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Malnourished children are susceptible to an increased risk of mortality owing to impaired immune functions. However, the underlying mechanism of altered immune functions and its interaction with malnutrition is poorly understood. This study investigates the immune function and evaluates the effect of a particular nutritional intervention on the immune cells of undernourished children. Stunted (LAZ <−2) and at‐risk of being stunted (length‐for‐age Z‐scores, LAZ <−1 to −2) children aged between 12 and 18 months were enrolled and were provided with the daily nutritional intervention of one egg and 150 mL cow's milk for 90 days. Peripheral blood mononuclear cells (PBMCs) were isolated at enrolment and upon completion of the intervention. Phenotypic profiles for CD3+ cells, CD4+ cells, CD8+ cells, NKT cells, and B cells were similar in both cohorts, both before and after the intervention. However, activated B cells (CD25+) were increased after nutritional intervention in the at‐risk of being stunted cohort. Several pro‐inflammatory cytokines, IL‐6, IFN‐γ, and TNF‐α, were elevated in the stunted children following the nutritional intervention. The results of the study indicate that nutritional intervention may have a role on activated B cells (CD25+) s in children who are at‐risk of being stunted and may alter the capacity of PBMC to produce inflammatory cytokines in stunted children. [ABSTRACT FROM AUTHOR]

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., Introduction The biological mechanisms by which malnutrition contributes to an estimated one-third of all deaths among children (1) and 60% of deaths due to diarrhea (2) are poorly understood (1). [...]

Dysregulations in the mammalian target of rapamycin (mTOR) pathway are associated with several human anomalies. We aimed to elucidate possible implications for potential aberrations in the mTOR pathway with childhood malnutrition. We analyzed the activity of phospho-mTORC1 and the expressions of several mTOR pathway genes, namely: MTOR, TSC1, LAMTOR2, RPS6K1 and RICTOR from peripheral blood mononuclear cells isolated from venous blood of children suffering from different forms of malnutrition and compared them with those from healthy children. Significant reduction in the phosphorylation of mTORC1 was noted, as well as a decrease in expression of LAMTOR2 gene and increase in TSC1 gene expression were observed between malnourished children in comparison to the healthy children. The deregulation in the activity of the TSC1 and LAMTOR2 gene was significantly associated with all forms of childhood malnutrition. Our findings provide key insights into possible down-modulation in the overall activity of the mTOR pathway in childhood malnutrition. Further studies focusing on the analysis of a multitude of components involved in the mTOR pathway both at the gene and protein expression levels are required for conclusive evidence for the aforementioned proposition. [ABSTRACT FROM AUTHOR]

Shigella species account for the second-leading cause of deaths due to diarrheal diseases among children of less than 5 years of age. The emergence of multi-drug-resistant Shigella isolates and the lack of availability of Shigella vaccines have led to the pertinence in the efforts made for the development of new therapeutic strategies against shigellosis. Consequently, designing small-interfering RNA (siRNA) candidates against such infectious agents represents a novel approach to propose new therapeutic candidates to curb the rampant rise of anti-microbial resistance in such pathogens. In this study, we analyzed 264 conserved sequences from 15 different conserved virulence genes of Shigella sp., through extensive rational validation using a plethora of first-generation and second-generation computational algorithms for siRNA designing. Fifty-eight siRNA candidates were obtained by using the first-generation algorithms, out of which only 38 siRNA candidates complied with the second-generation rules of siRNA designing. Further computational validation showed that 16 siRNA candidates were found to have a substantial functional efficiency, out of which 11 siRNA candidates were found to be non-immunogenic. Finally, three siRNA candidates exhibited a sterically feasible three-dimensional structure as exhibited by parameters of nucleic acid geometry such as: the probability of wrong sugar puckers, bad backbone confirmations, bad bonds, and bad angles being within the accepted threshold for stable tertiary structure. Although the findings of our study require further wet-lab validation and optimization for therapeutic use in the treatment of shigellosis, the computationally validated siRNA candidates are expected to suppress the expression of the virulence genes, namely: IpgD (siRNA 9) and OspB (siRNA 15 and siRNA 17) and thus act as a prospective tool in the RNA interference (RNAi) pathway. However, the findings of our study require further wet-lab validation and optimization for regular therapeutic use for treatment of shigellosis. [ABSTRACT FROM AUTHOR]

The prevalence of norovirus infections in different geographical locations and their attribution to childhood diarrhea is well established. However, there are no reports showing possible relationships of different norovirus genogroups with subsequent childhood malnutrition. In this study, we attempted to establish a potential association between asymptomatic norovirus infections with childhood growth faltering during. Non-diarrheal stools were collected from 1715 children enrolled in locations in a multi-county birth cohort study across eight different geographical locations and were assessed for norovirus genogroup I (GI) and norovirus genogroup II (GII). Asymptomatic norovirus GI infections were negatively associated with monthly length-for-age Z score/LAZ (β = −0.53, 95% CI: −0.73, −0.50) and weight-for-age Z score/WAZ (β = −0.39, 95% CI: −0.49, −0.28), respectively. The burden of asymptomatic norovirus GI infections was negatively associated with LAZ (β = −0.46, 95% CI: −0.67, −0.41) and WAZ (β = −0.66, 95% CI: −0.86, −0.53) at 2 years of age, whilst the burden of asymptomatic norovirus GII infections was negatively associated with WAZ (β = −0.27, 95% CI: −0.45, −0.25) at 2 years of age. Our findings warrant acceleration in attempts to develop vaccines against norovirus GI and norovirus GII, with the aim of minimizing the long-term sequelae on childhood growth. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The intestinal surface is directly exposed to both commensal microorganisms as well as pathogens with a single layer of epithelium separating luminal microorganisms from internal tissues. Antimicrobial peptides play a crucial role in allowing epithelial cells to contain in the lumen beneficial and pathogenic microorganisms. The commensal dependent, epithelial produced, Th2 cytokine IL-25 can induce IL-13 and potentially the antimicrobial peptide angiogenin-4. Here we show that IL-13 downstream of IL-25 is required to induce angiogenin-4. IL-25 mediated induction of angiogenin-4 is furthermore not dependent on IL-22 or IL-17. [ABSTRACT FROM AUTHOR]

Hydrogen polysulfides (H 2 S n ) have emerged as critical physiological mediators that are closely associated with hydrogen sulfide (H 2 S) signaling. H 2 S n exhibit greater nucleophilicity than H 2 S while also having electrophilic characteristics, enabling unique activities such as protein S-persulfidation. Despite their physiological importance, mechanisms and reactivities of H 2 S n remain inadequately explored due to their inherent instability in aqueous environments. Consequently, there is a need to develop biocompatible methods for controlled H 2 S n generation to elucidate their behaviors in biological contexts. Herein, we present a dual enzyme system (containing glucose oxidase (GOx) and chloroperoxidase (CPO)) with thioglucose as the substrate to facilitate the controlled release of H 2 S n . Fluorescence measurements with SSP4 and the trapping studies allowed us to confirm the production of H 2 S n . Such a method may be useful in elucidating the reactivity of hydrogen polysulfides in biological systems as well as provide a potential delivery of H 2 S n to target sites for biological applications., (© 2024 Wiley-VCH GmbH.)

Accurate and early diagnoses are prerequisites for prompt treatment. For coronavirus disease 2019 (COVID-19), it is even more crucial. Currently, choice of methods include rapid diagnostic tests and reverse transcription polymerase chain reaction (RT-PCR) using samples mostly of respiratory origin and sometimes saliva. We evaluated two rapid diagnostic tests with three specimen types using viral transport medium (VTM) containing naso-oropharyngeal (NOP) swabs, direct nasal and direct nasopharyngeal (NP) samples from 428 prospective patients. We also performed RT-PCR for 428 NOP VTM and 316 saliva samples to compare results. The sensitivity of the SD Biosensor Standard Q COVID-19 antigen (Ag) test kit drastically raised from an average of 65.55% (NOP VTM) to 85.25% (direct nasal samples), while RT-PCR was the gold standard. For the CareStart kit, the sensitivity was almost similar for direct NP swabs; the average was 84.57%. The specificities were ≥95% for both SD Biosensor Standard Q and CareStart COVID-19 Ag tests in all platforms. The kits were also able to detect patients with different variants as well. Alternatively, RT-PCR results from saliva and NOP VTM samples showed high sensitivities of 96.45% and 95.48% with respect to each other as standard. The overall results demonstrated high performance of the rapid tests, indicating the suitability for regular surveillance at clinical facilities when using direct nasal or direct NP samples rather than NOP VTM. Additionally, the analysis also signifies not showed that RT-PCR of saliva can be used as an choice of method to RT-PCR of NOP VTM, providing an easier, non-invasive sample collection method. IMPORTANCE There are several methods for the diagnosis of coronavirus disease 2019 (COVID-19), and the choice of methods depends mostly on the resources and level of sensitivity required by the user and health care providers. Still, reverse transcription polymerase chain reaction (RT-PCR) has been chosen as the best method using direct naso-oropharyngeal swabs. There are also other methods of fast detection, such as rapid diagnostic tests (RDTs), which offer result within 15 to 20 min and have become quite popular for self-testing and in the clinical setting. The major drawback of the currently used RT-PCR method is compliance, as it may cause irritation, and patients often refuse to test in such a way. RDTs, although inexpensive, suffer from low sensitivity due to technical issues. In this article, we propose saliva as a noninvasive source for RT-PCR samples and evaluate various specimen types at different times after infection for the best possible output from COVID-19 rapid tests.

Introduction: MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally regulate gene expression. Changes in miRNA expression have been reported in a number of intestinal diseases, in both tissue samples and readily accessible specimens like stools. Pathogenic infections, diet, toxins, and other environmental factors are believed to influence miRNA expression. However, modulation of miRNAs in humans is yet to be thoroughly investigated. In this study, we examined the expression levels of two human miRNAs (miRNA-122 and miRNA-21) in stool samples of a group of Bangladeshi children who had an altered/increased intestinal permeability (IIP). Methods: Stool samples were collected from children with IIP (L:M > 0.09) and normal intestinal permeability (NIP) (L:M ≤ 0.09). Quantitative PCR was performed to quantify the levels of miRNA-122 and miR-21 in stools. Commercial ELISA kits were used to measure gut inflammatory markers Calprotectin and REG1B. Serum samples were tested using Human Bio-Plex Pro Assays to quantify IL-1β, IL-2, IL-5, IL-10, IL-13, IFN-γ, and TNF-α. Total nucleic acid extracted from stool specimens were used to determine gut pathogens using TaqMan Array Card (TAC) system real-time polymerase chain reaction. Results: The expression levels of miRNA-122 (fold change 11.6; p < 0.001, 95% CI: 6.14-11.01) and miR-21 (fold change 10; p < 0.001, 95% CI: 5.05-10.78) in stool were upregulated in children with IIP than in children with normal intestinal permeability (NIP). Significant correlations were observed between stool levels of miR-122 and miR-21 and the inflammatory cytokines IL-1β, IL-2, IFN-γ, and TNF-α ( p < 0.05). Children with IIP were frequently infected with rotavirus, Campylobacter jejuni , Bacteroides fragilis , adenovirus, norovirus, astrovirus, and various Escherichia coli strains (ETEC&#95;STh, ETEC&#95;STp, EAEC&#95;aaiC, EAEC&#95;aatA) ( p < 0.001). miR-122 significantly correlated with the fecal inflammatory biomarkers REG1B ( p = 0.015) and Calprotectin ( p = 0.030), however miR-21 did not show any correlation with these fecal biomarkers., 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 Rashid, Siddiqua, Hossain, Siddique, Kabir, Noor, Alam, Ahmed and Haque.)

Chronic exposure to infectious agents results in environmental enteric dysfunction-a significant contributor to childhood stunting. Low plasma tryptophan (TRP), increased kynurenine (KYN), and KYN-TRP (KT) ratio are associated with infections and chronic immune activation. We postulated that both these conditions are interlinked, and therefore aimed to identify the association between KT ratio and the linear growth of Bangladeshi children. A total of 480 stunted and at risk of being stunted children aged 12-18 months were enrolled and provided nutrition intervention for 90 days. Plasma samples were assessed using liquid chromatography tandem mass spectrometry to measure TRP and KYN concentrations. Multivariable linear regression with generalized estimating equations was applied to analyze association between the KT ratio and linear growth. Tryptophan, KYN, and KT ratio were significantly higher in stunted children than in children at risk of being stunted both at baseline and at the end of nutrition intervention. Following intervention, the median (interquartile range [IQR]) KYN concentration was significantly reduced from 4.6 (3.6, 5.4) µmol/L to 3.9 (0.3, 7.6) µmol/L, and median (IQR) KT ratio decreased from 104 (80.9, 131) to 92.8 (6.6, 247) in stunted children. We also found KT ratio to be negatively associated (coefficient = -0.7; 95% CI = -1.13, -0.26; P-value = 0.002) with linear growth. In addition, KYN and KT ratio were positively correlated with fecal neopterin and plasma C-reactive protein, whereas TRP was negatively correlated with both of these biomarkers and alpha-1-acid glycoprotein. Our findings imply that KT ratio is associated in the pathophysiology of stunting as well as with biomarkers of inflammation in Bangladeshi children.

MicroRNAs (miRNAs) are a class of conserved endogenous, small non-coding RNA molecules with a length of 18-25 nucleotides that regulate gene expression by RNA interference processes, including mRNA chopping, mRNA deadenylation, and translation inhibition. miRNAs maintain the physiological functions of the intestine and are instrumental in gut pathogenesis. miRNAs play an important role in intercellular communication and are present in all body fluids, including stools with different composition and concentrations. However, under diseased conditions, miRNAs are aberrantly expressed and act as negative regulators of gene expression. The stable and differentially expressed miRNAs in stool enables miRNAs to be used as potential biomarkers for screening of various intestinal diseases. In this review, we summarize the expressed miRNA profile in stool and highlight miRNAs as biomarkers with potential clinical and diagnostic applications, and we aim to address the prospects for recent advanced techniques for screening miRNA in diagnosis and prognosis of intestinal disorders., (Copyright © 2020 Rashid, Hossain, Siddiqua, Kabir, Noor, Ahmed and Haque.)

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.

The parasite Entamoeba histolytica is a cause of diarrhea in infants in low-income countries. Previously, it was shown that tumor necrosis factor alpha (TNF-α) production was associated with increased risk of E. histolytica diarrhea in children. Interleukin-25 (IL-25) is a cytokine that is produced by intestinal epithelial cells that has a role in maintenance of gut barrier function and inhibition of TNF-α production. IL-25 expression was decreased in humans and in the mouse model of amebic colitis. Repletion of IL-25 blocked E. histolytica infection and barrier disruption in mice, increased gut eosinophils, and suppressed colonic TNF-α. Depletion of eosinophils with anti-Siglec-F antibody prevented IL-25-mediated protection. In contrast, depletion of TNF-α resulted in resistance to amebic infection. We concluded that IL-25 provides protection from amebiasis, which is dependent upon intestinal eosinophils and suppression of TNF-α. IMPORTANCE The intestinal epithelial barrier is important for protection from intestinal amebiasis. We discovered that the intestinal epithelial cytokine IL-25 was suppressed during amebic colitis in humans and that protection could be restored in the mouse model by IL-25 administration. IL-25 acted via eosinophils and suppressed TNF-α. This work illustrates a previously unrecognized pathway of innate mucosal immune response., (Copyright © 2017 Noor et al.)

Intestinal segmented filamentous bacteria (SFB) protect from ameba infection, and protection is transferable with bone marrow dendritic cells (BMDCs). SFB cause an increase in serum amyloid A (SAA), suggesting that SAA might mediate SFB's effects on BMDCs. Here we further explored the role of bone marrow in SFB-mediated protection. Transient gut colonization with SFB or SAA administration alone transiently increased the H3K27 histone demethylase Jmjd3, persistently increased bone marrow Csf2ra expression and granulocyte monocyte precursors (GMPs), and protected from ameba infection. Pharmacologic inhibition of Jmjd3 H3K27 demethylase activity during SAA treatment or blockade of granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling in SFB-colonized mice prevented GMP expansion, decreased gut neutrophils, and blocked protection from ameba infection. These results indicate that alteration of the microbiota and systemic exposure to SAA can influence myelopoiesis and susceptibility to amebiasis via epigenetic mechanisms. Gut microbiota-marrow communication is a previously unrecognized mechanism of innate protection from infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Clostridium difficile is a major, life-threatening hospital-acquired pathogen that causes mild to severe colitis in infected individuals. The tissue destruction and inflammation which characterize C. difficile infection (CDI) are primarily due to the Rho-glucosylating toxins A and B. These toxins cause epithelial cell death and induce robust inflammatory signaling by activating the transcription factor NF-κB, leading to chemokine and cytokine secretion. The toxins also activate the inflammasome complex, which leads to secretion of the pyrogenic cytokine IL-1β. In this study, we utilized glucosylation-deficient toxin A to show that activation of the inflammasome by this toxin is dependent on Rho glucosylation, confirming similar findings reported for toxin B. We also demonstrated that tissue destruction and in vivo inflammatory cytokine production are critically dependent on the enzymatic activity of toxin A, suggesting that inhibiting toxin glucosyltransferase activity may be effective in combating this refractory disease., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: There is an emerging paradigm that the human microbiome is central to many aspects of health and may have a role in preventing enteric infection. Entamoeba histolytica is a major cause of amebic diarrhea in developing countries. It colonizes the colon lumen in close proximity to the gut microbiota. Interestingly, not all individuals are equally susceptible to E. histolytica infection. Therefore, as the microbiota is highly variable within individuals, we sought to determine if a component of the microbiota could regulate susceptibility to infection. In studies utilizing a murine model, we demonstrated that colonization of the gut with the commensal Clostridia-related bacteria known as segmented filamentous bacteria (SFB) is protective during E. histolytica infection. SFB colonization in this model was associated with elevated cecal levels of interleukin 17A (IL-17A), dendritic cells, and neutrophils. Bone marrow-derived dendritic cells (BMDCs) from SFB-colonized mice had higher levels of IL-23 production in response to stimulation with trophozoites. Adoptive transfer of BMDCs from an SFB(+) to an SFB(-) mouse was sufficient to provide protection against E. histolytica. IL-17A induction during BMDC transfer was necessary for this protection. This work demonstrates that intestinal colonization with a specific commensal bacterium can provide protection during amebiasis in a murine model. Most importantly, this work demonstrates that the microbiome can mediate protection against an enteric infection via extraintestinal effects on bone marrow-derived dendritic cells., Importance: Entamoeba histolytica is the causative agent of amebiasis, an infectious disease that contributes significantly to morbidity and mortality due to diarrhea in the developing world. We showed in a murine model that colonization with the commensal members of the Clostridia known as SFB provides protection against E. histolytica and that dendritic cells from SFB-colonized mice alone can recapitulate protection. Understanding interactions between enteropathogens, commensal intestinal bacteria, and the mucosal immune response, including dendritic cells, will help in the development of effective treatments for this disease and other infectious and inflammatory diseases. The demonstration of immune-mediated protection due to communication from the microbiome to the bone marrow represents an emerging field of study that will yield unique approaches to the development of these treatments., (Copyright © 2014 Burgess et al.)

The role of leptin in the mucosal immune response to Clostridium difficile colitis, a leading cause of nosocomial infection, was studied in humans and in a murine model. Previously, a mutation in the receptor for leptin (LEPR) was shown to be associated with susceptibility to infectious colitis and liver abscess due to Entamoeba histolytica as well as to bacterial peritonitis. Here we discovered that European Americans homozygous for the same LEPR Q223R mutation (rs1137101), known to result in decreased STAT3 signaling, were at increased risk of C. difficile infection (odds ratio, 3.03; P = 0.015). The mechanism of increased susceptibility was studied in a murine model. Mice lacking a functional leptin receptor (db/db) had decreased clearance of C. difficile from the gut lumen and diminished inflammation. Mutation of tyrosine 1138 in the intracellular domain of LepRb that mediates signaling through the STAT3/SOCS3 pathway also resulted in decreased mucosal chemokine and cell recruitment. Collectively, these data support a protective mucosal immune function for leptin in C. difficile colitis partially mediated by a leptin-STAT3 inflammatory pathway that is defective in the LEPR Q223R mutation. Identification of the role of leptin in protection from C. difficile offers the potential for host-directed therapy and demonstrates a connection between metabolism and immunity.

Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites-Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis-were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites.

Objective: We sought to compare the tuberculin skin test (TST) to the QuantiFERON-TB Gold In-Tube assay (QFT-IT) and assess the effects of malnourishment and intestinal helminth infection on QFT-IT results., Methods: In this population-based cross-sectional study from Dhaka, Bangladesh, we screened children for latent tuberculosis infection with the QFT-IT and TST. We assess the agreement between the TST and QFT-IT, risk factors associated with indeterminate QFT-IT results, and magnitude of interferon γ (IFN-γ) production., Results: Three hundred and two children (aged 11-15.3 years) were enrolled, including 93 (30.8%) who were malnourished. Of 251 participants who provided stool samples, 117 (46.6%) were infected with Ascaris lumbricoides and/or Trichuris trichiura. TST results were positive (≥10 mm) for 101 (33.4%) children and negative for 201 (66.6%) children. QFT-IT results were positive for 107 (35.4%) children, negative for 121 (40.1%) children, and indeterminate for 74 (24.5%) children. Agreement between the tests was moderate (κ = 0.55 [95% confidence interval: 0.44-0.65]; P < .0001) when excluding indeterminate results. Children with indeterminate QFT-IT results were separately compared with children with positive and negative QFT-IT results; malnutrition (P = .0006 and .0003), and helminth infection (P = .05 and .02), and the statistical interaction between these 2 terms (P = .03 and .004) were associated with indeterminate results. Higher levels of IFN-γ in response to tuberculosis antigens were associated with positive TST results (P < .0001); lower levels were associated with malnutrition (P = .02)., Conclusions: Malnutrition and helminth infections were associated with indeterminate QFT-IT results. Therefore, the presence of such conditions may limit the interpretability of QFT-IT results in children.

The noninvasive diagnosis of amebic liver abscess is challenging, as most patients at the time of diagnosis do not have a concurrent intestinal infection with Entamoeba histolytica. Fecal testing for E. histolytica parasite antigen or DNA is negative in most patients. A real-time PCR assay was evaluated for detection of E. histolytica DNA in blood, urine, and saliva samples from amebic liver abscess as well as amebic colitis patients in Bangladesh. A total of 98 amebic liver abscess and 28 amebic colitis patients and 43 control subjects were examined. The real-time PCR assay detected E. histolytica DNA in 49%, 77%, and 69% of blood, urine, and saliva specimens from the amebic liver abscess patients. For amebic colitis the sensitivity of the real-time PCR assay for detection of E. histolytica DNA in blood, urine, and saliva was 36%, 61%, and 64%, respectively. All blood, urine, and saliva samples from control subjects were negative by the real-time PCR assay for E. histolytica DNA. When the real-time PCR assay results of the urine and saliva specimens were taken together (positive either in urine or saliva), the real-time PCR assay was 97% and 89% sensitive for detection of E. histolytica DNA in liver abscess and intestinal infection, respectively. We conclude that the detection of E. histolytica DNA in saliva and urine could be used as a diagnostic tool for amebic liver abscess.