*4.1. Study Context*

In Mozambique, the GEMS was conducted by the *Centro de Investigação em Saúde de Manhiça* (CISM), in six health facilities in the Manhiça District [55], which is located approximately 80 km north of the capital city Maputo in the country's Southern region. The district covers 2380 km2 and has a subtropical climate with two distinct seasons: a warm, rainy season from November to April, and the cool and dry season during the rest of the year [17,56]. Since 1996, CISM has been conducting a continuous Health and Demographic Surveillance System (HDSS) with regular update of demographic events for all surveyed population (current population followed: 203,132 uniquely identified individuals; 46,851

enumerated and geo-positioned households; 27,504 are children under 5 years). During the study period, the HDSS was covering approximately 95,000 inhabitants [17].

The rationale, study design, and methodology of the GEMS have been previously described elsewhere [57]; the study comprised three years of recruitment of acute moderateto-severe diarrhoea cases (MSD, GEMS1) and one additional year including less-severe diarrhoea (LSD, GEMS1A) [4,5]. In Manhiça, the GEMS collected samples uninterruptedly over 5 years, from December 2007 to October 2011, and GEMS1A from November 2011 to November 2012. The standardised epidemiological and clinical methods for the case-control study as well as the full definitions have been previously described elsewhere [18,55]. Briefly, all children aged 0–59 months (stratified in three age groups: 0–11 months, 12–23 months, and 24–59 months), presenting in the six sentinel health facilities with diarrhoea meeting inclusion criteria for the study were invited to participate. Community controls (up to three for MSD cases and one for LSD cases) matched to the index case by age, sex, and neighbourhood were identified using the HDSS databases and enrolled within 14 days after enrolment of the case, and the stool samples were collected and sent to the laboratory at CISM [58].

#### *4.2. Stool Collection and Initial Testing*

The stool samples collection and processing protocols were also standardised across GEMS sites as described elsewhere [55,58]. Samples were collected in sterile flasks and placed in a refrigerator or in a cool-box with a cooler block (2–8 ◦C) for up to 6 h until transported to the laboratory. Sample aliquots without preservatives were frozen at −80 ◦C for further testing. The CRYPTOSPORIDIUM II™ ELISA immunoassay (TECHLAB®, Blacksburg, VA, USA) was used as screening method for the specific detection of *Cryptosporidium* spp. following the manufacturer's instructions.
