Clade-specific multiplex digital PCR assay for monkeypox virus detection in spike-in wastewater samples

  1. Melissa Pitton
  2. Charles Gan
  3. Patrick Schmidhalter
  4. Christoph Ort
  5. Timothy Julian

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Abstract

Monkeypox virus (MPXV) is a zoonotic pathogen that has recently caused outbreaks in non-endemic areas. Wastewater-based surveillance is independent of individual testing and offers substantial potential for monitoring population-level disease dynamics of MPXV at the clade-level, as clades differ in transmissibility and mortality rate. Here, we report the validation of a four-plex digital PCR assay to detect and quantify the major clades and subclades of MPXV. The assay demonstrated strong linearity, analytical sensitivity (limits of detection ranged from 6.7 to 9.6 copies per reaction across targets), and specificity in distinguishing and quantifying MPXV by clade, which is advantageous for both clinical and wastewater applications.

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  1. Version published to 10.1099/acmi.0.001041.v2 on Access Microbiology
  2. Access Microbiology

    The article presents a multiplex digital PCR assay for the detection of monkeypox. This is a study that would be of interest to the field and community. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments, particularly concerns brought up about the completeness of the methodological description and it's reproducibility (additional details are required, as indication in the reviewer comments).

  3. Access Microbiology

    Comments to Author

    This manuscript presents an adaptation of singleplex assays into four-plex digital PCR assay for detecting and differentiating monkeypox virus clades, with potential application in wastewater-based surveillance. The work is timely, well-structured, and addresses an important public health need. However, several methodological and interpretive limitations weaken the paper's impact and generalizability. Major revisions: 46-69: Missing critical methodological details: description of the dPCR data analysis pipeline, including: The software and version used. How fluorescence thresholds were established to discriminate positive from negative partitions. How the common challenge of "rain" (indeterminate droplets) was addressed in the analysis. Furthermore, the description of the multiplex assay development is ambiguous. To clarify the experimental design: Assay Validation Conditions: Please specify whether the performance of the four-plex assay (lines 71-79) was evaluated using: (a) Individual target templates analyzed in separate reactions containing the full 4-plex primer/probe mix, or (b) Mixtures of multiple target templates analyzed in a single reaction with the 4-plex mix. This distinction is crucial for interpreting the specificity data and understanding if cross-reactivity or competition between targets in a mixed template scenario was assessed during validation. 78-79: The term "mean separation score" is a useful metric. Providing a brief definition or description of how this score was calculated would be very helpful for readers, as methodologies can vary between software packages and custom analyses. 83- Figure reorganization for improved flow. The authors should move Figure 1 to the Supplement as a demonstration of gating strategy. Include supplementary figures S1 (Linearity) and S2 (LoD) to the main manuscript as Figures 1 and 2. This immediately presents the most critical results to the reader. 94-95: LoD analysis: Testing concentrations (20-80 gc/rxn) that are all below the eventual LoD makes the probit regression an extrapolation, not an interpolation. The LoD is determined from a model fitted to data points where detection was inconsistent (p

    Please rate the manuscript for methodological rigour

    Poor

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  4. Access Microbiology

    Comments to Author

    I suggest amending the title to reflect the limited and synthetic/spike in nature of the wastewater expt. this a short paper and thus many important details missing in abstract, intro and discussion. abstract: why clades important? can you report numbers? Unclear here/ambiguous on nature of WW work intro: virology (dsDNA virus with genes X that are targets for assays, and is related to other orthopox viruses like variola/small pox viruses) discussion: limited comparison to other established assays. This really needs amended.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  5. Version published to 10.1099/acmi.0.001041.v1 on Access Microbiology