Myeloperoxidase as a biomarker for intestinal-brain axis dysfunction induced by malnutrition and Cryptosporidium infection in weanling mice

Abstract Cryptosporidiosis is a waterborne protozoal infection that may cause life-threatening diarrhea in undernourished children living in unsanitary environments. The aim of this study is to identify new biomarkers that may be related to gut-brain axis dysfunction in children suffering from the malnutrition/infection vicious cycle is necessary for better intervention strategies. Myeloperoxidase (MPO) is a well-known neutrophil-related tissue factor released during enteropathy that could drive gut-derived brain inflammation. We utilized a model of environmental enteropathy in C57BL/6 weanling mice challenged by Cryptosporidium and undernutrition. Mice were fed a 2%-Protein Diet (dPD) for eight days and orally infected with 107-C. parvum oocysts. C. parvum oocyst shedding was assessed from fecal and ileal-extracted genomic DNA by qRT-PCR. Ileal histopathology scores were assessed for intestinal inflammation. Prefrontal cortex samples were snap-frozen for MPO ELISA assay and NF-kb immunostaining. Blood samples were drawn by cardiac puncture after anesthesia and sera were obtained for serum amyloid A (SAA) and MPO analysis. Brain samples were also obtained for Iba-1 prefrontal cortex immunostaining. C. parvum-infected mice showed sustained stool oocyst shedding for six days post-infection and increased fecal MPO and inflammation scores. dPD and cryptosporidiosis led to impaired growth and weight gain. C. parvum-infected dPD mice showed increased serum MPO and serum amyloid A (SAA) levels, markers of systemic inflammation. dPD-infected mice showed greater MPO, NF-kB expression, and Iba-1 immunolabeling in the prefrontal cortex, an important brain region involved in executive function. Our findings suggest MPO as a potential biomarker for intestinal-brain axis dysfunction due to environmental enteropathy.

Clinical evaluation, biochemistry and genetic polymorphism analysis for the diagnosis of lactose intolerance in a population from northeastern Brazil

OBJECTIVE: This work aimed to evaluate and correlate symptoms, biochemical blood test results and single nucleotide polymorphisms for lactose intolerance diagnosis. METHOD: A cross-sectional study was conducted in Fortaleza, Ceará, Brazil, with a total of 119 patients, 54 of whom were lactose intolerant. Clinical evaluation and biochemical blood tests were conducted after lactose ingestion and blood samples were collected for genotyping evaluation. In particular, the single nucleotide polymorphisms C>T-13910 and G>A-22018 were analyzed by restriction fragment length polymorphism/polymerase chain reaction and validated by DNA sequencing. RESULTS: Lactose-intolerant patients presented with more symptoms of flatulence (81.4%), bloating (68.5%), borborygmus (59.3%) and diarrhea (46.3%) compared with non-lactose-intolerant patients (p<0.05). We observed a significant association between the presence of the alleles T-13910 and A-22018 and the lactose-tolerant phenotype (p<0.05). After evaluation of the biochemical blood test results for lactose, we found that the most effective cutoff for glucose levels obtained for lactose malabsorbers was <15 mg/dL, presenting an area under the receiver operating characteristic curve greater than 80.3%, with satisfactory values for sensitivity and specificity. CONCLUSIONS: These data corroborate the association of these single nucleotide polymorphisms (C>T-13910 and G>A-22018) with lactose tolerance in this population and suggest clinical management for patients with lactose intolerance that considers single nucleotide polymorphism detection and a change in the biochemical blood test cutoff from <25 mg/dL to <15 mg/dL.

Effects of glutamine alone or in combination with zinc and vitamin A on growth, intestinal barrier function, stress and satiety-related hormones in Brazilian shantytown children

OBJECTIVE: To determine the impact of supplemental zinc, vitamin A, and glutamine alone or in combination on growth, intestinal barrier function, stress and satiety-related hormones among Brazilian shantytown children with low median height-for-age z-scores. METHODS: A randomized, double-blind, placebo-controlled trial was conducted in children aged two months to nine years from the urban shanty compound community of Fortaleza, Brazil. Demographic and anthropometric information was assessed. The random treatment groups available for testing (a total of 120 children) were as follows: (1) glutamine alone, n = 38; (2) glutamine plus vitamin A plus zinc, n = 37; and a placebo (zinc plus vitamin A vehicle) plus glycine (isonitrogenous to glutamine) control treatment, n = 38. Leptin, adiponectin, insulin-like growth factor (IGF-1), and plasma levels of cortisol were measured with immune-enzymatic assays; urinary lactulose/mannitol and serum amino acids were measured with high-performance liquid chromatography. ClinicalTrials.gov: NCT00133406. RESULTS: Glutamine treatment significantly improved weight-for-height z-scores compared to the placebo-glycine control treatment. Either glutamine alone or all nutrients combined prevented disruption of the intestinal barrier function, as measured by the percentage of lactulose urinary excretion and the lactulose:mannitol absorption ratio. Plasma leptin was negatively correlated with plasma glutamine (p = 0.002) and arginine (p = 0.001) levels at baseline. After glutamine treatment, leptin was correlated with weight-for-age (WAZ) and weight-for-height z-scores (WHZ) (p≤0.002) at a 4-month follow-up. In addition, glutamine and all combined nutrients (glutamine, vitamin A, and zinc) improved the intestinal barrier function in these children. CONCLUSION: Taken together, these findings reveal the benefits of glutamine alone or in combination ...

Zinc, vitamin A, and glutamine supplementation in Brazilian shantytown children at risk for diarrhea results in sex-specific improvements in verbal learning

OBJECTIVE: To identify the impact of supplemental zinc, vitamin A, and glutamine, alone or in combination, on long-term cognitive outcomes among Brazilian shantytown children with low median height-for-age z-scores. METHODS: A randomized, double-blind, placebo-controlled trial was conducted in children aged three months to nine years old from the urban shanty compound community of Fortaleza, Brazil. Demographic and anthropometric information was assessed. The random treatment groups available for cognitive testing (total of 167 children) were: (1) placebo, n = 25; (2) glutamine, n = 23; (3) zinc, n = 18; (4) vitamin A, n = 19; (5) glutamine+zinc, n = 20; (6) glutamine+vitamin A, n = 21; (7) zinc+vitamin A, n = 23; and (8) glutamine+zinc+vitamin A, n = 18. Neuropsychological tests were administered for the cognitive domains of non-verbal intelligence and abstraction, psychomotor speed, verbal memory and recall ability, and semantic and phonetic verbal fluency. Statistical analyses were performed using SPSS, version 16.0. ClinicalTrials.gov: NCT00133406. RESULTS: Girls receiving a combination of glutamine, zinc, and vitamin A had higher mean age-adjusted verbal learning scores than girls receiving only placebo (9.5 versus 6.4, p = 0.007) and girls receiving zinc+vitamin A (9.5 versus 6.5, p = 0.006). Similar group differences were not found between male study children. CONCLUSIONS: The findings suggest that combination therapy offers a sex-specific advantage on tests of verbal learning, similar to that seen among female patients following traumatic brain injury.

Apolipoprotein E4 influences growth and cognitive responses to micronutrient supplementation in shantytown children from northeast Brazil

OBJECTIVE: Apolipoprotein E4 may benefit children during early periods of life when the body is challenged by infection and nutritional decline. We examined whether apolipoprotein E4 affects intestinal barrier function, improving short-term growth and long-term cognitive outcomes in Brazilian shantytown children. METHODS: A total of 213 Brazilian shantytown children with below-median height-for-age z-scores (HAZ) received 200,000 IU of retinol (every four months), zinc (40 mg twice weekly), or both for one year, with half of each group receiving glutamine supplementation for 10 days. Height-for-age z-scores, weight-for-age z-scores, weight-forheight z-scores, and lactulose:mannitol ratios were assessed during the initial four months of treatment. An average of four years (range 1.4-6.6) later, the children underwent cognitive testing to evaluate non-verbal intelligence, coding, verbal fluency, verbal learning, and delayed verbal learning. Apolipoprotein E4 carriage was determined by PCR analysis for 144 children. RESULTS: Thirty-seven children were apolipoprotein E4(+), with an allele frequency of 13.9 percent. Significant associations were found for vitamin A and glutamine with intestinal barrier function. Apolipoprotein E4(+) children receiving glutamine presented significant positive Pearson correlations between the change in height-for-age z-scores over four months and delayed verbal learning, along with correlated changes over the same period in weight-for-age z-scores and weight-for-height z-scores associated with non-verbal intelligence quotients. There was a significant correlation between vitamin A supplementation of apolipoprotein E4(+) children and improved delta lactulose/mannitol. Apolipoprotein E4(-) children, regardless of intervention, exhibited negative Pearson correlations between the change in lactulose-to-mannitol ratio over four months and verbal learning and non-verbal intelligence. CONCLUSIONS: During development, apolipoprotein E4 may function concomitantly with gut-tropic nutrients to benefit immediate nutritional status, which can translate into better long-term cognitive outcomes.

Intestinal barrier function and serum concentrations of rifampin, isoniazid and pyrazinamide in patients with pulmonary tuberculosis

Intestinal barrier function and serum concentrations of rifampin, isoniazid and pyrazinamide were studied in healthy controls and patients with active pulmonary tuberculosis. A case-control study of 29 controls and 30 cases attending at the Health Center, July, 2004 to December, 2005 was conducted. The body mass index was significantly reduced in cases compared to controls (p < 0.001). The intestinal paracellular transport of lactulose was significantly (p = 0.019) reduced in cases compared to controls. The transcellular transport of mannitol and the lactulose:mannitol ratio were not significantly (p = 0.0698) reduced in cases compared to controls. Low serum concentrations of rifampin, isoniazid and pyrazinamide were observed in 81 percent (48/59), 92 percent (54/59) and 28 percent (12/59), respectively, in all individuals. The results demonstrated a marked decrease on intestinal paracellular transport in patients with active pulmonary tuberculosis and reduced serum concentrations of rifampin and isoniazid in both groups.

Intestinal permeability and malabsorption of rifampin and isoniazid in active pulmonary tuberculosis

Low antimycobacterial drug concentrations have been observed in tuberculosis (TB) patients under treatment. The lactulose/mannitol urinary excretion test (L/M), normally used to measure intestinal permeability, may be useful to assess drug absorption. The objective of this research was to study intestinal absorptive function and bioavailability of rifampin and isoniazid in TB patients. A cross sectional study was done with 41 patients and 28 healthy controls, using the L/M test. The bioavailabilities of rifampin (R) and isoniazid (H) were evaluated in 18 patients receiving full doses. Urinary excretion of mannitol and lactulose, measured by HPLC, was significantly lower in TB patients. The serum concentrations of the drugs were below the expected range for R (8-24 mcg/mL) or H (3-6 mcg/mL) in 16/18 patients. Analyzing the drugs individually, 12/18 patients had low serum concentrations of R, 13/18 for H and 8/18 for both drugs. We suggest that there is a decrease in the functional absorptive area of the intestine in TB patients, which would explain the reduced serum concentrations of antituberculosis drugs. There is a need for new approaches to improve drug bioavailability in TB patients.

Association of vitamin A and zinc status with altered intestinal permeability: analyses of cohort data from northeastern Brazil.

To examine the association of intestinal barrier function with vitamin A deficiency and whether supplementation of micronutrients improves intestinal function and/or linear growth, height-for-age z-score (HAZ), concentrations of serum retinol and zinc, and intestinal permeability were determined in a cross-sectional sample of 75 children in northeastern Brazil. Effects of vitamin A and supplementation of zinc on intestinal permeability and growth were also determined comparing results before and after treatment in 20 children and age-matched controls. Lactulose:mannitol (L/M) permeability ratios inversely correlated with serum retinol concentrations (r = -0.55, p < 0.0005). Increased L/M permeability ratios with reduced concentrations of serum retinol were predominantly attributable to lower absorption of mannitol (r = 0.28, p = 0.02). L/M permeability ratios (p = 0.001) and HAZ scores (p = 0.007) improved with supplementation. It is concluded that impaired intestinal barrier function and linear growth shortfalls improve following supplementation of vitamin A and zinc in this setting.

Common infectious diseases and skin test anergy in children from an urban slum in Northeast Brazil

BACKGROUND: Acute respiratory infection (ARI), diarrheal disease (DD) and infective dermatitis (ID) are important causes of morbidity in children under five, in Northeast Brazil. Objectives: (a) to evaluate the morbidity of ARI, DD and ID; and (b) to determine their association with cellular immunity in poor urban children from Fortaleza, Brazil. MATERIALS AND METHODS: A prospective cohort study. At enrollment, multipuncture skin-tests (Multitest CMI) were performed and interpreted according to standard procedures. Children were followed for infectious diseases by weekly home visits. RESULTS: Seventy-one children aged 6 to 21 months were recruited in an ongoing cohort of newborns. A mean of 39 (6 to 63) home visits per child were made, which detected 184.5 symptomatic days per child-year of observation. ARI was present in 62 percent of the days of illness (6,378 out of 10,221), DD in 23 percent (2,296 days), ID in 6 percent (597) and other infections in 4 percent (373). Episodes per child-year were: 10 for ARI, 7 for DD and 1 for ID. Twelve (17 percent) out of 71 children were anergic. The incidences of ARI, DD and ID were similar in responsive versus anergic children. The mean duration of ID in anergy was 8.5 days, while it was 4.3 in the responsive group (P=0.007). Anergy was independent of age, sex and nutritional status. CONCLUSIONS: A high incidence of ARI and DD was found in these poor urban children. Skin-test responsiveness was not related to malnutrition, nor to morbidity due to ARI and DD, however anergic children had a longer duration of infective dermatitis.

AIDS-associated diarrhea and wasting in northeast Brazil is associated with subtherapeutic plasma levels of antiretroviral medications and with both bovine and human subtypes of Cryptosporidium parvum

Advanced HIV infection is frequently complicated by diarrhea, disruption of bowel structure and function, and malnutrition. Resulting malabsorption of or pharmacokinetic changes in antiretroviral agents might lead to subtherapeutic drug dosing and treatment failure in individual patients, and could require dose adjustment and/or dietary supplements during periods of diarrheal illness. We determined the plasma levels of antiretroviral medications in patients that had already been started on medication by their physicians in an urban infectious diseases hospital in northeast Brazil. We also obtained blood samples from patients hospitalized for diarrhea or AIDS-associated wasting, and we found reduced stavudine and didanosine levels in comparison with outpatients without diarrhea or wasting who had been treated at the same hospital clinic. There was a predominance of the protozoal pathogens Cryptosporidium and Isospora belli, typical opportunistic pathogens of AIDS-infected humans, in the stool samples of inpatients with diarrhea. We conclude that severe diarrhea and wasting in this population is associated with both protozoal pathogens and subtherapeutic levels of antiretroviral medications

Uso combinado del método cualitativo y del metanálisis en el enfoque diagnóstico de la diarrea infecciosa / Combined use of qualitative method and metanalysis on the diagnostic approach of infectious diarrhea

Objetivo: comparar la utilidad diagnóstica de pruebas índice en la identificación de la diarrea invasiva. Métodos: combinación de análisis cualitativo y cuantitativo (metanálisis). Identificación de datos y selección de los estudios: de 2603 referencias identificadas vía MEDLINE (1970-1994), listas de referencias y por correspondencia con expertos en el campo, 81 cumplieron los requerimientos de selección preestablecidos. De éstas, 25 contenían datos suficientes para un metanálisis adicional. Extracción de datos: en la revisión cualitativa, un autor extrajo información amplia sobre la utilidad diagnóstica de las pruebas índice y sobre las características metodológicas de los estudios. En el metanálisis, todos los datos de los artículos seleccionados fueron extraídos por un investigador, en tanto otro verificó su exactitud. Se calculó la tasa de verdaderos positivos (TPR) y de falsos positivos (FPR), a partir de cada tabla de contigencia de 2 por 2. Utilizando el método de transformación logística y regresión lineal ponderada de cuadros mínimos, se elaboró curvas resumen de las características operativas del receptor (SROC), para determinar la utilidad diagnóstica comparativa de las pruebas. Resultados del análisis de datos: el enfoque cualitativo no restrictivo identificó problemas metodológicos que fueron evaluados adicionalmente en el estudio cuantitativo. En el metanálisis, la graficación de TPR contra FPR, como curvas SROC, demostró puntos dispersos, indicando heterogeneidad. La curva de lactoferrina fecal mostró el mayor rendimiento y la de leucocitos fecales el menor rendimiento. Conclusiones: la lactoferrina fecal es la prueba con mayor utilidad diagnóstica en la identificación de la diarrea invasiva. Se propone la utilización del enfoque combinado cualitativo y metanalítico como un método adecuado de evaluación de utilidad de pruebas diagnósticas, tanto en la práctica clínica como en el proceso general de análisis de decisiones.

Diarréias hospitalares / Hospital diarrhea

Os autores revisam o assunto diarréias hospitalares e suas duas grandes causas: Clostridium difficile no adulto e rotavírus na criança. Pesquisa da citotoxina do C. difficile por cultura quantitativa em tecido ainda é a principal maneira do diagnóstico. Mais de 80 por cento dos pacientes estavam em uso de antibióticos por tempo prolongado, principalmente cefalosporinas de terceira geraçäo. De maneira geral, as coproculturas e os parasitológicos raramente foram positivos e ajudaram no diagnóstico das diarréias hospitalares. Salmonella permanece importante agente em surtos relacionados à ingestäo de alimentos contaminados.

Diarreias hospitalares / Nosocomial diarrhea

Os autores revisam o assunto Diarreias Hospitalares e suas duas grandes causas: Clostridium difficile no adulto e rotavirus na crianca. Pesquisa da citotoxina do C. difficile por cultura quantitativa em tecido ainda e a principal maneira do diagnostico. Mais de 80 por cento dos pacientes estavam em uso de antibioticos por tempo prolongado, principalmente cefalosporinas de terceira geracao. De maneira geral, as coproculturas e os parasitologicos raramemte foram positivos e ajudaram no diagnostico das diarreias hospitalares. Salmonella permanece importante agente em surtos relacionados a ingestao de alimentos contaminados. A magnitude e a importancia da diarreia hospitalar esta apenas comecando a ser apreciada: no passado, diarreia era frequentemente despercebida ou mesmo considerada entre as infeccoes hospitalares. Diarreia nosocomial e um importante problema em si mesmo e pode predispor a outras infeccoes hospitalares, como as infeccoes do trato urinario. Welliver e McLaughlin, relataram que num hospital pediatrico...

Emergence of sulfamethoxazole-trimethoprim resistant Shigella flexneri in Northeastern Brazil

Em contraste à experiência anterior, três de quatro cepas isoladas recentemente no Nordeste do Brasil em pacientes com diarréia inflamatória aguda, foram resistentes ao sulfametoxazol-trimetoprin. Foram realizados testes para averiguaçäo se a resistência era transferível: isolou-se e se caracterizou o plasmídio do DNA da Shigella resistente. Outras enterobacteriáceas foram simultaneamente isolados das fezes destes indivíduos e eram cepas transconjugantes. Cada cepa de Shigella resistente contém um grande plasmídio. Estes plasmídios têm peso molecular variando de 30 a 58 Mdal no seu tamanho. Dois destes plasmídios puderam ser transferidos para E. coli K-12. Estes dados mostram a limitaçäo do uso desta associaçäo de fármacos, largamente usados em enteroinfecçöes

Estudios prospectivos de los efectos de las enfermedades en una comunidad rural del Nordeste del Brasil / Prospective studies of the effects of diseases in a rural community Northeast of Brazil

Se hizo un estudio longitudinal de 78 semanas de duración de los residentes de tres vecindarios diferentes de Pacatuba, pueblo situado al nordeste del Brasil, para tratar de conocer la frecuencia de las enfermedades febriles, respiratorias y diarreicas entre esta población. Un miembro del equipo que llevó a cabo el estudio compiló la información mediante visitas que hacía una vez por semana a cada vivienda; los datos se basaban en lo que recordaba un adulto respecto a los síntomas manifestados durante la semana anterior por los miembros de la unidad familiar. En total, se obtuvieron datos de 48 familias por un período de tiempo suficiente y con bastante regularidad como para incluirlos en el análisis subsiguiente. Este análisis reveló que se habían notificado síntomas de enfermedades febriles, respiratorias o diarreicas en aproximadamente la tercera parte de todas las semanas del estudio entre niños de uno a dos años de edad residentes en dos vecindarios pobres comprendidos en la encuesta. De ahí en adelante esta tasa disminuyó constantemente, a medida que los grupos de edad se hacían progresivamente mayores, hasta alcanzar los niveles de adultos. Los números promedio de episodios de enfermedades febriles, respiratorias y diarreicas notificados en los niños menores de cinco años en los dos vecindarios pobres (Matadouro y Säo Joäo) fueron 6, 8, 5 y 3, 4 episodios por año en Matadouro y 4, 2, 7, 8 y 5, 1 por año en Säo Joäo. En contraste, parece que los niños estudiados en el "Centro" de clase media de Pacatuba acusaban una morbilidad coherentemente más baja en todos los grupos de edad estudiados. En general, los patrones de morbilidad de este vecindario mostraron la tendencia a parecerse a los notificados en Cleveland, Ohio y Charlottesville, Virginia. No se ha aclarado si la notificación incompleta podría ser en parte responsable de estas tasas aparentemente bajas. Sin embargo, es probable que los datos de los dos vecindarios pobres reflejen los patrones de morbilidad generales prevalecientes entre las grandes poblaciones rurales empobrecidas del nordeste del Brasil, que tiene una población rural de más de un millón de habitantes, y en la que más del 90% de los niños pertenecen a familias con ingresos por persona inferiores a US$ 30 mensuales. De ahí que los datos notificados ofrezcan una base provisional para visualizar un lastre de enfermedades de impresionante magnitud que soporta esta población mucho más numerosa

Etiologia de la diarrea infantil y terapia de rehidratacion oral en el nordeste de Brasil. / Etiology of infantile diarrhea and oral rehydration therapy in northeastern Brazil

La diarrea aguda es una causa importante de morbilidad y mortalidad en el nordeste de Brasil. El estudio aqui resenado documenta la presencia de E.coli enterotoxigeno y rotavirus en ninos con diarrea en un centro de rehidratacion del nordeste del Brasil y demuestra la aceptabilidad de la terapia de rehidratacion oral con glucosa y electrolitos administradas por las madres