*4.4. DNA Extraction and Purification*

Positive stool samples were defrosted and *G. duodenalis* cysts concentrated and purified using the Faust method [54]. Obtained supernatants were subjected to three freeze–thaw cycles to facilitate the mechanical breakage of the cyst wall [55]. Genomic DNA was extracted from the processed supernatants (ca 200 μL) using the PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Extracted and purified DNA samples in molecular grade water (200 μL) were kept at −20 ◦C and shipped to the Spanish National Centre for Microbiology (Health Institute Carlos III) in Majadahonda (Spain) for downstream genotyping analyses.

## *4.5. Molecular Confirmation of G. duodenalis*

Confirmation of *G. duodenalis* infection was achieved using a real-time PCR (qPCR) method targeting a 62-bp region of the gene codifying the small subunit ribosomal RNA (SSU rRNA) of the parasite [56]. Amplification reactions (25 μL) consisted of 3 μL of template DNA, 0.5 μM of each primer Gd-80F and Gd-127R, 0.4 μM of the probe (Table S12), and 12.5 μL TaqMan® Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA). Detection of parasitic DNA was performed on a Corbett Rotor GeneTM 6000 realtime PCR system (Qiagen, Hilden, Germany) using an amplification protocol consisting of 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.

## *4.6. Molecular Characterisation of G. duodenalis*

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), beta-giardin (bg), and triosephosphate isomerase (tpi) proteins of the parasite. A semi-nested PCR was used to amplify a ~432-bp fragment of the gdh gene [57]. 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 S12). 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 [58]. 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 S12). 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.

A nested PCR was used to amplify a ~530 bp-fragment of the *tpi* gene [59]. 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 S12). 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.

The semi-nested and nested PCR protocols described above were conducted on a 2720 Thermal Cycler (Applied Biosystems). Reaction mixes always included 2.5 units of MyTAQTM DNA polymerase (Bioline GmbH, Luckenwalde, Germany), and 5× MyTAQTM reaction buffer containing 5 mM dNTPs and 15 mM MgCl2. Laboratory-confirmed positive and negative DNA samples for each parasite species investigated were routinely used as controls and included in each round of PCR. PCR amplicons were visualised on 2% D5 agarose gels (Conda, Madrid, Spain) stained with Pronasafe nucleic acid staining solution (Conda). Positive PCR products were directly sequenced in both directions using appropriate internal primer sets (Table S12). DNA sequencing was conducted by capillary electrophoresis using BigDye® Terminator chemistry (Applied Biosystems) on an on ABI PRISM 3130 Genetic Analyser.
