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 | |||
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