**5. Conclusions**

This microscopy-based survey demonstrates that symptomatic and asymptomatic giardiasis are common in indigenous people from the Brazilian Amazon. Children under 15 years of age were particularly exposed to the infection, suggesting that acquired immunity plays a role in modulating the frequency and virulence of the disease. *G. duodenalis* infection rates varied largely among the surveyed tribes and sampling periods, suggesting that different pathways may be involved in the transmission of the parasite. Molecular sequence data indicated that the most likely source of infection was anthropic. The distribution of assemblages was independent of the occurrence of clinical manifestations, indicating that the genotype of the parasite was not associated with the outcome of the infection. Assemblage B accounted for near 75% of the infections detected and showed a high genetic diversity that impaired the correct identification of sub-assemblages BIII and BIV. This diversity was mainly associated with the presence of ambiguous positions (double peaks) at the chromatogram level, suggesting that coinfections and/or genetic recombination events were taking place, at unknown rates, in the investigated population. Further molecular epidemiological studies targeting animal (including domestic and wildlife) and environmental (drinking water) samples are needed to elucidate the transmission dynamics of *G. duodenalis* in this Brazilian geographical region.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2076-0 817/10/2/206/s1, Table S1: Prevalence and molecular diversity of *Giardia duodenalis* in humans in Brazil, Table S2: Prevalence and molecular diversity of *Giardia duodenalis* in domestic and wildlife animal species in Brazil, Table S3: Prevalence and molecular diversity of *Giardia duodenalis* in water samples in Brazil, Table S4: Prevalence and molecular diversity of *Giardia duodenalis* in fresh produce in Brazil, Table S5: Full dataset showing the molecular diversity of *G. duodenalis* at the *gdh*, *bg*, and *tpi* molecular markers, Table S6: Intra-assemblage B single nucleotide polymorphisms distribution and classification among *G. duodenalis* sequences at the *gdh*, *bg* and *tpi* loci. Hotspots for SNPs are identified and the summarised comparisons of frequencies between the hotspot and non-hotspot sites are highlighted with darker shades, Table S7: Univariable analysis comparing discontinuously *G. duodenalis*-positive results versus continuously *G. duodenalis*-positive results. *p*-values marked in bold indicate numbers that are significant on the 95% confidence limit, Table S8: Multivariable analysis comparing discontinuously *G. duodenalis*-positive results versus always *G. duodenalis*-positive results. *p*-values marked in bold indicate numbers that are significant on the 95% confidence limit, Table S9: Univariable analysis comparing always *G. duodenalis*-negative results versus always *G. duodenalis*-positive results. *p*-values marked in bold indicate numbers that are significant on the 95% confidence limit, Table S10: Multivariable analysis always comparing *G. duodenalis*-negative results versus always *G. duodenalis*-positive results. *p*-values marked in bold indicate numbers that are significant on the 95% confidence limit, Table S11: Multivariable analysis comparing always *G. duodenalis*-negative results versus always *G. duodenalis*-positive results and considering the presence

of coinfections. *p*-values marked in bold indicate numbers that are significant on the 95% confidence limit, Table S12: Oligonucleotides used for the molecular identification and characterisation of *G. duodenalis* in the present study, Figure S1: Maximum parsimony phylogenetic dendogram based on *bg* sequences of *G. duodenalis*. Numbers on nodes indicate the bootstrap/posterior probability values. GenBank accession numbers for all sequences used for the phylogenetic analysis were embedded in the tree, Figure S2: Maximum parsimony phylogenetic dendogram based on *tpi* sequences of *G. duodenalis*. Numbers on nodes indicate the bootstrap/posterior probability values. GenBank accession numbers for all sequences used for the phylogenetic analysis were embedded in the tree.

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

**Funding:** This research was funded by the São Paulo State Research Support Foundation (FAFESP, Brazil), the National Health Foundation (FUNASA, Brazil), and the Mato Grosso State Research Support Foundation (FAPEMAT, Brazil), grant number 0839/2006. Additional funding was obtained from 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 Institutional Review Board of Instituto de Ciências Biomédicas da Universidade de São Paulo (protocol code 14485—CONEP—on 17/06/2008).

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

**Data Availability Statement:** All relevant data are within the article and its additional files. The sequences obtained in this study have been deposited in GenBank under accession numbers MT542718- MT542765 (*gdh*), MT542766-MT542794 (*bg*), and MT542795-MT542829 (*tpi*).

**Acknowledgments:** S.M. Gennari, A. Marcili, and J. J. Shaw are in receipt of a Research Productivity Fellowship from the National Council for Scientific and Technological Development (CNPq, Brazil).

**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.

#### **References**

