Biological and Genetic Characterization of Cryptosporidium spp. and Giardia duodenalis Isolates from Five Hydrographical Basins in Northern Portugal

To understand the situation of water contamination with Cryptosporidium spp. and Giardia spp. in the northern region of Portugal, we have established a long-term program aimed at pinpointing the sources of surface water and environmental contamination, working with the water-supply industry. Here, we describe the results obtained with raw water samples collected in rivers of the 5 hydrographical basins. A total of 283 samples were analyzed using the Method 1623 EPA, USA. Genetic characterization was performed by PCR and sequencing of genes 18S rRNA of Cryptosporidium spp. and β-giardin of Giardia spp. Infectious stages of the protozoa were detected in 72.8% (206 of 283) of the water samples, with 15.2% (43 of 283) positive for Giardia duodenalis cysts, 9.5% (27 of 283) positive for Cryptosporidium spp. oocysts, and 48.1% (136 of 283) samples positive for both parasites. The most common zoonotic species found were G. duodenalis assemblages A-I, A-II, B, and E genotypes, and Cryptosporidium parvum, Cryptosporidium andersoni, Cryptosporidium hominis, and Cryptosporidium muris. These results suggest that cryptosporidiosis and giardiasis are important public health issues in northern Portugal. To the authors'' knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in raw water samples in the northern region of Portugal.     

Identical Assemblage of Giardia duodenalis in Humans,Animals and Vegetables in an Urban Area in Southern Brazil Indicates a Relationship among Them

Background

Giardia duodenalis infects humans and other mammals by ingestion of cysts in contaminated water or food, or directly in environments with poor hygiene. Eight assemblages, designated A–H, are described for this species.

Methodology/Principal Findings

We investigated by microscopy or by direct immunofluorescence technique the occurrence of G. duodenalis in 380 humans, 34 animals, 44 samples of water and 11 of vegetables. G. duodenalis cysts present in samples were genotyped through PCR-RFLP of β giardin and glutamate dehydrogenase (gdh) genes and sequencing of gdh. The gdh gene was amplified in 76.5% (26/34) of the human faeces samples with positive microscopy and in 2.9% (1/34) of negative samples. In 70.4% (19/27) of the positive samples were found BIV assemblage. In two samples from dogs with positive microscopy and one negative sample, assemblages BIV, C, and D were found. Cysts of Giardia were not detected in water samples, but three samples used for vegetable irrigation showed total coliforms above the allowed limit, and Escherichia coli was observed in one sample. G. duodenalis BIV was detected in two samples of Lactuca sativa irrigated with this sample of water. BIV was a common genotype, with 100% similarity, between different sources or hosts (humans, animals and vegetables), and the one most often found in humans.

Conclusions/Significance

This is the first study in Brazil that reports the connection among humans, dogs and vegetables in the transmission dynamics of G. duodenalis in the same geographic area finding identical assemblage. BIV assemblage was the most frequently observed among these different links in the epidemiological chain.     

Prevalence,Environmental Loading,and Molecular Characterization of Cryptosporidium and Giardia Isolates from Domestic and Wild Animals along the Central California Coast

The risk of disease transmission from waterborne protozoa is often dependent on the origin (e.g., domestic animals versus wildlife), overall parasite load in contaminated waterways, and parasite genotype, with infections being linked to runoff or direct deposition of domestic animal and wildlife feces. Fecal samples collected from domestic animals and wildlife along the central California coast were screened to (i) compare the prevalence and associated risk factors for fecal shedding of Cryptosporidium and Giardia species parasites, (ii) evaluate the relative importance of animal host groups that contribute to pathogen loading in coastal ecosystems, and (iii) characterize zoonotic and host-specific genotypes. Overall, 6% of fecal samples tested during 2007 to 2010 were positive for Cryptosporidium oocysts and 15% were positive for Giardia cysts. Animal host group and age class were significantly associated with detection of Cryptosporidium and Giardia parasites in animal feces. Fecal loading analysis revealed that infected beef cattle potentially contribute the greatest parasite load relative to other host groups, followed by wild canids. Beef cattle, however, shed host-specific, minimally zoonotic Cryptosporidium and Giardia duodenalis genotypes, whereas wild canids shed potentially zoonotic genotypes, including G. duodenalis assemblages A and B. Given that the parasite genotypes detected in cattle were not zoonotic, the public health risk posed by protozoan parasite shedding in cattle feces may be lower than that posed by other animals, such as wild canids, that routinely shed zoonotic genotypes.     

Multilocus typing of Cryptosporidium spp. and Giardia duodenalis from non-human primates in China

Non-human primates (NHPs) are commonly infected with Cryptosporidium spp. and Giardia duodenalis. However, molecular characterisation of these pathogens from NHPs remains scarce. In this study, 2,660 specimens from 26 NHP species in China were examined and characterised by PCR amplification of 18S rRNA, 70 kDa heat shock protein (hsp70) and 60 kDa glycoprotein (gp60) gene loci for Cryptosporidium; and 1,386 of the specimens by ssrRNA, triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) gene loci for Giardia. Cryptosporidium was detected in 0.7% (19/2660) specimens of four NHP species including rhesus macaques (0.7%), cynomolgus monkeys (1.0%), slow lorises (10.0%) and Francois’ leaf monkeys (6.7%), belonging to Cryptosporidium hominis (14/19) and Cryptosporidium muris (5/19). Two C. hominis gp60 subtypes, IbA12G3 and IiA17 were observed. Based on the tpi locus, G. duodenalis was identified in 2.2% (30/1,386) of specimens including 2.1% in rhesus macaques, 33.3% in Japanese macaques, 16.7% in Assam macaques, 0.7% in white-headed langurs, 1.6% in cynomolgus monkeys and 16.7% in olive baboons. Sequence analysis of the three targets indicated that all of the Giardia-positive specimens belonged to the zoonotic assemblage B. Highest sequence polymorphism was observed at the tpi locus, including 11 subtypes: three known and eight new ones. Phylogenetic analysis of the subtypes showed that most of them were close to the so-called subtype BIV. Intragenotypic variations at the gdh locus revealed six types of sequences (three known and three new), all of which belonged to so-called subtype BIV. Three specimens had co-infection with C. hominis (IbA12G3) and G. duodenalis (BIV). The presence of zoonotic genotypes and subtypes of Cryptosporidium spp. and G. duodenalis in NHPs suggests that these animals can potentially contribute to the transmission of human cryptosporidiosis and giardiasis.     

Cryptosporidium cuniculus and Giardia duodenalis in rabbits: genetic diversity and possible zoonotic transmission

Background

Cryptosporidium and Giardia are the two important zoonotic pathogens causing diarrhea of humans and animals worldwide. Considering the human cryptosporidiosis outbreak and sporadic cases caused by C. cuniculus, the important public health significance of G. duodenalis and little obtained information regarding rabbit infected with Cryptosporidium and Giardia in China, the aim of this study is to determine the prevalence and molecularly characterize Cryptosporidium and Giardia in rabbits in Heilongjiang Province, China.

Methodology/Principal Findings

378 fecal samples were obtained from rabbits in Heilongjiang Province. Cryptosporidium oocysts and Giardia cysts were detected using Sheather''s sugar flotation technique and Lugol''s iodine stain method, respectively. The infection rates of Cryptosporidium and Giardia were 2.38% (9/378) and 7.41% (28/378), respectively. Genotyping of Cryptosporidium spp. was done by DNA sequencing of the small subunit rRNA (SSU rRNA) gene and all the nine isolates were identified as Cryptosporidium cuniculus. The nine isolates were further subtyped using the 60-kDa glycoprotein (gp60) gene and two subtypes were detected, including VbA32 (n = 3) and a new subtype VbA21 (n = 6). G. duodenalis genotypes and subtypes were identified by sequence analysis of the triosephosphate isomerase (TPI) gene. The assemblage B (belonging to eight different subtypes B-I to B-VIII) was found in 28 G. duodenalis-positive samples.

Conclusions/Significance

The rabbits have been infected with Cryptosporidium and Giardia in Heilongjiang Province. The results show that the rabbits pose a threat to human health in the studied areas. Genotypes and subgenotypes of C. cuniculus and G. duodenalis in this study might present the endemic genetic characterization of population structure of the two parasites.     

A natural zoonotic giardiasis: Infection of a child via Giardia cysts in pet chinchilla droppings

Here, we report a case of direct zoonotic transmission of giardiasis between a pet chinchilla and a human. Microscopic and molecular examinations of stool samples from a child and samples of chinchilla droppings revealed cysts/DNA of Giardia intestinalis. The transmission from the chinchilla to the child has been confirmed as coprophagous after the 1-year-old toddler ingested pet chinchilla droppings. Molecular analysis of the gdh gene from both hosts classified the G. intestinalis cysts into the assemblage B genetic group, which has been previously shown to be characteristic of both human and chinchilla giardiasis. Both Giardia sub-assemblages BIII and BIV were present in the chinchilla droppings, whereas only the sub-assemblage BIV was isolated from the child's stool sample. To the best of our knowledge, this is the first report of a true zoonotic transmission of giardiasis, supporting the zoonotic potential of assemblage B.     

Molecular Characterization of Giardia Isolates from Calves and Humans in a Region in Which Dairy Farming Has Recently Intensified

Giardiasis is a notifiable disease of high prevalence in New Zealand, but there is limited knowledge about the sources of Giardia duodenalis genotypes that can potentially cause human infections. Dairy calves are one environmental source of Giardia isolates, but it is unknown whether they harbor genotypes that are potentially capable of causing infections in humans. To address these questions, 40 Giardia isolates from calves and 30 from humans, living in the same region and collected over a similar period, were genotyped using the β-giardin gene. The G. duodenalis genetic assemblages A and B were identified from both calves and humans, and genotype comparisons revealed a substantial overlap of identical genotypes from the two hosts for both assemblages. Significantly, no assemblage E (the genotype commonly found in cattle elsewhere in the world) has been detected in New Zealand livestock to date. Given recent and rapid land use conversions to dairy farming in many South Island regions of New Zealand, an increasingly large concentration of domestic cattle harboring genotypes potentially capable of causing infections in humans is particularly concerning.     

Occurrence of Cryptosporidium Oocysts and Giardia Cysts in Sewage in Norway

Samples of sewage influent from 40 sewage treatment works (STW) throughout Norway were examined for Cryptosporidium oocysts and Giardia duodenalis cysts. Both parasites were detected frequently (80% of STW were Cryptosporidium positive; 93% of STW were Giardia positive) and at maximum concentrations of >20,000 parasites/liter. The data suggest giardiasis is more widespread, and/or occurs with greater infection intensity, than cryptosporidiosis in Norway. STW serving higher person equivalents were more likely to be positive and had higher parasite concentrations. Parasite concentrations were used to estimate the proportion of contributing populations that could be clinically infected. For Cryptosporidium, the highest estimates were up to 5 per 100,000 individuals for two populations in eastern Norway. For Giardia, the highest estimate was 40 infected per 100,000 persons (approximately five times the usual national annual average) contributing to an STW in western Norway. As this population experienced a large waterborne giardiasis outbreak 6 months after sampling, it can be speculated that regular challenge with Giardia may occur here. Most Giardia isolates in sewage influent were assemblage A, although some assemblage B isolates were detected. There was substantial heterogeneity, but most samples contained isolates similar to genotype A3. Removal efficiencies at two STW with secondary treatment processes were estimated to be approximately 50% for Cryptosporidium and >80% for Giardia. An STW with minimal treatment had negligible removal of both parasites. Many STW in Norway have minimal treatment and discharge effluent into rivers and lakes, thus, risk of contamination of water courses by Cryptosporidium and Giardia is considerable.     

Presence of Cryptosporidium spp. and Giardia duodenalis in Drinking Water Samples in the North of Portugal

Cryptosporidium and Giardia are 2 protozoan parasites responsible for waterborne diseases outbreaks worldwide. In order to assess the prevalence of these protozoans in drinking water samples in the northern part of Portugal and the risk of human infection, we have established a long term program aiming at pinpointing the sources of surface water, drinking water, and environmental contamination, working with the water-supply industry. Total 43 sources of drinking water samples were selected, and a total of 167 samples were analyzed using the Method 1623. Sensitivity assays regarding the genetic characterization by PCR and sequencing of the genes, 18S SSU rRNA, for Cryptosporidium spp. and β,-giardin for G. duodenalis were set in the laboratory. According to the defined criteria, molecular analysis was performed over 4 samples. Environmental stages of the protozoa were detected in 25.7% (43 out of 167) of the water samples, 8.4% (14 out of 167) with cysts of Giardia, 10.2% (17 out of 167) with oocysts of Cryptosporidium and 7.2% (12 out of 167) for both species. The mean concentrations were 0.1-12.7 oocysts of Cryptosporidium spp. per 10 L and 0.1-108.3 cysts of Giardia duodenalis per 10 L. Our results suggest that the efficiency in drinking water plants must be ameliorated in their efficiency in reducing the levels of contamination. We suggest the implementation of systematic monitoring programs for both protozoa. To authors'' knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in drinking water samples in the northern part of Portugal.     

Cryptosporidium Source Tracking in the Potomac River Watershed

To better characterize Cryptosporidium in the Potomac River watershed, a PCR-based genotyping tool was used to analyze 64 base flow and 28 storm flow samples from five sites in the watershed. These sites included two water treatment plant intakes, as well as three upstream sites, each associated with a different type of land use. The uses, including urban wastewater, agricultural (cattle) wastewater, and wildlife, posed different risks in terms of the potential contribution of Cryptosporidium oocysts to the source water. Cryptosporidium was detected in 27 base flow water samples and 23 storm flow water samples. The most frequently detected species was C. andersoni (detected in 41 samples), while 14 other species or genotypes, almost all wildlife associated, were occasionally detected. The two common human-pathogenic species, C. hominis and C. parvum, were not detected. Although C. andersoni was common at all four sites influenced by agriculture, it was largely absent at the urban wastewater site. There were very few positive samples as determined by Environmental Protection Agency method 1623 at any site; only 8 of 90 samples analyzed (9%) were positive for Cryptosporidium as determined by microscopy. The genotyping results suggest that many of the Cryptosporidium oocysts in the water treatment plant source waters were from old calves and adult cattle and might not pose a significant risk to human health.     

Genetic characterization of Giardia duodenalis by sequence analysis in humans and animals in Pemba Island,Tanzania

Giardia duodenalis represents one of the most widespread human enteric parasites: about 200 million people in Asia, Africa and Latin America are infected. Giardia exerts a deep impact on public health because of high prevalence and possible effects on growth and cognitive functions in infected children. The major aim of this study was to detect and genetically characterize G. duodenalis in both human and animal fecal samples collected in Pemba Island, in the archipelago of Zanzibar (Tanzania), in order to deepen the knowledge of genotypes of Giardia in this area.Between October 2009 and October 2010, we collected 45 human fecal samples from children from 2 primary schools and 60 animal fecal samples: 19 from zebus (Bos primigenius indicus) and 41 from goats (Capra hircus). Detection and genetic identification were performed by multilocus analysis of ssu-rDNA and gdh genes. In humans we found a higher prevalence of assemblage B (sub-assemblage BIV), in goats of assemblage E and in zebus of assemblage A. Our study represents an important contribution to the epidemiological knowledge of G. duodenalis in this area of Tanzania.     

Genotyping Cryptosporidium andersoni in Cattle in Shaanxi Province,Northwestern China

The present study examined the prevalence and genotypes of Cryptosporidium andersoni in cattle in Shaanxi province, China. A total of 2071 fecal samples (847 from Qinchuan cattle and 1224 from dairy cattle) were examined for the presence of Cryptosporidium oocysts, and 70 samples (3.4%) were C. andersoni-positive and those positive samples were identified by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) and the Cryptosporidium oocyst wall protein (COWP) genes. C. andersoni was the only species found in the examined cattle in this province. Fifty-seven C. andersoni isolates were characterized into 5 MLST subtypes using multilocus sequence typing analysis, including a new subtype in the native beef breed Qinchuan cattle. All of these C. andersoni isolates presented a clonal genetic structure. These findings provide new insights into the genetic structure of C. andersoni isolates in Shaanxi province and basic data of Cryptosporidium prevalence status, which in turn have implications for controlling cryptosporidiosis in this province.     

Occurrence of Giardia duodenalis assemblages in alpacas in the Andean region

In this study, 352 fecal samples were analyzed for G. duodenalis from alpaca mothers and crias from three different areas of highland in Peru. The triosephosphate isomerase (TPI) gene of Giardia was amplified using a nested PCR protocol. Forty-six G. duodenalis-PCR positive samples were sequenced. G. duodenalis assemblage A was the most frequent followed by assemblage E. The former was seen in 37 animals whereas the latter was seen in nine. Most of the assemblage A infections were caused by the A1 subtype of sub-assemblage AI, except for three, which were caused by the A2 subtype of sub-assemblage AI. Assemblage A was found in all three geographic regions, while assemblage E was detected in crias from two regions. Among the four alpaca mothers positive for Giardia, three had assemblage AI and one had assemblage AII. Results of this study indicate that possible zoonotic transmission human to alpacas.     

Identification of zoonotic Cryptosporidium and Giardia genotypes infecting animals in Sydney's water catchments

To identify the animal sources for Cryptosporidium and Giardia contamination, we genotyped Cryptosporidium and Giardia spp. in wildlife from Sydney’s water catchments using sequence analysis at the 18S rRNA locus for Cryptosporidium and 18S rRNA and glutamate dehydrogenase (gdh) for Giardia. A total of 564 faecal samples from 16 different host species were analysed. Cryptosporidium was identified in 8.5% (48/564) samples from eight host species and Giardia was identified in 13.8% (78/564) from seven host species. Eight species/genotypes of Cryptosporidium were identified. Five G. duodenalis assemblages were detected including the zoonotic assemblages A and B.     

Molecular Genotyping of Giardia duodenalis Isolates from Symptomatic Individuals Attending Two Major Public Hospitals in Madrid,Spain

Background

The flagellate protozoan Giardia duodenalis is an enteric parasite causing human giardiasis, a major gastrointestinal disease of global distribution affecting both developing and industrialised countries. In Spain, sporadic cases of giardiasis have been regularly identified, particularly in pediatric and immigrant populations. However, there is limited information on the genetic variability of circulating G. duodenalis isolates in the country.

Methods

In this longitudinal molecular epidemiological study we report the diversity and frequency of the G. duodenalis assemblages and sub-assemblages identified in 199 stool samples collected from 184 individual with symptoms compatible with giardiasis presenting to two major public hospitals in Madrid for the period December 2013–January 2015. G. duodenalis cysts were initially detected by conventional microscopy and/or immunochomatography on stool samples. Confirmation of the infection was performed by direct immunofluorescence and real-time PCR methods. G. duodenalis assemblages and sub-assemblages were determined by multi-locus genotyping of the glutamate dehydrogenase (GDH) and β-giardin (BG) genes of the parasite. Sociodemographic and clinical features of patients infected with G. duodenalis were also analysed.

Principal findings

Of 188 confirmed positive samples from 178 giardiasis cases a total of 124 G. duodenalis isolates were successfully typed at the GDH and/or the BG loci, revealing the presence of sub-assemblages BIV (62.1%), AII (15.3%), BIII (4.0%), AI (0.8%), and AIII (0.8%). Additionally, 6.5% of the isolates were only characterised at the assemblage level, being all of them assigned to assemblage B. Discordant genotype results AII/AIII or BIII/BIV were also observed in 10.5% of DNA isolates. A large number of multi-locus genotypes were identified in G. duodenalis assemblage B, but not assemblage A, isolates at both the GDH and BG loci, confirming the high degree of genetic variability observed in other molecular surveys. BIV was the most prevalent genetic variant of G. duodenalis found in individuals with symptomatic giardiasis in the population under study.

Conclusions

Human giardiasis is an ongoing public health problem in Spain affecting primarily young children under four years of age but also individuals of all age groups. Our typing and sub-typing results demonstrate that assemblage B is the most prevalent G. duodenalis assemblage circulating in patients with clinical giardiasis in Central Spain. Our analyses also revealed a large genetic variability in assemblage B (but not assemblage A) isolates of the parasite, corroborating the information obtained in similar studies in other geographical regions. We believe that molecular data presented here provide epidemiological evidence at the population level in support of the existence of genetic exchange within assemblages of G. duodenalis.     

Molecular Characterization of Cryptosporidium Oocysts in Samples of Raw Surface Water and Wastewater

Recent molecular characterizations of Cryptosporidium parasites make it possible to differentiate the human-pathogenic Cryptosporidium parasites from those that do not infect humans and to track the source of Cryptosporidium oocyst contamination in the environment. In this study, we used a small-subunit rRNA-based PCR-restriction fragment length polymorphism (RFLP) technique to detect and characterize Cryptosporidium oocysts in 55 samples of raw surface water collected from several areas in the United States and 49 samples of raw wastewater collected from Milwaukee, Wis. Cryptosporidium parasites were detected in 25 surface water samples and 12 raw wastewater samples. C. parvum human and bovine genotypes were the dominant Cryptosporidium parasites in the surface water samples from sites where there was potential contamination by humans and cattle, whereas C. andersoni was the most common parasite in wastewater. There may be geographic differences in the distribution of Cryptosporidium genotypes in surface water. The PCR-RFLP technique can be a useful alternative method for detection and differentiation of Cryptosporidium parasites in water.     

Identification of Cryptosporidium from Dairy Cattle in Pahang,Malaysia

Cryptosporidium, a protozoan parasite, can cause cryptosporidiosis which is a gastrointestinal disease that can infect humans and livestock. Cattle are the most common livestock that can be infected with this protozoan. This study was carried out to determine the prevalence of Cryptosporidium infection in cattle in Kuantan, Pahang, Malaysia and to find out the association between the occurrence of infection and 3 different ages of cattle (calves less than 1 year, yearling, and adult cattle). The samples were processed by using formol-ether concentration technique and stained by modified Ziehl Neelsen. The results showed that 15.9% (24/151) of cattle were positive for Cryptosporidium oocysts. The occurrence of Cryptosporidium in calves less than 1 year was the highest with the percentage of 20.0% (11/55) followed by yearling and adult cattle, with the percentage occurrence of 15.6 % (7/45) and 11.8% (6/51), respectively. There was no significant association between the occurrence and age of cattle and presence of diarrhea. Good management practices and proper hygiene management must be taken in order to reduce the infection. It is highly important to control the infection since infected cattle may serve as potential reservoirs of the infection to other animals and humans, especially animal handlers.     

Molecular Epidemiology of Cryptosporidium spp. and Giardia spp. in Mussels (Mytilus californianus) and California Sea Lions (Zalophus californianus) from Central California

Cryptosporidium and Giardia are of public health importance, with recognized transmission through recreational waters. Therefore, both can contaminate marine waters and shellfish, with potential to infect marine mammals in nearshore ecosystems. A 2-year study was conducted to evaluate the presence of Cryptosporidium and Giardia in mussels located at two distinct coastal areas in California, namely, (i) land runoff plume sites and (ii) locations near sea lion haul-out sites, as well as in feces of California sea lions (CSL) (Zalophus californianus) by the use of direct fluorescent antibody (DFA) detection methods and PCR with sequence analysis. In this study, 961 individual mussel hemolymph samples, 54 aliquots of pooled mussel tissue, and 303 CSL fecal samples were screened. Giardia duodenalis assemblages B and D were detected in hemolymph from mussels collected near two land runoff plume sites (Santa Rosa Creek and Carmel River), and assemblages C and D were detected in hemolymph from mussels collected near a sea lion haul-out site (White Rock). These results suggest that mussels are being contaminated by protozoa carried in terrestrial runoff and/or shed in the feces of CSL. Furthermore, low numbers of oocysts and cysts morphologically similar to Cryptosporidium and Giardia, respectively, were detected in CSL fecal samples, suggesting that CSL could be a source and a host of protozoan parasites in coastal environments. The results of this study showed that Cryptosporidium and Giardia spp. from the feces of terrestrial animals and CSL can contaminate mussels and coastal environments.     

Giardia Cysts and Cryptosporidium Oocysts in Membrane-Filtered Municipal Wastewater Used for Irrigation

A wastewater tertiary treatment system based on membrane ultrafiltration and fed with secondary-treated municipal wastewater was evaluated for its Giardia cyst and Cryptosporidium oocyst removal efficiency. Giardia duodenalis (assemblages A and B) and Cryptosporidium parvum were identified in feed water but were found in filtered water only during occasional failure of the filtration system.     

Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay) were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh) gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61%) were classified as assemblage B (26 as BIII and 16 as BIV), 22 (32%) as assemblage A (3 as AI and 19 as AII) and five (7%) as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.