Multifaceted Assessment of Wastewater-Based Epidemiology for SARS-CoV-2 in Selected Urban Communities in Davao City, Philippines: A Pilot Study

  1. Maria Catherine B. Otero
  2. Lyre Anni E. Murao
  3. Mary Antoinette G. Limen
  4. Daniel Rev A. Caalim
  5. Paul Lorenzo A. Gaite
  6. Michael G. Bacus
  7. Joan T. Acaso
  8. Refeim M. Miguel
  9. Kahlil Corazo
  10. Ineke E. Knot
  11. Homer Sajonia
  12. Francis L. de los Reyes
  13. Caroline Marie B. Jaraula
  14. Emmanuel S. Baja
  15. Dann Marie N. Del Mundo

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Abstract

Over 60 countries have integrated wastewater-based epidemiology (WBE) in their COVID-19 surveillance programs, focusing on wastewater treatment plants (WWTP). In this paper, we piloted the assessment of SARS-CoV-2 WBE as a complementary public health surveillance method in susceptible communities in a highly urbanized city without WWTP in the Philippines by exploring the extraction and detection methods, evaluating the contribution of physico-chemical–anthropogenic factors, and attempting whole-genome sequencing (WGS). Weekly wastewater samples were collected from sewer pipes or creeks in six communities with moderate-to-high risk of COVID-19 transmission, as categorized by the City Government of Davao from November to December 2020. Physico-chemical properties of the wastewater and anthropogenic conditions of the sites were noted. Samples were concentrated using a PEG-NaCl precipitation method and analyzed by RT-PCR to detect the SARS-CoV-2 N, RdRP, and E genes. A subset of nine samples were subjected to WGS using the Minion sequencing platform. SARS-CoV-2 RNA was detected in twenty-two samples (91.7%) regardless of the presence of new cases. Cycle threshold values correlated with RNA concentration and attack rate. The lack of a sewershed map in the sampled areas highlights the need to integrate this in the WBE planning. A combined analysis of wastewater physico-chemical parameters such as flow rate, surface water temperature, salinity, dissolved oxygen, and total dissolved solids provided insights on the ideal sampling location, time, and method for WBE, and their impact on RNA recovery. The contribution of fecal matter in the wastewater may also be assessed through the coliform count and in the context of anthropogenic conditions in the area. Finally, our attempt on WGS detected single-nucleotide polymorphisms (SNPs) in wastewater which included clinically reported and newly identified mutations in the Philippines. This exploratory report provides a contextualized framework for applying WBE surveillance in low-sanitation areas.

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  1. Version published to 10.3390/ijerph19148789
  2. ScreenIT

    SciScore for 10.1101/2021.08.27.21262450: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsIRB: The study protocol was reviewed and approved by an independent Research Ethics Board (BMH-REC Code # 2020-08-02).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Linear regression analysis using the IBM SPSS Software (version 21) was used to assess if the Ct of the E, N, and RdRP genes were correlated with the RNA concentration and the computed weekly Attack Rate.
    SPSS
    suggested: (SPSS, RRID:SCR_002865)
    Whole Genome Sequencing: Eleven (11) wastewater samples that were positive for all targets (RdRP, N, and E genes) and with Ct values ranging from 29 to 39 were chosen for WGS (Supplemental Material, Sample details).
    WGS
    suggested: None
    Sequencing using a FLO-MIN106 flow cell on the MinION Mk1B device (Oxford Nanopore Technologies, United Kingdom) was allowed to run for approximately 24 hours.
    MinION
    suggested: (MinION, RRID:SCR_017985)
    Bioinformatic Analysis: Bioinformatic analysis was performed following the nCoV-2019 novel coronavirus bioinformatics protocol by the ARTIC Network (Loman et al., 2020), with several modifications made according to the actual experimental conditions.
    Bioinformatic
    suggested: (QFAB Bioinformatics, RRID:SCR_012513)
    Briefly, MinKNOW software was used to generate raw sequencing read files in FAST5 file format.
    MinKNOW
    suggested: None

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Even without testing resource limitations, COVID-19 infections in patients who purposely do not report having symptoms, and hence, do not get tested for COVID-19 because of fear of social stigma (Michael-Kordatou et al., 2020), or isolation and quarantine (Larsen and Wigginton, 2020) will not be captured by clinical surveillance and contact tracing. Wastewater-based epidemiology offers a complementary public health surveillance method that provides community infection data faster using less resources than individual RT-PCR testing (Daughton, 2020). With long-term periodic community wastewater testing for SARS-CoV-2, WBE can present an unbiased snapshot of COVID-19 cases at the community level, whether asymptomatic, pre-symptomatic, or symptomatic (Wu et al., 2020). WBE can also be useful in suburban and rural communities, especially in low-resource settings, where people may have limited access to healthcare (Larsen and Wiggington, 2020). Genomic surveillance in wastewater was attempted in this pilot work, and an average read depth of genome coverage was calculated to be ∼20x, which indicates that SARS-CoV-2 genomes were indeed sequenced reasonably and accurately (Goldman and Domschke, 2014). However, fragmented genomes were generated probably due to 1) the problematic nature of the sample, which may have been composed of degraded viral genetic material, and 2) the high Ct values (above 30) of the samples, which is known to be technically challenging for whole genome sequenci...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.08.27.21262450v1 on medRxiv