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Performance evaluation of multiplex PCR including Aspergillus-not so simple!

Alexandre Alanio1,2,3, Stéphane Bretagne4,2,3

  • 1Paris-Diderot, Sorbonne Paris Cité University.

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|September 25, 2016
PubMed
Summary

Multiplex PCR assays for invasive aspergillosis diagnosis can miss key fungal species. Validated, targeted quantitative PCRs for Aspergillus fumigatus offer a more reliable diagnostic approach.

Keywords:
Aspergillusazole resistance detectionelectrospray-ionization mass spectrometrymicroarraymultiplex PCRmultiplexed PCR and liquid-phase array

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Area of Science:

  • Medical Mycology
  • Molecular Diagnostics
  • Infectious Diseases

Background:

  • Invasive aspergillosis (IA) diagnosis relies on sensitive detection methods.
  • Multiplex PCR assays have been developed to detect multiple fungal species, including Aspergillus fumigatus, and azole resistance.

Purpose of the Study:

  • To evaluate the utility and limitations of multiplex PCR assays for diagnosing invasive aspergillosis and detecting azole resistance.
  • To compare multiplex PCR with validated quantitative PCR methods for IA diagnosis.

Main Methods:

  • Review of multiplex PCR techniques (microarrays, liquid-phase array, PCR/ESI MS) for fungal species identification.
  • Analysis of primer design challenges and limitations in amplifying diverse fungal species.
  • Assessment of multiplex assays for detecting Aspergillus fumigatus DNA and azole resistance genes.

Main Results:

  • Multiplex PCRs may fail to detect clinically relevant fungal species in complex samples due to primer amplification efficiencies.
  • Detection of azole resistance using multiplex assays is limited to high fungal loads because resistance genes are single-copy.
  • The specificity of multiplex PCRs is checked by probe hybridization or amplicon base composition analysis.

Conclusions:

  • Multiplex assays, while useful for mycobiome research, should be used cautiously for IA diagnosis or azole resistance detection.
  • Validated quantitative PCRs targeting Aspergillus fumigatus or a limited species set provide a safer and more sensitive option for IA diagnosis.