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.

Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC).

In the present study, we developed a new real-time PCR system based on the cycling probe technology (CPT), which is composed of two single tube real-time PCR assays: the Fusarium genus-specific assay and the Fusarium solani species complex (FSSC)-specific assay with primers targeting the 28s ribosomal RNA gene. The Fusarium genus-specific assay was shown to be highly specific, detecting all reference Fusarium strains with no cross-reaction with other reference fungal strains, such as Aspergillus spp. and human DNA. The FSSC-specific assay also reacted very specifically with FSSC, except for a cross-reaction with Fusarium lunatum. To validate the real-time PCR system, we tested 87 clinical isolates of Fusarium spp. Identification results from the real-time PCR system were found to be 100% concordant with those from DNA sequencing of EF-1α gene. The sensitivity testing also demonstrated high sensitivity, enabling detection of one copy of standard DNA with good reproducibility. Furthermore, both assays were shown to be extremely sensitive even when fungal cells were mixed with human cells, detecting 3 germinated conidia spiked in 3mL of human blood. To apply our new real-time PCR system to the molecular diagnosis of fusariosis, we evaluated its efficacy using a mouse model of invasive F. solani infection. Plasma and whole blood samples of infected mice were tested using the real-time PCR system. The sensitivity of the real-time PCR system was found to be 100% (n=4) in plasma samples. In contrast, no amplification signal was detected in whole blood samples. This system could provide a rapid and precise diagnostic tool for early diagnosis, which is necessary for appropriate treatment and improvement of prognosis of disseminated fusariosis.

Antifungal susceptibility of Aspergillus fumigatus clinical isolates collected from various areas in Japan.

Azole resistance among clinical isolates of Aspergillus fumigatus is becoming a serious problem in Europe, but the status in Japan is not yet known in detail. The aim of this study was to determine the present status of azole resistance in A. fumigatus in Japan. We employed 171 clinical isolates of A. fumigatus sensu stricto collected from 1987 to 2008 at the Medical Mycology Research Center, Chiba University, Japan for azole resistance determination. Identification of all isolates were re-examined both from the aspect of morphology and molecular phylogeny. The antifungal susceptibility of these isolates was tested based on the CLSI M38-A2 broth microdilution method. In our collection, only 1 (0.6%) and 2 isolates (1.2%) showed elevated MIC to voriconazole and itraconazole, respectively. Our study disclosed that the frequency of azole resistance in A. fumigatus still remains low in this collection.

Fetuin A, a serum component, promotes growth and biofilm formation by Aspergillus fumigatus.

Aspergillus fumigatus is an all-important pathogenic fungus and is known for its angiotropism. When it invades human organs, A. fumigatus makes direct contact with blood and its components by causing inflammation and invading vascular structures. To learn the effect of its contact with blood on the development of infection, we examined the effect of serum on A. fumigatus growth. In Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, hyphal tip growth was accelerated, forming a thickened and well-networked biofilm associated with extracellular matrix, and fetuin A was identified as the active component in the serum that accelerates fungal growth leading to formation of a community. These results suggest that fetuin A is a novel accelerator of the growth of A. fumigatus and that it participates in the formation of thick biofilm.

Evaluation of the Sensititre YeastOne and Etest in Comparison with CLSI M38-A2 for Antifungal Susceptibility Testing of Three Azoles, Amphotericin B, Caspofungin, and Anidulafungin, against Aspergillusfumigatus and Other Species, Using New Clinical Breakpoints and Epidemiological Cutoff Values.

Aspergillosis is an invasive fungal disease associated with high mortality. Antifungal susceptibility testing (AFST) is receiving increasing consideration for managing patients, as well as for surveilling emerging drug resistance, despite having time-consuming and technically complex reference methodologies. The Sensititre YeastOne (SYO) and Etest methods are widely utilized for yeasts but have not been extensively evaluated for Aspergillus isolates. We obtained Posaconazole (POS), Voriconazole (VCZ), Itraconazole (ITC), Amphotericin B (AMB), Caspofungin (CAS), and Anidulafungin (AND) minimum inhibitory concentrations (MICs) for both the Etest (n = 330) and SYO (n = 339) methods for 106 sequenced clinical strains. For 84 A. fumigatus, we analyzed the performance of both commercial methods in comparison with the CLSI-AFST, using available cutoff values. An excellent correlation could be demonstrated for Etest-AMB and Etest-VCZ (p < 0.01). SYO-MICs of AMB, VCZ, and POS resulted in excellent essential agreement (>93%), and >80% for AMB, VCZ, and ITC Etest-MICs. High categoric agreement was found for AMB, ITC, and CAS Etest-MICs (>85%) and AMB SYO-MICs (>90%). The considerable number of major/very major errors found using Etest and SYO, possibly related to the proposed cutoffs and associated with the less time-consuming processes, support the need for the improvement of commercial methods for Aspergillus strains.

Comparison of genotypes between environmental and clinical isolates of Cryptococcus neoformans var. grubii based on microsatellite patterns.

We applied multilocus microsatellite typing (MLMT) method to investigate the genetic relation between Cryptococcus neoformans var. grubii clinical and environmental isolates in São Paulo, Brazil. This MLMT method includes three functional gene sequences of C. neoformans var. grubii, which are dispersed on three chromosomes. In all, 89 strains (36 clinical and 53 environmental isolates) were analyzed. Of 36 clinical strains, 20 belonged to a major type of MLMT-13 (55.6%). They were mainly isolated from clinical specimens. About 52.8% of strains from the environment belong to a major type of MLMT-36, which are indigenous to environments and which were not isolated from clinical samples. Thus, we recognized two genotypes that distinguish majority of clinical and environmental strains. No differences were found in antifungal susceptibility and capsule size between major environmental and clinical MLMT types.

Airborne transmission of invasive fusariosis in patients with hematologic malignancies.

From 2006 to 2013, an increasing incidence of fusariosis was observed in the hematologic patients of our University Hospital. We suspected of an environmental source, and the indoor hospital air was investigated as a potential source of the fungemia. Air samplings were performed in the hematology and bone marrow transplant (BMT) wards using an air sampler with pre-defined air volumes. To study the molecular relationship among environmental and clinical isolates, 18 Fusarium spp. recovered from blood cultures were included in the study. DNA sequencing of a partial portion of TEF1α gene was performed for molecular identification. Molecular typing was carried out by multi-locus sequence typing (MLST) using a four-gene scheme: TEF1α, rDNA, RPB1 and RPB2. One hundred four isolates were recovered from the air of the hematology (n = 76) and the BMT (n = 28) wards. Fusarium isolates from the air were from five species complexes: Fusarium fujikuroi (FFSC, n = 56), Fusarium incarnatum-equiseti (FIESC, n = 24), Fusarium solani (FSSC, n = 13), Fusarium chlamydosporum (FCSC, n = 10), and Fusarium oxysporum (FOSC, n = 1). Fifteen Fusarium isolates recovered from blood belonged to FSSC, and three to FFSC. MLST identified the same sequence type (ST) in clinical and environmental isolates. ST1 was found in 5 isolates from blood and in 7 from the air, both identified as FSSC (Fusarium petroliphilum). STn1 was found in one isolate from blood and in one from the air, both identified as FFSC (Fusarium napiforme). F. napiforme was isolated from the air of the hospital room of the patient with fungemia due to F. napiforme. These findings suggested a possible clonal origin of the Fusarium spp. recovered from air and bloodcultures. In conclusion, our study found a diversity of Fusarium species in the air of our hospital, and a possible role of the air as source of systemic fusariosis in our immunocompromised patients.

[Antifungal activity of the novel adduct, GX-95, of silver with nanometer-scale particles to peptidic hydrolysates from collagen].

Silver has long been known to have an antimicrobial activity against bacteria and other microorganisms, and has been used as eating utensils, as dental fillings and so on. We developed a novel adduct, GX-95, of silver with nanometer-scale particles to peptidic hydrolysates from collagen. Antifungal activity of the adduct against pathogenic yeasts and filamentous fungi was examined in terms of minimal inhibitory concentrations (MICs). GX-95 was found to possess strong and broad antifungal activities against all fungi examined in the following MICs: 0.25 to 0.31 microg/ml for Candida albicans including resistant strains to fluconazole, itraconazole and flucytosine, 0.05 to 0.2 microg/ml for Cryptococcus neoformans strains, 0.025 to 0.4 microg/ml for Aspergillus fumigatus strains, 0.4 microg/ml for Trichophyton rubrum, and 0.05 microg/ml for Cladophialophora carrionii.

Effect of serum components on biofilm formation by Aspergillus fumigatus and other Aspergillus species.

Biofilm production by microorganisms is critical for their pathogenicity. Serum promotes biofilm production by Aspergillus fumigatus; however, its effects on other Aspergillus spp. have not been reported. We analyzed biofilm formation by five Aspergillus spp., i.e., A. fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus, and examined the effects of serum/serum proteins such as fetal bovine serum (FBS), fetuin A, and bovine serum albumin (BSA) on hyphal growth, hyphal branching, and extracellular matrix (ECM) formation. The antifungal susceptibility of A. fumigatus isolates that formed biofilms was also examined. All serum/serum proteins promoted the growth of all these fungal species; growth promotion was most evident with FBS, followed by fetuin A and BSA. This effect was most evident in case of A. fumigatus and least evident in case of A. terreus. Electron microscopy showed thick ECM layers surrounding fungal cell walls after culture with FBS, particularly in A. fumigatus. An increase in hyphal branching caused by fetuin A was the highest in case of A. fumigatus and A. nidulans. Biofilm-forming A. fumigatus showed resistance to most antifungal agents, although a synergism of micafungin and amphotericin B was suggested. Our results indicate that serum promotes biofilm formation, including thick ECM, by many Aspergillus spp., particularly A. fumigatus, and that this may be closely related to its virulence.

Antimicrobial effect of medical adhesive composed of aldehyded dextran and ε-Poly(L-Lysine).

Infection of surgical wounds is a severe problem. Conventional tissue reattachment methods have limits of incomplete sealing and high susceptibility to infection. Medical adhesives have several advantages over traditional tissue reattachment techniques, but still have drawbacks, such as the probability of infection, low adhesive strength, and high cytotoxicity. Recently, a new medical adhesive (new-adhesive) with high adhesive strength and low cytotoxicity, composed of aldehyded dextran and ε-poly(L-lysine), was developed. The antimicrobial activity of the new-adhesive was assayed using agar media and porcine skin. In the agar diffusion method, inoculated microorganisms that contacted the new-adhesive were inactivated, but this was not dependent on the amount of new-adhesive. Similar to the agar media results, the topical antimicrobial effect of new-adhesive was confirmed using a porcine skin antimicrobial assay, and the effect was not due to physical blocking based on comparison with the group whose wounds were wrapped.

In vitro antifungal activity of epigallocatechin 3-O-gallate against clinical isolates of dermatophytes.

Previously, we reported that epigallocatechin 3-O-gallate (EGCg) has growth-inhibitory effect on clinical isolates of Candida species. In this study, we investigated the antifungal activity of EGCg and antifungal agents against thirty-five of dermatophytes clinically isolated by the international guidelines (M38-A2). All isolates exhibited good susceptibility to EGCg (MIC50, 2-4 µg/mL, MIC90, 4-8 µg/mL, and geometric mean (GM) MICs, 3.36-4 µg/mL) than those of fluconazole (MIC50, 2-16 µg/mL, MIC90, 4-32 µg/mL, and GM MICs, 3.45-25.8 µg/mL) and flucytosin (MIC50, MIC90, and GM MICs, >64 µg/mL), although they were less susceptible to other antifungal agents, such as amphotericin B, itraconazole, and miconazole. These activities of EGCg were approximately 4-fold higher than those of fluconazole, and were 4 to 16-fold higher than flucytosin. This result indicates that EGCg can inhibit pathogenic dermatophyte species. Therefore, we suggest that EGCg may be effectively used solely as a possible agent or combined with other antifungal agents for antifungal therapy in dermatophytosis.

Oral candidiasis of HIV-infected children undergoing sequential HIV therapies.

Candida oral flora from 52 Brazilian HIV-infected children was characterized while they received antiviral monotherapy therapy and subsequently, HAART with the use of protease inhibitor. There was a significant increase in non-C. albicans Candida isolates from 9.6-28.8% (P=0.005) after the children were placed on protease inhibitor therapy. Although Candida albicans still remained the most commonly isolated species, relative presence of C. tropicalis (n=9) followed by C. parapsilosis (n=8) markedly increased in association with protease inhibitor therapy. Furthermore, rare Candida species including C. dubliniensis, C. norvegensis, C. humicula and C. rugosa also appeared after the onset of protease inhibitor therapy. Subsequent investigation of the antifungal sensitivity of these diverse isolates, derived during protease inhibitor therapy, demonstrated some variation in antifungal sensitivity. With notable exceptions, the majority were sensitive to amphotericin B while most C. albicans and non-C. albicans Candida isolates were also susceptible to fluconazole, itraconazole and ketoconazole. Amongst exceptions was a single C. tropicalis isolates which was resistant to fluconazole (MIC>64 microl/ml) and one C. albicans-B isolate which showed cross-resistance to all azoles and amphotericin.

Evaluation of the inhibitory effect of amphotericin B on the apical growth of F. solani using the BioCell-Tracer system.

The BioCell-Tracer (BCT) system is an automatic microscopic method used for measuring the growth rate of a single fungal hyphae, which has not yet been applied to study Fusarium spp. Considering the large resistance of Fusarium species to the available chemotherapy and that hyphae is the morphological fungal form most often seen in vivo, in this work, Amphotericin B MIC and MFC values for a Fusarium solani strain were obtained by the conventional assay method testing conidia and also by the BCT monitoring system. Both MIC and MFC values of AMB against F. solani determined by broth dilution method resulted in 4.0 microg ml(-1). By the BCT system, their values were 1.0 microg ml(-1), with an inhibition rate of 99.5% (Exp-GR) and 100.0% (Post-GR), showing that when testing hyphae directly, MIC and MFC were determined at two lower dilutions than the MIC and MFC values obtained with conidia. Using the BCT system, 4.0, 2.0 and 1.0 microg ml(-1) of AMB concentrations inhibited hyphae growth in 50 min whereas 0.5 microg ml(-1) of AMB needed 100 min to start hyphae growth inhibition. These findings lead us to conclude that antifungal susceptibility varies between conidia and hyphae. For this strain of Fusarium solani, hyphae were more susceptible to AMB than conidia.

Antifungal susceptibility of epigallocatechin 3-O-gallate (EGCg) on clinical isolates of pathogenic yeasts.

This is the first report to investigate the antifungal susceptibility of 21 clinical isolates of seven Candida species to epigallocatechin 3-O-gallate (EGCg) and to compare with six antifungal agents, amphotericin B (AMPH), fluconazole (FLCZ), flucytosin (5FC), itraconazole (ITCZ), micafungin (MCFG), and miconazole (MCZ), using a method following the National Committee for Clinical Laboratory Standards (NCCLS) M27-A guidelines. Among the tested species, Candida glabrata exhibited the highest susceptibility to EGCg (MIC50, 0.5-1 microg/ml and MIC90, 1-2 microg/ml) compared favorably with FLCZ, although they were slightly less susceptible than to AMPH, 5FC, MCFG, ITCZ, and MCZ. Candida guilliemondii and Candida parapsilosis (MIC50, 1-4 microg/ml and MIC90, 2-16 microg/ml) were also susceptible to EGCg, although they appear to be slightly less susceptible to EGCg than C. glabrata and the other antifungal agents tested. Moreover, the susceptibility of Candida krusei strains (MIC50, 2 microg/ml and MIC90, 4-8 microg/ml) to EGCg was approximately 2- to 8-fold higher than those of 5FC and FLCZ. Our data indicate that EGCg can inhibit clinically pathogenic Candida species, although the concentrations of EGCg for antifungal susceptibility were slightly higher than those of tested antifungal agents on the whole. Based on these results, we suggest that EGCg may be effectively used as a possible agent or adjuvant for antifungal therapy in Candidiasis.

HIV-1 anti-retroviral drug effect on the C. albicans hyphal growth rate by a Bio-Cell Tracer system

Declining incidence of oropharyngeal candidosis and opportunistic infections over recent years can be attributed to the use of highly active anti-retroviral therapy (HAART). Infection with C. albicans generally involves adherence and colonization of superficial tissues. During this process, budding yeasts are able to transform to hyphae and penetrate into the deep tissue. Using the biocell tracer system, C. albicans hyphal growth was dynamically observed at the cellular level. Ritonavir was effective in the inhibition of hyphal growth with growth rate of 0.8 mum/min. This study showed the in vitro effect of HIV anti-retroviral drug on the growth rate of the C. albicans hyphae.

O declínio na incidência de candidose orofaríngea e infecções oportunistas associadas a infecção pelo HIV tem sido atribuído a introdução da terapia antiretroviral combinada (HAART). Infecção por C. albicans envolve aderência e colonização da mucosa superficial. Durante este processo leveduras são capazes de transformar-se na forma de hifas e penetrar nos tecidos mais profundos. Usando o sistema "Bio-Cell Tracer", o crescimento de hifas de C. albicans foi observado dinamicamente a nível celular. Ritonavir, inibidor de protease do HIV, foi efetivo na inibição do crescimento de hifas com media de 0.8 mim/min.O presente estudo demonstrou o efeito in vitro de um agente anti-retroviral HIV sobre o crescimento de hifas de C. albicans.

Candida dubliniensis in a Brazilian family with an HIV 1- infected child: identification, antifungal susceptibility, drug accumulation and sterol composition

This study investigated the prevalence of C. dubliniensis in a Brazilian family with an HIV - infected child. A total of 42 oral isolates were obtained from eight family members. The identification of C. dubliniensis was performed by polymerase chain reactions (PCR) using primers against a specific sequence of the C. dubliniensis cytochrome b gene. Only the HIV-infected child and his grandmother were colonized by C. dubliniensis. In this study C. dubliniensis isolated from the HIV-infected child exhibited high susceptibility for azoles tested with MICs of 0.125 and 0.5 æg/mL for voriconazole and fluconazole, respectively. Accumulation of [³H] fluconazole in C. dubliniensis isolated from the HIV-infected child was slightly reduced in comparison to the reference susceptible strain. C. dubliniensis isolates had significantly lower ergosterol levels in comparison to C. albicans reference strains.

O presente estudo investigou a prevalência de C. dubliniensis em uma família brasileira com uma criança infectada pelo vírus HIV. Um total de 42 isolados orais foram obtidos de 8 membros da família. A identificação de C. dubliniensis foi realizada por polymerase chain reactions (PCR) usando primers contra a sequência específica para o gene C. dubliniensis cytochrome b. Apenas a criança infectada pelo vírus HIV e a avó estavam colonizados por C. dubliniensis. Neste estudo C. dubliniensis isolado da criança infectada pelo vírus HIV exibiu alta susceptibilidade para azoles com concentração mínima inibitória de 0.125 and 0.5 æg/mL para voriconazole and fluconazole respectivamente. Acúmulo de [³H] fluconazol intra-celular foi ligeiramente reduzido em C. dubliniensis isolado da criança infectada pelo vírus HIV em comparação com a cepa referência sensível ao fluconazole. Isolados de C. dubliniensis neste estudo apresentaram níveis significantemente reduzidos de ergosterol da membrane celular em comparação com C. albicans.