*2.3. Prevalence and Molecular Characterization of Cryptosporidium* spp.

Analyses of the 13 *ssu* rDNA sequences generated in the present study revealed the presence of four *Cryptosporidium* species circulating in the paediatric population under study, 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) (Table 3). Besides a case of cryptosporidiosis by *C. parvum* detected in a child with diarrhoea, the remaining cases were identified in asymptomatic children. Out of the four sequences characterised as *C. hominis*, two of them were identical to reference sequence AF108865, with the remaining two differing from it by one to two SNPs including a nucleotide deletion and ambiguous positions in the form of double peaks. Three of the sequences assigned to *C. parvum* corresponded to the "bovine genotype" of the parasite, characterised by a four-nucleotide (TAAT) deletion involving positions 686\_689 of reference sequence AF112571. These three sequences differed among them by 1–2 SNPs affecting positions 795, 837, and/or 892 of reference sequence AF112571. A fourth *C. parvum* sequence was not investigated further due to insufficient quality to accurately determine the presence of potential SNPs. All *C. hominis* and *C. parvum* isolates failed to be amplified at the *gp60* locus despite repeated attempts to do so. Therefore, their family subtypes remain unknown.

**Table 3.** Diversity, frequency, and main molecular features of *Cryptosporidium* spp. sequences at the *ssu* rRNA locus in infected symptomatic and asymptomatic children in Zambézia province, Mozambique. GenBank accession numbers are provided.


<sup>1</sup> Nucleotide(s) deletion. <sup>2</sup> Symptomatic child. <sup>3</sup> Sequence of insufficient quality to accurately determine the presence of potential single nucleotide polymorphisms. <sup>4</sup> Nucleotide insertion.

> Three of the *C. felis* sequences were identical among them but differed from reference sequence AF108862 by three SNPs including a deletion, an insertion, and a substitution (Table 3). The fourth *C. felis* sequence had an additional T insertion in position 826 of reference sequence AF108862. The only sequence assigned to *C. viatorum* was identical to positions 290–762 of reference sequence KX174309. Sequence analysis at the *gp60* locus revealed that this isolate belonged to the subtype XVaA3a of the parasite, showing 100% identity to positions 1–870 of reference sequence KP115936.

> The genetic relationships among *ssu* rRNA gene sequences generated in the present study, as inferred by a neighbor-joining analysis, were shown in Figure 2. All *Cryptosporidium* sequences clustered together (monophyletic groups) with different well-supported clades (58–99% of bootstrap) corresponding to appropriate reference sequences for *Cryptosporidium* species.

**Figure 2.** Phylogenetic relationships among *Cryptosporidium* species identified in infected SympTable 490. bp fragment (corresponding to position 538–1027 of reference sequence AF108865) of the *ssu* rRNA gene sequence. Genetic distances were calculated using the Kimura two-parameter model. Green filled squares represent sequences generated in the present study. Purple filled dots represent reference sequences. Bootstrap values lower than 50% are not displayed. *Cryptosporidium muris* was used as outgroup taxon to root the tree.
