Table 2. Risk assessment for Salmonella Typhimurium oral challenge.

Participants should be counselled for these risks in the participant information sheet and during screening. Biological safety and laboratory risk-assessments are not detailed. The following risks were highlighted as areas of focus during protocol development but are non-exhaustive.

Possible study risks	Comments	Risk mitigation strategies
Clinical Considerations
*Primary complications of Salmonella infection*
Severe gastroenteritis with dehydration	• Typical symptoms include watery diarrhoea, bloody diarrhoea, abdominal pain, nausea, vomiting, headache, and fever • Overall duration of symptoms typically ranges from 4 to 7 days • Total duration of illness is estimated to range between 3 to 19 days ⁴⁰.	• In-patient quarantine with continuous oversight from study physician and nurses (initial 7 days) • Daily stool output monitoring and culture • Daily assessment of symptoms and severity grading • Severe diarrhoea will be trigger antibiotic treatment • Oral rehydration for diarrhoea • Intravenous fluid replacement for severe hypovolaemia
Severe sepsis	• Complications of invasive Salmonella infection can include septicaemia, hypotension, tachycardia • Occur almost exclusively in clinically vulnerable and/or those who do not receive appropriate antibiotic treatment. • Estimated case-fatality rate 15-20% of iNTS in vulnerable patients ^9, 43. • ~5% of enteric infections with non-typhoidal Salmonella are thought to result in bacteraemia. • May increase to ~10% depending on underlying host risk factors and serotype ^6, 44. • Progression from enteric to systemic infection may occur more frequently in the context of altered gastric acid pH ⁴⁵, altered microbiota (including prior antibiotic treatment) ⁴⁶ and concomitant rotavirus infection ^47– 51.	• Exclusion of participants at higher risk of developing severe sepsis/ invasive disease • In-patient quarantine with continuous oversight from study physician and nurses. • Daily blood cultures to detect bloodstream infection • Early antimicrobial therapy in event of bloodstream infection or clinical signs/symptoms of developing sepsis. • Backup admission to medical inpatient unit and/or high-dependency unit and/or intensive care unit if required.
Deep-seated focal infection	• Bloodstream infection can lead to extra-intestinal focal infections at any site, including aortitis, osteomyelitis, meningitis, or arthritis. • Almost exclusively in clinically vulnerable and/or those who do not receive appropriate antibiotic treatment • Sickle cell disease is a recognised risk factor for osteomyelitis • Endovascular disease is associated with atherosclerotic disease, valvular disease, or endovascular prosthesis • Meningitis is a rare, but can occur in neonates, infants <12years of and in the context of advanced HIV infection.	• Exclusion of participants at risk e.g. underlying valvular heart disease, bone/joint disease (including prosthesis, metalwork), aneurysmal arterial disease • Clinical examination to exclude valvular heart disease. • Abdominal ultrasound during screening to detect asymptomatic biliary disease and aneurysmal disease of the abdominal aorta
*Post-infectious/Para-infectious complications*
Prolonged Salmonella shedding and/or microbiological relapse	• Convalescent shedding of Salmonella is common after symptomatic or asymptomatic NTS infection. • Microbiological relapse (with or without clinical symptoms) may develop post recovery and/or treatment • The precise duration of shedding is poorly understood. • In some instances, antibiotic treatment is associated with prolonged symptoms or higher rate of microbiological relapse. • Chronic carriage (shedding for >12months) is more common with host immunosuppression; in young children <5 and/or or in the context of biliary tract abnormalities. • Theoretical risk of secondary transmission if hygiene measures are not followed.	• Where required, use antibiotic classes not associated with prolonged shedding (e.g. fluoroquinolones) • Describe pattern of shedding by longitudinal collection of stool samples for detection of Salmonella spp. by PCR and culture. ○ Distinguish between PCR positive and culture positive samples. • Clearance stool cultures will be obtained upon completion of antibiotic therapy. ○ Follow guidance for typhoidal Salmonella i.e. three negative clearance stools. • Reinforce hygiene precautions after challenge. • Participants with chronic carriage (stool culture positive >4 weeks after antibiotics) can be referred to an Infectious Diseases specialist.
Clinical relapse	• Clinical relapse may develop post recovery and/or treatment • More common in immunocompromised patients. ○ Advanced HIV infection ⁵² ○ Chronic granulomatous disease ⁵³ ○ Defects in specific cytokine pathways ^10, 54 ○ Haematological malignancies ⁵⁵	• Exclusion of at-risk individuals • Follow up of participants for up to 1 year • Study team contactable 24/7 ○ Participants advised to contact study team in event of new symptoms
Reactive arthritis	• Gastrointestinal and genitourinary infections are commonly recognised triggers, including with Salmonella spp. • Typical symptoms - mono-arthritis or oligo-arthritis, often affecting the lower limbs, axial symptoms, enthesitis and/or dactylitis. • Symptom duration is highly variable – ○ most patients will have little-to-no symptoms at 6–12 months post onset. ○ A small proportion of patients may develop symptoms lasting >12 months ⁵⁶. • Prevalence of HLA-B27 increased in patients with reactive arthritis, although the estimates vary depending on illness definition and study design ^56– 58. • Presence of HLA-B27 is not essential for the development of reactive arthritis.	• HLA-B27 screening prior to enrolment • Participants who develop symptoms of reactive arthritis will be referred to a Rheumatology service
Post-infectious irritable bowel syndrome (IBS)	• Post-infectious irritable bowel syndrome is estimated to occur in 3–10% of patients following bacterial diarrhoea. • Symptoms generally resolve within 1 year ^59– 61.	• Exclusion of participants with pre-existing IBS • IBS questionnaire at baseline and post-challenge
Psychological impact of challenge and quarantine	• Study fatigue may occur due to the intense nature of the study procedures, especially during the quarantine period. • Participants may become anxious, lonely, or depressed by being confined to the quarantine unit for the minimum of 7 days.	• Anxiety and depression questionnaire during screening and after challenge. • Facilities to make phone and video calls with friends and family. • A small number of visitors may be allowed but would have to comply with a strict hygiene protocol.
*Risks associated with antibiotics*
Direct adverse effect of antibiotics	• Antibiotic intolerance: ○ gastro-intestinal upset, nausea, vomiting or other unspecified symptoms. • Antibiotic hypersensitivity ○ Rash, angio-oedema, difficulty in breathing anaphylaxis • Abnormal liver function tests or other biochemical abnormalities • Risk of antibiotic associated diarrhoea and/or Clostridioides difficile infection.	• Baseline ECG (QTC) • Regular measurements of liver function tests and electrolyte monitoring • Keep antibiotic treatment duration as short as possible • Exclude participants with known antibiotic hypersensitivity or allergy to either of the first-, second- and third-line antibiotics (ciprofloxacin, azithromycin, or other macrolide antibiotics and cephalosporins)
Carriage of multi-drug resistant organisms (MDRO)	• Antibiotic treatment may in theory increase the risk of carriage of drug resistant bacteria.	• Stool culture for carriage of multidrug resistant organisms (Carbapenem resistant organisms and Extended spectrum beta lactamase producing Enterobacterales)
Infection control considerations
Nosocomial spread of Salmonella between volunteers	• Theoretical risk of cross-transmission between volunteers impacting study endpoints	• Strict enteric precautions • Isolation of participants in side-rooms
Nosocomial spread of other pathogens, including SARS-CoV-2 (in-patient model)	• Theoretical risk of outbreak of SARS-CoV-2 (or other respiratory viruses) within quarantine facility.	• Use of personal protective equipment • Participants and investigators to have completed at least a primary SARS- CoV-2 vaccination course • Negative SARS-CoV-2 lateral flow test prior to admission to quarantine • Hospital infection prevention and control guidelines to be maintained throughout the quarantine phase • Investigators with relevant respiratory symptoms to isolate and avoid contact with volunteers.
Transmission to study investigators	• Theoretical risk of cross-transmission from study volunteers to study investigators	• Investigators will adhere to strict hygiene measures • Use of personal protective equipment per hospital guidelines for enteric infections
Public health considerations
Onward community transmission (ranging from household secondary cases to outbreak)	• Person-to-person spread has been described, especially when patients are symptomatic with diarrhoea. • Transmission within households can occur if the individual excreting S. Typhimurium fails to practice effective hand washing after defecation and is subsequently involved in uncooked food preparation. • Person-to-person spread has been described, especially when patients are symptomatic with diarrhoea. • Clearance cultures following NTS infection are not typically indicated according to UKHSA guidance ⁴². ○ This contrasts with UK public health guidance following infection with the typhoidal Salmonella serovars ( S. Typhi and Paratyphi)	• In-patient quarantine during putative infectious period. • Defined de-isolation criteria ○ Complete resolution of diarrhoea (Bristol stool type 6-7) for 48 hours • Participant counselling on stringent hygiene measures until microbiological clearance confirmed • Exclusion of participation from high-risk occupational groups ○ food handlers ○ ongoing contact with highly susceptible persons or young children ○ healthcare workers • Access to screening for household and other close contacts • Inform health protection services at time of challenge and time of confirmed clearance. • Engage with national reference laboratories to sequence isolates from participants to compare with any future community outbreaks.