Review 3: "Detection of Salmonella Typhi and blaCTX-M Genes in Drinking Water, Wastewater, and Environmental Biofilms in Sindh Province, Pakistan"

RR\ID Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review: This paper by Abraham et al. evaluated the feasibility and effectiveness of wastewater and clinical surveillance for S. Typhi in Vellore, India. The team collected both grab and Moore swab samples for a year and tested three genes of S. Typhi and HF183. Simultaneously they collected clinical cases and performed cross-sectional serosurvey from the area where the WW samples were collected. The study was well designed, and the results are informative. I have several minor suggestions that may improve the quality of the manuscript.

Abstract:

Results:

• 7.5% of grab and 15.3% “add paired” Moore swab …….

• The last sentence is not clear and needs to be rewritten. Monsoon season, membrane filtration, and Moore swab are three different things and using “-“ for membrane filtration and Moore swab seems not appropriate.

Conclusions:

• Replace “incidence” to “presence”

Keywords:

• Change “seroincidence” to “seroprevalence”

Author Summary:

• Change “seroincidence” to “seroprevalence”

Introduction:

• Better to use wastewater surveillance consistently through the whole paper and do not switch to environmental surveillance that is little different from WW surveillance.

• Last paragraph, …..ES as a “complement” to clinical surveillance

Methods:

• Needs detailed information for grab and Moore swab sample processing procedures like what volumes of grab and Moore swab samples were filtered, how Moore swab samples were processed, what temperature was used for culturing S. Typhi, and how many hours, etc.

• Need a paragraph for wastewater quality testing methods: pH, dissolved oxygen, total dissolved solids, and turbidity.

• Approximately one liter of grab samples was filtered? It much be a range of sample volumes since the filter is easy to be clogged.

• DNA extraction: is it a typo that 50-mL DNA was eluted?

• qPCR: suggest to have primers/probes sequencing information or what reference was used for the three gene detection; what positive control and negative controls were used; and how to define positive results?

• How to define HlyE-IgG positive?

• Weekly average rainfall: to replace “ “ with ‘  ‘

Results:

• S Typhi in WW samples: 517 using Moore swab? Moore swab is a sampling method not a concentration method. You need to revise the sentence.

• “The membrane filtration technique showed significantly elevated positivity in monsoon WW samples (aOR, 2.99; 95% CI, 1.03– 8.47). When you run the analysis for aOR estimate, what reference did you use for membrane filtration?

• Figure 2: need a legend for detailed description of the figure.

• Table 1: need to adjust the column and row and align them well and make the table nicer.

• Hospital cases of Typhoid fever: need case definition and inclusion/exclusion criteria

• Log-transformed HF183 in sewage (genome copies/μL): can you confirm the unit? Log10 GC/ul

• Figure 4 legend: need detailed description

• Table 2: need to adjust the rows and columns and make it nice

Discussion:

• The present study revealed a notable increase in WW positivity trends before the wetter months: can you use the raining mons instead of wetter months?