Jesus Emmanuel Sevilleja

Pediatric Diarrhea in Southern Ghana: Etiology and Association with Intestinal Inflammation and Malnutrition

Diarrhea is a major public health problem that affects the development of children. Anthropometric data were collected from 274 children with (N = 170) and without (N = 104) diarrhea. Stool specimens were analyzed by conventional culture, polymerase chain reaction for enteroaggregative Escherichia coli (EAEC), Shigella, Cryptosporidium, Entamoeba, and Giardia species, and by enzyme-linked immunosorbent assay for fecal lactoferrin levels. About 50% of the study population was mildly to severely malnourished. Fecal lactoferrin levels were higher in children with diarrhea (P = 0.019). Children who had EAEC infection, with or without diarrhea, had high mean lactoferrin levels regardless of nutritional status. The EAEC and Cryptosporidium were associated with diarrhea (P = 0.048 and 0.011, respectively), and malnourished children who had diarrhea were often co-infected with both Cryptosporidium and EAEC. In conclusion, the use of DNA-biomarkers revealed that EAEC and Cryptosporidium were common intestinal pathogens in Accra, and that elevated lactoferrin was associated with diarrhea in this group of children.

Cryptosporidium Infection Causes Undernutrition and, Conversely, Weanling Undernutrition Intensifies Infection

Abstract Cryptosporidium parvum is a leading pathogen in children in developing countries. To investigate whether early postnatal malnutrition leads to heavier C. parvum infections, we assessed intestinal adaptation and parasite load in suckling mice during the first 2 wk of life, analogous to the first postnatal yr in humans. Undernutrition was induced by daily C57BL6J pup separation from lactating dams. Half of the pups were separated daily, for 4 hr on day 4, 8 hr on day 5, and for 12 hr from day 6 until day 14. On day 6, each pup received an oral inoculum of 105 to 107 parasites in 10–25 μl of PBS. Littermate controls received PBS alone. Stools were assessed from days 8, 11, and 14 for oocyst counts. Mice were killed on day 14, 8 days postinoculation, at the peak of the infection. Ileal and colon segments were obtained for histology, real-time and reverse transcriptase PCR, and immunoassays. Villus and crypt lengths and cross-sectional areas were also measured. Undernourished and nourished mice infected with excysted 106 or 107 oocysts exhibited the poorest growth outcomes compared with their uninfected controls. Nourished 106-infected mice had comparable weight decrements to uninfected undernourished mice. Body weight and villi were additively affected by malnutrition and cryptosporidiosis. Hyperplastic crypts and heavier inflammatory responses were found in the ilea of infected malnourished mice. Undernourished infected mice exhibited greater oocyst shedding, TNF-α and IFN-γ intestinal levels, and mRNA expression compared to nourished mice infected with either 105 or 106 oocysts. Taken together, these findings show that Cryptosporidium infection can cause undernutrition and, conversely, that weanling undernutrition intensifies infection and mucosal damage.

Enteroaggregative Escherichia coli (EAEC) Impairs Growth while Malnutrition Worsens EAEC Infection: A Novel Murine Model of the Infection Malnutrition Cycle

BACKGROUND Enteroaggregative Escherichia coli is emerging as an increasingly recognized cause of persistent, mildly inflammatory diarrhea in the United States, especially among patients with AIDS, as well as among children, for whom it is accompanied by growth shortfalls. METHODS We describe a novel model of disease induced in neonatal and weaned C57BL/6 mice by pathogenic strains of enteroaggregative E. coli 042 and JM221. RESULTS Enteroaggregative E. coli caused growth impairment (up to 47%), persistent stool shedding (for >3 weeks), and a substantial tissue burden of organisms (150,000 organisms per milligram of tissue), as well as histopathological changes in the colonic epithelium (days 4 and 6 of infection) using the model. Undernutrition in neonatal mice, as well as in weaned mice, intensified infection by 1-4 logs, as assessed by quantitative polymerase chain reaction for fecal shedding of organisms. Growth impairment was dependent on both microorganism burden and challenge dose. CONCLUSIONS Both neonatal and weaned mice provide models for a vicious cycle of enteroaggregative E. coli infection that causes growth shortfalls and undernutrition, thus worsening infection. Hence, these neonatal and weaned mice provide the opportunity to dissect mechanisms of this cycle in childhood malnutrition, as well as to define the role played by innate and acquired host defenses in this important infection.

Cryptosporidium-Malnutrition Interactions: Mucosal Disruption, Cytokines, and TLR Signaling In A Weaned Murine Model

Abstract Cryptosporidiosis is a leading cause of persistent diarrhea in children in impoverished and developing countries and has both a short- and long-term impact on the growth and development of affected children. An animal model of cryptosporidial infection that mirrors closely the complex interaction between nutritional status and infection in children, particularly in vulnerable settings such as post-weaning and malnourishment, is needed to permit exploration of the pathogenic mechanisms involved. Weaned C57BL/6 mice received a protein-deficient (2%) diet for 3–12 days, then were infected with 5 × 107 excysted C. parvum oocyts, and followed for rate of growth, parasite stool shedding, and intestinal invasion/morphometry. Mice had about 20% reduction in weight gain over 12 days of malnutrition and an additional 20% weight loss after C. parvum challenge. Further, a significantly higher fecal C. parvum shedding was detected in malnourished infected mice compared to the nourished infected mice. Also, higher oocyst counts were found in ileum and colon tissue samples from malnourished infected mice, as well as a significant reduction in the villous height–crypt depth ratio in the ileum. Tissue Th1 cytokine concentrations in the ileum were significantly diminished by malnutrition and infection. mRNA for toll-like receptors 2 and 4 were diminished in malnourished infected mice. Treatment with nitazoxanide did not prevent weight loss or parasite stool shedding. These findings indicate that, in the weaned animal, malnutrition intensifies cryptosporidial infection, while cryptosporidial infection further impairs normal growth. Depressed TLR2 and 4 signaling and Th1 cytokine response may be important in the mechanisms underlying the vicious cycle of malnutrition and enteric infection.

Novel In Vitro and In Vivo Models and Potential New Therapeutics to Break the Vicious Cycle of Cryptosporidium Infection and Malnutrition

BACKGROUND Although several animal models of cryptosporidiosis have been reported, most involve genetically or pharmacologically immune-suppressed hosts. METHODS We report challenge with excysted (in vitro and in vivo) and unexcysted (in vivo) Cryptosporidium parvum oocysts in human colonic adenocarcinoma (HCT-8) cells and weaned nourished and malnourished C57BL/6 mice, following outcomes of growth rate, stool shedding, and tissue burden. We tested treatment with an oligodeoxynucleotide containing unmethylated CpG motif (CpG-ODN) and alanyl-glutamine in vivo and in vitro. RESULTS C. parvum-challenged mice showed prolonged weight loss (>10% over 4 days), robust stool shedding (>3 logs/d over 7 days), and epithelial infection in the ileum, cecum, and colon. Of 2 potential therapeutic compounds evaluated in the model, CpG-ODN reduced body weight loss (to <6% on days 3-7 after challenge), reduced shedding of organisms (by 25% on days 1 and 3 after challenge), and decreased the burden of parasites in the ileum. Alanyl-glutamine showed similar benefits. In vitro findings suggested that effects on the epithelial component of the mucosa probably likely responsible for beneficial effects seen in vivo. CONCLUSIONS Weaned mice provide a convenient and reproducible model of cryptosporidial disease, including its vicious cycle with body weight loss and heavier infection with malnutrition, and this model may be useful in exploring innovative therapeutic solutions for this challenging infectious disease.

Faecal contamination of drinking water in a Brazilian shanty town: importance of household storage and new human faecal marker testing

Worldwide, contaminated drinking water poses a major health threat, particularly to child development. Diarrhoea represents a large part of the water-related disease burden and enteric infections have been linked to nutritional and growth shortfalls as well as long-term physical and cognitive impairment in children. Previous studies detailed the frequency of infection and the consequences for child health in a shanty town in north-east Brazil. To determine the frequency of contaminated water, we measured faecal contamination in primary drinking water samples from 231 randomly selected households. Risk for contamination was compared across source and storage types. Nearly a third of the study households (70/231: 30.3%) had contaminated drinking water; the source with the highest frequency of contamination was well water (23/24: 95.8%). For tap water, the type of storage had a significant effect on the susceptibility to contamination (chi(2) = 12.090; p = 0.007). The observed pattern of contamination demonstrated the relative potential contributions of both source and storage. With evidence that supports the inclusion of source and storage in water quality surveys, this study, like others, suggests that contaminated drinking water in storage vessels may be an important factor for the documented diarrhoea disease burden in the Brazilian shanty town.

Evaluation of HIV protease and nucleoside reverse transcriptase inhibitors on proliferation, necrosis, apoptosis in intestinal epithelial cells and electrolyte and water transport and epithelial barrier function in mice

BackgroundProtease inhibitors (PIs) and reverse transcriptase drugs are important components of highly active antiretroviral therapy (HAART) for treating human acquired immunodeficiency syndrome (AIDS). Long-term clinical therapeutic efficacy and treatment compliance of these agents have been limited by undesirable side-effects, such as diarrhea. This study aims to investigate the effects of selected antiretroviral agents on intestinal histopathology and function in vivo and on cell proliferation and death in vitro.MethodsSelected antiretroviral drugs were given orally over 7 days, to Swiss mice, as follows: 100 mg/kg of nelfinavir (NFV), indinavir (IDV), didanosine (DDI) or 50 mg/kg of zidovudine (AZT). Intestinal permeability measured by lactulose and mannitol assays; net water and electrolyte transport, in perfused intestinal segments; and small intestinal morphology and cell apoptosis were assessed in treated and control mice. In vitro cell proliferation was evaluated using the WST-1 reagent and apoptosis and necrosis by flow cytometry analysis.ResultsNFV, IDV, AZT and DDI caused significant reductions in duodenal and in jejunal villus length (p < 0.05). IDV and AZT increased crypt depth in the duodenum and AZT increased crypt depth in the jejunum. NFV, AZT and DDI significantly decreased ileal crypt depth. All selected antiretroviral drugs significantly increased net water secretion and electrolyte secretion, except for DDI, which did not alter water or chloride secretion. Additionally, only NFV significantly increased mannitol and lactulose absorption. NFV and IDV caused a significant reduction in cell proliferation in vitro at both 24 h and 48 h. DDI and AZT did not alter cell proliferation. There was a significant increase in apoptosis rates in IEC-6 cells after 24 h with 70 ug/mL of NFV (control: 4.7% vs NFV: 22%) while IDV, AZT and DDI did not show any significant changes in apoptosis compared to the control group. In jejunal sections, IDV and NFV significantly increased the number of TUNEL positive cells.ConclusionThe PIs, NFV and IDV, increased cell apoptosis in vivo, water and electrolyte secretion and intestinal permeability and decreased villus length and cell proliferation. NFV was the only drug tested that increased cell apoptosis in vitro. The nucleoside reverse transcriptase inhibitors, AZT and DDI, did not affect cell apoptosis or proliferation. These findings may partly explain the intestinal side-effects associated with PIs.

Arginine decreases Cryptosporidium parvum infection in undernourished suckling mice involving nitric oxide synthase and arginase

OBJECTIVE This study investigated the role of L-arginine supplementation to undernourished and Cryptosporidium parvum-infected suckling mice. METHODS The following regimens were initiated on the fourth day of life and injected subcutaneously daily. The C. parvum-infected controls received L-arginine (200 mmol/L) or phosphate buffered saline. The L-arginine-treated mice were grouped to receive NG-nitro-arginine methyl ester (L-NAME) (20 mmol/L) or phosphate buffered saline. The infected mice received orally 10(6) excysted C. parvum oocysts on day 6 and were euthanized on day 14 at the infection peak. RESULTS L-arginine improved weight gain compared with the untreated infected controls. L-NAME profoundly impaired body weight gain compared with all other groups. Cryptosporidiosis was associated with ileal crypt hyperplasia, villus blunting, and inflammation. L-arginine improved mucosal histology after the infection. L-NAME abrogated these arginine-induced improvements. The infected control mice showed an intense arginase expression, which was even greater with L-NAME. L-arginine decreased the parasite burden, an effect that was reversed by L-NAME. Cryptosporidium parvum infection increased urine NO(3)(-)/NO(2)(-) concentrations compared with the uninfected controls, which was increased by L-arginine supplementation, an effect that was also reversed by L-NAME. CONCLUSION These findings show a protective role of L-arginine during C. parvum infection in undernourished mice, with involvement of arginase I and nitric oxide synthase enzymatic actions.

Diarrhea, Clostridium difficile, and Intestinal Inflammation in Residents of a Long-Term Care Facility

INTRODUCTION Long-term care facilities (LTCF) residents have been estimated to have the highest incidence of diarrheal illness among adults living in the developed world. This study describes undiagnosed diarrhea, intestinal inflammation, and Clostridium difficile colonization in a LTC population and explores whether these are associated with functional decline, as defined by weight loss or a change in cognitive or ADL status. METHODS An observational study of a convenience sampling of residents in a 180-bed LTCF was obtained; evaluation of stool and medical records was done. Stool specimens were evaluated for consistency, gross blood, inflammation (via quantitative fecal lactoferrin, IBD-SCAN), and C difficile (via PCR for gdh). SPSS and STATA were used and significance was set at P < .05. RESULTS There were 46 stools collected; 13 of the subjects were male, 28 were older than 65 years, and 35 were prescribed 5 to 15 medications. Twenty-six of the 46 stools collected had elevated quantitative fecal lactoferrin levels. Although only 5 subjects were reported to have diarrhea (4 with elevated lactoferrin), 28 stool specimens were observed to be liquid or semi-solid (19 with elevated lactoferrin), and these liquid/ semisolid stools were significantly correlated with lactoferrin positivity (P = .017). In analysis of functional status, there was no statistically significant association between change in ADL (n = 17) or cognitive status (n = 5) and elevated lactoferrin. However, all 3 subjects who had significant weight loss had elevated lactoferrin, although the mean fecal lactoferrin was not statistically different from those without weight loss. Of the 2 samples with C difficile, both were liquid and, when compared with all other liquid stools (n = 22), the mean lactoferrin was statistically higher (134.1 versus 28.8 microg/mL, P = .008). These 2 subjects had neither weight loss nor change in cognitive status, but 1 had a change in ADL status. DISCUSSION AND CONCLUSIONS Diarrhea in LTCF residents is underdiagnosed. Diarrhea and the presence of C difficile in the stool are associated with intestinal inflammation, as detected by fecal lactoferrin. With our small numbers, we were not able to identify a specific link; however, we were able to identify a correlation between weight loss and intestinal inflammation, but, with just 2 samples, not C difficile colonization. This relationship highlights the importance of larger studies to further examine the rate of diarrhea in LTCF; the effect of diarrhea and intestinal inflammation on weight loss; and the interaction of C difficile colonization with weight loss, malnutrition, and functional decline.

Clostridium difficile and Entamoeba histolytica infections in patients with colitis in the Philippines

Amoebiasis is a common cause of non-specific colitis in the Philippines. The prevalence of Clostridium difficile infection with colitis is unknown. Empiric use of metronidazole for colitis treatment is widely practiced. We investigated the association of C. difficile or Entamoeba histolytica infection with endoscopically/histopathologically proven colitis among adults in the Philippines. Two hundred and ten patients undergoing colonoscopy were enrolled. Demographic and clinical data were reviewed. Stool specimens were assayed for C. difficile and E. histolytica by ELISA. Microscopy was performed. The mean age of the patients was 53 y (range: 19-88 y) and 53% were male. Colitis was diagnosed in 39 of 205 patients. Clostridium difficile, E. histolytica and parasites were seen in 17 (43.6%), 10 (25.6%) and 11 (28.2%), respectively, of patients with colitis compared with 36 (21.7%; p=0.005), 13 (7.8%; p=0.001) and 56 (33.7%; p=0.51), respectively, of those without colitis. Diarrhoea and antibiotic intake history were significantly more common among patients with colitis than those without (43.6% and 20.5% vs 18.1% and 5.4%; p=0.001 and p=0.006, respectively). The mean duration of diarrhoea was 2.53 d shorter among patients with colitis. The most frequent antibiotics taken were fluoroquinolones and metronidazole (50% and 40% of antibiotic courses, respectively, in patients with colitis). This study suggests that C. difficile infection is common and might be overlooked in settings where amoebiasis and intestinal parasitism are endemic.