Evaluating the use of biobanked urine specimens for human urobiome studies

Case-control studies focused on the urinary tract microbiome, or urobiome, have consistently reported significant associations with disease. However, clinical urobiome studies have typically been small, averaging ~50 patients per study. While these sample sizes are sufficient to detect large effect sizes, they have not been able to differentiate disease phenotypes within a larger disease complex (e.g., different types of kidney stones), which have unique etiological origins. Biobanked urine specimens can help fill this void. However, since these specimens were not collected specifically for urobiome studies, they must be validated before drawing any strong conclusions. The objective of this study was to evaluate microbiome data derived from metagenomic analysis of biobanked urine specimens against the following criteria: (i) level of contaminants; (ii) retention of high-quality DNA; (iii) overgrowth of a few dominant bacteria; and (iv) preservation of sex-specific taxa. A total of 174 samples were assessed from biobanked or freshly collected specimens (<i>N</i> = 118 patients total), in addition to multiple positive and negative controls. While there were significant differences in diversity (alpha/beta; <i>P</i> < 0.001) based on whether or not samples were biobanked, these differences can largely be explained by study-specific variation. With these criteria, we find that biobanked urine specimens provide similar data to fresh specimens collected using standardized protocols and can be used for clinical urobiome studies.IMPORTANCEThe urinary tract microbiome, or urobiome, is an emerging field of study that has shown promise as an important contributor to urologic health and disease. However, since this field is relatively new, clinical studies to evaluate the urobiome in the context of urologic disease have been relatively small. The use of biobanked urine specimens would allow for much larger studies to be conducted in a relatively short period of time. However, the use of biobanked urine specimens must first be validated. In this study, we sought to evaluate the use of biobanked urine specimens through multiple metrics, compared to previous studies conducted specifically to assess the impact of the urobiome. Results of our study suggest that biobanked urine specimens produce similar data to urine samples collected under rigorously controlled conditions and can be used in casecontrol studies of urologic conditions.