This is the first time within the last twenty years that a review is being published on the occurrence of G. duodenalis in Brazil, addressing relevant issues such as prevalence, molecular epidemiology and analytical methods for parasite detection.

This systematic review identified research studies related to G. duodenalis in water, giardiasis in animals, prevalence of giardiasis across Brazilian regions, genotyping of strains isolated in humans, and giardiasis in indigenous populations. We also propose a network of G. duodenalis transmission in Brazil based on genotypes analyses.

This systematic review was performed according to recommendations established by Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). As databases for our searches, we have used PubMed, Embase, Scopus and the Brazilian database SciELO using the keywords «Giardia * » and «Brazil».

Giardiasis is an intestinal infection that affects more than two hundred million people annually worldwide; it is caused by the flagellated protozoan Giardia duodenalis. In tropical countries and in low or middle-income settings, like Brazil, its prevalence can be high. There is currently no systematic review on the presence of G. duodenalis in patients, animals or water sources in Brazil.

Giardiasis is an intestinal disease that affect millions of people worldwide, including children. Its main route of transmission is by ingestion of food or water contaminated with the protozoan G. duodenalis. Transmission does not require an animal host, although transmission from animals to human (zoonotic transmission) has been confirmed as an important vector of human giardiasis. This study is a comprehensive description of the impact of giardiasis in Brazil based on studies published in the country from the past 20 years. We describe Giardia prevalence in humans (including indigenous populations), animals and water supplies. In addition, we create a transmission network model for the disease, based on genotype data previously identified in animal and human hosts as well as in environmental samples. The data compiled here will be useful for design of policies to prevent giardiasis transmission in Brazil.

Funding: CHC is thankful to Brazilian National Council for Scientific and Technological Development (CNPq) for the postdoctoral fellowship. The funders have not played any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

In this study, the first systematic review on giardiasis in Brazil, we evaluated the studies published from 1995 to 2015 that address giardiasis as a concern for public health in the country. We describe prevalence of giardiasis across the states, the most frequent assemblages found in humans and animals, the geographic location and distribution of different parasitic assemblages, and contaminated water as a source of Giardia cysts.

Giardiasis is a disease closely related to low-income and lack of sanitation infrastructure [ 9 ]. Although Brazil has improved in infrastructure and educational levels in recent years, the country still presents a disparity among its regions. While in the Southwest region 82.3% of the houses have adequate sanitation systems, in the Northern region this coverage is only 22.4%. Much of this discrepancy is due to disparities in the sanitation services among the different social strata [ 10 ]. Brazil is a very large country (8,515,767,049 km 2 ) and currently has a population of more than 190 million people [ 10 ].

Eight genetic groups (or assemblages) of G. duodenalis (A to H) have been identified: assemblages A and B are considered zoonotic, infecting both humans and animals, including domestic animals, rodents and livestock. Other assemblages infect many species of animals. For example, assemblages C and D are usually infective to dogs and assemblage E is often found in ruminants [ 6 ]. Recent studies have shown that assemblages C and E are also able to infect humans [ 7 , 8 ], although this seems to be a rare occurrence.

Giardia duodenalis is a non-invasive protozoan that attaches to the mucosa of the small intestine in infected hosts leading to giardiasis—a disease that is characterized by a range of clinical symptoms (mild, moderate, or severe) or even asymptomatic infection in many cases. Giardiasis affects more than 280 million people annually worldwide [ 1 ] and its transmission occurs by the ingestion of cysts through contaminated water and food or through person-to-person contact (i.e., fecal-oral transmission). In low and middle-income countries, the prevalence of giardiasis can reach up to 30%. In many cases, lower socio-economic status is associated with a higher prevalence of the disease as there is a greater risk of exposure to contaminated water in poor communities [ 2 , 3 ]. Although the epidemiology regarding transmission is highly variable [ 4 ] and remains a contentious issue, giardiasis can be considered a zoonotic disease. [ 5 , 6 ].

Utilizing a similar approach as Janies [ 15 ] we used betweenness centrality to calculate the connectedness of hosts. To generate a transmission network, an apomorphy list (changes of host) was extracted from each phylogenetic tree. The apomorphy list holds the information of the shift from one host to another based on the relationships of ancestry between the different sequences, calculated on the phylogenetic trees, and the metadata (host) associated with it, thus, giving directionality to the graph. The graph generated is based on direction and frequency of transmission between the hosts observed on the phylogenetic tree and the relative size of each node is based on the Centrality Score of the given node within the network.

Phylogenetic analyses were performed on the three alignments using maximum likelihood as implemented in RAxML [ 14 ]. Host data was manually extracted from the original papers and categorized into seven different categories: Non-Human primates, Farm animals, Dogs, Cats, Humans, Environmental Samples and Wildlife Animals. Host data was associated with tree data utilizing a character matrix and data was mapped onto the phylogenies ( S3 , S4 and S5 Figs) .

For phylogenetic analysis, a bg gene from Giardia cati (KP798445.1), a gdh gene from Giardia psittaci (AB714978.1) and a tpi gene from Giardia microti (AY228649.1) were selected as outgroups. Nucleotide sequence data for 144 bg, 342 gdh and 148 tpi genes were aligned separately using MAFFT v.7.215 [ 12 ] under default settings. The alignments were visualized in Mesquite v.3.04 [ 13 ].

Multiple sequence analyses were performed on bg, gdh and tpi genes. For this analysis, two gdh sequences that were associated with vegetables rather than water, animals or humans were excluded (KJ741292 e KJ741293). A search for duplicate sequences was performed but no sequences were excluded to avoid loss of host data due to the presence of sequences with 100% identity in multiple hosts.

Based on the literature review of giardiasis studies that were performed in Brazil from 1995 to 2015, we extracted gene sequences deposited in Genbank from all the studies related to humans, animals and water, that were isolated in Brazil. Isolate sequences were extracted from Genbank on National Center for Biotechnology Information (NCBI; ncbi.nlm.nih.gov ) nucleotide database.

S1 Fig describes the procedure used to obtain the articles used in this study according to PRISMA. Table 1 shows the number of articles from each database included in the analyses. A map with Brazilian territory and its population density was constructed using the ArcGIS software.

Articles that either did not contain relevant information (a-e) or contained only information related to laboratory analysis such as morphological, molecular and biochemical analysis of Giardia were excluded. Articles without full text access were also excluded after attempts to search in other databases and direct contact with corresponding authors. The last date searched was November 1 st , 2016.

Searches for each topic were restricted to studies published between January 1995 and December 2015, in English, Portuguese, French or Spanish. Data were abstracted from each of the selected articles independently, using a standardized Excel sheet for the sub-themes: a) Detection of Giardia cysts in water samples in Brazil, b) Detection of Giardia in pets, farm animals and wild animals in Brazil, c) Prevalence of giardiasis in the Brazilian population across the states, d) Giardiasis in the Brazilian indigenous population, and e) Distribution of Giardia assemblages in human hosts across Brazil.

This systematic review was performed according to recommendations established by Preferred Reporting Items for Systematic and Meta-Analysis (PRISMA) [ 11 ], a statement of items for reporting systematic reviews. The authors searched in the U.S. National Institutes of Health's National Library (PubMed), Scopus, Embase and in the Brazilian database SciELO using the keywords «Giardia * » and «Brazil». This systematic review identified research studies related to Giardia in water, giardiasis in animals, prevalence of giardiasis across Brazilian regions, genotyping of strains isolated in humans and giardiasis in indigenous populations.

Results

Detection of Giardia cysts in water samples in Brazil The analyses of pathogenic protozoa in water samples in Brazil was initiated in the early 2000s. However, there have been no well-documented waterborne giardiasis outbreaks in the country through the period covered by this study. Contamination with G. duodenalis cysts was first documented in surface water samples from Brazil in 2001 by Franco et al [16]. Sampling was performed in the Atibaia River, in the city of Campinas, Southeastern Brazil during three consecutive weeks. The membrane filtration technique was employed to concentrate cysts using acetate cellulose membranes of 47 mm diameter and 3 μm porosity. For the recovery of this parasite, two different procedures were compared: either rinsing and scraping the membrane surface (RM method) or dissolving the membrane in acetone (ADM method). Cysts were visualized by a direct immunofluorescence assay (IFA) (Merifluor kit, Meridian Diagnostic, Cincinnati, Ohio). All water samples were positive for Giardia, despite the high turbidity. The RM method showed higher recovery rates in positive control assays. Although some studies [17–19] utilized the USEPA reference method 1623 (concentration by IDDEX FiltaMax followed by purification with Immunomagnetic Separation (IMS) and immunofluorescence assay (IFA) [20] or the calcium carbonate flocculation technique for concentration of Giardia cysts in different sources of water [17, 19, 21, 22], membrane filtration followed by immunofluorescence assay remains the most employed technique in the country for the detection of this parasite (Table 3). Sampling volumes ranged from 0.5 L to 1000 L. The USEPA Method 1623 was employed in only a few studies [17–19], probably due to its excessive cost being prohibitive for most Brazilian laboratories, except for those located in the Southeast region of the country. PPT PowerPoint slide

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larger image TIFF original image Download: Table 3. Detection of G. duodenalis cysts in water sources derived through different regions of Brazil. Only studies using the membrane filtration technique and immunofluorescence or molecular assays are included. https://doi.org/10.1371/journal.pntd.0006005.t003 It is relevant to emphasize that surface water is more commonly used for the supply of drinking water than underground or spring water sources in Brazil. Taken together, the data from Table 3, allied with other studies [17, 19, 22] denote the wide occurrence of G. duodenalis in surface waters in Brazil, and highlight the necessity for water treatment companies to comply with Ordinance 2914/2011 [23] to ensure a safe water supply for Brazilian population and to minimize public health risks. Moreover, few studies have shown recovery efficiency data: when Method 1623 was the chosen methodology, recoveries ranged from 34% to 39.4% [17–19] in accordance with USEPA’S recommendation. Matrix spiked sample assays conducted by membrane filtration and calcium carbonate flocculation, without or with IMS, showed an increase in recovery efficiencies when the IMS step was added to both methodologies [24]. Few studies have addressed the prevalence of G. duodenalis cysts in treated waters [17, 25, 26]. In the metropolitan region of São Paulo state, which has undergone accelerated population growth, G. duodenalis cysts were detected in treated water produced by a conventional water treatment system [17]. The detection of the parasite in water samples was performed using the USEPA Method 1623. Giardia cysts were detected in 41.7% of treated water samples, with concentration ranging from non-detected to 0.06 cysts/L. In other studies, all treated water samples were negative [25, 26]. In the past, most studies concerning G. duodenalis in water were performed in states of the Southeastern and Southern regions of Brazil. Very few studies were conducted in other regions, likely reflecting the social disparities existing throughout the country. Two studies [27, 28] were conducted in Pernambuco (a state in the Northeast region). The first study found a prevalence of 50.0% for Giardia cysts in samples from the Beberibe River which suffers anthropogenic and animal contamination as demonstrated by high levels of E. coli found in its water (from 50,000 to ≥ 160,000 NMP/100mL) [27]. The second study investigated bacterial and parasite contamination of rainwater stored in tanks and clay pots, in a semi-arid region of Northeast Brazil. G. duodenalis cysts were detected in 10.0% of rainwater samples conserved in clay pots and tanks, respectively [28]. In both studies, traditional methodologies such as spontaneous sedimentation and light microscopy were employed, and cysts were visualized on slides by Lugol staining. In Brazil, the presence of thermotolerant coliforms is still often used to verify water potability, and when combined with other water characteristics such as turbidity, it is an indirect indicator for the presence of potentially pathogenic protozoa [29]. In most studies, no correlation was found between the occurrence of G. duodenalis in raw water samples and bacteriological, physical, chemical or climate factors. However, in the Paraná state, a correlation was found between the presence of G. duodenalis and high mean value of turbidity– 1,198.85 NTU (r = 0.5809; p = 0.0029) [25]. The accurate detection of this protozoan and its molecular characterization is still incipient or non-existent in many parts of the country. To address the probability of G. duodenalis infection from water wells in a peri-urban area, genotypic characterization of the protozoan was performed. Sequence analysis from the gdh gene yielded predominantly genotype A–subgenotype II [18]. Additional genotypic characterization of G. duodenalis contaminating surface waters was conducted in two municipalities within the São Paulo state. Phylogenetic analyses based on gdh gene sequences also showed the predominance of G. duodenalis belonging to genotype AII, the most common genotype associated to human giardiasis in this region [30]. Evaluation of the genetic diversity of a set of environmental water samples (river and stream waters) from the metropolitan region of Campinas, São Paulo, found that most of the samples contained assemblages A and B. In addition, assemblages C and D were also identified at the water abstraction point of the city. Only one sample (from the Anhumas River), presented mixed assemblages (BIV and D) determined through amplification and sequencing reactions using the gdh and tpi genes [31]. Contamination by G. duodenalis cysts was monitored in an important mariculture production area in São Paulo state. The search for the protozoan was performed in brackish waters in all stages of oyster cultivation and treatment steps before to be sent to market. In addition, analyses included search for Giardia in a recreational area frequented by tourists. PCR amplification of the bg gene demonstrated contamination by G. duodenalis in all analyzed sites [32]. In Florianópolis, Santa Catarina state, the evaluation of tropical water sources in the main shellfish growing areas, revealed contamination by G. duodenalis (assemblage A) in one site of seawater highly impacted by domestic sewage [33]. Few studies have addressed the issue of microbiological risk for Giardia. A Quantitative Microbial Risk Assessment (QMRA) based on parasite concentration to estimate the probability of protozoan parasite infection associated with water ingestion was conducted in four densely urbanized regions of São Paulo state [19]. The estimated risk of Giardia infection ranged from 0.29% to 2.47% per year for adults, and from 0.08% to 0.70% for children. The infection risk by this parasite was higher than what is considerable tolerable by the USEPA for a yearly exposure. G. duodenalis risk infection was greater for adults than that observed for children, reflecting the higher water ingestion in adults compared to children. The study also concluded that the metropolitan region of Campinas exhibits the highest risk of G. duodenalis infection among all studied regions.

Detection of Giardia in pets, farm animals and wild animals in Brazil Although G. duodenalis is an important cause of gastrointestinal disease in humans, it has also been frequently diagnosed in wildlife and companion animals. The detection of this protozoan in animals has been widely documented in numerous hosts, including dogs, cats, calves, sheep, lambs, horses, pigs, non-human primates, and wildlife, in many regions of Brazil. Among the 61 studies that reported G. duodenalis cysts in these hosts between 1995 and 2015 (Fig 1), dogs were the most studied host (38%) followed by farm animals (23%) and wildlife (18%). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Proportion of studies of G. duodenalis in Brazil performed in different animal hosts. https://doi.org/10.1371/journal.pntd.0006005.g001 Conventional diagnosis based on optical microscopy was the only method in 68.8% (42/61) of the studies. The development of molecular markers has allowed the identification of specific assemblages in both animal hosts and human patients; the first studies with DNA-based approaches for assessing Giardia infection in animals were only published in 2007 [38, 39]. Examination of the last five years covered by this literature review, showed that molecular techniques such as Polymerase Chain Reaction (PCR) and DNA sequencing have been used in 53.8% (14/26) of the included studies and only studies published in the last two years of our analysis presented greater numbers of molecular diagnoses compared to techniques based on optical microscopy (Fig 2). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. Number of studies in which conventional and molecular methods were used for diagnosis of giardiasis in animals between 1995 and 2015. https://doi.org/10.1371/journal.pntd.0006005.g002 The Brazilian canine population is estimated at 28 million, including over 22 million stray dogs [40], which can be explained by the great availability of food in the streets (obtained from garbage) and the climate conditions [41–43]. The prevalence of Giardia cysts in dogs in Brazil ranged from 0.8% [44] to 45% [45]. In many regions of Brazil, this prevalence is between 8.4% to 11.1% based on microscopic examinations [46]. The genetic characterization studies using DNA-based approaches detected mainly host adapted genetic assemblages C and D, even though the assemblages AI, AII, BIII and BIV have also been reported [31, 34, 39, 47, 48]. Thus, zoonotic transmission could represent a public health problem in developing countries [41]. When compared to other countries, there is still relatively little information on G. duodenalis assemblages in dogs [49] even though the predominance of host adapted assemblages C and D is notable. Reports of assemblages A and B in dogs suggest that zoonotic transmission could represent a problem of public health in Brazil [41]. Both domestic and stray animals can be disseminators of zoonotic parasites. In Brazil, the prevalence of G. duodenalis in stray dogs is higher in comparison to household pets [41, 45, 50]. A statistically significant difference was also found between shelter dogs and household pets, probably due to the greater concentration of animals and exposure to environmental contamination [45], which can also be considered for stray dogs. A consistent program of sanitary education must be included in public health actions for the control of intestinal parasites in dogs [41]. Cats may also represent an important reservoir of G. duodenalis based on prevalence and the genotypes that have been identified in Brazilian studies. The prevalence ranges from 3.5% to 13.7% [51, 52] and most molecular studies detected the potential zoonotic genetic assemblages AI, BIII and BIV [31, 38, 39]. Wild felines may also represent a significant source of infection by G. duodenalis. The prevalence can reach 38.5% in captive felines with 23.1% having mixed infection with helminths [42, 53]. The presence of Giardia is also commonly reported in livestock, although most studies are restricted to cattle. These studies report that calves usually shed G. duodenalis cysts from the host adapted assemblage E. However the zoonotic assemblages A and B have also been identified [31, 54, 55]. Thus infected calves may also represent a public health risk [55]. The presence of G. duodenalis is rarely analyzed in goats, but in two recent studies in Brazil, prevalence of the parasite ranged between 22.6% and 29.3% and the dominant genotype was genetic assemblage E [56, 57]. A similar scenario is observed for studies with sheep. The prevalence of G. duodenalis in sheep ranged from 24% to 34% and the same host adapted assemblage was detected [54, 58]. Regarding equines, low prevalence was detected and no molecular studies have been published [59, 60]. The presence of cysts of G. duodenalis in wildlife animals has been evaluated by many studies in Brazil, most of them based on conventional diagnostic techniques. A single study with chinchillas, ostriches and a jaguar detected genetic assemblages AI and B [61]. In small wild rodents, a prevalence between 2.05 and 100% was reported. However, apart from the single exception just mentioned, there are no data regarding the genetic assemblages in wildlife [62–67]. A prevalence of 3.6% was found in captive snakes based on enzyme immunoassay [68]. In non-human primates, several studies have reported a prevalence between 0.5% and 44.4% [69–72]. The genetic assemblage A was detected in primates kept in a zoo, which highlights that, considering the zoonotic potential of the assemblage detected, regular coproparasitological surveys are necessary to safeguard the captive animals, their caretakers and people visiting zoos [69]. The genotype AI was also detected in captive Alouatta clamitans in the south of the country, indicating that these animals might be susceptible to infection with G. duodenalis strains of human origin [72, 73]. The states from the southeast region of Brazil are responsible for 52% (São Paulo 32%, Rio de Janeiro 15%, Minas Gerais 13% and Espírito Santo 2%) of the publications examined. In contrast, the North, Northeast and Middle West regions together are responsible for only 13.3% of all studies, which represents another reason for the inequality between regions.