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Objectives:Lactoferrin (LF) is a breast milk glycoprotein with protective effects against neonatal infections, mainly in premature and low-birth-weight (LBW) neonates. The aims of this study were to determine LF concentration in breast milk of mothers of LBW infants during the first 2 months postpartum, and to identify the factors associated with LF concentration.Study Design:Prospective study conducted as a part of an ongoing clinical trial in three Neonatal Units in Peru. We included 346 mothers of neonates with a birth weight <2000 g. We measured LF concentration in four stages of lactation using a commercial enzyme-linked immunosorbent assay kit. Multivariate analysis was performed to assess the association between maternal and neonatal factors, and LF concentration.Results:We collected 695 milk samples. LF mean concentration±standard deviation was 14.92±7.96 mg ml−1in colostrum (n=277), 10.73±5.67 in transitional milk (n=55), 10.34±6.27 at 1 month (n=259) and 8.52±6.47 at 2 months (n=104). There was a significant difference in LF concentration between different stages of lactation (P<0.001). Mothers with higher LF concentration in colostrum had higher values in the following 2 months. High maternal income and multiple gestation were significantly associated with higher LF levels; in contrast, maternal peripartum infections and male neonatal gender were associated with lower LF levels.Conclusions:LF concentration in breast milk of mothers of LBW infants was high and remained elevated even at 1 and 2 months postpartum. LF concentration in colostrum was higher in mothers with higher income and multiple pregnancies, and lower in mothers with peripartum infections.

ABSTRACTInfectious diarrhea can be classified based on its clinical presentation as noninflammatory or inflammatory disease. In developing countries, among inflammatory diarrhea cases, Shigellais the most common cause, followed by Campylobacterand Salmonella. Because the time frame in which treatment choices must be made is short and conventional stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive detection technique. The purpose of our study was to develop a multiplex real-time PCR procedure to simultaneously identify Campylobacterspp., Salmonellaspp., and Shigellaspp. Primers were designed to amplify the invA, ipaH, and 16S rRNA genes simultaneously in a single reaction to detect Salmonella, Shigella, and Campylobacter, respectively. Using this approach, we correctly identified 102 of 103 strains of the targeted enteropathogens and 34 of 34 other pathogens. The melting temperatures were 82.96 ± 0.05°C for invA, 85.56 ± 0.28°C for ipaH, and 89.21 ± 0.24°C for 16S rRNA. The limit of accurate quantification for the assay in stool samples was 104CFU g-1; however, the limit of detection was 103CFU g-1. This assay is a simple, rapid, inexpensive, and reliable system for the practical detection of these three enteropathogens in clinical specimens.

ABSTRACTEnterotoxigenic Escherichia coli(ETEC) is a major cause of childhood diarrhea. The present study sought to determine the prevalence and distribution of toxin types, colonization factors (CFs), and antimicrobial susceptibility of ETEC strains isolated from Peruvian children. We analyzed ETEC strains isolated from Peruvian children between 2 and 24 months of age in a passive surveillance study. Five E. colicolonies per patient were studied by multiplex real-time PCR to identify ETEC virulence factors. ETEC-associated toxins were confirmed using a GM1-based enzyme-linked immunosorbent assay. Confirmed strains were tested for CFs by dot blot assay using 21 monoclonal antibodies. We analyzed 1,129 samples from children with diarrhea and 744 control children and found ETEC in 5.3% and 4.3%, respectively. ETEC was more frequently isolated from children >12 months of age than from children <12 months of age (P< 0.001). Fifty-two percent of ETEC isolates from children with diarrhea and 72% of isolates from controls were heat-labile enterotoxin (LT) positive and heat-stable enterotoxin (ST) negative; 25% and 19%, respectively, were LT negative and ST positive; and 23% and 9%, respectively, were LT positive and ST positive. CFs were identified in 64% of diarrheal samples and 37% of control samples (P< 0.05). The most common CFs were CS6 (14% and 7%, respectively), CS12 (12% and 4%, respectively), and CS1 (9% and 4%, respectively). ST-producing ETEC strains caused more severe diarrhea than non-ST-producing ETEC strains. The strains were most frequently resistant to ampicillin (71%) and co-trimoxazole (61%). ETEC was thus found to be more prevalent in older infants. LT was the most common toxin type; 64% of strains had an identified CF. These data are relevant in estimating the burden of disease due to ETEC and the potential coverage of children in Peru by investigational vaccines.

ABSTRACTFive Escherichia colicolonies/patient were studied to evaluate the reliability of a multiplex real-time PCR assay for detection of diarrheagenic Escherichia coligroups, using a pool of five colonies rather than individual colonies. Sensitivity and specificity were 98% and 100%, respectively, at a fifth of the cost of the individual colony analysis.

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