Agents Of Invasive Fungal Infections Research Articles

High-throughput identification and determination of antifungal triazoles in human plasma using UPLC-QDa

Triazoles are common agents for invasive fungal infections, while therapeutic drug monitoring is needed to improve antifungal efficacy and reduce toxicity. This study aimed to exploit a simple and reliable liquid chromatography-mass spectrometry method for high-throughput monitoring of antifungal triazoles in human plasma using UPLC-QDa. Triazoles in plasma were separated by chromatography on a Waters BEH C18 column and detected using positive ions electrospray ionization fitted with single ion recording. M+ for fluconazole (m/z 307.11) and voriconazole (m/z 350.12), M2+ for posaconazole (m/z 351.17), itraconazole (m/z 353.13) and ketoconazole (m/z 266.08, IS) were selected as representative ions in single ion recording mode. The standard curves in plasma showed acceptable linearities over 1.25–40 μg/mL for fluconazole, 0.47–15 μg/mL for posaconazole and 0.39–12.5 μg/mL for voriconazole and itraconazole. The selectivity, specificity, accuracy, precision, recovery, matrix effect, and stability met acceptable practice standards under Food and Drug Administration method validation guidelines. This method was successfully applied to the therapeutic monitoring of triazoles in patients with invasive fungal infections, thereby guiding clinical medication.

Fungal Infections Caused by Kazachstania spp., Strasbourg, France, 2007-2020.

Rare fungal pathogens are emerging as agents of invasive fungal infections. We analyzed 13 cases of fungal infections caused by Kazachstania (Arxiozyma) spp. in Strasbourg University Hospital, Strasbourg, France. Among the cases, 4 patients had proven fungal disease (3 cases of invasive fungal disease and 1 mucocutaneous infection) and 9 were colonized by Kazachstania (Arxiozyma) spp. Candida albicans was also isolated from 11 of the 13 patients. None of the patients with proven invasive fungal disease met host criteria, but most had underlying diseases. All strains were identified as K. telluris by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and 3 were confirmed as K. bovina by internal transcribed spacer sequencing. For all tested strains, the MICs for fluconazole were >2 μg/mL. Emergence of this rare fungal infection might be explained by the increasing number of patients with immunocompromised conditions and gastroesophageal diseases.

Micafungin for Candida infections in Slovenia and Romania: A multicenter, observational, prospective study.

An echinocandin, such as micafungin, is recommended as first-line treatment for invasive Candida infections in immunocompromised patients. This multicenter, observational, prospective, non- interventional study evaluated the real-world use of micafungin in clinical practice in Slovenia and Romania, as this remains unexplored. The primary endpoint was evaluation of micafungin use, including rationale for prescription, treatment duration, and daily dose. Secondary endpoints included recordings of patient baseline characteristics and evaluations of efficacy and safety. Across 11 centers in two countries, 118 patients (18 children [< 16 years] and 100 adults [≥ 16 years]) received micafungin for the first time according to their clinic's standard practice. Micafungin was prescribed for treatment in 57.6% of patients and for prophylaxis in 40.7% of patients. The median (range) treatment duration was 9.0 (0 - 54) days and 13.0 (2 - 6)] days, respectively. The median dose of micafungin was higher than recommended for children receiving prophylaxis or treatment for invasive candidiasis and for adults receiving prophylaxis. Fever was the most commonly observed clinical sign at baseline (16 children [88.9%] and 31 adults [31%]) and hematologic malignancy was the most frequent primary diagnosis at admission (11 children [61.1%] and 40 adults [40%]). Candida species were the most commonly identified causal agents of invasive fungal infections (2 children [11.1%] and 48 adults [48%]). The efficacy and safety profiles of micafungin use in Slovenia and Romania based on clinician's own experiences in local clinical practice were consistent with those reported in other real-world studies.

Genetic diversity assessed using PFGE, MLP and MLST in Candida spp. candidemia isolates obtained from a Brazilian hospital.

Candida spp. are the main causative agents of invasive fungal infections in immunocompromised patients. Candidemia has attributable mortality rates of 15 to 35% and increases hospitalisation time and costs, thus making this disease a public health concern. This study aimed to use pulsed-field gel electrophoresis (PFGE), microsatellite length polymorphism (MLP) and multilocus sequence typing (MLST) to analyse the genetic relationships among 65 Candida spp. bloodstream isolates, including 35 Candida albicans, 15 Candida glabrata and 15 Candida tropicalis isolates, all of which were obtained from patients in a Brazilian hospital. Moreover, patient clinical data were assessed. All techniques resulted in high discriminatory indexes. C. albicans and C. tropicalis isolates showed high genetic variability, while C. glabrata isolates had relatively low genetic variability. Moreover, a cluster of C. glabrata isolates was identified in a hospital unit. New MLST sequence types, diploid sequence types and alleles are described. Relationships were not observed between the molecular typing results and clinical characteristics. The molecular typing of clinical strains increases our understanding of candidemia epidemiology and promotes the development of strategies that can reduce the incidence of this disease. Moreover, this study is the first to combine these techniques to genotype these three species in Brazil.

A Second-Generation Fungerp Analog, SCY-247, Shows Potent In Vitro Activity against Candida auris and Other Clinically Relevant Fungal Isolates.

Due to the increase of antifungal drug resistance and difficulties associated with drug administration, new antifungal agents for invasive fungal infections are needed. SCY-247 is a second-generation fungerp antifungal compound that interferes with the synthesis of the fungal cell wall polymer β-(1,3)-d-glucan. We conducted an extensive antifungal screen of SCY-247 against yeast and mold strains compared with the parent compound ibrexafungerp (IBX; formerly SCY-078) to evaluate the in vitro antifungal properties of SCY-247. SCY-247 demonstrated similar activity to IBX against all of the organisms tested. Moreover, SCY-247 showed a higher percentage of fungicidal activity against the panel of yeast and mold isolates than IBX. Notably, SCY-247 showed considerable antifungal properties against numerous strains of Candida auris Additionally, SCY-247 retained its antifungal activity when evaluated in the presence of synthetic urine, indicating that SCY-247 maintains activity and structural stability under environments with decreased pH levels. Finally, a time-kill study showed SCY-247 has potent anti-Candida, -Aspergillus, and -Scedosporium activity. In summary, SCY-247 has potent antifungal activity against various fungal species, indicating that further studies on this fungerp analog are warranted.

Phenotypic and Molecular Biological Analysis of Polymycovirus AfuPmV-1M From Aspergillus fumigatus: Reduced Fungal Virulence in a Mouse Infection Model.

The filamentous fungal pathogen Aspergillus fumigatus is one of the most common causal agents of invasive fungal infection in humans; the infection is associated with an alarmingly high mortality rate. In this study, we investigated whether a mycovirus, named AfuPmV-1M, can reduce the virulence of A. fumigatus in a mouse infection model. AfuPmV-1M has high sequence similarity to AfuPmV-1, one of the polymycovirus that is a capsidless four-segment double-stranded RNA (dsRNA) virus, previously isolated from the genome reference strain of A. fumigatus, Af293. However, we found the isolate had an additional fifth dsRNA segment, referred to as open reading frame 5 (ORF5), which has not been reported in AfuPmV-1. We then established isogenic lines of virus-infected and virus-free A. fumigatus strains. Mycovirus infection had apparent influences on fungal phenotypes, with the virus-infected strain producing a reduced mycelial mass and reduced conidial number in comparison with these features of the virus-free strain. Also, resting conidia of the infected strain showed reduced adherence to pulmonary epithelial cells and reduced tolerance to macrophage phagocytosis. In an immunosuppressed mouse infection model, the virus-infected strain showed reduced mortality in comparison with mortality due to the virus-free strain. RNA sequencing and high-performance liquid chromatography (HPLC) analysis showed that the virus suppressed the expression of genes for gliotoxin synthesis and its production at the mycelial stage. Conversely, the virus enhanced gene expression and biosynthesis of fumagillin. Viral RNA expression was enhanced during conidial maturation, conidial germination, and the mycelial stage. We presume that the RNA or translation products of the virus affected fungal phenotypes, including spore formation and toxin synthesis. To identify the mycovirus genes responsible for attenuation of fungal virulence, each viral ORF was ectopically expressed in the virus-free KU strain. We found that the expression of ORF2 and ORF5 reduced fungal virulence in the mouse model. In addition, ORF3 affected the stress tolerance of host A. fumigatus in culture. We hypothesize that the respective viral genes work cooperatively to suppress the pathogenicity of the fungal host.

268. Fungal NGS: Identification of Etiological Agents of Invasive Fungal Infection by High-throughput Sequencing

BackgroundInvasive fungal infections (IFI) cause severe symptoms that affect immunocompromised and transplant patient populations. Antifungal therapies vary depending on the pathogenic species, and delays in diagnosis can lead to graft loss and an increase in morbidity and mortality. Therefore, rapid identification of fungi causing IFI is critical for informing antifungal therapy. Such actionable genus/species information can be obtained quickly via Next-generation Sequencing (NGS). In this study, an NGS assay was developed to identify fungal species responsible for IFI, allowing for selection of effective antifungal therapies.MethodsInternal transcribed spacer (ITS) regions 1 and 2 were used for fungal identification. Primers were taken from published research and/or designed/modified by assessment in fungal sequence alignments. A DNA sequence database was compiled and a reference-assisted assembly approach utilizing % sequence ID and % coverage was developed for species identification. End-point PCR was conducted on DNA extracted from 19 pathogenic fungal species, and mixed communities (MC) for preliminary sensitivity and inclusivity. Sensitivity was assessed using dilutions of template DNA into the PCR reaction.ResultsNGS data of 14 individual species and 4 MC passed quality control checks. Using only ITS1 and ITS2, species identification was expected for 10 of 14 individuals. We observed species identification in 9 individual samples, and 13 were identified within the top 5 results. All individuals were identified to genus. In MC analyses, combinations of 3, 4, 6, and 10 fungal species resolved 100% of the genera present, but failed to resolve species adequately with only 2 loci evaluated. Unexpectedly, 3 tested Aspergillus spp. were correctly identified with this limited data in both single and MC samples. The lower limit of detection was assessed at 5,000 genomic equivalents/mL of eluate.ConclusionThe inclusivity and sensitivity demonstrated here of an NGS approach for identification of etiological agents of IFI support this assay’s potential utility as an aid in the treatment of IFI in at-risk patient groups. This assay allows for rapid identification (<4 days) of fungal species to aid clinicians in improving case outcomes.Disclosures All authors: No reported disclosures.

Dual DNA Barcoding for the Molecular Identification of the Agents of Invasive Fungal Infections.

Invasive fungal infections, such as aspergillosis, candidiasis, and cryptococcosis, have significantly increased among immunocompromised people. To tackle these infections the first and most decisive step is the accurate identification of the causal pathogen. Routine identification of invasive fungal infections has progressed away from culture-dependent methods toward molecular techniques, including DNA barcoding, a highly efficient and widely used diagnostic technique. Fungal DNA barcoding previously relied on a single barcoding region, the internal transcribed spacer (ITS) region. However, this allowed only for 75% of all fungi to be correctly identified. As such, the translational elongation factor 1α (TEF1α) was recently introduced as the secondary barcode region to close the gap. Both loci together form the dual fungal DNA barcoding scheme. As a result, the ISHAM Barcoding Database has been expanded to include sequences for both barcoding regions to enable practical implementation of the dual barcoding scheme into clinical practice. The present study investigates the impact of the secondary barcode on the identification of clinically important fungal taxa, that have been demonstrated to cause severe invasive disease. Analysis of the barcoding regions was performed using barcoding gap analysis based on the genetic distances generated with the Kimura 2-parameter model. The secondary barcode demonstrated an improvement in identification for all taxa that were unidentifiable with the primary barcode, and when combined with the primary barcode ensured accurate identification for all taxa analyzed, making DNA barcoding an important, efficient and reliable addition to the diagnostic toolset of invasive fungal infections.

Invasive Trichosporon infection in solid organ transplant patients: a report of two cases identified using IGS1 ribosomal DNA sequencing and a review of the literature

Trichosporon species are rare etiologic agents of invasive fungal infection in solid organ transplant (SOT) recipients. We report 2 well-documented cases of Trichosporon inkin invasive infection in SOT patients. We also conducted a detailed literature review of Trichosporon species infections in this susceptible population. We gathered a total of 13 cases of Trichosporon species infections. Any type of organ transplantation can be complicated by Trichosporon infection. Bloodstream infections and disseminated infections were the most common clinical presentations. Liver recipients with bloodstream or disseminated infections had poor prognoses. Although the most common species was formerly called Trichosporon beigelii, this species name should no longer be used because of the changes in the taxonomy of this genus resulting from the advent of molecular approaches, which were also used to identify the strains isolated from our patients. Antifungal susceptibility testing highlights the possibility of multidrug resistance. Indeed, Trichosporon has to be considered in cases of breakthrough infection or treatment failure under echinocandins or amphotericin therapy. Voriconazole seems to be the best treatment option.

Dynamics of Anidulafungin use in a Tertiary Care Hospital.

Dear Editor, Fungal agents have been reported to be more frequently responsible from health care associated infections in recent years [1]. New antifungal agents for invasive fungal infections are introduced to market and we wanted to evaluate our dynamics in use of one of them, the anidulafungin use in our patients with suspected invasive fungal infection. The study was designed as an observational retrospective single center study in a tertiary care hospital. The patients who were treated with anidulafungin, either empirically or culture based, even for one day were included. The pharmacy reports and infection control team consultation reports as well as hospital data base were used. A total of 27 patients included [Table/Fig-1]. Candida spp. was revealed from 14 (52%) patients: 4 (15%) C. albicans, 8 (30%) non-albicans Candida (NAC) and in 2 (7%) patients both C.albicans and NAC were revealed. From the culture positive 14 patients, only 9 (64%) had subsquent culture samples after initiating antifungal therapy, the remaining 5 patients either didn’t have a control blood culture because either they died (n=4) while they were under anidulafungin therapy or referred to another center and lost from follow-up (n=1). Culture negativity was achieved in a median of 3 days in all of the remaining 9 patients (100%) who have subsquent culture samples. Only one patient had another positive culture on the 12th day of therapy, after achieving negative blood culture on the 2nd therapy day. No source was found for persistence of candidemia and control blood culture was again turned to negative on the 19th day of therapy. Of total 27 patients, 22 (82%) patients had died because of underlying medical problems, 11 (44%) of the patients had died before they completed anidulafungin therapy and median anidulafungin therapy duration of these 11 patients were only 5 days (2-15days). As conclusion, anidulafungin was used in elder patients who have long hospitalization duration especially in ICUs; all the patients who have subsquent culture samples were achieved culture negativity under anidulafungin therapy; culture negativity was achieved in a median of 3 days. Attributable mortality cannot be calculated because of the study-design and controlled randomized studies are needed in these complicated patients of ICUs. [Table/Fig-1]: Demographic variables of the patients under anidulafungin therapy (n=27)

Identifizierung von Pilzen in Gewebeschnitten mit Fluoreszenz-in-situ-Hybridisierung

Deep fungal infections are associated with significant mortality despite the availability of new antifungal agents. The identification of causative fungi is important to define successful antifungal therapies as agents differ in the in vitro susceptibility. Characterization of tissue morphology and cultivation from tissue provide important clues to patient management. Molecular techniques such as PCR-based assays are increasingly being used to identify agents of invasive fungal infections. However, potential contamination limits the use when ubiquitous fungi are targeted. Hybridization with fluorescently labeled probes targeting the ribosomal RNA of fungi is emerging as an alternative identification strategy. Using conserved or variable regions of the rRNA as targets, group or species-specific probes can be synthesized to identify fungal pathogens and localize them in the infectious process. These techniques have been successfully applied to deep fungal infections due to different agents in various organ samples.

Genomics and Proteomics as Compared to Conventional Phenotypic Approaches for the Identification of the Agents of Invasive Fungal Infections

There is overwhelming evidence that prompt diagnosis coupled with timely instigation of appropriate antifungal therapy are critical determinants of clinical outcome in invasive fungal infections. However, since the clinical symptoms of infection are often nonspecific, the number and diversity of potential aetiological agents is vast, and many fungi exhibit species-specific differences in antifungal susceptibility, the accurate identification of the responsible pathogen is a cornerstone of the therapeutic decision pathway. Traditionally, identification was achieved by examination of the phenotypic characteristics of the fungus obtained in pure culture, ideally from a normally sterile site/sample. However, this standard culture-based approach lacks sensitivity and obtaining appropriate specimens for culture is often difficult. Moreover, numerous recent studies have demonstrated the existence of clinically relevant cryptic species within well-established morphospecies that can not be differentiated by phenotypic methods. Here we discuss recent advances in genomic and proteomic approaches for the rapid and accurate identification of the principal pathogenic fungi associated with invasive fungal infections.

Dry powder insufflation of crystalline and amorphous voriconazole formulations produced by thin film freezing to mice

Attention has begun to focus on the pulmonary delivery of antifungal agents for invasive fungal infections as inhalation of the fungal spores is often the initial step in the pathogenesis of many of these infections, including invasive pulmonary aspergillosis (IPA). IPA in immunocompromised patients has high mortality rates despite current systemic (oral or intravenous) therapies. In this study, particulate voriconazole (VRC) formulations were designed with suitable properties for inhalation using thin film freezing (TFF), a particle engineering process capable of producing low-density porous aggregate particles. Nanostructured amorphous morphology of VRC was less favorable in vitro and in vivo than microstructured crystalline morphology, despite being a poorly water-soluble compound. Using a Handihaler® dry powder inhaler (DPI), microstructured crystalline TFF-VRC and nanostructured amorphous TFF-VRC-PVP K25 (1:3) had fine particle fractions of 37.8% and 32.4% and mass median aerodynamic diameters of 4.2 and 5.2μm, respectively. Single dose 24-h pharmacokinetic studies were conducted in ICR mice. AUC0–24h in the lung tissue and plasma was 452.6μgh/g wet lung weight and 38.4μgh/mL, respectively, following a 10mg/kg insufflated dose of TFF-VRC directly into the lungs of the mice, while AUC0–24h in the lung tissue and plasma was 232.1μgh/g wet lung weight and 18.6μgh/mL, respectively, following a 10mg/kg insufflated dose of TFF-VRC-PVP K25 (1:3). High concentrations of VRC in lung tissue coupled with clinically relevant plasma concentrations suggest that pulmonary delivery of microstructured crystalline VRC could potentially be a beneficial strategy for administration of VRC to patients with invasive pulmonary fungal infections.

Results of a Prospective Non-Randomized Trial Comparing the Efficacy of Voriconazole (Vor) and Posaconazole (Pos) As Primary Prophylactic Agents of Invasive Fungal Infections (IFI) in Acute Leukemic Patients

Abstract 1120Between 2007–2010 we performed a monocentric non-randomized prospective study to compare Vor (orally or IV 200 mg b.i.d.) to Pos (orally 200 mg t.i.d.) as primary prophylactic antifungals in either de novo or relapsed leukemic patients. Most chemotherapy regimens used were those of the French collaborative group GOELAMS. The Vor arm was stopped mid-2008 because of available evidence in favor of Pos. The primary endpoint was the IFI incidence within 60 days after chemotherapy. IFI was diagnosed according to the 2008 EORTC criteria. Secondary endpoints were the 60 days post-chemotherapy death rate and frequency of empirical antifungal therapy. Variables potentially related to IFI incidence and to outcome as LAF rooms HEPA filtrated, cytogenetics risk-profile, diagnosis, disease status, age, duration of neutropenia or prophylaxis were also evaluated. Characteristics of patients at baseline are shown below. The unit of study was a cycle of chemotherapy. Among Vor and Pos treated patients 65.2 and 67% respectively corresponded to induction cycles.VoriconazolePosaconazolepN23115Age (years: median/mean, range)51.1/58 (26–72)59/57.6 (20–82)0.15Sex F/M9/1434/810.4AML2098n.s.ALL317n.s.Induction1577n.s.Consolidation838n.s.De novo20103n.s.Refractory/relapsed312n.s.Patients in LAF rooms, HEPA filtered with positive pressure YES/NOT8/1553/62n.s.ALL CYCLES *INDUCTION*5/10*34/43n.s.High/intermediate/low risk cytogenetics5/9/536/57/17n.s.Mean/Median duration of prophylaxis: days18.4/1624.1/210.1Mean-Median duration of ANC < 500/mm317.7–16/*19.5–18/**14.5–1217.9–17/*20.3–20/**13–13.5n.s.all cycles/*induction/**consolidation Results:IFI was diagnosed in 1/23 (4.4%) and 5/115 (4.4%) cycles respectively of Vor (1 proven C lusitaniae) and Pos (1 proven C krusei, 1 proven C albicans, 3 probable aspergillosis) arms (p 1.0). Five of 6 cases of IFI occurred during inductions and 1 on consolidations without significant difference between the two drugs. Most IFI were observed in patients not placed in LAF rooms. The IFI rate was 1.6% (1/62) and 6.5% (5/77) respectively for patients in LAF and conventional rooms (p 0.2). During induction patients in LAF rooms showed a trend to a lower incidence of IFI (0/39) compared to those in conventional rooms (5/48) (p 0.07), without superiority of Vor or Pos arms. Induction patients in LAF rooms presented respectively no IFI (0/8) and 1 (1/52) in the Vor and Pos arms (p 0.57). Among patients not located in LAF rooms both Pos and Vor arms presented a similar IFI rate, 6.4% (4/58) and 6.6% (1/14) respectively. Although patients with low-risk cytogenetics did not present any IFI, this was not significantly different to patients with intermediate and high-risk cytogenetics (p 0.5), conversely to an IFI incidence significantly higher in relapsed/refractory patients vs those who achieved or persisted in first complete remission (p 0.01). Empirical treatment was administered respectively to 8.7 and 16.5% of patients in the Vor and Pos arms (p 0.5) without difference in the IFI incidence between patients who received it or not. The 60 days death rate was 17.4 and 13 % respectively in the Vor and Pos arms (p=0.5). Patients deceased within 60 days were older than those who survived (p 0.08) and presented more frequently a poor prognosis cytogenetics (p 0.032). Death rate was higher but not significantly different during induction (17.4%) and consolidation (2.1%) (p 0.1). Patients in LAF rooms presented a significantly lower death rate (6.5%) than patients in conventional rooms (19.5%) (p 0.02). Induction patients in LAF rooms deceased less (7.7%) than those in conventional rooms (24.5%) (p 0.05). The 60 days death rate was not significantly different between patients placed or not in LAF rooms receiving either Pos or Vor, but it was significantly different between patients presenting or not an IFI (50 and 88% respectively) (p 0.033). This was confirmed in a multivariate analysis which identified diagnosis of IFI (0.004; OR 20.0) and age (0.02; OR 4.5) as significant prognostic variables of a 60 days death rate. Conclusions: both Pos and Vor present a good similar efficacy in primary prophylaxis of IFI in acute leukemic patients. Poor prognosis patients present a higher rate of IFI and death. Patients located in LAF rooms during induction tend to present a lower IFI incidence and a better survival. Disclosures:Off Label Use: Voriconazole use in primary prophylaxis of invasive fungal infections in leukemic patients.

Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in Formalin fixed, paraffin-embedded tissue specimens

BackgroundIdentification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients.MethodsWe designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broad-range fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach.ResultsPCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background.ConclusionsWhile PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples.

Evaluation of Antifungal Susceptibility Testing with Microdilution and Etest Methods of Candida Blood Isolates

Candida species that show an increasing number of clinical and/or microbiological resistance to several antifungals and are the most common agents of invasive fungal infections. The aim of this study was to investigate the in vitro susceptibility of Candida blood isolates to antifungal agents (amphotericin B, fluconazole, itraconazole, and voriconazole) by comparative use of the CLSI reference microdilution method and Etest. Four hundred Candida blood isolates (215 Candida albicans, 185 non-albicans Candida strains) were included in the study. The broth microdilution test was performed according to the CLSI M27 A2 document. Etest was carried out according to the manufacturer's instructions. The MIC results obtained with reference microdilution were compared with those obtained with the Etest by using percent and categorical agreements. According to MIK(90) values, voriconazole was the most active and itraconazole was the least active drug in vitro against all Candida species. Other than voriconazole, statistically significant differences were found when the susceptibility of Candida albicans and non-albicans Candida spp. to amphotericin B, fluconazole, and itraconazole were compared. These antifungal agents were found to be more active to C. albicans. Among the non-albicans Candida species, the lowest MIC values were obtained for Candida parapsilosis isolates. When the standard method was compared with Etest, the total agreement was higher for C. albicans than for non-albicans species, especially for fluconazole and voriconazole. In view of the findings, it was concluded that itraconazole showed the lowest activity against all Candida species. Etest could be an alternative method in assessing the in vitro antifungal susceptibility of Candida spp., but it is more convenient to use the microdilution method for studying in vitro susceptibility of non-albicans species, in particular for those possessing high MIC values against azoles.

Mycoviruses: future therapeutic agents of invasive fungal infections in humans?

Invasive fungal infections are relatively common opportunistic infections in immunocompromised patients and are still associated with a high mortality rate. Furthermore, these infections are often complicated by resistance or refractoriness to current antimicrobial agents. Therefore, an urgent need exists for new therapeutic strategies based on the identification of new microbial targets and novel antimicrobial agents. One such hypothetical therapeutic strategy may involve the use of mycoviruses that are able to selectively infect fungi. Current knowledge of mycoviruses of human pathogenic fungi and the scope for using (recombinant) mycoviruses as future biological control agents are reviewed here.

Newer antifungal agents for invasive fungal infections in patients with haematological malignancy

Since 2001 five new systemically administered antifungal agents have been approved for clinical use. This represents a major advance for antifungal therapy in haematological malignancy patients undergoing chemotherapy or haematopoietic stem cell transplant (HSCT). The echinocandins are a new class of antifungals with a novel mode of action. Capsofungin has already established itself as a valuable therapy for candidaemia and salvage therapy of invasive aspergillosis. Although both anidulafungin and micafungin are approved for treatment of candidiasis, their role in invasive aspergillosis requires more clinical trial evaluation. Of the two newer triazoles, voriconazole has been recommended in international guidelines as primary therapy for acute invasive aspergillosis. Posaconazole has a broad spectrum of activity in vitro and a potentially key role in antifungal prophylaxis in high-risk HSCT recipients and during prolonged neutropenia. Although some of these drugs have important interactions with other medications, and potential toxicities, they are safer to use and more efficacious than amphotericin B deoxycholate. Their arrival gives more choices to treat rarer mycoses and will facilitate clinical trial assessment of combination therapy of aspergillosis where single agent therapy gives less than 50% success rates.

Management of systemic fungal infections: alternatives to itraconazole

For many years, amphotericin B and flucytosine have been the only antifungal agents for invasive fungal infections. Amphotericin B was the standard of care for most of these infections. However, its use was often associated with low efficacy and poor tolerance. Fortunately, the antifungal armamentarium has increased during the past two decades with the addition of several new agents. In addition to itraconazole and fluconazole, lipid formulations of amphotericin B, voriconazole, caspofungin and micafungin have arrived on the market. Other agents are expected to be licensed shortly (anidulafungin, posaconazole). These various antifungal agents differ in their spectrum, pharmacokinetic profile, route of administration, efficacy in clinical trials, safety profile, drug-drug interactions and, importantly, their cost. There is no longer a unique standard agent for all or nearly all invasive fungal infections but a real choice among several agents. The characteristics of these new agents are reviewed to help clinicians in their decision to select an antifungal agent for their patients.