In Vitro Assessment of Antifungal Drugs and Sulfamethoxazole-Trimethoprim against Clinical Isolates of Conidiobolus lamprauges

LETTER

Conidiobolomycosis is an infection for which there are no sufficient clinical or in vitro data to support a consensus about the optimal treatment, and consequently, cases of therapeutic success and failure have been described for all forms of therapy (1–3). We have determined the in vitro activities of sulfamethoxazole, trimethoprim, and several antifungal drugs alone and in combination against Conidiobolus lamprauges, a pathogenic agent of nasopharyngeal conidiobolomycosis in animals (4, 5) and in one case of disseminated human disease (3).

Seven nonduplicate C. lamprauges isolates obtained from cases of sheep rhino-conidiobolomycosis from central-western Brazil, previously identified based on their macro- and microscopic characteristics (6) as well as the nucleotide sequences of their 18S ribosomal DNA (rDNA) genes (7), were used. The sequences obtained were analyzed, and multiple-sequence alignment was done with Conidiobolus coronatus, C. lamprauges, and Conidiobolus incongruus reference strains. The antimicrobials used in the susceptibility tests are described in Table 1. Broth microdilution susceptibility tests were performed by following the CLSI M38-A2 protocol (8, 9), except with the final inocula, which were diluted 1:10 to produce more consistent mycelial growth for the determination of MIC endpoints. The interactions between drugs were evaluated using a checkerboard test (10) and by a colorimetric 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) time-kill assay (11).

The MICs and minimum effective concentrations (MECs) of the antimicrobial drugs evaluated against C. lamprauges are presented in Table 1. Amphotericin B (MIC₁₀₀ range, 1 to 2 μg/ml), terbinafine (MIC₁₀₀ range, 1 to 4, and MIC₈₀, 0.06 to 2 μg/ml), and miconazole (MIC₁₀₀ range, 4 to 16 μg/ml) were considered the most effective drugs because they required the lowest concentrations for the in vitro inhibition of C. lamprauges. Checkerboard results (Table 1) showed synergistic interactions of 100% for the sulfamethoxazole-trimethoprim combination, 71% for the terbinafine-azole antifungal combinations, and 29% for the terbinafine-micafungin combination. All other interactions were indifferent. An XTT reduction assay showed significantly greater hyphal damage for the sulfamethoxazole-trimethoprim combination than for drugs used alone (Fig. 1A and B), but levels of hyphal viability after terbinafine treatment and after treatment with azole antifungals did not differ significantly (Fig. 1C).

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FIG 1

XTT reduction assay. Percentages of viable hyphae of C. lamprauges exposed to sulfamethoxazole (SMX), trimethoprim (TMP), terbinafine (TRB), miconazole (MCZ), posaconazole (POS), and voriconazole (VOR) at different concentrations (micrograms per milliliter) alone and in combination. Data represent the means ± standard errors of the means from seven strains in triplicate. Hyphal viability was determined at 12, 24, and 48 h of incubation at 37°C. Data were subjected to logarithmic transformation to obtain homogeneity between groups and were analyzed by one-way analysis of variance with the Holm-Sidak test for statistical comparisons between drug combination groups versus groups in which the drug was used alone. The statistics program SigmaPlot 12.5 was used for data analysis. An asterisk indicates a significant difference (P ≤ 0.01), and a hashtag indicates a significant difference (P < 0.001) from the groups in which a drug was used alone.

Susceptibility tests for C. lamprauges have previously been described in only a few reports (3, 9, 12), and these presented results similar to those obtained through this study. Previous works demonstrate the preventive and therapeutic potential of sulfamethoxazole-trimethoprim against Pneumocystis, Aspergillus, and Cryptococcus species (13, 14), suggesting that these drugs may also have potentially been used for the treatment of conidiobolomycosis.

In conclusion, the in vitro data reported here suggest that the combinations of sulfamethoxazole-trimethoprim and terbinafine alone or associated with azole antifungal drugs deserve attention as candidates for the treatment of conidiobolomycosis infections. Additional in vitro and in vivo studies with a large number of clinical isolates and antimicrobial combinations are needed to better understand the susceptibility of C. lamprauges to the antimicrobials described in this study.