Enhanced efficacy of synergistic combinations of antimicrobial peptides with caspofungin versus Candida albicans in insect and murine models of systemic infection

The objective of this study was to determine whether combinations of antimicrobial peptides (AMPs) with caspofungin display enhanced antifungal activity versus Candida albicans in vitro and in vivo. Three conventional AMPs that satisfied criteria favouring their potential development as novel antifungals were selected for investigation. Colistin sulphate was also included as a cyclic peptide antibiotic used in the clinic. Minimum inhibitory concentrations (MICs) were determined for each antifungal agent and checkerboard assays were used to determine fractional inhibitory concentration index (FICI) values for dual combinations of AMPs or colistin with caspofungin. Viability assays were performed for the same combinations in order to investigate fungicidal interactions. Synergistic antifungal combinations were then tested for efficacy in vivo and compared to monotherapies in wax moth larva and murine models of systemic C. albicans infection. In combination with caspofungin, each of the AMPs [hMUC7–12, DsS3(1–16), hLF(1–11)] and colistin were synergistic and candidacidal in vitro. The treatment of infected wax moth larvae with combinations of caspofungin with hMUC7–12, DsS3(1–16) or colistin resulted in significant enhancements in survival compared to treatment with monotherapies. Notably, the treatment of C. albicans-infected mice with a combination of caspofungin and DsS3(1–16) resulted in the enhancement of survival compared to groups treated with just the individual agents. This study demonstrates that combination therapies containing caspofungin and AMPs or colistin merit further development as potential novel treatments for C. albicans infections.     

Memory retention: potentiation of cholinergic drug combinations in mice

The amnesias characteristic of Alzheimer's disease and other age-related dementias are refractory to conventional pharmacotherapy. A recent strategy is to combined present drugs, to improve their memory enhancing effect. We utilize mice weakly trained on active avoidance in a T-maze in order to compare the effect on retention test performance of cholinergic drugs given alone and in two-drug and three-drug combinations. All drugs were injected intraventricularly immediately after training. Memory retention was tested one week later. A dose-response curve was determined for each of four drugs (arecoline, edrophonium, oxotremorine, deanol) and for several of their fixed-ratio combinations. The results indicate that each drug and each combination improved retention test performance up to an optimal dose; the improvement decreased with further increases in dose. A striking reduction (as much as 95%) in the optimal dose for enhanced retention was observed with these two-drug combinations, and further reduction with a three-drug combination. The practical implications of planned drug interactions as an improved means of treating amnesias associated with aging are under investigation.     

Antifungal Agents for Secondary Prophylaxis Based on Response to Initial Antifungal Therapy in Allogeneic Hematopoietic Stem Cell Transplant Recipients with Prior Pulmonary Aspergillosis

We performed a prospective study to evaluate the efficacy and safety of secondary antifungal prophylaxis (SAP) for patients with a history of invasive pulmonary aspergillosis (IPA) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, the prophylactic agents used were chosen based on treatment response to initial antifungal therapy. One hundred and thirty-six patients undergoing allo-HSCT with prior IPA were enrolled in this multicenter study. The agents of SAP included itraconazole in 24, voriconazole in 74, caspofungin in 32, and liposomal amphotericin B in 6. Eighty-eight patients had stable IPA and 48 had active IPA at the time of transplantation. The success rate of SAP was 91.2%. Twelve patients developed breakthrough invasive fungal disease (IFD), and none discontinued antifungal agents because drug-related adverse events. The incidence of breakthrough IFD was neither different among the different antifungal agents (P = .675) nor between patients with active and stable IPA (P = .080). The 1-year cumulative incidence of IFD and IPA relapse was 27.3% ± 4.5% and 24.7% ± 4.4%, respectively. Our data indicate that SAP with antifungal agents based on initial antifungal therapy has favorable efficacy and safety in allo-HSCT recipients with prior IPA. Active IPA might not increase the risk of breakthrough IFD after transplantation.     

Osteomyelitis caused by Aspergillus species: a review of 310 reported cases

Aspergillus osteomyelitis is a rare infection. We reviewed 310 individual cases reported in the literature from 1936 to 2013. The median age of patients was 43 years (range, 0–86 years), and 59% were males. Comorbidities associated with this infection included chronic granulomatous disease (19%), haematological malignancies (11%), transplantation (11%), diabetes (6%), pulmonary disease (4%), steroid therapy (4%), and human immunodeficiency virus infection (4%). Sites of infection included the spine (49%), base of the skull, paranasal sinuses and jaw (18%), ribs (9%), long bones (9%), sternum (5%), and chest wall (4%). The most common infecting species were Aspergillus fumigatus (55%), Aspergillus flavus (12%), and Aspergillus nidulans (7%). Sixty-two percent of the individual cases were treated with a combination of an antifungal regimen and surgery. Amphotericin B was the antifungal drug most commonly used, followed by itraconazole and voriconazole. Several combination or sequential therapies were also used experimentally. The overall crude mortality rate was 25%.     

Activities of eighteen antimicrobial regimens against Mycobacterium avium infection in beige mice

The therapeutic effect of 18 anti-Mycobacterium avium regimens was examined in beige mice after 91 days of infection. Treatments included monotherapy with clarithromycin (CLA), ethambutol (EMB), amikacin (AMI), rifabutin (RFB), ciprofloxacin (CIP), clofazimine (CLO), and combinations of CLA, CLA-EMB, or CLA-AMI with one of the other drugs. After monotherapy, only AMI and CLA displayed bacteriostatic and/or moderate bactericidal effects in spleens and lungs, while CIP and RFB were totally inactive and CLO and EMB showed intermediate effects against the isolate tested. Resistant mutants were isolated in spleens of mice treated with EMB, CIP, RFB, and CLO-Among two-drug combinations, CLA-RFB, CLA-CIP, and CLA-CLO were significantly more active than RFB, CIP, CLO, respectively, but not more active than CLA alone, in both organs; CLA-AMI and CLA-EMB were bactericidal in spleens and lungs, respectively. Although activity of CLA-EMB was significantly potentiated by RFB and CLO in spleens and lungs, that of CLA-AMI was significantly increased by RFB and CLO only in lungs. The most active regimen in spleens and lungs on day 91 was the combination of all three, namely CLA-AMI-EMB, which reduced the CFU numbers of 2.7 and 7.5 log10, in comparison with day 1 and day 91 counts in untreated control mice, respectively.     

Combination antifungal therapy against Candida species: the new frontier--are we there yet?

In the past decade, we have seen a significant increase in the incidence of invasive fungal infections. In addition, opportunistic fungal infections resistant to antifungal agents have become increasingly common and their frequency will more than likely continue to increase. The antifungal armamentarium for the treatment of serious fungal infections remains limited. A possible approach to overcoming antifungal drug resistance and high mortality rates seen in severe fungal infections is to combine two or three classes of antifungals, especially if the drugs have different mechanisms of action. The unique properties of newer antifungals now provide us with the opportunity to investigate antifungal combinations that may become the standard of care for serious fungal infections. Combinations of new agents along with more traditional antifungals have now been shown to possess some synergistic or at least additive activity against Candida in clinical trials. On the other hand, caution is still needed since other antifungal combinations have demonstrated antagonistic activity in vitro. Well-controlled clinical trials are needed to define the most efficacious antifungal regimen. Furthermore, these trials should also evaluate the side effect potential of combination regimens and the pharmacoeconomic impact these regimens may have. Thus, while much optimism exists for combination therapy, there is much yet to be done.     

In-vitro activity of nikkomycin Z alone and in combination with polyenes, triazoles or echinocandins against Aspergillus fumigatus

The in-vitro activity of nikkomycin Z was investigated in combination with polyenes, triazoles or echinocandins against 20 clinical isolates of Aspergillus fumigatus with the fractional inhibitory concentration index (FICI) method. The drug interactions were classified as synergic (FICI < or = 0.5), no interaction (FICI > 0.5, but FICI < or = 4) or antagonistic (FICI > 4). The fungicidal activity of nikkomycin Z alone and in combination with a representative echinocandin (caspofungin) or triazole (voriconazole) was also examined with time-kill experiments and fungal cell viability assays. Two-drug combinations of nikkomycin Z with amphotericin B (FICI 3.59 +/- 0.57), amphotericin B lipid complex (FICI 3.95 +/- 0.74), liposomal amphotericin B (FICI 3.62 +/- 0.98), itraconazole (FICI 2.0 +/- 0.0), voriconazole (FICI 1.07 +/- 0.37), posaconazole (FICI 2.20 +/- 0.44) or ravuconazole (FICI 1.76 +/- 0.44) showed no interactions, but the pairwise combination of nikkomycin Z with caspofungin (FICI 0.22 +/- 0.19) or micafungin (FICI 0.35 +/- 0.27) showed synergic activity against A. fumigatus. Time-kill studies and fungal cell viability assays showed that neither nikkomycin Z nor caspofungin alone possessed fungicidal activity against A. fumigatus, whereas a combination of these two drugs at concentrations > or = 2 mg/L (> or = 0.031 x the concentration of drug that produced no visible growth) killed germinated conidia within 24 h in a concentration-dependent manner. These data suggest that two-drug combinations of nikkomycin Z with echinocandins, but not with polyenes and triazoles, have a synergic effect against A. fumigatus.     

Antifungal and Antihepatotoxic Effects of Sepia Ink Extract Against Oxidative Stress as a Risk Factor of Invasive Pulmonary Aspergillosis in Neutropenic Mice

Background

There is a great need for novel strategies to overcome the high mortality associated with invasive pulmonary aspergillosis (IPA) in immunocompromised patients. To evaluate the antifungal and antihepatotoxic potentials of Sepia ink extract, its effect on liver oxidative stress levels was analyzed against IPA in neutropenic mice using amphotercin B as a reference drug.

Materials and Methods

Eighty neutropenic infected mice were randomly assigned into four main groups. The 1^st group was treated with saline, neutropenic infected (NI), the 2^nd group was treated with ink extract (200 mg/kg) (IE) and the 3^rd group was treated with amphotericin B (150 mg/kg) (AMB) and 4^th group was treated with IE plus AMB. Treatment was started at 24 h after fungal inoculation (1×10⁹ conidia/ml).

Results

The present study revealed good in vitro and in vivo antifungal activity of IE against A. fumigatus. IE significantly reduced hepatic fungal burden and returns liver function and histology to normal levels. Compared with the untreated infected group, mice in the IE, AMB, and IE+ AMB groups had increased glutathione reduced (GSH) and superoxide dismutase (SOD) and significantly reduced malondialdehyde (MDA) levels at 24 and 72 h after inoculation with A. fumigatus conidia.

Conclusion

It is then concluded that in combination with antifungal therapy (AMB), IE treatment can reduce hepatic fungal burden, alleviate hepatic granulomatous lesions and oxidative stress associated with IPA in neutropenic mice     

Epidemiology of invasive fungal infections in neonates and children

Invasive fungal infections are major causes of morbidity and mortality in neonates and in both immunocompromised and immunocompetent children. Although these infections have been well characterized in adults, the incidence and analysis of risk factors, diagnostic tools, treatments and outcomes have not been well described for large cohorts of paediatric or neonatal patients. Paediatric exclusion has limited our knowledge of the epidemiology and pathophysiology of paediatric fungal disease, and has also resulted in a paucity of data regarding the safety and efficacy of paediatric antifungal therapy. Previous paediatric cooperative models in other disciplines have successfully advanced our understanding and treatments of childhood diseases, but in the past there has not been a similar organization for paediatric invasive fungal infections. Although there are numerous other reviews outlining the differences in paediatric antifungal dosing pharmacokinetics, there are only smaller epidemiological reports depicting the exact distribution and outcomes of paediatric invasive fungal infections treated with these antifungals. This review will highlight some of the available epidemiological data on paediatric invasive fungal infections.     

Combination Antifungals as an Effective Means of Salvage in Paediatric Leukaemia Patients with Invasive Fungal Infections

Invasive fungal infections (IFIs) are an important cause of morbidity and mortality in paediatric leukaemias. Antifungal combinations to treat these patients are being explored. Fourteen children with leukaemias and IFIs were treated with a combination of antifungal agents at our centre. The first antifungal was amphotericin-B in 13 children and voriconazole in one child. In view of no improvement and clinical deterioration, in nine patients, voriconazole was added as the second antifungal agent and in four, it was caspofungin. All patients completed 4–6 weeks of antifungal therapy. The overall mortality attributable to IFI for the cohort was 4/14 (28%).     

Antifungal immunotherapy and immunomodulation: a double-hitter approach to deal with invasive fungal infections

In recent years, the incidence of life-threatening fungal infections has dramatically increased. Despite significant developments in antifungal chemotherapy, its efficacy remains limited by the inability to sterilize infected organs and the tendency to induce resistance and cause side effects. In response to these challenges, it is now recognized that several aspects of antifungal immunity can be modulated to better deal with fungal infections. Extensive work was carried out on the development and testing of preventive and therapeutic antifungal vaccines. The potential use of cytokines, adoptive T-cell transfer, monoclonal antibodies (MoAb) and antimicrobial peptides (AMP) as solo or adjunctive therapies is also receiving much attention. Although each of these immune-based treatment strategies has many advantages and some shortcomings, none on its own, has proven satisfactorily effective to deal with invasive fungal infections. Appropriate combinations that optimize the advantages and minimize the disadvantages of immune-based antifungals are still lacking mainly due to the immense difficulty in sorting out candidate combinations given the long list of choices. In this review, immune-based antifungals are divided into two general categories on the basis of the intended target being the host (immunomodulation through vaccines, cytokines, adoptive T-cell transfer or MoAb) or the pathogen (immunotherapy through MoAb or AMP). Potential advantages and disadvantages of immunotherapy and immunomodulation are tentatively discussed so as to facilitate the design of future studies that aim at devising more potent immune-based antifungal treatment combinations.     

Treatment of orogastrointestinal candidosis in SCID mice with fluconazole alone or in combination with recombinant granulocyte colony-stimulating factor or interferon-gamma.

Mucosal candidosis is common in acquired immune deficiency syndrome (AIDS) patients, where there is extensive mucosal involvement, but rarely dissemination. To mimic this disease, SCID mice were inoculated orally with Candida albicans, which could be recovered from standardized tissue samples of the esophagus, stomach, small intestine and caecum of all mice. Treatment with fluconazole at 5 or 10 mg kg(-1) per day were equivalent to each other and efficacious in reducing the fungal burden from all four tissues compared with no treatment or lower doses of fluconazole (P < 0.01-0.001). Fluconazole at 5 or 10 mg kg(-1) reduced fungal burden in the stomach by about 200 or 580-fold, respectively, and by approximately 25-fold in the other tissues, with 80 or 100% of mice cleared of esophageal infection, and 40 or 80% cleared of infection in the small intestine, respectively; the same doses cleared < or =20% of stomach infection and none of caecal infection. Treatment with recombinant human granulocyte colony-stimulating factor (G-CSF) up to 500 microg kg(-1) per day or 10(5) U of murine interferon-gamma (IFN-gamma) alone was ineffective, nor were combinations with a suboptimal dose fluconazole synergistic. Overall, fluconazole had dose-responsive efficacy, whereas neither G-CSF nor IFN-gamma alone or in combination with fluconazole improved efficacy. These studies demonstrate the utility of this model for examining antifungal efficacy in a situation that mimics clinical disease in AIDS patients.     

Clinical characteristics,radiologic findings,risk factors and outcomes of serum galactomannan-negative invasive pulmonary aspergillosis

Background

The sensitivity of galactomannan (GM) assay is suboptimal for detecting invasive pulmonary aspergillosis (IPA) in serum samples. However, the clinical characteristics, radiologic findings, and outcomes in patients with GM-negative IPA have not been fully elucidated.

Synergistic activity profile of griffithsin in combination with tenofovir, maraviroc and enfuvirtide against HIV-1 clade C

Griffithsin (GRFT) is possibly the most potent anti-HIV peptide found in natural sources. Due to its potent and broad-spectrum antiviral activity and unique safety profile it has great potential as topical microbicide component. Here, we evaluated various combinations of GRFT against HIV-1 clade B and clade C isolates in primary peripheral blood mononuclear cells (PBMCs) and in CD4⁺ MT-4 cells. In all combinations tested, GRFT showed synergistic activity profile with tenofovir, maraviroc and enfuvirtide based on the median effect principle with combination indices (CI) varying between 0.34 and 0.79 at the calculated EC₉₅ level. Furthermore, the different glycosylation patterns on the viral envelope of clade B and clade C gp120 had no observable effect on the synergistic interactions.Overall, we can conclude that the evaluated two-drug combination increases their antiviral potency and supports further clinical investigations in pre-exposure prophylaxis for GRFT combinations in the context of HIV-1 clade C infection.     

Experimental design and modelling approach to evaluate efficacy of β-lactam/β-lactamase inhibitor combinations

BackgroundA β-lactamase inhibitor (BLI) confers susceptibility of β-lactamase-expressing multidrug resistant (MDR) organisms to the partnering β-lactam (BL).AimsTo discuss the experimental design and modelling strategies for two-drug combinations, using ceftazidime- and aztreonam-avibactam combinations, as examples.SourcesThe information came from several publications on avibactam in vitro time-kill studies and corresponding pharmacodynamic models.ContentThe experimental design to optimally gather crucial information from constant-concentration time-kill studies is to use an agile matrix of two-drug concentration combinations that cover 0.25- to 4-fold BL minimum inhibitory concentration (MIC) relative to the BLI concentrations to be tested against the particular isolate. This shifting agile design can save substantial costs and resources, without sacrificing crucial information needed for model development. The complex synergistic BL/BLI interaction is quantitatively explored using a semi-mechanistic pharmacokinetic-pharmacodynamic (PK/PD) mathematical model that accounts for antimicrobial activities in the combination, bacteria-mediated BL degradation and inhibition of BL degradation by BLI. A predictive mathematical formulation for the two-drug killing effects preserves the correlation between the model-derived EC₅₀ of BL and the BL MIC. The predictive value of PK/PD model is evaluated against external data that were not used for model development, including but not limited to in vitro hollow fibre and in vivo murine infection models.ImplicationsAs a framework for translational predictions, the goal of this modelling strategy is to significantly decrease the decision-making time by running clinical trial simulations with MIC-substituted EC₅₀ function for isolates of comparable susceptibility through established correlation between BL MIC and EC₅₀ values.     

Synergistic effects of antimycobacterial drag combinations onMycobacterium avium complex determined radiometrically in liquid medium

A technique to determine the effects of combining antimycobacterial drugs in liquid medium employing the radiometric growth readings in the Bactec system was tested using 20 Mycobacterium avium complex strains. Ten of the strains had been isolated from children with lymphadenopathy and ten from adults with pulmonary disease. All isolates were resistant to streptomycin, rifampicin, isoniazid and ethambutol when tested with a conventional resistant ratio technique on Löwenstein-Jensen medium. Synergistic interactions were shown for the two-drug combinations streptomycin + ethambutol and ethambutol + rifampicin against all 20 strains. Good efficacy was also found for all three-drug combinations containing ethambutol. Thus, although most isolates of the Mycobacterium avium complex are resistant to first-line antituberculous drugs when tested individually, they are susceptible in vitro to certain combinations of these drugs. This rapid radiometric assay is an efficient means for detecting such synergy.     

Effect of the fluoride/calcium regimen on vertebral fracture occurrence in postmenopausal osteoporosis. Comparison with conventional therapy

We assessed the rates of vertebral fracture in patients with postmenopausal osteoporosis. Forty-five patients were not treated (91 person-years of observation); 59 were treated conventionally, with calcium (alone or combined with estrogen) or vitamin D or both (218 years); and 61 were treated with sodium fluoride combined with conventional therapy (251 years). The fracture rate (per thousand person-years) was 834 in untreated patients, 419 in those given calcium with or without vitamin D, 304 in those given fluoride and calcium with or without vitamin D, 181 in those given estrogen and calcium with or without vitamin D, and 53 in those given fluoride, estrogen, and calcium with or without vitamin D. It was reduced in all treatment groups (P less than 0.001 for calcium and P less than 1 x 10(-6) for other combinations); fluoride (one years of treatment) and estrogen (but not vitamin D) independently reduced the rate from that observed with calcium alone (P less than 0.001). The combination of calcium fluoride, and estrogen was more effective than any other combination (P less than 0.001). These results provide grounds for optimism about the efficacy of combinations of available agents with sodium fluoride for fracture in postmenopausal osteoporosis.     

Risk Factors for Invasive Pulmonary Aspergillosis and Hospital Mortality in Acute-On-Chronic Liver Failure Patients: A Retrospective-Cohort Study

Background: Invasive pulmonary aspergillosis (IPA) in acute-on-chronic liver failure (ACLF) patients is associated with a high mortality. But the clinical characteristics of and the risk factors for IPA among patients with ACLF remains unclear. This study was aimed at assessing clinical manifestation, the risk factors and antifungal medications for as well as the mortality due to IPA in ACLF patients at the First Affiliated Hospital, College of Medicine, Zhejiang University.Patients and Methods: Patients with ACLF who were diagnosed with proven or probable IPA by clinical and laboratory parameters from 1 December 2008 to 1 May 2012 were retrospectively evaluated to determine the risk factors for IPA and the clinical outcomes. The follow-up ended on 30 July 2012. Multivariate analysis was performed to identify the risk factors for mortality and the development of IPA.Results: In total, 787 patients with ACLF were enrolled, and 39 of these patients developed IPA. Thirty seven of these 39 patients died in spite of treatment with antifungal drugs. Controls included 48 patients who did not have a pulmonary infection. The survival rate of patients with IPA was significantly lower than that of those without IPA. IPA was found to be independently associated with age (p = 0.021), encephalopathy (p = 0.002), and steroid use (p = 0.000). There was significant difference in the prognosis between the patients treated with either voriconazole or itraconazole and those without antifungal treatment.Conclusions: Patients with ACLF and IPA have a high mortality rate. Patients with ACLF who present with encephalopathy should avoid steroids, as they increase the mortality rate. Azoles may prolong the survival time.     

Combination treatment of invasive fungal infections

The persistence of high morbidity and mortality from systemic fungal infections despite the availability of novel antifungals points to the need for effective treatment strategies. Treatment of invasive fungal infections is often hampered by drug toxicity, tolerability, and specificity issues, and added complications often arise due to the lack of diagnostic tests and to treatment complexities. Combination therapy has been suggested as a possible approach to improve treatment outcome. In this article, we undertake a historical review of studies of combination therapy and also focus on recent studies involving newly approved antifungal agents. The limitations surrounding antifungal combinations include nonuniform interpretation criteria, inability to predict the likelihood of clinical success, strain variability, and variations in pharmacodynamic/pharmacokinetic properties of antifungals used in combination. The issue of antagonism between polyenes and azoles is beginning to be addressed, but data regarding other drug combinations are not adequate for us to draw definite conclusions. However, recent data have identified potentially useful combinations. Standardization of assay methods and adoption of common interpretive criteria are essential to avoid discrepancies between different in vitro studies. Larger clinical trials are needed to assess whether combination therapy improves survival and treatment outcome in the most seriously debilitated patients afflicted with life-threatening fungal infections.