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Antifungal development has gained increasing attention due to its limited armamentarium and drug resistance. Drug repurposing holds great potential in antifungal discovery. In this study, we explored the antifungal activity of artemisinin and its derivatives, dihydroartemisinin, artesunate and artemether. We identified that artemisinins can inhibit the growth of Candida albicans, and can enhance the activity of three commonly used antifungals, amphotericin B, micafungin and fluconazole (FLC), on Candida albicans growth and filamentation. Artemisinins possess stronger antifungal effect with FLC than with other antifungals. Among artemisinins, artemether exhibits the most potent antifungal activity with FLC and can recover the susceptibility of FLC-resistant clinical isolates to FLC treatment. The combinatorial antifungal activity of artemether and FLC is broad-spectrum, as it can inhibit the growth of Candida auris, Candida tropicalis, Candida parapsilosis, Saccharomyces cerevisiae and Cryptococcus neoformans. Mechanistic investigation revealed that artemether might enhance azole efficacy through disrupting the function of Pdr5, leading to intracellular accumulation of FLC. This study identified artemether as a novel FLC potentiator, providing potential therapeutic insights against fungal infection and antifungal resistance., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cryptococcus neoformans is a facultative intracellular pathogen responsible for the most common cause of fungal meningioencephalitis, occurring primarily in immunocompromised individuals. Antiphagocytic protein 1 (App1) is a virulence factor produced by C. neoformans that inhibits phagocytosis of the yeast by host macrophages. Treatment of cryptococcosis includes amphotericin B, fluconazole, and flucytosine. Virulence factors have been shown to affect the susceptibility of the pathogen to antifungal drugs. In this study, we aimed to examine the relationship between App1 and antifungal drugs. We found that short-term exposure to amphotericin B downregulates APP1 expression while exposure to fluconazole upregulates APP1. In addition, App1 was found to increase the susceptibility of the yeast to amphotericin B and fluconazole. This study provides evidence of an intricate relationship between App1 and antifungal drugs., Competing Interests: The authors have declared that no competing interests exist.

Background: Cryptococcus neoformans, a basidiomycetous yeast, is a fungal pathogen that can colonize the lungs of humans causing pneumonia and fungal meningitis in severely immunocompromised individuals. Recent studies have implied that the antifungal drug fluconazole (FLC) can induce oxidative stress in C. neoformans by increasing the production of reactive oxygen species (ROS), as presence of the antioxidant ascorbic acid (AA) could reverse the inhibitory effects of FLC on C. neoformans. However, in Candida albicans, AA has been shown to stimulate the expression of genes essential for ergosterol biosynthesis. Hence, the contribution of ROS in FLC-mediated growth inhibition remains unclear., Results: In order to determine whether counteracting ROS generated by FLC in C. neoformans can contribute to diminishing inhibitory effects of FLC, we tested three other antioxidants in addition to AA, namely, pyrrolidine dithiocarbamate (PDTC), retinoic acid (RA), and glutathione (GSH). Our data confirm that there is an increase in ROS in the presence of FLC in C. neoformans. Importantly, all four antioxidants reversed FLC-mediated growth inhibition of C. neoformans to various extents. We further verified the involvement of increased ROS in FLC-mediated growth inhibition by determining that ROS-scavenging proteins, metallothioneins (CMT1 and CMT2), contribute to growth recovery by PDTC and AA during treatment with FLC., Conclusion: Our study suggests that ROS contributes to FLC-mediated growth inhibition and points to a complex nature of antioxidant-mediated growth rescue in the presence of FLC.

Cryptococcus neoformans is one of the most lethal fungi causing mortality across the globe. Immuno-competent patients and patients taking immuno-suppressive medications are extremely susceptible to its infection. For effective removal of cryptococcal burden, there is an urgent need for new forms of therapy. In the present study, we have explored the potential effects of amphotericin B (AMB) and fluconazole (FLC) in combination, against cryptococcosis in Swiss albino mice. To enhance the therapeutic potential of the tested drugs, they were entrapped into fibrin microspheres; a dual delivery vehicle comprising of poly-lactide co-glycolide (PLGA) microsphere that was additionally encapsulated into the fibrin cross-linked plasma bead. Dynamics of fibrin microspheres included survival and fungal burden in lung, liver and spleen of treated mice. While each drug was effective in combination or alone, prominent additive potential of AMB and FLC were clearly observed when used in fibrin microsphere. Significant reduction in fungal burden and increase in survival rate of AMB + FLC-fibrin microspheres treated mice shows an extensive accessibility of both tested drugs without any side-effects. A full potential of two-drug combination encapsulated in fibrin microspheres proposes an effective approach of safe delivery to the target site in their intact form and decrease the drug associated toxicities., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: Recent advances in the treatment and prevention of cryptococcal meningitis have the potential to decrease AIDS-related deaths. Areas covered: Targeted screening for asymptomatic cryptococcal antigenemia in persons with AIDS is a cost effective method for reducing early mortality in patients on antiretroviral therapy. For persons with symptomatic cryptococcal meningitis, optimal initial management with amphotericin and flucytosine improves survival compared to alternative therapies; however, amphotsericin is difficult to administer and flucytosine has not been available in middle or low income countries, where cryptococcal meningitis is most prevalent. Expert commentary: Improved care for cryptococcal meningitis patients in resource-limited settings is possible, and new treatment possibilities are emerging.

Cryptococcal antigen screening is recommended among people living with AIDS when entering HIV care with a CD4 count of <100 cells/μl, and preemptive fluconazole monotherapy treatment is recommended for those with subclinical cryptococcal antigenemia. Yet, knowledge is limited of current antimicrobial resistance in Africa. We examined antifungal drug susceptibility in 198 clinical isolates collected from Kampala, Uganda, between 2010 and 2014 using the CLSI broth microdilution assay. In comparison with two previous studies from 1998 to 1999 that reported an MIC50 of 4 μg/ml and an MIC90 of 8 μg/ml prior to widespread human fluconazole and agricultural azole fungicide usage, we report an upward shift in the fluconazole MIC50 to 8 μg/ml and an MIC90 value of 32 μg/ml, with 31% of isolates with a fluconazole MIC of ≥ 16 μg/ml. We observed an amphotericin B MIC50 of 0.5 μg/ml and an MIC90 of 1 μg/ml, of which 99.5% of isolates (197 of 198 isolates) were still susceptible. No correlation between MIC and clinical outcome was observed in the context of amphotericin B and fluconazole combination induction therapy. We also analyzed Cryptococcus susceptibility to sertraline, with an MIC50 of 4 μg/ml, suggesting that sertraline is a promising oral, low-cost, available, novel medication and a possible alternative to fluconazole. Although the CLSI broth microdilution assay is ideal to standardize results, limit human bias, and increase assay capacity, such assays are often inaccessible in low-income countries. Thus, we also developed and validated an assay that could easily be implemented in a resource-limited setting, with similar susceptibility results (P = 0.52)., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The incidence of invasive fungal infections is increasing in recent years. The present study mainly investigated glabridin (Gla) alone and especially in combination with fluconazole (FLC) against Cryptococcus neoformans and Candida species (Candida albicans, Candida tropicalis, Candida krusei, Candida parapsilosis and Candida Glabratas) by different methods. The minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) indicated that Gla possessed a broad-spectrum antifungal activity at relatively high concentrations. After combining with FLC, Gla exerted a potent synergistic effect against drug-resistant C. albicans and C. tropicalis at lower concentrations when interpreted by fractional inhibitory concentration index (FICI). Disk diffusion test and time-killing test confirming the synergistic fungicidal effect. Cell growth tests suggested that the synergistic effect of the two drugs depended more on the concentration of Gla. The cell envelop damage including a significant decrease of cell size and membrane permeability increasing were found after Gla treatment. Together, our results suggested that Gla possessed a synergistic effect with FLC and the cell envelope damage maybe contributed to the synergistic effect, which providing new information for developing novel antifungal agents.

Unlabelled: Cryptococcosis is an infectious disease of global significance for which new therapies are needed. Repurposing previously developed drugs for new indications can expedite the translation of new therapies from bench to beside. Here, we characterized the anti-cryptococcal activity and antifungal mechanism of estrogen receptor antagonists related to the breast cancer drugs tamoxifen and toremifene. Tamoxifen and toremifene are fungicidal and synergize with fluconazole and amphotericin B in vitro. In a mouse model of disseminated cryptococcosis, tamoxifen at concentrations achievable in humans combines with fluconazole to decrease brain burden by ~1 log10. In addition, these drugs inhibit the growth of Cryptococcus neoformans within macrophages, a niche not accessible by current antifungal drugs. Toremifene and tamoxifen directly bind to the essential EF hand protein calmodulin, as determined by thermal shift assays with purified C. neoformans calmodulin (Cam1), prevent Cam1 from binding to its well-characterized substrate calcineurin (Cna1), and block Cna1 activation. In whole cells, toremifene and tamoxifen block the calcineurin-dependent nuclear localization of the transcription factor Crz1. A large-scale chemical genetic screen with a library of C. neoformans deletion mutants identified a second EF hand-containing protein, which we have named calmodulin-like protein 1 (CNAG&#95;05655), as a potential target, and further analysis showed that toremifene directly binds Cml1 and modulates its ability to bind and activate Cna1. Importantly, tamoxifen analogs (idoxifene and methylene-idoxifene) with increased calmodulin antagonism display improved anti-cryptococcal activity, indicating that calmodulin inhibition can be used to guide a systematic optimization of the anti-cryptococcal activity of the triphenylethylene scaffold., Importance: Worldwide, cryptococcosis affects approximately 1 million people annually and kills more HIV/AIDS patients per year than tuberculosis. The gold standard therapy for cryptococcosis is amphotericin B plus 5-flucytosine, but this regimen is not readily available in regions where resources are limited and where the burden of disease is highest. Herein, we show that molecules related to the breast cancer drug tamoxifen are fungicidal for Cryptococcus and display a number of pharmacological properties desirable for an anti-cryptococcal drug, including synergistic fungicidal activity with fluconazole in vitro and in vivo, oral bioavailability, and activity within macrophages. We have also demonstrated that this class of molecules targets calmodulin as part of their mechanism of action and that tamoxifen analogs with increased calmodulin antagonism have improved anti-cryptococcal activity. Taken together, these results indicate that tamoxifen is a pharmacologically attractive scaffold for the development of new anti-cryptococcal drugs and provide a mechanistic basis for its further optimization.

Cryptococcus neoformans is an encapsulated fungus that causes cryptococcosis. Central nervous system infection is the most common clinical presentation followed by pulmonary, skin and eye manifestations. Cryptococcosis is primarily treated with amphotericin B (AMB), fluconazole (FLC) and itraconazole (ITC). In the present work, we evaluated the in vitro effect of terbinafine (TRB), an antifungal not commonly used to treat cryptococcosis. We specifically examined the effects of TRB, either alone or in conjunction with AMB, FLC and ITC, on clinical C. neoformans isolates, including some isolates resistant to AMB and ITC. Broth microdilution assays showed that TRB was the most effective drug in vitro. Antifungal combinations demonstrated synergism of TRB with AMB, FLC and ITC. The drug concentrations used for the combination formulations were as much as 32 and 16-fold lower than the minimum inhibitory concentration (MIC) values of FLC and AMB alone, respectively. In addition, calcofluor white staining revealed the presence of true septa in hyphae structures that were generated after drug treatment. Ultrastructural analyses demonstrated several alterations in response to drug treatment, such as cell wall alterations, plasma membrane detachment, presence of several cytoplasmic vacuoles and mitochondrial swelling. Therefore, we believe that the use of TRB alone or in combination with AMB and azoles should be explored as an alternative treatment for cryptococcosis patients who do not respond to standard therapies.

The yeast-like fungus Cryptococcus neoformans favours respiration as a mechanism of energy production, and thus depends heavily on mitochondrial function. Previous studies of a C. neoformans vad1Delta mutant revealed reduced expression of the mitochondrial elongation factor TUF1 and defects in glycerol utilization, consistent with mitochondrial dysfunction. In this study, we found that in trans expression of TUF1 in the vad1Delta mutant suppressed the mitochondrial defects, including growth on respiration-dependent carbon sources and fluconazole resistance, associated with VAD1 deletion. Tetracycline, an inhibitor of mitochondrial translation, was found to confer resistance to fluconazole in the wild-type and vad1Delta mutant, whereas the fluconazole susceptibility of the TUF1-overexpressing strain was unaffected by tetracycline treatment. In the presence of fluconazole, the vad1Delta mutant exhibited increased activation of the global transcriptional regulator Sre1. TUF1 overexpression failed to alter cleavage of Sre1 in response to fluconazole in the vad1Delta mutant, suggesting that TUF1 repression in the vad1Delta mutant is distal to Sre1, or that it occurs through an independent pathway.

Gomesin is an 18-residue cysteine-rich antimicrobial peptide produced by hemocytes of the spider Acanthoscurria gomesiana. In the present study, the antifungal properties of gomesin against Cryptococcus neoformans, the etiologic agent of cryptococcosis, were evaluated. Gomesin bound to the cell surface of cryptococci, which resulted in cell death associated with membrane permeabilization. Antifungal concentrations of gomesin were not toxic for human brain cells. Supplementation of cryptococcal cultures with the peptide (1 microM) caused a decrease in capsule expression and rendered fungal cells more susceptible to killing by human brain phagocytes. The possible use of gomesin in combination with fluconazole, a standard antifungal drug, was also evaluated. In association with fluconazole, gomesin concentrations with low antimicrobial activity (0.1-1 microM) inhibited fungal growth and enhanced the antimicrobial activity of brain phagocytes. These results reveal the potential of gomesin to promote inhibition of cryptococcal growth directly or by enhancing the effectiveness of host defenses.

Cryptococcal meningoencephalitis is a life-threatening infectious disease in immunocompromised patients. Unmethylated CpG-oligodeoxynucleotides (CpG-ODN) protects hosts in a mouse model. In the present study, we tested the adjuvant effect of CpG-ODN in anti-fungal chemotherapy. Administration of either fluconazole (FLCZ) or CpG-ODN was effective in extending survival, accelerating clearance of fungi and preventing disseminated infection. Combination of both agents provided more beneficial effect than either agent alone. Cytokine balance in the infected lungs was biased to Th1-type response by CpGODN, while FLCZ did not further promote. These results suggest that CpG-ODN is a promising adjuvant in chemotherapy against this infection.

We have recently demonstrated that upregulation of the ATP binding cassette (ABC) transporter-encoding gene AFR1 in Cryptococcus neoformans is involved in the in vitro resistance to fluconazole of this yeast. In the present study, we investigated the role of AFR1 in the in vivo response to fluconazole in a mouse model of systemic cryptococcosis. Mice were infected with a wild-type fluconazole-susceptible strain of C. neoformans, strain BPY22; an afr1 mutant, BPY444, which displayed hypersusceptibility to fluconazole in vitro; or an AFR1-overexpressing strain, BPY445, which exhibited in vitro resistance to the drug. In each of the three groups, infected animals were randomly assigned to fluconazole treatment or untreated-control subgroups. As expected, fluconazole prolonged survival and reduced fungal tissue burdens (compared with no treatment) in BPY22- and BPY444-infected mice, whereas it had no significant effects in mice infected with BPY445. When the pathogenicities of these strains in mice were investigated, strain BPY445 was significantly more virulent than BPY22 following inhalational or intravenous inoculation, but mice infected with BPY444 survived significantly longer than BPY22-infected animals only when infection was acquired via the respiratory tract. In in vitro macrophage infection studies, strain BPY445 also displayed enhanced intracellular survival compared with strains BPY22 and BPY444, suggesting that its increased virulence may be due to its reduced vulnerability to the antimicrobial factors produced by phagocytic cells. These findings indicate that the upregulation of the AFR1 gene is an important factor in either determining the in vivo resistance to fluconazole or influencing the virulence of C. neoformans.

This article reported the identification of heat shock protein 90 (hsp90) homologues by immunoblot in Cryptococcus neoformans. Mycograb, a genetically recombinant antibody against hsp90, was evaluated against 8 clinical isolates and the National External Quality Assessment Service for Microbiology strain of C. neoformans alone and in combination with amphotericin B, caspofungin, and fluconazole by checkerboard assay. At the end point of an optically clear well, the minimum inhibitory concentration (MIC) 0's ranged from 256 to 1024 microg/mL for Mycograb, from 0.5 to 1 microg/mL for amphotericin B, and from 16 to 32 microg/mL for caspofungin. The combination of Mycograb and amphotericin B produced a fractional inhibitory concentration index from 0.27 to 0.56, indicating a mainly synergistic effect, whereas for caspofungin, it varied from 0.5 to 2. At an end point of > or =50% inhibition, the MIC-2s varied from 16 to 128 microg/mL for Mycograb and from 0.125 to 16 microg/mL for fluconazole. The fractional inhibitory concentration index classified the combination as indifferent for 5 isolates, additive for 3 more isolates, and synergistic in a single isolate. Time-kill analysis on 2 isolates (F/7844 and F/10156), which had synergistic and additive results with amphotericin B, respectively, on checkerboard was performed with 4-16 microg/mL of Mycograb, 2-8 microg/mL of fluconazole, and 0.0625-2 microg/mL of amphotericin B. This demonstrated an increasingly static effect with augmenting concentrations of fluconazole and an initial static effect with amphotericin B at lower concentrations, which became fungicidal as the level of drug increased. The addition of either 4 or 8 microg/mL of Mycograb to 0.5 microg/mL of amphotericin B with C. neoformans F/7844 changed a static effect to a fungicidal effect at 8 h with an increased killing of 1.2 logs at 48 h. With C. neoformans F/10156, the addition of 16 microg/mL of Mycograb to 0.25 microg/mL of amphotericin B produced a difference in killing from 1 logarithm after 4 h to 1.5 logarithms after 48 h. These data suggest that the combination of amphotericin B and Mycograb would be worth exploring in the treatment of infection due to C. neoformans.

Purpose: The prophylactic role of liposomized chloroquine (lip-CQ) has been assessed against less susceptible Cryptococcus neoformans infection in murine model., Methods: In the current study, we investigated the antifungal activity of lip-CQ against C. neoformans in macrophages cell line (J 774) and murine model. Mice were pretreated with free as well as liposomized formulations of CQ at various doses. The anticryptococcal activity of fluconazole was compared in mice with or without CQ pretreatment. The efficacy of CQ prophylaxis was assessed by survival as well as colony forming units (cfu) in brain and lungs of treated mice., Results: Fluconazole alone was not found significantly effective against C. neoformans in both in vitro and in vivo studies. However, the antifungal activity of fluconazole increases in chloroquine-pretreated mice. Lip-CQ was found to be more effective in comparison to the same dose of free chloroquine in reducing fungal burden from macrophages in vitro and lungs and brain of C. neoformans infected mice., Conclusions: The enhanced prophylactic activity of lip-CQ seems due to rapid uptake of drug-containing liposomes by macrophages. The liposome-mediated accumulation of CQ in macrophages makes the environment unfavorable (alkaline) for the intracellular multiplication of C. neoformans. Moreover, the increased incidence of multi-drug resistance and diversity of pathogenic microorganisms inhibited or killed by CQ makes it the drug of choice for prophylactic therapy.

We evaluated the antifungal activities of amphotericin B, fluconazole, and flucytosine, alone and in combination, in a murine model of cryptococcal meningitis. The objectives were to determine the greatest antifungal effects achievable with these drugs alone or in combination. Meningitis was established in male BALB/c mice weighing 23 to 25 g by intracerebral injection of Cryptococcus neoformans. Treatment was started on day 2. Amphotericin B was tested at 0.3 to 1.3 mg/kg of body weight/day by slow intravenous injection. Fluconazole at 10 to 40 mg/kg/day and flucytosine at 20 to 105 mg/kg/day were administered in the sole source of drinking water. The mice were killed at 16 days, and the numbers of fungal colonies in the brain were quantified. The association between the response and the dose combination was evaluated by local nonparametric response surface methods; 99% confidence intervals were used to evaluate the antifungal effects. Ninety-five percent of the mice treated with amphotericin B at 0.5 mg/kg survived to the end of the experiment, regardless of the fluconazole or flucytosine dose used. The greatest activity was seen with amphotericin B plus fluconazole with or without flucytosine. However, the addition of flucytosine did not increase the antifungal activity. Given the widespread availability of amphotericin B and fluconazole and the relative safety profile of fluconazole compared to that of flucytosine, the full potential of this two-drug combination deserves further evaluation.

Objectives: In cryptococcosis, fluconazole is a standard prophylactic, therapeutic and maintenance option, particularly in the expanding HIV/AIDS group. However, its excessive use may lead to resistance in Cryptococcus neoformans. Variations in clinical response to fluconazole have already been noted elsewhere, and cases of post-therapy relapse are not uncommon. To assess azole antifungal susceptibility profiles of clinical cryptococcal isolates in India, the All India Institute of Medical Sciences (AIIMS) has recently initiated preliminary studies using NCCLS M27-A., Materials and Methods: Twenty-eight randomly chosen AIIMS clinical isolates (spanning 1997-2000), 16 isolates from other institutions in North India, and six reference strains of C. neoformans were subjected to susceptibility testing to fluconazole and itraconazole., Results: Among clinical isolates, susceptibilities to fluconazole and itraconazole were 84.1% and 93.2%, respectively. MICs for all clinical isolates were 0.25-32 mg/L for fluconazole and <0.03-0.25 mg/L for itraconazole. MIC50 and MIC90 values for fluconazole were 4 and 16 mg/L, respectively, and those for itraconazole were 0.032 and 0.125 mg/L, respectively. Out of 28 AIIMS clinical isolates, 22 had minimum fungicidal concentrations (MFCs) of fluconazole at 128 mg/L. Moderately high fluconazole MICs (16-32 mg/L) were observed in 16% of clinical isolates--probably the first such report from India. MIC/MFC ratios for fluconazole and itraconazole were 1:32 or more in 16 AIIMS clinical isolates, indicating possible azole tolerance. There was good agreement between MIC values obtained by the micro- and macro-broth dilution techniques of M27-A compared in this study., Conclusions: The observed MIC data warrant continued surveillance of susceptibility values of clinical cryptococcal isolates in India.

Background: The increasing frequency of systemic fungal infections and the emergency of secondary resistance to antifungals in the lasts years, has stimulated the use of methods for antifungal susceptibility testing. Etest is an easily performed quantitative method that has a good agreement with the broth microdilution reference method (NCCLS), if appropriate media are used., Aim: To compare the susceptibility to Amphotericin B (AmB) and Fluconazole (Flu) of 22 opportunistic yeast isolates (C albicans (7), C tropicalis (9), C parapsilosis (3) and Cryptococcus neoformans (3) by Etest, using three different media and to choose the best medium for each tested drug. The studied media were RPMI 1640, Casitone (Cas) and Sabouraud., Results: The interpretation of minimal inhibitory concentration (MIC) endpoints on Sabouraud was difficult for AmB. The American Type Culture Collection (ATCC) strains MICs were out of the acceptable range in this medium. RPMI and Cas were suitable media to test AmB and Flu, but best endpoints were obtained for AmB in RPMI and Flu in Cas., Conclusions: The use of appropriate media for each antifungal drug optimizes the MIC readings by Etest. AmB should be tested in RMPI and Flu in Cas. Sabouraud must not be used.

Resistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococ-cal meningitis, the most frequently encountered life-threatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans, a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole-resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridization procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole-resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter-encoding gene called C. neoformans AntiFungal Resistance 1 (CnAFR1). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced susceptibility of the knock-out mutant cnafr1 against fluconazole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in fluconazole resistance of C. neoformans. Our findings therefore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.

Cryptococcus neoformans isolates that exhibited unusual patterns of resistance to fluconazole and voriconazole were isolated from seven isolates from two different geographical regions: one isolate from an Israeli non-AIDS patient and six serial isolates from an Italian AIDS patient who had suffered six recurrent episodes of cryptococcal meningitis. Each isolate produced cultures with heterogeneous compositions in which most of the cells were susceptible, but cells highly resistant to fluconazole (MICs, >/=64 microg/ml) were recovered at a variable frequency (7 x 10(-3) to 4.6 x 10(-2)). Evidence showed that this type of resistance is innate and is unrelated to drug exposure since the Israeli patient had never been treated with azoles or any other antimycotic agents. Analysis of clonal subpopulations of these two strains showed that they exhibited heterogeneous patterns of resistance. The number of subpopulations which grew on fluconazole or voriconazole agar declined progressively with increasing azole concentration without a sharp cutoff point. For the Italian serial isolates, the number of clonal populations resistant to fluconazole (64 microg/ml) and voriconazole (1 microg/ml) increased steadily, yielding the highest number for the isolate from the last episode. Attempts to purify a sensitive subpopulation failed, but clones highly resistant to fluconazole (100 microg/ml) and moderately resistant to voriconazole (1 microg/ml) always produced a homogeneous population of resistant cells. Upon maintenance on drug-free medium, however, the majority of the homogeneously resistant cells of these subclones lost their resistance and returned to the stable initial heteroresistant phenotype. The pattern of heteroresistance was not affected by the pH or osmolarity of the medium but was influenced by temperature. The resistance appeared to be suppressed at 35 degrees C and was completely abolished at 40 degrees C. Although heterogeneity in azole resistance among subpopulations of single isolates has been reported for Candida species, the transient changes in expression of resistance under different growth conditions reported here have not been observed in fungal pathogens.

We compared the yeast nitrogen base (YNB) broth microdilution method with the National Committee for Clinical Laboratory Standards (NCCLS) M27-A microdilution reference method for measuring the in vitro susceptibility of Cryptococcus neoformans isolates to fluconazole. A total of 149 isolates of C. neoformans var. neoformans from Ugandan AIDS patients was tested by both methods. An overall agreement of 88% between the two microdilution methods was observed. All isolates grew well in both RPMI 1640 and YNB media, and MICs could be read after 48 h of incubation by both methods. The range of fluconazole MICs obtained with the YNB method was broader than that obtained with the NCCLS method. The extended range of MICs provided by the YNB method may be of clinical value, as it appears that the clinical outcome may be better among patients infected with strains inhibited by lower concentrations of fluconazole as determined by the YNB method. The YNB method appears to be a viable option for testing C. neoformans against fluconazole.

Anticryptococcal activity of human serum and apotransferrin in RPMI 1640 was studied in vitro. The effects of varying concentrations of FeCl3 on this activity was investigated. Possible synergy of serum and apotransferrin with fluconazole was also measured. The fungistatic activity of human serum, whether lyophilized, stored at 4 degrees C, fresh frozen or purchased from commercial sources vs. Cryptococcus neoformans was comparable. There was no significant loss of fungistatic activity after freezing and thawing the serum up to 10 times. The fungistatic activity of human serum was similar when tested in different tissue culture media with the exception of Medium 199. The addition of apotransferrin (2.0 or 0.2 mg/ml) to RPMI 1640 had an inhibitory effect on cryptococcal growth. This effect was reversed by 20 microM, of FeCl3 at both apotransferrin concentrations. By contrast, addition of FeCl3 to human serum and RPMI 1640 did not reverse inhibition of growth. Fluconazole synergized with the human serum preparations described, but not with pooled commercial serum, for fungicidal activity. Synergistic activity of fluconazole and human serum was not affected by the addition of FeCl3. Apotransferrin did not show any synergistic fungicidal activity with fluconazole.

The inhibitory effect of human serum on the multiplication of Cryptococcus neoformans and the interaction with fluconazole were studied. Compared with cryptococcal multiplication in RPMI 1640 medium alone, 5% human serum in medium inhibited multiplication by 76% +/- 6% (n = 8). The inhibitory effect of human serum was donor independent, [corrected] heat stable (56 degrees C, 30 min), and not due to albumin or globulin. Bovine and murine sera were not inhibitory at that concentration. A fungistatic concentration of fluconazole (5.0 micrograms/ml) in medium plus 5% human serum resulted in 40% +/- 5% (n = 8) killing (reduction of inoculum CFU) in a 24-h assay. Bovine or murine sera did not have the enhancing effect, and this human serum activity was heat stable and donor independent. At 2.5 micrograms of fluconazole per ml, fungistasis by fluconazole plus human serum was significantly greater than with either alone. Higher serum concentrations [corrected] potentiated fluconazole more. At higher fluconazole concentrations (e.g., 20 micrograms/ml) fluconazole alone could kill, but serum potentiated this. A fluconazole-resistant isolate (MIC, 100 micrograms/ml) was not killed by fluconazole (5.0 micrograms/ml) in 5% human serum, but human serum potentiated the partial fluconazole inhibition. When human serum was dialyzed (molecular weight cutoff, 6,000 to 8,000) against phosphate-buffered saline, it lost the ability to synergize with fluconazole for killing Cryptococcus organisms but not the capacity to inhibit multiplication. Filtration of serum suggested the filtrate with a molecular weight of < 10,000 could interact synergistically with fluconazole for killing but could not inhibit cryptococcal multiplication. These findings indicate that human serum has two components, one (macromolecular) with a unique ability to inhibit C. neoformans and a low-molecular-weight component that enhances fluconazole anticryptococcal activity.

This study aimed to compare the susceptibilities of fluconazole, obtained in two laboratories, using three RPMI-1640 formulations (manufacturers') and inhibition standards (80%). A total of 39 selected Candida species (in vitro susceptible and resistant) and Cryptococcus neoformans isolates were tested in each medium by broth macro- and microdilution procedures following the National Committee for Clinical Laboratory Standards proposed reference method (M27-P). Macrodilution minimum inhibitory concentrations (MIC80%) were the lowest drug concentrations with turbidity (growth) less than or equal to that of the specific 80% inhibition standards (1:5 growth control). Microdilution MIC-2 were the lowest drug concentrations in which there was prominent decrease in growth. In general, the three formulations of RPMI-1640 medium provided similar MICs for most of the yeasts tested in both laboratories and by each test.

The purpose of this study was to evaluate 1:5 growth control dilutions (80% inhibition standards) to determine fluconazole minimum inhibitory concentration (MIC) end points in three laboratories. We tested 39 selected Candida species (in vitro susceptible: fluconazole MIC of < or = 1 microgram/ml, and resistant: fluconazole MIC of > or = 8 micrograms/ml) and Cryptococcus neoformans isolates by broth macro- and microdilution procedures following the National Committee for Clinical Laboratory Standards proposed reference method for yeasts (M27-P). Macrodilution MIC80% were the lowest drug concentrations with turbidity (growth) less than or equal to that of specific 1:5 dilutions of the growth control. Microdilution MICs-2 were the lowest drug concentrations in which there was prominent decrease of growth. A total of 1608 MICs were evaluated. C. krusei, C. parapsilosis, and C. tropicalis strains had reproducible fluconazole MICs by both tests (24 and 48 h). Fluconazole MIC80% and MIC-2 end points were consistent at 24 h (with C. albicans) and more variable at 48 h. MICs of C. neoformans were more reproducible at 72 h than at 48 h by both tests. This study suggests that the determination of fluconazole MICs is dependent on the length of incubation and the yeast being tested, and that antifungal testing of the yeasts may be performed by either test.