Incidence of Cyp51 A key mutations in Aspergillus fumigatus-a study on primary clinical samples of immunocompromised patients in the period of 1995-2013

PLoS One. 2014 Jul 29;9(7):e103113. doi: 10.1371/journal.pone.0103113. eCollection 2014.

Abstract

As the incidence of azole resistance in Aspergillus fumigatus is rising and the diagnosis of invasive aspergillosis (IA) in immunocompromised patients is rarely based on positive culture yield, we screened our Aspergillus DNA sample collection for the occurrence of azole resistance mediating cyp51 A key mutations. Using two established, a modified and a novel polymerase chain reaction (PCR) assays followed by DNA sequence analysis to detect the most frequent mutations in the A. fumigatus cyp51 A gene conferring azole resistance (TR34 (tandem repeat), L98H and M220 alterations). We analyzed two itraconazole and voriconazole and two multi-azole resistant clinical isolates and screened 181 DNA aliquots derived from clinical samples (blood, bronchoalveolar lavage (BAL), biopsies, cerebrospinal fluid (CSF)) of 155 immunocompromised patients of our Aspergillus DNA sample collection, previously tested positive for Aspergillus DNA and collected between 1995 and 2013. Using a novel PCR assay for the detection of the cyp51 A 46 bp tandem repeat (TR46) directly from clinical samples, we found the alteration in a TR46/Y121F/T289A positive clinical isolate. Fifty stored DNA aliquots from clinical samples were TR46 negative. DNA sequence analysis revealed a single L98H mutation in 2010, two times the L98H alteration combined with TR34 in 2011 and 2012 and a so far unknown N90K mutation in 1998. In addition, four clinical isolates were tested positive for the TR34/L98H combination in the year 2012. We consider our assay of epidemiological relevance to detect A. fumigatus azole resistance in culture-negative clinical samples of immunocompromised patients; a prospective study is ongoing.

Trial registration: ClinicalTrials.gov NCT01695512.

MeSH terms

  • Antifungal Agents / pharmacology
  • Aspergillosis / diagnosis
  • Aspergillosis / etiology
  • Aspergillosis / microbiology*
  • Aspergillus fumigatus / drug effects
  • Aspergillus fumigatus / genetics*
  • Aspergillus fumigatus / immunology
  • Azoles / pharmacology
  • Cytochrome P-450 Enzyme System / genetics*
  • Drug Resistance, Fungal / genetics
  • Fungal Proteins / genetics*
  • Humans
  • Immunocompromised Host*
  • Microbial Sensitivity Tests
  • Mutation*
  • Polymerase Chain Reaction
  • Sensitivity and Specificity

Substances

  • Antifungal Agents
  • Azoles
  • Fungal Proteins
  • Cytochrome P-450 Enzyme System
  • cytochrome P-450 CYP51A, Aspergillus

Associated data

  • ClinicalTrials.gov/NCT01695512

Grants and funding

The development of the multi-azole resistance PCR assays was supported by a grant of the German Cancer Aid/Dr. Mildred Scheel Foundation for Cancer Research (Project 109 411). An ongoing multicenter prospective clinical study focusing the combined molecular and serologic diagnosis of invasive aspergillosis and multi-azole PCR based detection of cyp51 A alterations in Aspergillus DNA positive clinical samples is supported by a research grant of GILEAD Sciences (ClinicalTrials.gov Identifier: NCT01695512). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.