Azole antifungals are widely used for therapy and prophylaxis of Candida infections. A better understanding of the mechanisms of resistance to these agents as well as early detection of resistance are essential for patient management. Azole resistance is often due to a combination of factors including increased expression of efflux pumps and missense mutations in ERG11 [3–5, 15]. The latter have been linked to clinically-relevant increases in the MICs, not only to fluconazole, but also to the newer azoles voriconazole and posaconazole [4, 5, 10, 15] This proof of principle study highlights the great potential of a simple rapid (2 h) and highly-specific RCA-based SNP detection assay that can be readily be performed in the clinical laboratory for the detection and/or surveillance for ERG11 mutations. Using this method, we identified Erg11p amino acid substitutions in 24 of 25 previously-uncharacterised Australian isolates with reduced susceptibility to fluconazole.
The sensitivity and reproducibility of the RCA assay was established by determining its ability to detect known ERG11 mutations in "reference" isolates (Table 1) in comparison with DNA sequencing. The padlock probes designed for this study also accurately identified and distinguished between SNPs within the ERG11 genes in the test isolates. These included SNPs that were located close together such as those at nucleotides 1343, 1346 and 1349 corresponding to the amino acid substitutions G448E, F449S and G450E, respectively (Additional file 1). Importantly, identification of ERG11 mutations by the RCA assay was concordant with sequencing in all cases where an ERG11 mutation-specific probe was used. An additional finding was that even though probes (or pairs of probes) were not designed to detect heterozygous nucleotide substitutions per se, the RCA assay detected such changes in isolates containing an ERG11 mutation in only one allele, as demonstrated by their identification in fluconazole-susceptible isolates.
A large number (n = 20) of amino acid substitutions were identified in test isolates with reduced susceptibility/resistance to fluconazole. In agreement with a prior report, all but one isolate had at least one, and often multiple missense mutations in ERG11 . Substitutions also varied widely between individual isolates. Similar results have been reported by Perea et al. who detected 13 ERG11 mutations in 20 C. albicans isolates with high level fluconazole resistance of which 11 were linked to resistance . In contrast, just a single ERG11 mutation profile (comprising the same two mutations) was found in 14 of 15 fluconazole-resistant isolates in another study .
To our knowledge the G450V amino acid substitution has not been previously identified among isolates with reduced susceptibility to azoles. Most of the other substitutions described here have previously been seen in azole-resistant isolates [5, 15, 17, 20] In particular, the substitutions G464S, G307S and G448E, known to confer azole resistance [5, 12, 15], were identified in three or more isolates. However, it is notable that the substitutions Y132H, S405F and R467K which appear to be prevalent in the United States and Europe were rare in Australian isolates [5, 12, 13, 15]. Nineteen of the 20 amino acid substitutions, including G450V, present in the test isolates were clustered into the three "hot-spot" regions as described previously . These hot spots include the residues 105–165 near the N-terminus of the protein, region 266–287 and region 405–488 located towards the C terminus of the protein. The exception was the G307S substitution (n = 3 isolates). However, in a computer-generated model of Erg11p, G307S is located close to the heme cofactor binding site. As such, substitutions at this residue might be expected to impact negatively on the binding of the azole .
In contrast to the fluconazole-resistant strains described above, 22% of fluconazole-susceptible isolates contained no ERG11 mutations and of those that did, substantially fewer (five compared with 20) amino acid substitutions were detected. Also of interest, all Erg11p amino acid substitutions from isolates with reduced azole susceptibility phenotypes were homozygous whereas with one exception (E266D), those in fluconazole-susceptible isolates were present as heterozygous substitutions. While these two observations support the general notion that ERG11 mutations are linked to azole resistance, the presence of ERG11 mutations in susceptible isolates is not readily explained. Development of "resistance" requires prolonged exposure to an azole [3, 4]; however previous studies have not attempted to relate mutations in susceptible isolates to fluconazole exposure. Due to the retrospective nature of the present study we were unable to test this association.
The limitations of this study are recognised. Given the small numbers of isolates in our collection and that the presence of ERG11 mutations are not necessarily functionally related to resistance, we were unable to determine the clinical relevance of the ERG11 mutations identified. Since the substitutions E266D, D116E and V347I were present in both fluconazole-susceptible and, resistant isolates, it could be argued that they are unlikely to have contributed to reduced azole susceptibility [5, 12, 17, 19]. On the other hand, with one exception, all identified mutations were heterozygous in fluconazole-susceptible isolates; the finding supports the contention that loss of heterozygosity in a diploid species such as C. albicans is a step in the development of the azole-resistant phenotype [3, 20, 29]. It is also possible that many ERG11 polymorphisms whilst not conferring resistance per se, may play a role in increasing the level of resistance [12, 21].
Conversely, the absence of substitutions G307S, G448E, G464S, Y132H, S405F and R467K, in susceptible isolates strongly suggests they have contributed to the resistant phenotype. This hypothesis can be tested by site-directed mutagenesis and expression studies of specific ERG11 alleles in Saccharomyces cerevisiae. Using this approach, Sanglard and co-workers demonstrated that the substitutions G464S, Y132H, S405F and R467K were linked to azole resistance among their collection of isolates ; similar studies are warranted to determine if the new substitution G450V is associated with resistance. Testing matched, susceptible and resistant, isolates from the same patient for ERG11 mutations may also assist in determining if particular mutations impact on azole resistance; unfortunately, matched isolates were not available in the present study. In general, neither the type or number of mutations in isolates sequentially obtained from the same patient correlated with azole MICs (Table 2), emphasising the need to assess additional genes to understand the contribution of each to the resistance phenotype. As such, methods that detect polymorphisms are well-placed to screen large numbers of isolates from different sources for mutations and to guide functional testing of these isolates for resistance.
This study demonstrates a new application of a simple RCA-based technique for the rapid and accurate detection of SNPs in the ERG11 gene as potential markers of resistance and for the tracking of resistant strains. Other sequencing-independent methods include conventional real time PCR and/or other probe-based technologies eg. molecular beacons or TaqMan probes [30, 31]. Results using conventional real time PCR are well-known to be highly-dependent on the physical characteristics of the platform. Molecular beacons and TaqMan probe methods are conveniently available in the form of commercial kits. Although able to detect SNPs with good sensitivity [30, 31], strict attention to the Tm of the probes is required to ensure adequate specificity. The RCA-based method described here offers several advantages over other amplification techniques in that ligation of the probe ends by DNA ligase requires perfectly-matched target-probe complexes preventing nonspecific amplification generated by conventional PCR and resulting in very high specificity. It is also rapid (2 h compared to 1–2 days for DNA sequencing following DNA extraction). Whilst the set-up costs of the assay are relatively high, (AUD 300 per probe), a typical commercial batch of each probe provides sufficient material for up to 5000 assays. Running costs are estimated at no more than AUD 2 per assay compared to AUD 15 for DNA sequencing. The limitations of RCA in the primary identification of resistance are acknowledged (see above). However, the technique is well-suited as an epidemiological tool for high throughput screening for commonly-encountered ERG11 SNPs to assist in the detection of potentially-resistant strains and to track the movement of such strains. Further, its utility in detecting SNPs in other genes that have been linked to azole resistance in C. albcians such as those encoding for the transcriptional activator of CDR1 (TAC1) and the transcriptional activator Upc2 (UPC2) [32, 33] warrant consideration.