*4.5. Molecular Detection and Characterization of Blastocystis* sp.

Identification of *Blastocystis* sp. was achieved by a direct PCR targeting the *ssu* rRNA gene of the parasite [61]. This protocol uses the pan-*Blastocystis*, barcode primers RD5 and BhRDr (Table S4) to amplify a PCR product of 600 bp. Amplification reactions (25 μL) included 5 μL of template DNA and 0.5 μM of the primer set RD5/BhRDr. Amplification conditions consisted of one step of 95 ◦C for 3 min, followed by 30 cycles of 1 min each at 94, 59 and 72 ◦C, with an additional 2 min final extension at 72 ◦C.

All the direct, 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 MyTAQ™ DNA polymerase (Bioline GmbH, Luckenwalde, Germany), and 5× MyTAQ™ Reaction Buffer containing 5 mM dNTPs and 15 mM MgCl2. Laboratory-confirmed positive and negative DNA isolates for each parasitic species investigated were routinely used as controls and included in each round of PCR. PCR amplicons were visualized 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 the internal primer set described above. DNA sequencing was conducted by capillary electrophoresis using the BigDye® Terminator chemistry (Applied Biosystems) on an ABI PRISM 3130 automated DNA sequencer.

#### *4.6. Sequence and Phylogenetic Analyses*

Raw sequencing data in both forward and reverse directions were viewed using the Chromas Lite version 2.1 sequence analysis program (https://technelysium.com.au/wp/ chromas/, accessed on 23 February 2021). The BLAST tool (http://blast.ncbi.nlm.nih.gov/ Blast.cgi, accessed on 23 February 2021) was used to compare nucleotide sequences with sequences retrieved from the NCBI GenBank database. Generated DNA consensus sequences were aligned to appropriate reference sequences using the MEGA 6 software [62] to identify *G. duodenalis* assemblages/sub-assemblages and *Cryptosporidium* species. *Blastocystis* sequences were submitted at the *Blastocystis* 18S database (http://pubmlst.org/blastocystis/, accessed on 23 February 2021) for sub-type confirmation and allele identification.

The evolutionary relationships among the identified *G. duodenalis* assemblages/subassemblages at the three loci investigated and the *Cryptosporidium* species found were inferred by a phylogenetic analysis using the neighbor-joining method in MEGA 6. Only sequences with unambiguous (no double peak) positions were used in the analyses. The evolutionary distances were computed using the Kimura 2-parameter method and modelled with a gamma distribution. The reliability of the phylogenetic analyses at each branch node was estimated by the bootstrap method using 1000 replications. Representative sequences of different *G. duodenalis* assemblages and sub-assemblages and *Cryptosporidium* species were retrieved from the NCBI database and included in the phylogenetic analyses for reference and comparative purposes.

Representative *G. duodenalis* sequences obtained in this study have been deposited in GenBank under accession numbers MW508361–MW508394 (*gdh* locus), MW508394– MW508410 (*bg* locus) and MW556751–MW556764 (*tpi* locus). Representative *Cryptosporidium* spp. sequences were deposited under accession numbers MW563962–MW563970 (*ssu* rRNA locus) and MW574004 (*gp60* locus). Representative *Blastocystis* sp. sequences were deposited under accession numbers MW564221–MW564233.

#### **5. Conclusions**

This PCR-based epidemiological study provides novel data on the molecular diversity of *G. duodenalis*, *Cryptosporidium* spp., and *Blastocystis* sp. in children with and without diarrhoea in Zambézia province, a Mozambican region where this information was completely lacking. Generated results complement and expand those obtained previously by the GEMS project in paediatric populations in Maputo province. A high intra-species molecular diversity was observed among the three parasites investigated, a finding compatible with an epidemiological scenario of high endemicity where infections and reinfections

were common. No obvious differences in the distribution and frequency of parasite' species/genotypes were observed between apparently healthy children and children with clinical manifestations, suggesting that virulence/pathogenicity was not associated to a given genetic variant. Transmission of giardiosis and blastocystosis was primarily of anthropic nature, but strong molecular evidence indicated that a significant number of cryptosporidiosis cases were the result of zoonotic events. Data presented here highlight the need to conduct new molecular surveys in animal and environmental (drinking water) samples to complete our understanding of the transmission dynamics of these protist species in Mozambique. Measures directed to improve access to safe drinking water, sanitation facilities, and personal hygiene (e.g., hand washing) practices would definitively help in minimizing the transmission of diarrhoea-causing pathogens in highly endemic areas in Mozambique.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2076-0 817/10/3/255/s1, Table S1: Diversity, frequency, and main molecular features of *Giardia duodenalis* sequences at the *gdh* locus in infected symptomatic and asymptomatic children in Zambézia province, Mozambique. GenBank accession numbers are provided. Superscript numbers identify single nucleotide polymorphisms involving amino acid change, Table S2: Diversity, frequency, and main molecular features of *Giardia duodenalis* sequences at the *bg* locus in infected symptomatic and asymptomatic children in Zambézia province, Mozambique. GenBank accession numbers are provided. Superscript numbers identify single nucleotide polymorphisms involving amino acid change, Table S3: Diversity, frequency, and main molecular features of *Giardia duodenalis* sequences at the *tpi* locus in infected symptomatic and asymptomatic children in Zambézia province, Mozambique. GenBank accession numbers are provided. Superscript numbers identify single nucleotide polymorphisms involving amino acid change, Table S4: Oligonucleotides used for the molecular identification and/or characterization of *Giardia duodenalis*, *Cryptosporidium* spp., and *Blastocystis* sp. in the present study, Figure S1: Phylogenetic relationships among *Giardia duodenalis* assemblages and sub-assemblages identified in infected symptomatic and asymptomatic children in Zambézia province, Mozambique. The analysis was conducted by a neighbor-joining method of a 502-bp fragment (corresponding to position 101–602 of reference sequence AY072727) of the *bg* 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. Because *bg* is a *Giardia*-specific protein, no outgroup taxon was used to root the tree, Figure S2: Phylogenetic relationships among *Giardia duodenalis* assemblages and sub-assemblages identified in infected symptomatic and asymptomatic children in Zambézia province, Mozambique. The analysis was conducted by a neighbor-joining method of a 479-bp fragment (corresponding to position 1–479 of reference sequence AF069560) of the *tpi* 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. *Giardia muris* was used as outgroup taxon to root the tree.

**Author Contributions:** Conceptualization, A.S.M., S.B. and D.C.; methodology, S.B. and D.C.; software, P.C.K. and A.D.; validation, S.B. and D.C.; formal analysis, A.S.M., P.C.K., A.D. and D.C.; investigation, A.S.M., P.C.K., A.D., B.B. and M.H.-d.-M.; resources, D.C.; data curation, D.C.; writing original draft preparation, D.C.; writing—review and editing, A.S.M., P.C.K., A.D., S.B. and D.C.; visualization, S.B. and D.C.; supervision, S.B. and D.C.; project administration, D.C.; funding acquisition, D.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Health Institute Carlos III (ISCIII), Ministry of Economy and Competitiveness (Spain), grant number PI16CIII/00024.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Health Institute Carlos III (CEI PI17\_2017-v3; date of approval: 23 October 2017) and the National Bioethics Committee for Health of the Republic of Mozambique (52/CNBS/2017; date of approval: 11 September 2017).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** All relevant data are within the paper and its Supplementary Materials.

**Acknowledgments:** The authors are grateful to the participating children and their parents/legal guardians, and to school principals and teachers at the participating schools for their logistic assistance.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
