Study setting

The study aims to recruit patients in the community (primary care or public health responses) and in public hospitals. The mode of collection will either be a self-collect rectal swab (done by the patient themselves) and bulk faeces sample or clinician collect rectal swab and bulk faeces sample within a clinical environment. The generalisability of this trial is supported by its relatively broad inclusion and exclusion criteria, which are designed to reflect routine clinical practice. Because the trial aims to evaluate the use of rectal swabs as an alternative to bulk faeces sampling, eligibility is not restricted (e.g., based on symptom presentation), allowing for the inclusion of participants with diverse clinical profiles. This inclusive approach will mimic routine clinical practice, and facilitate recruitment across a broad range of demographic, clinical, and socioeconomic backgrounds, enhancing the external validity of the findings. Vulnerable groups, such as comorbid or immunocompromised individuals, are identified in the recruitment phase and this data is recorded to be used in the analyses. The lower age limit of 18 years has been set due to ethical and regulatory considerations in recruiting children and performing rectal swabs on a vulnerable group. While children are not included in this study, the trial findings relevant to rectal swab accuracy are expected to be applicable to all ages.

Inclusion criteria:

• Persons aged 18 and older.
• Able to consent to and perform the additional rectal swab test.
• Close contact or person exposed, suspected, or confirmed case associated with an outbreak of an enteric organism. E.g., hepatitis A outbreak, foodborne outbreak.
• Chronic carrier of an enteric organism that requires testing.
• Person involved in a clinical encounter where they are or are likely suffering from a disease caused by an enteric organism. (E.g., person presenting to the GP or Emergency Department with diarrhoea or jaundice)

Exclusion criteria:

• Persons unlikely (as judged by the clinician) to be harbouring an enteric organism.
• Persons where collection of a rectal swab is medically contra-indicated.

Consent

In all study settings, the potential participant will be asked by the treating clinician for consent to be contacted by the local study coordinator. After which the local study coordinator/study nurse will contact the person to obtain consent for inclusion into the study.

There will be two ways that a participant can consent to participating (verbal, written). Verbal consent will be obtained over the phone with a study coordinator or study nurse, as is commonly done in outbreak situations. The study nurse will read the patient information sheet to the participant over the phone, ensure the participant understands the process and document this in a database held on Queensland Health data servers. The testing pack will be sent to the participant and will include the patient information sheet about the study and all contact details for questions about the study. Furthermore, implicit consent will occur when the participant obtains both samples (the participant can decline to participate by not providing the rectal swab) and returns one or both samples to the pathology laboratory.

If the testing is done in person (a study clinician is present), verbal, written and implicit consent (the patient will obtain the sample themselves and return it to the study coordinator) will be obtained using the patient information sheet, and the signature document will be kept, with a copy offered to the participant.

Opting out of the study will be possible for participants at all stages of the study, up until the analysis of the research. For example, a participant may agree to participation over the phone, then, once they receive the full pack with the Patient Information Sheet, and they read over it in detail, they can withdraw from the study by calling us and withdrawing, or, not sending the rectal swab, and then withdrawing after we check in with them after the rectal swab was not returned.

Risks and harm

Risks associated with the rectal swab sample collection process include discomfort or pain, and in rare cases, infection. In some cases, participants may experience minimal bleeding. There is a low to medium risk of these events occurring if sample collection is conducted properly. These risks are outlined in the patient information and consent form, ensuring that participants are fully informed prior to their involvement. To mitigate these risks, strict adherence to hygiene and sterile techniques is emphasized during both self-collection and clinician-guided collection.

Trial process

The site study coordinator will provide participants with oral and written information about the procedure, including its purpose, what to expect (2 tests being performed), and potential risks and then obtain verbal or written informed consent. The participant will ensure all necessary materials are present in the pre-prepared packs which will include sterile rectal swabs, bulk faeces collection sample container, gloves, pre-filled pathology form and labeling supplies. The participant or clinician will conduct the procedure in a private, clean, and comfortable setting to maintain participant dignity and comfort (preferably a toilet).

Outcomes

Primary Outcomes: Assess sensitivity, specificity, positive and negative predictive value, positive and negative agreement, accuracy, and receiver operating curves (at various CT value cutoffs), to determine the efficacy the new rectal swab approach.

Secondary outcomes: Acceptability of the rectal swabs in comparison to the gold standard of bulk faeces testing. This will be measured by asking participants which sampling method they preferred, and collecting information from participants in relation to any adverse events that may have occurred.

Timeline

Participant recruitment is expected to commence in early 2025. Recruitment and data collection are expected to be completed within two years. Results are expected shortly after, placing the trial completion date in late 2027 or early 2028. It is expected that an average of 3–6 swabs will be obtained per week across collection sites. Some situations, such as outbreaks or mass gathering events, will allow for greater numbers of swabs to be collected in a small timeframe. After participants return the test samples, they will receive a follow-up phone call. Results of the bulk faeces test will be returned to the patient by the clinician that requested the test.

Sample size calculation

A sample size analysis with a wide range of input parameters, as described by Hajian-Tilaki 2014, demonstrates that less than 600 samples will be sufficient to cover a range of diseases (Fig 2) [16]. Assuming that 30% of samples will contain an organism (“proportion positive”) with an expected sensitivity of at least 95% (“sensitivity priors”) means we will require a sample size of no more than 61 paired samples. As the prevalence of disease in samples decreases, the sample size required is higher. For a sensitivity of 95% and a prevalence of 20%, the sample size increases from 61 to 92. These estimates are based on general sensitivity assumptions and a simplified disease prevalence model, with the literature reporting non-specific prevalence to be approximately 25%, and sensitivity estimates typically >90% [17–20]. These estimates may change slightly as recruitment progresses and the distribution of pathogens becomes clearer. To address potential variability across different pathogens, stratified analyses will be conducted. Power calculations for key pathogen subgroups indicate that, for a sensitivity of 95% (with a range of 90–99%), a minimum of 73 positive samples is required per pathogen subgroup.

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Fig 2. Sample size analysis.

Includes a range of parameters. The “Sensitivity priors” represents the approximate expected sensitivity of our method, and the “proportion positive” represents the proportion of samples that will contain an organism, which also means that should test positive.

https://doi.org/10.1371/journal.pone.0329643.g002

Constrained resources (e.g., funding), demands a higher pre-test probability at the time of testing to estimate with confidence. For example, if we only have funding for 40 persons tested, we will elect to test persons with clear known linkages to a person with the illness (symptomatic family member of someone diagnosed with norovirus), or we would perform clearance testing in persons with known disease using these methods (the guidelines call for clearance testing for a variety of illnesses). With greater funding, testing can approximate normal clinical encounters and have less restrictive criteria for testing. If the funding is limited to 40 samples, we will endeavour to control the testing method, aiming for in-clinic self-testing.

Recruitment

The recruitment strategy will be multifaceted to obtain samples from a range of representative clinical settings, e.g., outbreaks, community, and in-hospital. The strategy will involve a central study coordinator supporting acquisition of samples, coordinating the multiple sources. For outbreaks, the study coordinator will be able to send sample collection packs to the patient, family, and/or symptomatic members of an outbreak via express post. In community settings, the study coordinator can obtain samples from GP practices that are seeing patients who were recently inpatients in hospital – identified from admission lists, or our Infectious Diseases Physician study collaborators. Inpatients, especially those which represent persons with a high pre-test probability, will be identified, recruited via their treating practitioner, and then consented to take part in the study. Consent will be performed verbally over the phone by a study coordinator (records kept locally), and/or with a physical form sent in a return envelope.

Laboratory testing

Faeces will be collected in standard collection containers and rectal swabs utilised will be dry cotton swabs. Samples will be registered and transported chilled using existing referral pathways. Biological materials will be stored in the laboratory in accordance with NPAAC minimum requirements and will be discarded once all required testing on these samples has been completed for assay validation, research and testing required for public health and/or clinical investigations. Faeces and rectal swabs samples will be subjected to nucleic acid extraction using the QIAGEN EZ or KingFisher FLEX instruments. Real-time RT-PCR will be performed to identify hepatitis A or E virus-positive specimens, using existing methods at the Public Health Virology laboratory, Queensland Health. Extracted RNA from hepatitis A or E virus RT-PCR positive samples will then be used to perform partial or whole genome sequencing. Paired rectal swabs and faeces samples from individual patients will be sequenced in parallel on the same sequencing run, using either Nanopore or Illumina sequencing chemistries and instruments. The partial or whole genome sequences generated from each patient will be analysed and firstly aligning with references sequences to determine genotype and then their nucleotide homology and phylogenetic relatedness will be compared to determine whether variation between the sequences from each respective sample type (i.e., faeces vs rectal swab) exists.

Data collection and management

Laboratory diagnostics will provide the majority of the outcomes using established testing methods. Pathologists will provide the diagnosis for samples. We will be collecting information about demographics, symptomatology, setting it was collected in, type of collect (self-collect vs clinician collect), and two patient reported outcomes by firstly asking the patient if they had an adverse event during or after the testing, and the nature of the event if present. Secondly, we will ask the patient which test they preferred and follow-up with why. Data analysis will be conducted by members of the research team.

Participant-level dataset will not be publicly accessible. Statistical code and full protocol will be publicly accessible.

Statistical methods

We will summarise safety and efficacy data using descriptive statistics, including means, medians, and frequencies. We will use statistical tests to compare groups (e.g., chi-square tests for categorical data, t-tests for continuous data). We will also adjust for multiple comparisons to control for type I error.

Logistic regression models will be used to assess accuracy in the presence of potential confounders. This includes sex (female/male), age (by age group), person collecting sample (self-collect/clinician collect), comorbidities, symptomatology (symptomatic/asymptomatic), duration of symptoms, and whether blood was present in the sample. Subgroup analysis will be conducted according to disease type (e.g., viral, or bacterial).

Oversight and monitoring

The Coordinating Centre manages the operational and logistical aspects, ensuring smooth trial execution, and the overall trial implementation. Protocol evaluation and amendments will be discussed at regular Coordinating Centre trial conduct meetings.

The roles outlined here may be performed by more than one person. i.e., the Project Manager may also do the role of the Data Manager. The roles include:

• Principal Investigator (PI): Provides scientific leadership and oversees the entire trial. Ensures the trial adheres to regulatory requirements, supervises data analysis, and maintains overall responsibility for the study’s conduct.
• Project Manager: Manages day-to-day operations of the trial. Coordinates activities across sites, manages timelines and budgets, and oversees staff at the coordinating center.
• Data Manager: Handles data collection, management, and analysis. Ensures data quality, integrity, and compliance with data protection regulations. Manages databases and performs statistical analyses.
• Clinical Research Coordinators: This role supports the PI and Project Manager in trial implementation. They are based on-site in a clinical setting and are responsible for the handling patient recruitment, informed consent, data entry, and follow-up activities.
• Regulatory Affairs Specialist: Ensures compliance with regulatory requirements. Manages submissions to ethics committees and regulatory bodies and maintains regulatory documentation.
• Quality Assurance (QA) Officer: Monitors trial conduct to ensure compliance with protocols and regulations. Conducts audits, manages deviations, and implements corrective actions.
• Statisticians: Provide statistical expertise for study design and analysis. Develop statistical analysis plans, perform interim and final analyses, and assist with interpretation of results.
• Administrative Staff: Provide administrative support to the coordinating center team. Handle documentation, scheduling, and communication tasks.

The Trial Steering Committee (TSC) provides independent oversight of the clinical trial. It ensures the trial is conducted according to the protocol and addresses any issues that arise during the study. The TSC provides independent oversight, ensuring scientific integrity, regulatory compliance, and participant safety. The TSC will have oversight of the interim analyses and key decisions such as a stop-early decision. Trial conduct will be discussed at TSC meetings and on demand by local ethics committees. The TSC will include:

• Independent Chairperson: Leads the TSC meetings and ensures unbiased oversight. Facilitates discussions, ensures all viewpoints are considered, and guides decision-making processes.
• Principal Investigator: Provides insights on trial progress and scientific aspects. Updates the TSC on trial status, protocol adherence, and emerging results.
• Independent Clinicians: Offer clinical expertise and independent perspectives. Review safety data, assess clinical implications, and provide recommendations based on their expertise.
• Regulatory Affairs Specialist: Ensures regulatory compliance and addresses regulatory issues. Provides updates on regulatory submissions, compliance, and any regulatory changes affecting the trial.

Adverse event reporting

An adverse event (AE) is any untoward medical occurrence in a patient or clinical trial participant administered a medicinal product and that does not necessarily have a causal relationship with this treatment. An adverse reaction is any untoward and unintended response to an investigational medicinal product related to any dose administered. Probable and/or definitely related AEs are defined as adverse reactions for this trial.

A serious adverse event (SAE)/serious adverse reaction (SAR) is any adverse event/adverse reaction that results in death, is life-threatening, requires hospitalisation or prolongation of existing hospitalisation, results in persistent or significant disability or incapacity.

AEs will be classified according to:

1. Severity
   • Mild: Adverse events that are easily tolerated and do not interfere with daily activities. These could include minor discomfort or irritation at the site of the rectal swab.
   • Moderate: Events that cause some interference with daily activities but are not dangerous. Examples may include moderate pain, cramping, or a temporary increase in bowel movement frequency.
   • Severe: Events that significantly interfere with daily activities or pose a risk to health. Severe adverse events might involve intense pain, bleeding, or infection requiring medical intervention.
2. Expectedness
   • Expected Adverse Events: These are events that are known to occur based on previous studies or the nature of the procedures. For rectal swabs, this could include expected mild discomfort or minor bleeding.
   • Unexpected Adverse Events: Events that are not anticipated based on prior knowledge. Unexpected events could be severe allergic reactions, systemic infections, or unusual gastrointestinal symptoms.
3. Relatedness to Procedure
   • Definitely Related: There is a clear, direct link between the adverse event and the rectal swab procedure. An example is rectal bleeding immediately following the swab.
   • Probably Related: There is a reasonable likelihood that the event is related to the procedure, but other factors could be involved. For instance, discomfort that occurs after the procedure and subsides soon afterward.
   • Possibly Related: The event could be related to the procedure but could also be attributed to other causes. This might include abdominal pain occurring days after the procedure.
   • Unrelated: The event has no apparent connection to the procedure. An example is an adverse event like a headache, which is likely due to other factors.
4. 4. Type of Adverse Event
   • Local Adverse Events: These are events occurring at the site of the rectal swab. They might include irritation, redness, or bleeding.
   • Systemic Adverse Events: Events affecting the body beyond the local site, such as fever, nausea, or generalised infection.
   • Procedure-Related Adverse Events: Specific to the process of specimen collection, like discomfort during the swab insertion or difficulties with the bulk faeces collection method.
   • Non-Procedure-Related Adverse Events: Unrelated to the procedure itself, such as a common cold or unrelated illness occurring during the trial period.

Dissemination plan

We will aim for a comprehensive local dissemination plan (including national conferences, local news networks, maximum reach for local population), progressing to international journals and conferences. Overall, this dissemination plan will reach consumers and the general public, clinicians, pathologists that may perform these tests and academics that may use this in their future work.