Establishing a National Molecular Surveillance Program for the Detection of Plasmodium falciparum Markers of Resistance to Antimalarial Drugs in Haiti.

Chloroquine remains the first-line treatment for uncomplicated malaria in Haiti, and until recently, sulfadoxine-pyrimethamine was the second-line treatment. A few studies have reported the presence of molecular markers for resistance in Plasmodium falciparum parasites, and in vivo therapeutic efficacy studies (TESs) have been limited. Recognizing the history of antimalarial resistance around the globe and the challenges of implementing TESs in low-endemic areas, the Ministry of Health established a surveillance program to detect molecular markers of antimalarial resistance in Haiti. Sentinel sites were purposefully selected in each of Haiti's 10 administrative departments; an 11th site was selected in Grand'Anse, the department with the highest number of reported cases. Factors considered for site selection included the number of malaria cases identified, observed skills of laboratory technicians conducting rapid diagnostic tests (RDTs), stock and storage conditions of RDTs, accuracy of data reporting to the national surveillance system, and motivation to participate. Epidemiologic data from 2,437 patients who tested positive for malaria from March 2016 to December 2018 and consented to provide samples for molecular sequencing are presented here. Of these, 936 (38.4%) patients reported self-treatment with any medication since the onset of their illness before diagnosis; overall, 69 (2.8%) patients reported taking an antimalarial. Ten patients (0.4%) reported travel away from their home for at least one night in the month before diagnosis. Establishing a molecular surveillance program for antimalarial drug resistance proved practical and feasible in a resource-limited setting and will provide the evidence needed to make informed treatment policy decisions at the national level.

Use of Bead-Based Serologic Assay to Evaluate Chikungunya Virus Epidemic, Haiti.

The index case of chikungunya virus (CHIKV) in Haiti was reported during early 2014; the vector, the pervasive Aedes aegypti mosquito, promoted rapid spread throughout the country. During December 2014-February 2015, we collected blood samples from 4,438 persons at 154 sites (62 urban, 92 rural) throughout Haiti and measured CHIKV IgG by using a multiplex bead assay. Overall CHIKV seroprevalence was 57.9%; differences between rural (mean 44.9%) and urban (mean 78.4%) areas were pronounced. Logistic modeling identified the urban environment as a strong predictor of CHIKV exposure (adjusted odds ratio 3.34, 95% CI 2.38-4.69), and geographic elevation provided a strong negative correlation. We observed no correlation between age and antibody positivity or titer. Our findings demonstrated through serologic testing the recent and rapid dissemination of the arbovirus throughout the country. These results show the utility of serologic data to conduct epidemiologic studies of quickly spreading mosquitoborne arboviruses.

Correlation of Biomarker Expression in Colonic Mucosa with Disease Phenotype in Crohn's Disease and Ulcerative Colitis.

BACKGROUND: Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are characterized by chronic intestinal inflammation due to immunological, microbial, and environmental factors in genetically predisposed individuals. Advances in the diagnosis, prognosis, and treatment of IBD require the identification of robust biomarkers that can be used for molecular classification of diverse disease presentations. We previously identified five genes, RELA, TNFAIP3 (A20), PIGR, TNF, and IL8, whose mRNA levels in colonic mucosal biopsies could be used in a multivariate analysis to classify patients with CD based on disease behavior and responses to therapy. AIM: We compared expression of these five biomarkers in IBD patients classified as having CD or UC, and in healthy controls. RESULTS: Patients with CD were characterized as having decreased median expression of TNFAIP3, PIGR, and TNF in non-inflamed colonic mucosa as compared to healthy controls. By contrast, UC patients exhibited decreased expression of PIGR and elevated expression of IL8 in colonic mucosa compared to healthy controls. A multivariate analysis combining mRNA levels for all five genes resulted in segregation of individuals based on disease presentation (CD vs. UC) as well as severity, i.e., patients in remission versus those with acute colitis at the time of biopsy. CONCLUSION: We propose that this approach could be used as a model for molecular classification of IBD patients, which could further be enhanced by the inclusion of additional genes that are identified by functional studies, global gene expression analyses, and genome-wide association studies.

Secretory IgA is Concentrated in the Outer Layer of Colonic Mucus along with Gut Bacteria.

Antibodies of the secretory IgA (SIgA) class comprise the first line of antigen-specific immune defense, preventing access of commensal and pathogenic microorganisms and their secreted products into the body proper. In addition to preventing infection, SIgA shapes the composition of the gut microbiome. SIgA is transported across intestinal epithelial cells into gut secretions by the polymeric immunoglobulin receptor (pIgR). The epithelial surface is protected by a thick network of mucus, which is composed of a dense, sterile inner layer and a loose outer layer that is colonized by commensal bacteria. Immunofluorescence microscopy of mouse and human colon tissues demonstrated that the SIgA co-localizes with gut bacteria in the outer mucus layer. Using mice genetically deficient for pIgR and/or mucin-2 (Muc2, the major glycoprotein of intestinal mucus), we found that Muc2 but not SIgA was necessary for excluding gut bacteria from the inner mucus layer in the colon. Our findings support a model whereby SIgA is anchored in the outer layer of colonic mucus through combined interactions with mucin proteins and gut bacteria, thus providing immune protection against pathogens while maintaining a mutually beneficial relationship with commensals.

Lessons from mother: Long-term impact of antibodies in breast milk on the gut microbiota and intestinal immune system of breastfed offspring.

From birth to adulthood, the gut microbiota matures from a simple community dominated by a few major bacterial groups into a highly diverse ecosystem that provides both benefits and challenges to the host. Currently there is great interest in identifying environmental and host factors that shape the development of our gut microbiota. Breast milk is a rich source of maternal antibodies, which provide the first source of adaptive immunity in the newborn's intestinal tract. In this addendum, we summarize our recent data demonstrating that maternal antibodies in breast milk promote long-term intestinal homeostasis in suckling mice by regulating the gut microbiota and host gene expression. We also discuss important unanswered questions, future directions for research in this field, and implications for human health and disease.

Secretory antibodies in breast milk promote long-term intestinal homeostasis by regulating the gut microbiota and host gene expression.

Maintenance of intestinal homeostasis requires a healthy relationship between the commensal gut microbiota and the host immune system. Breast milk supplies the first source of antigen-specific immune protection in the gastrointestinal tract of suckling mammals, in the form of secretory IgA (SIgA). SIgA is transported across glandular and mucosal epithelial cells into external secretions by the polymeric Ig receptor (pIgR). Here, a breeding scheme with polymeric Ig receptor-sufficient and -deficient mice was used to study the effects of breast milk-derived SIgA on development of the gut microbiota and host intestinal immunity. Early exposure to maternal SIgA prevented the translocation of aerobic bacteria from the neonatal gut into draining lymph nodes, including the opportunistic pathogen Ochrobactrum anthropi. By the age of weaning, mice that received maternal SIgA in breast milk had a significantly different gut microbiota from mice that did not receive SIgA, and these differences were magnified when the mice reached adulthood. Early exposure to SIgA in breast milk resulted in a pattern of intestinal epithelial cell gene expression in adult mice that differed from that of mice that were not exposed to passive SIgA, including genes associated with intestinal inflammatory diseases in humans. Maternal SIgA was also found to ameliorate colonic damage caused by the epithelial-disrupting agent dextran sulfate sodium. These findings reveal unique mechanisms through which SIgA in breast milk may promote lifelong intestinal homeostasis, and provide additional evidence for the benefits of breastfeeding.

Multifactorial patterns of gene expression in colonic epithelial cells predict disease phenotypes in experimental colitis.

BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) is complex and the need to identify molecular biomarkers is critical. Epithelial cells play a central role in maintaining intestinal homeostasis. We previously identified five "signature" biomarkers in colonic epithelial cells (CEC) that are predictive of disease phenotype in Crohn's disease. Here we investigate the ability of CEC biomarkers to define the mechanism and severity of intestinal inflammation. METHODS: We analyzed the expression of RelA, A20, pIgR, tumor necrosis factor (TNF), and macrophage inflammatory protein (MIP)-2 in CEC of mice with dextran sodium sulfate (DSS) acute colitis or T-cell-mediated chronic colitis. Factor analysis was used to combine the five biomarkers into two multifactorial principal components (PCs). PC scores for individual mice were correlated with disease severity. RESULTS: For both colitis models, PC1 was strongly weighted toward RelA, A20, and pIgR, and PC2 was strongly weighted toward TNF and MIP-2, while the contributions of other biomarkers varied depending on the etiology of inflammation. Disease severity was correlated with elevated PC2 scores in DSS colitis and reduced PC1 scores in T-cell transfer colitis. Downregulation of pIgR was a common feature observed in both colitis models and was associated with altered cellular localization of pIgR and failure to transport IgA. CONCLUSIONS: A multifactorial analysis of epithelial gene expression may be more informative than examining single gene responses in IBD. These results provide insight into the homeostatic and proinflammatory functions of CEC in IBD pathogenesis and suggest that biomarker analysis could be useful for evaluating therapeutic options for IBD patients.

Regulation of the polymeric immunoglobulin receptor in intestinal epithelial cells by Enterobacteriaceae: implications for mucosal homeostasis.

The commensal microbiota of the human colon profoundly impacts host gene expression and mucosal homeostasis. Secretory IgA antibodies, which influence the composition of the intestinal microbiota and provide immunity against pathogens, are transported across intestinal epithelial cells (IEC) by the polymeric immunoglobulin receptor (pIgR). To compare the effects of different colonic bacteria on pIgR expression, the human IEC line HT-29 was stimulated with various species representing the 4 major phyla of colonic bacteria. Only bacteria from the family Enterobacteriaceae (phylum Proteobacteria) induced expression of pIgR and other target genes of bacterial pattern recognition receptors. HT-29 cells responded to purified ligands for Toll-like receptor (TLR)4 but not TLR2. Expression of pIgR and transport of IgA were significantly reduced in colons of mice deficient in the TLR adaptor MyD88, consistent with a role for TLR signaling in the regulation of pIgR by colonic bacteria. Induction of pIgR expression in HT-29 cells required NF-kappaB signaling but not MAPK signaling, in contrast to the requirement for both NF-kappaB and MAPK signaling for induction of pro-inflammatory genes. These results suggest that commensal Enterobacteriaceae may promote intestinal homeostasis by enhancing pIgR expression in IEC.