Molecular characterization of virulence and antimicrobial resistance profile of Shigella species isolated from children with moderate to severe diarrhea in northeastern Brazil.

Molecular characterization of virulence and antimicrobial resistance profiles were determined for Shigella species isolated from children with diarrhea in Fortaleza, Brazil. Fecal specimens were collected along with socioeconomic and clinical data from children with moderate to severe diarrhea requiring emergency care. Shigella spp. were isolated by standard microbiological techniques, and we developed 4 multiplex polymerase chain reaction assays to detect 16 virulence-related genes (VRGs). Antimicrobial susceptibility tests were performed using disk diffusion assays. S. flexneri and S. sonnei were the predominant serogroups. S. flexneri was associated with low monthly incomes; more severe disease; higher number of VRGs; and presence of pic, set, and sepA genes. The SepA gene was associated with more intense abdominal pain. S. flexneri was correlated with resistance to ampicillin and chloramphenicol, whereas S. sonnei was associated with resistance to azithromycin. Strains harboring higher numbers of VRGs were associated with resistance to more antimicrobials. We highlight the correlation between presence of S. flexneri and sepA, and increased virulence and suggest a link to socioeconomic change in northeastern Brazil. Additionally, antimicrobial resistance was associated with serogroup specificity in Shigella spp. and increased bacterial VRGs.

Combination of different methods for detection of Campylobacter spp. in young children with moderate to severe diarrhea.

Campylobacter spp. were detected - using culture, ELISA, PCR, and qPCR - among children (0-36months) with moderate to severe diarrhea in Northeastern Brazil. Our data showed that either the qPCR alone or PCR along with ELISA might be an alternative to culture to diagnose Campylobacter due to their enhanced sensitivity.

Prevalence of enteroaggregative Escherichia coli and its virulence-related genes in a case-control study among children from north-eastern Brazil.

Enteroaggregative Escherichia coli (EAEC) is an important agent that causes endemic and epidemic diarrhoeal diseases worldwide. Several EAEC virulence-related genes (VRGs) have been described but their role in the clinical outcome of infection is not completely defined. This study investigated the prevalence of EAEC and potential associations of its VRGs with risk of or protection from diarrhoeal diseases in children from urban communities in north-eastern Brazil. The case-control study included 166 children, who had their stools evaluated for the EAEC diagnostic genes (aaiC and aatA) using PCR. Positive samples were further analysed by multiplex PCR and identified 18 VRGs. EAEC was found in the same proportion in both groups (41%). The plasmid-borne gene encoding a hexosyltransferase homologue (capU) was the most frequently detected (89.6%), followed by dispersin protein (aap, 58.2%) and EAEC HilA homologue (eilA, 57.8%). The AAF/III fimbrial subunit (agg3A) gene was observed at lower frequency (1.5%). Plasmid-encoded toxin (pet) or AAF/II fimbrial subunit (aafA) was associated significantly with disease. AAF/IV fimbrial subunit (agg4A) or hypothetical plasmid-encoded haemolysin (orf61) was detected significantly more in controls than in children with diarrhoea. In addition, one set of genes in combination, aaiC and agg3/4C but lacking agg4A and orf61, was associated with diarrhoea cases; and another one, orf61 in the absence of pet and aafA, was correlated with control children. These data confirm a high prevalence, endemicity and heterogeneity of EAEC strains in the developing urban areas of north-eastern Brazil. Statistical correlation between cases and controls was seen with either isolated or combined sets of genes, suggesting that the pathophysiology of EAEC infection involves a complex and dynamic modulation of several VRGs.

Enteroaggregative Escherichia coli quantification in children stool samples using quantitative PCR.

Enteroaggregative Escherichia coli (EAEC) is a common cause of infectious diarrhea, especially in children living in poor-resource countries. In this article, we present a SYBR Green-based real-time polymerase chain reaction (qPCR) method for quantitative detection of EAEC in DNA directly extracted from human stool samples. To test the proposed qPCR system, we examined specificity, sensitivity, repeatability, and also the degree of DNA extraction efficiency using EAEC strain 042 spiked into EAEC-free stool sample. The specificity of this assay was proved using six strains of EAEC, seven strains of other E. coli types, and one strain of Shigella. The detection limit of qPCR was 67 CFU/reaction. In naturally infected stool samples, we found EAEC in quantities varying from 6.7 × 10(5) to 2 × 10(9 ) CFU/g of feces. We could not detect any reduction after stool DNA extraction for the amounts of 10(7) and 10(6) CFU/mL of spiked EAEC. This qPCR assay is simple, rapid, reproducible, sensitive, specific, and allows rapid EAEC quantification to be used in a variety of further EAEC studies. This new quantitative method provides a relatively simple means to quantify EAEC, which will likely be key to understanding the pathophysiology and impact of EAEC infection.

Campylobacter jejuni and Campylobacter coli in children from communities in Northeastern Brazil: molecular detection and relation to nutritional status.

This study determined the prevalence of Campylobacter jejuni/coli and its relation with nutritional status in children from Northeastern Brazil. This was a case-control study design. Stool samples were evaluated for hipO (C. jejuni), ask (C. coli), and cdtABC (C. jejuni's cytolethal distending toxin) genes. The nutritional status from these children was assessed by anthropometric measures and z-scores. C. jejuni and C. coli were detected in 9.6% (8/83) and 6.0% (5/83) in the diarrhea group and in 7.2% (6/83) and 1.2% (1/83) of the nondiarrhea group, respectively. Children with positive molecular detection of C. jejuni showed significantly lower z-scores than children without C. jejuni. The cdtABC operon was found in 57% of hipO(+) samples. C. jejuni/coli prevalence was similar in diarrhea and nondiarrhea groups. There was a significant association of C. jejuni infection with lower nutritional status.

Genes codificadores de fatores de virulência, inflamação e avaliação nutricional da infecção intestinal associada com Escherichia coli enteroagregativa em crianças de Fortaleza, Ceará, Brasil [manuscrito] / Genes coding for virulence factors, nutritional evaluation of intestinal inflammation and infection associated with Escherichia coli enteroaggregative in children in Fortaleza, Ceará, Brazil [manuscript]

Escherichia coli enteroagregativa (EAEC) é um patótipo de E. coli que tem sido cada vez mais identificado como agente etiológico das doenças diarreicas. Esse trabalho teve como objetivos determinar a prevalência de EAEC e investigar a importância de alguns genes associados à virulência no grau de severidade das doenças diarreicas causadas pelo microorganismo, além de avaliar o impacto dessas infecções na inflamação intestinal e no estado nutricional de crianças carentes de Fortaleza, Ceará. Crianças na faixa etária entre 2 e 36 meses, com histórico ou não de diarreia nos últimos 14 dias, tiveram suas medidas antropométricas avaliadas e suas amostras fecais coletadas. O diagnóstico de EAEC foi realizado por reação de polimerase em cadeia (PCR) dos genes aaiC (cromossomal) e aatA (plasmidial). Amostras positivas foram pesquisadas quanto à presença dos genes de virulência aggR (regulador transcricional), aap (dispersina), pic (enterotoxina), pet (enterotoxina) e astA (enterotoxina). A sequência nucleotídica do gene aggR foi analisada através de sequenciamento. Alíquotas fecais foram submetidas à quantificação de lactoferrina (LFF) e citocinas (IL-4, IL-10, TNF-α e IFN-γ) através de reação imunoenzimática (ELISA). Esse estudo analisou 83 crianças com diarreia (casos) e 83 crianças sem diarreia (controles). EAEC foi encontrada na mesma proporção entre ambos os grupos (41,0%). Crianças com diarreia apresentaram redução significativa na espessura da prega cutânea, índice de massa corporal e escores-z peso-por-idade (WAZ) e peso-por-altura (WHZ), mas a presença da bactéria não foi associada com alterações nos índices antropométricos analisados. Entre as amostras positivas para EAEC, não houve diferença quanto à presença isolada dos fatores de virulência pesquisados nas crianças que desenvolveram ou não diarreia...