Evaluation of paper gradient concentration strips for antifungal combination testing of Candida spp.

Abstract

In vitro combination testing with broth microdilution chequerboard (CHEQ) method is widely used although it is time-consuming, cumbersome and difficult to apply in routine setting of clinical microbiology laboratory. A new gradient concentration paper strip method, the Liofilchem(®) MIC test strips (MTS), provides an alternative easy and fast method enabling the simultaneous diffusion of both drugs in combination. We therefore tested a polyene+azole and an azole+echinocandin combination against 18 Candida isolates with the CHEQ method based on EUCAST guidelines and the MTS method in research and routine settings. Fractional inhibitory concentration (FIC) indices were calculated after 24 and 48 h of incubation based on complete and prominent (FIC-2) growth inhibition endpoints. Reproducibility and agreement within 1 twofold dilution was assessed. The FICs of the two methods were correlated quantitatively with t-test and Pearson analysis and qualitatively with Chi-squared test. The reproducibility of the CHEQ and MTS method was 88-100% and their agreement was 80% with 62-77% of MTS FICs being higher than the corresponding CHEQ FICs. A statistically significant Pearson correlation (r = 0.86, P = 0.0003) and association (χ(2) = 17.05, df = 4, P = 0.002) was found between MTS FIC and CHEQ FIC-2 after 24 h. Categorical agreement was 63% with no very major or major errors. All MTS synergistic interactions were also synergistic with the CHEQ method.

Antifungal Combinations against Candida Species: From Bench to Bedside

“…Among them, we excluded 714 reports for the following reasons: combinations other than antifungals (n = 438), reviews (n = 138), off-topics (n = 103), languages other than English (n = 19), no fungal identification (n = 12) and unspecified combination therapy (n = 4). Therefore, a total of 92 studies were included in this review [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 ...…”

“…Synergy with ITZ was observed for one isolate of C. glabrata , and antagonism with KTZ was noted in four isolates of C. tropicalis . Siopi et al, compared the classic checkerboard method with a gradient assay and found that combination of VRC and CAS yielded variable results which were species-dependent with antagonism occurring in C. parapsilosis , C. krusei and C. tropicalis , additivism in C. albicans and C. kefyr and synergism in C. glabrata [ 15 ]. The last triazole introduced in clinical practice, ISV, was tested in combination with all three echinocandins [ 16 , 17 ].…”

“…A later study addressing the in vitro effects of AMB plus FLU against C. parapsilosis (n = 60) confirmed that antagonism was not a rare event occurring in 33% of the cases, while indifference/additivism characterized most of the cases [ 31 ]. The addition of VRC to AMB produced variable results which were either species- or methodology-dependent [ 15 , 22 , 31 ]. One study investigated this interaction against multiple clinical isolates belonging to six Candida species by two methods: the classical checkerboard and the gradient concentration strips diffusion [ 32 ].…”

Techniques for the Assessment of In Vitro and In Vivo Antifungal Combinations

“…This protocol has been used to test antibiotic combinations against gram-negative and gram-positive bacteria [36], but also to test voriconazole combined with either caspofungin or amphotericin B against Candida spp. [37], and to test various combinations against Candida glabrata [38][39][40].…”