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Differential diagnosis for pythiosis using thermophilic helicase DNA amplification and restriction fragment length

Navaporn Worasilchai1, Piyasak Chaumpluk2, Arunaloke Chakrabarti3

  • 1Interdisciplinary Program, Medical Microbiology, Chulalongkorn University, Bangkok, Thailand.

Medical Mycology
|May 20, 2017
PubMed

Insights

Diagnosing Pythiosis is difficult due to its similarity to fungal infections. A new thermophilic helicase DNA amplification (tHDA) assay rapidly and accurately identifies Pythium insidiosum, aiding clinical diagnosis.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Mycology

Background:

  • Pythiosis is caused by Pythium insidiosum, a fungus-like organism.
  • Accurate diagnosis is challenging due to similarities with fungal pathogens like those causing mucormycosis.
  • Identifying P. insidiosum in clinical labs is difficult.

Purpose of the Study:

  • To develop a rapid and accurate species-specific identification method for P. insidiosum.
  • To differentiate P. insidiosum from closely related pathogenic fungi.
  • To evaluate the potential of the assay for direct use on clinical samples.

Main Methods:

  • Development of a thermophilic helicase DNA amplification (tHDA) technique.
  • Utilized a COX2 primer to amplify a 91 base-pair (bp) DNA fragment.
  • Employed restriction fragment length polymorphism (RFLP) with the CviKI-1 enzyme.

Main Results:

  • The tHDA assay successfully identified 39 isolates of P. insidiosum among 67 fungal isolates.
  • The assay demonstrated high sensitivity, with limiting concentrations of 100 picograms and 100 femtograms for one- and two-step protocols, respectively.
  • RFLP analysis accurately distinguished P. insidiosum from other fungal species using the amplified 91 bp fragment.

Conclusions:

  • The developed tHDA-RFLP assay is a rapid and accurate method for identifying P. insidiosum.
  • This assay can effectively differentiate P. insidiosum from similar fungal species.
  • The assay holds potential for direct application in clinical diagnostics.
Keywords:
Pythium insidiosumrestriction fragment length polymorphismthermophilic helicase DNA amplification

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