Comparative study of the efficacy of liposomal amphotericin B and amphotericin B deoxycholate against six species of Zygomycetes in a murine lethal infection model

The objective of this study was to investigate the efficacy of liposomal amphotericin B (L-AMB) at a clinical dose (3 mg/kg) against six species (5 genera) of Zygomycetes in a murine lethal infection model, and to compare findings with those for deoxycholate amphotericin B (D-AMB). The correlation between in-vitro activity and in-vivo efficacy of L-AMB was also investigated. Cyclophosphamide-treated mice were inoculated intravenously with conidial suspensions. Four hours or 1 day after inoculation, a single dose of L-AMB or D-AMB was administered intravenously. The number of mice that survived for 14 days was recorded. L-AMB at a dose of at least ≥1 mg/kg significantly prolonged the survival time of infected mice compared with the control group. The ED₅₀ of L-AMB was nearly equivalent to that of D-AMB, except for the treatment initiated on day 1 in the Rhizopus oryzae model. At the maximum tolerated dose (MTD) of each agent, survival percentages with L-AMB (10 mg/kg) were equal to or higher than those with D-AMB (1 mg/kg). The ED₅₀ of L-AMB decreased as the MIC against the infecting strain decreased. In conclusion, L-AMB was effective at a clinical dosage, and at the MTD the efficacy of L-AMB was equal or superior to that of D-AMB in a murine model of disseminated zygomycosis. The in-vivo activity of L-AMB was correlated with its in-vitro activity.     

Intraocular penetration of liposomal amphotericin B after intravenous injection in inflamed human eyes

PurposeTo determine the intraocular penetration of amphotericin B (AMPH-B) after an intravenously injection of liposomal amphotericin B (L-AMB) in inflamed human eyes.MethodsSeven eyes of 5 patients with fungal eye diseases (endophthalmitis in 6 eyes and keratitis in 1 eye) were treated with intravenous injections of 100–250 mg/day of L-AMB. Samples of blood, corneal button, aqueous humor, and vitreous humor were collected and assessed for AMPH-B.ResultsThe AMPH-B level in the cornea (604.0 μg/g) of the case with fungal keratitis exceeded the minimum inhibitory concentration. However, the levels in the aqueous and vitreous humors of the cases with fungal endophthalmitis were lower, e.g., 0.02 ± 0.01 μg/ml (0.09% of serum level) in the aqueous humor and 0.05 ± 0.08 μg/ml (0.17% of serum level) in the vitreous humor.ConclusionsThe AMPH-B levels administered intravenously were very low in the aqueous and vitreous humors. Our findings indicate that intravenous L-AMB can be considered only for patients with mild endogenous fungal endophthalmitis, e.g., isolated chorioretinitis without vitreous extensions.     

Efficacy and safety of liposomal amphotericin B for deep mycosis in patients with connective tissue disease

The efficacy and safety of liposomal amphotericin B (L-AMB) in the treatment of invasive fungal infections (IFIs) were retrospectively evaluated for patients with connective tissue diseases (CTDs) during treatment with immunosuppressive therapy. Subjects were 13 patients with CTDs complicated by IFI, on the basis of clinical symptoms, imaging findings, and microbiological and histological examinations. All patients were treated with L-AMB. Efficacy and safety were evaluated before and after administration of L-AMB. Underlying diseases were systemic lupus erythematosus for 4 patients, rheumatoid arthritis for 3, microscopic polyangiitis for 2, adult-onset Still disease for 1, dermatomyositis for 1, and mixed connective tissue disease for 1. Eight patients were resistant to other antifungal drugs. Prednisolone was given to 11 patients and the median dose was 10 mg/day. Immunosuppressants were used for 8 patients. The median duration of administration of L-AMB was 8.5 days (range 4–38 days). In proven and probable diagnosis patients (n = 5), the treatment was effective for 3 patients and ineffective for 2 (efficacy rate 60 %). Serum 1,3-β-d-glucan antigenemia (BG) levels decreased after treatment in the 2 patients who were positive for BG. Serum Aspergillus galactomannan antigen levels decreased in 3 of 4 patients with Aspergillus infection. No patient died of IFI. Regarding potential adverse reactions, there were no significant changes in serum creatinine and potassium levels. L-AMB is effective and well-tolerated for treatment of IFI in patients with CTDs.     

The evaluation of frequency of nephrotoxicity caused by liposomal amphotericin B

Background

Liposomal amphotericin B (L-AmB) was developed to reduce nephrotoxicity and maximize the therapeutic utility of amphotericin B in the treatment of invasive fungal infections. However, there is little investigation into the safety of L-AmB in patients with several renal functions. Therefore, we retrospectively evaluated the clinical safety of L-AmB among patients with several renal functions.

Efficacy of aerosolized liposomal amphotericin B against murine invasive pulmonary mucormycosis

Invasive pulmonary mucormycosis is a life-threatening fungal infection encountered in immunocompromised patients. An intravenous high-dose lipid formulation of amphotericin B, such as liposomal amphotericin B (L-AMB), is the recommended treatment. The efficacy of inhaled L-AMB against mucormycosis has not been evaluated.We evaluated the efficacy of inhaled aerosolized L-AMB in murine invasive pulmonary mucormycosis. ICR female mice were immunosuppressed with cortisone acetate and cyclophosphamide and challenged on day 0 with 1 × 10⁶ conidia of Rhizopus oryzae (TIMM 1327) intratracheally. Infected mice were assigned to one of the following 3 treatment groups: (i) control, (ii) treatment only (aerosolized L-AMB from day 1–5 after challenge), and (iii) prophylaxis followed by treatment (aerosolized L-AMB from day −2 to 5 before and after challenge). Survival was monitored until 12 days after challenge. For fungal-burden and histopathological examination, mice were sacrificed 4 h after treatment on day 3. Numbers of colony-forming units per lung were calculated. To study the distribution of AMB after inhalation of L-AMB, immunohistochemical studies using AMB antibody were performed.Aerosolized L-AMB significantly improved survival rate and decreased fungal burden compared with control group, and histopathology findings were superior to those of control group. However, no significant differences were detected between the treatment-only and prophylaxis followed by treatment groups. Immunohistochemical analysis showed that L-AMB was promptly distributed in lung tissue after inhalation therapy.Aerosolized L-AMB showed modest efficacy against R. oryzae infection in mice treated after fungal challenge. Prophylaxis with aerosolized L-AMB was not effective in this animal model.     

Interaction of granulocyte colony-stimulating factor and high doses of liposomal amphotericin B in the treatment of systemic murine scedosporiosis

Because human infections by Scedosporium prolificans are difficult to treat and show a very poor outcome, new therapeutic strategies are needed. Liposomal amphotericin B (LAMB) (40 mg/kg/day) increased significantly the mean survival time in immunosuppressed mice compared with a control group (22.6 vs. 8.8 days). Amphotericin B deoxycholate (1.5 mg/kg/day) and granulocyte colony-stimulating factor (G-CSF) (300 μg/kg/day) were ineffective. The combination of LAMB (40 mg/kg/day) and G-CSF (150 or 300 μg/kg/day) did not improve the results obtained with LAMB alone.     

Evaluation of the safety and efficacy of liposomal amphotericin B (L-AMB) in children

A multicenter, uncontrolled clinical study has been conducted to evaluate the safety, efficacy, and pharmacokinetics of liposomal amphotericin B (L-AMB) in children. In this article, the safety and efficacy of L-AMB are discussed. Subjects were diagnosed with invasive fungal infection (definitely diagnosed cases), possible fungal infection (clinically diagnosed cases), and febrile neutropenia with suspected fungal infection (febrile neutropenia cases). Of the 39 subjects treated with L-AMB, 18 received a definite (11) or clinical (7) diagnosis of invasive fungal infection. In these subjects, excluding one unevaluable subject, L-AMB was effective in nine out of 17 subjects(52.9%). Of 12 febrile neutropenia cases, improvement in clinical symptoms, etc., was observed for six but these were excluded from the efficacy analysis because they concomitantly used medications that may have affected efficacy. The causative fungus was identified in four out of 39 subjects and confirmed to be eliminated by treatment with L-AMB in one subject. Adverse events possibly related to L-AMB (adverse drug reactions) were reported in 36 out of 39 subjects (92.3%). The most commonad verse drug reaction was decreased potassium in 20 out of 39 subjects (51.3%), but all these subjects recovered with appropriate treatment, for example potassium supplementation.In a Japanese Phase II clinical study of adult patients, the incidence of adverse drug reactions was 95.3%(82/86 subjects) and the efficacy was 63.6% (42/66). Taken together, these data indicate that the safety and efficacy of L-AMB are almost the same in pediatric and adult patients.     

Antifungal activity of amphotericin B cochleates against Candida albicans infection in a mouse model

Cochleates are lipid-based supramolecular assemblies composed of natural products, negatively charged phospholipid, and a divalent cation. Cochleates can encapsulate amphotericin B (AmB), an important antifungal drug. AmB cochleates (CAMB) have a unique shape and the ability to target AmB to fungi. The minimal inhibitory concentration and the minimum lethal concentration against Candida albicans are similar to that for desoxycholate AmB (DAMB; Fungizone). In vitro, CAMB induced no hemolysis of human red blood cells at concentrations of as high as 500 microg of AmB/ml, and DAMB was highly hemolytic at 10 microg of AmB/ml. CAMB protect ICR mice infected with C. albicans when the agent is administered intraperitoneally at doses of as low as 0.1 mg/kg/day. In a tissue burden study, CAMB, DAMB, and AmBisome (liposomal AmB; LAMB) were effective in the kidneys, but in the spleen CAMB was more potent than DAMB at 1 mg/kg/day and was equivalent to LAMB at 10 mg/kg/day. In summary, CAMB are highly effective in treating murine candidiasis and compare well with AmBisome and AmB.     

A real-world prospective observational study on the efficacy and safety of liposomal amphotericin B in 426 patients with persistent neutropenia and fever

IntroductionInvasive fungal diseases are crucial causes of morbidity and mortality among patients with febrile neutropenia (FN). Though liposomal amphotericin B (L-AMB) is one of the agents recommended for first-line empirical antifungal therapy in patients with FN, large-scale clinical studies have not been performed in Japan.MethodsAn open-label prospective multi-center study was carried out to evaluate the safety and efficacy of L-AMB in Japanese patients with FN suspected of having fungal infection.ResultsOf the 426 patients registered, safety and efficacy evaluations were conducted for 424 and 399, respectively. By clinical response criteria using 5 composite endpoints, the response rate was 46.6% (186/399). The response rate by age were 54.5% (child: 30/55), 47.5% (adult: 97/204), 42.1% (elderly: 59/140) respectively. Regarding the composite endpoints, resolution of fever was observed in 61.2% (244/399), no breakthrough fungal infection in 99.0% (395/399), survival for 7 days or longer after the completion of treatment in 83.7% (334/399), no discontinuation of treatment due to toxicity or lack of efficacy in 60.9% (243/399), and successful treatment of any baseline fungal infection in 10/18. Adverse drug reactions (ADRs) developed in 61.1% (259/424), and frequent ADRs were hypokalemia, kidney dysfunction, and liver dysfunction, as previously reported.ConclusionsThe safety and efficacy profile of L-AMB in Japanese patients with FN suspected of having fungal infection were elucidated for the first time, through the analysis of a large number of cases including pediatric patients under real-world clinical settings collected in this nationwide study.     

Efficacy of escalating doses of liposomal amphotericin B (AmBisome) against hematogenous Candida lusitaniae and Candida krusei infection in neutropenic mice.

Immunosuppressed CF1 mice were infected intravenously with two strains of Candida krusei and four strains of Candida lusitaniae (two of which were resistant to amphotericin B). Mice were treated with 1 or 2 mg of amphotericin B desoxycholate per kg of body weight per day or escalating doses of liposomal amphotericin B (8 to 30 mg/kg/day) or were left untreated. Higher doses of liposomal amphotericin B were as effective as standard dose of amphotericin B desoxycholate in prolonging survival but were significantly more effective in reducing the fungal burden in the kidneys of animals infected with both C. krusei strains and the C. lusitaniae strains that were susceptible to amphotericin B desoxycholate. This advantage of liposomal amphotericin B therapy could not be demonstrated in mice infected with the C. lusitaniae strains that were resistant to amphotericin B desoxycholate.     

In vitro antifungal combination effects of micafungin with fluconazole, voriconazole, amphotericin B, and flucytosine against clinical isolates of Candida species

Micafungin (MCFG) is an echinocandin antifungal agent that exhibits potent activity against most species of Candida and Aspergillus. We investigated the in vitro antifungal combination effects of MCFG with four other antifungal agents — fluconazole (FLCZ), voriconazole (VRCZ), amphotericin B, and flucytosine — against clinical isolates of 54 Candida spp. by checkerboard analysis. The synergistic antifungal effects of MCFG-FLCZ and MCFG-VRCZ were 11% and 15%, respectively, and the latter displayed a synergistic activity of 63% against Candida glabrata. Antagonism was not observed in any of the combinations tested.     

Comparison of antifungal and cytotoxicity activities of titanium dioxide and zinc oxide nanoparticles with amphotericin B against different Candida species: In vitro evaluation

Background Candida species are known to cause serious fungal infections that produce cutaneous, mucosal, and systemic infections. Nowadays, mortality and morbidity candidiasis in immunocompromised patients have increased. Nanotechnology is a new world‐known technology and includes particles ranging from about 1 to 100 nanometers. The purpose of this study was to evaluate the antifungal and cytotoxicity activities of titanium dioxide nanoparticles (TiO2‐NPs) and zinc oxide nanoparticles (ZnO‐NPs) compared to amphotericin B (AmB) on different Candida spp in in vitro conditions.MethodsIn the present study, susceptibility of different Candida species to TiO2‐NPs and ZnO‐NPs compared to AmB was determined by broth microdilution (BMD) and agar well diffusion methods. Cytotoxicity of TiO2‐NPs and ZnO‐NPs and amphotericin B was measured by MTT (3‐(4, 5‐Dimethylthiazol‐2‐yl)‐2, 5‐Diphenyltetrazolium Bromide) assay.ResultsThe results indicated that the TiO2‐NPs and ZnO‐NPs showed antifungal activities against pathogenic Candida spp. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of TiO2‐NP ranges against Candida spp. were 128‐256 µg/mL and 256‐512 µg/mL, respectively. The MIC and MFC values of ZnO‐NPs were 64‐128 µg/mL and 256‐512 µg/mL, respectively. However, MICs and MFCs of AmB were 8‐16 µg/mL and 16‐32 µg/mL, respectively. The MTT assay results showed that the CC50% belonged to ZnO‐NPs 706.2 μg/mL, for TiO2‐NPs 862.1 μg/mL, and for AmB 70.19 μg/mL, respectively.ConclusionOur findings showed that TiO2‐NPs and ZnO‐NPs had antifungal effects against all Candida species, yet the antifungal properties of TiO2‐NPs and ZnO‐NPs were significantly less than those of AmB. The CC50% of AmB was significantly lower than ZnO‐NPs and TiO2‐NPs.     

米卡芬净单药及联合两性霉素B对小鼠侵袭性肺曲霉病的治疗作用

目的评价米卡芬净单药及联合两性霉素B对小鼠侵袭性肺曲霉病(IPA)的治疗作用。方法采用环磷酰胺骨髓抑制、烟曲霉孢子滴鼻接种构建中性粒细胞减少小鼠IPA模型。实验动物随机分为为4组:模型对照组(NS+5%GS,A组)、米卡芬净治疗组[5 mg/(kg.d),B组]、两性霉素B治疗组[1 mg/(kg.d),C组]和米卡芬净[5 mg/(kg.d)]加两性霉素B[1mg/(kg.d)]治疗组(D组),治疗从接种第2天开始,每天1次,共7 d。①动物生存期观察:每组15~16只小鼠,接种后每天观察1次至第21天。共进行2批次实验,分别给予2×105和6×106个孢子/小鼠。②肺的真菌负荷:每组9只小鼠,给予1.5×104个孢子/小鼠。治疗结束后取肺,匀浆后梯度稀释培养,记取菌落数并计算肺的真菌负荷量。结果①生存分析:第1批次,米卡芬净、两性霉素B单药及联合用药治疗均能延长小鼠生存期,但3种治疗方案在延长生存期方面差异无显著性(B组、C组分别与A组比较均有P<0.05,D组与A组比较P<0.01)。第2批次,联合用药组生存期长于其它各组,米卡芬净及两性霉素B单药治疗组与模型组比较差异无显著性(D组与A组比较P<0.01;D组分别与B组及C组比较均有P<0.05)。②肺的真菌负荷:两性霉素B单药及联合用药均能降低肺的真菌负荷,米卡芬净单药不降低肺的真菌负荷(C组与A组比较P<0.01;D组与A组比较P<0.05;B组与C组比较P<0.05;B组与D组比较P<0.01)。结论单独应用米卡芬净或两性霉素B及联合用药均能够延长中性粒细胞减少IPA小鼠生存期,当孢子接种量为6×106/小鼠时,联合用药优于单独应用米卡芬净或两性霉素B。单独应用米卡芬净不降低肺的真菌负荷,联合用药或两性霉素B单药在降低肺的真菌负荷方面均优于对照组及米卡芬净组。     

Successful treatment of proven coronavirus disease 2019-associated pulmonary aspergillosis with liposomal amphotericin B in a patient with bronchiolitis obliterans syndrome after allogeneic hematopoietic stem cell transplantation

Coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) is being increasingly recognized as a severe complication that contributes to poor prognoses among patients with COVID-19. However, little is known regarding the clinical course of CAPA with hematological malignancies, especially after allogeneic hematopoietic stem cell transplantation (HSCT). A 29-year-old woman was diagnosed with proven CAPA with an Aspergillus fumigatus identified by cultures of bronchoalveolar lavage and lung biopsy four years after haploidentical HSCT for acute myelogenous leukemia. She had been taking oral prednisolone for bronchiolitis obliterans syndrome that developed after HSCT. Although prolonged RT-PCR positivity for SARS-CoV-2 (133 days after the onset of COVID-19) without shedding of viable virus was observed, the COVID-19 was treated with favipiravir, remdesivir, dexamethasone, and enoxaparin. However, the CAPA did not respond to combination therapy, which included triazole (voriconazole, itraconazole, posaconazole) and echinocandin (caspofungin, micafungin), even though the Aspergillus fumigatus isolate was found to be susceptible to these agents in vitro. Nevertheless, a total of 16 weeks of liposomal amphotericin B (L-AMB) therapy led to a favorable response, and the patient was discharged from the hospital on day 213. This case provided essential experience of CAPA treated with L-AMB in a recipient with chronic respiratory disease after HSCT.     

Failure of liposomal amphotericin B therapy in patients with severe pancreatitis complicated by Candida lusitaniae infection

Candida lusitaniae is an uncommon pathogen that accounts for approximately 1% of patients with candidiasis. In this report, we present the case of a 24-year-old woman with severe pancreatitis who was emergently admitted to Northern Yokohama Hospital. We started treating the pancreatitis and infections according to her culture results. However, her symptoms, accompanied by a necrotic pancreas, did not improve. Finally, C. lusitaniae was detected in the blood and catheter samples. We started antifungal treatment according to the culture results, but the patient died.Generally, the mortality rate for acute pancreatitis ranges from 3% for patients with interstitial edematous pancreatitis to 17% for those who develop pancreatic necrosis. Although we chose appropriate antibiotics and antifungal agents based on the culture results, the treatments failed. Early detection, sufficient doses of antimicrobials and frequent monitoring using sample culture are crucial because infection control may be inadequate, especially in tissues with low blood flow, such as necrotic tissues.     

Occurrence and improvement of renal dysfunction and serum potassium abnormality during administration of liposomal amphotericin B in patients with hematological disorders: A retrospective analysis

Liposomal amphotericin B (L-AMB) has the potential to cause two major adverse events, renal dysfunction and serum potassium abnormality; however, appropriate clinical management of these events remains unclear. We retrospectively analyzed data regarding 128 hematology patients who received L-AMB in our institute and examined the association between clinical characteristics and renal dysfunction or serum potassium abnormality. We found that the median weight-normalized dose of L-AMB was 2.69 mg/kg and the median administration period was 16 days. The overall occurrence rates of renal dysfunction and hypokalemia were 55.7% and 76.6%, respectively. Multivariate analysis revealed that pre-existing renal dysfunction (P = 0.017) and concomitant use of nephrotoxic (P < 0.0001) or antifungal drugs (P = 0.012) were independent risk factors for renal dysfunction. A higher infusion volume did not mitigate the risk of renal dysfunction. Hypokalemia occurred significantly less often in men (P = 0.028) and in patients who concomitantly used nephrotoxic drugs (P = 0.013). Approximately 40% of the adverse events were improved at 30 days after L-AMB termination and there was no significant association between these adverse events improvement and L-AMB dosage or infusion volume. Of note, hyperkalemia was observed in more patients who received allogeneic hematopoietic stem cell transplantation (P = 0.0303) and concomitant treatment with nephrotoxic drugs (P = 0.0281). These results suggest that imprudent reduction of L-AMB dose or redundant intravenous infusion may have minimal benefit for critical patients with suspected invasive fungal infection.     

Fungemia due to Fusarium solani under low-dose liposomal amphotericin B in a patient after cord blood transplantation

The introduction of the prophylactic use of antifungal drugs caused the increased occurrence of invasive fungal infections due to previously rare molds, such as fusariosis, after allogeneic hematopoietic stem cell transplantation. We herein report the case of a patient with diffuse large B-cell lymphoma who developed fungemia due to Fusarium solani during liposormal amphotericin B on day 25 after cord blood transplantation (CBT). Because Fusarium species might differ in virulence and drug susceptibility, the sequencing of the internal transcribed spacer region of the ribosomal RNA gene accurately identified Fusarium solani to be the cause of fungemia at the species level. This case highlights Fusarium solani as the cause of fungemia in a patient under liposormal amphotericin B treatment after CBT.     

小剂量两性霉素B治疗恶性血液病侵袭性真菌感染的效果观察

目的 探讨小剂量两性霉素B治疗恶性血液病侵袭性真菌感染的有效性及安全性.方法 我院2008年1月至2012年6月接受治疗的恶性血液病合并侵袭性真菌感染患者98例,应用计算机随机分为对照组和实验组,对照组47例,两性霉素B维持剂量为50 ～ 60 mg/d;实验组51例,两性霉素B维持剂量为25～ 30 mg/d.比较两组的疗效和不良反应发生率.结果 两性霉素B中位累计剂量对照组为725(175,1595) mg,实验组为735(225,1485) mg,差异无统计学意义(P =0.834);治疗中位时间对照组为19(8,34)d,实验组为29(11,58)d,差异有统计学意义(P=0.000).治疗14 d对照组与实验组可评估疗效者分别为37例和48例,两组总有效率、无进展率、热退率差异均无统计学意义(P均＞0.05).因不良反应终止治疗者对照组14例(29.8％),实验组6例(11.8％),差异有统计学意义(P=0.027);对照组肝肾功能损害发生率高于实验组[27.7％ (13/47)与11.8％(6/51),P=0.047].结论 两性霉素B维持剂量25 ～ 30 mg/d临床疗效不低于维持剂量50～60 mg/d,不良反应少,临床可操作性强,具有可实践性.     

Antifungal activity of amphotericin B,fluconazole, and voriconazole in an in vitro model of Candida catheter-related bloodstream infection

The activity of five simulated antifungal regimens for eradication of catheter-related bloodstream Candida infection was evaluated with an in vitro pharmacodynamic model. Single-lumen central venous catheters were colonized with Candida species by sequentially incubating central venous catheters in plasma and then in growth medium (RPMI plus morpholinepropanesulfonic acid) containing a standardized suspension (10(5) CFU/ml) of Candida albicans, Candida glabrata, or slime-producing Candida parapsilosis. Colonized central venous catheters were then placed in a one-compartment pharmacodynamic model where five antifungal regimens (plus control) were simulated: amphotericin B, 1.0 mg/kg every 24 h; amphotericin B, 0.5 mg/kg every 24 h; fluconazole, 400 mg every 24 h; fluconazole, 800 mg every 24 h; and voriconazole, 4 mg/kg every 12 h. During exposure to the simulated clinical regimens, samples were serially removed from the model over 48 h for quantitation of viable organisms. All antifungal regimens suppressed fungal counts by both peripheral and catheter sampling versus control (P = 0.001). Overall, antifungal activity ranked amphotericin B (1 mg/kg) > amphotericin B (0.5 mg/kg) > or = voriconazole > fluconazole (800 mg) > or = fluconazole (400 mg). No regimen, however, completely eradicated (by culture and electron microscopy) central venous catheter colonization. Regrowth was noted in the model during therapy against C. glabrata and C. parapsilosis but was not associated with an increase in the MICs for the isolates. Lack of in vitro antifungal activity against biofilm-encased organisms appeared to be the primary reason for mycological failure of antifungal regimens in the model.