive samples also required detection of human RNase P; Gel electrophoresis and Sanger sequencing of a subset of qPCR-positive samples (n = 6)
Bovine coronavirus (>10% recovery) Solids: 131; Liquids: 15 N/A Negative and positive extraction controls; Negative and positive PCR controls; Measured pepper mild mottle virus in samples; Re-analyzed a subset of generic MPXV assay-positive samples with West African assay
NR 9 Average of 38.25 ± 1.15; samples positive if Ct < 40 Measured pepper mild mottle virus in samples
Murine gammaherpesvirus (22 ± 10% recovery) 8 Pre-amplified samples: 5.5 – 10.0; non-pre-amplified samples: 38.1 – 46.9 Pre-outbreak samples as negative controls; Process blanks and negative DNA extraction controls; Negative pre-amplification controls; Positive and negative qPCR controls; Gel electrophoresis of qPCR products; PCR followed by gel electrophoresis and Sanger sequencing of qPCR-positive samples (88% of qPCR-positive samples confirmed by sequencing)
Other notes Source
Liquid wastewater concentrates were also analyzed for MPXV DNA; solids gave better results and therefore used for the study (de Jonge et al., 2022)
(Girón-Guzmán et al., 2023)
Authors describe work to optimize qPCR assays and establish LOD50 values; One additional sample tested negative via qPCR but positive via nested PCR and Sanger sequencing (La Rosa et al., 2023)
Only study of MPXV RNA in wastewater to date (Sharkey et al., 2022)
(Wurtzer et al., 2022)
Median effective volume of wastewater processed was 128.6 mL due to volume limitations in DNA extraction step; Report assay and sample limits of detection and quantification (Mejia et al., 2022)
(Wannigama et al., 2023)
Compared concentrations of MPXV DNA in settled solids versus liquid wastewater samples and found significantly higher concentrations in solids on a per mass basis (Wolfe et al., 2023)
(Gazecka et al., 2023)
MPXV DNA target was pre-amplified prior to performing qPCR This study
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a

This information was not always stated explicitly, so these values represent our best estimation based on the information provided in the manuscript, figures, and supplementary information.

b

Also included samples from before the 2022 mpox outbreak as negative controls NR = Not reported; N/A = Not applicable

Overall, captured solids that were either collected at the WWTP (Wolfe et al., 2023) or prepared in the laboratory either by centrifugation (de Jonge et al., 2022; Mejia et al., 2022; Wurtzer et al., 2022) or filtration (Sharkey et al., 2022; this study) were preferred over the liquid portion of raw wastewater for MPXV DNA detection. Wolfe et al. reported that MPXV DNA was ~103-fold more concentrated in wastewater solids as compared to liquids on a per mass basis (2023), and de Jonge et al. also commented that wastewater solids performed better for MPXV DNA detection as compared to liquids (2022). Similar observations have been made for SARS-CoV-2, where the viral RNA was found to be ~102–104-fold more concentrated in wastewater solids on a per mass basis (Graham et al., 2021; Kim et al., 2022; Li et al., 2021). Sample processing and DNA extraction/purification methods differed amongst the reported studies. Precipitation methods (Girón-Guzmán et al., 2023; La Rosa et al., 2023) were reported, however, as stated, centrifugation or filtration to isolate wastewater solids were the most popular approaches. These different sample preparation methods resulted in varied recovery rates. Methods which involved the extraction of DNA from wastewater solids had reported recoveries of >10% (Wolfe et al., 2023), 22% (this study), and 75% (Wurtzer et al., 2022). Both Wolfe et al. and Wurtzer et al. used a bovine coronavirus surrogate to estimate viral recovery, while this study employed a murine gammaherpesvirus (MHV68) surrogate, as it is an enveloped DNA virus with a size similar to MPXV (diameters of ~150–200 nm and ~200–250 nm, respectively (Bortz et al., 2003; Cho and Wenner, 1973; Stewart et al., 1996)). Recovery was also assessed for an aluminum-based adsorption-precipitation sample preparation method, reporting a range of 32–46% recovery using inactivated MPXV (Girón-Guzmán et al., 2023). For detection and quantification, qPCR assays that targeted the G2R region of the MPXV genome were the most commonly reported, with high Ct values suggesting relatively low concentrations of MPXV DNA in wastewater. As previously discussed, these high Ct values near the limits of qPCR detection and quantification may increase the potential for false negative detections, which prompted our use of pre-amplification of MPXV DNA prior to qPCR in order to enhance confidence in the reported results.

This literature analysis revealed a few overarching themes that could inform future monitoring of low-titer pathogens, such as MPXV, in community wastewater. First, sample collection should occur on a regular basis, such as multiple times weekly or daily if budgetary and logistical allowances can accommodate such a cadence. Regular sampling not only enhances the chances of capturing the pathogen signal in wastewater, but also provides greater temporal resolution to support real-time, actionable data. Second, as is the recommendation for most WBE studies regardless of the target, the use of composite samples rather than grab samples is recommended. There is ample evidence that composite samples provide a more representative snapshot of the community of interest than grab samples, which are more biased towards the material collected at one specific moment (Ahmed et al., 2021). Third, as evidenced by this pilot study as well as others, the use of captured solids rather than the liquid portion of wastewater may provide higher concentrations of pathogen nucleic acid and, in certain circumstances, may be the only way to detect MPXV DNA in wastewater. Of course, the relevance of this point will depend on the route(s) by which the pathogen of interest is shed into wastewater (e.g., urine, feces, etc.). Fourth, sample concentration and extraction methods varied across the studies analyzed here, yielding different recovery rates and ranges of MPXV DNA quantified (where reported). As a result of this variability and the limited number of reports to date, it is difficult at this time to pinpoint any particular method which will provide the greatest benefit across all metrics. Rather, this variability highlights the need for further experimentation in order to determine the most robust procedure for capturing low-abundance viruses in such a complex matrix. However, one post-DNA extraction step that was reported amongst multiple studies was PCR inhibitor removal (de Jonge et al., 2022; La Rosa et al., 2023; Sharkey et al., 2022; Wolfe et al., 2023; Wurtzer et al., 2022; this study), with one study specifically noting better results after inhibitor removal (de Jonge et al., 2022). Thus, the use of such a kit may be beneficial for future studies. Finally, the use of either a surrogate virus or, preferably, the actual virus of interest to evaluate the recovery of any method is essential. Given safety considerations of working with MPXV and the need for rapid turnaround to provide data to communities, several groups utilized surrogate viruses that were already in-house and actively in use for SARS-CoV-2 monitoring, such as bovine coronavirus. While useful, a more comparable virus (e.g., a DNA virus) would likely provide better insight into how MPXV would behave using the same method. One group reported the use of inactivated MPXV to assess recovery (Girón-Guzmán et al., 2023), and the study presented here introduced the use of a murine gammaherpesvirus (MHV68) as a surrogate. Since recovery is an essential parameter that must be evaluated for any method/pathogen of interest, and as the field of WBE continues to expand to monitor for other targets, it is essential that a suite of commercial products be developed that can mimic viral targets of interest and be safely handled by laboratory personnel.

For the experimental portion of this study, it is worth noting a few limitations. First, as this work utilized an existing SARS-CoV-2 monitoring network, sample collection frequencies (once every two weeks) were already established. As stated, a more frequent sample collection schedule (e.g., weekly or daily) may have afforded better chances for capturing the presence of mpox virus in wastewater, assuming the mpox virus was actively circulating in the community at that time. Second, given that the reported pilot study acted as a secondary analysis to a major analytical pipeline (SARS-CoV-2), at times there was inadequate sample volume to support both protocols. This was particularly a problem for Texas, and Washington to a lesser extent, in the months of July and August (Figure 1), which unfortunately resulted in a lack of mpox virus data for those weeks. Third, while the reported method was successful at detecting MPXV DNA in wastewater, it may not be amenable for high-throughput applications given that the filter on which solids are captured during vacuum filtration was subsequently removed and then subjected to nucleic acid extraction. Finally, though the pre-amplification protocol presented here helped to reduce false negative results, we note that the introduction of this step does not allow for quantification of MPXV DNA via qPCR, and therefore quantification was not attempted here.

5. Conclusions

The 2022 mpox outbreak rapidly spread to non-endemic regions of the world in the midst of the ongoing COVID-19 global pandemic. This sudden onset of cases prompted the investigation of WBE as a means to assess community-level infection and disease spread in support of public health decision-making. This study reports on a method for the extraction and detection of MPXV DNA from raw wastewater that is distinct from what is currently reported in the literature. The method reported herein can be utilized as a reliable approach to qualitatively assess the presence of mpox in communities given its ability to reduce false negatives as compared to a more traditional qPCR detection workflow. Moreover, results from a state of the science literature review revealed both trends and variability in the protocols currently used for MPXV detection in wastewater, identifying a need for further optimization of WBE methods for MPXV and, by extension, other low-abundance viruses. Overall, this study presents a comprehensive assessment of the current landscape of MPXV detection in wastewater and should be leveraged as a resource to inform subsequent investigation.

Supplementary Material

1
2

Highlights.

  • Mpox virus DNA detected in wastewater from four states across the U.S.

  • Pre-amplification of mpox virus DNA prior to qPCR reduced false negatives by 87%

  • Wastewater detections of mpox virus occurred at various stages of the 2022 outbreak

  • Literature review identified need for WBE method optimization for low-titer pathogens

Acknowledgements:

The authors would like to thank Dr. Xiaofang Jiang for her support as part of NIH NLM grant U01LM013129, Dr. Efrem Lim and LaRinda Holland for providing resources to support MHV68 recovery experiments, and Dr. Temitope O. C. Faleye for helpful conversations about Sanger sequencing. The Arizona State University Genomics Facility performed the sequencing described herein. The U.S. map displayed in Figure 1 and in the graphical abstract was created using MapChart.net.

Funding:

This study was made possible with funding from the NIH NLM (U01LM013129) and the Virginia G. Piper Charitable Trust (LTR 05/01/12).

Footnotes

Competing interests: EMD is a managing member of AquaVitas, LLC, a company working in the field of wastewater-based epidemiology. RUH is also a managing member of AquaVitas, LLC and founder of the ASU non-profit project OneWaterOneHealth operating in the same intellectual space.

Declaration of interests

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Rolf U. Halden reports financial support was provided by Virginia G Piper Charitable Trust. Rolf U. Halden reports financial support was provided by National Institutes of Health. Matthew Scotch reports financial support was provided by National Institutes of Health. Arvind Varsani reports financial support was provided by National Institutes of Health. Rolf U. Halden reports a relationship with AquaVitas, LLC that includes: board membership. Rolf U. Halden reports a relationship with OneWaterOneHealth Nonprofit Project that includes: board membership. Erin M. Driver reports a relationship with AquaVitas, LLC that includes: board membership.

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Data and materials availability:

All data needed to evaluate the conclusions in the paper are present in the main text and/or the supplementary materials. Special requests can be made to the corresponding author.

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Associated Data

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Supplementary Materials

1
NIHMS1926972-supplement-1.pdf (267.2KB, pdf)
2
NIHMS1926972-supplement-2.xlsx (19.9KB, xlsx)

Data Availability Statement

All data needed to evaluate the conclusions in the paper are present in the main text and/or the supplementary materials. Special requests can be made to the corresponding author.

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