Dataset Information

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

ABSTRACT: The underestimation of Shigella 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 Shigella via a metagenomics approach. Three groups of samples were selected from diarrheal cases from the Global Enteric Multicenter Study: nine Shigella 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 "Shigella" among the three groups. The proportions of Shigella-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, P = 0.627) and distinct from the culture- qPCR- group (0.17 ± 0.15%, P < 0.05). The read counts of sequences previously targeted by Shigella/enteroinvasive Escherichia coli (EIEC) 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 were 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 for detecting Shigella.

SUBMITTER: Liu J

PROVIDER: S-EPMC5786726 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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

Liu Jie J Almeida Mathieu M Kabir Furqan F Shakoor Sadia S Qureshi Shahida S Zaidi Anita A Li Shan S Tamboura Boubou B Sow Samba O SO Mandomando Inacio I Alonso Pedro L PL Ramamurthy Thandavarayan T Sur Dipika D Kotloff Karen K Nataro James J Levine Myron M MM Stine O Colin OC Houpt Eric E

Journal of clinical microbiology 20180124 2

The underestimation of Shigella 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 Shigella via a metagenomics approach. Three groups of samples were selected from diarrheal cases from the Global Enteric Multicenter Study: nine Shigella culture-positive and qPCR-positive (culture+ qPCR+) samples, nine culture- ...[more]

PMID: 29118177

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Project description:Background: The relationship between asymptomatic Salmonella exposure within the gastrointestinal tract and Salmonella bacteraemia is poorly understood, in part due to the low sensitivity of stool culture, and the lack of validated molecular diagnostic tests for the detection of Salmonella in stool. The study aimed to determine a reliable molecular diagnostic test for Salmonella in stool specimens. Methods: We optimized an in-house monoplex real time polymerase chain reaction (PCR) for the detection of SalmonellaTTR and InvA genes in stool by including a selenite broth pre-culture step for Salmonella before DNA extraction, and validated their specificity against other local common pathogens. Then we assessed their performance against a well-validated multiplex PCR targeting the same TTR and InvA genes, and against stool culture using clinical stool specimens collected from a cohort of 50 asymptomatic healthy Malawian children that were sampled at 1-month intervals over a period of 12 months. We employed a latent Markov model to estimate the specificities and sensitivities of PCR methods. Results: TTR and InvA primers were both able to detect all the different Salmonella serovars tested, and had superior limits of detection if DNA was extracted after selenite pre-culture. TTR sensitivity and specificity for monoplex-PCR were (99.53%, 95.46%) and for multiplex-PCR (90.30%, 99.30%) respectively. InvA specificity and specificity for using monoplex-PCR was (95.06%, 90.31%) and multiplex-PCRs (89.41%, 98.00%) respectively. Sensitivity and specificity for standard stool culture were 62.88% and 99.99% respectively. Culture showed the highest PPV (99.73%) and mono-TTR had the highest NPV (99.67%). Conclusion: Test methods demonstrated high concordance although stool culture and monoplexed TTR primers had superior specificity and sensitivity respectively. The use of selenite pre-enrichment step increased Salmonella detection rate. Taken together, molecular detection methods used here could be used to reveal the true extent of both asymptomatic and symptomatic Salmonella exposure events.

Dataset Information

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

Publications

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

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