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Epidemiology of Giardia duodenalis assemblages in Brazil: there is still a long way to go

Abstract

Giardia duodenalis infection is distributed worldwide and can achieve prevalence around 60%, especially in developing countries. This protozoan is divided into eight assemblages, in which A and B have high zoonotic potential, whereas C to H are host-specific. This scenario is changing as molecular studies progress, highlighting that knowledge on host-specificity still has a long way to go. Understanding the players involved in transmission routes enables rational designs of control strategies. Considering the high prevalence of giardiasis, this review aims to gather together the data on available studies on the distribution of G. duodenalis assemblages in Brazil until September 2020.

Key words:
Giardia duodenalis; assemblage; genotyping; human; animals; water and soil; Brazil


Giardia is an intestinal protozoan found in a wide spectrum of animal hosts (birds, reptiles and mammals). In mammals the infection occurs by G. duodenalis, synonymous with G. lamblia and G. intestinalis. Although there is no consensus on the most appropriate name, in this review we will use G. duodenalis.11. Simner PJ. Medical parasitology taxonomy update: January 2012 to December 2015. J Clin Microbiol. 2016; 5(1): 43-7.

G. duodenalis present worldwide distribution, showing prevalence of 2-7% in developed countries and can reach over 30% in low and middle-income countries.22. Minetti C, Chalmers RM, Beeching NJ, Probert C, Lamden K. Giardiasis. BMJ. 2016; 355: i5369. High frequencies are observed in poorer countries as they are closely associated with poor sanitation infrastructure. Although the water supply system is the main source of G. duodenalis transmission in outbreaks, in endemic areas direct transmission has great epidemiological relevance.

The great genomic diversity found in the G. duodenalis species meant that its subspecies were initially organised into groups, which are currently called assemblages or genotypes. The first divisions in G. duodenalis assemblages were carried out according to specificity by the host from which the isolate originated.33. Monis PT, Andrews RH, Mayrhofer G, Ey PL. Molecular systematics of the parasitic protozoan Giardia intestinalis. Mol Biol Evol. 1999; 16(9): 1135-44.,44. Thompson RC, Hopkins RM, Homan WL. Nomenclature and genetic groupings of Giardia infecting mammals. Parasitol Today. 2000; 16(5): 210-3.,55. Adam RD. Biology of Giardia lamblia. Clin Microbiol Rev. 2001; 14(3): 447-75. This subdivision was corroborated in intrinsic characteristics of the parasite, such as antigenic factors and isoenzymes, but mainly by DNA analysis, which confirmed the heterogeneity of G. duodenalis.44. Thompson RC, Hopkins RM, Homan WL. Nomenclature and genetic groupings of Giardia infecting mammals. Parasitol Today. 2000; 16(5): 210-3.,55. Adam RD. Biology of Giardia lamblia. Clin Microbiol Rev. 2001; 14(3): 447-75.

Nowadays, the G. duodenalis species is phylogenetically divided into eight assemblages, classified from A to H.44. Thompson RC, Hopkins RM, Homan WL. Nomenclature and genetic groupings of Giardia infecting mammals. Parasitol Today. 2000; 16(5): 210-3.,66. Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev. 2011; 24(1): 110-40. Classically, assemblages A and B are considered potentially zoonotic, as they infect humans and a wide variety of mammals, while assemblages C-H are considered host-specific. Assemblages C and D are associated with infection in dogs, assemblage F in felines, assemblage G in rodents and assemblage H in marine mammals.55. Adam RD. Biology of Giardia lamblia. Clin Microbiol Rev. 2001; 14(3): 447-75.,77. Lasek-Nesselquist E, Welch DM, Sogin ML. The identification of a new Giardia duodenalis assemblage in marine vertebrates and a preliminary analysis of G. duodenalis population biology in marine systems. Int J Parasitol. 2010; 40(9): 1063-74. The assemblage E was considered only infecting ungulate and biungulate animals, but the human infection has been reported.88. Fantinatti M, Bello AR, Fernandes O, Da-Cruz AM. Identification of Giardia lamblia assemblage E in humans points to a new anthropozoonotic cycle. J Infect Dis. 2016; 214(8): 1256-9.,99. Zahedi A, Field D, Ryan U. Molecular typing of Giardia duodenalis in humans in Queensland - first report of assemblage E. Parasitology. 2017; 144(9): 1154-61.,1010. Ryan U, Zahedi A. Molecular epidemiology of giardiasis from a veterinary perspective. Adv Parasitol. 2019; 106: 209-54. Yet, the affinity of these assemblages for the hosts has been constantly discussed and updated.1111. Cacciò SM, Lalle M, Svärd SG. Host specificity in the Giardia duodenalis species complex. Infect Genet Evol. 2018; 66: 335-45.

When symptomatic, the most frequent symptoms of giardiasis are consequence of acute or chronic diarrhoea, such as abdominal colic, flatulence, dehydration, nausea, vomiting and fatigue.66. Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev. 2011; 24(1): 110-40.,1212. Thompson RC, Reynoldson JA, Mendis AH. Giardia and giardiasis. Adv Parasitol. 1993; 32: 71-160. There is no proven association between clinical manifestations and the infecting assemblage. Symptomatic and asymptomatic cases were already observed for all assemblages, especially in humans.1313. Einarsson E, Ma'ayeh S, Svärd SG. An up-date on Giardia and giardiasis. Curr Opin Microbiol. 2016; 34: 47-52. However, studies correlating specific symptoms, as well as disease severity, differ in its results. Some authors report assemblage B as more associated with clinical manifestations,1414. Gelanew T, Lalle M, Hailu A, Pozio E, Cacciò SM. Molecular characterization of human isolates of Giardia duodenalis from Ethiopia. Acta Trop. 2007; 102(2): 92-9.,1515. Al-Mohammed HI. Genotypes of Giardia intestinalis clinical isolates of gastrointestinal symptomatic and asymptomatic Saudi children. Parasitol Res. 2011; 108(6): 1375-81.,1616. Pijnacker R, Mughini-Gras L, Heusinkveld M, Roelfsema J, van Pelt W, Kortbeek T. Different risk factors for infection with Giardia lamblia assemblages A and B in children attending day-care centres. Eur J Clin Microbiol Infect Dis. 2016; 35(12): 2005-13.,1717. Puebla LJ, Núñez FA, García AB, Rivero LR, Millán IA, Prado RC. Prevalence of Giardia duodenalis among children from a central region of Cuba: molecular characterization and associated risk factors. J Parasit Dis. 2017; 41(2): 405-13. while others find the same association with assemblage A.1818. Read C, Walters J, Robertson ID, Thompson RC. Correlation between genotype of Giardia duodenalis and diarrhoea. Int J Parasitol. 2002; 32(2): 229-31.,1919. Haque R, Roy S, Kabir M, Stroup SE, Mondal D, Houpt ER. Giardia assemblage A infection and diarrhea in Bangladesh. J Infect Dis. 2005; 192(12): 2171-3.,2020. Cordón GP, Soldan OCP, Vásquez FV, Velasco Soto JR, Bordes LS, Moreno MS, et al. Prevalence of enteroparasites and genotyping of Giardia lamblia in Peruvian children. Parasitol Res. 2008; 103(2): 459-65. Similarly, no relationship between the immune response profile and a specific infecting assemblage was observed, although assemblages A, B, and E are associated with damage to the intestinal mucosa in humans.2121. Pacheco FTF, Carvalho SS, Cardoso LS, Andrade LS, das Chagas GMT, Gomes DC, et al. Immune response markers in sera of children infected with Giardia duodenalis AI and AII subassemblages. Immunobiology. 2019; 224(4): 595-603.,2222. Cascais-Figueiredo T, Austriaco-Teixeira P, Fantinatti M, Silva-Freitas ML, Santos-Oliveira JR, Coelho CH, et al. Giardiasis alters intestinal fatty acid binding protein (I-FABP) and plasma cytokines levels in children in Brazil. Pathogens. 2020; 9(1): 7.

The different molecular characterisation tools have played an important role not only to better understand the characteristics of the assemblages, but also to understand the zoonotic cycles of transmission. Further genotyping studies are needed in order to elucidate the complexity involved in the transmission dynamics of G. duodenalis assemblages, especially in regions with high frequencies of infection.

Brazilian studies about the epidemiology of giardiasis indicates that the G. duodenalis frequency in children can be higher than 60%.2323. Guimarães S, Sogayar MI. Occurrence of Giardia lamblia in children of municipal day-care centers from Botucatu, São Paulo State, Brazil. Rev Inst Med Trop São Paulo. 1995; 37(6): 501-6.,2424. Machado ER, Costa-Cruz JM. Strongyloides stercoralis and other enteroparasites in children at Uberlândia city, state of Minas Gerais, Brazil. Mem Inst Oswaldo Cruz. 1998; 93(2): 161-4.,2525. Machado RC, Marcari EL, Cristante SFV, Carareto CMA. Giardíase e helmintíases em crianças de creches e escolas de 1° e 2° graus (públicas e privadas) da cidade de Mirassol (SP, Brasil). Rev Soc Bras Med Trop. 1999; 32(6): 697-704.,2626. Guimarães S, Sogayar MI. Detection of anti-Giardia lamblia serum antibody among children of day care centers. Rev Saude Publica. 2002; 36(1): 63-8.,2727. Lima Jr OA, Kaiser J, Catisti R. High occurrence of giardiasis in children living on a 'landless farm workers' settlement in Araras, São Paulo, Brazil. Rev Inst Med Trop São Paulo. 2013; 55(3): S0036-46652013000300185. The numbers demonstrate the need for control strategies for this infection. However, in view of the low perspective of implementing the universal sanitation, it is essential to know the sources of contamination and the possible transmission routes in order to search strategies of prophylaxis. Thus, understanding the epidemiology of G. duodenalis assemblages could contribute to reducing giardiasis. In this context, this review aimed to describe the distribution of G. duodenalis assemblages in Brazil between 2007-2020.

MATERIALS AND METHODS

A review was carried out based on an exploratory and descriptive bibliographic survey in September, 2020, with the main theme: “Giardia duodenalis assemblages circulating in Brazil”. The electronic database, Medical Literature Analysis and Retrieval System on-line (Medline), was used.

Medical subject headings (MeSH) terms were used to define the search for: (1) etiological agent: “Giardia”; (2) characteristic of the etiological agent: “assemblage” or “genotype”; (3) geographical area: “Brazil”. To search with the aforementioned descriptors, the Boolean operator “and” was used. Thus, we obtained the following research strategy: (1) [(Giardia) AND assemblage] AND Brazil; (2) [(Giardia lamblia) OR (Giardia intestinalis) OR (Giardia duodenalis) AND assemblage] AND Brazil; (3) [(Giardia) AND (genotype)] AND Brazil; (4) [(Giardia intestinalis) OR (Giardia lamblia) OR (Giardia duodenalis) AND (genotype)] AND Brazil (Fig. 1).

Fig. 1:
methodological search strategy for Medline bibliographic survey on Giardia duodenalis assemblages in Brazil.

The following inclusion criteria were used for the selection of manuscripts: articles retrieved in full, published in English until the year 2020. The following were excluded: studies that did not perform genotyping of the Giardia isolates (n = 5); studies that performed analysis in silico or from data available from databases (n = 2); studies that were not performed with isolates from Brazil (n = 2); in vitro and experimental model studies that did not perform genotyping of original isolates (n = 4); studies that used DNA from single cysts of G. duodenalis separated using a micromanipulation technique (n = 1); studies on archaeological material (n = 2); studies that did not use original G. duodenalis genotyping results and that had already been addressed in previous papers (n = 2); studies review (n = 1).

RESULTS AND DISCUSSION

Frequency and distribution of genotyping studies in Brazil

Knowledge of the geographic distribution of parasitosis, such as giardiasis, is essential to target specific control measures. In Brazil, giardiasis is a public health problem with an unknown prevalence. Most of the studies in this field results from research by individual groups, and are usually sectional and aimed at local scope parasitological surveys. The lack of information about the transmission dynamics of G. duodenalis in the country makes it difficult to implement strategic control actions that are targeted to potential sources of transmission. The scarcity of studies associated with the absence of a national infection survey makes it hard to understand the evolution of parameters related to the frequency of giardiasis in the population. Currently, it is not possible to say whether the G. duodenalis distribution in Brazil is changing upwards or downwards.

Although there are many areas with high frequencies of G. duodenalis infection (over 40%), all genotyping studies are relatively recent, with the first one dating back to 2007.1212. Thompson RC, Reynoldson JA, Mendis AH. Giardia and giardiasis. Adv Parasitol. 1993; 32: 71-160. We have observed a modest progress in these studies but with a low annual frequency (Fig. 2). In the period 2007-2020, only 41 studies were identified on Medline (Table I). This search tool has limitations as journals fulfilling technical criteria to be available in the Medline research base, are considered with the predetermined MeSH terms. Then, an underestimated number of studies published in the grey literature was not considered herein. However, the strategy used ensures reproducibility and guarantees a sample of studies carried out in Brazil.

Fig. 2:
articles published on Medline about Giardia assemblage from Brazil. The points represent the number of published articles found in the search.

Brazil is a country of large continental proportions (8,516,000 km²), composed of 27 states and a federal district divided into five regions (North, Northeast, Midwest, Southeast and South). Of the Brazilian regions, one of them (Midwest) does not present any studies of G. duodenalis genotyping, while two others (North and Northeast) share only two studies, each carried out from human samples. Most of the studies were developed in the Southeast region (30/41). This region is responsible for the main share of the country’s gross domestic product (GDP), and despite its small geographic dimension, it concentrates a large number of research institutes and postgraduate courses (48.45%).2929. IBGE - Instituto Brasileiro de Geografia e Estatística. 2013. Available from: https://www.ibge.gov.br/ accessed in 20 April 2020.
https://www.ibge.gov.br/...

Brazil has the greatest biodiversity on the planet. It is estimated that about 11,000 known vertebrate animal species and 210 species of mammals have already been observed.3030. ICMBio - Instituto Chico Mendes de Conservação da Biodiversidade. 2018. Available from: https://portaldabiodiversidade.icmbio.gov.br/portal/ accessed in 20 April 2020.
https://portaldabiodiversidade.icmbio.go...
However, little research has been performed on Giardia and its genotypes in wild animal studies. All studies were carried out using samples of wild animals that had some proximity to humans (captive animals or animals in parks) which would facilitate transmission.3131. Volotão AC, Júnior JC, Grassini C, Peralta JM, Fernandes O. Genotyping of Giardia duodenalis from southern brown howler monkeys (Alouatta clamitans) from Brazil. Vet Parasitol. 2008; 158(1-2): 133-7.,3232. Soares RM, de Souza SL, Silveira LH, Funada MR, Richtzenhain LJ, Gennari SM. Genotyping of potentially zoonotic Giardia duodenalis from exotic and wild animals kept in captivity in Brazil. Vet Parasitol. 2011; 180(3-4): 344-8.,3333. da Cunha MJR, Cury MC, Santín M. Molecular identification of Enterocytozoon bieneusi, Cryptosporidium, and Giardia in Brazilian captive birds. Parasitol Res. 2017; 116(2): 487-93. There are no studies on the circulation of G. duodenalis assemblages in these animals in a natural environment.

Most studies (34/41) of G. duodenalis genotyping are carried out with samples of humans, domestic animals or farm animals (Table I). Up to now, it is not possible to establish a relationship between the G. duodenalis assemblage and the pathogenesis of giardiasis. Infections in humans have been further explored due to its importance in public health. The approach of man to pets increases the importance of research on transmission routes, including research by this group. Although farm animals have less affective proximity to humans, infection by G. duodenalis can impact the weight gain of these animals. Then, studies with these hosts are also relevant for the economy.3434. Niine T, Peetsalu K, Tummeleht L, Kuks A, Orro T. Acute phase response in organic lambs associated with colostrum serum amyloid A, weight gain, and Cryptosporidium and Giardia infections. Res Vet Sci. 2018; 121: 117-23.,3535. Shivley CB, Lombard JE, Urie NJ, Kopral CA, Santin M, Earleywine TJ, et al. Preweaned heifer management on US dairy operations: Part VI. Factors associated with average daily gain in preweaned dairy heifer calves. J Dairy Sci. 2018; 101(10): 9245-58.

Despite the high prevalence of Giardia in humans, and the largest diversity of animals in the world, the Brazilian contribution to the understanding of G. duodenalis diversity is scarce. In relation to developed countries, such as Portugal, Spain, Italy and China, Brazil has few studies of G. duodenalis genotyping, and most of them are concentrated in a single region. Further studies on the topic should be promoted, both in humans and in domestic, farm and wild animals, in order to assist in understanding the dynamics of transmission of G. duodenalis.

TABLE I
Genotyping studies of Giardia duodenalis in Brazil up to September 2020

Genotyping strategies of G. duodenalis in Brazil

Many genes have been proposed, and 16 targets for molecular characterisation are described, divided according to the potential to discriminate species of the genus Giardia and/or discriminate G. duodenalis assemblages.3636. Lujan HD, Svard S. Giardia a model organism. Wien: Springer Wien New York; 2011. 1140 pp. However, due to the high frequency of single nucleotide polymorphisms, there are few genes used in the literature for genotyping. Initially, the most used gene was small subunit ribosomal ribonucleic acid (SSU rRNA), as it is extremely conserved;3737. Abe N, Read C, Thompson RC, Iseki M. Zoonotic genotype of Giardia intestinalis detected in a ferret. J Parasitol. 2005; 91(1): 179-82. however, the amplification of the locus by polymerase chain reaction (PCR) can present difficulties.66. Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev. 2011; 24(1): 110-40. Thus, for G. duodenalis typing, the genes most widely used are those that encode the proteins Triose Phosphate Isomerase (tpi),3838. Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, et al. Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg Infect Dis. 2003; 9(11): 1444-52. Glutamate Dehydrogenase (gdh)33. Monis PT, Andrews RH, Mayrhofer G, Ey PL. Molecular systematics of the parasitic protozoan Giardia intestinalis. Mol Biol Evol. 1999; 16(9): 1135-44. and Beta Giardin (βg)3939. Cacciò SM, De Giacomo M, Pozio E. Sequence analysis of the beta-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int J Parasitol. 2002; 32(8): 1023-30.. In Brazil, 83% of the G. duodenalis genotyping studies carried out from 2007-2019 (29/35) used the sequencing of regions of classically-used genes. βg, gdh and/or tpi were used in all studies, with the exception of one research study that used only the target SSU-rRNA.3333. da Cunha MJR, Cury MC, Santín M. Molecular identification of Enterocytozoon bieneusi, Cryptosporidium, and Giardia in Brazilian captive birds. Parasitol Res. 2017; 116(2): 487-93. Six studies that used the SSU-rRNA marker in a multiplex approach with bg, gdh or tpi (Table I).3232. Soares RM, de Souza SL, Silveira LH, Funada MR, Richtzenhain LJ, Gennari SM. Genotyping of potentially zoonotic Giardia duodenalis from exotic and wild animals kept in captivity in Brazil. Vet Parasitol. 2011; 180(3-4): 344-8.,4040. Paz e Silva FM, Lopes RS, Araújo Jr JP. Genetic characterisation of Giardia duodenalis in dairy cattle in Brazil. Folia Parasitol (Praha). 2012; 59(1): 15-20.,4141. Ferreira FP, Caldart ET, Freire RL, Mitsuka-Breganó R, Freitas FM, Miura AC, et al. The effect of water source and soil supplementation on parasite contamination in organic vegetable gardens. Rev Bras Parasitol Vet. 2018; 27(3): 327-37.,5959. Yamashiro S, Foco MLR, Pineda CO, José J, Nour EAA, Siqueira-Castro ICV, et al. Giardia spp. and Cryptosporidium spp. removal efficiency of a combined fixed-film system treating domestic wastewater receiving hospital effluent. Environ Sci Pollut Res Int. 2019; 26(22): 22756-71.,6060. de Aquino MCC, Harvey TV, Inácio SV, Nagata WB, Ferrari ED, Oliveira BCM, et al. First description of Giardia duodenalis in buffalo calves (Bubalus bubalis) in southwest region of São Paulo State, Brazil. Food Waterborne Parasitol. 2019; 16: e00062.,6161. Pineda CO, Leal DAG, Fiuza VRDS, Jose J, Borelli G, Durigan M, et al. Toxoplasma gondii oocysts, Giardia cysts and Cryptosporidium oocysts in outdoor swimming pools in Brazil. Zoonoses Public Health. 2020; 67(7): 785-95.

The low investment in tools with discriminatory potential results that sequencing is still the most widely used methodology, although it may present divergences according to the gene target used. The whole genome sequencing (WGS) is suggested for genotyping;1010. Ryan U, Zahedi A. Molecular epidemiology of giardiasis from a veterinary perspective. Adv Parasitol. 2019; 106: 209-54. however, the high cost and the delay in obtaining results prevent its use on a large scale, especially in developing countries, such as Brazil.

In Brazil, assemblage-specific PCR was used in four studies, mainly with the gdh and tpi markers, but orfC4 and SSu-rRNA were also observed.4242. Durigan M, Abreu AG, Zucchi MI, Franco RM, de Souza AP. Genetic diversity of Giardia duodenalis: multilocus genotyping reveals zoonotic potential between clinical and environmental sources in a metropolitan region of Brazil. PLoS One. 2014; 9(12): e115489.,4343. Faria CP, Zanini GM, Dias GS, da Silva S, Sousa MC. Molecular characterization of Giardia lamblia: first report of assemblage B in human isolates from Rio de Janeiro (Brazil). PLoS One. 2016; 11(8): e0160762.,4444. Ulloa-Stanojlovic FM, Aguiar B, Jara LM, Sato MI, Guerrero JA, Hachich E, et al. Occurrence of Giardia intestinalis and Cryptosporidium sp. in wastewater samples from São Paulo State, Brazil, and Lima, Peru. Environ Sci Pollut Res Int. 2016; 23(21): 22197-205.,4545. de Araújo RS, Aguiar B, Dropa M, Razzolini MTP, Sato MIZ, Lauretto MS, et al. Detection and molecular characterization of Cryptosporidium species and Giardia assemblages in two watersheds in the metropolitan region of São Paulo, Brazil. Environ Sci Pollut Res Int. 2018; 25(15): 15191-203.,4646. Gomes KB, Fernandes AP, Menezes A, Amorim Jr R, Silva EF, Rocha MO. Giardia duodenalis: genotypic comparison between a human and a canine isolates. Rev Soc Bras Med Trop. 2011; 44(4): 508-10. PCR or quantitative PCR (qPCR) were used to identify assemblages A and B in isolates from human clinical samples, surface raw water or wastewater.4343. Faria CP, Zanini GM, Dias GS, da Silva S, Sousa MC. Molecular characterization of Giardia lamblia: first report of assemblage B in human isolates from Rio de Janeiro (Brazil). PLoS One. 2016; 11(8): e0160762.,4444. Ulloa-Stanojlovic FM, Aguiar B, Jara LM, Sato MI, Guerrero JA, Hachich E, et al. Occurrence of Giardia intestinalis and Cryptosporidium sp. in wastewater samples from São Paulo State, Brazil, and Lima, Peru. Environ Sci Pollut Res Int. 2016; 23(21): 22197-205.,4545. de Araújo RS, Aguiar B, Dropa M, Razzolini MTP, Sato MIZ, Lauretto MS, et al. Detection and molecular characterization of Cryptosporidium species and Giardia assemblages in two watersheds in the metropolitan region of São Paulo, Brazil. Environ Sci Pollut Res Int. 2018; 25(15): 15191-203. These results were identical to those of PCR restriction fragment length polymorphism (PCR-RFLP), and no divergence or inconsistencies in the assemblages were found among the four different loci (qPCR: gdh, tpi, orfC4; PCR-RFLP: βg, gdh).4343. Faria CP, Zanini GM, Dias GS, da Silva S, Sousa MC. Molecular characterization of Giardia lamblia: first report of assemblage B in human isolates from Rio de Janeiro (Brazil). PLoS One. 2016; 11(8): e0160762. In isolates from dog samples, some divergences between assemblage-specific PCR were found for C and D, using the tpi target, when compared to gene sequencing.

PCR-RFLP was used for genotyping in ten studies, most of which were accompanied mainly by gene sequencing (8/10).2121. Pacheco FTF, Carvalho SS, Cardoso LS, Andrade LS, das Chagas GMT, Gomes DC, et al. Immune response markers in sera of children infected with Giardia duodenalis AI and AII subassemblages. Immunobiology. 2019; 224(4): 595-603.,2828. Volotão AC, Costa-Macedo LM, Haddad FS, Brandão A, Peralta JM, Fernandes O. Genotyping of Giardia duodenalis from human and animal samples from Brazil using beta-giardin gene: a phylogenetic analysis. Acta Trop. 2007; 102(1): 10-9.,4040. Paz e Silva FM, Lopes RS, Araújo Jr JP. Genetic characterisation of Giardia duodenalis in dairy cattle in Brazil. Folia Parasitol (Praha). 2012; 59(1): 15-20.,4343. Faria CP, Zanini GM, Dias GS, da Silva S, Sousa MC. Molecular characterization of Giardia lamblia: first report of assemblage B in human isolates from Rio de Janeiro (Brazil). PLoS One. 2016; 11(8): e0160762.,4747. Paz e Silva FM, Monobe MM, Lopes RS, Araujo Jr JP. Molecular characterization of Giardia duodenalis in dogs from Brazil. Parasitol Res. 2012; 110(1): 325-34.,4848. Colli CM, Bezagio RC, Nishi L, Ferreira ÉC, Falavigna-Guilherme AL, Gomes ML. Food handlers as a link in the chain of transmission of Giardia duodenalis and other protozoa in public schools in southern Brazil. Trans R Soc Trop Med Hyg. 2015; 109(9): 601-3.,4949. Colli CM, Bezagio RC, Nishi L, Bignotto TS, Ferreira ÉC, Falavigna-Guilherme AL, et al. Identical assemblage of Giardia duodenalis in humans, animals and vegetables in an urban area in southern Brazil indicates a relationship among them. PLoS One. 2015; 10(3): e0118065.,5050. Paz e Silva FM, Lopes RS, Bresciani KD, Amarante AF, Araujo Jr JP. High occurrence of Cryptosporidium ubiquitum and Giardia duodenalis genotype E in sheep from Brazil. Acta Parasitol. 2014; 59(1): 193-6.,5151. Rafael K, Marchioro AA, Colli CM, Tiyo BT, Evangelista FF, Bezagio RC, et al. Genotyping of Giardia duodenalis in vegetables cultivated with organic and chemical fertilizer from street markets and community vegetable gardens in a region of Southern Brazil. Trans R Soc Trop Med Hyg. 2017; 111(12): 540-5.,5252. Quadros RM, Weiss PH, Marques SM, Miletti LC. Potential cross-contamination of similar Giardia duodenalis assemblage in children and pet dogs in southern Brazil, as determined by PCR-RFLP. Rev Inst Med Trop São Paulo. 2016; 58: 66. The most used target in PCR-RFLP was gdh, and sometimes appeared combined with the target βg.2121. Pacheco FTF, Carvalho SS, Cardoso LS, Andrade LS, das Chagas GMT, Gomes DC, et al. Immune response markers in sera of children infected with Giardia duodenalis AI and AII subassemblages. Immunobiology. 2019; 224(4): 595-603.,4343. Faria CP, Zanini GM, Dias GS, da Silva S, Sousa MC. Molecular characterization of Giardia lamblia: first report of assemblage B in human isolates from Rio de Janeiro (Brazil). PLoS One. 2016; 11(8): e0160762.,4747. Paz e Silva FM, Monobe MM, Lopes RS, Araujo Jr JP. Molecular characterization of Giardia duodenalis in dogs from Brazil. Parasitol Res. 2012; 110(1): 325-34.,4848. Colli CM, Bezagio RC, Nishi L, Ferreira ÉC, Falavigna-Guilherme AL, Gomes ML. Food handlers as a link in the chain of transmission of Giardia duodenalis and other protozoa in public schools in southern Brazil. Trans R Soc Trop Med Hyg. 2015; 109(9): 601-3.,4949. Colli CM, Bezagio RC, Nishi L, Bignotto TS, Ferreira ÉC, Falavigna-Guilherme AL, et al. Identical assemblage of Giardia duodenalis in humans, animals and vegetables in an urban area in southern Brazil indicates a relationship among them. PLoS One. 2015; 10(3): e0118065.,5050. Paz e Silva FM, Lopes RS, Bresciani KD, Amarante AF, Araujo Jr JP. High occurrence of Cryptosporidium ubiquitum and Giardia duodenalis genotype E in sheep from Brazil. Acta Parasitol. 2014; 59(1): 193-6.,5151. Rafael K, Marchioro AA, Colli CM, Tiyo BT, Evangelista FF, Bezagio RC, et al. Genotyping of Giardia duodenalis in vegetables cultivated with organic and chemical fertilizer from street markets and community vegetable gardens in a region of Southern Brazil. Trans R Soc Trop Med Hyg. 2017; 111(12): 540-5.,5252. Quadros RM, Weiss PH, Marques SM, Miletti LC. Potential cross-contamination of similar Giardia duodenalis assemblage in children and pet dogs in southern Brazil, as determined by PCR-RFLP. Rev Inst Med Trop São Paulo. 2016; 58: 66. Only one study used only the βg target for genotyping in PCR-RFLP.2828. Volotão AC, Costa-Macedo LM, Haddad FS, Brandão A, Peralta JM, Fernandes O. Genotyping of Giardia duodenalis from human and animal samples from Brazil using beta-giardin gene: a phylogenetic analysis. Acta Trop. 2007; 102(1): 10-9. In these studies, PCR-RFLP showed a satisfactory result in identifying the assemblages circulating, with the exception of just one study where PCR-RFLP data obtained by sequencing did not determine the assemblage of isolates from samples of humans, dogs and vegetables.4949. Colli CM, Bezagio RC, Nishi L, Bignotto TS, Ferreira ÉC, Falavigna-Guilherme AL, et al. Identical assemblage of Giardia duodenalis in humans, animals and vegetables in an urban area in southern Brazil indicates a relationship among them. PLoS One. 2015; 10(3): e0118065.

The relevance of the whole genetic sequence for a better understanding of G. duodenalis isolates is undeniable; however, all other sequencing brings an extremely relevant contribution to the scientific literature. The choice of the genotyping tool must be determined according to the investigation focus. PCR-RFLP proved to be a good strategy for an initial survey of circulating assemblages. Assemblage-specific PCR can be of great value in places where infectious assemblages are already known. Due to the divergences between the gene targets used in the sequencing, the use of more than one marker is recommended, as data from multi-locus sequencing brings more robustness to understanding the assemblage transmission dynamics. However, new gene markers and new financially viable tools are urgently needed to stimulate the expansion of genotyping studies and, consequently, enable a better understanding of the real zoonotic potential of G. duodenalis assemblages.

Circulation of G. duodenalis assemblages in environmental samples from Brazil

The presence of intestinal parasites dates from antiquity. In Brazil, the findings in coprolites and mummies point to the existence of these infections for more than 7,000 years and the existence of Giardia, for more than 5,300 years.5353. Frías L, Leles D, Araújo A. Studies on protozoa in ancient remains - A Review. Mem Inst Oswaldo Cruz. 2013; 108(1): 1-12. However, isolating cysts from archaeological material is an essential but difficult task, and techniques continue to be improved.5454. Leles D, Cascardo P, Pucu E, Brener B, Sudré A, Alves E, et al. Methodological innovations for the study of irreplaceable samples reveal giardiasis in extinct animals (Nothrotherium maquinense and Palaeolama maior). Parasitol Int. 2018; 67(6): 776-80.,5555. Leles D, Frías L, Araújo A, Brener B, Sudré A, Chame M, et al. Are immunoenzymatic tests for intestinal protozoans reliable when used on archaeological material? Exp Parasitol. 2019; 205: 107739. Thus, further studies are needed to determine the genetic characteristics of the G. duodenalis isolates circulating at that time.

The occurrence of Giardia in water and food is associated with contamination by faeces from humans and/or animals, so different species and assemblages can be found. The main form of G. duodenalis infection, reported mainly in cases of outbreaks, is via waterborne transmission.5656. Torgerson PR, Devleesschauwer B, Praet N, Speybroeck N, Willingham AL, Kasuga F, et al. World Health Organization estimates of the global and regional disease burden of 11 foodborne parasitic diseases, 2010: a data synthesis. PLoS Med. 2015; 12(12): e1001920. Thus, assessing the quality of drinking water sources is an excellent measure of giardiasis control, and genotyping the water isolates and the individuals who consume it helps to outline the transmission cycles.

In Brazil, genotyping studies of G. duodenalis in water are basically concentrated in the State of São Paulo,4242. Durigan M, Abreu AG, Zucchi MI, Franco RM, de Souza AP. Genetic diversity of Giardia duodenalis: multilocus genotyping reveals zoonotic potential between clinical and environmental sources in a metropolitan region of Brazil. PLoS One. 2014; 9(12): e115489.,4444. Ulloa-Stanojlovic FM, Aguiar B, Jara LM, Sato MI, Guerrero JA, Hachich E, et al. Occurrence of Giardia intestinalis and Cryptosporidium sp. in wastewater samples from São Paulo State, Brazil, and Lima, Peru. Environ Sci Pollut Res Int. 2016; 23(21): 22197-205.,4545. de Araújo RS, Aguiar B, Dropa M, Razzolini MTP, Sato MIZ, Lauretto MS, et al. Detection and molecular characterization of Cryptosporidium species and Giardia assemblages in two watersheds in the metropolitan region of São Paulo, Brazil. Environ Sci Pollut Res Int. 2018; 25(15): 15191-203.,5757. Fernandes LN, de Souza PP, de Araújo RS, Razzolini MT, Soares RM, Sato MI, et al. Detection of assemblages A and B of Giardia duodenalis in water and sewage from São Paulo State, Brazil. J Water Health. 2011; 9(2): 361-7.,5858. Leal DAG, Souza DSM, Caumo KS, Fongaro G, Panatieri LF, Durigan M, et al. Genotypic characterization and assessment of infectivity of human waterborne pathogens recovered from oysters and estuarine waters in Brazil. Water Res. 2018; 137: 273-80.,5959. Yamashiro S, Foco MLR, Pineda CO, José J, Nour EAA, Siqueira-Castro ICV, et al. Giardia spp. and Cryptosporidium spp. removal efficiency of a combined fixed-film system treating domestic wastewater receiving hospital effluent. Environ Sci Pollut Res Int. 2019; 26(22): 22756-71.,6161. Pineda CO, Leal DAG, Fiuza VRDS, Jose J, Borelli G, Durigan M, et al. Toxoplasma gondii oocysts, Giardia cysts and Cryptosporidium oocysts in outdoor swimming pools in Brazil. Zoonoses Public Health. 2020; 67(7): 785-95. and in the State of Paraná, which presents a single study4141. Ferreira FP, Caldart ET, Freire RL, Mitsuka-Breganó R, Freitas FM, Miura AC, et al. The effect of water source and soil supplementation on parasite contamination in organic vegetable gardens. Rev Bras Parasitol Vet. 2018; 27(3): 327-37. (Table II) (Fig. 3). The presence of G. duodenalis assemblages A, B, C, D and E suggests the contamination of water sources by faeces from different groups of hosts.

TABLE II
Giardia duodenalis assemblages found in animals and in the environment in Brazilian states

Fig. 3:
distribution of Giardia duodenalis assemblage in Brazil. The states where G. duodenalis genotyping were not found are presented whithout colour. The arrows indicate the hosts and the respective assemblages observed.

The two studies that genotyped G. duodenalis isolates from sewage samples identified the presence of assemblages A, B and C (Table I).5757. Fernandes LN, de Souza PP, de Araújo RS, Razzolini MT, Soares RM, Sato MI, et al. Detection of assemblages A and B of Giardia duodenalis in water and sewage from São Paulo State, Brazil. J Water Health. 2011; 9(2): 361-7.,5959. Yamashiro S, Foco MLR, Pineda CO, José J, Nour EAA, Siqueira-Castro ICV, et al. Giardia spp. and Cryptosporidium spp. removal efficiency of a combined fixed-film system treating domestic wastewater receiving hospital effluent. Environ Sci Pollut Res Int. 2019; 26(22): 22756-71. More studies like this should be encouraged, because quantifying the presence of intestinal parasites in sewage could be a strategy to predict the contamination index of the human and animal population by agents such as G. duodenalis.

In addition to waterborne transmission, giardiasis is also a foodborne disease. In Brazil, genotyping studies of G. duodenalis from food have only been observed in the State of Paraná (Tables I-II) (Fig. 3).4141. Ferreira FP, Caldart ET, Freire RL, Mitsuka-Breganó R, Freitas FM, Miura AC, et al. The effect of water source and soil supplementation on parasite contamination in organic vegetable gardens. Rev Bras Parasitol Vet. 2018; 27(3): 327-37.,4949. Colli CM, Bezagio RC, Nishi L, Bignotto TS, Ferreira ÉC, Falavigna-Guilherme AL, et al. Identical assemblage of Giardia duodenalis in humans, animals and vegetables in an urban area in southern Brazil indicates a relationship among them. PLoS One. 2015; 10(3): e0118065.,5151. Rafael K, Marchioro AA, Colli CM, Tiyo BT, Evangelista FF, Bezagio RC, et al. Genotyping of Giardia duodenalis in vegetables cultivated with organic and chemical fertilizer from street markets and community vegetable gardens in a region of Southern Brazil. Trans R Soc Trop Med Hyg. 2017; 111(12): 540-5.,6262. Tiyo R, de Souza CZ, Piovesani AFA, Tiyo BT, Colli CM, Marchioro AA, et al. Predominance of Giardia duodenalis assemblage AII in fresh leafy vegetables from a market in southern Brazil. J Food Prot. 2016; 79(6): 1036-9. The presence of assemblages A, B and E on the surface of vegetables was also investigated. It is common to use horse and cattle faeces as vegetable fertilisation strategies, and this could justify the presence of isolates of assemblage E in these foods. However, it is worth mentioning that the plants undergo a constant irrigation process and that the assemblage (E) has already been observed in water source.4141. Ferreira FP, Caldart ET, Freire RL, Mitsuka-Breganó R, Freitas FM, Miura AC, et al. The effect of water source and soil supplementation on parasite contamination in organic vegetable gardens. Rev Bras Parasitol Vet. 2018; 27(3): 327-37. As assemblages A and B can also be found in these vegetables,3333. da Cunha MJR, Cury MC, Santín M. Molecular identification of Enterocytozoon bieneusi, Cryptosporidium, and Giardia in Brazilian captive birds. Parasitol Res. 2017; 116(2): 487-93.,3535. Shivley CB, Lombard JE, Urie NJ, Kopral CA, Santin M, Earleywine TJ, et al. Preweaned heifer management on US dairy operations: Part VI. Factors associated with average daily gain in preweaned dairy heifer calves. J Dairy Sci. 2018; 101(10): 9245-58.,4040. Paz e Silva FM, Lopes RS, Araújo Jr JP. Genetic characterisation of Giardia duodenalis in dairy cattle in Brazil. Folia Parasitol (Praha). 2012; 59(1): 15-20. the interpretation can be equivalent. However, in Brazil, the cultivation of vegetables is still predominately artisanal and familiar, although carried out in large fields, which results in human contact and contact with other animals that have been raised on the land.

Giardia duodenalis assemblage’ circulation in host from Brazil

Classically, G. psittaci and G. ardeae species are commonly reported in bird droppings and G. duodenalis in mammal infections.55. Adam RD. Biology of Giardia lamblia. Clin Microbiol Rev. 2001; 14(3): 447-75.,66. Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev. 2011; 24(1): 110-40. However, recent reports have demonstrated the circulation of the following G. duodenalis assemblages in bird species: A, B, D and F from Spain,6363. Reboredo-Fernández A, Ares-Mazás E, Cacciò SM, Gómez-Couso H. Occurrence of Giardia and Cryptosporidium in wild birds in Galicia (Northwest Spain). Parasitology. 2015; 142(7): 917-25.,6464. Cano L, de Lucio A, Bailo B, Cardona GA, Muadica AS, Lobo L, et al. Identification and genotyping of Giardia spp. and Cryptosporidium spp. isolates in aquatic birds in the Salburua wetlands, Álava, northern Spain. Vet Parasitol. 2016; 221: 144-8. A from Italy,6565. Papini R, Girivetto M, Marangi M, Mancianti F, Giangaspero A. Endoparasite infections in pet and zoo birds in Italy. Scientific World Journal. 2012; 2012: 253127. and A and B from the Ivory Coast.6666. Berrilli F, D’Alfonso R, Giangaspero A, Marangi M, Brandonisio O, Kaboré Y, et al. Giardia duodenalis genotypes and Cryptosporidium species in humans and domestic animals in Côte d’Ivoire: occurrence and evidence for environmental contamination. Trans R Soc Trop Med Hyg. 2012; 106(3): 191-5. In Brazil, the first report of this parasite in birds occurred in toco toucan which was infected by assemblage A.3333. da Cunha MJR, Cury MC, Santín M. Molecular identification of Enterocytozoon bieneusi, Cryptosporidium, and Giardia in Brazilian captive birds. Parasitol Res. 2017; 116(2): 487-93. As this is a bird that lives in captivity, the infection may have occurred through contact with human faeces, which is corroborated by the finding of this assemblage (A) (Tables I-II) (Fig. 3). The possibility of G. duodenalis infection in birds could greatly increase its potential for dispersion.

Although the division of G. duodenalis assemblage by association with the host it infects is still maintained today, the findings indicate that its current distribution may be much more complex. Many doubts persist when we observe species infected by assemblages outside the expected host-specific species: (1) Is it possible that the circulation of these assemblages has always occurred and the scarcity of studies has hindered their previous identification? (2) Is it possible that the high frequency of interspecific contact could increase the pressure of infection and consequently select individuals capable of infecting this new host?

In Brazil, assemblages A, B, C and E have already been identified in isolates from human faeces samples distributed in 21 studies carried out in the nine states.88. Fantinatti M, Bello AR, Fernandes O, Da-Cruz AM. Identification of Giardia lamblia assemblage E in humans points to a new anthropozoonotic cycle. J Infect Dis. 2016; 214(8): 1256-9.,1212. Thompson RC, Reynoldson JA, Mendis AH. Giardia and giardiasis. Adv Parasitol. 1993; 32: 71-160.,4242. Durigan M, Abreu AG, Zucchi MI, Franco RM, de Souza AP. Genetic diversity of Giardia duodenalis: multilocus genotyping reveals zoonotic potential between clinical and environmental sources in a metropolitan region of Brazil. PLoS One. 2014; 9(12): e115489.,4343. Faria CP, Zanini GM, Dias GS, da Silva S, Sousa MC. Molecular characterization of Giardia lamblia: first report of assemblage B in human isolates from Rio de Janeiro (Brazil). PLoS One. 2016; 11(8): e0160762.,4646. Gomes KB, Fernandes AP, Menezes A, Amorim Jr R, Silva EF, Rocha MO. Giardia duodenalis: genotypic comparison between a human and a canine isolates. Rev Soc Bras Med Trop. 2011; 44(4): 508-10.,4848. Colli CM, Bezagio RC, Nishi L, Ferreira ÉC, Falavigna-Guilherme AL, Gomes ML. Food handlers as a link in the chain of transmission of Giardia duodenalis and other protozoa in public schools in southern Brazil. Trans R Soc Trop Med Hyg. 2015; 109(9): 601-3.,4949. Colli CM, Bezagio RC, Nishi L, Bignotto TS, Ferreira ÉC, Falavigna-Guilherme AL, et al. Identical assemblage of Giardia duodenalis in humans, animals and vegetables in an urban area in southern Brazil indicates a relationship among them. PLoS One. 2015; 10(3): e0118065.,5252. Quadros RM, Weiss PH, Marques SM, Miletti LC. Potential cross-contamination of similar Giardia duodenalis assemblage in children and pet dogs in southern Brazil, as determined by PCR-RFLP. Rev Inst Med Trop São Paulo. 2016; 58: 66.,5959. Yamashiro S, Foco MLR, Pineda CO, José J, Nour EAA, Siqueira-Castro ICV, et al. Giardia spp. and Cryptosporidium spp. removal efficiency of a combined fixed-film system treating domestic wastewater receiving hospital effluent. Environ Sci Pollut Res Int. 2019; 26(22): 22756-71.,6767. Souza SL, Gennari SM, Richtzenhain LJ, Pena HF, Funada MR, Cortez A, et al. Molecular identification of Giardia duodenalis isolates from humans, dogs, cats and cattle from the state of São Paulo, Brazil, by sequence analysis of fragments of glutamate dehydrogenase (gdh) coding gene. Vet Parasitol. 2007; 149(3-4): 258-64.,6868. Kohli A, Bushen OY, Pinkerton RC, Houpt E, Newman RD, Sears CL, et al. Giardia duodenalis assemblage, clinical presentation and markers of intestinal inflammation in Brazilian children. Trans R Soc Trop Med Hyg. 2008; 102(7): 718-25.,6969. Volotão AC, Ramos NM, Fantinatti M, Moraes MV, Netto HA, Storti-Melo LM, et al. Giardiasis as zoonosis: between proof of principle and paradigm in the Northwestern region of São Paulo State, Brazil. Braz J Infect Dis. 2011; 15(4): 382-3.,7070. Santos CK, Grama DF, Limongi JE, Costa FC, Couto TR, Soares RM, et al. Epidemiological, parasitological and molecular aspects of Giardia duodenalis infection in children attending public daycare centers in southeastern Brazil. Trans R Soc Trop Med Hyg. 2012; 106(8): 473-9.,7171. David ÉB, Guimarães S, de Oliveira AP, de Oliveira-Sequeira TCG, Bittencourt GN, Nardi ARM, et al. Molecular characterization of intestinal protozoa in two poor communities in the State of São Paulo, Brazil. Parasit Vectors. 2015; 8: 103.,7272. Nunes BC, Pavan MG, Jaeger LH, Monteiro KJ, Xavier SC, Monteiro FA, et al. Spatial and molecular epidemiology of Giardia intestinalis deep in the Amazon, Brazil. PLoS One. 2016; 11(7): e0158805.,7373. Oliveira-Arbex AP, David EB, Oliveira-Sequeira TC, Bittencourt GN, Guimarães S. Genotyping of Giardia duodenalis isolates in asymptomatic children attending daycare centre: evidence of high risk for anthroponotic transmission. Epidemiol Infect. 2016; 144(7): 1418-28.,7474. Scalia LA, Fava NM, Soares RM, Limongi JE, da Cunha MJ, Pena IF, et al. Multilocus genotyping of Giardia duodenalis in Brazilian children. Trans R Soc Trop Med Hyg. 2016; 110(6): 343-9.,7575. Faria CP, Zanini GM, Dias GS, da Silva S, Sousa MDC. New multilocus genotypes of Giardia lamblia human isolates. Infect Genet Evol. 2017; 54: 128-37.,7676. Nunes BC, Calegar DA, Pavan MG, Jaeger LH, Monteiro KJL, Dos Reis ERC, et al. Genetic diversity of Giardia duodenalis circulating in three Brazilian biomes. Infect Genet Evol. 2018; 59: 107-12.,7777. Seguí R, Muñoz-Antoli C, Klisiowicz DR, Oishi CY, Köster PC, de Lucio A, et al. Prevalence of intestinal parasites, with emphasis on the molecular epidemiology of Giardia duodenalis and Blastocystis sp., in the Paranaguá Bay, Brazil: a community survey. Parasit Vectors. 2018; 11(1): 490.,7878. Corrêa CRT, Oliveira-Arbex AP, David ÉB, Guimarães S. Genetic analysis of Giardia duodenalis isolates from children of low-income families living in an economically successful region in southeastern Brazil. Rev Inst Med Trop São Paulo. 2020; 62: e20. Assemblages A and B are classically associated with infection in humans (Table I) (Fig. 3).55. Adam RD. Biology of Giardia lamblia. Clin Microbiol Rev. 2001; 14(3): 447-75.,66. Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev. 2011; 24(1): 110-40. However, the identification of assemblages C and E points to the possibility of man’s participation in the transmission cycles of these assemblages. Assemblage E was reported in humans by our group in Rio de Janeiro,88. Fantinatti M, Bello AR, Fernandes O, Da-Cruz AM. Identification of Giardia lamblia assemblage E in humans points to a new anthropozoonotic cycle. J Infect Dis. 2016; 214(8): 1256-9. and also by researchers from Minas Gerais.7474. Scalia LA, Fava NM, Soares RM, Limongi JE, da Cunha MJ, Pena IF, et al. Multilocus genotyping of Giardia duodenalis in Brazilian children. Trans R Soc Trop Med Hyg. 2016; 110(6): 343-9. However, assemblage C has only been reported in São Paulo.4242. Durigan M, Abreu AG, Zucchi MI, Franco RM, de Souza AP. Genetic diversity of Giardia duodenalis: multilocus genotyping reveals zoonotic potential between clinical and environmental sources in a metropolitan region of Brazil. PLoS One. 2014; 9(12): e115489. Other authors have reported the occurrence of assemblages C7979. Soliman RH, Fuentes I, Rubio JM. Identification of a novel Assemblage B subgenotype and a zoonotic Assemblage C in human isolates of Giardia intestinalis in Egypt. Parasitol Int. 2011; 60(4): 507-11.,8080. Štrkolcová G, Maďar M, Hinney B, Goldová M, Mojžišová J, Halánová M. Dog’s genotype of Giardia duodenalis in human: first evidence in Europe. Acta Parasitol. 2015; 60(4): 796-9.,8181. Cao S, Xu M, Jiang Y, Liu H, Yuan Z, Sun L, et al. Prevalence and genetic characterization of Cryptosporidium, Giardia and Enterocytozoon in chickens from Ezhou, Hubei, China. Front Vet Sci. 2020; 7: 30. and E99. Zahedi A, Field D, Ryan U. Molecular typing of Giardia duodenalis in humans in Queensland - first report of assemblage E. Parasitology. 2017; 144(9): 1154-61.,8282. Abdel-Moein KA, Saeed H. The zoonotic potential of Giardia intestinalis assemblage E in rural settings. Parasitol Res. 2016; 115(8): 3197-202. in humans from other countries. Of the assemblages already identified in humans, only assemblage F was not yet identified in Brazil.1111. Cacciò SM, Lalle M, Svärd SG. Host specificity in the Giardia duodenalis species complex. Infect Genet Evol. 2018; 66: 335-45. Possibly, the low frequency of this assemblage, even among felines in the country, has not favoured the occurrence of human infection.

Domestic animals have great relevance in G. duodenalis zoonotic transmission, mainly anthropozoonotic. The two genotyping studies of G. duodenalis in cats carried out in São Paulo4242. Durigan M, Abreu AG, Zucchi MI, Franco RM, de Souza AP. Genetic diversity of Giardia duodenalis: multilocus genotyping reveals zoonotic potential between clinical and environmental sources in a metropolitan region of Brazil. PLoS One. 2014; 9(12): e115489.,6767. Souza SL, Gennari SM, Richtzenhain LJ, Pena HF, Funada MR, Cortez A, et al. Molecular identification of Giardia duodenalis isolates from humans, dogs, cats and cattle from the state of São Paulo, Brazil, by sequence analysis of fragments of glutamate dehydrogenase (gdh) coding gene. Vet Parasitol. 2007; 149(3-4): 258-64. and one in Rio de Janeiro (one animal),2828. Volotão AC, Costa-Macedo LM, Haddad FS, Brandão A, Peralta JM, Fernandes O. Genotyping of Giardia duodenalis from human and animal samples from Brazil using beta-giardin gene: a phylogenetic analysis. Acta Trop. 2007; 102(1): 10-9. point to the circulation of assemblage A (Table II) (Fig. 3). In Brazil, the feline host-specific assemblage (F) was only observed in São Paulo,6767. Souza SL, Gennari SM, Richtzenhain LJ, Pena HF, Funada MR, Cortez A, et al. Molecular identification of Giardia duodenalis isolates from humans, dogs, cats and cattle from the state of São Paulo, Brazil, by sequence analysis of fragments of glutamate dehydrogenase (gdh) coding gene. Vet Parasitol. 2007; 149(3-4): 258-64. where the circulation of assemblages B and D was also observed.4242. Durigan M, Abreu AG, Zucchi MI, Franco RM, de Souza AP. Genetic diversity of Giardia duodenalis: multilocus genotyping reveals zoonotic potential between clinical and environmental sources in a metropolitan region of Brazil. PLoS One. 2014; 9(12): e115489.

In dogs, infection by host-specific assemblages (C and D) was observed in Minas Gerais, São Paulo, Santa Catarina and Paraná (Table II) (Fig. 3).4242. Durigan M, Abreu AG, Zucchi MI, Franco RM, de Souza AP. Genetic diversity of Giardia duodenalis: multilocus genotyping reveals zoonotic potential between clinical and environmental sources in a metropolitan region of Brazil. PLoS One. 2014; 9(12): e115489.,4747. Paz e Silva FM, Monobe MM, Lopes RS, Araujo Jr JP. Molecular characterization of Giardia duodenalis in dogs from Brazil. Parasitol Res. 2012; 110(1): 325-34.,4949. Colli CM, Bezagio RC, Nishi L, Bignotto TS, Ferreira ÉC, Falavigna-Guilherme AL, et al. Identical assemblage of Giardia duodenalis in humans, animals and vegetables in an urban area in southern Brazil indicates a relationship among them. PLoS One. 2015; 10(3): e0118065.,5252. Quadros RM, Weiss PH, Marques SM, Miletti LC. Potential cross-contamination of similar Giardia duodenalis assemblage in children and pet dogs in southern Brazil, as determined by PCR-RFLP. Rev Inst Med Trop São Paulo. 2016; 58: 66.,6767. Souza SL, Gennari SM, Richtzenhain LJ, Pena HF, Funada MR, Cortez A, et al. Molecular identification of Giardia duodenalis isolates from humans, dogs, cats and cattle from the state of São Paulo, Brazil, by sequence analysis of fragments of glutamate dehydrogenase (gdh) coding gene. Vet Parasitol. 2007; 149(3-4): 258-64.,7171. David ÉB, Guimarães S, de Oliveira AP, de Oliveira-Sequeira TCG, Bittencourt GN, Nardi ARM, et al. Molecular characterization of intestinal protozoa in two poor communities in the State of São Paulo, Brazil. Parasit Vectors. 2015; 8: 103.,8484. Fava NM, Soares RM, Scalia LA, Cunha MJ, Faria ES, Cury MC. Molecular typing of canine Giardia duodenalis isolates from Minas Gerais, Brazil. Exp Parasitol. 2016; 161: 1-5. The infection by assemblage E, which is host-specific for farm animals, was reported in Minas Gerais.8383. Fantinatti M, Caseca AC, Bello AR, Fernandes O, Da-Cruz AM. The presence of Giardia lamblia assemblage A in dogs suggests an anthropozoonotic cycle of the parasite in Rio de Janeiro, Brazil. Infect Genet Evol. 2018; 65: 265-9. Although they used multilocus genotyping, some studies observed a large divergence of the data obtained from the two markers used (tpi and gdh). This could explain the identification of many different assemblages in these dog samples4242. Durigan M, Abreu AG, Zucchi MI, Franco RM, de Souza AP. Genetic diversity of Giardia duodenalis: multilocus genotyping reveals zoonotic potential between clinical and environmental sources in a metropolitan region of Brazil. PLoS One. 2014; 9(12): e115489.,8484. Fava NM, Soares RM, Scalia LA, Cunha MJ, Faria ES, Cury MC. Molecular typing of canine Giardia duodenalis isolates from Minas Gerais, Brazil. Exp Parasitol. 2016; 161: 1-5. (Table I).

In Rio de Janeiro, only assemblage A was identified in dogs.2828. Volotão AC, Costa-Macedo LM, Haddad FS, Brandão A, Peralta JM, Fernandes O. Genotyping of Giardia duodenalis from human and animal samples from Brazil using beta-giardin gene: a phylogenetic analysis. Acta Trop. 2007; 102(1): 10-9.,8383. Fantinatti M, Caseca AC, Bello AR, Fernandes O, Da-Cruz AM. The presence of Giardia lamblia assemblage A in dogs suggests an anthropozoonotic cycle of the parasite in Rio de Janeiro, Brazil. Infect Genet Evol. 2018; 65: 265-9. However, dogs infected by this assemblage (A) were also observed in other states: São Paulo,4242. Durigan M, Abreu AG, Zucchi MI, Franco RM, de Souza AP. Genetic diversity of Giardia duodenalis: multilocus genotyping reveals zoonotic potential between clinical and environmental sources in a metropolitan region of Brazil. PLoS One. 2014; 9(12): e115489.,6969. Volotão AC, Ramos NM, Fantinatti M, Moraes MV, Netto HA, Storti-Melo LM, et al. Giardiasis as zoonosis: between proof of principle and paradigm in the Northwestern region of São Paulo State, Brazil. Braz J Infect Dis. 2011; 15(4): 382-3.,7171. David ÉB, Guimarães S, de Oliveira AP, de Oliveira-Sequeira TCG, Bittencourt GN, Nardi ARM, et al. Molecular characterization of intestinal protozoa in two poor communities in the State of São Paulo, Brazil. Parasit Vectors. 2015; 8: 103. Minas Gerais,4646. Gomes KB, Fernandes AP, Menezes A, Amorim Jr R, Silva EF, Rocha MO. Giardia duodenalis: genotypic comparison between a human and a canine isolates. Rev Soc Bras Med Trop. 2011; 44(4): 508-10.,8484. Fava NM, Soares RM, Scalia LA, Cunha MJ, Faria ES, Cury MC. Molecular typing of canine Giardia duodenalis isolates from Minas Gerais, Brazil. Exp Parasitol. 2016; 161: 1-5. Santa Catarina.5252. Quadros RM, Weiss PH, Marques SM, Miletti LC. Potential cross-contamination of similar Giardia duodenalis assemblage in children and pet dogs in southern Brazil, as determined by PCR-RFLP. Rev Inst Med Trop São Paulo. 2016; 58: 66. Assemblage B was observed in Minas Gerais,8484. Fava NM, Soares RM, Scalia LA, Cunha MJ, Faria ES, Cury MC. Molecular typing of canine Giardia duodenalis isolates from Minas Gerais, Brazil. Exp Parasitol. 2016; 161: 1-5. Santa Catarina5252. Quadros RM, Weiss PH, Marques SM, Miletti LC. Potential cross-contamination of similar Giardia duodenalis assemblage in children and pet dogs in southern Brazil, as determined by PCR-RFLP. Rev Inst Med Trop São Paulo. 2016; 58: 66., São Paulo4242. Durigan M, Abreu AG, Zucchi MI, Franco RM, de Souza AP. Genetic diversity of Giardia duodenalis: multilocus genotyping reveals zoonotic potential between clinical and environmental sources in a metropolitan region of Brazil. PLoS One. 2014; 9(12): e115489. and Paraná4949. Colli CM, Bezagio RC, Nishi L, Bignotto TS, Ferreira ÉC, Falavigna-Guilherme AL, et al. Identical assemblage of Giardia duodenalis in humans, animals and vegetables in an urban area in southern Brazil indicates a relationship among them. PLoS One. 2015; 10(3): e0118065. (Table II) (Fig. 3)

In Brazil, all assemblages (A, B, C, D, E and F) were identified in domestic animals, demonstrating the epidemiological importance of pets in maintaining cysts in the environment. The identification of assemblages A or B circulating in pets suggests that hosts other than dogs and cats may be involved in the transmission cycles. However, the genotyping of G. duodenalis isolates from these domestic animals still remains an unknown field.

Even considering the scarce knowledge of G. duodenalis in infecting wild animals, assemblages with high anthropozoonotic potential (A and B) were already identified in these animals (non-human primates, ostriches, chinchillas, jaguar, toucan).3131. Volotão AC, Júnior JC, Grassini C, Peralta JM, Fernandes O. Genotyping of Giardia duodenalis from southern brown howler monkeys (Alouatta clamitans) from Brazil. Vet Parasitol. 2008; 158(1-2): 133-7.,3232. Soares RM, de Souza SL, Silveira LH, Funada MR, Richtzenhain LJ, Gennari SM. Genotyping of potentially zoonotic Giardia duodenalis from exotic and wild animals kept in captivity in Brazil. Vet Parasitol. 2011; 180(3-4): 344-8.,3333. da Cunha MJR, Cury MC, Santín M. Molecular identification of Enterocytozoon bieneusi, Cryptosporidium, and Giardia in Brazilian captive birds. Parasitol Res. 2017; 116(2): 487-93.,8585. David ÉB, Patti M, Coradi ST, Oliveira-Sequeira TC, Ribolla PE, Guimarães S. Molecular typing of Giardia duodenalis isolates from nonhuman primates housed in a Brazilian zoo. Rev Inst Med Trop São Paulo. 2014; 56(1): 49-54. All animals surveyed had some proximity to humans, which could justify the circulation of these assemblages.

In farm animals (cattle, sheep, pig, and buffalo), the assemblages A, B and E were reported, as expected (Tables I-II) (Fig. 3).4040. Paz e Silva FM, Lopes RS, Araújo Jr JP. Genetic characterisation of Giardia duodenalis in dairy cattle in Brazil. Folia Parasitol (Praha). 2012; 59(1): 15-20.,4242. Durigan M, Abreu AG, Zucchi MI, Franco RM, de Souza AP. Genetic diversity of Giardia duodenalis: multilocus genotyping reveals zoonotic potential between clinical and environmental sources in a metropolitan region of Brazil. PLoS One. 2014; 9(12): e115489.,5050. Paz e Silva FM, Lopes RS, Bresciani KD, Amarante AF, Araujo Jr JP. High occurrence of Cryptosporidium ubiquitum and Giardia duodenalis genotype E in sheep from Brazil. Acta Parasitol. 2014; 59(1): 193-6.,6060. de Aquino MCC, Harvey TV, Inácio SV, Nagata WB, Ferrari ED, Oliveira BCM, et al. First description of Giardia duodenalis in buffalo calves (Bubalus bubalis) in southwest region of São Paulo State, Brazil. Food Waterborne Parasitol. 2019; 16: e00062.,6767. Souza SL, Gennari SM, Richtzenhain LJ, Pena HF, Funada MR, Cortez A, et al. Molecular identification of Giardia duodenalis isolates from humans, dogs, cats and cattle from the state of São Paulo, Brazil, by sequence analysis of fragments of glutamate dehydrogenase (gdh) coding gene. Vet Parasitol. 2007; 149(3-4): 258-64.,8686. Fava NM, Soares RM, Scalia LA, Kalapothakis E, Pena IF, Vieira CU, et al. Performance of glutamate dehydrogenase and triose phosphate isomerase genes in the analysis of genotypic variability of isolates of Giardia duodenalis from livestocks. Biomed Res Int. 2013; 2013: 875048. In these animals, the identification of assemblages A and B, as well as assemblage E, highlights the possible participation of humans, and even domestic animals such as dogs, in the transmission cycles.

In Brazil, assemblages G and H were not yet identified, probably due to the scarcity of studies in rodents and marine mammals. From marine waters, only isolates found in oysters were phylogenetically classified as assemblage A.5858. Leal DAG, Souza DSM, Caumo KS, Fongaro G, Panatieri LF, Durigan M, et al. Genotypic characterization and assessment of infectivity of human waterborne pathogens recovered from oysters and estuarine waters in Brazil. Water Res. 2018; 137: 273-80. As these molluscs function as filtering organisms, it cannot be ruled out that assemblage A comes from contaminated environments. It is worth noting that pseudoparasitism cannot be excluded as a possibility in isolated cases of unexpected assemblages in certain hosts.

Final considerations

Brazil is a country with extensive territorial proportions, which means that it presents geographical, climatical biomes with cultural and GDP differences. However, there is a lack of knowledge about the G. duodenalis assemblages circulating in the country, and most of the regions still need to be explored. G. duodenalis genotyping is an important strategy to learn about the genetic variability of the species, mainly, to help understand potential transmission cycles. Despite the high frequency of this protozoa, especially in Brazilian children, little has been explored about the epidemiology of assemblages in humans and animals. In wild animals, we do not know whether G. duodenalis infection occurs in the natural environment and which circulating assemblages would be involved in this case. By sequencing or PCR strategies, and its variations, the circulation of assemblages A, B, C, D, E and F are being reported. Assemblage A in particular, and also assemblage B, have a high anthropozoonotic potential, so their occurrence is expected in different host species. The others assemblages (C-H) are considered host-specific, but can cause the infection of different, atypical hosts, as observed in human infection by assemblage C (host-specific for dogs) and E (considered host-specific for farm animals), in canine infection by assemblage E and in toucan infection by assemblage A. Despite these data, given that few areas of the country were studied and also that few hosts were investigated, knowledge about the real epidemiology of G. duodenalis assemblages in Brazil is still a long way to go.

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Publication Dates

  • Publication in this collection
    25 Jan 2021
  • Date of issue
    2020

History

  • Received
    22 Aug 2020
  • Accepted
    25 Nov 2020
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