Colonizing and Virulence Factors in Enterotoxigenic Escherichia coli from Peru.

Enterotoxigenic Escherichia coli (ETEC) ranks among the most relevant diarrheagenic pathogens. Efforts to design vaccines to fight ETEC have been focused on colonizing factors (CFs) and atypical virulence factors (AVF). An effective vaccine must account for differences in the regional prevalence of these CFs and AVFs to be truly effective in a given area. In the present study, the presence of 16 CFs and 9 AVFs, as well as the heat-stable (ST) variants (STh or STp), was established by polymerase chain reaction in 205 Peruvian ETEC isolates (120 from diarrhea cases and 85 from healthy controls). Ninety-nine (48.3%) isolates were heat-labile, 63 (30.7%) ST, and 43 (21.0%) presented both toxins. Of ST isolates, 59 (28.8%) possessed STh, 30 (14.6%) STp, five (2.4%) both STh and STp, and 12 (5.8%) were not amplified for any variant tested. The presence of CFs was associated with diarrhea (P < 0.0001). The presence of eatA as well as concomitant presence of CSI, CS3, and CS21 and of C5 and C6 was statistically related to diarrhea cases. The present results suggests that, if effective, a vaccine considering CS6, CS20, and CS21, together with EtpA, would provide protection against 64.4% of the isolates analyzed, whereas the addition of CS12 and EAST1 would lead to 83.9% coverage. Large studies are needed to establish both the ideal candidates to be considered to develop a vaccine effective in the area, and continuous surveillance is needed to detect displacement of circulating isolates that may compromise future vaccines.

Supervisión de ensayos clínicos en hospitales del Seguro Social de Salud del Perú: enfoque administrativo y regulatorio / Supervision of clinical trials in Social Health Insurance at Peruvian Hospitals: administrative and regulatory approach

RESUMEN El objetivo del estudio fue describir las características de los ensayos clínicos (EC) supervisados por el Instituto de Evaluación de Tecnologías en Salud e Investigación en EsSalud entre el 2015 y 2018 y las principales observaciones de las supervisiones realizadas. Se realizó un estudio descriptivo de 82 ensayos clínicos supervisados entre 2015 y 2018. La mayoría de los ensayos clínicos fueron estudios de fase III (81,7%), la vía de administración más frecuente de los productos de estudio fue oral (47,6%) y la mayoría fueron patrocinados por la industria farmacéutica (96,3%). Las observaciones más frecuentes fueron las relacionadas al contrato de estudio (83,8%), al pago por concepto de overhead (57,3%) y a la falta de documentos regulatorios (47,6%). Estos hallazgos permiten la identificación de oportunidades de mejora en la regulación y gestión de la investigación.

ABSTRACT The objective of the study was to describe the characteristics of the Clinical Trials (CT) supervised by the Institute of Health Technology Assessment and Research carried out in EsSalud between 2015 and 2018 and the main observations of the supervisions completed. A descriptive study of 82 supervised clinical trials was conducted between 2015 and 2018. Most of the clinical trials were phase III studies (81.7%); the most frequent route of administration of the study products was oral (47.6%), and most were sponsored by the pharmaceutical industry (96.3%). The most frequent observations were those related to the study contract (83.8%), overhead payment (57.3%), and the lack of regulatory documents (47.6%). These findings allow the identification of opportunities for improvement in research regulation and management.

[Supervision of clinical trials in Social Health Insurance at Peruvian Hospitals: administrative and regulatory approach]. / Supervisión de ensayos clínicos en hospitales del Seguro Social de Salud del Perú: enfoque administrativo y regulatorio.

The objective of the study was to describe the characteristics of the Clinical Trials (CT) supervised by the Institute of Health Technology Assessment and Research carried out in EsSalud between 2015 and 2018 and the main observations of the supervisions completed. A descriptive study of 82 supervised clinical trials was conducted between 2015 and 2018. Most of the clinical trials were phase III studies (81.7%); the most frequent route of administration of the study products was oral (47.6%), and most were sponsored by the pharmaceutical industry (96.3%). The most frequent observations were those related to the study contract (83.8%), overhead payment (57.3%), and the lack of regulatory documents (47.6%). These findings allow the identification of opportunities for improvement in research regulation and management.

El objetivo del estudio fue describir las características de los ensayos clínicos (EC) supervisados por el Instituto de Evaluación de Tecnologías en Salud e Investigación en EsSalud entre el 2015 y 2018 y las principales observaciones de las supervisiones realizadas. Se realizó un estudio descriptivo de 82 ensayos clínicos supervisados entre 2015 y 2018. La mayoría de los ensayos clínicos fueron estudios de fase III (81,7%), la vía de administración más frecuente de los productos de estudio fue oral (47,6%) y la mayoría fueron patrocinados por la industria farmacéutica (96,3%). Las observaciones más frecuentes fueron las relacionadas al contrato de estudio (83,8%), al pago por concepto de overhead (57,3%) y a la falta de documentos regulatorios (47,6%). Estos hallazgos permiten la identificación de oportunidades de mejora en la regulación y gestión de la investigación.

High Prevalence and Increased Severity of Norovirus Mixed Infections Among Children 12-24 Months of Age Living in the Suburban Areas of Lima, Peru.

In an active diarrhea surveillance study of children aged 12-24 months in Lima, Peru, norovirus was the most common pathogen identified. The percentage of mixed (bacterial and noroviral) infections was significantly higher among norovirus-positive samples (53%) than among norovirus-negative samples (12%). The combination of norovirus with the most common bacterial pathogens was associated with increased clinical severity over that of either single-pathogen norovirus or single-pathogen bacterial infections.

Prevention of renal damage caused by Shiga toxin type 2: Action of Miglustat on human endothelial and epithelial cells.

Typical hemolytic uremic syndrome (HUS) is responsible for acute and chronic renal failure in children younger than 5 years old in Argentina. Renal damages have been associated with Shiga toxin type 1 and/or 2 (Stx1, Stx2) produced by Escherichia coli O157:H7, although strains expressing Stx2 are highly prevalent in Argentina. Human glomerular endothelial cells (HGEC) and proximal tubule epithelial cells are very Stx-sensitive since they express high levels of Stx receptor (Gb3). Nowadays, there is no available therapy to protect patients from acute toxin-mediated cellular injury. New strategies have been developed based on the Gb3 biosynthesis inhibition through blocking the enzyme glucosylceramide (GL1) synthase. We assayed the action of a GL1 inhibitor (Miglustat: MG), on the prevention of the renal damage induced by Stx2. HGEC primary cultures and HK-2 cell line were pre-treated with MG and then incubated with Stx2. HK- 2 and HGEC express Gb3 and MG was able to decrease the levels of this receptor. As a consequence, both types of cells were protected from Stx2 cytotoxicity and morphology damage. MG was able to avoid Stx2 effects in human renal cells and could be a feasible strategy to protect kidney tissues from the cytotoxic effects of Stx2 in vivo.

Comparative analysis of antimicrobial resistance in enterotoxigenic Escherichia coli isolates from two paediatric cohort studies in Lima, Peru.

BACKGROUND: Antibiotic resistance is increasing worldwide, being of special concern in low- and middle-income countries. The aim of this study was to determine the antimicrobial susceptibility and mechanisms of resistance in 205 enterotoxigenic Escherichia coli (ETEC) isolates from two cohort studies in children <24 months in Lima, Peru. METHODS: ETEC were identified by an in-house multiplex real-time PCR. Susceptibility to 13 antimicrobial agents was tested by disk diffusion; mechanisms of resistance were evaluated by PCR. RESULTS: ETEC isolates were resistant to ampicillin (64%), cotrimoxazole (52%), tetracycline (37%); 39% of the isolates were multidrug-resistant. Heat-stable toxin producing (ETEC-st) (48%) and heat-labile toxin producing ETEC (ETEC-lt) (40%) had higher rates of multidrug resistance than isolates producing both toxins (ETEC-lt-st) (21%), p<0.05. Only 10% of isolates were resistant to nalidixic acid and none to ciprofloxacin or cefotaxime. Ampicillin and sulfamethoxazole resistance were most often associated with blaTEM (69%) and sul2 genes (68%), respectively. Tetracycline resistance was associated with tet(A) (49%) and tet(B) (39%) genes. Azithromycin inhibitory diameters were ≤15 mm in 36% of isolates, with 5% of those presenting the mph(A) gene. CONCLUSIONS: ETEC from Peruvian children are often resistant to older, inexpensive antibiotics, while remaining susceptible to ciprofloxacin, cephalosporins and furazolidone. Fluoroquinolones and azithromycin remain the drugs of choice for ETEC infections in Peru. However, further development of resistance should be closely monitored.

Bovine lactoferrin decreases cholera-toxin-induced intestinal fluid accumulation in mice by ganglioside interaction.

Secretory diarrhea caused by cholera toxin (CT) is initiated by binding of CT's B subunit (CTB) to GM1-ganglioside on the surface of intestinal cells. Lactoferrin, a breast milk glycoprotein, has shown protective effect against several enteropathogens. The aims of this study were to determine the effect of bovine-lactoferrin (bLF) on CT-induced intestinal fluid accumulation in mice, and the interaction between bLF and CT/CTB with the GM1-ganglioside receptor. Fluid accumulation induced by CT was evaluated in the mouse ileal loop model using 56 BALB/c mice, with and without bLF added before, after or at the same time of CT administration. The effect of bLF in the interaction of CT and CTB with GM1-ganglioside was evaluated by a GM1-enzyme-linked immunosorbent assay. bLF decreased CT-induced fluid accumulation in the ileal loop of mice. The greatest effect was when bLF was added before CT (median, 0.066 vs. 0.166 g/cm, with and without bLF respectively, p<0.01). We conclude that bLF decreases binding of CT and CTB to GM1-ganglioside, suggesting that bLF suppresses CT-induced fluid accumulation by blocking the binding of CTB to GM1-ganglioside. bLF may be effective as adjunctive therapy for treatment of cholera diarrhea.

Genotypic characterization of enterotoxigenic Escherichia coli strains causing traveler's diarrhea.

This study aims to characterize the presence of virulence factors of enterotoxigenic Escherichia coli (ETEC) causing traveler's diarrhea. Among 52 ETEC isolates, the most common toxin type was STh, and the most frequent colonization factors (CFs) were CS21, CS6, and CS3. On the other hand, the nonclassical virulence factors EAST1 and EatA were frequently present.

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.

[Frequency and pathotypes of diarrheagenic Escherichia coli in Peruvian children with and without diarrhea]. / Frecuencia y patotipos de Escherichia coli diarrogénicas en niños peruanos con y sin diarrea.

UNLABELLED: INTRODUCTION; Diarrheagenic E. coli (DEC) are a major cause of diarrhea in children in developing countries. However, they are not part of routine diagnosis in clinical laboratories. OBJECTIVES: To determine the DEC prevalence in Peruvian children and to describe the genetic variability of these strains. MATERIALS AND METHODS: A total of 8 003 E. coli strains previously isolated from eight different studies of diarrhea in children, mainly from peri-urban areas of Lima, were analyzed. Diagnosis of DEC was done with Multiplex real-time PCR using genes for each of the 6 DEC groups. Conventional PCR was performed for the detection of additional virulence genes. RESULTS: Globally, the mean prevalence in diarrhea samples (n=4,243) was: enteroaggregative E. coli (EAEC) 9.9%, enteropathogenic E. coli (EPEC) 8.5%, enterotoxigenic E. coli (ETEC) 6.9%, diffusely adherent E. coli (DAEC) 4.8%, Shiga toxin-producing E. coli (STEC) 0.8% and enteroinvasive E. coli (EIEC) 0.6%. The relative frequency of each pathogen varies according to the age and the type of study. The main pathotypes in control samples (n=3,760) were EPEC (10.9%) and EAEC (10.4%). An important variability in the virulence genes frequency and molecular resistance mechanisms for each pathotype was found, without differences between diarrhea and control groups. CONCLUSIONS: DEC are a major cause of diarrhea in Peruvian children. These pathogens are highly heterogeneous. Additional studies are required to determine the prevalence in rural areas of Peru and in severe diarrhea cases.

[Adhesion patterns in diffusely adherent Escherichia coli (DAEC) strains isolated from children with and without diarrhea]. / Patrones de adherencia de cepas de Escherichia coli Difusamente adherente (DAEC) provenientes de niños con y sin diarrea.

INTRODUCTION: Diffusely adherent E. coli (DAEC) is the sixth recognized group of diarrheagenic E. coli. However, its association with diarrhea remains controversial. Variability in the adherence patterns of clinical strains is unknown. OBJECTIVES: To compare the adherence patterns between strains isolated from children with and without diarrhea. MATERIALS AND METHODS: A total of 31 DAEC strains were analyzed, 25 from children with diarrhea and 6 from asymptomatic (control) children, isolated from a cohort study of children under one year of age in the southern districts of Lima. DAEC were identified by PCR (daaD gene). The pattern and adherence score in HEp-2 cell culture were evaluated, Actin polimerization was determined by fluorescence actin staining (FAS) and motility was evaluated by conventional microbiology methods. RESULTS: Diffuse adherence pattern was found in 88% of diarrhea samples and in the total of control strains. The number of bacteria adhered per cell was significantly lower in diarrhea samples (p<0.05). However, actin polymerization was greater in diarrhea samples (60% vs. 17%). Motility test was positive in 60% of the diarrhea samples and in all control samples. CONCLUSIONS: Our findings suggest a difference between adherence patterns, actin polymerization and motility between DAEC strains corresponding to diarrhea and control groups. The significance of these results must be confirmed with a bigger number of strains and determining the presence of virulence genes in the strains.

Frecuencia y patotipos de Escherichia coli diarrogénicas en niños peruanos con y sin diarrea / Frequency and pathotypes of diarrheagenic Escherichia coli in peruvian children with and without diarrhea

Introducción. Las E. coli diarrogénicas (DEC) son una de las principales causas de diarrea en niños en países en vías de desarrollo. Sin embargo, no son rutinariamente diagnosticadas en los laboratorios clínicos. Objetivos. Determinar la prevalencia de las DEC en niños peruanos y describir la variabilidad genética de estas cepas. Materiales y métodos. Se utilizaron 8 003 cepas de E. coli previamente aisladas de ocho estudios previos de diarrea en niños, mayormente en zonas periurbanas de Lima. El diagnóstico de las DEC fue a través de un PCR múltiple a tiempo real para los seis grupos de DEC. Se empleó PCR para la determinación de genes adicionales de virulencia. Resultados. La prevalencia promedio global en muestras de diarrea (n=4 243) fue: E. coli enteroagregativa (EAEC) 9,9 por ciento, enteropatogénica (EPEC) 8,5 por ciento, enterotoxigénica (ETEC) 6,9 por ciento, difusamente adherente (DAEC) 4,8 por ciento, productora de toxina shiga (STEC) 0,8 por ciento y enteroinvasiva (EIEC) 0,6 por ciento. La frecuencia relativa de cada patógeno varía según la edad y tipo de estudio. Los principales patotipos en muestras control (n=3 760) fueron EPEC (10,9 por ciento) y EAEC (10,4 por ciento). Se encontró una gran variabilidad en la frecuencia de genes de virulencia para cada patotipo, así como en los mecanismos moleculares de resistencia, sin diferencias significativas entre muestras de diarrea y control. Conclusiones. Las DEC son causa importante de diarrea en niños peruanos. Estos patógenos son altamente heterogéneos. Se requieren estudios adicionales para determinar la prevalencia en zonas rurales del Perú, así como en casos graves de diarrea.

Introduction. Diarrheagenic E. coli (DEC) are a major cause of diarrhea in children in developing countries. However, they are not part of routine diagnosis in clinical laboratories. Objectives. To determine the DEC prevalence in Peruvian children and to describe the genetic variability of these strains. Materials and methods. A total of 8 003 E. coli strains previously isolated from eight different studies of diarrhea in children, mainly from peri-urban areas of Lima, were analyzed. Diagnosis of DEC was done with Multiplex real-time PCR using genes for each of the 6 DEC groups. Conventional PCR was performed for the detection of additional virulence genes. Results. Globally, the mean prevalence in diarrhea samples (n=4,243) was: enteroaggregative E. coli (EAEC) 9.9 percent, enteropathogenic E. coli (EPEC) 8.5 percent, enterotoxigenic E. coli (ETEC) 6.9 percent, diffusely adherent E. coli (DAEC) 4.8 percent, Shiga toxin-producing E. coli (STEC) 0.8 percent and enteroinvasive E. coli (EIEC) 0.6 percent. The relative frequency of each pathogen varies according to the age and the type of study. The main pathotypes in control samples (n=3,760) were EPEC (10.9 percent) and EAEC (10.4 percent). An important variability in the virulence genes frequency and molecular resistance mechanisms for each pathotype was found, without differences between diarrhea and control groups. Conclusions. DEC are a major cause of diarrhea in Peruvian children. These pathogens are highly heterogeneous. Additional studies are required to determine the prevalence in rural areas of Peru and in severe diarrhea cases.

Patrones de adherencia de cepas de Escherichia coli Difusamente adherente (DAEC) provenientes de niños con y sin diarrea / Adhesion patterns in diffusely adherent Escherichia coli (DAEC) strains isolated from children With and without diarrhea

Introducción. Las E. coli de adherencia difusa (DAEC) son el sexto grupo de E. coli diarrogénicas reconocidas. Su asociación con diarrea es controversial. No se conoce la variabilidad en los patrones de adherencia de cepas clínicas. Objetivos. Comparar los patrones de adherencia entre cepas aisladas de niños con y sin diarrea. Materiales y métodos. Se analizó 31 cepas DAEC, 25 de diarrea y 6 de niños asintomáticos (control) aislados de un estudio de cohorte de niños menores de 12 meses en el cono sur de Lima. Las DAEC fueron identificadas por PCR (gen daaD). Se evaluó el patrón y grado de adherencia en cultivos de células HEp-2; la polimerización de actina se evaluó por la prueba de coloración de fluorescencia de actina (FAS); y la motilidad se evaluó por métodos convencionales microbiológicos. Resultados. El patrón de adherencia difusa se encontró en el 88 por ciento de muestras de diarrea y en el 100 por ciento de muestras control. La cantidad de bacterias adheridas por célula fue significativamente menor en las muestras de diarrea (p<0,05). Sin embargo, la polimerización de actina fue mayor en las muestras de diarrea (60 por ciento frente a 17 por ciento). La prueba de motilidad fue positiva en el 60 por ciento de las cepas de diarrea y en el total de muestras control. Conclusiones. Nuestros hallazgos sugieren la existencia de diferencia en los patrones de adherencia, polimerización de actina y motilidad entre cepas de DAEC correspondientes a los grupos de diarrea y control. La significancia de estos resultados debe confirmarse con mayor número de cepas, así como la determinación de los genes de virulencia en las cepas.

Introduction. Diffusely adherent E. coli (DAEC) is the sixth recognized group of diarrheagenic E. coli. However, its association with diarrhea remains controversial. Variability in the adherence patterns of clinical strains is unknown. Objectives. To compare the adherence patterns between strains isolated from children with and without diarrhea. Materials and methods. A total of 31 DAEC strains were analyzed, 25 from children with diarrhea and 6 from asymptomatic (control) children, isolated from a cohort study of children under one year of age in the southern districts of Lima. DAEC were identified by PCR (daaD gene). The pattern and adherence score in HEp-2 cell culture were evaluated, Actin polimerization was determined by fluorescence actin staining (FAS) and motility was evaluated by conventional microbiology methods. Results. Diffuse adherence pattern was found in 88 percent of diarrhea samples and in the total of control strains. The number of bacteria adhered per cell was significantly lower in diarrhea samples (p<0.05). However, actin polymerization was greater in diarrhea samples (60 percent vs. 17 percent). Motility test was positive in 60 percent of the diarrhea samples and in all control samples. Conclusions. Our findings suggest a difference between adherence patterns, actin polymerization and motility between DAEC strains corresponding to diarrhea and control groups. The significance of these results must be confirmed with a bigger number of strains and determining the presence of virulence genes in the strains.

Detection of the CS20 colonization factor antigen in diffuse-adhering Escherichia coli strains.

We analyzed a randomly selected group of 30 diffusely adherent (DAEC), 30 enteropathogenic, 30 enteroaggregative, and five Shiga toxin-producing Escherichia coli strains isolated from children with diarrhea. Enterotoxigenic E. coli (ETEC) colonization factors (CFs) were evaluated by a dot-blot assay using 21 CF-specific monoclonal antibodies. Out of 95 non-ETEC strains, three DAEC were found to express coli surface antigen 20 (CS20). No other E. coli expressed CFs. We confirmed the three CS20-positive strains as ETEC-negative by repeat PCR and as toxin-negative by ganglioside-GM1-enzyme-linked immunosorbent assay. To our knowledge, this is the first study that has identified currently recognized CFs in non-ETEC diarrheagenic E. coli strains identified using molecular methods. CFs may be an unrecognized relevant adherence factor in other E. coli, which may then play a role in pathogenesis and the immune response of the host.

Diarrheagenic Escherichia coli in human immunodeficiency virus (HIV) pediatric patients in Lima, Peru.

We conducted a prospective study in three hospitals in Lima in human immunodeficiency virus (HIV) children to determine the frequency of diarrheagenic Escherichia coli. Five E. coli colonies/patients were studied by a multiplex real-time polymerase chain reaction to identify the six currently recognized groups of diarrhea-associated E. coli. We have analyzed 70 HIV-associated diarrheal and 70 control samples from HIV-infected children without diarrhea. Among the diarrheal episodes 19% were persistent, 3% dysenteric, and 33% were associated with moderate or severe dehydration. The diarrheagenic E. coli were the most commonly isolated pathogens in diarrhea (19%) and control samples (26%) (P = 0.42), including enteroaggregative (6% versus 10%), enteropathogenic (6% versus 10%), and enterotoxigenic E. coli (4% versus 3%), respectively. The HIV-infected children with diarrhea had the worse age-related immunosuppression, higher viral loads, and were on highly active antiretroviral treatment (HAART) less often than HIV-infected children without diarrhea. Diarrheagenic E. coli were highly resistant to ampicillin (74%) and cotrimoxazole (70%).