Sensitivity and performance characteristics of a direct PCR with stool samples in comparison to conventional techniques for diagnosis of Shigella andenteroinvasive Escherichia coli infection in children with acute diarrhoeain Calcutta, India

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 1 0(2) cfu) of the PCR technique was obtained by artificially spiking negativ e stool samples with a standard strain of S. flexneri type 2, then determin ing the detection limit, Specificity (100%) of the method was determined by testing a number of known Shigella and ETEC strains and organisms other th an 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 ETEC from 1% (3 of 300) patients, All enriched stool samples were subjected to P CR to amplify the target sequence of invasive plasmid antigen (ipa)H locus, a multicopy element found on the chromosome and invasion plasmid, The stoo l PCR was positive in 24 of the 26 culture-positive and in 22 culture-negat ive 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 hybrid istion with enriched stool cultures blotted on to membranes, 9.6% (29 of 30 0) of dysentery cases were identified as being caused by Shigella spp, or E IEC, Thus the sensitivity of enriched stool culture, colony hybridisation a nd enriched stool PCR was found to be 54%, 60% and 96%, respectively, when each of the methods was compared to the total microbiologically confirmed c ases of dysentery, It was also observed that only 38% (48 of 126) of acute bloody dysentery cases actually had shigella or EIEC infection, as confirme d by laboratory methods. Moreover, this PCR assay could identify a number o f untypable Shigella strains (Sh OUT), which would have remained undiagnose d had this assay not been used.