Detection of Shigella in direct stool specimens using a metagenomic approach

Abstract

The underestimation of Shigella species as a cause of childhood diarrhea disease has become increasingly apparent with quantitative (q)PCR based diagnostic methods versus culture. We sought to confirm qPCR-based detection of Shigella via a metagenomics approach. Three groups of samples were selected from diarrheal cases from the Global Enteric Multicenter Study: 9 Shigella culture positive and qPCR positive (culture+/qPCR+), 9 culture negative but qPCR positive (culture-/qPCR+), 9 culture negative and qPCR negative (culture-/qPCR-). Fecal DNA was sequenced using paired-end Illumina HiSeq, whereby 3.26 x 108  5.6 x 107 high quality reads were generated for each sample. We used Kraken software to compare the read counts specific to “Shigella” among the three groups. The proportion of Shigella-specific non-human sequence reads between culture+/qPCR+ (0.65±0.42%) and culture-/qPCR+ (0.55±0.31%) was similar (Mann-Whitney U test, P = 0.769) and distinct from the culture-/qPCR- group (0.17±0.15%, P = 0.001). The read counts of sequences previously targeted by Shigella/enteroinvasive E. coli qPCR assays, namely ipaH, virA, virG, ial, ShET2, and ipaH3, were also similar between the culture+/qPCR+ and culture-/qPCR+ groups, and distinct from the culture-/qPCR- groups (P < 0.001). Kraken performed well versus other methods: its precision and recall of Shigella was excellent at the genus level but variable at the species level. In summary, metagenomic sequencing indicates that Shigella/EIEC qPCR positive samples are similar to those of Shigella culture positive samples in Shigella sequence composition, thus supporting qPCR as an accurate method to detect Shigella.