title: Sensitivity and performance characteristics of a direct PCR with stool samples in comparison to conventional techniques for diagnosis of Shigella and enteroinvasive Escherichia coli infection in children with acute diarrhoea in Calcutta, India.
abstract: As the sensitivity of the conventional techniques for identifying Shigella spp. and enteroinvasive Escherichia coli (EIEC) causing dysentery cases is low, a PCR assay was evaluated in this study. Analytical sensitivity (2 x 10(2) cfu) of the PCR technique was obtained by artificially spiking negative stool samples with a standard strain of S. flexneri type 2, then determining the detection limit. Specificity (100%) of the method was determined by testing a number of known Shigella and EIEC strains and organisms other than Shigella spp. A total of 300 stool samples collected from children with acute diarrhoea was plated on to two selective agar media after enrichment in Luria broth. Shigella spp. were isolated from 7.7% (23 of 300) and EIEC from 1% (3 of 300) patients. All enriched stool samples were subjected to PCR to amplify the target sequence of invasive plasmid antigen (ipa)H locus, a multicopy element found on the chromosome and invasion plasmid. The stool PCR was positive in 24 of the 26 culture-positive and in 22 culture-negative stools, thus detecting the presence of Shigella spp. or EIEC in 15.3% (46 of 300) of diarrhoea cases. When an ial probe was used for colony hybridistion with enriched stool cultures blotted on to membranes, 9.6% (29 of 300) of dysentery cases were identified as being caused by Shigella spp. or EIEC. Thus the sensitivity of enriched stool culture, colony hybridisation and enriched stool PCR was found to be 54%, 60% and 96%, respectively, when each of the methods was compared to the total microbiologically confirmed cases of dysentery. It was also observed that only 38% (48 of 126) of acute bloody dysentery cases actually had shigella or EIEC infection, as confirmed by laboratory methods. Moreover, this PCR assay could identify a number of untypable Shigella strains (Sh OUT), which would have remained undiagnosed had this assay not been used.
authors.forename: Shanta
authors.initials: S
authors.lastname: Dutta
authors.affiliation: National Institute of Cholera and Enteric Diseases, Beliaghata, Calcutta 700010 and *University College of Medicine, Calcutta 700024, India.
authors.forename: A
authors.initials: A
authors.lastname: Chatterjee
authors.forename: P
authors.initials: P
authors.lastname: Dutta
authors.forename: K
authors.initials: K
authors.lastname: Rajendran
authors.forename: S
authors.initials: S
authors.lastname: Roy
authors.forename: K C
authors.initials: KC
authors.lastname: Pramanik
authors.forename: S K
authors.initials: SK
authors.lastname: Bhattacharya
mesh_terms: D000208:Acute Disease; D015373:Bacterial Typing Techniques / Q000379:methods*; D001483:Base Sequence; D002675:Child, Preschool; D015169:Colony Count, Microbial; D003470:Culture Media; D003967:Diarrhea / Q000382:microbiology*; D004405:Dysentery, Bacillary / Q000175:diagnosis* / Q000453:epidemiology; D004926:Escherichia coli / Q000235:genetics* / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000175:diagnosis* / Q000453:epidemiology; D005243:Feces / Q000382:microbiology; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007194:India / Q000453:epidemiology; D007223:Infant; D008297:Male; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012760:Shigella / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D003470:Culture Media