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• W2020179542 abstract "Diarrheal diseases are the leading cause of childhood mortality in Nicaragua1 and in many other Latin American, African and Asian countries. In children <2 years of age, repeated and prolonged episodes of diarrhea are also significant factors in the pathogenesis of malnutrition.2, 3 In developing countries 20 to 40% of diarrheal disease episodes among children are associated with invasive bacterial pathogens such as Shigella, Salmonella, Campylobacter and enteropathogenic Escherichia coli. Definitive diagnosis of bacterial gastroenteritis requires stool culture, which is frequently not available, prohibitive because of cost and not clinically useful for initial management because of delays in obtaining results. Recent investigations have described methods to detect invasive bacterial diarrhea by direct stool agglutination assays, allowing for rapid initiation of therapy. The most widely used assay is the Leukotest (TechLab, Blacksburg, VA), which detects fecal lactoferrin as a marker of fecal leukocytes.4, 5 The Leukotest was shown to be more sensitive than direct staining of fecal leukocytes for diagnosis of invasive bacterial enteritis in a recent study in Brazil, where a positive result predicted the presence of invasive pathogens on stool culture with a sensitivity of 94%, a specificity of 60% and a negative predictive value of 94%.6 The presence of a negative lactoferrin agglutination test therefore correlated highly with a negative stool culture. The test requires refrigeration during storage but no sophisticated equipment for testing, so it is applicable to developing country clinic situations. Test results are interpreted with the naked eye along with simultaneous positive and negative control panels. Several cofactors potentially influence the presence of lactoferrin in stools of children, and the impact of these cofactors on interpretation of the Leukotest has not been well-studied. Fecal lactoferrin is present in high concentrations in human milk and is found in the stool of normal breast-fed infants,7 but the impact of breast-feeding on the interpretation of this test in children with diarrhea is unknown. Helminth infections are very common in the tropics and are estimated to infect up to one-fourth of the world's population. These infections are also associated with colonic infiltration of eosinophils, lymphocytes and other leukocytes, and many of these cells secrete or bind lactoferrin.8, 9 Materials and methods. The goals of this project were to investigate the impact of breast-feeding on Leukotest results in breast-fed and non-breast-fed children with diarrhea and to examine differences in results in groups stratified according to presence or absence of helminth infections. Stool samples from 147 pediatric patients younger than 7 years of age with diarrhea meeting entry criteria (presence of 3 or more soft stools or 1 liquid stool during the last 24 h) were enrolled with parental informed consent. After enrollment limited demographic and clinical data (including a dietary history of breast milk consumption) were collected by parent interview, and a stool specimen was immediately sent for analysis by a trained laboratory technician who was unaware of clinical data about the patients. Assays performed on these specimens were: (1) methylene blue stain for fecal leukocytes, read by examining 10 high power fields within the area of fecal mucus. The presence of fecal leukocytes were quantitated by the methods of Harris et al.10; (2) Leukotest agglutination assay, performed according to manufacturer's instructions, within 1 h of stool specimen collection. One drop of stool specimen was mixed with 2.5 ml of diluent, and a drop of diluted specimen was reacted with a drop of sensitized latex, along with positive and negative control wells. Assays were read as negative (−) through highly positive (4+). For purposes of this study all positive tests (1+ through 4+) were categorized as positive. All stool specimens were also analyzed by stool culture11 and direct parasite screen. Specimens that could not be cultured within 1 h were preserved in Cary Blair transport medium. Salmonella, Shigella and Vibrio cholerae were isolated by direct inoculation of MacConkey agar, SS agar and TCBS agar. Salmonella antisera and Shigella polyvalent antisera were used for confirmation. Limitations in funding and laboratory resources precluded the isolation of Campylobacter jejuni or tests for viral enteropathogens such as rotavirus. Unconcentrated and zinc sulfate-concentrated stools were also examined microscopically for the presence of Ascaris, Trichuris, Giardia and other significant parasites. Cryptosporidium and Cyclospora were sought by acid-fast staining using carbol fuchsin with methylene blue counterstain after fixation of stool smears for 1 min in 10% NaOH. Data from questionnaires and laboratory assays were entered into an Access 97 database file by the project data manager. Cases were grouped by history of breast-feeding or presence of helminths for subanalyses, and within each group the sensitivity, specificity and predictive values of the corresponding agglutination test were determined by standard statistical methods.12 Results of methylene blue stain and Leukotest were also compared. Data were analyzed by SPSS, and statistical comparisons were made by chi square tests. The study protocol was approved by the Institutional Review Board of Tulane University Medical Center and by an Ethical Review Committee of the Sistema Local de Atencion Integral en Salud of Carazo. Results. Between October and December of 1995, 147 patients were enrolled. All children were from low income urban and periurban communities in and around Diriamba, Nicaragua (population, 50 000). Seventy-nine subjects were female (53.8%), and distribution by age groups was as follows: <12 months, 43 (29.3%); 12 to 24 months, 35 (23.8%); 25 to 36 months, 29 (19.7%); 37 to 71 months, 39 (26.5%); >71 months, 1 (0.7%). All but 2 of the 47 children (32%) who consumed breast milk were breast-fed at least twice daily. The mean duration of diarrhea before enrollment was 2.9 days, and subjects averaged 3.9 stools in the 24 h before enrollment. Mucus was present in almost one-half of cases, and blood was present in 12.2% of cases. In 19 cases (12.9%) antibiotic treatment had been given within the preceding 2 weeks. Giardia lamblia was the most commonly identified organism (47 cases), followed by Ascaris lumbricoides (16 cases), Shigella spp. (8 cases), Trichuris trichiura (5 cases) and V. cholerae (2 cases). Salmonella spp. were sought but were not isolated from any specimen. Among the 19 children who had received antibiotics in the prior 2 weeks, 18 had a negative stool culture and V. cholerae was isolated in one case. Fecal leukocytes were detected by methylene blue stain in 41 cases (27.9%), and Leukotest results were positive in 48 cases (32.7%). In the entire study population Leukotest results were positive in a higher proportion of breast-fed children (23 of 47 cases; 48.9%) than non-breast-fed children (25 of 100 cases; 25%). There was a very high correlation between results of methylene blue stain and Leukotest assay (Table 1). With the methylene blue test as a standard for comparison, the sensitivity and positive predictive value of the Leukotest vs. methylene blue stain did not vary with breast-feeding status, but substantial decreases in both specificity (from 91.1% to 77.8%) and negative predictive value (from 96% to 87.5%) were observed for breast-fed children compared with non-breast-fed children.TABLE 1: Correlations between Leukotest results vs. methylene blue stain in Nicaraguan children with diarrhea In the 8 cases that were culture positive for Shigella spp., methylene blue stain and Leukotest showed identical sensitivity (75%) and negative predictive value (98%). Among the 18 subjects who had received antibiotics and who had negative stool cultures, 8 (44.4%) had positive Leukotest results and 7 (38.8%) had positive methylene blue results. It is likely that some of these were cases of shigellosis that were not detected because of antibiotic use, or they were cases caused by agents not identified by our laboratory. The presence of fecal leukocytes, detected by either methylene blue stain or Leukotest, was compared in groups stratified according to the presence or absence of helminth infection, i.e. infection with either A. lumbricoides or T. trichiura. There were no significant differences in the prevalence of positive methylene blue stain or positive Leukotest results in these 2 groups, suggesting that helminth infection is not an important consideration in interpretation of these tests in this population with high rates of intestinal helminthiasis. In the case of the methylene blue stain, positive results were found in 3 of 16 (18.7%) cases with intestinal helminths and in 39 of 131 (29.7%) cases without helminths (P = 0.55, two-tailed Fisher exact test). In the case of the Leukotest, positive results were found in 5 of 16 (31.2%) cases with intestinal helminths and in 43 of 131 (32.8%) cases without helminths (P = 0.89, Mantel-Haenzel test). Discussion. The major findings of this project were: (1) there is a high correlation between results of methylene blue stain and Leukotest assay in Nicaraguan children with diarrhea, although specificity and negative predictive value of the Leukotest were better in non-breast-fed than in breast-fed children; (2) neither methylene blue stain or Leukotest results were related to the presence of intestinal helminths. To our knowledge this is the first published study comparing results of the Leukotest assay in groups stratified by breast milk intake, because other studies have either not examined this cofactor or have excluded breast-fed children. It is also the first published analysis of Leukotest results according to presence of intestinal helminths. In clinical practice the methylene blue stain and Leukotest appear to be roughly equivalent, and both may have advantages in specific situations. Although the specificity of the Leukotest for detection of fecal leukocytes was diminished in breast-fed children vs. non-breast-fed children, in our study population the correlation between results of these tests was high. The relative loss of specificity and negative predictive value in breast fed children probably reflects the effect of fecal lactoferrin from human milk in the diet, and is probably not significant in practical terms because the major goal of a screening test is high sensitivity. Although specimens were examined soon after collection, it is likely that the Leukotest is more specific than indicated by our data because it may detect cases with low numbers of leukocytes or leukocytes that have degraded, both of which are usually missed by microscopy. Infections with Ascaris and Trichuris can produce intense inflammatory reactions with mixed eosinophilic infiltrates in the distal colon and rectal bleeding; therefore the presence of these helminths could conceivably confound the interpretation of tests that detect fecal lactoferrin. In our study intestinal helminth infections were prevalent but were not related to the presence of positive results by either methylene blue stain or Leukotest. This is an important consideration in many developing countries, where infectious diarrhea and intestinal helminths are found together in a significant number of patients. These infections are generally not associated with diarrhea, and we assumed that the prevalence of invasive enteropathogens (including both those which we could detect, such as Shigella, and those we could not, such as Campylobacter) would be similar in both groups. Limitations of funding and laboratory resources limited the data we could collect, so we were unable to examine results according to presence or absence of certain pathogens, such as Campylobacter. For this reason no attempt was made to stratify results by the presence or absence of bacterial enteropathogens, which would be limited to those we were able to detect. We are also limited in our ability to extrapolate the lack of association between Leukotest results and helminth infections to the few severe cases with very high parasite burdens, which may provide different results. However, these results extend our understanding of two important cofactors that must be considered when interpreting results of fecal lactoferrin detection kits. Acknowledgments. We thank the British Embassy in Nicaragua for providing laboratory equipment and the Gorgas Memorial Fellowship Fund of the American Society of Tropical Medicine and Hygiene for providing additional funding to support this project. We also thank TechLab, Inc., for generously providing the Leukotest kits used in this project, Drs. Oswaldo Mercado and Rodolfo Correa of the Sistema Local de Atencion Integral en Salud of Carazo and Mr. Mario Villa of New Orleans for their enthusiastic support and encouragement. Richard A. Oberhelman, M.D. S. Efrain Guerrero, M.D. Doris Mercado, B.A. Marta Lucia Fernandez, M.D. Robertino Mera, M.D., Ph.D. Department of Tropical Medicine; Tulane University School of Public Health and Tropical Medicine (RAO); Louisiana State University Medical Center (RM); New Orleans, LA CICEN (Centro para Investigaciones Clinicas de Enfermedades de la Niñez) Project (SEG, DM); Sistema Local de Atencion Integral en Salud of Carazo; Ministry of Health (DM, MLF); Diriamba, Carazo, Nicaragua" @default.
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• W2020179542 title "OBSERVATIONS ON THE IMPACT OF BREAST-FEEDING AND OF INTESTINAL HELMINTHIASIS ON A RAPID AGGLUTINATION ASSAY FOR FECAL LACTOFERRIN IN NICARAGUAN CHILDREN WITH DIARRHEA" @default.
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