*4.4. Data Collection*

Demographic, clinical and epidemiological data were assessed by interviewing children's caregivers with a semi-standardized questionnaire. Information regarding sex, age, animal contact (defined as having physical contact with an animal or their excrements) [16] and caregiver education status were collected. HIV status was self-reported by the children's caregivers and confirmed in the children's vaccination cards. If unknown, permission was asked to collect blood samples and tested according to the national testing algorithm. The children newly diagnosed as HIV-positive were followed by the physician at hospital and referred to their neighborhood health facilities for routine assistance after discharge.

#### *4.5. Sample Collection and Management*

A single stool sample from each child was collected after inclusion. In cases of liquid diarrhea, non-absorbent diapers were used instead of ordinary diapers. Samples were transferred to sterile polystyrene tubes, kept refrigerated in cooler boxes (approximately 2 ◦C to 8 ◦C), without preservative and sent to the Laboratory of Parasitology in INS, Maputo.

A smear was made from fresh stool, and an aliquot was kept in the original tube under 2 to 8 ◦C for concentration and subsequent microscopic examination for *Cryptosporidium* spp. oocysts. A second aliquot was placed in a vial without preservative, stored under −40 ◦C and was specifically intended for extracting and purifying genomic deoxyribonucleic acid (DNA) for molecular analysis. A set of previously frozen sub-samples (approximately 0.5 mL) was shipped to *Laboratório Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz/Fundação Oswaldo Cruz* in Rio de Janeiro, Brazil, under dry ice for DNA extraction, detection and genetic characterization of *Cryptosporidium* species.
