*Article* **Molecular Diversity of** *Giardia duodenalis***,** *Cryptosporidium* **spp., and** *Blastocystis* **sp. in Symptomatic and Asymptomatic Schoolchildren in Zambézia Province (Mozambique)**

**Aly S. Muadica 1,2, Pamela C. Köster 1, Alejandro Dashti 1, Begoña Bailo 1, Marta Hernández-de-Mingo 1, Sooria Balasegaram <sup>3</sup> and David Carmena 1,\***


**Abstract:** Infections by the protist enteroparasites *Giardia duodenalis*, *Cryptosporidium* spp., and, to a much lesser extent, *Blastocystis* sp. are common causes of childhood diarrhoea in low-income countries. This molecular epidemiological study assesses the frequency and molecular diversity of these pathogens in faecal samples from asymptomatic schoolchildren (*n* = 807) and symptomatic children seeking medical attention (*n* = 286) in Zambézia province, Mozambique. Detection and molecular characterisation of pathogens was conducted by polymerase chain reaction (PCR)-based methods coupled with Sanger sequencing. *Giardia duodenalis* was the most prevalent enteric parasite found [41.7%, 95% confidence interval (CI): 38.8–44.7%], followed by *Blastocystis* sp. (14.1%, 95% CI: 12.1–16.3%), and *Cryptosporidium* spp. (1.6%, 95% CI: 0.9–2.5%). Sequence analyses revealed the presence of assemblages A (7.0%, 3/43) and B (88.4%, 38/43) within *G. duodenalis*-positive children. Four *Cryptosporidium* species were detected, including *C. hominis* (30.8%; 4/13), *C. parvum* (30.8%, 4/13), *C. felis* (30.8%, 4/13), and *C. viatorum* (7.6%, 1/13). Four *Blastocystis* subtypes were also identified including ST1 (22.7%; 35/154), ST2 (22.7%; 35/154), ST3 (45.5%; 70/154), and ST4 (9.1%; 14/154). Most of the genotyped samples were from asymptomatic children. This is the first report of *C. viatorum* and *Blastocystis* ST4 in Mozambique. Molecular data indicate that anthropic and zoonotic transmission (the latter at an unknown rate) are important spread pathways of diarrhoea-causing pathogens in Mozambique.

**Keywords:** *Giardia*; *Cryptosporidium*; *Blastocystis*; enteric parasites; children; diarrhoea; PCR; molecular epidemiology; genotyping; Mozambique
