Luzia Lyra

Is the incidence of candidemia caused by Candida glabrata increasing in Brazil? Five-year surveillance of Candida bloodstream infection in a university reference hospital in southeast Brazil

From 2006 to 2010, a retrospective study was conducted in a university referral tertiary care hospital to study the frequency and distribution of Candida species in different medical specialties. The use of mechanical ventilation, central venous catheter, and urinary catheter were recorded per 1,000 patient-days and the use of antifungals was calculated using defined daily dose (DDD). A total of 313 episodes were identified and the overall incidence was 0.54 (0.41-0.71) episodes per 1,000 patient-days. Candida albicans caused 44% of the overall episodes, followed by C. tropicalis (21.7%), C. parapsilosis (14.4%), C. glabrata (11.2%), and C. krusei (3.5%). The incidence of C. glabrata significantly increased from 2006-2010 (range: 4.8-23.5%) (P = 0.024). Candida glabrata was associated with malignancies (P = 0.004) and C. krusei with hematologic malignancies (P < 0.0001). The use of antifungals was higher in the hematology/bone marrow transplant units and represented 40% of all fluconazole prescription in the hospital. There was no correlation with the use of fluconazole and the increasing ratio of C. glabrata (r = 0.60). The use of invasive devices was significantly higher in the intensive care units (ICUs) than the medical and surgical emergencies units (P < 0.001). In contrast, the emergencies had higher incidence of candidemia (2-2.1 episodes/1,000 patient-days) than the ICUs (1.6 episodes 1,000 patient-days). Candida glabrata candidemia showed a significant increase in contrast to the current national literature where C. parapsilosis remained the most important non-C. albicans Candida species in Brazilian hospitals. Our findings suggested that the increasing incidence of C. glabrata was not associated with use of fluconazole and other risk factors might play an important role.

Susceptibility testing of terbinafine alone and in combination with amphotericin B, itraconazole, or voriconazole against conidia and hyphae of dematiaceous molds.

Studies have demonstrated excellent in vivo efficacy of terbinafine combined with other antifungal agents against dematiaceous molds; however, there is a lack of in vitro studies. Most studies evaluated conidia inocula, but susceptibility testing of hyphae could mimic the fungal status in infected tissues and might reflect the therapeutic potential of the agent. We investigated the in vitro susceptibility of terbinafine alone and in combination with amphotericin B, itraconazole, or voriconazole against conidia by microdilution and dynamic measurement of hyphae growth of dematiaceous molds. The MIC values for hyphae were, until 3 dilutions, below the MIC obtained for conidia. The results indicated 100% synergistic interactions between terbinafine and azoles or amphotericin B in all tests, but lower MICs for hyphae. In conclusion, our findings allow us to say that the hyphal form of tested dematiaceous molds showed high susceptibility to all antifungal agents evaluated, alone and in combination with terbinafine.

In Vitro Evaluation of the Type of Interaction Obtained by the Combination of Terbinafine and Itraconazole, Voriconazole, or Amphotericin B against Dematiaceous Molds

ABSTRACT In vitro associations using the checkerboard microdilution method indicated lower MIC ranges and MIC median values for each drug (terbinafine, itraconazole, voriconazole, and amphotericin B) in association than those obtained for each single drug. Fractional inhibitory concentration index (FIC) results showed 100% synergism in the association of terbinafine with voriconazole, 96.5% in the association of terbinafine with amphotericin B, and 75.9% in the association of terbinafine with itraconazole. Drug combinations may be useful for treatment of dematiaceous mold infections as an alternative treatment to enhance the effectiveness of each drug.

Fusarium napiforme systemic infection: case report with molecular characterization and antifungal susceptibility tests.

IntroductionDuring the last decades, Fusarium spp. has been reported as a significant cause of disease in humans, especially in immunocompromised patients, who have high risk of invasive life-threatening disease. Fusarium species usually reported as cause of human disease are F. solani, F. oxysporum and F. verticillioides.Case descriptionWe describe the second case in the literature of disseminated fusariosis caused by Fusarium napiforme, that occurred in a 60-year-old woman with multiple myeloma after subsequent cycles of chemotherapy.Discussion and EvaluationWe identified the F. napiforme not only by standard morphologic criteria by macroscopic and microscopic characteristics, but also confirmed by molecular biology methods, including sequencing. The antifungal susceptibility of the F. napiforme isolates were tested to seven antifungal drugs; the azoles were the most active drug against all the isolates tested.ConclusionsFusarium spp. are of relevance in medical mycology, and their profiles of low susceptibility to antifungal drugs highlight the importance for faster and more accurate diagnostic tests, what can contribute to an earlier and precise diagnosis and treatment.

Standardization of Hyphal Growth Inhibition Rate as a Means of Evaluating Microsporum spp. in vitro Susceptibility to Terbinafine, Griseofulvin, and Ciclopiroxolamine

Reference methods for antifungal susceptibility tests recommend the use of conidia as inoculum. However, some isolates produce few conidia, while the invasive form of filamentous fungi in general is hyphae making susceptibility tests infeaseble. These facts suggest that other than conidia broth dilution method is required for susceptibility tests. The aim of this study was to clarify if the hyphal growth inhibition rate could be used as a method of determining the antifungal susceptibility of genus Microsporum. For this reason, a method which traces hyphal tips automatically and measures their growth rate was standardized for Microsporum spp. Control growth curves and test growth curves obtained by real-time observation of the hyphae groups responses to different concentrations of terbinafine, griseofulvin, and ciclopiroxolamine were used to compare with minimum inhibitory concentrations (MICs) obtained by conidia broth microdilution method. A visible reduction in the growth inhibition rate was observed when hyphal activity was evaluated using the third or fourth serial two-fold dilution below the MIC determined by broth microdilution for terbinafine and ciclopiroxolamine. For griseofulvin, this reduction occurred after the fifth dilution below the MIC. This study highlights the importance of the inoculum type used to determine the in vitro susceptibility of Microsporum strains. We conclude that measurement of hyphal growth inhibition, despite being time consuming, could be a suitable method for evaluating antifungal susceptibility, particularly for fungi as Microsporum spp. that produce a small (or not at all) number of conidia.

Visual analysis of DNA microarray data for accurate molecular identification of non-albicans Candida isolates from patients with candidemia episodes.

ABSTRACT The performance of a visual slide-based DNA microarray for the identification of non-albicans Candida spp. was evaluated. Among 167 isolates that had previously been identified by Vitek 2, the agreement between DNA microarray and sequencing results was 97.6%. This DNA microarray platform showed excellent performance.

Expression of activation and cytotoxic molecules by peripheral blood lymphocytes of patients with paracoccidioidomycosis

In a previous study, we reported an increased number of T CD8(+) cells in the bronchoalveolar lavage (BAL) of patients with pulmonary paracoccidioidomycosis, suggesting a role for these cells in the local immune response. The aims of this study were to verify, by flow cytometry, the activation state, as well as the production of cytotoxic molecules by peripheral blood lymphocytes (CD8(+) and CD4(+)). Specimens were obtained from patients with paracoccidioidomycosis (PCM), individuals with PCM-infection, i.e., healthy individuals with demonstrated strong cellular response against the fungus (PI) and controls, with studies conducted both ex-vivo and in vitro, after stimulation with Paracoccidioides brasiliensis yeast cells. The ex-vivo analysis demonstrated that PCM patients presented a lower frequency of granzyme A, B and perforin-positive cells, as compared to individuals with PCM infection (PI). P. brasiliensis stimulation led to a discrete increase in CD69(+) cells and a reduction in cytotoxic granule expression in all groups. The addition of IL-15 induced an increase in the frequency of CD69(+) cells only in PI individuals and controls. The effect of IL-15 on granzyme A and B expression was low, but a higher frequency of CD8(+) perforin(+) was detected in PI individuals than in patients with active PCM. IL-15Ralpha expression was lower in CD4(+) T cells from patients, in relation to the PI group. Furthermore, low levels of granulysin were detected in sera from PCM patients, but a tendency for an increase in these levels was observed after antifungal therapy. Taken together, these results indicate that lymphocytes from PCM patients are poorly activated, express low levels of IL-15Ralpha and produce basal levels of cytotoxic granules. These findings may account for the defective cytotoxic activity in patients and, consequently, a low capacity to kill the fungus.

Preanalytical conditions for broth microdilution antifungal susceptibility of Microsporum spp.

Dermatophytoses caused by the genus Microsporum require a long‐duration therapy compared to infections caused by other genera. Treatment of these cutaneous infections includes topical and systemic antifungal agents. Tinea capitis and tinea unguium caused by M. canis and M. gypseum are the most difficult‐to‐treat dermatophytoses. There are few specific studies about corresponding antifungal susceptibility in vitro. Recently, the Clinical and Laboratory Standards Institute proposed the M38A document as standard to determine the minimum inhibitory concentrations (MICs) of several antifungal agents against conidium‐forming filamentous fungi; however, dermatophytes were not included in this document. This study aimed to contribute to continuing investigations concerning the optimal antifungal susceptibility testing conditions of Microsporum spp. to terbinafine, ciclopiroxolamine and griseofulvin. The results pointed out potato dextrose agar as the best culture medium for inducing conidia sporulation, inoculum density amounting to 1 × 103 conidia ml−1, containing only microconidia, with an incubation time of 7 days at 28 °C and 100% growth inhibition serving as an endpoint. The minimum fungicidal concentration values were in accordance with the MICs values, showing a fungicidal activity of these drugs towards the tested strains. According to our results, in general, terbinafine was more active than griseofulvin and ciclopiroxolamine.

Resistance Surveillance in Candida albicans: A Five-Year Antifungal Susceptibility Evaluation in a Brazilian University Hospital

Candida albicans caused 44% of the overall candidemia episodes from 2006 to 2010 in our university tertiary care hospital. As different antifungal agents are used in therapy and also immunocompromised patients receive fluconazole prophylaxis in our institution, this study aimed to perform an antifungal susceptibility surveillance with the C.albicans bloodstream isolates and to characterize the fluconazole resistance in 2 non-blood C.albicans isolates by sequencing ERG11 gene. The study included 147 C. albicans bloodstream samples and 2 fluconazole resistant isolates: one from oral cavity (LIF 12560 fluconazole MIC: 8μg/mL) and one from esophageal cavity (LIF-E10 fluconazole MIC: 64μg/mL) of two different patients previously treated with oral fluconazole. The in vitro antifungal susceptibility to amphotericin B (AMB), 5-flucytosine (5FC), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), caspofungin (CASP) was performed by broth microdilution methodology recommended by the Clinical and Laboratory Standards Institute documents (M27-A3 and M27-S4, CLSI). All blood isolates were classified as susceptible according to CLSI guidelines for all evaluated antifungal agents (MIC range: 0,125–1.00 μg/mL for AMB, ≤0.125–1.00 μg/mL for 5FC, ≤0.125–0.5 μg/mL for FLC, ≤0.015–0.125 μg/mL for ITC, ≤0.015–0.06 μg/mL for VRC and ≤0.015–0.125 μg/mL for CASP). In this study, we also amplified and sequenced the ERG11 gene of LIF 12560 and LIF-E10 C.albicans isolates. Six mutations encoding distinct amino acid substitutions were found (E116D, T128K, E266D, A298V, G448V and G464S) and these mutations were previously described as associated with fluconazole resistance. Despite the large consumption of antifungals in our institution, resistant blood isolates were not found over the trial period. Further studies should be conducted, but it may be that the very prolonged direct contact with the oral antifungal agent administered to the patient from which was isolated LIF E-10, may have contributed to the development of resistance.

Evaluation of antifungal combination against Cryptococcus spp.

The second cause of death among systemic mycoses, cryptococcosis treatment represents a challenge since that 5‐flucytosine is not currently available in Brazil. Looking for alternatives, this study evaluated antifungal agents, alone and combined, correlating susceptibility to genotypes. Eighty Cryptococcus clinical isolates were genotyped by URA5 gene restriction fragment length polymorphism. Antifungal susceptibility was assessed following CLSI‐M27A3 for amphotericin (AMB), 5‐flucytosine (5FC), fluconazole (FCZ), voriconazole (VRZ), itraconazole (ITZ) and terbinafine (TRB). Drug interaction chequerboard assay evaluated: AMB + 5FC, AMB + FCZ, AMB + TRB and FCZ + TRB. Molecular typing divided isolates into 14 C. deuterogattii (VGII) and C. neoformans isolates were found to belong to genotype VNI (n = 62) and VNII (n = 4). C. neoformans VNII was significantly less susceptible than VNI (P = 0.0407) to AMB; C. deuterogattii was significantly less susceptible than VNI and VNII to VRZ (P < 0.0001). C. deuterogattii was less susceptible than C. neoformans VNI for FCZ (P = 0.0170), ITZ (P < 0.0001) and TRB (P = 0.0090). The combination FCZ + TRB showed 95.16% of synergistic effect against C. neoformans genotype VNI isolates and all combinations showed 100% of synergism against genotype VNII isolates, suggesting the relevance of cryptococcal genotyping as it is widely known that the various genotypes (now species) have significant impact in antifungal susceptibilities and clinical outcome. In difficult‐to‐treat cryptococcosis, terbinafine and different antifungal combinations might be alternatives to 5FC.