Simple, low-cost molecular assays for TR34/L98H mutations in the cyp51A gene for rapid detection of triazole-resistant Aspergillus fumigatus isolates

Abstract

Simple, low-cost PCR/PCR-restriction fragment length polymorphism (RFLP) assays targeting cyp51A promoter and codon 98 regions were developed for the detection of triazole-resistant Aspergillus fumigatus strains carrying TR34/L98H mutations. The assays were evaluated using 40 itraconazole-susceptible isolates and 35 itraconazole-resistant isolates. The prevalence of TR34/L98H mutations in clinical/environmental A. fumigatus isolates may now be determined easily from resource-poor settings.

FIG 1

Agarose gels of PCR amplicons with AFCYP98F and AFCYP98R primers from 7 A. fumigatus isolates (lanes 1 to 7) (a) and RFLP patterns obtained from 8 triazole-resistant (lanes 1 to 8) and 6 triazole-susceptible (lanes 9 to 14) A. fumigatus isolates (b). The positions of migration of diagnostic fragments of 289 bp (for the L98H mutation) and 189 bp (for the wild-type codon at cyp51A98) are indicated by arrows. The minor band at 350 bp in some lanes represents the undigested full-length amplicon. In both panels, lane M is a 100-bp DNA ladder, and the positions of migration of 100-bp, 300-bp, and 600-bp fragments are marked.

FIG 2

Agarose gel of PCR products obtained with AFCYPPF and AFCYPPR primers from 8 A. fumigatus isolates. The amplicon of ∼139 bp in lanes 1, 2, 5, and 6 indicates the presence of TR₃₄, while the amplicon of ∼105 bp in lanes 3, 4, 7, and 8 indicates the absence of TR₃₄ (wild-type sequence) in the promoter region of cyp51A. Lane M is a 100-bp DNA ladder, and the positions of migration of 100-bp, 300-bp, and 600-bp fragments are marked.