Different culture media applied to the study of Cryptococcus neoformans susceptibility to amphotericin B and fluconazole

ABSTRACT Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus cause life-threatening candidiasis, cryptococcosis, and aspergillosis, resulting in several hundred thousand deaths annually. The patients at the greatest risk of developing these life-threatening invasive fungal infections have weakened immune systems. The vulnerable population is increasing due to rising numbers of immunocompromised individuals as a result of HIV infection or immunosuppressed individuals receiving anticancer therapies and/or stem cell or organ transplants. While patients are treated with antifungals such as amphotericin B, all antifungals have serious limitations due to lack of sufficient fungicidal effect and/or host toxicity. Even with treatment, 1-year survival rates are low. We explored methods of increasing drug effectiveness by designing fungicide-loaded liposomes specifically targeted to fungal cells. Most pathogenic fungi are encased in cell walls and exopolysaccharide matrices rich in mannans. Dectin-2 is a mammalian innate immune membrane receptor that binds as a dimer to mannans and signals fungal infection. We coated amphotericin-loaded liposomes with monomers of Dectin-2’s mannan-binding domain, sDectin-2. sDectin monomers were free to float in the lipid membrane and form dimers that bind mannan substrates. sDectin-2-coated liposomes bound orders of magnitude more efficiently to the extracellular matrices of several developmental stages of C. albicans, C. neoformans, and A. fumigatus than untargeted control liposomes. Dectin-2-coated amphotericin B-loaded liposomes reduced the growth and viability of all three species more than an order of magnitude more efficiently than untargeted control liposomes and dramatically decreased the effective dose. Future efforts focus on examining pan-antifungal targeted liposomal drugs in animal models of fungal diseases. IMPORTANCE Invasive fungal diseases caused by Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals in current use have serious limitations due to host toxicity and/or insufficient fungal cell killing that results in recurrent infections. Few new antifungal drugs have been introduced in the last 2 decades. Hence, there is a critical need for improved antifungal therapeutics. By targeting antifungal-loaded liposomes to α-mannans in the extracellular matrices secreted by these fungi, we dramatically reduced the effective dose of drug. Dectin-2-coated liposomes loaded with amphotericin B bound 50- to 150-fold more strongly to C. albicans, C. neoformans, and A. fumigatus than untargeted liposomes and killed these fungi more than an order of magnitude more efficiently. Targeting drug-loaded liposomes specifically to fungal cells has the potential to greatly enhance the efficacy of most antifungal drugs.

Download Full-text

Trends in Antifungal Drug Susceptibility of Cryptococcus neoformans Isolates Obtained through Population-Based Surveillance in South Africa in 2002-2003 and 2007-2008

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00048-11 ◽

2011 ◽

Vol 55 (6) ◽

pp. 2606-2611 ◽

Cited By ~ 43

Author(s):

Nelesh P. Govender ◽

Jaymati Patel ◽

Marelize van Wyk ◽

Tom M. Chiller ◽

Shawn R. Lockhart ◽

...

Keyword(s):

South Africa ◽

Cryptococcus Neoformans ◽

Amphotericin B ◽

Fungal Infections ◽

Antifungal Susceptibility ◽

Drug Susceptibility ◽

Population Based ◽

Content Type ◽

Microdilution Method ◽

Broth Microdilution Method

ABSTRACTCryptococcus neoformansis the most common cause of meningitis among adult South Africans with HIV infection/AIDS. Widespread use of fluconazole for treatment of cryptococcal meningitis and other HIV-associated opportunistic fungal infections in South Africa may lead to the emergence of isolates with reduced fluconazole susceptibility. MIC testing using a reference broth microdilution method was used to determine if isolates with reduced susceptibility to fluconazole or amphotericin B had emerged among cases of incident disease. Incident isolates were tested from two surveillance periods (2002-2003 and 2007-2008) when population-based surveillance was conducted in Gauteng Province, South Africa. These isolates were also tested for susceptibility to flucytosine, itraconazole, voriconazole, and posaconazole. Serially collected isolate pairs from cases at several large South African hospitals were also tested for susceptibility to fluconazole. Of the 487 incident isolates tested, only 3 (0.6%) demonstrated a fluconazole MIC of ≥16 μg/ml; all of these isolates were from 2002-2003. All incident isolates were inhibited by very low concentrations of amphotericin B and exhibited very low MICs to voriconazole and posaconazole. Of 67 cases with serially collected isolate pairs, only 1 case was detected where the isolate collected more than 30 days later had a fluconazole MIC value significantly higher than the MIC of the corresponding incident isolate. Although routine antifungal susceptibility testing of incident isolates is not currently recommended in clinical settings, it is still clearly important for public health to periodically monitor for the emergence of resistance.

Download Full-text

In vitro activities of Traganum nudatum and Mentha pulegium extracts combined with amphotericin B against Candida albicans in production of hydrolytic enzymes

Current Medical Mycology ◽

10.18502/cmm.6.3.4499 ◽

2020 ◽

Author(s):

Ikram Tefiani ◽

Sidi Mohammed Lahbib Seddiki ◽

Moustafa Yassine Mahdad

Keyword(s):

Candida Albicans ◽

Minimum Inhibitory Concentration ◽

Amphotericin B ◽

Inhibitory Concentration ◽

Culture Media ◽

Hydrolytic Enzymes ◽

Normal Flora ◽

Mentha Pulegium ◽

Hydrolytic Activities

Background and Purpose: Candida albicans is an important microorganism in the normal flora of a healthy subject; however, it has an expedient pathogenic character that induces hydrolytic virulence. Regarding this, the present study aimed to find an in vitro alternative that could reduce the virulence of this yeast. Materials and Methods: For the purpose of the study, the effect of amphotericin B (AmB) combined with the extract of Traganum nudatum (E1) or Mentha pulegium (E2) was evaluated against the hydrolytic activities of esterase, protease, and phospholipase. This effect was determined by calculating the minimum inhibitory concentration (MIC), used to adjust the extract/AmB mixtures in culture media. Results: The evaluated Pz values, which corresponded to the different enzymatic activities, showed a decrease in the hydrolytic activities of C. albicans strains after the addition of E1/AmB and E2/AmB combinations at descending concentrations (lower than the obtained MICs). Conclusion: Based on the findings, it would be possible to reduce the pathogenesis of this species without destabilizing the balance of the flora.

Download Full-text

Darunavir inhibits Cryptococcus neoformans/Cryptococcus gattii species complex growth and increases the susceptibility of biofilms to antifungal drugs

Journal of Medical Microbiology ◽

10.1099/jmm.0.001194 ◽

2020 ◽

Vol 69 (6) ◽

pp. 830-837

Author(s):

Raimunda Sâmia Nogueira Brilhante ◽

José Alexandre Telmos Silva ◽

Géssica dos Santos Araújo ◽

Vandbergue Santos Pereira ◽

Wilker Jose Perez Gotay ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Amphotericin B ◽

Metabolic Activity ◽

Biofilm Formation ◽

Species Complex ◽

Cryptococcus Gattii ◽

Antifungal Drugs ◽

Planktonic Cells ◽

Species Activity ◽

Checkerboard Assay

Introduction. Cryptococcus species are pathogens commonly associated with cases of meningoencephalitis in individuals who are immunosuppressed due to AIDS. Aim. The aim was to evaluate the effects of the antiretroviral darunavir alone or associated with fluconazole, 5-flucytosine and amphotericin B against planktonic cells and biofilms of Cryptococcus species. Methodology. Susceptibility testing of darunavir and the common antifungals against 12 members of the Cryptococcus neoformans/Cryptococcus gattii species complex was evaluated by broth microdilution. The interaction between darunavir and antifungals against planktonic cells was tested by a checkerboard assay. The effects of darunavir against biofilm metabolic activity and biomass were evaluated by the XTT reduction assay and crystal violet staining, respectively. Results. Darunavir combined with amphotericin B showed a synergistic interaction against planktonic cells. No antagonistic interaction was observed between darunavir and the antifungals used. All Cryptococcus species strains were strong biofilm producers. Darunavir alone reduced biofilm metabolic activity and biomass when added during and after biofilm formation (P<0.05). The combination of darunavir with antifungals caused a significant reduction in biofilm metabolic activity and biomass when compared to darunavir alone (P<0.05). Conclusion. Darunavir presents antifungal activity against planktonic cells of Cryptococcus species and synergism with amphotericin B. In addition, darunavir led to reduced biofilm formation and showed activity against mature biofilms of Cryptococcus species. Activity of the antifungals against mature biofilms was enhanced in the presence of darunavir.

Download Full-text

CRYPTOCOCCUS NEOFORMANS INFECTION OF THE CENTRAL NERVOUS SYSTEM: A CASE TREATED BY AMPHOTERICIN B, WITH POSTMORTEM EXAMINATION

Annals of Internal Medicine ◽

10.7326/0003-4819-52-2-445 ◽

1960 ◽

Vol 52 (2) ◽

pp. 445 ◽

Cited By ~ 6

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Cryptococcus Neoformans ◽

Amphotericin B ◽

Postmortem Examination ◽

System A ◽

The Central Nervous System

Download Full-text

New Triazole NT-a9 Has Potent Antifungal Efficacy against Cryptococcus neoformans In Vitro and In Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01628-19 ◽

2019 ◽

Vol 64 (2) ◽

Cited By ~ 1

Author(s):

Ren-Yi Lu ◽

Ting-Jun-Hong Ni ◽

Jing Wu ◽

Lan Yan ◽

Quan-Zhen Lv ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Amphotericin B ◽

Pathogenic Fungi ◽

Control Group ◽

Survival Times ◽

Content Type ◽

Fungal Burden ◽

Wide Range

ABSTRACT In the past decades, the incidence of cryptococcosis has increased dramatically, which poses a new threat to human health. However, only a few drugs are available for the treatment of cryptococcosis. Here, we described a leading compound, NT-a9, an analogue of isavuconazole, that showed strong antifungal activities in vitro and in vivo. NT-a9 showed a wide range of activities against several pathogenic fungi in vitro, including Cryptococcus neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, with MICs ranging from 0.002 to 1 μg/ml. In particular, NT-a9 exhibited excellent efficacy against C. neoformans, with a MIC as low as 0.002 μg/ml. NT-a9 treatment resulted in changes in the sterol contents in C. neoformans, similarly to fluconazole. In addition, NT-a9 possessed relatively low cytotoxicity and a high selectivity index. The in vivo efficacy of NT-a9 was assessed using a murine disseminated-cryptococcosis model. Mice were infected intravenously with 1.8 × 106 CFU of C. neoformans strain H99. In the survival study, NT-a9 significantly prolonged the survival times of mice compared with the survival times of the control group or the isavuconazole-, fluconazole-, or amphotericin B-treated groups. Of note, 4 and 8 mg/kg of body weight of NT-a9 rescued all the mice, with a survival rate of 100%. In the fungal-burden study, NT-a9 also significantly reduced the fungal burdens in brains and lungs, while fluconazole and amphotericin B only reduced the fungal burden in lungs. Taken together, these data suggested that NT-a9 is a promising antifungal candidate for the treatment of cryptococcosis infection.

Download Full-text

Efficacy of Oral Encochleated Amphotericin B in a Mouse Model of Cryptococcal Meningoencephalitis

mBio ◽

10.1128/mbio.00724-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 8

Author(s):

R. Lu ◽

C. Hollingsworth ◽

J. Qiu ◽

A. Wang ◽

E. Hughes ◽

...

Keyword(s):

Mouse Model ◽

Cryptococcus Neoformans ◽

Amphotericin B ◽

Oral Absorption ◽

Oral Formulation ◽

Significant Saving ◽

Content Type ◽

Cryptococcal Meningoencephalitis ◽

Cryptococcal Disease ◽

The Impact

ABSTRACT Cryptococcus neoformans is an encapsulated yeast responsible for approximately a quarter of a million deaths worldwide annually despite therapy, and upwards of 11% of HIV/AIDS-related deaths, rivaling the impact of tuberculosis and malaria. However, the most effective antifungal agent, amphotericin B, requires intravenous delivery and has significant renal and hematopoietic toxicity, making it difficult to utilize, especially in resource-limited settings. The present studies describe a new nanoparticle crystal encapsulated formulation of amphotericin B known as encochleated amphotericin B (CAmB) that seeks to provide an oral formulation that is low in toxicity and cost. Using a 3-day delayed model of murine cryptococcal meningoencephalitis and a large inoculum of a highly virulent strain of serotype A C. neoformans, CAmB, in combination with flucytosine, was found to have efficacy equivalent to parental amphotericin B deoxycholate with flucytosine and superior to oral fluconazole without untoward toxicity. Transport of fluorescent CAmB particles to brain as well as significant brain levels of amphotericin drug was demonstrated in treated mice, and immunological profiles were similar to those of mice treated with conventional amphotericin B. Additional toxicity studies using a standardized rat model showed negligible toxicity after a 28-day treatment schedule. These studies thus offer the potential for an efficacious oral formulation of a known fungicidal drug against intrathecal cryptococcal disease. IMPORTANCE Cryptococcus neoformans is a significant global fungal pathogen that kills an estimated quarter of a million HIV-infected individuals yearly and has poor outcomes despite therapy. The most effective therapy, amphotericin B, is highly effective in killing the fungus but is available only in highly toxic, intravenous formulations that are unavailable in most of the developing world, where cryptococcal disease in most prevalent. For example, in Ethiopia, reliance on the orally available antifungal fluconazole results in high mortality, even when initiated as preemptive therapy at the time of HIV diagnosis. Thus, alternative agents could result in significant saving of lives. Toward this end, the present work describes the development of a new formulation of amphotericin B (CAmB) that encapsulates the drug as a crystal lipid nanoparticle that facilitates oral absorption and prevents toxicity. Successful oral absorption of the drug was demonstrated in a mouse model that, in combination with the antifungal flucytosine, provided efficacy equal to a parental preparation of amphotericin B plus flucytosine. These studies demonstrate the potential for CAmB in combination with flucytosine to provide an effective oral formulation of a well-known, potent fungicidal drug combination.

Download Full-text