An alternative host model of a mixed fungal infection by azole susceptible and resistant Aspergillus spp strains

Figures & data

Table 1. Primers and Probes Sequences designed for the Multiplex Nested Real Time PCR assay.

Primer Name	Sequence (5’-3’)^a
OliNestCyp51A1 (Forward. First PCR)	CTYGCTCAATGTYGTTTATCAAYTATTCTTYCGGC
OliNestCyp51A2 (Reverse. First PCR)	CGCMGACTGAGTYAAGCCGTACTTGATGAACTTC
OliCyp51int-3 (Forward, second PCR)	CAGAACCGCCAATGGTCT
OliCyp51int-2 (Reverse, second PCR)	GACRTCCTTGAGCTTGCC
Cyp51int-AF (A.fumigatus probe)	FAM-CGCGATCAGATTGTAGTTTGACATTCATTCCTGGGGATCGCG-BHQ1
Cyp51int-AL (A. lentulus probe)	HEX-CGCGATCAAGCTAGCGTCTGACATTTATCCCGGGATCGCG-BHQ1

^a Underlining regions indicates the PCR target sequence

Figure 1. (A) Survival rate of G. mellonella infected with A. fumigatus (CM-237) or A. lentulus (CM-1290) (10⁵ spores/larvae). Control group (dotted line) represent survival of larvae inoculated with saline only. (B) Histopathology of G. mellonella infected with A. fumigatus and A. lentulus, at different times points of the infection (Day +2 and +4). 20 x magnification, PAS stained. Differences between the A. fumigatus and A. lentulus infection pattern: larvae showed branched invasive hyphal growth of A. fumigatus (steady arrows) while A. lentulus was encapsulated by immune cells (dash arrows).

Figure 2. (A) Killing rate comparison of G. mellonella infected with A. fumigatus with or without VRC treatment. (B) Killing rate comparison of G. mellonella infected with A. lentulus with or without VRC treatment. (C) Fungal burden determination in G. mellonella infected with A. fumigatus (AF) or A. lentulus (AL), untreated (NT) or treated with VRC. Statistical significance by unpaired t test of VRC treated vs. untreated data for each time point is marked as (*P < 0.01 or **P < 0.002).

Figure 3. Histopathology of G. mellonella infected with A. fumigatus and A. lentulus and treated with VRC, at different time points of the infection (Day +2, +4 and +6). 20 x magnification, PAS stained. Upper panel: when the larvae were infected with A. fumigatus and treated with VRC, the fungus was almost cleared with tissues appearing healthy. Lower panel: A. lentulus CM-1290 strain persisted in the tissues with an increase of fungal burden after 4 and 6 d post-infection (black arrows).

Figure 4. (A) Survival rate of G. mellonella infected with a mixture of A. fumigatus (AF):A. lentulus (AL) (1:1) compared to single infections. (B) Comparative analysis of VRC treated and untreated larvae (This data is the same that was used for Fig. 4A). (C) Fungal burden determination in G. mellonella infected with a mixed inocula of both A. fumigatus and A. lentulus. (D) Fungal burden determination and VRC treatment efficacy in a mixed fungal infection. Statistical significance by unpaired t test of A. fumigatus versus A. lentulus at 6 d is marked as (***P <0.003).