Evaluation of Gradient Concentration Strips for Detection of Terbinafine Resistance in Trichophyton spp.

The number of dermatophytosis cases resistant to terbinafine is increasing all over the world. Therefore, there is a need for antifungal susceptibility testing of dermatophytes for better management of the patients. In the present study, we have evaluated a gradient test (GT) method for testing the susceptibility of dermatophytes to terbinafine. MIC values to terbinafine determined by the EUCAST reference technique and by gradient test were compared for 79 Trichophyton spp. isolates. Overall, MICs were lower with gradient test (MIC50 of 0.002 µg/mL) than with EUCAST (MIC50 of 0.016 µg/mL). Good categorical agreement (>90%) between the 2 techniques was obtained but the essential agreement was variable depending on the batch of gradient test.

Reliability of a terbinafine agar containing method for the screening of dermatophyte resistance.

The increase in terbinafine resistance worldwide due to Trichophyton indotineae underlies the need for surveillance networks, deploying easy to perform methods to correctly identify resistant isolates and thereby reduce their spread. In the present study, we evaluated the performances of the terbinafine containing agar method (TCAM). Different technical parameters, such as culture medium (RPMI agar [RPMIA] or Sabouraud dextrose agar [SDA]) and inoculum size, were evaluated. Our study showed that terbinafine susceptibility determined using the TCAM was reliable and independent of the inoculum or medium used. We then performed a multicenter, blinded study. 5 isolates of T. indotineae and 15 of genotype I or II of T. interdigitale, including 5 terbinafine-resistant isolates (4 T. indotineae and 1 T. interdigitale), were sent to eight clinical microbiology laboratories. Each laboratory analyzed the 20 isolates' terbinafine susceptibility by the TCAM using both culture media. TCAM allowed all participants to correctly determine the terbinafine susceptibility of analyzed isolates without prior training. All participants agreed that the dermatophyte tested, regardless of species or genotype, grew better on SDA than on RPMIA medium but accumulated fungal growth after 14 days eventually minimized the effect of this difference. In conclusion, TCAM is a reliable, easy to perform screening method for assessing terbinafine resistance. However, despite good performances, TCAM is a qualitative method and minimal inhibitory concentrations must be determined by the European Committee for Antimicrobial Susceptibility Testing standardized method to follow the evolution of terbinafine resistance levels.

The increase in terbinafine resistance worldwide due to Trichophyton indotineae underlies the need for surveillance networks. The terbinafine containing agar method is a reliable and easy-to-use tool in clinical microbiology laboratories. It can be used to rapidly screening resistant isolates, thereby reducing their spread.

Infectious native aortic aneurysm with negative blood cultures: do not forget the pneumococcal urinary antigen test!

Infectious native aortic aneurysm (INAA) are rare but life-threatening infections. Early microbiological identification is crucial to initiate adequate therapy and decrease the peri-operative risk, but can be challenging when blood cultures remain negative. We describe two cases of pneumococcal INAA with negative blood cultures, diagnosed in the with the pneumococcal urinary antigen test.

TIVAP-related infection due to Gram-negative aerobic bacilli: should TIVAP stay or should it go?

We aimed to describe the outcome of totally implantable venous-access port (TIVAP)-related infections due to Gram-negative aerobic bacilli (Pseudomonas aeruginosa and other Pseudomonas spp., Acinetobacter spp., and Stenotrophomonas maltophilia), or GNAB, and assess the safety of conservative treatment. We conducted a retrospective study in a French teaching hospital, from January 2016 to December 2020, including adult patients treated for TIVAP-related infection due to GNAB. Success of conservative treatment was defined as a functional TIVAP 3 months after infection with no recurrence. We performed a bivariate analysis and analyzed causes for treatment failure. We included 68 patients (53 TIVAP-related bloodstream infections, 11 TIVAP-related infections, and 4 probable TIVAP-related infections) due to GNAB, mostly P. aeruginosa (50/68, 74%). TIVAP removal was initially decided for 49/68 patients (72%). Among the 19/68 (28%) patients with conservative treatment (all for infections caused by P. aeruginosa), 5/19 (26%) had successful treatment, 7/19 (37%) experienced failure (without sepsis or septic shock), 6/19 (32%) died within 3 months without TIVAP removal and no signs of infection recurrence, and 1 patient had TIVAP removal as it was no longer required. TIVAP-related infections caused by GNAB frequently require TIVAP removal. Conservative treatment can be performed in selected patients with a non-complicated infection caused by P. aeruginosa, who can benefit from the continuation of antineoplastic chemotherapy or palliative care. Treatment failures were not associated with sepsis or septic shock.

In Vitro Synergy of Isavuconazole Combined With Colistin Against Common Candida Species.

Interactions of isavuconazole and colistin were evaluated against 57 common Candida strains belonging to the species Candida albicans (n = 10), Candida glabrata (n = 10), Candida kefyr (n = 8), Candida krusei (n = 10), Candida parapsilosis (n = 9), and Candida tropicalis (n = 10) by a broth microdilution checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference methodology for antifungal susceptibility testing. Results were analyzed with the fractional inhibitory concentration index and by the response surface analysis. Interpretation by the fractional inhibitory concentration index showed synergy for 50%, 80%, 90%, and 90% of the C. kefyr, C. krusei, C. glabrata, and C. tropicalis strains, respectively. Combination of isavuconazole with colistin against C. albicans and C. parapsilosis exhibited only indifference for 100% and 90% of the strains, respectively. The results were confirmed by response surface analysis for all species except for C. glabrata, for which an indifferent interaction was found for the majority of strains. Antagonistic interaction was never seen regardless of the interpretation model was used.

Terbinafine Resistance in Dermatophytes: A French Multicenter Prospective Study.

In recent years, we have moved from the sporadic description of terbinafine-resistant (TerR) Trichophyton spp. isolates to the Indian outbreak due to T. indotineae. Population flows have spread TerR worldwide, altering local epidemiology. We conducted a prospective multicentric study to determine the relative frequency of TerR isolates in France (Paris area) and of the newly introduced T. indotineae species. TerR isolates were screened by the terbinafine-containing-agar-medium (TCAM) method and confirmed by EUCAST. Sequencing methods were used to identify isolates to the species/genotype level and to analyze substitutions in the squalene epoxidase gene (SQLE). In total, 3 isolates out of 580 (T. rubrumn = 1; T. interdigitalen = 1; T. indotineaen = 1) grew on TCAM, showed terbinafine resistance by EUCAST and harbored the Phe397Leu (n = 2) or Leu393Ser (n = 1) substitution in the SQLE. ITS-sequencing of isolates of the T. mentagrophytes/interdigitale complex (n = 125) revealed a relative frequency of 4.8% for T. indotineae and the presence of T. mentagrophytes genotype VII. Despite the detection of terbinafine resistance, isolates from this complex remained susceptible to itraconazole, voriconazole and amorolfine. Terbinafine resistance is present in France and the dermatophyte epidemiology is changing. Efficient systems must be implemented to survey the evolution of newly introduced species and to identify TerR isolates.

In Vitro Activity of Amphotericin B in Combination with Colistin against Fungi Responsible for Invasive Infections.

The in vitro interaction of amphotericin B in combination with colistin was evaluated against a total of 86 strains comprising of 47 Candida species (10 Candida albicans, 15 Candida auris, five Candida glabrata, three Candida kefyr, five Candida krusei, four Candida parapsilosis and five Candida tropicalis), 29 Aspergillus species (five Aspergillus flavus, 10 Aspergillus fumigatus, four Aspergillus nidulans, five Aspergillus niger, and five Aspergillus terreus), and 10 Rhizopus species (seven Rhizopus arrhizus, one Rhizopus delemar and two Rhizopus microsporus) strains. For the determination of the interaction, a microdilution checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference method for antifungal susceptibility testing was used. Results of the checkerboard technique were evaluated by the fractional inhibitory concentration index (FICI) based on the Loewe additivity model for all isolates. Different inhibition endpoints were used to capture both the interaction at MIC and sub-MIC levels. Additionally, checkerboard technique results for Candida species were evaluated by response surface analysis based on the Bliss independence model. Against common Candida species, the combination was synergistic for 75% of the strains by FICI and for 66% of the strains by response surface analysis. For C. tropicalis, the interaction was antagonistic for three isolates by FICI, but antagonism was not confirmed by response surface analysis. Interestingly, synergistic and antagonistic FICIs were simultaneously present on checkboard microplates of all three strains. Against C. auris the combination was synergistic for 73% of the strains by response surface analysis and for 33% of the strains by FICI. This discrepancy could be related to the insensitivity of the FICI to detect weak interactions. Interaction for all other strains was indifferent. For Aspergillus and Rhizopus species combination exhibited only indifferent interactions against all tested strains.

In Vitro Antifungal Combination of Terbinafine with Itraconazole against Isolates of Trichophyton Species.

Terbinafine is used as the first-line therapy for dermatophytosis, but the incidence of terbinafine resistance is increasing. A combination of terbinafine with itraconazole was tested using the checkerboard method based on the EUCAST methodology for antifungal susceptibility testing against 9 terbinafine-susceptible and 7 terbinafine-resistant clinical isolates of Trichophyton spp. from India. Synergistic interactions were observed for 4/9 of the susceptible isolates, with fractional inhibitory concentration index (FICI) values of 0.3125 to 0.5, and for 4/7 of the resistant isolates, with FICI values of 0.032 to 0.3125.

In vitro and in vivo evaluation of antifungal combinations against azole-resistant Aspergillus fumigatus isolates.

Azole resistance in Aspergillus fumigatus (Af) has become a widespread threat and a major concern for optimal management of patients with invasive aspergillosis (IA). Combination of echinocandins with azoles is an attractive alternative option for the treatment of IA due to azole-resistant Af strains. The aim of this study was to evaluate the in vitro and in vivo combination of caspofungin (CAS) with either voriconazole (VRZ) or posaconazole (PSZ). In vitro interactions were assessed by two methods, and an animal model of IA in Galleria mellonella was used for in vivo evaluation. Assessment of efficacy was based on larvae mortality. Groups of 10 larvae were infected by 3 clinical strains of Af (azole susceptible, AfS; PSZ resistant, AfR1; VRZ and PSZ resistant strain, AfR2). In vitro, combination of CAS and azoles was indifferent against AfS, and AfR2, and a synergy was found for AfR1. When compared to VRZ monotherapy, the combination of VRZ at 4 µg/larva with CAS at 4 µg/larva improved survival of AfR2-infected larvae (p=0.0066). Combination of PSZ at 4µg/larva with CAS at 4 µg/larva improved survival of AfR1-infected larvae compared to CAS (p=0.0002) and PSZ (0.0024) monotherapy. Antagonism was never observed. In conclusion, the combination of caspofungin with azoles is a promising alternative for the treatment of azole resistant strains of Af.

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

Systemic fungal infections are associated with high mortality rates despite adequate treatment. Moreover, acquired resistance to antifungals is increasing, which further complicates the therapeutic management. One strategy to overcome antifungal resistance is to use antifungal combinations. In vitro, several techniques are used to assess drug interactions, such as the broth microdilution checkerboard, agar-diffusion methods, and time-kill curves. Currently, the most widely used technique is the checkerboard method. The aim of all these techniques is to determine if the interaction between antifungal agents is synergistic, indifferent, or antagonistic. However, the interpretation of the results remains difficult. Several methods of analysis can be used, based on different theories. The most commonly used method is the calculation of the fractional inhibitory concentration index. Determination of the usefulness of combination treatments in patients needs well-conducted clinical trials, which are difficult. It is therefore important to study antifungal combinations in vivo, in experimental animal models of fungal infections. Although mammalian models have mostly been used, new alternative animal models in invertebrates look promising. To evaluate the antifungal efficacy, the most commonly used criteria are the mortality rate and the fungal load in the target organs.

In vitro synergy of isavuconazole in combination with colistin against Candida auris.

The in vitro interactions of isavuconazole with colistin were evaluated against 15 clinical Candida auris isolates by a microdilution checkerboard technique based on the EUCAST reference method for antifungal susceptibility testing and by agar diffusion using isavuconazole gradient concentration strips with or without colistin incorporated RPMI agar. Interpretation of the checkerboard results was done by the fractional inhibitory concentration index and by response surface analysis based on the Bliss model. By checkerboard, combination was synergistic for 93% of the isolates when interpretation of the data was done by fractional inhibitory concentration index, and for 80% of the isolates by response surface analysis interpretation. By agar diffusion test, although all MICs in combination decreased compared to isavuconazole alone, only 13% of the isolates met the definition of synergy. Essential agreement of EUCAST and gradient concentration strip MICs at +/- 2 log2 dilutions was 93.3%. Antagonistic interactions were never observed for any technique or interpretation model used.