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Abstract 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.

Objectives: Clostridioides difficile infection (CDI) is characterized by neutrophilia in blood, with a high leukocyte count accompanying severe infection. In this study, we characterized peripheral blood neutrophil activation and maturity in CDI by (i) developing a method to phenotype stored neutrophils for disease-related developmental alterations and (ii) assessing neutrophil-associated biomarkers., Methods: We stored fixed leukocytes from blood collected within 24 h of diagnosis from a cohort of hospitalized patients with acute CDI. Additional study cohorts included recurrent CDI patients at time of and two months after FMT therapy and a control healthy cohort. We assessed levels of neutrophil surface markers CD66b, CD11b, CD16 and CD10 by flow cytometry. Plasma neutrophil elastase and lipocalin-2 were measured using ELISA, while G-CSF, GM-CSF and cytokines were measured using O-link Proteomic technology., Results: CD66b + neutrophil abundance assessed by flow cytometry correlated well with complete blood counts, establishing that neutrophils in stored blood are sufficiently well-preserved for phenotyping by flow cytometry. Neutrophil abundance was significantly increased in CDI patients compared to healthy controls. Emergency granulopoiesis in acute CDI patients was evidenced by lower neutrophil surface expression of CD10, CD11b and CD16. CD10 + staining of neutrophils started to recover within 3-7 days of CDI treatment. Neutrophil activation and degranulation were higher in acute CDI as assessed by plasma neutrophil elastase and lipocalin-2. Biomarker levels in immunocompetent subjects were associated with recurrence and fatal outcomes., Conclusions: Neutrophil activation and emergency granulopoiesis characterize the early immune response in acute CDI, with plasma degranulation biomarkers predictive of disease severity., Competing Interests: Declaration of competing interest WAP has a conflict of interest in that he is a consultant for TechLab, Inc, which makes diagnostic tests for C. difficile infection. The other authors have no conflicts of interest to disclose., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Whānau Pakari is a healthy lifestyle assessment and intervention programme for children and adolescents with obesity in Taranaki (Aotearoa/New Zealand), which, in this region, replaced the nationally funded Green Prescription Active Families (GRxAF) programme. We compared national referral rates from the GRxAF programme (age 5-15 years) and the B4 School Check (B4SC, a national preschool health and development assessment) with referral rates in Taranaki from Whānau Pakari. We retrospectively analysed 5 years of clinical data (2010-2015), comparing referral rates before, during, and after the Whānau Pakari clinical trial, which was embedded within the programme. We also surveyed programme referrers and stakeholders about their experiences of Whānau Pakari, analysing their responses using a multiple-methods framework. After the Whānau Pakari trial commenced, Taranaki GRxAF referral rates increased markedly (2.3 pretrial to 7.2 per 1000 person-years), while NZ rates were largely unchanged (1.8-1.9 per 1000 person-years) ( p < 0.0001 for differences during the trial). Post-trial, Taranaki GRxAF referral rates remained higher irrespective of ethnicity, being 1.8 to 3.2 times the national rates ( p < 0.001). Taranaki B4SC referrals for obesity were nearly complete at 99% in the last trial year and 100% post-trial, compared with national rates threefold lower (31% and 32%, respectively; p < 0.0001), with Taranaki referral rates for extreme obesity sustained at 80% and exceeding national rates for both periods (58% and 62%, respectively; p < 0.01). Notably, a referral was 50% more likely for referrers who attended a Whānau Pakari training half-day (RR = 1.51; p = 0.009). Stakeholders credited the success of Whānau Pakari to its multidisciplinary team, family-centred approach, and home-based assessments. However, they highlighted challenges such as navigating multidisciplinary collaboration, engaging with families with complex needs, and shifting conventional healthcare practices. Given its favourable referral trends and stakeholder endorsement, Whānau Pakari appears to be a viable contemporary model for an accessible and culturally appropriate intervention on a national and potentially international scale.

Cryptosporidium species are a major cause of diarrhea and associated with growth failure. There is currently only limited knowledge of the parasite's genomic variability. We report a genomic analysis of Cryptosporidium parvum isolated from Bangladeshi infants and reanalysis of sequences from the United Kingdom. Human isolates from both locations shared 154 variants not present in the cattle-derived reference genome, suggesting host-specific adaptation of the parasite. Remarkably 34.6% of single-nucleotide polymorphisms unique to human isolates were nonsynonymous and 8.2% of these were in secreted proteins. Linkage disequilibrium decay indicated frequent recombination. The genetic diversity of C. parvum has potential implications for vaccine and therapeutic design. Clinical Trials Registration. NCT02764918., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

There is no vaccine to protect from cryptosporidiosis, a leading cause of diarrhea in infants in low- and middle-income countries. Here, we comprehensively identified parasite antigens associated with protection from reinfection. A Cryptosporidium protein microarray was constructed by in vitro transcription and translation of 1,761 C. parvum, C. hominis, or C. meleagridis antigens, including proteins with a signal peptide and/or a transmembrane domain. Plasma IgG and/or IgA from Bangladeshi children longitudinally followed for cryptosporidiosis from birth to 3 years of age allowed for identification of 233 seroreactive proteins. Seven of these were associated with protection from reinfection. These included Cp23, Cp17, Gp900, and 4 additional antigens - CpSMP1, CpMuc8, CpCorA and CpCCDC1. Infection in the first year of life, however, often resulted in no detectable antigen-specific antibody response, and antibody responses, when detected, were specific to the infecting parasite genotype and decayed in the months after infection. In conclusion, humoral immune responses against specific parasite antigens were associated with acquired immunity. While antibody decay over time and parasite genotype-specificity may limit natural immunity, this work serves as a foundation for antigen selection for vaccine design.

Aim: Following trials of inhaled antibiotics in adults, this study investigates the efficacy of nebulised gentamicin to improve respiratory function in children with bronchiectasis., Methods: This is a randomised, double-blind, placebo-controlled, crossover trial of 12-week nebulised placebo/gentamicin, 6-week washout, 12-week gentamicin/placebo. Participants were children (5-15 years) with bronchiectasis, chronic infection (any pathogen), and able to perform spirometry from a hospital bronchiectasis clinic. Primary outcomes were change in forced expiratory volume in 1 s (FEV 1 ) and hospitalisation days. Secondary outcomes included sputum bacterial density, sputum inflammatory markers, additional antibiotics and symptom severity. Analyses were on an intention-to-treat basis., Results: Fifteen children (mean 11.7-years-old) completed the study. There was no significant change in mean FEV 1 (56%/55%, P = 0.38) or annual rate of hospital admissions (1.1/0, P = 0.12) between gentamicin and placebo, respectively. However, Haemophilus influenzae sputum growth (27% vs. 80%, P = 0.002) and bacterial density (2.4 log 10 cfu/mL lower P < 0.001) improved with gentamicin. Sputum inflammatory markers interleukin-1β (P < 0.001), interleukin-8 (P < 0.001) and tumour necrosis factor-α (P = 0.003) were lower with gentamicin. Poor recruitment limited study power and treatment adherence was challenging for this cohort., Conclusions: In this crossover study of nebulised gentamicin in children with bronchiectasis, there was a reduction in sputum bacterial density and inflammation. However, there were no major improvements in clinical outcomes and adherence was a challenge., (© 2022 The Authors. Journal of Paediatrics and Child Health published by John Wiley & Sons Australia, Ltd on behalf of Paediatrics and Child Health Division (The Royal Australasian College of Physicians).)

Aim: Breastfeeding is a fundamental aspect of tikanga Māori (Māori cultural traditions/practices) requiring protection and promotion. This study identifies determinants of exclusive breastfeeding in wāhine Māori., Methods: Wāhine Māori enrolled in the Growing Up in New Zealand child cohort study participated (n=1060). Exclusive breastfeeding duration was self-reported. Hierarchical regression analyses were framed by a model of Māori health and wellbeing., Results: Most wāhine Māori initiated breastfeeding (96%), with 12% exclusively breastfeeding for six or more months. Wāhine Māori had increased odds of exclusively breastfeeding for six or more months if they: thought it best to breastfeed for >6 months (adjusted odds ratio (aOR)=1.94, 95% confidence interval (CI)=1.05-3.78); thought returning to work would not (aOR=2.17, 95% CI=1.17-4.24) or may (aOR=4.25, 95% CI=1.86-9.85) limit breastfeeding; were experienced mothers (aOR=2.55, 95% CI=1.35-5.06); or were undecided about vaccination (aOR=3.16, 95% CI=1.55-6.39). Exclusive breastfeeding for six or more months was less likely if mothers experienced depression during pregnancy (aOR=0.47, 95% CI=0.20-0.99) or viewed cultural traditions/practices as "fairly important" (aOR=0.53, 95% CI=0.27-0.99), compared to "very important"., Conclusion: Determinants of exclusive breastfeeding in wāhine Māori are knowledge of breastfeeding recommendations, return to work, motherhood experience, connection to Te Ao Māori (Māori worldview) and tikanga Māori, antenatal depression and vaccine indecision. Interventions delivered within a Kaupapa Māori framework will best address breastfeeding inequities in Aotearoa New Zealand., Competing Interests: Nil

Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess in humans. The parasitic lifestyle and the virulence of the protist require elaborate biological processes, including vesicular traffic and stress management against a variety of reactive oxygen and nitrogen species produced by the host immune response. Although the mechanisms for intracellular traffic of representative virulence factors have been investigated at molecular levels, it remains poorly understood whether and how intracellular traffic is involved in the defense against reactive oxygen and nitrogen species. Here, we demonstrate that EhArfX2, one of the Arf family of GTPases known to be involved in the regulation of vesicular traffic, was identified by comparative transcriptomic analysis of two isogenic strains: an animal-passaged highly virulent HM-1:IMSS Cl6 and in vitro maintained attenuated avirulent strain. EhArfX2 was identified as one of the most highly upregulated genes in the highly virulent strain. EhArfX2 was localized to small vesicle-like structures and largely colocalized with the marker for the trans-Golgi network SNARE, EhYkt6, but neither with the endoplasmic reticulum (ER)-resident chaperon, EhBip, nor the cis-Golgi SNARE, EhSed5, and Golgi-luminal galactosyl transferase, EhGalT. Expression of the dominant-active mutant form of EhArfX2 caused an increase in the number of lysosomes, while expression of the dominant-negative mutant led to a defect in lysosome formation and cysteine protease transport to lysosomes. Expression of the dominant-negative mutant in the virulent E. histolytica strain caused a reduction of the size of liver abscesses in a hamster model. This defect in liver abscess formation was likely at least partially attributed to reduced resistance to nitrosative, but not oxidative stress in vitro . These results showed that the EhArfX2-mediated traffic is necessary for the nitrosative stress response and virulence in the host., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Saito-Nakano, Makiuchi, Tochikura, Gilchrist, Petri and Nozaki.)

The information fathers receive about infant vaccination may influence their decision to vaccinate. We describe fathers' sources of vaccination information and paternal determinants of timely infant vaccinations. Participants were from a child cohort study in New Zealand. The child cohort was established by enrolling pregnant women and their partners. During pregnancy, fathers (n = 4017) of the cohort children born 2009-2010 described information sources that encouraged or discouraged infant vaccination. The National Immunization Register provided infant vaccination data. Independent associations of the vaccination information received by fathers with the timeliness of their infant's vaccination were determined using multivariable logistic regression. Associations were described using adjusted odds ratios and 95% confidence intervals. One-third of fathers (1430/4017 [36%]) recalled receiving vaccination information, 64% of which encouraged vaccination. Most infants (2900/4017 [72%]) received all their vaccinations on time, however only 58% of Māori infants were vaccinated on time. Paternal determinants of vaccination timeliness were the father receiving discouraging or conflicting information about vaccination, father's ethnicity, father's vaccination hesitancy, and whether the mother received vaccination information. To improve vaccination uptake and timeliness, a vaccination conversation with mothers, fathers and whānau could be included in routine antenatal care, informing and supporting decision-making, and addressing concerns. Vaccination education should address present and historic distrust of the health system. Framing vaccination within a Māori model of health and including fathers and whānau in decision-making will address vaccination inequities in New Zealand.

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., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

Aim: The incidence of childhood empyema has been increasing in some developed countries despite the introduction of pneumococcal vaccination. This study aimed to document the incidence, bacterial pathogens, and morbidity/mortality of parapneumonic effusion/empyema in New Zealand., Methods: A prospective study of 102 children <15 years of age requiring hospitalization with parapneumonic effusion/empyema between May 1, 2014 and May 31, 2016 notified via the New Zealand Paediatric Surveillance Unit. Parapneumonic effusion/empyema was defined as pneumonia and pleural effusion persisting ≥7 days, and/or any pneumonia, and pleural effusion necessitating drainage. Notifying pediatricians completed standardized questionnaires., Results: Annual pediatric parapneumonic effusion/empyema incidence was 5.6/100,000 (95% confidence interval [CI]: 4.7-6.9). Most children (80%) required surgical intervention and 31% required intensive care. A causative organism was identified in 71/102 (70%) cases. Although Staphylococcus aureus (25%) and Streptococcus pneumoniae (25%) infection rates were equal, prolonged hospitalization and intensive care admission were more common in children with S. aureus PPE/E. Māori and Pasifika children were over-represented at 2.2 and 3.5 times, their representation in the New Zealand pediatric population. Pneumococcal vaccination was incomplete, with only 61% fully immunized and 30% unimmunized. Haemophilus influenzae type b vaccine uptake was near complete at 89/94 (95%), with influenza immunization only 3/78 (4%)., Conclusions: New Zealand has a high incidence of pediatric complicated parapneumonic effusion/empyema with significant morbidity. S. aureus was a significant cause of severe empyema in New Zealand, particularly for Māori and Pasifika children. Improvements in vaccine coverage are needed along with strategies to reduce S. aureus disease morbidity., (© 2021 Wiley Periodicals LLC.)

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<0.0001) and were more likely to be sub-clinical (Chi square test for trend; p = 0.01). Repeat infections were associated with the development of growth faltering (Pearson correlation = -0.18; p = 0.0004). High levels of fecal IgA antibodies against the Cryptosporidium Cp23 sporozoite protein at one year of life were associated with a delay in reinfection and amelioration of growth faltering through three years of life (HAZ IgA high responders -1.323 ± 0.932 versus HAZ -1.731 ± 0.984 p = 0.0001). We concluded that nonsterile immunity to cryptosporidiosis in young children was associated with high levels of mucosal IgA anti-Cp23 and protection from diarrhea and growth faltering. Trial Registration: NCT02764918., Competing Interests: The authors have declared that no competing interests exist.

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 .

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

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., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Aim: To determine if the routine use of automatically calculated birthweight centiles prior to discharge from the delivery unit is associated with improved adherence to the neonatal hypoglycaemia guideline., Methods: We conducted retrospective audits of adherence to the neonatal hypoglycaemia guideline in a tertiary maternity hospital in Auckland, New Zealand in a randomly selected cohort of newborn infants at risk of neonatal hypoglycaemia before (2011) and after (2015) the introduction of routine use of calculated birthweight centiles for all infants. The primary outcome was adherence to the guideline, defined as (i) blood glucose concentration screening in the first 48 h after birth; (ii) the initial measurement taken 1-2 h after birth; and (iii) at least three consecutive blood glucose concentrations ≥2.6 mmol/L, over 12 h, prior to cessation of screening., Results: The audits examined the records of 400 infants (200 each in 2011 and 2015) to determine guideline adherence. Adherence improved from 2011 to 2015 (59/200 (30%) vs. 95/200 (48%), P < 0.001), with the largest improvement in large-for-gestational age infants (7/50 (14%) vs. 25/50 (50%), P = <0.001). Screened infants whose care was adherent to the guideline had a higher incidence of hypoglycaemia detection (adherent, 64/154 (42%) vs. non-adherent, 34/246 (14%), P < 0.001)., Conclusions: The routine use of calculated birthweight centiles was associated with improved adherence to the neonatal hypoglycaemia guideline and increased detection of neonatal hypoglycaemia in at-risk infants. Thus, identifying practices that improve guideline adherence may improve the detection of hypoglycaemia in asymptomatic at-risk infants., (© 2019 Paediatrics and Child Health Division (The Royal Australasian College of Physicians).)

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 Objective: Serological 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., Methods: Serum samples were collected from three different cohorts: stunted (length-for-age Z-scores (LAZ) <-2) and at risk of stunting children (LAZ <-1 to -2) and malnourished adults (body mass index <18.5 kg/m 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., Results: A 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., Conclusion: The 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., Competing Interests: Competing interests: None declared.

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 <2 years were tested by Giardia/Cryptosporidium QUIK CHEK, enzyme-linked immunosorbent assay (ELISA), and quantitative polymerase chain reaction (qPCR) to compare the performance of the individual techniques. We also tested for the presence of other diarrheal pathogens in qPCR-positive samples with a TaqMan Array Card (TAC) to assess whether Cryptosporidium was the sole causative agent for the diarrheal episodes., Results: Of 832 samples, 4.4% were found positive for Cryptosporidium by QUIK CHEK, 3.6% by ELISA, and 8.8% by qPCR. Using TAC-attributed Cryptosporidium diarrhea as the gold standard, the sensitivities of QUIK CHEK, ELISA, and qPCR were 92.3%, 71.8%, and 100%, respectively; the specificities were 97.1%, 94.3%, and 0%, respectively. Analysis of the qPCR-positive and QUIK CHEK-negative samples by TAC identified other enteropathogens as more likely than Cryptosporidium to be the causative agents of diarrhea., Conclusions: QUIK CHEK was more sensitive and specific than ELISA. While qPCR detected Cryptosporidium in more samples than QUIK CHEK, most of these were instances of qPCR detecting small quantities of Cryptosporidium DNA in a diarrheal episode caused by another enteropathogen. We concluded that QUIK CHEK was comparable in sensitivity and superior in specificity to qPCR for the diagnosis of Cryptosporidium diarrhea.

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.

We studied the genetic diversity of Cryptosporidium hominis infections in slum-dwelling infants from Dhaka over a 2-year period. Cryptosporidium hominis infections were common during the monsoon, and were genetically diverse as measured by gp60 genotyping and whole-genome resequencing. Recombination in the parasite was evidenced by the decay of linkage disequilibrium in the genome over <300 bp. Regions of the genome with high levels of polymorphism were also identified. Yet to be determined is if genomic diversity is responsible in part for the high rate of reinfection, seasonality, and varied clinical presentations of cryptosporidiosis in this population.

Background: Infectious diseases are the leading cause of hospital admissions in young children. Hospitalisation with an infectious disease is a recurrent event for some children. Our objective was to describe risk factors for infectious disease readmission following hospital admission with an infectious disease in the first two years of life., Methods: We performed a national cohort study of New Zealand children, born 2005-2009, with an infectious disease admission before age 24 months. Children readmitted with an infectious disease within 12 months of the first infectious disease admission were identified. Every infectious disease admission was categorised as a respiratory, enteric, skin and soft tissue, urinary or other infection. Independent associations of demographic and child health factors with infectious disease readmission were determined using multiple variable logistic regression., Results: From 2005 to 2011, there were 69,902 infectious disease admissions for 46,657 children less than two years old. Of these 46,657 children, 10,205 (22%) had at least one infectious disease readmission within 12 months of their first admission. The first infectious disease admission was respiratory (54%), enteric (15%), skin or soft tissue (7%), urinary (4%) or other (20%). Risk of infectious disease readmission was increased if the first infectious disease admission was respiratory (OR = 1.87, 95% CI 1.78-1.95) but not if it was in any other infectious disease category. Risk factors for respiratory infectious disease readmission were male gender, Pacific or Māori ethnicity, greater household deprivation, presence of a complex chronic condition, or a first respiratory infectious disease admission during autumn or of ≥3 days duration. Fewer factors (younger age, male gender, presence of a complex chronic condition) were associated with enteric infection readmission. The presence of a complex chronic condition was the only factor associated with urinary tract infection readmission and none of the factors were associated with skin or soft tissue infection readmission., Conclusions: In children less than two years old, infectious disease readmission risk is increased if the first infectious disease admission is a respiratory infectious disease but not if it is another infectious disease category. Risk factors for respiratory infectious disease readmission are different from those for other infectious disease readmissions.

Objectives: Pregnant women routinely receive information in support of or opposing infant immunization. We aimed to describe immunization information sources of future mothers' and determine if receiving immunization information is associated with infant immunization timeliness., Methods: We analyzed data from a child cohort born 2009-2010 in New Zealand. Pregnant women ( N = 6822) at a median gestation of 39 weeks described sources of information encouraging or discouraging infant immunization. Immunizations received by cohort infants were determined through linkage with the National Immunization Register ( n = 6682 of 6853 [98%]). Independent associations of immunization information received with immunization timeliness were described by using adjusted odds ratios (ORs) and 95% confidence intervals (CIs)., Results: Immunization information sources were described by 6182 of 6822 (91%) women. Of these, 2416 (39%) received information encouraging immunization, 846 (14%) received discouraging information, and 565 (9%) received both encouraging and discouraging information. Compared with infants of women who received no immunization information (71% immunized on-time), infants of women who received discouraging information only (57% immunized on time, OR = 0.49, 95% CI 0.38-0.64) or encouraging and discouraging information (61% immunized on time, OR = 0.51, 95% CI 0.42-0.63) were at decreased odds of receiving all immunizations on time. Receipt of encouraging information only was not associated with infant immunization timeliness (73% immunized on time, OR = 1.00, 95% CI 0.87-1.15)., Conclusions: Receipt, during pregnancy, of information against immunization was associated with delayed infant immunization regardless of receipt of information supporting immunization. In contrast, receipt of encouraging information is not associated with infant immunization timeliness., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2017 by the American Academy of Pediatrics.)

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.

Parasitic protozoan infections represent a major health burden in the developing world and contribute significantly to morbidity and mortality. These infections are often associated with considerable variability in clinical presentation. An emerging body of work suggests that the intestinal microbiota may help to explain some of these differences in disease expression. The objective of this minireview is to synthesize recent progress in this rapidly advancing field. Studies of humans and animals and in vitro studies of the contribution of the intestinal microbiota to infectious disease are discussed. We hope to provide an understanding of the human-protozoal pathogen-microbiome interaction and to speculate on how that might be leveraged for treatment., (Copyright © 2017 Burgess et al.)

Background: Uncontrolled hemorrhage is the leading cause of potentially preventable traumatic death. Bleeding victims must receive immediate medical attention to save lives, and the first opportunity to control bleeding after trauma often comes from bystanders. Educating the general public is important for improving outcomes for hemorrhaging victims, and it is imperative for all people, including those with no clinical training, to have the knowledge to respond until trained medical specialists arrive., Study Design: An 8-minute educational module was deployed to all hospital employees and included information on the location and contents of hemorrhage control bags in the hospital and how to use the materials in the bags to respond to uncontrolled hemorrhage. A pre-post questionnaire was administered with the module to evaluate effectiveness. McNemar tests were used to compare the responses and evaluate effectiveness of the education., Results: Eighty-four percent of eligible employees (n = 4,845) completed the module and all items on the questionnaires. Three-quarters of respondents provided direct or ancillary care to patients, and one-quarter worked in nonclinical roles. On average, 57% of questions were answered correctly in the pre-questionnaire and 98% were answered correctly in the post-questionnaire. The module was effective for all employees regardless of clinical training., Conclusions: There is currently no succinct hemorrhage control education available that can be deployed across a large workplace environment. Results demonstrate that the brief learning module was effective in educating all employees in the basics of hemorrhage control. The module could be deployed in clinical and nonclinical settings., (Copyright © 2017 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: Pertussis immunization programs aim to prevent severe infant disease. We investigated temporal trends in infant pertussis deaths and pediatric intensive care unit (PICU) admissions and associations of changes in disease detection and vaccines used with death and PICU admission rates., Methods: Using national data from New Zealand (NZ), we described infant pertussis deaths and PICU admissions from 1991 to 2013, over which time national immunization coverage at 2 years of age increased from <80% to 92%. In NZ, pertussis became a notifiable disease with polymerase chain reaction (PCR) diagnosis available in 1997 and acellular replaced whole-cell vaccine in 2000. We used Poisson regression to model temporal trends and compared rates in time intervals using rate ratios (RRs) with 95% confidence intervals (CIs)., Results: There were 10 pertussis deaths and 159 infant PICU admissions with pertussis from 1991 to 2013. The annual number of infant pertussis PICU admissions increased from 1991 to 2013 (P = 0.02) but the number of pertussis deaths did not (P = 0.09). The risk of PICU admission during infancy with pertussis was increased in the notification/PCR versus the non-notification/PCR era (RR: 1.12; 95% CI: 1.02-1.19) and when acellular replaced whole-cell vaccine (RR: 1.19; 95% CI: 1.06-1.31). Median Pediatric Index of Mortality scores during 2001-2013 were lower than during 1991-1999 (P < 0.001)., Conclusions: Infant PICU pertussis admission rates have increased in NZ despite improvements in immunization coverage. Higher rates have occurred since pertussis notification/PCR became available and since acellular replaced whole-cell vaccine. The severity of disease in infants admitted to PICU with pertussis has decreased in recent years.

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 ≥0.1IU/mL., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

Background: An estimated 1 million children die each year before their fifth birthday from diarrhea. Previous population-based surveys of pediatric diarrheal diseases have identified the protozoan parasite Entamoeba histolytica, the etiological agent of amebiasis, as one of the causes of moderate-to-severe diarrhea in sub-Saharan Africa and South Asia., Methods: We prospectively studied the natural history of E. histolytica colonization and diarrhea among infants in an urban slum of Dhaka, Bangladesh., Results: Approximately 80% of children were infected with E. histolytica by the age of 2 years. Fecal anti-galactose/N-acetylgalactosamine lectin immunoglobulin A was associated with protection from reinfection, while a high parasite burden and expansion of the Prevotella copri level was associated with diarrhea., Conclusions: E. histolytica infection was prevalent in this population, with most infections asymptomatic and diarrhea associated with both the amount of parasite and the composition of the microbiota., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America.)

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.

Background: Most women decide about infant immunisation during pregnancy. However, we have limited knowledge of the immunisation intentions of their partners. We aimed to describe what pregnant women and their partners intended for their future child's immunisations, and to identify associations between parental intentions and the subsequent timeliness of infant immunisation., Methods: We recruited a cohort of pregnant New Zealand (NZ) women expecting to deliver between April 2009 and March 2010. The cohort included 11% of births in NZ during the recruitment period and was generalisable to the national birth cohort. We completed antenatal interviews independently with mothers and partners. We determined immunisation receipt from the National Immunisation Register and defined timely immunisation as receiving all vaccines (scheduled at 6-weeks, 3- and 5-months) within 30 days of their due date. We described independent associations of immunisation intentions with timeliness using adjusted odds ratios (OR) and 95% confidence intervals (CI)., Results: Of 6172 women, 5014 (81%) intended full immunisation, 245 (4%) partial immunisation, 140 (2%) no immunisation and 773 (13%) were undecided. Of 4152 partners, 2942 (71%) intended full immunisation, 208 (5%) partial immunisation, 83 (2%) no immunisation and 921 (22%) were undecided. Agreement between mothers and partners was moderate (Kappa=0.42). Timely immunisation occurred in 70% of infants. Independent of their partner's intentions, infants of pregnant women who decided upon full immunisation were more likely to be immunised on time (OR=7.65, 95% CI: 4.87-12.18). Independent of the future mother's intentions, infants of partners who had decided upon full immunisations were more likely to be immunised on time (OR=3.33, 95% CI: 2.29-4.84)., Conclusions: During pregnancy, most future parents intend to fully immunise their child; however, more partners than mothers remain undecided about immunisation. Both future mothers' and future fathers' intentions are independently associated with the timeliness of their infant's immunisations., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: In children, community-acquired pneumonia is a frequent cause of emergency department (ED) presentation and hospital admission. Quality primary care may prevent some of these hospital visits., Aims: The aim of this study was to identify primary care factors associated with ED presentation and hospital admission of preschool-aged children with community-acquired pneumonia., Methods: A case-control study was conducted by enrolling three groups: children presenting to the ED with pneumonia and admitted (n = 326), or discharged home (n = 179), and well-neighbourhood controls (n = 351). Interviews with parents and primary care staff were conducted and health record review was performed. The association of primary care factors with ED presentation and hospital admission, controlling for available confounding factors, was determined using logistic regression., Results: Children were more likely to present to the ED with pneumonia if they did not have a usual general practitioner (GP) (odds ratio (OR) = 2.50, 95% confidence interval (CI) = 1.67-3.70), their GP worked ⩽ 20 h/week (OR = 1.86, 95% CI = 1.10-3.13) or their GP practice lacked an immunisation recall system (OR = 5.44, 95% CI = 2.26-13.09). Lower parent ratings for continuity (OR=1.63, 95% CI = 1.01-2.62), communication (OR = 2.01, 95% CI = 1.29-3.14) and overall satisfaction (OR = 2.16, 95% CI = 1.34-3.47) increased the likelihood of ED presentation. Children were more likely to be admitted when antibiotics were prescribed in primary care (OR = 2.50, 95% CI = 1.43-4.55). Hospital admission was less likely if children did not have a usual GP (OR = 0.22, 95% CI = 0.11-0.40) or self-referred to the ED (OR = 0.48, 95% CI = 0.26-0.89)., Conclusions: Accessible and continuous primary care is associated with a decreased likelihood of preschool-aged children with pneumonia presenting to the ED and an increased likelihood of hospital admission, implying more appropriate referral. Lower parental satisfaction is associated with an increased likelihood of ED presentation.

Aim: To describe iron status at birth in a population sample of children., Method: Cord blood samples were obtained at birth from 131 infants enrolled in the cohort study Growing Up in New Zealand. Cord blood serum ferritin (SF) and haemoglobin (Hb) concentrations were measured and associations of SF and Hb with maternal and birth characteristics were determined., Results: Demographics were comparable to the larger cohort, except for having a higher pre-pregnancy body mass index (26.9 vs. 25.4 kg/m2, P=0.005), lower frequency of cigarette smoking during pregnancy (2% vs. 11%, P=0.0004), and smaller proportion with birth-weight <2500 g (0% vs. 5%, P=0.03). Median (interquartile range) SF was 135 (88-180) mcg/L and mean (plus or minus SD) Hb was 160 plus or minus 17 g/L. Eight newborns (7%) had cord SF levels indicative of iron deficiency (SF <35 mcg/L), two newborns were anaemic (Hb <130 g/L) and none had iron deficiency anaemia. Median SF was lower in newborns whose mothers consumed greater than or equal to 3 servings of milk/day during the pregnancy (131 vs. 151 mcg/L, P=0.04). No other associations with SF or Hb were observed., Conclusion: Iron deficiency is present in 7% of newborns in New Zealand. Newborns whose mothers consumed more milk during pregnancy had a lower median SF concentration.

Molecular tools have the potential to differentiate microscopically similar gut micro-eukaryotes that may have significantly different relationships with the human host. Using broad range Entamoeba primers to amplify a section of the eukaryotic 18S small subunit ribosomal RNA gene a novel member of the Entamoeba family (Entamoeba bangladeshi) has recently been identified. The goal of this review is to place this species in the context of what is already known about this genus and to discuss the tools and data needed to elucidate the host-microbe relationship.

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., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Entamoeba histolytica possesses a family of approximately 100 putative transmembrane kinases (TMKs), indicating that the parasite has an extensive means of environmental sensing. The TMKs have been divided into nine sub-groups based on the sequence composition of their intracellular kinase as well as extracellular cysteine-rich domains. EhTMKB1-9 has been recently shown to be expressed in proliferating trophozoites and induced by serum. Interference with EhTMKB1-9 by antisense RNA knockdown or expression of a truncated protein diminished proliferation, adhesion and cytotoxicity. Here we report the involvement of EhTMKB1-9 in phagocytosis and its virulence function in the formation of amebic colitis. Trophozoites induced to express higher levels of wild type EhTMKB1-9 showed increased capacity for endocytosis. In contrast, cells compromised for the EhTMKB1-9 expression through antisense inhibition showed significantly lower levels of phagocytosis and endocytosis under the experimental conditions. The role of EhTMKB1-9 as a virulence factor was studied using animal models of amebiasis. Trophozoites expressing high levels of mutant protein lacking the kinase domain showed a competitive disadvantage with regard to survival as well as invasive phenotype in the murine model of amebic colitis. The same parasites however, were not compromised in their ability to generate abscess in the gerbil model of invasive liver amebiasis. EhTMKB1-9 is the second member from the "B" group of EhTMKs which seems to be deployed by the parasite during intestinal infection. TMKs are attractive targets for drug development because of their requirement in virulence and proliferation.

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.

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.

The ubiquitin-proteasome system (UPS) and autophagy provide major cellular pathways for protein degradation. Since the p53 pathway controls autophagy, we investigated whether p53 regulates UPS in ovarian tumour cell lines. A reporter cell line (SKOV3-EGFPu) was established to measure UPS function against a constant genetic background. Transient expression of either wild type or mutant p53 in SKOV3-EGFPu cells reduced UPS activity as compared to vector control. These results, together with those from endogenous p53 expression in seven ovarian cancer cell lines, suggest that expression of both wild-type and mutant p53 protein impairs UPS function. Thus, p53 expression may regulate protein homeostasis by down-regulating UPS function in response to cellular stress., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Entamoeba histolytica contains a large and novel family of transmembrane kinases (TMKs). The expression patterns of the E. histolytica TMKs in individual trophozoites and the roles of the TMKs for sensing and responding to extracellular cues were incompletely characterised. Here we provide evidence that single cells express multiple TMKs and that TMK39 and TMK54 likely serve non-redundant cellular functions. Laser-capture microdissection was used in conjunction with microarray analysis to demonstrate that single trophozoites express more than one TMK gene. Anti-peptide antibodies were raised against unique regions in the extracellular domains of TMK39, TMK54 and PaTMK, and TMK expression was analysed at the protein level. Flow cytometric assays revealed that populations of trophozoites homogeneously expressed TMK39, TMK54 and PaTMK, while confocal microscopy identified different patterns of cell surface expression for TMK39 and TMK54. The functions of TMK39 and TMK54 were probed by the inducible expression of dominant-negative mutants. While TMK39 co-localised with ingested beads and expression of truncated TMK39 interfered with trophozoite phagocytosis of apoptotic lymphocytes, expression of a truncated TMK54 inhibited growth of amoebae and altered the surface expression of the heavy subunit of the E. histolytica Gal/GalNAc lectin. Overall, our data indicates that multiple members of the novel E. histolytica TMK family are utilised for non-redundant functions by the parasite., ((c) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

It is not understood why only some infections with Entamoeba histolytica result in disease. The calcium-regulated transcription factor upstream regulatory element 3-binding protein (URE3-BP) was initially identified by virtue of its role in regulating the expression of two amebic virulence genes, the Gal/GalNac lectin and ferredoxin. Here we tested whether this transcription factor has a broader role in regulating virulence. A comparison of in vivo to in vitro parasite gene expression demonstrated that 39% of in vivo regulated transcripts contained the URE3 motif recognized by URE3-BP, compared to 23% of all promoters (P < 0.0001). Amebae induced to express a dominant positive mutant form of URE3-BP had an increase in an elongated morphology (30% +/- 6% versus 14% +/- 5%; P = 0.001), a 2-fold competitive advantage at invading the intestinal epithelium (P = 0.017), and a 3-fold increase in liver abscess size (0.1 +/- 0.1 g versus 0.036 +/- 0.1 g; P = 0.03). These results support a role for URE3-BP in virulence regulation.

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 muM 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 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 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&#95;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 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.