Dissection of Barrier Dysfunction in Organoid-Derived Human Intestinal Epithelia Induced by Giardia duodenalis.

BACKGROUND & AIMS: The protozoa Giardia duodenalis is a major cause of gastrointestinal illness worldwide, but underlying pathophysiological mechanisms remain obscure, partly due to the absence of adequate cellular models. We aimed at overcoming these limitations and recapitulating the authentic series of pathogenic events in the primary human duodenal tissue by using the human organoid system. METHODS: We established a compartmentalized cellular transwell system with electrophysiological and barrier properties akin to duodenal mucosa and dissected the events leading to G. duodenalis-induced barrier breakdown by functional analysis of transcriptional, electrophysiological, and tight junction components. RESULTS: Organoid-derived cell layers of different donors showed a time- and parasite load-dependent leak flux indicated by collapse of the epithelial barrier upon G. duodenalis infection. Gene set enrichment analysis suggested major expression changes, including gene sets contributing to ion transport and tight junction structure. Solute carrier family 12 member 2 and cystic fibrosis transmembrane conductance regulator-dependent chloride secretion was reduced early after infection, while changes in the tight junction composition, localization, and structural organization occurred later as revealed by immunofluorescence analysis and freeze fracture electron microscopy. Functionally, barrier loss was linked to the adenosine 3',5'-cyclic monophosphate (cAMP)/protein kinase A-cAMP response element-binding protein signaling pathway. CONCLUSIONS: Data suggest a previously unknown sequence of events culminating in intestinal barrier dysfunction upon G. duodenalis infection during which alterations of cellular ion transport were followed by breakdown of the tight junctional complex and loss of epithelial integrity, events involving a cAMP/protein kinase A-cAMP response element-binding protein mechanism. These findings and the newly established organoid-derived model to study G. duodenalis infection may help to explore new options for intervening with disease and infection, in particular relevant for chronic cases of giardiasis.

Prevalence and molecular characterization of Cryptosporidium and Giardia in pre-weaned native calves in the Republic of Korea.

Cryptosporidium spp. and Giardia duodenalis are protozoan parasites that cause diarrhea in humans and animals. Molecular data on Cryptosporidium spp. and G. duodenalis in calves in the Republic of Korea (ROK) is limited; thus, we investigated the prevalence of Cryptosporidium and Giardia in pre-weaned calves, analyzed the association between these parasites and diarrhea, and identified their zoonotic potential for human infection. Fecal samples were collected from 315 pre-weaned calves aged 1-60 days from 10 different regions in the ROK and screened for Cryptosporidium spp. and G. duodenalis using PCR. Overall prevalence of Cryptosporidium spp. and G. duodenalis was 4.4% (n = 14) and 12.7% (n = 40), respectively. Co-infection was not detected. All Cryptosporidium-positive samples were identified as C. parvum after sequence analysis of a small subunit rRNA fragment and further subtyped into zoonotic IIaA15G2R1 (n = 13) and IIaA18G3R1 (n = 1) by DNA sequencing of the 60-kDa glycoprotein gene. To our knowledge, this is the first report of C. parvum IIaA15G2R1 subtype in calves in the ROK. Based on ß-giardin (bg) gene, G. duodenalis-positive samples belonged to assemblages E (n = 36) and A (n = 4), with the latter belonging to subtype A1, the zoonotic genotype. Six subtypes of assemblage E were identified at the bg locus: E1 (n = 6), E2 (n = 3), E3 (n = 13), E5 (n = 1), E8 (n = 1), and E11 (n = 1). The occurrence of C. parvum and G. duodenalis was not associated with diarrhea in pre-weaned Korean native calves. The present results suggest that the prevalence of C. parvum is not related to calf age; in contrast, the prevalence of G. duodenalis was significantly higher in 41-50-day-old calves (odds ratio = 9.90, 95% confidence interval 2.37-41.34; P = 0.001) than in 1-10-day-old calves. Therefore, calves are a potential source of zoonotic transmission, which may have significant public health implications.

Giardia Alters Commensal Microbial Diversity throughout the Murine Gut.

Giardia lamblia is the most frequently identified protozoan cause of intestinal infection. Over 200 million people are estimated to have acute or chronic giardiasis, with infection rates approaching 90% in areas where Giardia is endemic. Despite its significance in global health, the mechanisms of pathogenesis associated with giardiasis remain unclear, as the parasite neither produces a known toxin nor induces a robust inflammatory response. Giardia colonization and proliferation in the small intestine of the host may, however, disrupt the ecological homeostasis of gastrointestinal commensal microbes and contribute to diarrheal disease associated with giardiasis. To evaluate the impact of Giardia infection on the host microbiota, we used culture-independent methods to quantify shifts in the diversity of commensal microbes throughout the gastrointestinal tract in mice infected with Giardia We discovered that Giardia's colonization of the small intestine causes a systemic dysbiosis of aerobic and anaerobic commensal bacteria. Specifically, Giardia colonization is typified by both expansions in aerobic Proteobacteria and decreases in anaerobic Firmicutes and Melainabacteria in the murine foregut and hindgut. Based on these shifts, we created a quantitative index of murine Giardia-induced microbial dysbiosis. This index increased at all gut regions during the duration of infection, including both the proximal small intestine and the colon. Giardiasis could be an ecological disease, and the observed dysbiosis may be mediated directly via the parasite's unique anaerobic fermentative metabolism or indirectly via parasite induction of gut inflammation. This systemic alteration of murine gut commensal diversity may be the cause or the consequence of inflammatory and metabolic changes throughout the gut. Shifts in the commensal microbiota may explain observed variations in giardiasis between hosts with respect to host pathology, degree of parasite colonization, infection initiation, and eventual clearance.

Synergistic effects of fenbendazole and metronidazole against Giardia muris in Swiss mice naturally infected.

In this study were proposed different protocols for the treatment of mice naturally infected with Giardia muris. Male Swiss mice were divided into seven groups, with five animals each, in a blind, controlled, randomized by drawing lots and once-repeated experiment. Parasite detection and cure control were performed using the Faust method and search by trophozoites in the intestinal mucosa. Clinical parameters (weight, water and feed consumption, elimination of excreta, aspect of the fur and feces) were also evaluated. All animals were treated with metronidazole (M), fenbendazole (F), and probiotics (P), administered intragastrically, during 7 days. M1, FM1, and F1 groups were treated 1×/day; M3, FM3, and PM3 groups 3×/day; and ST (control group) received only water. After the 5th and 7th days of treatment, the animals in FM1/FM3 and PM3/M3 groups presented, respectively, negative results and remained negative in the following 10 days. Animals in F1 group consumed less water (p = 0.00010) compared with FM1/FM3/PM3. The animals in M1 group compared with FM3/M3, F1 compared with M3, and ST compared with FM1/FM3/M3/PM3 consumed a larger amount of feed (p = 0.00001). The animals in F1 group compared with FM3/M1/M3/PM3, FM1 compared with FM3, and ST compared with FM3/M1/M3/PM3 eliminated lower volume of excreta (p = 0.00001). The results show that the association between F and M potentiates the effects, indicating a synergistic action of these two drugs, and FM1 is the best protocol due to early negativity in the animals, lower concentrations of the drugs, lower risk of toxicity and stress, and less alterations in clinical parameters.

Ethanol and isopropanol in concentrations present in hand sanitizers sharply reduce excystation of Giardia and Entamoeba and eliminate oral infectivity of Giardia cysts in gerbils.

Enteric protozoan parasites, which are spread by the fecal-oral route, are important causes of diarrhea (Giardia duodenalis) and amebic dysentery (Entamoeba histolytica). Cyst walls of Giardia and Entamoeba have a single layer composed of fibrils of ß-1,3-linked GalNAc and ß-1,4-linked GlcNAc (chitin), respectively. The goal here was to determine whether hand sanitizers that contain ethanol or isopropanol as the active microbicide might reduce transmission of these parasites. We found that treatment with these alcohols with or without drying in a rotary evaporator (to model rapid evaporation of sanitizers on hands) kills 85 to 100% of cysts of G. duodenalis and 90 to 100% of cysts of Entamoeba invadens (a nonpathogenic model for E. histolytica), as shown by nuclear labeling with propidium iodide and failure to excyst in vitro. Alcohols with or without drying collapsed the cyst walls of Giardia but did not collapse the cyst walls of Entamoeba. To validate the in vitro results, we showed that treatment with alcohols eliminated oral infection of gerbils by 1,000 G. duodenalis cysts, while a commercial hand sanitizer (Purell) killed E. invadens cysts that were directly applied to the hands. These results suggest that expanded use of alcohol-based hand sanitizers might reduce the transmission of Giardia and Entamoeba.

[Comparative evaluation of clinical effectiveness of treatment of giardiasis].

Giardia is the most common causes of protozoan diarrhea that leads to significant morbidity and mortality worldwide. The purpose of this study was to determine the clinical efficiency of different scheme of therapy giardiasis with new original plant preparation "Sausalin" (Kazakhstan). We conducted open clinical trial with participation of 93 patients with giardiasis. According the method of treatment the patients were divided into three groups.  Group I - Sausalin at the dose 300 mg/day; group II - Metronidazole at 750 mg/ day; group III - combination of Sausalin 300 mg/day and Metronidazole 750 mg/ day. The treatment was conducted during 10 days. The protozoal clearance rate and clinical symptoms were assessed. There were no significant differences in the efficiency of treatments in group I and group II. The protozoal clearance rate was 68% in group I (Sausalin); in group II -  42,1% (metronidazole). In group III - 83,2% (combination therapy) (р=0.001; 95% CI 54,6-89,7). There was no negative effect on clinical and biochemical blood analysis. We detected statistically significant differences in the dynamics of clinical symptoms (defecation disorders, dyspepsia, abdominal pain, asthenia) of giardiasis in a group of patients receiving Sausalin.The scheme with new drug Sausalin can be used as alternative treatment of Giardiasis. Moreover, the use of the Sausalin is improved the clinical symptoms and safety of therapy.

Clinical and diagnostic features and treatment of giardiasis.

Giardia is the most common causes of protozoan diarrhea that lead to significant morbidity and mortality worldwide. Giardiasis can be cause of disturbance of host immune response. The treatment of Giardiasis is unsuccessful in some cases. The purpose of this study was to determine the clinical features and the content of secretory immunoglobulin A (sIgA) among adults and to evaluate efficiency of new plant preparation "Sausalin". The clinical studies were conducted in Karaganda Regional Infection Hospital (Kazakhstan). 250 patients with giardiasis were randomly assigned to receive sausalin at a dose 720 mg/day or ornidazole at 1500 mg/day. Clinical symptoms of giardisis and efficiency of treatment were evaluated. Protozoal clearance rate and clinical symptoms were assessed. Stool samples were collected from 40 patients and examined the content of sIgA. Our study found the prevalence of abdominal pain, dyspeptic syndrome and the symptoms of intoxication in patients with giardiasis. The increase the level of sIgA was detected, especially in females (88 mg/l). Sausalin was more effectiveness than ornidazole. After the treatment, the clearance rate of giardia (85.71% vs. 42.19%; P<0.05) and the clinical efficacy were significantly higher in the sausalin-treated group than in the ornidazole-treated group. The features of clinic manifestations of giardiasis were identified in population of Kazakhstan. Our data suggest the higher level of sIgA was significantly associated with features of clinic manifestations that the participant had. Treatment with sausalin was more effective than treatment with ornidazole. Further research is needed to explain the existence relationship between Giardia infection and host immune response.

GIARDIA DUODENALIS GENOTYPING NOT LINKED TO CLINICAL SYMPTOMATOLOGY AND NUTRITIONAL STATUS OF SCHOOL-AGED CHILDREN OF SOLEDAD AND GALAPA MUNICIPALITY SCHOOLS, ATLÁNTICO, COLOMBIA.

Giardia duodenalis genotypes A and B have been reported in Colombia. The population consisted of 235 schoolchildren whose ages ranged from 2 to 10 yr of age from the municipalities of Soledad and Galapa in the department of Atlántico, Colombia. Fecal samples were obtained and then analyzed in triplicate using the sedimentation in formalin-ether (Ritchie's method) and direct examination techniques. Of the 235 fecal samples, 35 samples were positive for G. duodenalis; positive samples were concentrated in a sucrose gradient and sonicated for 3 cycles of 20 sec. DNA extraction was performed, and the parasites were genotyped by conventional PCR amplifying a region of the ß-giardin gene. A general prevalence of G. duodenalis of 13.2% was found, and of these genotyped samples, 13 (56.7%) and 7 (20%) corresponded to genotype A, 1 (4.3%), and 3 (25%) corresponded to genotype B, and 9 (39.1%) and 2 (16.7%) were not defined, in the municipalities Soledad and Galapa, respectively. Additionally, 23 children were diagnosed with symptomatologic giardiasis, and 12 were asymptomatic; the most relevant symptoms were abdominal pain (7, 20%) and diarrhea (13, 56.7%). The nutritional status of children with Giardia genotypes A and B were as follows: 3 in a state of malnutrition (10%), 10 normal (33.3%), and 6 overweight and obese (20%) with genotype A, and 1 in a state of malnutrition (3.3%) and 3 normal (10%) with genotype B. The genotypes found in G. duodenalis did not show an association with nutritional status or with the clinical manifestations evaluated in schoolchildren.

Probiotics in the management of Giardia duodenalis: an update on potential mechanisms and outcomes.

Giardia duodenalis is a common cause of infection in children and travelers. The most frequent symptom is diarrhea in these patients. G. duodenalis trophozoites use a highly specialized adhesive disc to attach the host intestinal epithelium to induce intestinal damages. Pathological features of the small intestine following giardiasis include villous atrophy; infiltration of granulocytes, lymphocytes, and plasma cells into the lamina propria; and nodular lymphoid hyperplasia. The disturbed intestinal microbiota has been observed in patients with giardiasis. Therefore, a growing body of evidence has emphasized restoring the gut microbiome by probiotics in giardiasis. This study aimed to review the literature to find the pathologic features of giardiasis and its relationship with imbalanced microbiota. Then, benefits of probiotics in giardiasis and their potential molecular mechanisms were discussed. It has been illustrated that using probiotics (e.g., Lactobacillus and Saccharomyces) can reduce the time of gastrointestinal symptoms and repair the damages, particularly in giardiasis. Probiotics' capability in restoring the composition of commensal microbiota may lead to therapeutic outcomes. According to preclinical and clinical studies, probiotics can protect against parasite-induced mucosal damages via increasing the antioxidant capacity, suppressing oxidative products, and regulating the systemic and mucosal immune responses. In addition, they can reduce the proportion of G. duodenalis load by directly targeting the parasite. They can destroy the cellular architecture of parasites and suppress the proliferation and growth of trophozoites via the production of some factors with anti-giardial features. Further researches are required to find suitable probiotics for the prevention and treatment of giardiasis.

Anti-Giardia Drug Discovery: Current Status and Gut Feelings.

Giardia parasites are ubiquitous protozoans of global importance that impact a wide range of animals including humans. They are the most common enteric pathogen of cats and dogs in developed countries and infect ∼1 billion people worldwide. While Giardia infections can be asymptomatic, they often result in severe and chronic diseases. There is also mounting evidence that they are linked to postinfection disorders. Despite growing evidence of the widespread morbidity associated with Giardia infections, current treatment options are limited to compound classes with broad antimicrobial activity. Frontline anti-Giardia drugs are also associated with increasing drug resistance and treatment failures. To improve the health and well-being of millions, new selective anti-Giardia drugs are needed alongside improved health education initiatives. Here we discuss current treatment options together with recent advances and gaps in drug discovery. We also propose criteria to guide the discovery of new anti-Giardia compounds.

Impact of intestinal permeability, inflammation status and parasitic infections on infant growth faltering in rural Bangladesh.

A longitudinal study of 298 rural Bangladeshi infants found evidence of growth faltering starting at 3 months of age. Anthropometric status declined substantially in the first 2 years of life, with weight-for-height (WHZ) falling from - 0.49 to - 1.75, weight-for-age (WAZ) from - 1.18 to - 2.87 and height-for-age (HAZ) from - 1.00 to - 1.88. Higher concentrations of the acute-phase protein alpha-1-acid glycoprotein (AGP) and higher gut mucosal damage (as signified by raised lactulose:mannitol (L:M) ratios) were both associated with chronic malnutrition as indicated by poorer HAZ and WAZ scores (P = 0.011 and 0.005 for AGP and 0.039 and 0.019 for L:M ratio, respectively). Higher Hb levels were related to improved z-scores, while elevation of Giardia-specific IgM titre (GSIgM) was associated with poor WAZ and WHZ (P = 0.015 and 0.039, respectively). IgG did not show any significant association with z-scores and the L:M ratio did not correlate with any of the inflammation markers or Giardia infection. The prevalence of geohelminth infections was low (only 4 % in the total study period). However, the level of GSIgM indicated high endemicity of Giardia infection from early in life, although very few cysts were detected from stool samples. These findings suggest that rural Bangladeshi infants are being exposed to high levels of infection with concomitant gut damage and growth faltering.

Encystation commitment in Giardia duodenalis: a long and winding road.

Cholesterol and bile salts are relevant modulators of Giardia encystation. Although several molecules within signaling cascades have been identified, and changes in their expression observed during giardial encystation, their underlying interactions leading to expression of cyst wall markers (CWPs and precursors of the GalNAc homopolymer) are not well defined. Recent experimental data and the completion of the Giardia Genome Project Database (GiardiaDB) allow us now to consider the role of bile salts as "natural stimuli" and the potential involvement of a Raf/MEK/ERK pathway mediating cholesterol-regulated expression of cyst-specific genes. These new findings may provide promising targets for diagnostics, drug design and prophylactic intervention against giardiasis.

Giardia duodenalis: Role of secreted molecules as virulent factors in the cytotoxic effect on epithelial cells.

During the course of giardiasis in humans and experimental models, G. duodenalis trophozoites express and secrete several proteins (ESPs) affecting structural, cellular and soluble components of the host intestinal milieu. These include the toxin-like molecules CRP136 and ESP58 that induce intestinal hyper-peristalsis. After the completion of the Giardia genome database and using up-to date transcriptomic and proteomic approaches, secreted 'virulence factors' have also been identified and experimentally characterized. This repertoire includes arginine deiminase (ADI) that competes for arginine, an important energy source for trophozoites, some high-cysteine membrane proteins (HCMPs) and VSP88, a versatile variant surface protein (VSP) that functions as an extracellular protease. Another giardial protein, enolase, moonlights as a metabolic enzyme that interacts with the fibrinolytic system and damages host epithelial cells. Other putative Giardia virulence factors are cysteine proteases that degrade multiple host components including mucin, villin, tight junction proteins, immunoglobulins, defensins and cytokines. One of these proteases, named giardipain-1, decreases transepithelial electrical resistance and induces apoptosis in epithelial cells. A putative role for tenascins, present in the Giardia's secretome, is interfering with the host epidermal growth factor. Based on the roles that these molecules play, drugs may be designed to interfere with their functions. This review presents a comprehensive description of secreted Giardia virulence factors. It further describes their cytotoxic mechanisms and roles in the pathophysiology of giardiasis, and then assesses their potential as targets for drug development.

Giardia duodenalis infection and anthropometric status in preschoolers in Salvador, Bahia State, Brazil.

The aim of this study was to estimate the association between Giardia duodenalis infection and anthropometric deficits, as measured by weight-for-age and height-for-age. This cross-sectional study included 629 children from 12 to 48 months of age, selected from 30 geographic areas in the city of Salvador, Bahia State, Brazil. Poisson regression and linear regression were used for the multivariate statistical analyses. G. duodenalis was diagnosed in 13.5% of the children. The children's breastfeeding duration and living conditions (garbage collection and paved streets or sidewalks) modified the effect of G. duodenalis infection on anthropometric status. Among infected children, there were statistically significant associations between weight deficit and shorter breastfeeding (PR=2.22; 95%CI: 1.56-3.14) and inadequate paving of streets and sidewalks (PR=2.00; 95%CI: 1.37-2.92), while height deficit was associated with deficient public garbage collection (PR=2.21; 95%CI: 1.31-2.51). In the linear regression, the association with the anthropometric indicators remained positive and statistically significant. The child's unhealthy living environment aggravated the negative effect of G. duodenalis infection on anthropometric status, and breastfeeding was a protective factor in the outcome.

Outbreak of giardiasis and cryptosporidiosis associated with a neighborhood interactive water fountain--Florida, 2006.

An outbreak of giardiasis and cryptosporidiosis was identified in central Florida in September 2006. Environmental and epidemiological investigations indicated the likely source was a neighborhood interactive water fountain in a large upscale urban neighborhood. Forty-nine cases meeting the case definition were identified, of which 38 were giardiasis, nine were cryptosporidiosis, and two were co-infections. The median age of those affected was four years old, and 32 (65.3%) cases were male. This outbreak and other similar occurrences highlight the need to design and implement more stringent disinfection practices and filtration requirements for treated interactive water venues. Giardia cysts and Cryptosporidium oocysts are small and chlorine-resistant, and they may require supplemental disinfection methods, such as ultraviolet light irradiation, ozonation, or chlorine dioxide. Individuals who use these types of venues also need to change their behavior to prevent disease transmission. This is the first documentation of a giardiasis outbreak associated with exposure to an interactive water fountain in the United States.

Infection of dogs by experimental inoculation with human isolates of Giardia duodenalis: clinical and laboratory manifestations.

The susceptibility of dogs to experimental inoculation with trophozoites and cysts of human isolates of Giardia duodenalis and the clinical and laboratory profiles of infection of these animals were studied. Two groups (A and B), each comprising three dogs, were inoculated with G. duodenalis trophozoites and cysts, respectively. A third group of two dogs was not inoculated and remained as control. After inoculation feces were collected daily to determine the pre-patent period, by flotation in 33% zinc sulfate solution. Blood samples (5mL) were collected from animals at 15-day intervals during the 165 days of the experimental period and were used to carry out the hemogram and biochemical evaluation of the levels of total protein, albumin, alanine aminotransferase, gamma glutamyltransferase, urea and creatinine. A prepatent period was observed at 5-6 days post-inoculation (p.i.) in the inoculated dogs, with cysts eliminated for approximately 3 months. No alterations were seen in the clinical parameters evaluated. Anemia was observed at 15 p.i. in the inoculated dogs. The mean eosinophil count of inoculated groups was higher than that of the control (p< or =0.05) but none of the biochemical parameters analyzed presented significant differences. The results of this study show that G. duodenalis from human isolates is able to infect dogs with minimal systemic manifestations without producing clinical signs of giardiasis.

[Experimental model of activated Lamblia (Giardia) muris infection in albino mice].

Experimental L. muris infection was reproduced in 100% of the intact albino mice intragastrically given levomycin in an average total dose of 15.88-34.84 or 0.88-1.02 g/kg for 18-34 days. With levomycin administration, the intensity of giardiasis was 1121.6-8540.1 (mean 4830.9) thousand L. muris trophozoites per animal. The total number of trophozoites per animal decreased to 302.2-3481.4 (mean 1546.4) thousand and 28.1-324.0 (mean 109.4) thousand specimens 5-8 and 11-13 days after discontinuation of the antibiotic, respectively. The maximum number of L. muris trophozoites was observed in the proximal and middle portions of the murine small intestine during and after the administration oflevomycin. The highest isolation of cysts was seen 12-14 days after the initiation of administration of the antibiotic. Following 8-10 days of terminations of a course of levomycin therapy the native smear of animal feces showed no Lamblia cysts. In mice with activated infection, the isolation rate of Lamblia cysts was directly related to the intensity of intestinal infection with trophozoites of the parasite.

Pathogenic Mechanisms of Cryptosporidium and Giardia.

Intestinal protozoa are important etiological agents of diarrhea, particularly in children, yet the public health risk they pose is often neglected. Results from the Global Enteric Multicenter Study (GEMS) showed that Cryptosporidium is among the leading causes of moderate to severe diarrhea in children under 2 years. Likewise, Giardia infects approximately 200 million individuals worldwide, and causes acute diarrhea in children under 5 years. Despite this recognized role as pathogens, the question is why and how these parasites cause disease in some individuals but not in others. This review focuses on known pathogenic mechanisms of Cryptosporidium and Giardia, and infection progress towards disease.