Direct Detection of Shigella in Stool Specimens by Use of a Metagenomic Approach

Abstract

The underestimation of <i>Shigella</i> species as a cause of childhood diarrhea disease has become increasingly apparent with quantitative PCR (qPCR)-based diagnostic methods versus culture. We sought to confirm qPCR-based detection of <i>Shigella</i> via a metagenomics approach. Three groups of samples were selected from diarrheal cases from the Global Enteric Multicenter Study: nine <i>Shigella</i> culture-positive and qPCR-positive (culture⁺ qPCR⁺) samples, nine culture-negative but qPCR-positive (culture^- qPCR⁺) samples, and nine culture-negative and qPCR-negative (culture^- qPCR^-) samples. Fecal DNA was sequenced using paired-end Illumina HiSeq, whereby 3.26 × 10⁸ ± 5.6 × 10⁷ high-quality reads were generated for each sample. We used Kraken software to compare the read counts specific to "<i>Shigella</i>" among the three groups. The proportions of <i>Shigella</i>-specific nonhuman sequence reads between culture⁺ qPCR⁺ (0.65 ± 0.42%) and culture^- qPCR⁺ (0.55 ± 0.31%) samples were similar (Mann-Whitney U test, <i>P</i> = 0.627) and distinct from the culture^- qPCR^- group (0.17 ± 0.15%, <i>P</i> < 0.05). The read counts of sequences previously targeted by <i>Shigella/</i>enteroinvasive <i>Escherichia coli</i> (EIEC) qPCR assays, namely, <i>ipaH</i>, <i>virA</i>, <i>virG</i>, <i>ial</i>, <i>ShET2</i>, and <i>ipaH3</i>, were also similar between the culture⁺ qPCR⁺ and culture^- qPCR⁺ groups and distinct from the culture^- qPCR^- groups (<i>P</i> < 0.001). Kraken performed well versus other methods: its precision and recall of <i>Shigella</i> were excellent at the genus level but variable at the species level. In summary, metagenomic sequencing indicates that <i>Shigella</i>/EIEC qPCR-positive samples are similar to those of <i>Shigella</i> culture-positive samples in <i>Shigella</i> sequence composition, thus supporting qPCR as an accurate method for detecting <i>Shigella</i>.