Combination Of Amphotericin Research Articles

Molecular epidemiology and antifungal susceptibility profiles of clinical Cryptococcus neoformans/Cryptococcus gattii species complex.

Introduction. Limited data regarding the epidemiology and susceptibility profiles of cryptococcosis are available in the Middle East.Aim. Our study aimed to evaluate the molecular diversity, mating types and antifungal susceptibility pattern of Cryptococcus species (n=14) isolated from 320 suspected patients with cryptococcosis.Methodology. The URA5 gene was subjected to restriction fragment length polymorphism and sequence analysis. In addition, in vitro antifungal susceptibility testing was performed by Clinical and Laboratory Standards Institute (CLSI) M27-A4 and M59 guidelines.Results. Overall, 14 (4.4 %) patients were confirmed as cryptococcosis. Based on molecular type, 85.7 and 14.3 % of the isolates were C. neoformans VN I and VN II, respectively. Phylogenetic analysis of URA5 gene sequences revealed clustering of VN I and VN II isolates into two distinct clades with a substantial difference within each molecular type. Voriconazole and 5-fluorocytosine, respectively, had the lowest (0.031 μg ml-1) and highest (8 µg ml-1) MICs. The epidemiological cutoff values (ECVs) for amphotericin B, fluconazole, voriconazole and 5-fluorocytosine encompassed ≥97 % of all 14 C. neoformans VN I species. However, according to the CLSI document M59, ECVs for itraconazole (7; 50 % of the isolates) and for posaconazole (1; 7.1 % of the isolate), were one log2 dilution higher than the wild type range. Combinations of amphotericin B with 5-fluorocytosine, amphotericin B with fluconazole and fluconazole with 5-fluorocytosine exhibited synergistic effects against 37, 31 and 12.5 % of the isolates, respectively.Conclusion. Our findings may significantly contribute to the development of management strategies for patients at a higher risk of cryptococcosis, particularly HIV-positive individuals.

Antifungal Activity, Toxicity, and Membranolytic Action of a Mastoparan Analog Peptide.

Invasive fungal infections, such as cryptococcosis and paracoccidioidomycosis are associated with significant rates of morbidity and mortality. Cryptococcosis, caused by Cryptococcus neoformans, is distributed worldwide and has received much attention as a common complication in patients with HIV. Invasive fungal infections are usually treated with a combination of amphotericin B and azoles. In addition, 5-fluorocytosine (5-FC) is applied in cryptococcosis, specifically to treat central nervous system infection. However, host toxicity, high cost, emerging number of resistant strains, and difficulty in developing new selective antifungals pose challenges. The need for new antifungals has therefore prompted a screen for inhibitory peptides, which have multiple mechanisms of action. The honeycomb moth Galleria mellonella has been widely used as a model system for evaluating efficacy of antifungal agents. In this study, a peptide analog from the mastoparan class of wasps (MK58911) was tested against Cryptococcus spp. and Paracoccidioides spp. In addition, peptide toxicity tests on lung fibroblasts (MRC5) and glioblastoma cells (U87) were performed. Subsequent tests related to drug interaction and mechanism of action were also performed, and efficacy and toxicity of the peptide were evaluated in vivo using the G. mellonella model. Our results reveal promising activity of the peptide, with an MIC in the range of 7.8–31.2 μg/mL, and low toxicity in MRC and U87 cells (IC50 > 500 μg/mL). Taken together, these results demonstrate that MK58911 is highly toxic in fungal cells, but not mammalian cells (SI > 16). The mechanism of toxicity involved disruption of the plasma membrane, leading to death of the fungus mainly by necrosis. In addition, no interaction with the drugs amphotericin B and fluconazole was found either in vitro or in vivo. Finally, the peptide showed no toxic effects on G. mellonella, and significantly enhanced survival rates of larvae infected with C. neoformans. Although not statistically significant, treatment of larvae with all doses of MK58911 showed a similar trend in decreasing the fungal burden of larvae. These effects were independent of any immunomodulatory activity. Overall, these results present a peptide with potential for use as a new antifungal drug to treat systemic mycoses.

A Highly Active Triterpene Derivative Capable |of Biofilm Damage to Control Cryptococcus spp.

Cryptococcus neoformans is an encapsulated yeast responsible for more than 180,000 deaths per year. The standard therapeutic approach against cryptococcosis is a combination of amphotericin B with flucytosine. In countries where cryptococcosis is most prevalent, 5-fluorocytosine is rarely available, and amphotericin B requires intravenous administration. C. neoformans biofilm formation is related to increased drug resistance, which is an important outcome for hospitalized patients. Here, we describe new molecules with anti-cryptococcal activity. A collection of 66 semisynthetic derivatives of ursolic acid and betulinic acid was tested against mature biofilms of C. neoformans at 25 µM. Out of these, eight derivatives including terpenes, benzazoles, flavonoids, and quinolines were able to cause damage and eradicate mature biofilms. Four terpene compounds demonstrated significative growth inhibition of C. neoformans. Our study identified a pentacyclic triterpenoid derived from betulinic acid (LAFIS13) as a potential drug for anti-cryptococcal treatment. This compound appears to be highly active with low toxicity at minimal inhibitory concentration and capable of biofilm eradication.

Exploiting Lipids to Develop Anticryptococcal Vaccines

Cryptococcus spp. are responsible for life-threatening infections in humans causing mortality rates of 70% in developing countries. Antifungal therapy to combat cryptococcosis is based on the combination of amphotericin B, azoles, and 5-flucytosine. However, treatment failure is frequently triggered by antifungal resistance, drug-drug interactions, and toxicity. New alternatives to prevent cryptococcosis are imperative. Here, we discuss the roles of lipids in the immunological control of the disease caused by Cryptococcus spp. Recently, remarkable advances on immunology of fungal infections have been made and a number of studies indicated the potential of vaccine formulations to combat cryptococcosis. New formulations exploiting virulence regulators and genetically modified attenuated strains have been tested. In this context, lipids have emerged as virulence regulators and immunogens to be explored. Glucosylceramide (GlcCer), sterylglycosides (SGs), and lipid-containing extracellular vesicles have been recently tested in vaccine formulations and their anticryptococcal efficacy was confirmed in vivo. Together, the data discussed here encourage the use of fungal lipids in anticryptococcal vaccinal strategies.

Combination of amphotericin B and chitosan platelets for the treatment of experimental cutaneous leishmaniasis: Histological and immunohistochemical examinations

Cutaneous leishmaniasis (CL) is a chronic infectious disease caused by protozoa of the genus Leishmania. Although amphotericin B-deoxycholate (AmB-DOC) showed its ability to partially cure the infection after intravenous administration, it can lead to dose-limiting side-effects (mainly nephorotoxicity). Previous data from our group showed that chitosan particles with typical flat surfaces, called platelets acted as a booster of AmB-DOC activity against Candida albicans and C. glabrata. Platelets were obtained by a hierarchical self-assembly process between chitosan hydrophobically-modified with oleic acid and α-cyclodextrin in water. The objective of this work is to investigate whether chitosan platelets are active against CL, alone or in combination with AmB-DOC. In vitro evaluations showed that platelets exhibited intrinsic antiparasitic activity, while native chitosan was inactive. When combined with AmB-DOC, chitosan platelets exerted an additive effect against Leishmania major amastigote (Fractional inhibitory concentration index FICI = 1.213). Histological and immunohistochemical examinations of skin lesions of mice infected with L. major and treated with the combination showed a significant decrease of the inflammatory granuloma and a reduction of the parasitic load, in comparison with AmB-DOC alone. Our work provides histological and immunohistochemical evidences that the combination of chitosan platelets and AmB could be considered as a strategy to cure CL.

In vitro evaluation of antifungal combination against Cryptococcus neoformans

This study evaluated combinations of amphotericin B with anidulafungin, caspofungin, and micafungin against 30 clinical isolates of Cryptococcus neoformans following the CLSI M27-A3 and the checkerboard microdilution method. The combination amphotericin B + micafungin showed 60% of synergistic effect against C. neoformans, while most of the other interactions were indifferent.

Clinical high risk factors and prognosis of 25 pulmonary mucormycosis cases

Objective: To analyze high risk factors, therapeutic regimen and prognosis of patients with pulmonary mucormycosis. Methods: A retrospective analysis was performed on the clinical data of 25 patients who were diagnosed as pulmonary mucormycosis in the First Affiliated Hospital, School of Medicine, Zhejiang University, between January 2009 and December 2017. Results: Univariate analysis revealed that no risk factor had significant effect on prognosis of these 25 cases, while multivariate logistic regression analysis confirmed that the exposure to antifungal drugs was correlated to worse prognosis(P=0.046). All 4 patients who had received surgical lesionectomy were cured, and 8 patients who had not received any therapy targeted to mucormycosis were all dead. Seven of 13 patients received antifungal drug to mucormycosis were effective, with an effective rate of 54.0%. The mortality rate of the operative group was lower than that of the non-operative group (P=0.026), and the prognosis of the patients received drug treatment was better than patients without treatment(P=0.022). There was no significant difference in the prognosis between groups of single drug or combination of amphotericin B and posaconazole. Conclusions: Pulmonary mucormycosis is more likely to occur in patients with tumor of the hematopoietic and lymphoid tissues, immunosuppression after solid organ transplantation, long-term glucocorticoid usage and diabetes. Surgical lesionectomy can improve the survival rate, and the treatment with amphotericin B and /or posaconazole can also improve the prognosis of the patients.

Perfil epidemiológico e terapêutico de pacientes com criptococose atendidos em hospital escola de Santa Maria/RS

Criptococose é uma doença fúngica causada por leveduras do complexo Cryptococcus neoformans e Cryptococcus gattii. A infecção geralmente ocorre através da inalação dos basidiósporos do fungo presente no ambiente. Casos graves ocorrem devido à infecção do sistema nervoso central. A enfermidade possui distribuição mundial, associada principalmente aos indivíduos imunocomprometidos, portadores do vírus da imunodeficiência humana (HIV). Este estudo objetivou verificar o perfil epidemiológico e os protocolos terapêuticos dos pacientes diagnosticados com criptococose no Hospital Universitário de Santa Maria (Santa Maria, RS), no período de março de 2010 a março de 2017. Avaliaram-se 46 prontuários clínicos, sendo que o perfil dos pacientes observados foi predominantemente do sexo masculino, etnia branca, faixa etária de 31 a 50 anos, portadores de HIV, apresentando taxa de células CD4+<100 células/µL e com forma clínica prevalente, a neurocriptococose. A associação de anfotericina B e fluconazol foi o protocolo terapêutico adotado na maioria dos pacientes estudados.

Variation in chemical composition of Eucalyptus globulus essential oil under phenological stages and evidence synergism with antimicrobial standards

Bacterial resistance to antibiotics is a serious public health problem which is due to the immoderate and often inappropriate prescription of antibiotics. As a result, the exploitation of new bioactive molecules with limited or no side effects from natural sources and their adoption as a therapeutic alternative to synthetic molecules have become priorities. It is in this perspective that our study was undertaken with the objective was to investigate the phenological stage effect on the yield and chemical composition of essential oils extracted from Tunisian Eucalyptus (Eucalyptus globulus L.) aerial parts. The synergitic effect of this essential oil with conventional antimicrobials was also evaluated against nine pathogenic bacteria and Candida albicans ATCC 10231. The cytotoxicity of these combinations was tested on Vero cells. Gas chromatography–mass spectrometry analysis showed two different chemotypes depending on the growth stage which were characterized as 1,8-cineole (13.23%) at vegetative stage and p-cymene at full flowering (32.19%) and fructification (37.82%) stages. A predominance of monoterpene hydrocarbons (72.84%) during the fructification stage was detected with p-cymene (12.58%–37.82%) and α-pinene (10.41%–13.39%) as the determinants of this class. In relation to the phenological stage, Eucalyptus essential oil showed potent capacity to reduce stable free radical DPPH (IC50 up to 740 μg/mL) and moderate ability to reduce the concentration of iron ions (EC50 and IC50 up to 2 and 12 mg/mL, respectively). Eucalyptus globulus essential oil was active against the different bacterial strains especially during the vegetative and full flowering stages (MIC = 2 mg/mL) against Bacillus cereus ATCC 14579 and Enterococcus faecalis ATCC 29212. Candida albicans ATCC 10231 was more sensitive than bacteria during the fructification stage. The combination of Eucalyptus essential oil with ampicillin revealed a partial synergy effect toward Methicillin-resistant Staphylococcus aureus (FICI of 0.53). Nevertheless, an addition effect was recorded under the combination of amphotericin B and Eucalyptus essential oil. The cytototoxity activity showed that these associations enhanced cell viability and made Eucalyptus essential oil non toxic toward Vero cells. The results showed that the precious Eucalyptus oil in association with antimicrobial agents could be not only a promising alternative but also a potential molecular complex which is hardly recognizable by microorganisms.

C. auris: What do we know about the emergence of this multidrug resistant yeast?

Candida auris has been reported as emerging yeast with variable antifungal susceptibility profile and development of antifungal resistance. Infections are associated with a variety of invasive fungal diseases observed in critically ill patients such those with hematological malignances, in intensive care units, with catheter devices, those undergoing invasive procedures. Genetic analysis showed the emergence of separate clades in different geographical regions. C auris was reported in Japan, India, South Korea, Pakistan, Oman, South Africa, USA, United Kingdom, Norway, Germany, Spain, Venezuela, Colombia and Brazil. The identification is not easy with conventional diagnostic tools and missidentification was reported since C auris is closely related with C. haemulonii, C lusitaniae and others. However it is important to have a suspition if an isolation of a yeast that forms pink to beige colonies in chromogenic agar, growing at 42 C but not in the presence of cycloheximede, microscopic oval to elongated yeast without pseudohyphe, is encountored. There is not a breakpoint for C auris and reduce susceptibility were reported for fluconazole, voriconazole, itraconazole, isavuconazole, amphotercin B. Echinocandins are proposed to use, although due their more widespread use, some isolates were reported with low susceptibility. In vitro the new 1-3 B glucan inhibitor SCY-078 has been demonstrated potent activity. Suggested therapy include echinocandins, combination of amphotericin B plus flucytosin, micafungin plus voriconazole, depending the site of infection. Thus, it is important to have the knowledge about the emerging of this yeast that becomes a potential dominant opportunistic pathogen in immunocompromised hosts.

In vitro activity of antifungal combinations against planktonic and sessile cells of Candida albicans isolated from medical devices in an intensive care department

BackgroundInvasive fungal infections are an emerging health problem worldwide. They are responsible for a significant rate of morbidity and mortality. Infections caused by Candida albicans involve proliferation of biofilms on biotic or abiotic surface. These adherent communities exhibit characteristics distinct from planktonic cells such as the ability to tolerate high concentrations of antifungal. ObjectiveThe object of our study was focused on the determination of the susceptibility to amphotericin B, caspofungin, voriconazole and two antifungal combinations (amphotericin B/caspofungin and amphotericin B/voriconazole) of both planktonic and sessile cells of C. albicans, which were isolated from catheters. Material and methodsThe susceptibility of C. albicans to antifungals was determined using the broth microdilution method according to Clinical Laboratory Standards Institute CLSI (2008). A Checkerboard assay was employed to evaluate the efficacy of drugs combinations. Biofilm susceptibility was determined using a metabolic [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] (XTT) reduction assay. ResultsThe minimal inhibitory concentrations of individual antifungal drugs determined against C. albicans biofilms (SMICs) were significantly higher (P<0.05) than planktonic ones (MICs). They went from 2 to 64μg/mL for amphotericin B, from 1 to 64μg/mL for caspofungin and from 2 to 128μg/mL for voriconazole. The combination of amphotericin B to caspofungin or to voriconazole decreased significantly the MIC values for planctonic (P<0.0001) and sessile cells (P=0.0016). Based on Fractional Inhibitory Concentration Index (FICI), no antagonistic interaction was observed. ConclusionThe obtained results showed that the combination of amphotericin B with either caspofungin or voriconazole can be used as a new strategy for management of systemic mycoses associated to medical devices.

Rash in an Extremely Low Birth Weight Infant

Primary cutaneous aspergillosis (PCA) is one of several invasive fungal infections that have increased in incidence in the last four decades. We present a case of an extremely low birth weight infant born at 24 weeks gestation diagnosed with cutaneous aspergillosis, highlighting the risk factors and clinical findings associated with neonatal PCA. Additionally, we discuss use of serum galactomannan testing as well as combination amphotericin B and voriconazole therapy.

Combination of Amphotericin B and Terbinafine against Melanized Fungi Associated with Chromoblastomycosis.

Our in vitro studies showed that a combination of amphotericin B and terbinafine had synergistic effects against the majority of melanized fungi associated with chromoblastomycosis (CBM) and similar infections, including those with Cladophialophora carrionii, Cladophialophora arxii, Exophialadermatitidis, Exophialaspinifera, Fonsecaea monophora, Fonsecaea nubica, Fonsecaea pedrosoi, and Phialophora verrucosa. This drug combination could provide an option for the treatment of severe or unresponsive cases of CBM, particularly in cases due to species of Fonsecaea and Cladophialophora.

Chapter 11 - Cryptococcal meningitis in AIDS

Cryptococcal meningitis remains a significant cause of morbidity and mortality amongst patients living with human immunodeficiency virus (HIV). The prevalence in the developed world has decreased as HIV is being diagnosed earlier, but is still significant, and the prevalence in resource-limited settings is exceedingly high. The presenting symptoms usually include a headache, fever, and, less often, cranial nerve abnormalities. Space-occupying lesions do occur, but are rare. Once diagnosed, patients should be treated with a combination of amphotericin and flucytosine, with step-down therapy to fluconazole for a minimum of a year, or until the CD4 count is above 100 cells/μL, whichever is longer. In the acute phase of treatment increased opening pressure is common, which should be managed aggressively with frequent lumbar punctures, or through neurosurgical interventions (lumbar drains, ventriculoperitoneal shunts) if those fail. Antiretrovirals should be delayed at least 2 weeks, but maybe as many as 10 weeks, after initiation of antifungal therapy in order to prevent clinical or subclinical immune reconstitution inflammatory syndrome (IRIS), which may lead to increased mortality. However, if IRIS does develop, there is no role for antiretroviral interruption, and the condition should be managed supportively by use of anti-inflammatories and aggressive management of elevated opening pressure, if present. Steroids should be administered for specific indications only (IRIS or cryptococcoma with cerebral edema and risk of herniation) as routine use of steroids increases mortality in cryptococcal meningitis.

Combining two antifungal agents does not enhance survival of Galleria mellonella larvae infected with Madurella mycetomatis.

To determine whether combination therapy would improve therapeutic outcome in eumycetoma caused by Madurella mycetomatis. Survival, colony-forming units (CFU), melanisation and histopathology in M. mycetomatis-infected Galleria mellonella larvae treated with amphotericin B, itraconazole, terbinafine or combinations thereof were determined. Compared to larvae treated with 5% glucose, enhanced survival was obtained when M. mycetomatis-infected larvae were treated with amphotericin B, but not when they were treated with itraconazole or terbinafine. Combination therapy did not increase survival compared to 5% glucose-treated larvae, itraconazole-treated larvae or terbinafine-treated larvae. Compared to amphotericin B monotreatment, a significant decrease in survival was noted when this therapy was combined with either itraconazole or terbinafine. CFU, melanisation and histopathology did not differ between monotherapy, combination therapy or 5% glucose-treated larvae. Combining different classes of antifungal agents did not enhance the survival of M. mycetomatis-infected G. mellonella larvae. Instead of improving the therapeutic outcome, combining either itraconazole or terbinafine with amphotericin B resulted in significantly lower survival rates of infected larvae than amphotericin B monotherapy. This experimental study does not provide support for the use of combined amphotericin B and itraconazole, combined itraconazole and terbinafine or combined terbinafine and amphotericin B and should be confirmed in other animal models.

The Anti-helminthic Compound Mebendazole Has Multiple Antifungal Effects against Cryptococcus neoformans.

Cryptococcus neoformans is the most lethal pathogen of the central nervous system. The gold standard treatment of cryptococcosis, a combination of amphotericin B with 5-fluorocytosine, involves broad toxicity, high costs, low efficacy, and limited worldwide availability. Although the need for new antifungals is clear, drug research and development (R&D) is costly and time-consuming. Thus, drug repurposing is an alternative to R&D and to the currently available tools for treating fungal diseases. Here we screened a collection of compounds approved for use in humans seeking for those with anti-cryptococcal activity. We found that benzimidazoles consist of a broad class of chemicals inhibiting C. neoformans growth. Mebendazole and fenbendazole were the most efficient antifungals showing in vitro fungicidal activity. Since previous studies showed that mebendazole reaches the brain in biologically active concentrations, this compound was selected for further studies. Mebendazole showed antifungal activity against phagocytized C. neoformans, affected cryptococcal biofilms profoundly and caused marked morphological alterations in C. neoformans, including reduction of capsular dimensions. Amphotericin B and mebendazole had additive anti-cryptococcal effects. Mebendazole was also active against the C. neoformans sibling species, C. gattii. To further characterize the effects of the drug a random C. gattii mutant library was screened and indicated that the antifungal activity of mebendazole requires previously unknown cryptococcal targets. Our results indicate that mebendazole is as a promising prototype for the future development of anti-cryptococcal drugs.

One-Step Continuous Flow Synthesis of Antifungal WHO Essential Medicine Flucytosine Using Fluorine

In Africa around 625 000 mortalities per annum (20% of HIV/AIDS related deaths) are due to the affects of the Cryptococcal meningitis (CM) fungal infection. Recently, the World Health Organisation (WHO) and the Infectious Disease Society of America (IDSA) recommended that the first line treatment for CM is a combination of amphotericin B and flucytosine, both now WHO Essential Medicines. However, flucytosine is not even registered for use in any African nation due, in part, to its relatively high cost of manufacture and lack of generic manufacturers. Currently, flucytosine is manufactured by an expensive four-step manufacturing process. Here we report a one-step continuous flow process involving the reaction of inexpensive cytosine with fluorine gas using stainless steel tubular laboratory and pilot-scale silicon carbide reactor devices which is readily scaleable to a manufacturing process with a low initial capital expenditure.

Bone and joint infections caused by mucormycetes: A challenging osteoarticular mycosis of the twenty-first century.

Osteomyelitis and arthritis caused by mucormycetes are rare diseases that rank among the most challenging complications in orthopedic and trauma surgery. The aim of this work is to review the epidemiological, clinical, diagnostic, and therapeutic aspects of the osteoarticular mucormycosis with particular emphasis on high-risk patients. A systematic review of osteoarticular mucormycosis was performed using PUBMED and EMBASE databases from 1978 to 2014. Among 34 patients with median age 41 (0.5-73 years), 24 (71%) were males. While 12 (35%) were immunocompromised patients, 14 (41%) had prior surgery, and seven (21%) suffered trauma. Other underlying conditions included diabetes mellitus, hematological malignancies, transplantation, and corticosteroid therapy. The median diagnostic delay from onset of symptoms and signs was 60 (10-180) days. The principal mechanism of the infection was direct inoculation (n = 19; 56%), and in immunocompromised patients was usually hematogenous disseminated. The long bones were infected by trauma or surgery, while a wide variety of bones were involved by hematogenous dissemination. Combined surgery and amphotericin B treatment were implemented in 28 (82%) and eight (23%) had an unfavorable outcome. Osteoarticular mucormycosis occurs most frequently after trauma or surgical procedures. These infections are progressively destructive and more virulent in individuals with impaired immune systems. Early diagnosis, timely administration of amphotericin B, control of underlying conditions, and surgical debridement of infected tissue are critical for successful management of osteoarticular mucormycosis.

Treatment of Rhino-Orbito-Cerebral Aspergillosis with Combination of Amphotericin, Posaconazole and Amphotericin Irrigation: A Case Report

Treatment of Rhino-Orbito-Cerebral Aspergillosis with Combination of Amphotericin, Posaconazole and Amphotericin Irrigation: A Case Report

A Trichosporon asahii Bloodstream Infection in a Preterm Newborn with a Successful Outcome: A Case Report

Trichosporon asahii is a rare opportunistic fungal pathogen that causes fatal systemic infections in immunocompromised patients. We have reported a case of T. asahii fungemia in a low birth weight preterm newborn baby in the neonatal intensive care unit (NICU). This case of T. asahii fungemia was unresponsive to treatment with a combination of Amphotericin B and Fluconazole. However, Treatment with a combination of Voriconazole and Caspofungin was successfully eliminated the fungus from the blood of the infected patient.