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

J Med Microbiol. 2001 Aug;50(8):667-674. doi: 10.1099/0022-1317-50-8-667.

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.

Publication types

  • Comparative Study
  • Evaluation Study

MeSH terms

  • Acute Disease
  • Bacterial Typing Techniques / methods*
  • Base Sequence
  • Child, Preschool
  • Colony Count, Microbial
  • Culture Media
  • Diarrhea / microbiology*
  • Dysentery, Bacillary / diagnosis*
  • Dysentery, Bacillary / epidemiology
  • Escherichia coli / genetics*
  • Escherichia coli / isolation & purification
  • Escherichia coli Infections / diagnosis*
  • Escherichia coli Infections / epidemiology
  • Feces / microbiology
  • Female
  • HeLa Cells
  • Humans
  • India / epidemiology
  • Infant
  • Male
  • Microbial Sensitivity Tests
  • Molecular Sequence Data
  • Plasmids / genetics
  • Polymerase Chain Reaction
  • Sensitivity and Specificity
  • Shigella / genetics*
  • Shigella / isolation & purification

Substances

  • Culture Media