Wastewater PMMOV Normalization

Normalize wastewater gene copies by assayed/extraction volumes, shared recovery, and PMMoV concentration; not a case-count estimate.

Description

Convert wastewater assay copies to recovery-adjusted copies per liter and a pathogen-to-PMMoV ratio using shared processing volumes.

A wastewater assay measures genetic material in the small portion of an extract placed in a reaction. Copies per reaction therefore depend on how much wastewater was processed, the final extract volume, and the volume tested. Converting those copies to copies per liter puts measurements on a common sample-volume basis. A recovery fraction describes the share of material recovered during processing; dividing by that fraction adjusts for the assumed loss.^[1]

Pepper mild mottle virus, usually written PMMoV, is a plant virus used as a fecal marker in wastewater surveillance. Dividing a pathogen concentration by its PMMoV concentration produces a unitless ratio that can help account for differences in fecal content. Its usefulness depends on the sample and assay context.^[2]

Wastewater PMMOV Normalization returns concentrations for both targets and their ratio. It applies the same volume and recovery multiplier to each target, so that multiplier cancels from the ratio. The outputs describe the submitted assay measurements and assumptions; they do not estimate community case counts.

When to normalize wastewater assay measurements

Use this tool after an assay has supplied pathogen and PMMoV copy estimates from the same processing workflow. It helps reconcile per-reaction results with the original wastewater volume and compare a marker-normalized signal across samples.

Convert copies per reaction to copies per liter of processed wastewater.
Compare concentration changes with changes in the pathogen-to-PMMoV ratio.
Explore how an assumed shared recovery fraction changes concentrations while leaving the ratio unchanged.

How to use the tool and calculate normalized concentrations

Enter pathogen and PMMoV copies per reaction from matched assays.
Enter the total extract volume and reaction template volume in microliters, and the processed wastewater volume in milliliters.
Enter recovery as a fraction greater than zero and at most one; for example, 0.5 means 50% recovery.
Run the calculation and read both copies-per-liter outputs alongside the unitless pathogen PMMoV ratio.

Copies per reaction: Pathogen copies may be zero. PMMoV copies must be positive because they are the ratio denominator. Copy estimates may be fractional.
Processing volumes: All three volumes must be positive. The template volume cannot exceed the extract volume.
Recovery fraction: A shared positive recovery estimate applied to both targets. Use 1 when representing complete recovery in the supplied assumptions.

M = (extractVolumeUl / templateVolumeUl) × (1000 / processedWastewaterMl) / recoveryFraction

The extract-to-template ratio scales the assayed portion to the whole extract. The factor 1000 converts the wastewater volume basis from milliliters to liters. Recovery correction then scales for the supplied loss estimate.

pathogenCopiesPerLiter = pathogenCopiesPerReaction × M; pmmovCopiesPerLiter = pmmovCopiesPerReaction × M; pathogenPmmovRatio = pathogenCopiesPerReaction / pmmovCopiesPerReaction

The implementation calculates the ratio directly from reaction copies because both concentrations share M.

json

{
  "input": {
    "pathogenCopiesPerReaction": 10,
    "pmmovCopiesPerReaction": 100,
    "extractVolumeUl": 100,
    "templateVolumeUl": 5,
    "processedWastewaterMl": 100,
    "recoveryFraction": 0.5
  },
  "output": {
    "pathogenCopiesPerLiter": 4000,
    "pmmovCopiesPerLiter": 40000,
    "pathogenPmmovRatio": 0.1
  }
}

A supplied pathogen value of zero produces zero pathogen concentration and ratio. Missing fields, invalid types, nonfinite values, invalid volumes, zero PMMoV, or zero recovery produce an input error rather than normalized results.

Limitations of shared-recovery normalization

Both targets must be represented by the same extraction, template, wastewater-volume, and recovery assumptions. Separate target recoveries or additional dilution steps are not modeled.
A zero pathogen value is treated as a measured numeric zero. The tool does not distinguish nondetection from a true zero or apply detection-limit substitutions.
Copy counts measure genetic targets, and the tool does not determine infectivity, shedding per person, population size, flow-adjusted load, or case counts.
The calculation does not propagate assay or recovery uncertainty. Small PMMoV denominators can make ratios unstable.

PMMoV normalization does not consistently improve agreement with clinical indicators in every sewershed. Preserve the original concentrations and assay metadata when evaluating whether the ratio is useful for a particular site.

Alternative or Complementary assay quality checks

Review assay calibration, detection limits, and process-control recovery before comparing normalized signals. An internal-control Ct delay can flag a possible inhibition issue, but it does not supply the recovery fraction or repair the assay result. When targets have different processing factors, calculate their concentrations separately before forming a ratio.

References

Wastewater Surveillance Data Reporting and Analytics: concentration, recovery, and normalization — Centers for Disease Control and Prevention (archived guidance)
Omicron COVID-19 Case Estimates Based on Previous SARS-CoV-2 Wastewater Load, Regional Municipality of Peel, Ontario, Canada — Emerging Infectious Diseases / Centers for Disease Control and Prevention (2023)

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