Spread and persistence of antimicrobial resistance genes in wastewater from human and animal sources in São Paulo, Brazil.

The spread of antimicrobial resistance (AMR) through multiple reservoirs is a global concern. Wastewater is a critical AMR dissemination source, so this study aimed to assess the persistence of resistance genetic markers in wastewater using a culture-independent approach. Raw and treated wastewater samples (n = 121) from a wastewater treatment plant (WWTP), a human hospital, a veterinary hospital, and a pig farm were monthly collected and concentrated by filtration. DNA was extracted directly from filter membranes, and PCR was used in the qualitative search of 32 antimicrobial resistance genes (ARGs). Selected genes (blaCTX-M, blaKPC, qnrB, and mcr-1) were enumerated by quantitative real-time PCR (qPCR). Twenty-six ARGs were detected in the qualitative ARGs search, while quantitative data showed a low variation of the ARG's relative abundance (RA) throughout the months, especially at the human hospital and the WWTP. At the WWTP, despite significantly reducing the absolute number of gene copies/L after each treatment stage (p < 0.05), slight increases (p > 0.05) in the RAs of genes blaCTX-M, qnrB, and mcr-1 were observed in reused water (tertiary treatment) when compared with secondary effluent. Although the increase is not statistically significant, it is worth noting that there was some level of ARGs concentration after the disinfection process. No significant absolute or relative after-treatment quantification reductions were observed for any ARGs at the veterinary hospital or the pig farm. The spread of ARGs through sewage needs to be continuously addressed, because their release into natural environments may pose potential risks of exposure to resistant bacteria and impact local ecosystems.

Increased susceptibility to encephalitozoonosis associated with mixed Th1/Th2 profile and M1/M2 profile in mice immunosuppressed with cyclophosphamide.

Encephalitozoon cuniculi is a unicellular, spore-forming, obligate intracellular eukaryote belonging to the phylum Microsporidia. It is known to infect mainly immunocompromised and immunocompetent mammals, including humans. The parasite-host relationship has been evaluated using both in vitro cell culturing and animal models. For example, Balb/c and C57BL/6 mouse strains have been used interchangeably, although the latter has been considered more susceptible due to the higher fungal load observed after infection. In the present study, we identified the characteristics of the immune response of C57BL/6 mice treated or not with the immunosuppressant cyclophosphamide (Cy) and challenged with E. cuniculi by intraperitoneal route. After 14 days of infection, serum was collected to analyze Th1, Th2, and Th17 cytokine levels. In addition, peritoneal washes were performed, and the spleen sample was collected for immune cell phenotyping, whereas liver, spleen, kidney, lung, intestine, and central nervous system (CNS) samples were collected for histopathological analysis. Although infected mice displayed a reduced absolute number of macrophages, they showed an M1 profile, an elevated number of CD4+T, CD8+T, B-1, and B-2 lymphocytes, with a predominance of Th1 inflammatory cytokines (interferon [IFN]-γ, tumor necrosis factor [TNF]-α, and interleukin [IL]-2) and Th17. Furthermore, Cy-Infected mice showed a reduced absolute number of macrophages with an M1 profile but a reduced number of CD4+T, CD8+T, B-1, and B-2 lymphocytes, with a predominance of Th1 inflammatory cytokines (IFN-γ, TNF-α, and IL-2) and Th2 (IL-4). This group displayed a higher fungal burden as well and developed more severe encephalitozoonosis, which was associated with a reduced number of T and B lymphocytes and a mixed profile of Th1 and Th2 cytokines.

Parasitological, microbiological, and antimicrobial resistance profiles of raw and drinking water in a tourist city in the tri-border region of South America.

Despite the large amounts of freshwater available in Brazil, the deterioration of surface water can represent a risk of waterborne disease for national and international tourists. The main goal of this study was to assess the quality of drinking water in the triple border region of Brazil before and after being treated in water treatment plants (WTPs) and in Municipal Early Childhood Education Centers (MECECs), in terms of parasitological, microbiological, and physical-chemical aspects. Different water samples were monitored: raw water (RW), treated water (TW), and tap water from the MECECs, giving 60 samples in total, to investigate the presence of Giardia and Cryptosporidium, microbiological indicators, Pseudomonas aeruginosa, and antimicrobial resistance profiles using conventional microbiological assays and parasitological, immunological, and molecular techniques. The results obtained were compared with the reference values recommended by the legislation of drinking water in Brazil. For the first time, contamination by Cryptosporidium and Giardia was demonstrated in RW used to supply WTPs, in TW of Foz do Iguaçu, and in water destined for consumption by children. A total of 52 bacterial isolates were obtained, with high percentages of multidrug resistance to antibiotics, including a carbapenem-resistant profile, highlighting the need to improve quality control standards.

One-year surveillance of SARS-CoV-2 in wastewater from vulnerable urban communities in metropolitan São Paulo, Brazil.

The current COVID-19 pandemic has emphasized the vulnerability of communities living in the urban outskirts and informal settlements. The lack of reliable COVID-19 case data highlights the importance and application of wastewater-based epidemiology. This study aimed to monitor the COVID-19 trends in four vulnerable urban communities (slums and low-income neighborhoods) in metropolitan São Paulo by assessing the SARS-CoV-2 RNA viral load in wastewater. We analyzed 160 samples from May 2020 to June 2021 with weekly or fortnightly samplings. The samples were ultracentrifuged with glycine elution and quantified by N1/N2 SARS-CoV-2 RT-qPCR. The results of positivity were 100% (Paraisópolis, Heliópolis and Cidade Tiradentes) and 76.9% (Vila Brasilândia). The new case numbers of COVID-19, counted from the onset of symptoms, positively correlated with SARS-CoV-2 N1 viral loads from the two largest communities (p<0.001). SARS-CoV-2 infectivity was tested in Vero E6 cells after concentration with the two techniques, ultrafiltration (Centricon® Plus-70 10 kDa) and sucrose cushion ultracentrifugation, but none of the evaluated samples presented positive results. Next-generation sequencing (NGS) analysis from samples collected in March and August 2021 revealed the presence of the clade 20 J (lineage P.1) belonging to the most prevalent circulating variant in the country. Our results showed that wastewater surveillance data can be used as complementary indicators to monitor the dynamics and temporal trends of COVID-19. The infectivity test results strengthened the evidence of low risk of infection associated with SARS-CoV-2 in wastewater.

Successful use of albendazole and fenbendazole therapy in a cat with persistent diarrhea due Enterocytozoon bieneusi.

Enterocytozoon bieneusi, also known as microsporidia, is an obligate, opportunistic, and neglected intracellular pathogen that causes diarrhea in humans. Although identified in the cat feces by epidemiological studies, no association with diarrhea has been demonstrated. We demonstrated a case of chronic enteritis by E. bieneusi in a 1-year-old male Maine Coon cat, neutered with diarrhea for nine months, by histopathological analysis of gastrointestinal biopsies and PCR of feces. The treatment with albendazole (10 days) followed by fenbendazole (5 days) proved to be effective and safe after diagnosis. This description highlights the need to investigate these pathogens in cases of diarrhea due to their importance in public health since they are zoonotic agents.

Opportunistic pneumonia caused by E. cuniculi in mice immunosuppressed with cyclophosphamide

Opportunistic fungal pneumonia is a cause of concern in immunocompromised patients due to its high morbidity and mortality rates. One such opportunistic agent affecting immunocompromised patients is the microsporidia called Encephalitozoon cuniculi. This study aimed to evaluate pneumonia caused by E. cuniculi in mice treated with the immunosuppressive agent cyclophosphamide (Cy). This study also aimed to describe the immune cells associated with the microsporidial pneumonia. C57BL/6 mice were infected intravenously with E. cuniculi spores and treated with Cy (75 mg/kg/week, intraperitoneally). Thirty days post-infection, the fungal burden (qPCR), histopathological lesions, cytokine production, and the phenotype of the immune cells in the lung parenchyma were evaluated. Histologically, interstitial pneumonia with lymphocytic infiltrate was observed in the infected animals. The infiltrate mainly consisted of CD8+ and CD4+ T lymphocytes, with reduced populations of B lymphocytes and macrophages. The production of tumor necrosis factor-alpha (TNF-α) was significant in the animals of the infected groups. Also, the fungal burden was higher in the Cy-treated animals, which was confirmed by the immunohistochemical observation of spores. These results demonstrated that E. cuniculi infection of C57BL/6 mice caused lymphocytic interstitial pneumonia (characterized by a predominant lymphocytic infiltrate), which was aggravated by Cy-induced immunosuppression. Thus, these results can be used to understand the different pathological, immunological, and therapeutic aspects of lymphocytic interstitial pneumonia.

Hepatitis A Outbreaks and Environmental Circulation of Genotype IA Strains in the São Paulo City, 2017-2018.

Hepatitis A virus (HAV) is the major cause of enterically transmitted infectious hepatitis. Between 2016 and 2017, the number of confirmed cases of hepatitis A virus (HAV) increased from 64 to 786 in São Paulo affecting mainly adults aged between 18 and 39 years (80%) and males (88%). To support epidemiological surveillance, the present study monitored the presence of HAV in urban sewage samples collected bimonthly for 1 year (November 2017-November 2018) in the central region of the city, where most of cases were detected. Sewage samples were concentrated by polyethylene glycol precipitation and HAV RNA was quantified by RT-qPCR. Nucleotide sequencing targeting the VP1/2A junction region was carried out to genotype the HAV strains. HAV was detected in 76.9% (40/52) of the samples, with a geometric mean viral load of 5.09 × 104 (± SD 4.51 × 105) genome copies (GC/L) (Mauá Street) and 5.27 × 104 (± SD 1.26 × 106) GC/L (Prestes Maia Avenue). Of the 40 positive samples, 8 were typed as HAV subgenotype IA [100% nucleotide (nt) identity with HAV strain VRD_521_2016]. Highest homology was obtained with sequences from European countries (Italy, Spain) and Israel, all of which had reported recent HAV outbreaks associated with men who have sex with men. Our results highlight that wastewater surveillance is a useful tool to support investigating HAV outbreaks in the community, including circulating genotypes.

Real-time PCR detection of Toxoplasma gondii in surface water samples in São Paulo, Brazil.

Water is considered an important vehicle for the spread of human toxoplasmosis in several countries. Toxoplasma gondii oocysts can persist in the environment for long periods, being highly resistant to the various chemical inactivation processes commonly used by water supply systems, distinctly from simple filtration and flocculation that are efficient in removing oocysts from drinking water. The existing methodologies for identification and quantification of this parasite in water samples are not standardized and have limitations. This study aimed to evaluate the presence of T. gondii oocysts in surface water samples used as a source for the production of drinking water in the State of São Paulo, through the implementation of a specific methodology using real-time PCR technique (qPCR). Volumes of 20 L of the sample were concentrated by filtration in Envirocheck® HV capsules. For DNA extraction, the PowerSoil DNA isolation® kit (currently DNeasy PowerSoil®) was used. The target sequence selected for qPCR was a 62-base-pair fragment of the B1 gene. In the initial recovery evaluation of the method in four replicates of reverse osmosis water, the mean recovery was 48.5% (SD ± 11.5), while the mean recovery for method performance in matrices was 3.2% (SD ± 3.2) (rainy season) and 62.0% (SD ± 6.2) (dry period), suggesting that the characteristics of the samples and the climatic conditions interfere in the recovery efficiency. Of the 39 samples analyzed (May to December 2015), 7.7% (3/39) were positive for T. gondii, and among the ten sources studied; the occurrence of the oocysts was detected in 30% (3/10).

Detection and molecular characterization of Cryptosporidium species and Giardia assemblages in two watersheds in the metropolitan region of São Paulo, Brazil.

Cryptosporidium and Giardia are associated with cases of water and foodborne outbreaks in the world. This study included 50 samples of surface raw water collected from two watersheds in the state of São Paulo, Brazil. The isolation of (oo)cysts was performed in accordance with the U.S. Environmental Protection Agency's methods 1623 and genotypic characterization and quantification were carried out by Nested PCR and qPCR assays based on 18S rRNA and gdh genes, respectively. U.S. EPA 1623 method showed the presence of (oo)cysts in 40% ([Formula: see text] = 0.10 oocysts/L) and 100% ([Formula: see text] = 7.6 cysts/L) of samples from São Lourenço River, respectively, and 24% ([Formula: see text] = 0.8 oocysts/L) and 60% ([Formula: see text] = 1.64 cysts/L) of Guarapiranga Reservoir, respectively. The qPCR assay detected C. hominis/parvum in 52% (0.06 to 1.85 oocysts/L) of São Lourenço River and 64% (0.09 to 1.4 oocysts/L) of Guarapiranga Reservoir samples. Presence/absence test for Giardia intestinalis was positive in 92% of São Lourenço River and 8% of Guarapiranga Reservoir samples. The assemblage A was detected in 16% (0.58 to 2.67 cysts/L) in São Lourenço River and no positive samples were obtained for assemblage B in both water bodies. The characterization of anthroponotic species C. parvum/hominis, G. intestinalis, and assemblage A was valuable in the investigation of possible sources of contamination in the watersheds studied confirming the need of expanding environmental monitoring measures for protection of these water sources in our country.

Occurrence of Giardia intestinalis and Cryptosporidium sp. in wastewater samples from São Paulo State, Brazil, and Lima, Peru.

The objectives of the study were to detect and genotype Cryptosporidium spp. and Giardia intestinalis in wastewater samples obtained from five cities with high transit of people in the State of São Paulo, Brazil, and at the entrance of a Wastewater Treatment Plant (WWTP) in Lima, Peru. Samples were collected and concentrated by centrifugation. The genomic DNA was extracted for molecular characterization by nested PCR for Cryptosporidium and double nested PCR for Giardia, followed by sequencing and phylogenetic analysis. G. intestinalis was found in 63.6 % of the samples, and the human assemblages A and B were identified. Cryptosporidium sp. was found in 36.4 % of the samples, and the species were corresponding to Cryptosporidium hominis, Cryptosporidium cuniculus, and Cryptosporidium muris. Results revealed the presence of human pathogenic Cryptosporidium species and G. intestinalis human pathogenic assemblages. Molecular tools highlight the importance to map the genetic diversity of these parasites, as well as to detect their epidemiological circulation pathway in the environment.

INTESTINAL AND PULMONARY INFECTION BY Cryptosporidium parvum IN TWO PATIENTS WITH HIV/AIDS.

We describe two patients with HIV/AIDS who presented pulmonary and intestinal infection caused by Cryptosporidium parvum, with a fatal outcome. The lack of available description of changes in clinical signs and radiographic characteristics of this disease when it is located in the extra-intestinal region causes low prevalence of early diagnosis and a subsequent lack of treatment.

Quantificação e caracterização genotípica de Cryptosporidium spp. isolados de água bruta superficial e esgoto bruto para a monitorização em mananciais de abastecimento público na Região Metropolitana de São Paulo (RMSP) / Quantification and molecular characterization of Cryptosporidium spp. from raw surface water and raw sewage for the monitoring of public water supply sources in the metropolitan region of São Paulo

As doenças de veiculação hídrica, sobretudo aquelas causadas por protozoários intestinais, emergiram como um dos principais problemas de saúde pública. Diferentes aspectos são abordados sobre a biologia e a epidemiologia dos principais protozoários parasitas de transmissão hídrica. Cryptosporidium está descrito como um importante parasita associado a casos de surtos de veiculação hídrica e alimentos no mundo. A epidemiologia complexa desse protozóario e o fato de que a maioria das espécies e genótipos não pode ser diferenciada morfologicamente, aumentam o interesse por metodologias sensíveis e rápidas na detecção de espécies responsáveis pela infecção em humanos. Neste estudo foram avaliadas 50 amostras de água bruta superficial, coletadas no Rio São Lourenço da Serra (P1A) e Represa de Guarapiranga (P2A) e 50 de esgoto bruto coletadas em São Lourenço da Serra (P1E) e no poço vertical de Taboão da Serra (P2E) entre os meses de janeiro e dezembro de 2013. O isolamento dos oocistos na água foi realizado pelo Método 1623.1 e as amostras de esgoto bruto por centrifugação, separação imunomagnética (IMS). A caracterização genotípica ocorreu por meio da nested PCR, clonagem e sequenciamento com base no gene 18S rRNA comum a todas as espécies de Cryptosporidium. O ensaio de PCR em tempo real (qPCR) foi avaliado simultaneamente para detecção e quantificação de oocistos nas amostras. De acordo com os resultados obtidos pela nested PCR, Cryptosporidium foi detectado na água bruta superficial em 12 por cento (3/25) no manancial P1A e 16 por cento (4/25) no P2A. No esgoto bruto o parasito foi detectado em 20 por cento (5/25) das amostras no ponto P1E e 24 por cento (6/25) no poço vertical P2E. A qPCR detectou 52 por cento (0,79 a 1,85 oocistos/L) de amostras positivas no manancial P1A e o parasito foi detectado em 64 por cento (0,72 a 1,4 oocistos/L) no manancial P2A. No esgoto bruto 72 por cento das amostras foram positivas tanto no ponto P1E (7 a 655 oocistos/L) como no P2E (5 a 519 oocistos/L). A caracterização molecular permitiu a identificação de C. parvum e C. hominis na água bruta superficial, e C. hominis, C. parvum, e C. muris no esgoto bruto. As espécies do gênero Cryptosporidium identificadas neste estudo apresentam expressiva relevância para o desenvolvimento da doença humana. Neste sentido, as metodologias de concentração e caracterização empregadas nas análises demonstraram no geral, o potencial para aplicação em estudos de vigilância ambiental e foram úteis na diferenciação de espécies patogênicas. A presença de C. muris associada às espécies antroponóticas identificadas auxiliou na investigação de prováveis fontes de contaminação no ambiente confirmando a necessidade da expansão de medidas efetivas para proteção destes mananciais.

Waterborne diseases, especially those caused by intestinal protozoa, have emerged as a major public health problem. Different aspects are addressed on the biology and epidemiology of most waterborne protozoan parasites. Cryptosporidium is described as an important parasite associated with cases of waterborne and food outbreaks in the world. The complex epidemiology of this protozoan, as well as the fact that most species and genotypes cannot be differentiated morphologically, increase the interest for sensitive and rapid methods for the detection of species responsible for infection in humans. In this study, 50 samples of raw surface water were collected from São Lourenço River (P1A) and Guarapiranga Dam (P2A), and 50 samples of raw sewage were collected from São Lourenço da Serra (P1E) and from Taboão da Serras vertical well (P2E), between January and December of 2013. The isolation of oocysts in water was carried out by the USEPA Method 1623.1 and raw sewage samples were processed by centrifugation, immunomagnetic separation (IMS). Genotypic characterization occurred by nested PCR, cloning and sequencing based on 18S rRNA gene, which is common to all Cryptosporidium species. Real time PCR assays (qPCR) were carried out both for detection and quantification of oocysts simultaneously in the samples. According to the results obtained by nested PCR, Cryptosporidium was detected in raw surface water in 12 per cent (3/25) of samples at P1A and in 16 per cent (4/25) at P2A. In raw sewage the parasite was detected in 20 per cent (5/25) of samples at P1E and 24 per cent (6/25) in P2E vertical well. qPCR detected 52 per cent (0.79 to 1.85 oocysts/L) of positive samples at P1A, while the parasite was detected in 64 per cent (0.72 to 1.4 oocysts/L) of samples at P2A water supply. Regarding raw sewage, 72 per cent of samples were positive both at P1E (7 to 655 oocysts/L) and P2E (5 to 519 oocysts/L Molecular characterization allowed the identification of C. parvum and C. hominis in raw surface water, and C. hominis, C. parvum and C. muris in raw sewage. Cryptosporidium species identified herein belong to a group of organisms of significant relevance in waterborne diseases. Therefore, concentration and characterization methodologies applied in our analyses showed to be useful for environmental surveillance studies, as well as they were useful in the differentiation of human pathogens. The presence of C. muris associated to anthroponotic species helped in the investigation of likely contamination sources in the environment, confirming the need of expansion in effective measures to protect these water supplies.

Detection of assemblages A and B of Giardia duodenalis in water and sewage from São Paulo state, Brazil.

Giardia duodenalis is a protozoan that parasitizes humans and other mammals and causes giardiasis. Although its isolates have been divided into seven assemblages, named A to G, only A and B have been detected in human faeces. Assemblage A isolates are commonly divided into two genotypes, Al and All. Even though information about the presence of this protozoan in water and sewage is available in Brazil, it is important to verify the distribution of different assemblages that might be present, which can only be done by genotyping techniques. A total of 24 raw and treated sewage, surface and spring water samples were collected, concentrated and purified. DNA was extracted, and a nested PCR was used to amplify an 890 bp fragment of the gdh gene of G. duodenalis, which codes for glutamate dehydrogenase. Positive samples were cloned and sequenced. Ten out of 24 (41.6%) samples were confirmed to be positive for G. duodenalis by sequencing. Phylogenetic analysis grouped most sequences with G. duodenalis genotype All from GenBank. Only two raw sewage samples presented sequences assigned to assemblage B. In one of these samples genotype All was also detected. As these assemblages/genotypes are commonly associated to human giardiasis, the contact with these matrices represents risk for public health.

A modified method for detecting Cryptosporidium oocysts using DNA templates extracted from environmental samples / Método modificado para detecção de oocistos de Cryptosporidium utilizando DNA extraído de amostras ambientais

The protozoan parasite Cryptosporidium has emerged as one of the most important water contaminants, causing outbreaks of waterborne diarrhea worldwide. In order to assess the importance for public health of this pathogen’s presence in environmental samples, several methods have been developed to isolate and detect Cryptosporidium oocysts. In the present study, a reliable and reproducible method has been standardized for detecting and identifying Cryptosporidium oocysts in water samples in the State of São Paulo, Brazil as the first step for future genotyping studies. Water samples were concentrated by filtration, and then subjected to ultrasound in Tween 80 0.1%, the obtained sediment was transferred into micro tubes containing 1.0 ml of distilled water and stored at -20ºC. DNA was extracted with the addition of 1% PVP in lysis buffer, the organic extraction was performed in Phase Lock GelHeavy®. There was a 214 bp amplification on the expected fragment in five out of the 11 water samples analyzed. The results of this study demonstrated the application usefulness of the standardized test in epidemiological studies and surveillance programs because the technology allowed to increase significantly the amount of amplified product.

O protozoário parasito Cryptosporidium tem emergido como um dos mais importantes contaminantes da água, causando surtos de diarreia de veiculação hídrica em todo mundo. Para avaliar o significado, para a saúde pública,da presença desse agente patogênico em amostras ambientais, vários métodos têm sido desenvolvidos para isolar e detectar oocistos de Cryptosporidium. No presente estudo foi padronizado um método confiável e reprodutível para detectar e identificar oocistos de Cryptosporidium em amostras de água no Estado de São Paulo, Brasil, comoo primeiro passo para futuros estudos de genotipagem. Amostras de água foram concentradas por filtração,submetidas a ultrasom em solução de Tween 80 a 0.1%; o sedimento obtido foi transferido para microtuboscontendo 1,0 ml de água destilada e conservado a -20ºC. O DNA foi extraído com adição de 1% de PVP no tampão de lise; a extração foi realizada em tubo Phase Lock Gel Heavy®. Houve amplificação do fragmento esperado de 214 bp em cinco das 11 amostras de água analisadas. Os resultados deste estudo demonstraram a utilidade deaplicação do teste padronizado em estudos epidemiológicos e em programas de vigilância, em virtude da técnica ter apresentado sensibilidade para incrementar significativamente a quantidade de produto amplificado.

Genotipagem de Cryptosporidium spp. provenientes de amostras de águas superficiais e recreacionais como fonte de informação da dispersão de espécies no ambiente, 2006-2008 / Genotyping of Cryptosporidium spp. from superficial and recreational waters samples as source of information of species dispersion in the environment, 2006-2008

As doenças causadas por patógenos emergentes e oportunistas são uma grande preocupação para os profissionais de Saúde Pública. A criptosporidiose é uma doença cosmopolita, cujo agente etiológico é o protozoário coccídia Cryptosporidium. Este parasita emergiu como um importante patógeno de veiculação hídrica, responsável por surtos em diferentes países e atualmente é considerado um problema para indivíduos imunocomprometidos e imunocompetentes. A taxonomia do gênero, baseada em aspectos morfológicos, é de difícil realização devido ao reduzido tamanho dos oocistos e também devido à perda de suas características morfológicas, principalmente em amostras ambientais. Essa limitação estimulou a aplicação de métodos moleculares na identificação das espécies destes microrganismos. Neste estudo, amostras de águas superficiais foram submetidas à ested-PCR para detecção de Cryptosporidium. No total 30 amostras de águas de 10 pontos de coleta situados no estado de São Paulo foram analisadas. Cryptosporidium foi detectado em 30 por cento das amostras analisadas e a genotipagem permitiu a identificação de C. hominis, C. meleagridis e C. andersoni. Embora a identificação de Cryptosporidium em amostras ambientais seja uma tarefa complexa, os métodos moleculares são essenciais para determinação de espécies, ajudando a elucidar a epidemiologia deste protozoário em nosso país.