Lucie Ecker

Age-Related Susceptibility to Infection with Diarrheagenic Escherichia coli among Infants from Periurban Areas in Lima, Peru

BACKGROUND Diarrheagenic Escherichia coli strains are being recognized as important pediatric enteropathogens worldwide. However, it is unclear whether there are differences in age-related susceptibility to specific strains, especially among infants. METHODS We conducted a passive surveillance cohort study of diarrhea that involved 1034 children aged 2-12 months in Lima, Peru. Control stool samples were collected from randomly selected children without diarrhea. All samples were analyzed for common enteric pathogens and for diarrheagenic E. coli with use of multiplex real-time polymerase chain reaction. RESULTS The most frequently isolated pathogens in 1065 diarrheal episodes were diarrheagenic E. coli strains (31%), including enteroaggregative (15.1%) and enteropathogenic E. coli (7.6%). Diarrheagenic E. coli, Campylobacter species, and rotavirus were more frequently isolated from infants aged >or=6 months. Among older infants, diffusely adherent E. coli and enterotoxigenic E. coli were more frequently isolated from diarrheal samples than from control samples (P <.05). Children aged >or=6 months who were infected with enterotoxigenic E. coli had a 4.56-fold increased risk of diarrhea (95% confidence interval, 1.20-17.28), compared with younger children. Persistent diarrhea was more common in infants aged <6 months (13.5% vs 3.6%; P <.001). Among children with diarrheagenic E. coli-positive samples, coinfections with other pathogens were more common in children with diarrhea than in control children (40.1% vs 15.6%; P <.001). CONCLUSIONS Diarrheagenic E. coli strains were more frequently isolated in samples from older infants. In this setting with high frequency of pathogen exposure and high frequency of breastfeeding, we hypothesize that the major age-related differences result from decreased exposure to milk-related protective factors and from increased exposure to contaminated food and water.

Quantitative Real-time Polymerase Chain Reaction for Enteropathogenic Escherichia coli: A Tool for Investigation of Asymptomatic Versus Symptomatic Infections

BACKGROUND Enteropathogenic Escherichia coli (EPEC) strains are pediatric pathogens commonly isolated from both healthy and sick children with diarrhea in areas of endemicity. The aim of this study was to compare the bacterial load of EPEC isolated from stool samples from children with and without diarrhea to determine whether bacterial load might be a useful tool for further study of this phenomenon. METHODS EPEC was detected by polymerase chain reaction (PCR) of colonies isolated on MacConkey plates from 53 diarrheal and 90 healthy children aged <2 years. DNA was isolated from stool samples by cetyltrimethylammonium bromide extraction. To standardize quantification by quantitative real-time PCR (qRT-PCR), the correlation between fluorescence threshold cycle and copy number of the intimin gene of EPEC E2348/69 was determined. RESULTS The detection limit of qRT-PCR was 5 bacteria/mg stool. The geometric mean load in diarrhea was 299 bacteria/mg (95% confidence interval [CI], 77-1164 bacteria/mg), compared with 29 bacteria/mg (95% CI, 10-87 bacteria/mg) in control subjects (P = .016). Bacterial load was significantly higher in children with diarrhea than in control subjects among children <12 months of age (178 vs 5 bacteria/mg; P = .006) and among children with EPEC as the sole pathogen (463 vs 24 bacteria/mg; P = .006). CONCLUSIONS EPEC load measured by qRT-PCR is higher in diarrheal than in healthy children. qRT-PCR may be useful to study the relationship between disease and colonization in settings of endemicity.

Genotypic and Phenotypic Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Peruvian Children

ABSTRACT Enterotoxigenic 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. coli colonies 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.

Validation of five-colony pool analysis using multiplex real-time PCR for detection of diarrheagenic Escherichia coli.

ABSTRACT Five Escherichia coli colonies/patient were studied to evaluate the reliability of a multiplex real-time PCR assay for detection of diarrheagenic Escherichia coli groups, 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.

Allelic variability of critical virulence genes (eae, bfpA and perA) in typical and atypical enteropathogenic Escherichia coli in Peruvian children.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhoea in developing countries. The aim of this study was to describe the allelic diversity of critical EPEC virulence genes and their association with clinical characteristics. One hundred and twenty EPEC strains isolated from a cohort diarrhoea study in Peruvian children were characterized for the allele type of eae (intimin), bfpA (bundlin pilin protein of bundle-forming pilus) and perA (plasmid encoded regulator) genes by PCR-RFLP. Atypical EPEC strains (eae+, bfp-) were the most common pathotype in diarrhoea (54/74, 73 %) and control samples from children without diarrhoea (40/46, 87 %). Overall, there were 13 eae alleles; the most common were beta (34/120, 28 %), theta (24/120, 20 %), kappa (14/120, 12 %) and mu (8/120, 7 %). There were five bfpA alleles; the most common were beta1/7 (10/26), alpha3 (7/26) and beta5 (3/26). There were three perA alleles: beta (8/16), alpha (7/16) and gamma (1/16). The strains belonged to 36 distinct serogroups; O55 was the most frequent. The gamma-intimin allele was more frequently found in diarrhoea episodes of longer duration (>7 days) than those of shorter duration (3/26, 12 % vs 0/48, 0 %, P<0.05). The kappa-intimin allele had the highest clinical severity score in comparison with other alleles (P<0.05). In Peruvian children, the virulence genes of EPEC strains are highly variable. Further studies are needed to evaluate additional virulence markers to determine whether relationships exist between specific variants and clinical features of disease.

Factors Affecting Caregivers' Use of Antibiotics Available Without a Prescription in Peru

OBJECTIVE: To determine factors that affect caregivers’ decisions about antibiotic use in children in settings where antibiotics are available without prescription. METHODS: In a house-to-house survey, 1200 caregivers in 3 periurban districts of Lima, Peru, were asked about antibiotic use in young children. RESULTS: In this sample, 87.2% of children aged <5 years had received an antibiotic drug in their lives; 70.3% had received antibiotics before 1 year of age, and 98.8% of those had been prescribed by a physician. Given hypothetical cases of common cold and nondysenteric diarrhea, caregivers would seek medical advice in 76.4% and 87.1%, respectively, and 84.6% of caregivers said they respected medical decisions even if an antibiotic was not prescribed. Caregivers with high school–level education accepted 80% more medical decisions of not using an antibiotic and used fewer pharmacist-recommended antibiotics. For each additional year of life, the risk of self-medicated antibiotic use and the use of pharmacist-recommended antibiotics increased in 30%. (OR: 1.3, 95% CI: 1.1–1.4, P = .001 and OR: 1.3, 95% CI: 1.2–1.5, P < .001, respectively). Caregivers respected a medical decision of not prescribing an antibiotic 5 times more when physicians had explained the reason for their advice (OR: 5.0, 95% CI: 3.2–7.8, P < .001). CONCLUSIONS: Prescribed antibiotic use in these young children is common. Even if they are available without prescription, caregivers usually comply with medical advice and follow physicians’ recommendations when antibiotics are not prescribed. Improving physician prescribing habits could reduce irrational antibiotic use, decreasing future caregiver-driven misuse.

Fecal Leukocytes in Children Infected with Diarrheagenic Escherichia coli

ABSTRACT The purpose of this study was to determine the presence and quantity of fecal leukocytes in children infected with diarrheagenic Escherichia coli and to compare these levels between diarrhea and control cases. We analyzed 1,474 stool samples from 935 diarrhea episodes and 539 from healthy controls of a cohort study of children younger than 2 years of age in Lima, Peru. Stools were analyzed for common enteric pathogens, and diarrheagenic E. coli isolates were studied by a multiplex real-time PCR. Stool smears were stained with methylene blue and read by a blinded observer to determine the number of polymorphonuclear leukocytes per high-power field (L/hpf). Fecal leukocytes at >10 L/hpf were present in 11.8% (110/935) of all diarrheal episodes versus 1.1% (6/539) in controls (P < 0.001). Among stool samples with diarrheagenic E. coli as the only pathogen isolated (excluding coinfection), fecal leukocytes at >10 L/hpf were present in 8.5% (18/212) of diarrhea versus 1.3% (2/157) of control samples (P < 0.01). Ninety-five percent of 99 diarrheagenic E. coli diarrhea samples were positive for fecal lactoferrin. Adjusting for the presence of blood in stools, age, sex, undernutrition, and breastfeeding, enterotoxigenic E. coli (ETEC) isolation as a single pathogen, excluding coinfections, was highly associated with the presence of fecal leukocytes (>10 L/hpf) with an odds ratio (OR) of 4.1 (95% confidence interval [CI], 1.08 to 15.51; P < 0.05). Although diarrheagenic E. coli was isolated with similar frequencies in diarrhea and control samples, clearly it was associated with a more inflammatory response during symptomatic infection; however, in general, these pathogens elicited a mild inflammatory response.

Safety and immunogenicity of an investigational fully liquid hexavalent DTaP-IPV-Hep B-PRP-T vaccine at two, four and six months of age compared with licensed vaccines in Latin America.

Objective: This trial assessed the safety of a fully liquid investigational hexavalent DTaP-IPV-Hep B-PRP-T vaccine containing 10 &mgr;g Hansenula polymorpha–derived recombinant hepatitis B (hep B) antigen for primary vaccination of infants at 2, 4 and 6 months of age compared with licensed comparators. Methods: Participants received the DTaP-IPV-Hep B-PRP-T vaccine (group 1, N = 1422) or licensed DTwP-Hep B//Hib (Tritanrix-Hep B/Hib) and oral poliovirus vaccines (group 2, N = 711). The incidence of severe fever (≥39.6°C rectal equivalent) in the 2 groups was compared statistically; reactogenicity was evaluated from parental reports. Anti-Hep B antibody titers were measured in a subset of participants (no hepatitis B vaccination at birth) 1 month after dose 3. Results: The investigational vaccine was well tolerated. After any dose, fever (rectal equivalent temperature ≥38°C) was observed in 74.8% and 92.7% of participants in groups 1 and 2; severe fever was observed in 4.0% and 5.5% of participants. Solicited injection site and systemic reactions were numerically less frequent in group 1 than group 2, although this difference was not assessed statistically. In both groups, all participants included in the immunogenicity analysis achieved anti-Hep B ≥10 mIU/mL and ≥96.2% of participants achieved anti-Hep B ≥100 mIU/mL, although geometric mean titer was approximately 3-fold lower for the investigational vaccine. Conclusion: This new, fully liquid acellular pertussis hexavalent vaccine demonstrated less reactogenicity than the licensed comparator whole cell pertussis vaccine and was highly immunogenic for the new Hep B valence.

Niveles de resistencia a quinolonas y otros antimicrobianos en cepas de Escherichia coli comensales en niños de la zona periurbana de Lima, Perú

El objetivo principal del estudio fue establecer el nivel de resistencia a antimicrobianos en un total de 222 cepas comensales de E. coli de origen fecal, en Peru. Las frecuencias de resistencia encontrados, frente los antimicrobianos evaluados, fueron: ampicilina (62,6%), cotrimoxazol (48,6%), tetraciclina (43,0%) y cloranfenicol (15,8%). Destacan los elevados niveles de resistencia a quinolonas: 32% al acido nalidixico (NAL) y 12% a ciprofloxacino (CIP). Estos elevados niveles hacia las quinolonas en cepas comensales aisladas en ninos de esta franja de edad, realzan el uso extendido y el impacto de consumo de este tipo de antimicrobianos en la comunidad, mostrando el riesgo potencial de su perdida de utilidad en el area.

Norovirus prevalence in 'pathogen negative' gastroenteritis in children from periurban areas in Lima, Peru.

Norovirus was detected in 17.4% of 224 diarrhoeal samples from children younger than 24 months of age in Lima, in whom all common pathogens had been excluded (pathogen negative). Norovirus was identified more frequently in children older than 12 months of age than in younger children (34% vs 8%, P<0.001). Among norovirus-positive samples, genogroup II was the predominant group (92%). Compared with rotavirus, norovirus episodes tended to be of shorter duration and less severe. The role of norovirus as a cause of diarrhoea and the ascertainment of its severity in developing countries needs further confirmation by future epidemiological studies.