Key result
Correction for 20-fold pre-dilution increases RSV neutralization titers and reduces variance to reveal cohort differences.
Correcting a dilution error in RSV neutralization assay data strengthened the statistical significance of differences in antibody titers between recently infected individuals and the general population.
Assay pre-dilution correction sharpens RSV nAb titer comparisons; leaves open immunity debt in recent outbreak cohorts.
We discovered that calculation of titers for 10 serum samples run in the same batch on the same day failed to account for a 20-fold pre-dilution. All 10 specimens were from RSV PCR-positive individuals in the emergency department (ED), including 7 immunocompetent and 3 immunosuppressed individuals. Correction of the values for the pre-dilution leads to a 20-fold increase in the final titers for specimens from Individuals in Table S6: 06B4, 131F, 0646, 0CF5, 06E0, 029D, 11DB, 085B, 130E, and 0DEE. While comprehensively reviewing all data due to pre-dilution issue, we also found one specimen for individual 0786 from the RSV PCR-positive immunocompetent cohort that should not have passed QC and subsequently removed this specimen’s data from the analysis. The increased titers in these individuals significantly reduced the variance of RSV nAb titers in these cohorts and led to more statistically significant differences among individuals recently infected with RSV compared to the general population and individuals who later developed RSV infection, as one would hypothesize. We have updated Figure 3,
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Piliper et al. (2025) studied this question. Correction for a 20-fold pre-dilution significantly increased RSV neutralization assay titers, reducing variance and revealing statistically significant differences in cohorts.
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