Erik Mercado

New insights into the epidemiology of enteropathogenic Escherichia coli infection

Enteropathogenic Escherichia coli (EPEC) are among the most important pathogens infecting children worldwide and are one of the main causes of persistent diarrhea. EPEC were originally serogroup-defined E. coli associated with infantile diarrhea. As various mechanisms of pathogenesis have been discovered, EPEC classification has come to be based on the presence of specific genes. The eae (intimin) and bfpA (bundle-forming pilus) genes have both been used for identification of EPEC and for subdivision of this group of bacteria into typical and atypical strains. For many years typical EPEC have been considered to be the leading cause of infantile diarrhea in developing countries and were considered rare in industrialized countries. However, current data suggests that atypical EPEC are more prevalent than typical EPEC in both developing and developed countries. Moreover, the duration of diarrhea in patients infected with atypical EPEC is significantly longer than that caused by other pathogens. When comparing the isolation rates of EPEC among children with diarrhea and healthy controls without diarrhea, in general, there is a higher isolation rate in diarrhea, although not significantly higher in all studies. These inconsistencies probably are related to the study patient populations, reflecting a possible age-related susceptibility to infection.

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.

Frecuencia y patotipos de Escherichia coli diarrogénica 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.

Multiplex Real-Time PCR for Detection of Campylobacter, Salmonella, and Shigella

ABSTRACT Infectious diarrhea can be classified based on its clinical presentation as noninflammatory or inflammatory disease. In developing countries, among inflammatory diarrhea cases, Shigella is the most common cause, followed by Campylobacter and 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 Campylobacter spp., Salmonella spp., and Shigella spp. 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 104 CFU g−1; however, the limit of detection was 103 CFU g−1. This assay is a simple, rapid, inexpensive, and reliable system for the practical detection of these three enteropathogens in clinical specimens.

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.

Diarrheagenic Escherichia coli in Human Immunodeficiency Virus (HIV) Pediatric Patients in Lima, Perú

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%).

First report of Streptococcus pneumoniae serotype 6D in South America

Streptococcus pneumoniae includes the two serotypes 6A and 6B as well as two recently discovered serotypes, 6C and 6D, in which the wciNα gene is replaced by wciNβ within the cps locus. Serotype 6D occurrence in Asia (2, 4), the Fiji islands (5) and Europe (7, 9) has recently been reported. To our knowledge, we describe here the first description of serotype 6D isolates identified within the Americas. Using the CDC serogroup 6 serotyping scheme (8), we identified 155 serogroup 6 isolates within a larger collection (n = 693) taken from nasopharyngeal carriage in children <2 years old in Peru (2007 to 2009; 541 strains) (unpublished data) and invasive pneumococcal disease (IPD) surveillance in children in Lima hospitals (2006 to 2009; 152 strains) (reference 10 and unpublished data). Among the 155 serogroup 6 isolates, 26, 105, and 22 isolates were identified as serotypes, 6A, 6B, and 6C, respectively (Table 1). We tentatively identified two 6D isolates on the basis of positive reactions with factor sera for serotype 6B (factor 6c) and serotype 6C (factor 6d) and a negative reaction with factor serum for serotype 6A (factor 6b). We verified the serologic results for all 155 serogroup 6 isolates using a recently developed PCR scheme that efficiently resolves all four serogroup 6 serotypes (6). This scheme discriminates between serotypes 6A/6C and 6B/6D using wciP allele-specific reactions. The presence or absence of wciNβ determined by a second reaction subsequently allowed resolution into all four serotypes (Table 1). Table 1. Quellung and PCR results for 155 serogroup 6 isolates The two 6D isolates, both from carriage, shared the new multilocus sequence typing (MLST) profile ST6148 (ST6148, 8-8-263-12-6-104-14) and were both fully susceptible to a variety of antimicrobial agents by broth microdilution. ST6148 is unrelated to previously described 6D genotypes on the MLST website (http://spneumoniae.mlst.net/, last accessed on 5 January 2011). It is, however, highly related to serotype 6A and 6B isolates both from carriage and invasive disease that were characterized from this same study, differing only at the recP allele. These data, together with data published elsewhere, show identical or related multilocus sequence types shared between serotype 6C or 6D strains containing wciNβ and 6A or 6B strains that contain wciNα (3, 4, 7). This suggests that the two different wciN genes are frequently horizontally transferred between different serogroup 6 strains in nature to effect intraserogroup 6 capsular switch events. On the basis of these genotype observations it also circumstantially appears likely that in some cases wciP is cotransferred with wciN, which could effect, for example, a serotype 6A to serotype 6D capsular switch. In summary, we believe that this is the first reported detection of serotype 6D among pneumococcal isolates recovered in South America or elsewhere in the western hemisphere, despite the fact that our U.S.-based surveillance has been vigilant for its appearance using serologic and PCR-based approaches for 6D detection (3, 8). The work presented here validates serotype 6D as the 92nd serotype identifiable by using CDC pneumococcal typing sera (Table 1). Serotype 6C was not effectively targeted by the pneumococcal 7-valent conjugate vaccine according to recent IPD surveillance data (3); however, there are no existing comparable data for serotype 6D. It is hoped that the recently implemented 13-valent conjugate vaccine (1) will effectively target all serogroup 6 pneumococcal diseases.

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.

In Vitro Development and Analysis of Escherichia coli and Shigella boydii Azithromycin–Resistant Mutants

The aim of this study was to develop and analyze in vitro azithromycin (AZM)-resistant mutants of Escherichia coli and Shigella boydii. Three clinical isolates of E. coli and one S. boydii isolated from feces samples collected from children under 5 years of age with diarrhea in Lima, Peru were inoculated onto Mueller-Hinton plates containing increasing serial dilutions of AZM ranging from their specific minimal inhibitory concentration (2 or 4 mg/l) to 64 mg/l. From these plates, 16 AZM-resistant mutants were selected to determine the stability of the resistance and the presence of cross resistance with other antibiotics. The role of Phe-Arg-β-Naphthylamide (PAβN)-inhibitible efflux pumps as well as the presence of mutations in the rplV, rplD, and rrlH (23S rRNA) genes and alterations in the outer membrane profiles were determined in these 16 mutants. The rate of mutation ranged from < 2.70×10(-10) to 2.17×10(-7) for E. coli and from < 9.58×10(-10) to 1.05×10(-8) for S. boydii. E. coli mutants showed an increase in the AZM-MIC up to sixfold with one strain achieving a MIC >256 mg/l. In contrast, S. boydii only presented increases of up to twofold in MIC levels. All the strains obtained, but one showed stable AZM resistance. In the presence of PAβN, the AZM MICs decreased to parental levels in Shigella mutants, while no MIC returned to parental levels among the E. coli mutants. No cross resistance to other classes of antibiotics was found. These results show the relevance of PAβN-inhibitible efflux pumps in the basal levels and development of AZM resistance. Further studies to characterize the remaining unidentified mechanisms of AZM resistance are needed.