**4. Materials and Methods**

#### *4.1. Study Area and Stool Sample Collection*

A prospective cross-sectional molecular epidemiological study of diarrhoea-causing enteric parasites including the protozoan *G. duodenalis* and *Cryptosporidium* spp. and the stramenopile *Blastocystis* sp., was conducted with children aged 3–14 from 10 of the 22 districts of Zambézia province, central Mozambique, between October 2017 and February 2019. Stool samples were collected from participating schoolchildren attending 18 primary schools or children seeking medical attention at seven primary healthcare centres (Figure 4). In school settings (range: 35–2111; mean: 651 schoolchildren) informative meetings were held for interested families. Schoolchildren volunteering to participate were given sampling

kits to obtain stool samples during school attendance. In primary health clinics, children with gastrointestinal complaints (chronic or acute diarrhoea, bloating, abdominal pain) were invited to participate in the survey. Samples were collected by members of the research team at scheduled times and an aliquot (2–3 g) transferred to REAL Minisystem devices (Durviz, Valencia, Spain) for stool sample conservation and concentration. Preserved samples were maintained at room temperature up to three months before being transported to the Spanish National Centre for Microbiology (Majadahonda, Spain) for processing and analysis. Inclusion and exclusion criteria to participate in the study, school features, and sampling procedures as well as the detailed analysis of potential associations linked with enteric parasite infections in the paediatric populations surveyed here were thoroughly described elsewhere (Muadica et al., 2020) [27].

**Figure 4.** Map showing the geographical location of the Zambézia province in Mozambique (upper left corner) and the primary school and healthcare centres sampled in the present study.

#### *4.2. DNA Extraction and Purification*

Genomic DNA was isolated from about 200 mg of each faecal specimen by using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, except that samples mixed with InhibitEX buffer were incubated for 10 min at 95 ◦C. Extracted and purified DNA samples (200 μL) were kept at −20 ◦C until further molecular analysis. A water extraction control was included in each sample batch processed.

#### *4.3. Molecular Detection and Characterization of Giardia duodenalis*

*Giardia duodenalis* DNA was detected by qPCR amplification of a 62 bp-fragment of the small subunit ribosomal RNA (*ssu* rRNA) gene of the parasite [55]. Amplification reactions (25 μL) consisted of 3 μL of template DNA, 0.5 μM of primers Gd-80F and Gd127R, 0.4 μM of probe (Additional file 1: Table S1), and 12.5 μL TaqMan® Gene Expression Master Mix (Applied Biosystems, CA, USA). Detection of parasitic DNA was performed on a Corbett Rotor Gene™ 6000 real-time PCR system (Qiagen) using an amplification protocol consisting on an initial hold step of 2 min at 55 ◦C and 15 min at 95 ◦C followed by 45 cycles of 15 s at 95 ◦C and 1 min at 60 ◦C. Water (no-template) and genomic DNA (positive) controls were included in each PCR run.

*Giardia duodenalis* isolates with a qPCR-positive result were re-assessed by sequencebased multi-locus genotyping of the genes encoding for the glutamate dehydrogenase (*gdh*), ß-giardin (*bg*) and triose phosphate isomerase (*tpi*) proteins of the parasite. A semi-nested PCR was used to amplify a 432-bp fragment of the *gdh* gene [56]. PCR reaction mixtures (25 μL) included 5 μL of template DNA and 0.5 μM of the primer pairs GDHeF/GDHiR in the primary reaction and GDHiF/GDHiR in the secondary reaction (Table S4). Both amplification protocols consisted of an initial denaturation step at 95 ◦C for 3 min, followed by 35 cycles of 95 ◦C for 30 s, 55 ◦C for 30 s and 72 ◦C for 1 min, with a final extension of 72 ◦C for 7 min. A nested PCR was used to amplify a 511 bp fragment of the bg gene [57]. PCR reaction mixtures (25 μL) consisted of 3 μL of template DNA and 0.4 μM of the primers sets G7\_F/G759\_R in the primary reaction and G99\_F/G609\_R in the secondary reaction (Table S4). The primary PCR reaction was carried out with the following amplification conditions: one step of 95 ◦C for 7 min, followed by 35 cycles of 95 ◦C for 30 s, 65 ◦C for 30 s, and 72 ◦C for 1 min with a final extension of 72 ◦C for 7 min. The conditions for the secondary PCR were identical to the primary PCR except that the annealing temperature was 55 ◦C. Finally, a nested PCR was used to amplify a 530 bp-fragment of the *tpi* gene [58]. PCR reaction mixtures (50 μL) included 2-2.5 μL of template DNA and 0.2 μM of the primer pairs AL3543/AL3546 in the primary reaction and AL3544/AL3545 in the secondary reaction (Table S4). Both amplification protocols consisted of an initial denaturation step at 94 ◦C for 5 min, followed by 35 cycles of 94 ◦C for 45 s, 50 ◦C for 45 s and 72 ◦C for 1 min, with a final extension of 72 ◦C for 10 min.

#### *4.4. Molecular Detection and Characterization of Cryptosporidium* spp.

The presence of *Cryptosporidium* spp. was assessed using a nested PCR to amplify a 587-bp fragment of the *ssu* rRNA gene of the parasite [59]. Amplification reactions (50 μL) included 3 μL of DNA sample and 0.3 μM of the primer pairs CR-P1/CR-P2 in the primary reaction and CR-P3/CPB-DIAGR in the secondary reaction (Table S4). Both PCR reactions were carried out as follows: one step of 94 ◦C for 3 min, followed by 35 cycles of 94 ◦C for 40 s, 50 ◦C for 40 s and 72 ◦C for 1 min, concluding with a final extension of 72 ◦C for 10 min.

Isolates identified as *C. hominis* or *C. parvum* by *ssu*-PCR (and Sanger sequencing, see below) were reanalysed at the 60 kDa glycoprotein (*gp60*) gene for subtyping purposes. Briefly, a nested PCR was conducted to amplify a 870 bp fragment of the *gp60* locus [60]. PCR reaction mixtures (50 μL) included 2-3 μL of template DNA and 0.3 μM of the primer pairs AL-3531/AL-3535 in the primary reaction and AL-3532/AL-3534 in the secondary reaction (Table S4). The primary PCR reaction consisted of an initial denaturation step of 94 ◦C for 5 min, followed by 35 cycles of 94 ◦C for 45 s, 59 ◦C for 45 s, and 72 ◦C for 1 min with a final extension of 72 ◦C for 10 min. The conditions for the secondary PCR were identical to the primary PCR except that the annealing temperature was 50 ◦C. Similarly, samples identified as *C. viatorum* by *ssu*-PCR were reanalysed at the *gp60* locus using a nested PCR protocol specifically developed for this *Cryptosporidium* species [45]. This protocol amplifies a 950 bp fragment of the *gp60* gene. Amplification reactions (50 μL) included 1–2 μL of DNA sample and 0.25 μM of the primer pair CviatF2/CviatR5 in the primary reaction and 0.5 μM of the primer pair CviatF3/CviatR8 in the secondary reaction (Table S4). Both PCR reactions were carried out as follows: one step of 95 ◦C for 4 min, followed by 35 cycles of 95 ◦C for 30 s, 58 ◦C for 30 s and 72 ◦C for 1 min, concluding with a final extension of 72 ◦C for 7 min.
