PREreview of Wastewater detection of emerging arbovirus infections: Case study of Dengue in the United States

This review reflects comments and contributions from Femi Arogundade.

The study investigates the use of wastewater monitoring for detecting Dengue virus RNA in a community in Miami-Dade County, Florida. The research finds consistent detection of Dengue virus serotype 3 (DENV-3) in wastewater solids during a period of identified Dengue cases, suggesting the feasibility of using wastewater surveillance for early detection of emerging arbovirus infections, even in regions with low incidence rates. General comments include the robustness of the methodology, the potential for wastewater monitoring to supplement traditional surveillance efforts, and the need for further research to understand the relationship between measured concentrations of DENV RNA in wastewater and infections in the community.

General Comments:

Positive Aspects of the Paper

• The study introduces an innovative approach to disease surveillance by utilizing wastewater monitoring to detect Dengue virus RNA, offering a novel method for the early detection of arbovirus infections.

• The research demonstrates methodological rigour, with well-designed assays for Dengue virus serotypes, stringent quality control measures, and thorough testing of assay specificity and sensitivity, enhancing the reliability of the findings.

• The study addresses the challenge of under-reporting in Dengue cases, particularly in regions with low incidence rates, and highlights the potential of wastewater monitoring as a supplementary tool for public health surveillance.

• Wastewater analysis provides a population-level perspective, potentially capturing asymptomatic or mild cases that may be missed by traditional clinical surveillance, contributing to a more comprehensive understanding of the disease burden.

• The study demonstrates the feasibility of implementing wastewater monitoring even in regions with emerging arbovirus infections and low incidence rates, suggesting the broad applicability of this approach.

• The authors have made the data openly accessible through the Stanford Digital Repository, promoting transparency and enabling further scrutiny and validation of the findings. The provision of data in an openly accessible repository promotes transparency and allows for independent verification of results, a key strength in scientific research.

• The paper appropriately acknowledges limitations, such as the inability to distinguish between locally-acquired and travel-associated cases, emphasizing the need for further research to enhance the utility of wastewater surveillance.

• The inclusion of supporting information, including methodological details and additional figures and tables, enhances the paper's comprehensibility and allows for a more in-depth understanding of the research process.

Major Comments:

• The methodology demonstrates robustness through the validation of the Dengue virus assays for specificity and sensitivity, both in silico and in vitro. This strengthens the reliability of the results by ensuring that the assays accurately target the intended viral sequences.

• The incorporation of negative and positive controls in the environmental sample testing, as well as the assessment of BCoV recoveries and PMMoV values, indicates a rigorous quality control process. This enhances the validity of the experimental results and minimizes the potential for false positives or negatives.

• The detailed description of pre-analytical methods, including dewatering and RNA extraction, contributes to the transparency of the study. However, the potential impact of variations in sample processing time (1-3 days) on RNA degradation should be discussed and addressed.

• The use of digital droplet RT-PCR for measuring viral RNA concentrations is a strength, providing a sensitive and quantitative approach. However, additional details regarding the reproducibility and precision of this technique would further strengthen the methodological description.

• The conversion of RNA targets to concentrations per gram dry weight of solids is appropriate. However, more detailed information on the error calculations, especially associated with the Poisson distribution, would enhance the transparency of the reported concentrations.

• The study establishes a correlation between wastewater detection of DENV-3 RNA and reported Dengue cases, supporting the conclusion that wastewater monitoring could serve as an early indicator of arbovirus circulation. The consistent detection over multiple weeks strengthens this correlation.

• Adherence to EMMI (Environmental Microbiology Minimal Information) guidelines demonstrates a commitment to transparency and standardization in reporting environmental microbiology research, reinforcing the study's methodological reliability.

• While the study demonstrates the feasibility of wastewater monitoring in the studied region, caution should be exercised in generalizing the findings to other locations with potentially different demographic and environmental factors.

Minor Comments:

• Provide more details on the concurrent mosquito surveillance results, especially the consistent capture of Aedes aegypti without any mosquitoes testing positive for DENV. Discuss potential reasons for this discrepancy between human cases and mosquito surveillance.

• The study collected samples approximately three times per week over a three-month period. Was this frequency sufficient to capture potential variations in Dengue virus RNA concentrations over time, considering the dynamic nature of infectious disease outbreaks?

• How was the training and standardization of WWTP staff ensured during the sample collection process? Were there any efforts to minimize potential biases introduced by different staff members?

• Consider providing a concise summary or visual representation of the sampling dates and frequency for each WWTP to enhance the reader's understanding of the temporal distribution of samples.

• While the paper mentions the samples being collected using sterile containers, it might be useful to elaborate on additional quality control measures implemented during the field collection to ensure the integrity and representativeness of the samples.

• The Digital droplet RT-PCR analytical methods mention the labeling of probes with different fluorescent molecules for each Dengue virus type. Confirm if the choice of fluorescent labels was based on considerations such as compatibility and distinctiveness during multiplexing.

• Offer insights into the significance of consistently detecting DENV-3 RNA in wastewater samples. Discuss whether this suggests a dominance of DENV-3 in the community and its implications for public health interventions.

• Clarify the interpretation of the range of incidence rates (0.77 - 4.23 cases/1 million people) based on wastewater results. Discuss the implications of these rates in terms of the potential utility of wastewater monitoring for early outbreak detection.

• Given the consistent detection of DENV-3 RNA in wastewater samples, what implications does this have for the understanding of Dengue virus prevalence and transmission dynamics in the community? How does this relate to the reported cases of Dengue in the study area?

• Considering the inability to distinguish between locally-acquired and travel-associated cases in wastewater samples, how might this limitation impact the specificity of the findings, and what recommendations or considerations are suggested for future studies to address this challenge?

• Could you provide more details on the inhibitor removal kit used in the RNA extraction process, and how its application addresses potential issues in downstream RT-PCR? Additionally, what considerations were taken into account for the drying process of the dewatered solids?

• How was the decision made to use hydrolysis-probe RT-PCR assays for DENV Types 1, 2, 3, and 4? Were alternative methods considered, and what factors influenced the selection of this specific assay?

• The study collected three samples of wastewater solids per week from each of the three wastewater treatment plants. What considerations led to this sampling frequency, and how does it contribute to the robustness of the data? Were there challenges or limitations associated with this frequency?

• The three selected wastewater treatment plants are estimated to serve 95% of the county's population. How representative are these locations, and what potential biases or limitations might arise from not covering the entire geographic area?

• The study used bovine coronavirus (BCoV) vaccine as an RNA recovery control. Can you elaborate on the rationale behind choosing BCoV and how its use contributes to the accuracy of RNA recovery assessment?

• The use of digital droplet RT-PCR for measuring viral RNA concentrations is a key aspect of the methodology. Can you discuss the advantages and limitations of this method in the context of the study's objectives?

• What considerations were taken into account during the drying process of the dewatered solids to determine dry weight? How might variations in dry weight impact the accuracy of RNA concentration measurements?

• What potential sources of bias or confounding factors were considered during the design and execution of the study, particularly concerning the representativeness of the wastewater samples and their correlation with reported Dengue cases?

Comments on Statistical Analysis:

• The paper effectively communicates the statistical aspects of the study, such as the use of standard deviations to report errors associated with the Poisson distribution and variability among replicates. This clarity enhances the transparency of the statistical methodology.

• The methodology mentions the requirement of at least 3 positive droplets for a sample to be recorded as positive. Could the paper provide further justification for this threshold, and how was it determined? Discussing the rationale behind this criterion would strengthen the understanding of the results.

Suggestions for Future Studies:

• Undertake a comparative analysis between wastewater monitoring and traditional clinical surveillance methods. Assess the concordance between detected DENV RNA in wastewater and reported clinical cases, providing a more comprehensive understanding of the utility of wastewater surveillance.

• Conduct a more extended longitudinal study to further investigate the temporal dynamics of Dengue virus RNA in wastewater. This could provide insights into seasonal variations, patterns of virus shedding, and the potential for early detection of outbreaks.

• Expand the geographic coverage of wastewater sampling to cover a broader area within the study region. This could help in assessing the representativeness of the selected wastewater treatment plants and provide a more comprehensive picture of Dengue virus circulation in the community.

• Develop methods to differentiate between serotypes of Dengue virus in wastewater samples. This would enhance the ability to identify specific virus strains circulating in the population and improve the accuracy of surveillance data.

• Conduct further research on the characteristics of wastewater solids, including the persistence of viral RNA, to better understand the factors influencing the detectability and longevity of Dengue virus in this matrix.

• Integrate environmental variables such as temperature, rainfall, and land use into the analysis. This could provide a more comprehensive understanding of the factors influencing the presence and concentration of Dengue virus in wastewater.

• Stay abreast of technological advancements in wastewater analysis, such as improved RNA extraction methods or novel detection techniques. Adopting cutting-edge technologies could enhance the sensitivity and specificity of the surveillance approach.

Competing interests

The author declares that they have no competing interests.