Development of a simple, rapid, and sensitive diagnostic assay for enterotoxigenic E. coli and Shigella spp applicable to endemic countries.

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  • Author(s): Chakraborty S;Chakraborty S; Connor S; Connor S; Velagic M; Velagic M
  • Source:
    PLoS neglected tropical diseases [PLoS Negl Trop Dis] 2022 Jan 28; Vol. 16 (1), pp. e0010180. Date of Electronic Publication: 2022 Jan 28 (Print Publication: 2022).
  • Publication Type:
    Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: Public Library of Science Country of Publication: United States NLM ID: 101291488 Publication Model: eCollection Cited Medium: Internet ISSN: 1935-2735 (Electronic) Linking ISSN: 19352727 NLM ISO Abbreviation: PLoS Negl Trop Dis Subsets: MEDLINE
    • Publication Information:
      Original Publication: San Francisco, CA : Public Library of Science
    • Subject Terms:
      Dysentery, Bacillary/*diagnosis ; Escherichia coli Infections/*diagnosis ; Molecular Diagnostic Techniques/*methods ; Nucleic Acid Amplification Techniques/*methods; DNA, Bacterial/analysis ; Dysentery, Bacillary/microbiology ; Enterotoxigenic Escherichia coli/genetics ; Enterotoxigenic Escherichia coli/isolation & purification ; Escherichia coli Infections/microbiology ; Feces/microbiology ; Humans ; Molecular Diagnostic Techniques/instrumentation ; Nucleic Acid Amplification Techniques/instrumentation ; Shigella/genetics ; Shigella/isolation & purification
    • Abstract:
      Enterotoxigenic E. coli (ETEC) and Shigella spp (Shigella) are complex pathogens. The diagnostic assays currently used to detect these pathogens are elaborate or complicated, which make them difficult to apply in resource poor settings where these diseases are endemic. The culture methods used to detect Shigella are not sensitive, and the methods used to detect ETEC are only available in a few research labs. To address this gap, we developed a rapid and simple diagnostic assay-"Rapid LAMP based Diagnostic Test (RLDT)." The six minutes sample preparation method directly from the fecal samples with lyophilized reaction strips and using established Loop-mediated Isothermal Amplification (LAMP) platform, ETEC [heat labile toxin (LT) and heat stable toxins (STh, and STp) genes] and Shigella (ipaH gene) detection was made simple, rapid (<50 minutes), and inexpensive. This assay is cold chain and electricity free. Moreover, RLDT requires minimal equipment. To avoid any end user's bias, a battery-operated, handheld reader was used to read the RLDT results. The results can be read as positive/negative or as real time amplification depending on the end user's need. The performance specifications of the RLDT assay, including analytical sensitivity and specificity, were evaluated using fecal samples spiked with ETEC and Shigella strains. The limit of detection was ~105 CFU/gm of stool for LT, STh, and STp and ~104 CFU/gm of stool for the ipaH gene, which corresponds to about 23 CFU and 1 CFU respectively for ETEC and Shigella per 25uL reaction within 40 minutes. The RLDT assay from stool collection to result is simple, and rapid and at the same time sufficiently sensitive. RLDT has the potential to be applied in resource poor endemic settings for the rapid diagnosis of ETEC and Shigella.
      Competing Interests: The authors have declared that no competing interests exist.
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    • Grant Information:
      R01 AI108695 United States AI NIAID NIH HHS; R21 AI137804 United States AI NIAID NIH HHS
    • Accession Number:
      0 (DNA, Bacterial)
    • Subject Terms:
      LAMP assay
    • Publication Date:
      Date Created: 20220128 Date Completed: 20220225 Latest Revision: 20221001
    • Publication Date:
      20240829
    • Accession Number:
      PMC8827434
    • Accession Number:
      10.1371/journal.pntd.0010180
    • Accession Number:
      35089927