Quantitative norovirus viral load is not affected by home storage of stool

Abstract In preparation of a clinical trial of norovirus treatment, there were concerns raised by FDA about risk of self‐storage of stool from patients infected with norovirus affecting quantitative assessments of norovirus RNA. Specifically, most home freezers are frost‐free and may expose the samples to multiple rounds of freeze‐thaw. Stool samples collected by the study team were stored at different lengths in a frost‐free freezer and at −80°C. Quantitative PCRs of norovirus were performed on all samples using the same assay. By all measures, there was no significant change in measured viral load with home storage.

to predict the infectivity of human norovirus. [6][7][8] Such surrogate-based studies have estimated that human norovirus could stay potentially infectious on frozen foods (less than or equal to −20 • C) and refrigerated foods (≤10 • C) for up to 6 months and up to 7 days. [5][6][7] While conducting a clinical trial of nitazoxanide against norovirus in transplant recipients (NNITS, ClinicalTrials.gov Identifier: NCT03395405), enrollment was challenged, in part, by patients need to frequently present to clinic to provide stool specimens for quantitative polymerase chain reaction (PCR) of norovirus, which was a key secondary outcome measure of the study. It was felt one way to reduce the need for visits would be to have patients collect and store stool using standard methods and containers at home. In reviewing our proposal, the Food and Drug Administration (FDA) noted that viral RNA in any clinical matrix, but particularly in stool, is not stable, especially if it is subjected to multiple rounds of freeze-thaw that could result in the breakdown of viral particles. Further, home self-storage would require use of self-defrosting home freezers, which would further expose the samples to multiple rounds of freeze-thaw and compromise the integrity of the viral RNA samples.
Given the known stability of virus in traditional freezers and to address the concerns raised by the FDA, we utilized residual samples collected and processed in our existing protocol and studied quantitative viral measurements after freezing at −80 • and after freezing in a frost-free freezer for 2 weeks and 2 months.

METHODS
Samples for this study were conducted as part of our IRB-approved

RESULTS
Stool samples were collected from two subjects at five different study visits (Subject 02ENW004 on visit 4; Subject 02ENW005 on visits 1, 3, and 8). The norovirus loads in the samples were determined by qPCRs TA B L E 1 GII noroviral loads in Ct, genome copies/PCR reaction, and genome copies/ml 10% stool of stool stored at −80 • C or in a frost-free freezer for indicated times Note: The multiplier 214.28571 is a factor to convert the value of genome copies/PCR reaction in the genome copies/ml 10% stool that is required by the MOP of the study. This multiplier was calculated based on number 6.022 × 10 23 , Avogadro's constant.
that were run in triplicates for each specimen. Only GII noroviruses were detected, and the results are shown for each sample and storage condition in Table 1. For each of the four samples, there is far less than 0.5 log difference in the genome copies/ml 10% stool across the three storage conditions (i.e., 6.2 log 10 copies/ml in all three conditions for sample #1). In no sample was there a reduction in the viral titer comparing samples stored at −80 • or stored in a frost-free freezer for up to 2 months. DISCUSSION These data suggest that there is not significant loss of viral load with the storage of stool in frost-free freezers. In fact, the variability of the data over time in storage is within 0.5 logs, which is the expected range of values in replicate. Further, there was no decline in detectable virus with longer storage in a frost-free freezer. As such, these data support the ability for patients to collect and store samples at home and reduce the number of in person visits to potentially improve enrollment of the study. Importantly, such an approach should not jeopardize virologic endpoints and may inform design of future studies.
In our current study, we proposed to have subjects present to clinic for samples to be collected at week 0, 1, 2, 3, 4, 8, 12, and 24 weeks. This represents significant burden on subjects to travel to clinic, mostly to deliver samples to the sites. Most other clinical data can be collected by a verbal discussion with the patient, which could be done at a distance for most appointments. Only visits 0, 1, 4, and 24 require in person visits to also collect blood. In designing the study, we selected 2 week and 2 month storage in a frost-free freezer as a surrogate of short and longer-term storage at home with the goal of collecting data quickly to address FDA concerns. If there had been any evidence of loss of agreement between samples stored at −80 • C and 2 week or 2 month storage, additional timepoints would have been studied.
Given the lack of significant change in viral load under various storage conditions in addition to prior studies in nonclinical settings demonstrating stability of norovirus for up to 6 months, we did not do additional studies to assess stability beyond 2 months. [5][6][7] We did note an increase in genome copies after 2 months of storage in samples 3 and 4. It is felt that this increase is small and likely the result of experimental variation.
One limitation is that this study did not assess levels of infectious A key challenge to conducting clinical studies is the frequency of in person visits. Approaches to minimize in person visits should improve patient enrollment and retention. We have demonstrated that selfstorage of stool specimens for quantitative norovirus PCR in frost-free freezers does not result in change in detection of viral load with storage. The data suggest that such a patient-focused approach will not jeopardize virologic endpoints and may improve enrollment. As such, it should be considered in future study designs of trials of norovirus epidemiology, prevention, or treatment.