Investigation of giardiasis in captive animals in zoological gardens with strain typing of assemblages in China.

Giardia duodenalis is a common zoonotic intestinal pathogen. It has been increasingly reported in humans and animals; however, genotyping information for G. duodenalis in captive animals is still limited. This study was conducted to assess the prevalence and multilocus genotyping of G. duodenalis in captive animals in zoological gardens in Shanghai, China. A total of 678 fresh fecal samples were randomly collected from captive animals including non-human primates (NHPs) (n = 190), herbivores (n = 190), carnivores (n = 151), birds (n = 138) and reptiles (n = 9) in a zoo and were examined for the presence of G. duodenalis using nested polymerase chain reaction (nested PCR). All G. duodenalis positive samples were assayed with PCR followed by sequencing at ß-giardin (bg), glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes. In this study, 42 specimens (6.2%) were tested G. duodenalis-positive of the 678 fecal samples examined based on a single locus. A total of 30 (4.4%), 30 (4.4%) and 22 (3.2%) specimens were successfully amplified and sequenced at gdh, tpi and bg loci, respectively. Assemblages A and B were identified with assemblage B dominating in NHPs. Sequence analysis demonstrated that one, two and five new isolates were identified at bg, gdh and tpi loci. DNA sequences and new assemblage-subtypes of zoonotic G. duodenalis assemblages A and B were identified in the current study. Our data indicate the occurrence and molecular diversity of G. duodenalis and the potential zoonotic transmission in captive animals in China.

Household finished flooring and soil-transmitted helminth and Giardia infections among children in rural Bangladesh and Kenya: a prospective cohort study.

BACKGROUND: Soil-transmitted helminths and Giardia duodenalis are responsible for a large burden of disease globally. In low-resource settings, household finished floors (eg, concrete floors) might reduce transmission of soil-transmitted helminths and G duodenalis. METHODS: In a prospective cohort of children nested within two randomised trials in rural Bangladesh and Kenya, we estimated associations between household finished flooring and soil-transmitted helminths and G duodenalis prevalence. In 2015-16, we collected stool samples from children aged 2-16 years in rural Bangladesh and Kenya. We detected soil-transmitted helminth infection using quantitative PCR (qPCR; Bangladesh n=2800; Kenya n=3094), and G duodenalis using qPCR in Bangladesh (n=6894) and ELISA in Kenya (n=8899). We estimated adjusted prevalence ratios (aPRs) using log-linear models adjusted for potential confounders. FINDINGS: 7187 (92·2%) of 7795 children in Bangladesh and 9077 (93·7%) of 9686 children in Kenya provided stool specimens that were analysed by qPCR. At enrolment, 691 (10%) households in Bangladesh and 471 (5%) households in Kenya had finished floors. In both countries, household finished flooring was associated with lower Ascaris lumbricoides prevalence (Bangladesh aPR 0·33, 95% CI 0·14-0·78; Kenya 0·62, 0·39-0·98) and any soil-transmitted helminths (Bangladesh 0·73, 0·52-1·01; Kenya 0·57, 0·37-0·88). Household finished floors were also associated with lower Necator americanus prevalence in Bangladesh (0·52, 0·29-0·94) and G duodenalis prevalence in both countries (Bangladesh 0·78, 0·64-0·95; Kenya 0·82, 0·70-0·97). INTERPRETATION: In low-resource settings, living in households with finished floors over a 2-year period was associated with lower prevalence of G duodenalis and some soil-transmitted helminths in children. FUNDING: Bill & Melinda Gates Foundation and Task Force for Global Health.

Observation of Giardia sp. in the termite gut of Heterotermes tenuis.

Giardia comprises one genus with several morphologically distinct species described in mammals (including humans, marsupials, rodents), birds, and amphibians. This group of protists provokes diarrhoea diseases in humans and animals worldwide. Transmission of the parasite occurs through the faecal-oral route. Regarding the presence of Giardia in invertebrates, some works have shown that flies can transmit Giardia cysts by contact and transport between contaminated faeces and food. In this way, flies would eventually transmit this parasite. To date, Giardia's presence in the gut of other invertebrates has not been described in the literature. Here we show by first time, using scanning electron microscopy, the presence of Giardia-like trophozoites in the gut of termite Heterotermes tenuis. Two groups of Giardia were found based exclusively on the size and the flange shape of the protozoa: one presented eight flagella, a ventral disc, size, and shape very similar to Giardia intestinalis. In contrast, other cells were smaller and showed some differences in the external morphology. We cannot exclude the possibility that they correspond to the same species and that these differences result from protozoan heterogeneity.

Giardia duodenalis in humans and animals - Transmission and disease.

Giardia duodenalis is a protozoan parasite infecting the upper intestinal tract of humans, as well as domestic and wild animals worldwide. Transmission of giardiasis occurs through the faecal-oral route, and may be either direct (i.e., person-to-person, animal-to-animal or zoonotic) or indirect (i.e., waterborne or foodborne). While asymptomatic infections are common in both humans and animals, a wide range of enteric symptoms have been reported, along with extra-intestinal and post-infectious complications. A definitive diagnosis of giardiasis is generally made by detection of cysts in stool specimens through microscopical examination of wet mounts, or through the use of permanent or fluorescent antibody stains. More recently, molecular methods have become popular for diagnosis and for testing environmental samples. Symptomatic giardiasis is often treated to reduce the duration of symptoms, to prevent complications, and to minimize transmission of the parasite to other hosts. Direct faecal-oral transmission of giardiasis can be largely controlled thorough improved hygiene and sanitation. In the case of waterborne transmission, a multiple barrier approach, including limiting access of people and animals to watersheds and reservoirs, and treatment using flocculation, filtration and disinfection, is necessary to minimize the risk. Since foodborne transmission is often associated with the consumption of fresh produce, a number of control measures can be taken during pre- and post-harvest, as well as at the food handler/consumer level to minimize the risk of contamination, or for removing or inactivating parasites. Good husbandry and farm management practices are important in controlling the spread of giardiasis in livestock and companion animals.

Frequency of Giardia duodenalis infection and its genetic variability in dogs in Cuiabá, Midwest Brazil.

INTRODUCTION: Giardia duodenalis, a unicellular, eukaryotic, and flagellated protozoan, presents two evolutionary forms in its life cycle, namely, trophozoites and cysts. It causes diarrhea in humans, dogs, cats, rodents, and ungulates. Despite being morphologically similar, the isolates of G. duodenalis are genetically diverse, affecting the stability and unanimity of taxonomic classification. Since different Giardia assemblages may occur within one isolate, multilocus genotyping is recommended for the genetic identification. METHODOLOGY: To determine the frequency of G. duodenalis infections in domiciled dogs in Cuiabá Municipality (State of Mato Grosso, Midwestern Brazil) and characterize its genetic variability, fecal samples were collected from 147 dogs. RESULTS: Overall, 6.8% (10/147) of the samples presented cysts of G. duodenalis, which sequencing and genotypic characterization using tpi and gluD revealed assemblages C and A, genetic grouping of G. duodenalis. Only three samples amplified by tpi and one sample amplified by gluD. CONCLUSIONS: The risk factors age, gender, breed, diet and the presence of other dogs in the same house were not correlationated with giardiasis. The host-specific and zoonotic genotype warns of the risk of inter and intraspecies transmission and it provides, for the first time, information about genetic characterization of G. duodenalis isolates in dogs in Cuiabá, Midwest region of Brazil.

Cryptosporidium and Giardia in dam water on sheep farms - An important source of transmission?

Cryptosporidium and Giardia infections can negatively impact livestock health and reduce productivity, and some species and genotypes infecting livestock have zoonotic potential. Infection occurs via the faecal-oral route. Waterborne infections are a recognised source of infection for humans, but the role of livestock drinking water as a source of infection in livestock has not been described. This study aimed to determine whether contaminated drinking water supplies, such as farm dams, are a likely transmission source for Cryptosporidium and Giardia infections for extensively managed sheep. Dam water samples (n = 47) were collected during autumn, winter and spring from 12 farm dams located on six different farms in south west Western Australia, and faecal samples (n = 349) were collected from sheep with access to these dams. All samples were initially screened for Cryptosporidium spp. at the 18S locus and Giardia spp. at the gdh gene using qPCR, and oocyst numbers were determined directly from the qPCR data using DNA standards calibrated by droplet digital PCR. Cryptosporidium-positive sheep faecal samples were typed and subtyped by sequence analysis of 18S and gp60 loci, respectively. Giardia-specific PCR and Sanger sequencing targeting tpi and gdh loci were performed on Giardia- positive sheep faecal samples to characterise Giardia duodenalis assemblages. To identify Cryptosporidium and Giardia spp. in dam water samples, next-generation sequencing analysis of 18S and gdh amplicons were performed, respectively. Two species of Cryptosporidium (Cryptosporidium xiaoi and Cryptospordium ubiquitum (subtype family XIIa)) were detected in 38/345 sheep faecal samples, and in water from 9/12 farm dams during the study period, with C. xiaoi the species most frequently detected in both faeces and dam water overall. Giardia duodenalis assemblages AI, AII and E were detected in 36/348 faecal samples and water from 10/12 farm dams. For dam water samples where oo/cysts were detected by qPCR, Cryptosporidium oocyst concentration ranged from 518-2429 oocysts/L (n = 14), and Giardia cyst concentration ranged from 102 to 1077 cysts/L (n = 17). Cryptosporidium and Giardia with zoonotic potential were detected in farm dam water, including C. ubiquitum, C. hominis, C. parvum, C. cuniculus, C. xiaoi, and G. duodenalis assemblages A, B and E. The findings suggest that dam water can be contaminated with Cryptosporidium species and G. duodenalis assemblages that may infect sheep and with zoonotic potential, and farm dam water may represent one source of transmission for infections.

The Predominance of Giardia duodenalis AII sub-assemblage in young children from Salvador, Bahia, Brazil. / Predominio del subconjunto AII de Giardia duodenalis en niños pequeños de Salvador, Bahía, Brasil

INTRODUCTION: Giardia duodenalis is an intestinal protozoan with a high prevalence in children of developing countries. Molecular studies revealed a great genetic diversity of G. duodenalis, with assemblages A and B found mainly in humans. Despite its importance, the information on the molecular epidemiology of human giardiasis is still limited in Brazil. OBJECTIVE: To characterize G. duodenalis molecular isolates in children from Salvador, Bahia, Brazil. MATERIALS AND METHODS: Giardia duodenalis positive fecal samples were obtained from 71 children from two day care centers and 39 users of a clinical analysis laboratory. Samples were analyzed by PCR-RFLP of the glutamate dehydrogenase (gdh) and beta-giardin genes and by the sequencing of beta-giardin. RESULTS: Of the 110 G. duodenalis samples, 80 (72.7%) amplified one or both target genes. Of these, 62 (77.5 %) were identified as assemblage A and 18 (22.5%) as assemblage B. The subassemblage AII was identified in 58.8% (n=47) of isolates followed by the sub-assemblage AI (18.8%, n=15), BIV (11.2%, n=9), and BIII (5.0%, n=4). The AII sub-assemblage was the most frequent in children of both day care centers whereas AI was found only in the group attended at the clinical laboratory. Sub-assemblage AII predominated in children under two years. CONCLUSIONS: The higher frequency of AII sub-assemblage suggests that anthroponotic transmission is more common in Salvador, but that zoonotic transmission pathways are also present and a change in susceptibility to different molecular patterns of Giardia may occur during child growth.

Introducción. Giardia duodenalis es un protozoo intestinal de gran prevalencia en los niños de los países en desarrollo. En estudios moleculares se ha evidenciado la gran diversidad genética de G. duodenalis y se han identificado los conjuntos A y B, principalmente en humanos. A pesar de su importancia, el conocimiento de la epidemiología molecular de la giardiasis humana aún es limitado en Brasil. Objetivo. Caracterizar los aislamientos moleculares de G. duodenalis de muestras tomadas a niños de Salvador, Bahía, Brasil. Materiales y métodos. Las muestras fecales positivas para G. duodenalis se obtuvieron de 71 niños de dos guarderías y de 39 usuarios de un laboratorio de análisis clínicos. Las muestras se analizaron mediante PCR-RFLP de los genes gdh y beta-giardin, y secuenciación de beta-giardin. Resultados. De las 110 muestras de G. duodenalis, en 80 (72,7 %) se amplificaron uno o ambos genes. De estos, 62 (77,5 %) se identificaron como pertenecientes al conjunto A y 18 (22,5 %) al B. El subconjunto AII se identificó en el 58,8 % (n=47) de los aislamientos, seguido del AI en el 18,8% (n=15), el BIV en el 11,2% (n=9) y el BIII en el 5,0% (n=4). El subconjunto AII fue el más frecuente en los niños de ambas guarderías, en tanto que el AI solo se encontró en el grupo atendido en el laboratorio clínico. El subconjunto AII predominó en los niños menores de dos años. Conclusiones. La mayor frecuencia del subconjunto AII sugiere que la transmisión antroponótica es más común en Salvador, pero también existen vías de transmisión zoonóticas, y que pueden ocurrir cambios en la sensibilidad frente a diferentes patrones moleculares de Giardia durante el crecimiento infantil.

Companion animals as a potential source of Giardia intestinalis infection in humans in the Czech Republic - A pilot study.

Giardia intestinalis is a common enteric single-celled parasite infecting both humans and animals. Its eight morphologically identical but genetically distinct groups called assemblages differ from each other in host range. While assemblages A and B infect a wide range of hosts, including humans, the other assemblages (C to H) limit their host preferences to particular animal groups only. In companion animals as Giardia hosts, genotyping data have previously shown various results depending on pet species, location, environmental or breeding conditions, and the study design. To strengthen available epidemiological data from developed countries and to evaluate the role of pets in Giardia zoonotic transmission, we investigated Giardia-positive stool samples of three pet species (54 dogs, 18 cats, and 18 chinchillas) by a sequence-based analysis of three Giardia genes (ß-giardin, glutamate dehydrogenase and triose phosphate isomerase). In dog samples, we confirmed assemblage C (21/54), assemblage D (32/54), and one case of a mixed infection C + D (1/54). In cats, we found assemblage F (16/18) and assemblage A, specifically sub-assemblage AI (2/18). All Giardia samples from chinchillas were characterised as assemblage B, specifically sub-assemblage BIV (18/18). These results indicate that in the Czech Republic, pet dogs may not represent a source of Giardia infection for humans because of the presence of only canid-specific genotypes C and D. In contrast, other pets, namely, chinchillas and, to a lesser extent, cats, may pose a potential risk of Giardia transmission to owners or breeders because they can host zoonotic Giardia genotypes.

Update on the major imported protozoan infections in travelers and migrants.

Globalization has contributed to the emergence of specific parasitic diseases in novel geographical areas, and in these regions, these infections in travelers and immigrants may cause a considerable burden of disease. Timely diagnosis and treatment of protozoan infections to decrease mortality and prevent associated complications are essential. In this respect, the increased availability of specific DNA-detection procedures has improved the diagnosis of many imported parasitic infections. Travelers and immigrants with associated comorbidities or immunosuppression may pose a special challenge regarding management. An updated review of the main protozoan infections in mobile populations (malaria, Chagas disease, leishmaniasis, enteric protozoan infections) is provided, focusing on the changing epidemiology of these diseases, recent developments in diagnosis and management and the possibility of local transmission of imported infections.

Investigations from Northern Greece on mussels cultivated in areas proximal to wastewaters discharges, as a potential source for human infection with Giardia and Cryptosporidium.

Marine bivalves are usually cultivated in shallow, estuarine waters where there is a high concentration of nutrients. Many micro-pollutants, including the protozoan parasites Giardia duodenalis and Cryptosporidium spp., which also occur in such environments, may be concentrated in shellfish tissues during their feeding process. Shellfish can thus be considered as vehicles for foodborne infections, as they are usually consumed lightly cooked or raw. Therefore, the main objective of this study was to investigate the presence of both parasites in Mediterranean mussels, Mytilus galloprovincialis that are cultivated in Thermaikos Gulf, North Greece, which is fed by four rivers that are contaminated with both protozoa. Moreover, the occurrence of these protozoa was monitored in treated wastewaters from 3 treatment plants that discharge into the gulf. In order to identify potential sources of contamination and to estimate the risk for human infection, an attempt was made to genotype Giardia and Cryptosporidium in positive samples. Immunofluorescence was used for detection and molecular techniques were used for both detection and genotyping of the parasites. In total, 120 mussel samples, coming from 10 farms, were examined for the presence of both protozoa over the 6-month farming period. None of them were found positive by immunofluorescence microscopy for the presence of parasites. Only in 3 mussel samples, PCR targeting the GP60 gene detected Cryptosporidium spp. DNA, but sequencing was not successful. Thirteen out of 18 monthly samples collected from the 3 wastewater treatment plants, revealed the presence of Giardia duodenalis cysts belonging to sub-assemblage AII, at relatively low counts (up to 11.2 cysts/L). Cryptosporidium oocysts (up to 0.9 oocysts/L) were also detected in 4 out of 8 samples, although sequencing was not successful at any of the target genes. At the studied location and under the sampling conditions described, mussels tested were not found to be harboring Giardia cysts and the presence of Cryptosporidium was found only in few cases (by PCR detection only). Our results suggest that the likelihood that mussels from these locations act as vehicles of human infection for Giardia and Cryptosporidium seems low.

Detection methods and prevalence of transmission stages of Toxoplasma gondii, Giardia duodenalis and Cryptosporidium spp. in fresh vegetables: a review.

One of the ways of human parasitic infection is the accidental ingestion of vegetables contaminated with parasites, which represents a major human health hazard. This non-exhaustive review aims to evaluate studies carried out on five types of vegetables (lettuce, parsley, coriander, carrot and radish) since 2000, particularly the methods used for recovery, concentration, detection and identification of protozoan parasites such as Toxoplasma gondii, Giardia duodenalis and Cryptosporidium spp., and the results of each work. Various studies have determined the presence of pathogenic parasites in fresh vegetables with different rates; this variation in rate depends particularly on the detection method used which is related to each parasite and each vegetable type. The variation in parasitic prevalence in food could be due to different factors such as the geographical location, the size of analysed samples and the methods used for parasite detection.

Occurrence and multilocus genotyping of Giardia duodenalis from post-weaned dairy calves in Sichuan province, China.

Giardia duodenalis is a zoonotic parasitic protist and poses a threat to human and animal health. This study investigated the occurrence of G. duodenalis infection in post-weaned calves from Sichuan province, China. Faecal samples were collected from a total of 306 post-weaned calves (3-12 months old) from 10 farms, including 4 intensive feeding farms and 6 free-ranging farms. The overall infection rate of G. duodenalis was 41.2% (126/306) based on the PCR results at any of the three genetic loci: beta-giardin (bg), triose-phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. Giardia duodenalis assemblages E (n = 115, 91.3%), A (n = 3, 2.4%), and A mixed with E (n = 8, 6.3%) were identified among the 126 positive specimens. Multilocus sequence typing of G. duodenalis revealed 34 assemblage E multilocus genotypes (MLGs), 1 assemblage A MLG and 7 mixed assemblage (A and E) MLGs. The eBURST data showed a high degree of genetic diversity within assemblage E MLGs. The phylogenetic tree revealed that MLG E3 was the primary MLG subtype in Sichuan province and also the most widely distributed in China.

A cytonaut's guide to protein trafficking in Giardia lamblia.

Over the past years, the subcellular organization of the Excavata member Giardia lamblia (syn. duodenalis, intestinalis) has been investigated in considerable detail. There are several reasons for this endeavour which go beyond this parasite's medical importance and are mostly concerned with its reduced subcellular complexity and debated evolutionary status. One may say that simplification has emerged as a paradigm for the evolution of Giardia's subcellular architecture. However, a complete appreciation of the evolutionary and ecological significance of this phenomenon is far from complete. In this chapter, we present and discuss the most recent data on the main trafficking pathways in G. lamblia which include endo- and exo-cytosis, organellar import and function. We provide perspectives on open questions concerning organelle replication and inheritance and include a technical outlook on methods and approaches to genetic manipulations in G. lamblia. A better understanding of G. lamblia subcellular organization at the morphological and molecular level complements any effort aimed at elucidating this parasitic species' evolutionary status and could provide us with the basis for novel strategies to interfere with parasite transmission and/or pathogenesis.

Molecular epidemiology of giardiasis from a veterinary perspective.

A total of eight Giardia species are accepted. These include: Giardia duodenalis (syn. Giardia intestinalis and Giardia lamblia), which infects humans and animals, Giardia agilis, Giardia ardeae, Giardia psittaci, Giardia muris, Giardia microti, Giardia peramelis and G. cricetidarum, which infect non-human hosts including amphibians, birds, rodents and marsupials. Giardia duodenalis is a species complex consisting of eight assemblages (A-H), with assemblages A and B the dominant assemblages in humans. Molecular studies to date on the zoonotic potential of Giardia in animals are problematic and are hampered by lack of concordance between loci. Livestock (cattle, sheep, goats and pigs) are predominantly infected with G. duodenalis assemblage E, which has recently been shown to be zoonotic, followed by assemblage A. In cats and dogs, assemblages A, B, C, D and F are commonly reported but relatively few studies have conducted molecular typing of humans and their pets and the results are contradictory with some studies support zoonotic transmission but the majority of studies suggesting separate transmission cycles. Giardia also infects a broad range of wildlife hosts and although much less well studied, host-adapted species as well as G. duodenalis assemblages (A-H) have been identified. Fish and other aquatic wildlife represent a source of infection for humans with Giardia via water contamination and/or consumption of undercooked fish and interestingly, assemblage B and A predominated in the two molecular studies conducted to date. Our current knowledge of the transmission dynamics of Giardia is still poor and the development of more discriminatory typing tools such as whole genome sequencing (WGS) of Giardia isolates is therefore essential.

Multilocus genotyping and risk factor analysis of Giardia duodenalis in dogs in Korea.

Giardia duodenalis (syn. G. intestinalis, G. lamblia), a protozoan parasite that infects most mammals (including humans), has recently been classified into eight different groups/assemblages (A to H) based on host range and genetic characteristics. Assemblages A and B have been reported in humans and in many other animals, including dogs. This study analyzed Giardia infections, implementing multilocus genotyping, including the 18S rRNA, gdh, and ß-giardin genes, in sheltered, companion, and special-purpose dogs throughout Korea. Of 640 dog stool samples, 99 (15.5%) were G. duodenalis-positive; this prevalence was similar to that previously reported in Korean calves. In addition, the prevalence was significantly higher in the southern region, and lower in non-sheltered dogs, older dogs, and in the autumn season. Phylogenetic analysis revealed that all G. duodenalis-positive samples belonged to assemblages C and D. Although no G. duodenalis assemblages A and B (mainly found in humans) were identified in this study, assemblage C was detected in the main types of dogs in contact with humans. Assemblage C, thought to be dog-specific, was recently identified in humans. As Giardia (including zoonotic assemblages) is distributed throughout Korea, dog living environments and the potential for dog-human transmission should be monitored.

Effects of single and integrated water, sanitation, handwashing, and nutrition interventions on child soil-transmitted helminth and Giardia infections: A cluster-randomized controlled trial in rural Kenya.

BACKGROUND: Helminth and protozoan infections affect more than 1 billion children globally. Improving water quality, sanitation, handwashing, and nutrition could be more sustainable control strategies for parasite infections than mass drug administration, while providing other quality of life benefits. METHODS AND FINDINGS: We enrolled geographic clusters of pregnant women in rural western Kenya into a cluster-randomized controlled trial (ClinicalTrials.gov NCT01704105) that tested 6 interventions: water treatment, improved sanitation, handwashing with soap, combined water treatment, sanitation, and handwashing (WSH), improved nutrition, and combined WSH and nutrition (WSHN). We assessed intervention effects on parasite infections by measuring Ascaris lumbricoides, Trichuris trichiura, hookworm, and Giardia duodenalis among children born to the enrolled pregnant women (index children) and their older siblings. After 2 years of intervention exposure, we collected stool specimens from 9,077 total children aged 2 to 15 years in 622 clusters, including 2,346 children in an active control group (received household visits but no interventions), 1,117 in the water treatment arm, 1,160 in the sanitation arm, 1,141 in the handwashing arm, 1,064 in the WSH arm, 1,072 in the nutrition arm, and 1,177 in the WSHN arm. In the control group, 23% of children were infected with A. lumbricoides, 1% with T. trichiura, 2% with hookworm, and 39% with G. duodenalis. The analysis included 4,928 index children (median age in years: 2) and 4,149 older siblings (median age in years: 5); study households had an average of 5 people, <10% had electricity access, and >90% had dirt floors. Compared to the control group, Ascaris infection prevalence was lower in the water treatment arm (prevalence ratio [PR]: 0.82 [95% CI 0.67, 1.00], p = 0.056), the WSH arm (PR: 0.78 [95% CI 0.63, 0.96], p = 0.021), and the WSHN arm (PR: 0.78 [95% CI 0.64, 0.96], p = 0.017). We did not observe differences in Ascaris infection prevalence between the control group and the arms with the individual interventions sanitation (PR: 0.89 [95% CI 0.73, 1.08], p = 0.228), handwashing (PR: 0.89 [95% CI 0.73, 1.09], p = 0.277), or nutrition (PR: 86 [95% CI 0.71, 1.05], p = 0.148). Integrating nutrition with WSH did not provide additional benefit. Trichuris and hookworm were rarely detected, resulting in imprecise effect estimates. No intervention reduced Giardia. Reanalysis of stool samples by quantitative polymerase chain reaction confirmed the reductions in Ascaris infections measured by microscopy in the WSH and WSHN groups. Trial limitations included imperfect uptake of targeted intervention behaviors, limited power to detect effects on rare parasite infections, and that it was not feasible to blind participants and sample collectors to treatment status. However, lab technicians and data analysts were blinded to treatment status. The trial was funded by the Bill & Melinda Gates Foundation and the United States Agency for International Development. CONCLUSIONS: Integration of improved water quality, sanitation, and handwashing could contribute to sustainable control strategies for Ascaris infections, particularly in similar settings with recent or ongoing deworming programs. Combining nutrition with WSH did not provide further benefits, and water treatment alone was similarly effective to integrated WSH. Our findings provide new evidence that drinking water should be given increased attention as a transmission pathway for Ascaris. TRIAL REGISTRATION: ClinicalTrials.gov NCT01704105.