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. 2023 Sep 10:890:164289.
doi: 10.1016/j.scitotenv.2023.164289. Epub 2023 May 20.

Monkeypox viral nucleic acids detected using both DNA and RNA extraction workflows

Mark E Sharkey  1 , Kristina M Babler  2 , Bhavarth S Shukla  1 , Samantha M Abelson  3 , Bader Alsuliman  3 , Ayaaz Amirali  2 , Samuel Comerford  1 , George S Grills  4 , Naresh Kumar  3 , Jennifer Laine  5 , Jisue Lee  6 , Walter E Lamar  7 , Christopher E Mason  8 , Johnathon Penso  3 , Brian D Reding  5 , Stephan C Schürer  9 , Mario Stevenson  1 , Dušica Vidović  10 , Helena M Solo-Gabriele  11
Affiliations

Monkeypox viral nucleic acids detected using both DNA and RNA extraction workflows

Mark E Sharkey et al. Sci Total Environ. .

Abstract

Molecular methods have been used to detect human pathogens in wastewater with sampling typically performed at wastewater treatment plants (WWTP) and upstream locations within the sewer system. A wastewater-based surveillance (WBS) program was established at the University of Miami (UM) in 2020, which included measurements of SARS-CoV-2 levels in wastewater from its hospital and within the regional WWTP. In addition to the development of a SARS-CoV-2 quantitative PCR (qPCR) assay, qPCR assays to detect other human pathogens of interest were also developed at UM. Here we report on the use of a modified set of reagents published by the CDC to detect nucleic acids of Monkeypox virus (MPXV) which emerged during May of 2022 to become a concern worldwide. Samples collected from the University hospital and from the regional WWTP were processed through DNA and RNA workflows and analyzed by qPCR to detect a segment of the MPXV CrmB gene. Results show positive detections of MPXV nucleic acids in the hospital and wastewater treatment plant wastewater which coincided with clinical cases in the community and mirrored the overall trend of nationwide MPXV cases reported to the CDC. We recommend the expansion of current WBS programs' methods to detect a broader range of pathogens of concern in wastewater and present evidence that viral RNA in human cells infected by a DNA virus can be detected in wastewater.

Keywords: COVID-19; MPXV; Monkeypox virus; Mpox; SARS-CoV-2 wastewater-based surveillance; Wastewater.

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Conflict of interest statement

Declaration of competing interest The authors declare no competing interests.

Figures

Unlabelled Image
Graphical abstract
Fig. 1
Fig. 1
Number of clinical MPXV cases during May 2022 through January 2023 for UHealth Tower, Miami-Dade County, Florida, and the U.S.
Fig. 2
Fig. 2
MPXV genomic DNA was detected in concentrated wastewater collected weekly at the Miami-Dade Central District Wastewater Treatment Plant. MPXV target was first detected by quantitative PCR in mid-July of 2022 and remained detectable until testing negative in September. All weekly samples analyzed from June 8 through July 6, plus September 7 to September 21 were below detection limits of 180 gc/L.
Fig. 3
Fig. 3
MPXV RNA was detected in concentrated wastewater collected daily at the Miami-Dade Central District Wastewater Treatment Plant. All daily samples analyzed for MPXV from June 1 to July 9, 2022 (data not shown below) were below detection limits of 180 gc/L. The MPXV target was first detected by quantitative PCR in mid-July of this year and increased over the sampling period (a). Amplified RNA products from two dates were sequenced and aligned with an MPXV genomic sequence submitted to GenBank (ON563414) in May 2022 (b) for confirmation. Flanking MPXV-US sequences are the PCR primer binding sites.
See this image and copyright information in PMC

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