Development of a multiplex polymerase chain reaction assay for diarrheagenic Escherichia coli and Shigella spp. and its evaluation on colonies, culture broths, and stool

Taniuchi, Mami; Walters, Carol C.; Gratz, Jean; Maro, Athanasia; Kumburu, Happiness; Serichantalergs, Oralak; Sethabutr, Orntipa; Bodhidatta, Ladaporn; Kibiki, Gibson; Toney, Denise M.; Berkeley, Lynette Y.; Nataro, James P.; Houpt, Eric R.

doi:10.1016/j.diagmicrobio.2012.03.008

Cited by 48 publications

(37 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…A correlation between the aatA gene and CFU of EAEC in stool was found. Further research on this method, combined with clinical information, could reveal a possible correlation between the degree of illness and the EAEC burden (156).…”

Section: Discussionmentioning

confidence: 99%

Epidemiology and Clinical Manifestations of Enteroaggregative Escherichia coli

et al. 2014

View full text Add to dashboard Cite

SUMMARY Enteroaggregative Escherichia coli (EAEC) represents a heterogeneous group of E. coli strains. The pathogenicity and clinical relevance of these bacteria are still controversial. In this review, we describe the clinical significance of EAEC regarding patterns of infection in humans, transmission, reservoirs, and symptoms. Manifestations associated with EAEC infection include watery diarrhea, mucoid diarrhea, low-grade fever, nausea, tenesmus, and borborygmi. In early studies, EAEC was considered to be an opportunistic pathogen associated with diarrhea in HIV patients and in malnourished children in developing countries. In recent studies, associations with traveler's diarrhea, the occurrence of diarrhea cases in industrialized countries, and outbreaks of diarrhea in Europe and Asia have been reported. In the spring of 2011, a large outbreak of hemolytic-uremic syndrome (HUS) and hemorrhagic colitis occurred in Germany due to an EAEC O104:H4 strain, causing 54 deaths and 855 cases of HUS. This strain produces the potent Shiga toxin along with the aggregative fimbriae. An outbreak of urinary tract infection associated with EAEC in Copenhagen, Denmark, occurred in 1991; this involved extensive production of biofilm, an important characteristic of the pathogenicity of EAEC. However, the heterogeneity of EAEC continues to complicate diagnostics and also our understanding of pathogenicity.

show abstract

Section: Discussionmentioning

confidence: 99%

Epidemiology and Clinical Manifestations of Enteroaggregative Escherichia coli

et al. 2014

View full text Add to dashboard Cite

show abstract

“…The gene encoding the regulator AggR (Dudley et al, 2006) is a good target for detecting EAEC and has long been used for this purpose since it regulates the AAF PAI, governing the enteroaggregative adhesion along with those specifying other AA-associated plasmid-encoded factors (Dudley et al, 2006). Because aagR is plasmid-located and may not be detected in the event of plasmid loss, the concomitant detection of the chromosomal gene aaiC, encoding a secreted protein of EAEC and harboured by the PAI AAI (Taniuchi et al, 2012), has been proposed by the authors to circumvent this possibility.…”

Section: Detection Of Additional Virulence Factorsmentioning

confidence: 99%

Scientific Opinion on VTEC‐seropathotype and scientific criteria regarding pathogenicity assessment

Publication¹

2013

EFS2

137

View full text Add to dashboard Cite

During 2007During -2010 545 confirmed human VTEC infections and 777 haemolytic uraemic syndrome (HUS) cases were reported in the EU; isolates from 85 % of cases were not fully serotyped and therefore could not be classified using the Karmali seropathotype concept. Seropathotype group D covered 5 % of isolates from fully serotyped cases; 14 cases (0.7 %) belonged to seropathotype group E, defined by Karmali et al. (2003) as nonhuman only. Isolates from around 27 % of cases could not be assigned. There were no HUS cases reported for the serotypes in groups D and E but 17 HUS cases could not be assigned. The health outcome was reported for only a fraction of confirmed cases. About 64 % of patients presented with only diarrhoea; VTEC infection resulted in HUS in around 10 % of cases. The new ISO/TS 13136:2012 standard improves the detection of VTEC in food. An alternative concept based on the detection of verocytotoxins alone or genes encoding such verocytotoxins does not provide a sound scientific basis on which to assess risk to the consumer because there is no single or combination of marker(s) that fully define a "pathogenic" VTEC. Strains positive for verocytotoxin 2 gene (vtx2)-and eae (intimin production)-or [aaiC (secreted protein of EAEC) plus aggR (plasmid-encoded regulator)] genes are associated with higher risk of more severe illness than other virulence gene combinations. The 2011 O104:H4 outbreak demonstrated the difficulty of predicting the emergence of "new" pathogenic VTEC types by screening only for the eae gene or by focusing on a restricted panel of serogroups. A molecular approach utilising genes encoding virulence characteristics additional to the presence of vtx genes has been proposed. The approach adopted entailed a summary of the types of pathogenic E. coli which have been associated with cases of human disease, and the putative virulence factors therein; the use of data from the European Surveillance System (TESSy data) as provided by the ECDC (European Centre for Disease Prevention and Control) and data available in the EU Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2011 for assessing the current situation regarding human infections with VTEC in the EU; a review of methods for the isolation and identification of VTEC, including detection of virulence factors and characterisation and typing of VTEC strains and virulence genes therein; hazard characterisation, including illnesses associated with VTEC and identification of predictive factors for VTEC that may contribute to human disease; evaluation of the seropathotype concept using the Karmali approach, a modification of the Karmali approach based on the health outcome of reported confirmed human VTEC cases in the EU during 2007-2010, and a molecular approach based on the identification of known or putative colonization genes and additional toxins; and finally exposure assessment, including EU monitoring data on occurrence of VTEC in RTE food. © EuropeanThe BIOHAZ Panel concluded ...

show abstract

“…Moreover, even in experienced hands, the cultivation of Shigella spp. is impeded by inconsistent bacterial load, loss of bacterial viability during specimen transport and storage, and requirements for specific reagents that are labile and sometimes unavailable (2). Nevertheless, advantages of cultivation are its validation and verification of a bacterium's identity and its availability for downstream characterization.…”

mentioning

confidence: 99%

Quantitative PCR for Detection of Shigella Improves Ascertainment of Shigella Burden in Children with Moderate-to-Severe Diarrhea in Low-Income Countries

et al. 2013

Self Cite

View full text Add to dashboard Cite

Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 ؋ 10 4 ipaH copies per 100 ng of stool DNA for set 1 (n ‫؍‬ 877). One hundred fifty-eight (18%) specimens yielded >2.9 ؋ 10 4 ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with >2.9 ؋ 10 4 ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 ؋ 10 4 ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH, a 90% increase from the 172 cases detected by culture in both samples. Among a subset (n ‫؍‬ 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% (n ‫؍‬ 129) for culture to 17.6% (n ‫؍‬ 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 ؋ 10 4 ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella.

show abstract

Development of a multiplex polymerase chain reaction assay for diarrheagenic Escherichia coli and Shigella spp. and its evaluation on colonies, culture broths, and stool

Cited by 48 publications

References 43 publications

Epidemiology and Clinical Manifestations of Enteroaggregative Escherichia coli

Epidemiology and Clinical Manifestations of Enteroaggregative Escherichia coli

Scientific Opinion on VTEC‐seropathotype and scientific criteria regarding pathogenicity assessment

Quantitative PCR for Detection of Shigella Improves Ascertainment of Shigella Burden in Children with Moderate-to-Severe Diarrhea in Low-Income Countries

Contact Info

Product

Resources

About