Study design

The ORIGINS Project (ORIGINS) is a longitudinal birth cohort of Western Australian families recruited during pregnancy with the children currently being followed up to the age of 5 years (protocol described in the cohort profile [16]. ORIGINS collects longitudinal biological specimens, the details of which can be found in the biobank profile [17]. The study was approved by the Joondalup Health Campus Human Research Ethics Committee (ref:1440), and all caregivers give written informed consent for themselves and on behalf of their children [16, 17].

Participants

The 3948 participant families included in this report represent all ORIGINS caregivers who enrolled between 2017 and 2022, and consented to provide biological samples. Data presented in this analysis are for children whose caregivers provided consent for their child after birth by December 2022 (n = 3713). Participants who withdrew consent prior to this report and had specifically requested their data be destroyed were excluded.

Data collection

ORIGINS participant data were recorded in REDCap databases [18, 19]. Specimen data were recorded on a linked OpenSpecimen database (v9.0, Krishagni Solutions Pt.Ltd, Pune, Maharashtra, India). Data presented in this analysis were extracted between February and April 2023.

Urine sample collection schedule

Child urine samples were requested from participants at ages 2, 6 and 12 months, 3 and 5 years of age (Fig. 1) [17]. Reasons for urine collection were explained to participants during recruitment and as part of the written instructions included with each sample kit. Caregivers were instructed not to collect urine samples when their child was acutely unwell, such as with fever or diarrhea. Samples were to be collected on weekdays only, kept cool and out of direct sunlight, and returned to ORIGINS on the same day of collection.

Fig. 1

Child urine sampling protocol and timeline

Kit preparation

Kits included detailed instructions and paperwork for recording the collection date, time and method used. Contents of kits are presented in Supplementary Table 2 and Supplementary Fig. 1. The total cost of each collection kit at ages 2 to 12 months was $1.00AUD, and at ages 3 to 5 years was $0.32AUD.

Collection methodsClean catch (all ages)

The clean catch method was a collection option for all ages. Caregivers were instructed to complete hand hygiene and use the gloves provided when handling the contents of their kits. Using the wipe, caregivers cleaned their infant or child’s urethral area before placing the specimen jar in place. After collection, samples were immediately sealed, labelled with the date, time, and method of collection, and placed in a biohazard bag.

Cotton pad (2, 6 and 12 months of age only)

The cotton-pad collection method was an option for infants aged 2, 6 and 12 months only. Caregivers were instructed to complete hand hygiene and use the gloves provided when handling the contents of their collection kits. Caregivers then place a Tegaderm sticker and sterile cotton balls into a clean nappy and wait until the child passed urine. Once wet, caregivers removed the nappy and transferred the wet cotton balls to the sterile specimen jar. Samples were then sealed and labelled with the date, time and method of collection and placed in a biohazard bag. If the cotton balls had fecal contamination, caregivers were instructed to discard the sample and contact the ORIGINS team for another collection kit.

For both methods of collection, ORIGINS staff were available to discuss the protocol over the phone and anecdotal tips shared by caregivers during phone calls were passed on to others requesting further support. This included waiting until after the infant or child had passed a stool to begin collecting the urine to minimise fecal contamination, or placing a warm damp towel on the infant or child’s belly to prompt the passing of urine when the towel was removed [20].

Return of samples

Sample return options were outlined on the kit instructions and ORIGINS website. These options changed over the project duration to reflect movement of the central study site location, and in response to SARS-CoV-2 public health measures that limited access to hospital sites where the study was originally centered. A QR code system linked to the project website was adopted in later stages of the study to allow for rapid dissemination of changes to sample return options, and to minimise circulation of outdated information.

Sample return options were:

1)

In person drop-off by participants to designated Western Diagnostic Pathology collection centers across the Perth metropolitan area where 95% of active participants resided.

2)

In-person drop-off by participants to the study clinic site at Joondalup Health Campus or, beginning in November 2022, at the relocated study clinic site in the nearby suburb of Edgewater.

3)

Courier collection from participant’s homes (if within 100km from the central study site in Joondalup), which was offered from October 2019 to December 2022.

The courier service was introduced for the 2 and 6 month timepoints in response to participant feedback that in-person drop-off of samples was difficult for families with young infants. This service was expanded to all timepoints and proved essential for continuity of sample collection during 2020 and 2022 when appointments were performed virtually due to SARS-COV-2 public health measures. The cost per sample collection by courier was $25.00AUD inclusive of external courier fees and staff time to arrange and administrate the service. After samples were received by ORIGINS, they were transported to a central Western Diagnostic Pathology laboratory dedicated to ORIGINS’s sample processing and storage.

Processing and biobanking procedures

Samples were considered of acceptable quality in the laboratory if they met the following criteria: 1) sample was processed within 36 h from collection; 2) sample contained a minimum volume of 0.5 ml urine; 3) cotton pad was still wet (if applicable); 4) sample was free from visible fecal or other contamination; 5) sample was not knowingly frozen at any point after collection; 6) sample container was not compromised (e.g. cracked or leaking). Any sample not processed for any of these reasons was recorded. Method of processing and sample storage are outlined in the ORIGINS biobank paper [17].

Statistical analysis

Analyses of the data were performed between February and July 2023 using GraphPad Prism. Descriptive statistics are reported as means ± SD or frequencies and percentages. Unpaired, two-tailed t-tests for continuous variables and Chi-squared tests for categorical variables were used to assess differences between participants who complied and were classified as “responders” (returned at least one sample) or did not comply (did not return any samples) with the sample collection schedule. Participants with children who had not yet reached the first sample collection time point as of December 2022 were excluded from this analysis of compliance but are represented in the full cohort demographics data.