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Cryptosporidiosis is common in early childhood, and both diarrheal and subclinical infections are associated with adverse developmental outcomes. Improved therapeutic medications may help reduce the burden of cryptosporidial diarrhea; however, an effective vaccine would be better able to prevent the detrimental impact of both diarrheal and subclinical disease. A more complete understanding of naturally occurring immunity may further inform strategies to develop an effective vaccine. In this prospective cohort study of Bangladeshi children, greater fecal IgA at 12 months, but not plasma IgG, directed against two sporozoite-expressed, immunodominant and vaccine candidate antigens was associated with delayed time to subsequent cryptosporidiosis to 3 years of life. These findings extend prior work and further support the role of mucosal antibody responses in naturally developing protective immunity to Cryptosporidium.

In this prospective cohort study of Bangladeshi children, greater fecal immunoglobulin A, but not plasma immunoglobulin G, directed against the Cryptosporidium sporozoite-expressed antigen Cp23 at 12 months of age was associated with delayed time to subsequent cryptosporidiosis. This finding suggests a protective role for mucosal antibody-mediated immunity in naturally exposed children.

We conducted a longitudinal study of cryptosporidiosis from birth to three years of age in an urban slum of Dhaka Bangladesh. Fecal DNA was extracted from monthly surveillance samples and diarrheal stool samples collected from 392 infants from birth to three years. A pan-Cryptosporidium qPCR assay was used to identify sub-clinical and symptomatic cryptosporidiosis. Anthropometric measurements were collected quarterly to assess child nutritional status. 31% (121/392) of children experienced a single and 57% (222/392) multiple infections with Cryptosporidium. Repeat infections had a lower burden of parasites in the stool (Cq slope = -1.85; p, Author summary Cryptosporidium is one of the top causes of diarrhea and growth faltering in Bangladesh infants. We discovered that a prior infection resulted in incomplete immunity that protected from diarrhea and growth faltering but not infection and was associated with mucosal IgA against a sporozoite surface protein Cp23. The most important implication of these findings is that a cryptosporidiosis vaccine may not need to achieve complete protection from infection to have a beneficial impact on child health.

Background The protozoan parasites in the Cryptosporidium genus cause both acute diarrheal disease and subclinical (ie, nondiarrheal) disease. It is unclear if the microbiota can influence the manifestation of diarrhea during a Cryptosporidium infection. Methods To characterize the role of the gut microbiota in diarrheal cryptosporidiosis, the microbiome composition of both diarrheal and surveillance Cryptosporidium-positive fecal samples from 72 infants was evaluated using 16S ribosomal RNA gene sequencing. Additionally, the microbiome composition prior to infection was examined to test whether a preexisting microbiome profile could influence the Cryptosporidium infection phenotype. Results Fecal microbiome composition was associated with diarrheal symptoms at 2 timepoints. Megasphaera was significantly less abundant in diarrheal samples compared with subclinical samples at the time of Cryptosporidium detection (log2 [fold change] = –4.3; P = 10–10) and prior to infection (log2 [fold change] = –2.0; P = 10–4); this assigned sequence variant was detected in 8 children who had diarrhea and 30 children without diarrhea. Random forest classification also identified Megasphaera abundance in the pre- and postexposure microbiota as predictive of a subclinical infection. Conclusions Microbiome composition broadly, and specifically low Megasphaera abundance, was associated with diarrheal symptoms prior to and at the time of Cryptosporidium detection. This observation suggests that the gut microenvironment may play a role in determining the severity of a Cryptosporidium infection. Clinical Trials Registration. NCT02764918., Megasphaera abundance in the stool of Bangladeshi infants is associated with the development of diarrhea upon infection with the Cryptosporidium parasite.

Background Cryptosporidium is a leading cause of diarrhea in Sub-Saharan Africa and is associated with substantial morbidity and mortality in young children. Methods We analyzed data from children aged 6–71 months presenting to 2 public hospitals in Western Kenya with acute diarrhea and their primary caregivers, including detection of Cryptosporidium by quantitative polymerase chain reaction (PCR) and immunoassay analysis in stool samples from both children and their caregivers. Associations between potential transmission sources and child/caregiver Cryptosporidium infection were evaluated using prevalence ratios (PRs). Secondary analyses evaluated host and clinical risk factors of child/caregiver Cryptosporidium infection. Results Among 243 child–caregiver pairs enrolled, 77 children (32%) and 57 caregivers (23%) had Cryptosporidium identified by either immunoassay or PCR. Twenty-six of the 243 child–caregiver pairs (11%) had concordant detection of Cryptosporidium. Cryptosporidium infection in children was associated with detection of Cryptosporidium in caregivers (adjusted PR [aPR], 1.8; 95% CI, 1.2 to 2.6; P = .002) and unprotected water source (aPR, 2.0; 95% CI, 1.3 to 3.2; P = .003). Risk factors for Cryptosporidium detection in caregivers included child Cryptosporidium infection (aPR, 2.0; 95% CI, 1.3 to 3.0; P = .002) as well as cow (aPR, 3.1; 95% CI, 1.4 to 7.0; P = .02) and other livestock ownership (aPR, 2.6; 95% CI, 1.1 to 6.3; P = .03) vs no livestock ownership. Recent diarrhea in caregivers and children was independently associated with child and caregiver Cryptosporidium infections, respectively. Conclusions Our results are consistent with the hypothesis that Cryptosporidium transmission can occur directly between child–caregiver dyads as well as through other pathways involving water and livestock. Additional research into caregivers as a source of childhood Cryptosporidium infection is warranted.

In humans in addition to diarrheal disease and sub-clinical infections the Cryptosporidium parasite can cause developmental delays and growth failure in malnourished infants. We hypothesized that genetic variants may be responsible for the differences in pathogen phenotypes. Resequencing thirty-two Bangladesh C. hominis isolates identified both polymorphic regions and evidence of frequent genetic recombination. This increased the genetic diversity of the parasites. Additional studies are now needed to identify the genetic changes predisposing the parasite to the different phenotypes.

The gut microbiome provides resistance to infection. However, the mechanisms for this are poorly understood. Colonization with the intestinal bacteriumClostridium scindensprovided protection from the parasiteEntamoeba histolyticavia innate immunity. Introduction ofC. scindensinto the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte-progenitors (GMPs) and provided neutrophil-mediated protection against subsequent challenge withE. histolytica. Adoptive transfer of bone-marrow fromC. scindenscolonized-mice into naïve-mice protected against ameba infection and increased intestinal neutrophils. Because of the known ability ofC. scindensto metabolize the bile salt cholate, we measured deoxycholate and discovered that it was increased in the sera ofC. scindenscolonized mice, as well as in children protected from amebiasis. Administration of deoxycholate alone (in the absence ofC. scindens) increased the epigenetic mediator JMJD3 and GMPs and provided protection from amebiasis. In conclusion the microbiota was shown to communicate to the bone marrow via microbially-metabolized bile salts to train innate immune memory to provide antigen-nonspecific protection from subsequent infection. This represents a novel mechanism by which the microbiome protects from disease.One Sentence SummaryIntroduction of the human commensal bacteriaClostridium scindensinto the intestinal microbiota epigenetically alters bone marrow and protects from future parasite infection.

Background Glucose hydrogen breath testing is a noninvasive test for small intestine bacterial overgrowth (SIBO). A positive glucose hydrogen breath test is common in children from low-income countries and has been found to be associated with malnutrition as measured by stunted growth. The microbiome associated with positive breath testing is relatively unstudied. Methods We performed 16 S V4 rDNA microbiome analysis on the stool of 90 Bangladeshi children aged 2 years from an impoverished neighborhood who were tested at the same time for SIBO by glucose hydrogen breath testing. Data were analyzed by linear discriminant analysis effect size with SIBO as the outcome. Any selected genera were tested individually by Wilcoxon’s rank-sum test to ensure that linear discriminant analysis effect size results were not outlier-skewed. Results Linear discriminant analysis effect size analysis identified Lactobacillus (linear discriminate analysis score, 4.59; P = .03) as over-represented in 15 out of the 90 children who were SIBO positive. Conclusions These results suggest that glucose hydrogen breath test positivity in children from low-income settings may be due to an upper intestinal Lactobacillus bloom, potentially explaining the association of SIBO with the gut damage and inflammation that leads to malnutrition.

Background and objectiveSerological screening with a confirmation through biopsy has improved the understanding of coeliac disease (CD) epidemiology worldwide. Prevalence of CD in Bangladesh is not yet explored and therefore, we aimed to assess the seroprevalence of CD in slum-dwelling malnourished children and adults in Dhaka.MethodsSerum samples were collected from three different cohorts: stunted (length-for-age Z-scores (LAZ) 2). Samples from all the participants were assessed for anti-tissue transglutaminase antibody (tTG-IgA) and total serum IgA by ELISA. Positive tTG-IgA and randomly selected low IgA values were reconfirmed using anti-tTG-IgG and gliadin IgG ELISA. CD was diagnosed when second screening tests were found positive and the participants were further investigated by small bowel biopsy.ResultsA total of 818 participants (240 stunted, 272 at risk of stunting children and 306 malnourished adults) were enrolled in the study. Overall, anti-tTG-IgA was positive in 5/818 (0.6%; 95% CI 0.25% to 1.46%). Of the five positive cases, anti-tTG-IgG and gliadin IgG were found positive in only one participant. Duodenal biopsy of positive participant revealed characteristic lesions of CD. Randomly selected low IgA values were found negative in tTG-IgG and gliadin IgG for all the participants. No participant was found total IgA deficient.ConclusionThe incidence of coeliac autoimmunity is low in malnourished slum dwellers regardless of age in Bangladesh. It is important to investigate the nationwide prevalence to reveal the definite picture.

The outcome of an Entamoeba histolytica infection is variable, and the contribution of genetic diversity within E. histolytica to human disease is not fully understood. The information provided by the whole genome sequence of the E. histolytica reference laboratory strain (HM-1:IMSS), and 13 additional laboratory strains have been made publically available. In this review, theories on the source of the unexpected level of structural diversity found in E. histolytica will be discussed.

Background Glucose hydrogen breath testing is a noninvasive test for small intestine bacterial overgrowth (SIBO). A positive glucose hydrogen breath test is common in children from low-income countries and has been found to be associated with malnutrition as measured by stunted growth. The microbiome associated with positive breath testing is relatively unstudied. Methods We performed 16 S V4 rDNA microbiome analysis on the stool of 90 Bangladeshi children aged 2 years from an impoverished neighborhood who were tested at the same time for SIBO by glucose hydrogen breath testing. Data were analyzed by linear discriminant analysis effect size with SIBO as the outcome. Any selected genera were tested individually by Wilcoxon’s rank-sum test to ensure that linear discriminant analysis effect size results were not outlier-skewed. Results Linear discriminant analysis effect size analysis identified Lactobacillus (linear discriminate analysis score, 4.59; P = .03) as over-represented in 15 out of the 90 children who were SIBO positive. Conclusions These results suggest that glucose hydrogen breath test positivity in children from low-income settings may be due to an upper intestinal Lactobacillus bloom, potentially explaining the association of SIBO with the gut damage and inflammation that leads to malnutrition.

BackgroundCryptosporidium, an apicomplexan protozoa, is a leading contributor to diarrheal morbidity and mortality in children under five years old worldwide. As there is no vaccine and no approved drug for Cryptosporidium spp. in young children, preventing parasite transmission is crucial. We undertook a pilot case-control study to define the extent of person-to-person transmission of cryptosporidiosis within families in an urban and rural community in Bangladesh.MethodsWe enrolled 48 case families with a Cryptosporidium-infected child aged 6-18 months. Controls were age-sex matched Cryptosporidium-negative children (n=12). Once children were identified, we enrolled all household members. We then followed these individuals for 8 weeks, with weekly surveillance stools and testing with qPCR for Cryptosporidium spp.FindingsIn the 48 case families, the rate of secondary infections with Cryptosporidium was 18.6% (22/118) compared to 0 new infections (0/35) in the 12 control families. In the 22 urban Mirpur households, the secondary attack rate was 30% (18/60) in cases compared to 0% (0/14) in controls (chi-square p = 0.018). In contrast, in the 21 rural Mirzapur households, the secondary attack rate was 6.9% (4/58) in case households compared to 0% (0/21) in controls (chi-square p = 0.22). Genotyping by gp60 demonstrated infection with the same subspecies in five of six families. Serologic response to Cryptosporidium infection was associated with younger age, longer duration of infection, and C hominis gp60_IbA9G3R2 infection.InterpretationThe high rate of secondary infection in Mirpur suggests that person-to-person transmission is likely a major source of Cryptosporidium infection for young children living in this region. GP 60 genotyping demonstrated direction of infection in 2 households, and concurrent infection in five households. Further work is needed to understand the differences in parasite transmissibility and immunity to different genotypes.

BACKGROUND: Cryptosporidium is a major cause of childhood diarrhea. Current modes of cryptosporidiosis diagnosis involve procedures that are costly and require both a well-equipped laboratory and technical expertise. Therefore, a cost-effective, user-friendly, and rapid method for point-of-care detection of Cryptosporidium is desirable. METHODS: A total of 832 diarrheal stool specimens collected from 200 children aged

This birth cohort study describes the early childhood burden of cryptosporidiosis in rural and urban Bangladesh. Important findings include an association of repeated and subclinical cryptosporidiosis with growth faltering, and disparate predominant Cryptosporidium species in the urban vs rural site., Background Cryptosporidiosis is a major cause of childhood diarrhea in low- and middle-income countries and has been linked to impairment of child growth. This study investigated the burden of cryptosporidiosis and its impact on child growth in both a rural and an urban site in Bangladesh. Methods Pregnant women in the second trimester were identified at 2 sites in Bangladesh, 1 urban and 1 rural. Their offspring were enrolled at birth into the study (urban, n = 250; rural, n = 258). For 2 years, the children were actively monitored for diarrhea and anthropometric measurements were obtained every 3 months. Stool samples were collected monthly and during diarrheal episodes with Cryptosporidium infection and causative species determined by quantitative polymerase chain reaction assays. Results Cryptosporidium infections were common at both sites and mostly subclinical. In the urban site, 161 (64%) children were infected and 65 (26%) had ≥2 infections. In the rural site, 114 (44%) were infected and 24 (9%) had multiple infections. Adjusted for potential confounders, cryptosporidiosis was associated with a significantly greater drop in the length-for-age z score (LAZ) at 24 months from LAZ at enrollment (Δ-LAZ), an effect greatest in the children with multiple episodes of cryptosporidiosis. The most common species in Mirpur was Cryptosporidium hominis, whereas Cryptosporidium meleagridis predominated in Mirzapur. Conclusions Cryptosporidiosis is common in early childhood and associated with early growth faltering in Bangladeshi children. Predominant Cryptosporidium species differed between the 2 sites, suggesting different exposures or modes of transmission but similar consequences for child growth. Clinical Trials Registration NCT02764918.

Background Diarrhea is frequent in communities without clean water, which include low-income South African populations in Giyani and Pretoria. In these populations, the amount of diarrhea caused by Entamoeba histolytica, inclusive of all ages, sexes, and human immunodeficiency virus status, is uncertain. Infection with E. histolytica can modulate the host microbiota, and a key species indicative of this is the Prevotella copri pathobiont. Methods A cross-sectional study of patients attending gastroenterology clinics was conducted to determine the frequency and burden of 4 Entamoeba species and P. copri. Results Entamoeba species were present in 27% of patients (129/484), with E. histolytica detected in 8.5% (41), E. dispar in 8% (38), E. bangladeshi in 4.75% (23), and E. moshkovskii in 0%. This is the first description of E. bangladeshi outside Bangladesh. In E. histolytica-positive samples, the levels of both the parasite and P. copri were lower in nondiarrheal samples, validating the results of a study in Bangladesh (P = .0034). By contrast, in E. histolytica-negative samples positive for either of the nonpathogenic species E. dispar or E. bangladeshi, neither P. copri nor Entamoeba levels were linked to gastrointestinal status. Conclusions Nonmorphologic identification of this parasite is essential. In South Africa, 3 morphologically identical Entamoeba were common, but only E. histolytica was linked to both disease and changes in the microbiota.

The disease severity of Entamoeba histolytica infection ranges from asymptomatic to life-threatening. Recent human and animal data implicate the gut microbiome as a modifier of E. histolytica virulence. Here we have explored the association of the microbiome with susceptibility to amebiasis in infants and in the mouse model of amebic colitis. Dysbiosis occurred symptomatic E. histolytica infection in children, as evidenced by a lower Shannon diversity index of the gut microbiota. To test if dysbiosis was a cause of susceptibility, wild type C57BL/6 mice (which are innately resistant to E. histiolytica infection) were treated with antibiotics prior to cecal challenge with E. histolytica. Compared with untreated mice, antibiotic pre-treated mice had more severe colitis and delayed clearance of E. histolytica. Gut IL-25 and mucus protein Muc2, both shown to provide innate immunity in the mouse model of amebic colitis, were lower in antibiotic pre-treated mice. Moreover, dysbiotic mice had fewer cecal neutrophils and myeloperoxidase activity. Paradoxically, the neutrophil chemoattractant chemokines CXCL1 and CXCL2, as well as IL-1β, were higher in the colon of mice with antibiotic-induced dysbiosis. Neutrophils from antibiotic pre-treated mice had diminished surface expression of the chemokine receptor CXCR2, potentially explaining their inability to migrate to the site of infection. Blockade of CXCR2 increased susceptibility of control non-antibiotic treated mice to amebiasis. In conclusion, dysbiosis increased the severity of amebic colitis due to decreased neutrophil recruitment to the gut, which was due in part to decreased surface expression on neutrophils of CXCR2., Author summary Amebiasis, caused by intestinal infection of Entamoeba histolytica, is one of the leading causes of parasite infection-related mortality and morbidity around the world. However, pathogenesis, such as determinant factors of infection outcome, is still unclear although recent data indicate that the gut microbiome plays an important role. In the present study, we firstly found that dysbiosis, which was represented by a lower Shannon diversity index of the gut microbiota, occurred symptomatic E. histolytica infection in children living in endemic area. In mouse model, we demonstrated that dysbiosis induced by antibiotic pre-treatment increased the severity of amebic colitis due to decreased neutrophil activity as well as decreased IL-25 associated mucosal defense in the gut. Moreover, we demonstrated surface expression on neutrophils of CXCR2 was diminished in mice with dysbiosis, which resulted in decreased neutrophil recruitment to the gut. This study is of fundamental importance in amebiasis research for the discovery of a mechanism of microbiome-mediated resistance to amebiasis via neutrophil trafficking to the gut. The work is importantly of broad interest in infectious diseases and immunology for the discovery that neutrophil mediated protection can be disturbed by dysbiosis.

The outcome of an Entamoeba histolytica infection is variable and the contribution of genetic diversity within E. histolytica to human disease is not fully understood. The information provided by the whole genome sequence of the E. histolytica reference laboratory strain (HM-1:IMSS) and thirteen additional laboratory strains have been made publically available. In this review theories on the source of the unexpected level of structural diversity found in E. histolytica will be discussed.

Anti-tetanus antibodies in biological samples are typically detected using an enzyme-linked immunosorbent assay based on toxoided tetanus neurotoxin as antigen. We demonstrate that recombinantly produced fragment C of the toxin heavy chain is an effective alternative antigen for assessment of tetanus-immune status in plasma samples.

Mouse strain-specific differences in the carbohydrate composition of intestinal mucins were hypothesized to account for strain-dependent susceptibility to Entamoeba histolytica. To test this hypothesis, intestinal mucins from susceptible and resistant inbred strains of mice were analyzed for their O-glycan content and for their ability to inhibit amoebic adherence to (GalNAc)12-27-HSA neo-glycoproteins. The results showed that the colorectal mucin O-glycan of susceptible CBA mice was lower in sialic acid content than that of resistant C57BL/6 and BALB/c mice. Mucins from CBA mice were more potent inhibitors of E. histolytica adherence to neo-glycoproteins than were mucins from C57BL/6 or BALB/c mice. Consistent with the role of terminal Gal/GalNAc as a receptor for amoebic adherence, sialidase treatment of C57BL/6 and BALB/c colorectal mucins increased their ability to inhibit E. histolytica adherence to the neo-glycoproteins. These results provide evidence of mouse strain-specific differences in the sialic acids content of mucin O-glycans. These dissimilarities likely contribute to the differential susceptibility of the three mouse strains to E. histolytica infection., Biochemical and Biophysical Research Communications, 436(2), pp.252-258; 2013

We have developed recombinant fragment C based rapid point of care dipstick devices to assess tetanus immunization status using plasma or whole blood. The devices demonstrated specificity of 0.90 and sensitivity of 0.90 (whole blood) / 0.94 (plasma) at field sites in Bangladesh and The Gambia when compared to a commercial ELISA with the immune cut-off titer set as ≥ 0.1 IU/mL.

Entamoeba histolytica causes intestinal disease in endemic settings throughout the world. Diagnosis of E. histolytica infection would be improved by the identification of biomarkers that are expressed by cysts of E. histolytica, but not by cysts of closely related commensal species of Entamoeba. Herein, we describe two novel monoclonal antibodies (1A4 and 1D3) produced against a spacer region of the E. histolytica Jacob2 lectin, an outer cyst wall protein. These reagents demonstrated no cross-reaction to E. dispar recombinant antigen and low picomolar molecular detection limits when paired in ELISA sandwich assays. In an immunofluorescence microscopy assay, the α-Jacob2 murine antibodies labeled cysts of three xenically cultured E. histolytica isolates but did not label cysts of three E. bangladeshi isolates. Monoclonal antibody 1A4 did not cross-react with xenic cultures of three E. dispar isolates, demonstrating specificity to E. histolytica, while monoclonal antibody 1D3 cross-reacted with two out of three E. dispar isolates. Both antibodies labeled cysts in formalin-fixed slides, a potential logistical advantage in some settings. The monoclonal antibody 1A4 was also used in an immunofluorescence microscopy assay with formalin-fixed stool specimens. Seven out of ten ELISA-positive stool specimens exhibited 1A4-labeled cyst-like objects, compared to one out of seven ELISA-negative specimens. These results demonstrate that antibodies generated against the flexible spacer of E. histolytica Jacob2 lectin recognize and bind to Jacob2 protein in whole cysts and are capable of differentiating Entamoeba species in fixed specimens. Thus, Jacob2 is a promising biomarker for use in diagnosing E. histolytica infection., Author Summary Entamoeba histolytica is a prevalent human parasite requiring sensitive and specific detection. Assays available for Entamoeba detection utilize antibodies to detect parasite protein in stool, and they distinguish E. histolytica from nonpathogenic commensal amoeba. However, these tests have exhibited suboptimal sensitivity in some studies. This may have occurred because the tests do not detect the cyst stage of the parasite, which is more prevalent and stable in stool. Moreover, they cannot be used on formalin-fixed material, which creates logistical problems in some settings. Here, we have generated antibodies against a region of an E. histolytica cyst surface protein that has a unique sequence relative to nonpathogenic amoeba. One of these antibodies bound to E. histolytica cysts in fixed, xenic cultures without cross-reacting to nonpathogenic E. dispar and E. bangladeshi, while another antibody bound to both E. histolytica and E. dispar. The antibody that selectively bound to E. histolytica cysts also labeled cyst-like objects in fixed stool specimens from E. histolytica-positive patients. As they detected fixed organisms, these antibodies could ultimately make specimen collection and processing for Entamoeba diagnosis a simpler process. Additionally, this study indicates that the antibodies’ cyst protein target may be a useful biomarker for E. histolytica detection.

The release of the Entamoeba histolytica genome has facilitated the development of techniques to survey rapidly and to relate gene expression with biology. The association and potential contribution of differential gene expression to the life cycle and the virulence of this protozoan parasite of humans are reviewed here.

The unicellular eukaryote Entamoeba histolytica is a human parasite that causes amebic dysentery and liver abscess. A genome-wide analysis of gene expression modulated by intestinal colonization and invasion identified an upregulated transcript that encoded a putative high-mobility-group box (HMGB) protein, EhHMGB1. We tested if EhHMGB1 encoded a functional HMGB protein and determined its role in control of parasite gene expression. Recombinant EhHMGB1 was able to bend DNA in vitro, a characteristic of HMGB proteins. Core conserved residues required for DNA bending activity in other HMGB proteins were demonstrated by mutational analysis to be essential for EhHMGB1 activity. EhHMGB1 was also able to enhance the binding of human p53 to its cognate DNA sequence in vitro, which is expected for an HMGB1 protein. Confocal microscopy, using antibodies against the recombinant protein, confirmed its nuclear localization. Overexpression of EhHMGB1 in HM1:IMSS trophozoites led to modulation of 33 transcripts involved in a variety of cellular functions. Of these, 20 were also modulated at either day 1 or day 29 in the mouse model of intestinal amebiasis. Notably, four transcripts with known roles in virulence, including two encoding Gal/GalNAc lectin light chains, were modulated in response to EhHMGB1 overexpression. We concluded that EhHMGB1 was a bona fide HMGB protein with the capacity to recapitulate part of the modulation of parasite gene expression seen during adaptation to the host intestine.

Children in developing countries are continually exposed to enteric pathogens [1–4]. This results in high rates of morbidity and mortality, especially in infants [5]. The outcome of an Entamoeba histolytica exposure can be diarrhea, dysentery, or amebic liver abscess, with the latter occurring mainly in men [6]. Disease, however, occurs in a minority of E. histolytica infections (approximately 20%) [6]. While both host and parasite factors contribute to outcome, they do not entirely explain susceptibility [7–9]. A prospective longitudinal study of enteric pathogens in a slum community in Dhaka, Bangladesh, has provided a rich data set to identify parameters important for protection and disease. It has been previously shown that children in this study cohort who are malnourished at birth are more susceptible to amebiasis and in general experience more-severe diarrhea [2]. There is evidence in support of both passive and active acquired immunity. Infants with mothers with high levels of breast milk anti-galactose/N-acetylgalactosamine (Gal/GalNAc) lectin immunoglobulin A (IgA) had fewer E. histolytica infections, and children with fecal IgA anti-Gal/GalNAc lectin IgA also had a lower incidence of new infection [10, 11]. Weaning and the introduction of supplementary food into the diet is known to trigger changes in the bacterial composition of the microbiome that include increases in Prevotellaceae and Bacteroides species [12, 13]. The first 2 years of life in general is a period of rapid immune and microbiome maturation, which, in this population, may be disrupted by malnutrition and environmental insult [13–15]. Here we describe the natural history of amebiasis in the first 2 years of life in infants from an urban slum in Dhaka. The cumulative incidence of amebic infection and diarrhea, the association of protection with anti-Gal/GalNAc lectin IgA, the impact of parasite burden on symptoms, and potential role of Prevotella copri in E. histolytica diarrhea are described.

Background Cryptosporidiosis is a common cause of infectious diarrhea in young children worldwide, and is a significant contributor to under-five mortality. Current treatment options are limited in young children. In this study, we describe the natural history of Cryptosporidium spp. infection in a birth cohort of children in Bangladesh and evaluate for association with malnutrition. Methodology/Principal Findings This is a longitudinal birth cohort study of 392 slum-dwelling Bangladeshi children followed over the first two years of life from 2008 to 2014. Children were monitored for diarrheal disease, and stool was tested for intestinal protozoa. Anthropometric measurements were taken at 3-month intervals. A subset of Cryptosporidium positive stools were genotyped for species and revealed that C. hominis was isolated from over 90% of samples. In the first two years of life, 77% of children experienced at least one infection with Cryptosporidium spp. Non-diarrheal infection (67%) was more common than diarrheal infection (6.3%) although 27% of children had both types of infection. Extreme poverty was associated with higher rates of infection (chi-square, 49.7% vs 33.3%, p = 0.006). Malnutrition was common in this cohort, 56% of children had stunted growth by age two. Children with Cryptosporidium spp. infection had a greater than 2-fold increased risk of severe stunting at age two compared to uninfected children (odds ratio 2.69, 95% CI 1.17, 6.15, p = 0.019) independent of sex, income, maternal body-mass index, maternal education and weight for age adjusted z (WAZ) score at birth. Conclusions/Significance Cryptosporidium infection is common (77%) in this cohort of slum-dwelling Bangladeshi children, and both non-diarrheal and diarrheal infections are significantly associated with a child’s growth at 2 years of age., Author Summary Diarrheal disease is a leading cause of death in young children worldwide. Cryptosporidium species are responsible for a large proportion of global burden of diarrhea. This study describes the natural history of cryptosporidiosis in a birth cohort of impoverished Bangladeshi children. Children were enrolled at birth and monitored for diarrhea twice a week for two years. Stool samples were tested for enteric protozoa. Children in this cohort had significant rates of malnutrition compared to the W.H.O. reference population, and extreme poverty was common. A majority of children were infected with Cryptosporidium spp, and we found that children who had at least one infection with Cryptosporidium spp during the two year follow up period were significantly more likely to have growth faltering by age 24 months. Cryptosporidiosis is a common infection in this cohort, and is associated with poverty and reduced growth during the first two years of life.

SUMMARY In addition to the ever-present concern of medical professionals about epidemics of infectious diseases, the relative ease of access and low cost of obtaining, producing, and disseminating pathogenic organisms or biological toxins mean that bioterrorism activity should also be considered when facing a disease outbreak. Utilization of whole-genome sequencing (WGS) in outbreak analysis facilitates the rapid and accurate identification of virulence factors of the pathogen and can be used to identify the path of disease transmission within a population and provide information on the probable source. Molecular tools such as WGS are being refined and advanced at a rapid pace to provide robust and higher-resolution methods for identifying, comparing, and classifying pathogenic organisms. If these methods of pathogen characterization are properly applied, they will enable an improved public health response whether a disease outbreak was initiated by natural events or by accidental or deliberate human activity. The current application of next-generation sequencing (NGS) technology to microbial WGS and microbial forensics is reviewed.

Establishment of intestinal infection withEntamoeba histolyticadepends on the mouse strain; C57BL/6 mice are highly resistant, and C3H/HeJ mice are relatively susceptible. We found that resistance to intestinal infection was independent of lymphocyte activity or H-2 haplotype and occurred in the first hours to days postchallenge according to in vivo imaging. At 18 h postchallenge, the ceca of resistant C57BL/6 mice were histologically unremarkable, in contrast to the severe inflammation observed in susceptible C3H/HeJ mice. Comparison of cecal gene expression in C3H/HeJ and C57BL/6 mice demonstrated that there was parasite-induced upregulation of proinflammatory and neutrophil chemotaxis transcripts and there was downregulation of transforming growth factor β signaling molecules. Pretreatment with dexamethasone abrogated the partial resistance of C3H/HeJ or CBA mice through an innate, lymphocyte-independent mechanism, but it had no effect on the high-level resistance of C57BL/6 mice. Similarly, administration of a neutrophil-depleting anti-Gr-1 monoclonal antibody (RB6-8C5) decreased the partial resistance of CBA mice and led to severe pathology compared to control antibody-treated mice, but it had no effect on C57BL/6 resistance. These data indicate that there are discrete mechanisms of innate resistance toE. histolyticadepending on the host background and, in contrast to other reports, imply that neutrophils are protective and not damaging in intestinal amebiasis.

Two major genotypic assemblages of Giardia lamblia infect humans; the epidemiologic significance of this phenomenon is poorly understood. We developed a single-vessel multiplex real-time PCR (qPCR) assay that genotypes Giardia infections into assemblages A and/or B directly from fecal samples. The assay utilized Scorpion probes that combined genotype-specific primers and probes for the 18S rRNA gene into the same molecule. The protocol was capable of detecting as few as 20 trophozoites per PCR on fecal DNA isolated using a commercial method or 1.25 trophozoites per PCR on fecal DNA isolated using a G. lamblia -specific oligonucleotide capture technique. The assay was specific for fecal specimens, with no amplification of the discordant genotype with the opposite Scorpion probe. When 97 clinical specimens from Bangladesh were used, the multiplex PCR assay detected 95% (21 of 22) of Giardia microscopy-positive specimens and 18% (13 of 74) of microscopy-negative specimens. Microscopy-negative and qPCR-positive specimens had higher average cycle threshold values than microscopy-positive and qPCR-positive specimens, suggesting that they represented true low-burden infections. Most (32 of 35) infections were assemblage B infections. This single-reaction multiplex qPCR assay distinguishes assemblage A Giardia infections from assemblage B infections directly on fecal samples and may aid epidemiologic investigation.

TO THE EDITOR: Diarrheal diseases have a major effect on global health, particularly the health of malnourished children (1). The enteric parasites Entamoeba histolytica and E. moshkovskii are potential causes of diarrheal disease in children (2). For the past 20 years, we have been studying Entamoeba infections in children from the urban slum of Mirpur in Dhaka, Bangladesh (3).

Transcriptional control of the hgl5 gene of Entamoeba histolytica is mediated through an unusual core promoter composed of TATA, GAAC and Initiator elements. In the hgl5 promoter the GAAC element (AATGAACT) determines the site and rate of transcription initiation. Here we tested the role of the GAAC element in transcription activation from upstream regulatory elements (UREs) in the hgl5 promoter. We also examined the function of the GAAC element in the ferredoxin (fdx) promoter and characterized the protein binding to the GAAC element. Electrophoretic mobility shift assays (EMSA) demonstrated that the GAAC region is necessary for higher-order nuclear protein complex assembly. The function of the GAAC element in transcription activation mediated by UREs revealed that mutation of the GAAC element did not affect transcription activation mediated by the hgl5 URE4 but abrogated activation by the hgl5 URE3. We compared the role of the GAAC elements in the hgl5 and fdx promoters. Competitive gel shift assays were consistent with the same nuclear protein binding to the GAAC elements in both genes. Mutation of the GAAC element in the fdx gene decreased reporter gene expression, however, in contrast to hgl5 gene, had no effect on the site of transcription initiation. These results support a role for the GAAC element in assembly of nuclear proteins at the core promoter and in transcription activation mediated by URE3. The differing effect on transcription initiation in the hgl5 and fdx genes upon mutation of the GAAC element suggests a context-dependence of the GAAC-binding protein in gene expression.

Data from whole genome sequencing of Entamoeba histolytica strains have been used to identify polymorphic loci. Sixteen of these loci were used to genotype 63 E. histolytica strains obtained from an area in Bangladesh where amebiasis is endemic. These markers seemed to segregate autonomously and exhibit extensive haplotype diversity. Despite the genetic heterogeneity, a strong association was observed between two single-nucleotide polymorphisms (SNPs) in the cylicin-2 gene and disease phenotype. This observation suggests that there are genetic differences between virulent and avirulent parasites and that, because of the naturally occurring high background reassortment, multilocus sequence typing in E. histolytica will permit us to link genetic markers with disease outcome in clinical samples.

Ayeh-Kumi, P. F., Ali, I. M., Lockhart, L. A., Gilchrist, C. A., Petri, W. A., Jr., and Hague, R. 2001. Entamoeba histolytica: Genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene Experimental Parasitology 99, 80–88. The varied organ tropisms and clinical presentations of infection by Entamoeba histolytica have stimulated interest in the role of parasite genetic diversity in virulence. We investigated genetic diversity among 54 E. histolytica isolates from Bangladesh by analyzing polymorphism in the serine-rich gene by nested PCR on DNA extracted from stool and liver aspirate pus. We detected both size and restriction site polymorphisms among the isolates within this endemic area. A combination of the nested PCR results and the Alu I digestion of the PCR products examined yielded 25 distinct DNA banding patterns among the 42 stool isolates and an additional 9 distinct patterns among the 12 liver abscess isolates. Approximately half of the isolates had unique polymorphisms. Interestingly, the majority of E. histolytica from the liver had polymorphisms which were not present in intestinal isolates from the same geographic area. These data are consistent with the existence of genetic differences between E. histolytica which cause intestinal and those which cause hepatic disease. We conclude that there is genetic diversity within E. histolytica isolates from an endemic population as reflected in serine-rich E. histolytica protein gene polymorphism. The correlation of genetic differences with the pathogenic potential of E. histolytica strains and the implications of genetic diversity for the immunoprophylaxis of amebiasis require further study.

Killing by Entamoeba histolytica requires parasite adherence to host galactose- and N -acetyl- d -galactosamine (Gal/GalNAc)-containing cell surface receptors. A 260-kDa heterodimeric E. histolytica Gal/GalNAc lectin composed of heavy (Hgl) and light (Lgl) subunits has been previously described. Here we present the cloning and characterization of Igl, a 150-kDa intermediate subunit of the Gal/GalNAc lectin. Igl, Hgl, and Lgl colocalized on the surface membrane of trophozoites. Two unlinked copies of genes encoding Igl shared 81% amino acid sequence identity (GenBank accession no. AF337950 and AF337951 ). They encoded cysteine-rich proteins with amino- and carboxy-terminal hydrophobic signal sequences characteristic of glycosylphosphatidylinositol (GPI)-anchored membrane proteins. The igl genes lacked carbohydrate recognition domains but were members of a large family of amebic genes containing CXXC and CXC motifs. These data indicate that Igl is part of the parasite's multimolecular Gal/GalNAc adhesin required for host interaction.

The hgl5 gene of Entamoeba histolytica is negatively regulated through the upstream regulatory element 3 (URE3) DNA motif TATTCTATT. This motif is also present and significant in the function of the E. histolytica fdx gene promoter. A yeast one-hybrid screen was used to identify an E. histolytica cDNA encoding a protein (URE3-BP) that recognized this DNA motif. Analysis of the predicted amino acid sequence demonstrated the presence of two EF-hand motifs but identified no canonical DNA binding motifs. URE3-BP, expressed in bacteria, demonstrated Ca2+-dependent and sequence-specific recognition of the URE3 DNA sequence as assessed by electrophoretic mobility shift assays. Antibodies raised against URE3-BP blocked the formation of the URE3 DNA-protein complex by native nuclear extracts. The URE3-BP protein was present in theE. histolytica nucleus and cytoplasm with an apparent molecular mass of 22.6 kDa. Our results represent the first use of a yeast genetic screen to identify, on the basis of function, a DNA-binding protein of an early branching eukaryote. Since the URE3 DNA can modulate gene expression in both a positive and negative manner, this protein may have more than one mechanism of interaction with transcriptional machinery. Characterization of URE3-BP should provide insight into transcription regulation and virulence control in this parasite.

The early branching eukaryote Entamoeba histolytica is a human parasite that is the etiologic agent of amebic dysentery and liver abscess. The sequencing of the E. histolytica genome combined with the development of an E. histolytica microarray has resulted in the identification of several distinct gene expression profiles associated with virulence. The function of many modulated transcripts is unknown and their role in pathogenicity is unclear. They however represent a pool of potential virulence factors that could be targets for the development of novel therapeutics. Efficient tools and methods to characterize these novel virulence-associated genes and proteins would be beneficial. Here we report the use of the Gateway((R)) cloning system to generate the E. histolytica expression vector pAH-DEST. To test the usefulness of this system, the vector was used to construct a plasmid containing a recombinant version of the locus EHI_144490, which encoded a protein of unknown function. The recombinant gene was expressed and the recombinant protein, which was strep-myc-tagged, showed a cytoplasmic localization in transfected trophozoites. This expression vector with the Gateway((R)) system should facilitate investigation into the functions of novel proteins in E. histolytica.

The ferredoxin ( fdx ) and lectin ( hgl 5) promoters of Entamoeba histolytica contain the DNA sequence motif TATTCTATT (URE3). Previously we showed that mutation of the URE3 motif in the hgl 5 lectin promoter results in an increase in promoter reporter activity. Mutation of this motif in the fdx promoter led to a 40-to-50% decrease in fdx promoter activity as measured by reporter gene activity and abundance of mRNA. E. histolytica nuclear proteins exhibited sequence-specific binding to the URE3 motif in electrophoretic mobility shift assays. These results support the regulation of both ferredoxin and lectin promoters by a nuclear protein(s) which recognizes the URE3 motif.

Background The outcome of an Entamoeba histolytica infection is variable and can result in either asymptomatic carriage, immediate or latent disease (diarrhea/dysentery/amebic liver abscess). An E. histolytica multilocus genotyping system based on tRNA gene-linked arrays has shown that genetic differences exist among parasites isolated from patients with different symptoms however, the tRNA gene-linked arrays cannot be located in the current assembly of the E. histolytica Reference genome (strain HM-1:IMSS) and are highly variable. Results To probe the population structure of E. histolytica and identify genetic markers associated with clinical outcome we identified in E. histolytica positive samples selected single nucleotide polymorphisms (SNPs) by multiplexed massive parallel sequencing. Profile SNPs were selected which, compared to the reference strain HM-1:IMSS sequence, changed an encoded amino acid at the SNP position, and were present in independent E. histolytica isolates from different geographical origins. The samples used in this study contained DNA isolated from either xenic strains of E. histolytica trophozoites established in culture or E. histolytica positive clinical specimens (stool and amebic liver abscess aspirates). A record of the SNPs present at 16 loci out of the original 21 candidate targets was obtained for 63 of the initial 84 samples (63% of asymptomatically colonized stool samples, 80% of diarrheal stool, 73% of xenic cultures and 84% of amebic liver aspirates). The sequences in all the 63 samples both passed sequence quality control metrics and also had the required greater than 8X sequence coverage for all 16 SNPs in order to confidently identify variants. Conclusions Our work is in agreement with previous findings of extensive diversity among E. histolytica isolates from the same geographic origin. In phylogenetic trees, only four of the 63 samples were able to group in two sets of two with greater than 50% confidence. Two SNPs in the cylicin-2 gene (EHI_080100/XM_001914351) were associated with disease (asymptomatic/diarrhea p = 0.0162 or dysentery/amebic liver abscess p = 0.0003). This study demonstrated that there are genetic differences between virulent and avirulent E. histolytica strains and that this approach has the potential to define genetic changes that influence infection outcomes.

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Malnutrition substantially increases susceptibility to Entamoeba histolytica in children. Leptin is a hormone produced by adipocytes that inhibits food intake, influences the immune system, and is suppressed in malnourished children. Therefore we hypothesized that diminished leptin function may increase susceptibility to E. histolytica infection. We prospectively observed a cohort of children, beginning at preschool age, for infection by the parasite E. histolytica every other day over 9 years and evaluated them for genetic variants in leptin (LEP) and the leptin receptor (LEPR). We found increased susceptibility to intestinal infection by this parasite associated with an amino acid substitution in the cytokine receptor homology domain 1 of LEPR. Children carrying the allele for arginine (223R) were nearly 4 times more likely to have an infection compared with those homozygous for the ancestral glutamine allele (223Q). An association of this allele with amebic liver abscess was also determined in an independent cohort of adult patients. In addition, mice carrying at least 1 copy of the R allele of Lepr were more susceptible to infection and exhibited greater levels of mucosal destruction and intestinal epithelial apoptosis after amebic infection. These findings suggest that leptin signaling is important in mucosal defense against amebiasis and that polymorphisms in the leptin receptor explain differences in susceptibility of children in the Bangladesh cohort to amebiasis.

The production of substance P (SP) and the mRNA encoding its precursor preprotachykinin (PPT) is regulated by nerve growth factor (NGF) in dorsal root ganglion neurons (1). We have shown previously that two regions of the bovine PPT promoter are capable of mediating the induction by NGF of the downstream structural gene in transfected PC12 cells (2). Both regions contained a sequence element, similar to a known transcription factor binding site, which is present in several other NGF-regulated genes. We show here that PC12 cells contain a single-stranded DNA binding protein (SSBP-PC12) which can interact specifically with this site. Binding activity was increased by NGF treatment of PC12 cells.

The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) is a transcription factor that binds DNA in a Ca 2+ -inhibitable manner. The protein is located in both the nucleus and the cytoplasm but has also been found to be enriched in the plasma membrane of amebic trophozoites. We investigated the reason for the unusual localization of URE3-BP at the amebic plasma membrane. Here we identify and characterize a 22-kDa Ca 2+ -dependent binding partner of URE3-BP, EhC2A, a novel member of the C2-domain superfamily. Immunoprecipitations of URE3-BP and EhC2A showed that the proteins interact and that such interaction was enhanced in the presence of Ca 2+ . Recombinant and native EhC2A bound phospholipid liposomes in a Ca 2+ -dependent manner, with half-maximal binding occurring at 3.4 μM free Ca 2+ . A direct interaction between EhC2A and URE3-BP was demonstrated by the ability of recombinant EhC2A to recruit recombinant URE3-BP to phospholipid liposomes in a Ca 2+ -dependent manner. URE3-BP and EhC2A were observed to translocate to the amebic plasma membrane upon an increase in the intracellular Ca 2+ concentration of trophozoites, as revealed by subcellular fractionation and immunofluorescent staining. Short hairpin RNA-mediated knockdown of EhC2A protein expression significantly modulated the mRNA levels of URE3-BP-regulated transcripts. Based on these results, we propose a model for EhC2A-mediated regulation of the transcriptional activities of URE3-BP via Ca 2+ -dependent anchoring of the transcription factor to the amebic plasma membrane.

The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

A genome-wide transcriptional analysis of Entamoeba histolytica was performed on trophozoites isolated from the colon of six infected mice and from in vitro culture. An Affymetrix platform gene expression array was designed for this analysis that included probe sets for 9435 open reading frames (ORFs) and 9066 5 " and 3 " flanking regions. Transcripts were detected for >80% of all ORFs. A total of 523 transcripts (5.2% of all E. histolytica genes) were significantly changed in amebae isolated from the intestine on Days 1 and 29 after infection: 326 and 109 solely on Days 1 and 29, and 88 on both days. Quantitative real-time reverse transcriptase PCR confirmed these changes in 11/12 genes tested using mRNA isolated from an additional six mice. Adaptation to the intestinal environment was accompanied by increases in a subset of cell signaling genes including transmembrane kinases, ras and rho family GTPases, and calcium binding proteins. Significant decreases in mRNA abundance for genes involved in glycolysis and concomitant increases in lipases were consistent with a change in energy metabolism. Defense against bacteria present in the intestine (but lacking from in vitro culture) was suggested by alterations in mRNA levels of genes similar to the AIG1 plant antibacterial proteins. Decreases in oxygen detoxification pathways were observed as expected in the anaerobic colonic lumen. Of the known virulence factors the most remarkable changes were a 20‐35-fold increase in a cysteine proteinase four-like gene, and a 2‐3-fold decrease in two members of the Gal/GalNAc lectin light subunit family. Control of the observed changes in mRNA abundance in the intestine might potentially rest with four relatedproteinswithDNAbindingdomainsthatweredown-regulated6‐16-foldintheintestinalenvironment.Inconclusion,thefirstgenome-wide analysis of the transcriptome of E. histolytica demonstrated that the vast majority of genes are transcribed in trophozoites, and that in the host intestine trophozoites altered the expression of mRNAs for genes implicated in metabolism, oxygen defense, cell signaling, virulence, antibacterial activity, and DNA binding. © 2006 Elsevier B.V. All rights reserved.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.