Background Due to diagnostic challenges in childhood tuberculosis (TB), the World Health Organization (WHO) has recommended the use of non-sputum-based samples, including stool. This study evaluated the feasibility and acceptability of different TB diagnostic sampling procedures.

Methods In a prospective observational cohort study, we collected clinical data from children presenting with presumptive pulmonary tuberculosis (PTB). At enrolment, collection of TB diagnostics samples included respiratory samples (gastric aspirate, induced sputum and expectorated sputum), blood, urine and stool. Questionnaires on the acceptability and feasibility were collected from caregivers and healthcare workers (HCWs). A social scientist observed the collection of samples and performed qualitative interviews with HCWs.

Results We conducted 59 diagnostic and acceptability questionnaires of children’s experiences of TB diagnostic sample collection. Sample collection was successful in 59% for urine, 36% for stool, 74% for blood and 72% for any respiratory sample. Overall, more than half of the caregivers felt that stool (86%), urine (75%), blood samples (67%) and respiratory samples (57%) were convenient for their children.

We observed sample collection in 32 children. HCWs had specific challenges with collecting urine samples from young girls due to the leaking urine bags. Children of all ages were resistant when collecting respiratory samples. In children aged 7–12 years, HCWs faced difficulties with collecting stool samples. These children felt embarrassed providing stool samples due to increased self-awareness. HCWs found blood samples easiest to collect, followed by respiratory samples, urine and stool. Even though blood sample collection was observed to cause more discomfort and pain.

Conclusion Although urine and stool samples seem a good non-invasive alternative sample for TB diagnosis in children, remaining challenges hamper the feasibility and acceptability of these specimens, which will need to be considered for future studies.