Comparison of Broth Dilution and Semisolid Agar Dilution for In Vitro Susceptibility Testing of Cryptococcus neoformans

Summary

We compared the in vitro activity of amphotericin B, flucytosine, itraconazole, fluconazole, ketoconazole and miconazole against 18 strains of Cryptococcus neoformans by using two methods: microbroth dilution and semisolid agar dilution.

By both of the methods minimum inhibitory concentrations (MICs) showed a wide range for all antifungal agents but not for amphotericin B. Statistically significant differences between the two methods were observed only with amphotericin B and flucytosine, p = 0.048 and p = 0.045 respectively.

Our study suggests that azole susceptibility testing for C. neoformans may be performed by the broth microdilution as well as the semisolid agar test. The choice of the method when testing amphotericin B and flucytosine is more problematic.     

Comparison of Disk Diffusion,E-Test and Broth Microdilution Test in Determination of Susceptibility of Aspergillus Species to Amphotericin B,Itraconazole and Voriconazole

Abstract

In response to the recent increase in Aspergillus infections, new antifungal agents have become available accompanied by studies on antifungal susceptibility tests for epidemiological follow-up. The aim of this study was to compare the efficacy of Clinical Laboratory Standards Institute (CLSI) M 38-A broth microdilution test with the disk diffusion and E-test in determining the susceptibility of Aspergillus spp. to amphotericin B, itraconazole and voriconazole.

The study was carried out on 18 A. fumigatus, 7 A. flavus, 5 A. niger and 2 A. versicolor strains isolated from clinical samples. The microdilution method was performed by following the instructions of CLSI M 38-A. The E-test and disk diffusion tests were performed according to the instructions of their manufacturers.

The percent agreement between the E-test and CLSI M38-A broth microdilution test at 24 (48) h within ± 2 dilutions was, respectively, 81% (69%) for amphotericin B, 75% (72%) for itraconazole and 85% (81%) for voriconazole. The disk diffusion test showed good correlation with the E-test but poor correlation with the broth microdilution test for the three antifungal agents we tested.

In conclusion, E-test and disk diffusion test have their advantages such as ease of application and interpretation, but their correlation with the broth microdilution should be improved.     

Antifungal activity of amphotericin B,caspofungin and posaconazole on Candida albicans biofilms in intermediate and mature development phases

The aim of this study was to examine the antifungal activity of amphotericin B, caspofungin and posaconazole on Candida albicans biofilms in the intermediate and mature development phases. Candida albicans biofilms, previously grown for either 24, 48 or 72 h in 96‐well microtitre plates, were treated for 48 h with amphotericin B, caspofungin or posaconazole in increasing concentrations according to the respective minimal inhibitory concentration (MIC) determined for planktonic cells (1–128 × MIC). The biofilms were quantified using the mean optical density (OD) determined by XTT assay. Antifungal activities were expressed as percentage of reduction in OD of drug‐treated biofilms compared to untreated biofilms. To test the fungicidal activity of antifungal agents, the unfixed biofilms were scraped off and seeded to Sabouraud agar. Caspofungin and amphotericin B showed higher activity against C. albicans biofilm grown for 24 h and 72 h (≥50% reduction of OD) than biofilms grown for 48 h, whereas posaconazole showed similar, but reduced activity against all phases of C. albicans biofilm (≤50% reduction of OD). Caspofungin at 1–4 × MIC achieved the greatest decrease in the biofilm OD grown for 24, 48 and 72 h, whereas amphotericin B showed dose‐dependent activity. However, all tested antifungals failed to reach fungicidal activity in all biofilm development phases.     

In Vitro Effects of Ampicillin,Ciprofloxacin and Ofloxacin on Salmonella typhi within Human Monocyte-Derived Macrophages

Abstract

The incidence of invasive fungal infections (IFIs) caused by both common and uncommon opportunistic fungi is increasing along with emerging fungal resistance. Since traditional agents (amphotericin B, fluconazole, itraconazole) are limited by an inade-quate spectrum of activity, drug resistance or toxicity, there is a great interest in the development of new antifungal agents for treatment of IFIs in high-risk populations. In recent years a number of systemic antifungal drugs have become available and options for treatment of IFIs have expanded. A new generation of triazole agents (voriconazole, posaconazole, isavuconazole, ravuconazole and albaconazole), with a broad spectrum of activity and sufficient improvements in potency to overcome resistance have emerged and represent an alternative to conventional antifungals for the prevention and treatment of IFIs. This article focuses on the microbiology, pharmacology, clinical efficacy and safety of the new antifungal triazole generation.     

In vitro interactions between amphotericin B and other antifungal agents and rifampin against Fusarium spp.

Fusarium species are common hyaline soil saprophytes and plant pathogens that are opportunistic fungal pathogens of immunocompromised patients. The treatment for fusariosis remains uncertain with an unfavourable prognosis; new possibilities for treatment, such as various synergistic drug interactions, must be uncovered. In this study, we evaluated the in vitro interactions of amphotericin B with caspofungin, ketoconazole, 5‐flucytosine, itraconazole, miconazole, rifampin, fluconazole, terbinafine and voriconazole against isolates of Fusarium spp. using the chequerboard method with interactions evaluated by fractional inhibitory concentration indices. The highest percentages of synergistic interactions were observed for the combinations of amphotericin B and caspofungin (68.7%), amphotericin B and rifampin (68.7%), amphotericin B plus 5‐flucytosine (59.3%) and amphotericin B with voriconazole (37.5%). The pattern of susceptibility to antifungal agents among Fusarium species and their consequence on the effects of drug combinations are also discussed.     

In Vitro Evaluation of the Antimicrobial Activity of Meropenem in Combination with Polymyxin B and Gatifloxacin Against Pseudomonas aeruginosa and Acinetobacter baumannii

Abstract

The antimicrobial activity of meropenem combined with either polymyxin B or gatifloxacin was evaluated by the checkerboard method against Pseudomonas aeruginosa (10 strains) and Acinetobacter baumannii (10 strains). In addition, the triple combination of polymyxin B, gatifloxacin, and meropenem was also studied as well as the polymyxin B and gatifloxacin combination. A partial synergism interaction between meropenem and polymyxin B was observed for 80% of the A. baumannii strains. In contrast, this combination showed an indifferent effect for 80% of the P. aeruginosa strains tested. The combination of meropenem and gatifloxacin showed synergism only for two strains of A. baumannii, and partial synergism and additive effect for seven strains and indifference for four strains of both species. For the strains of P. aeruginosa, the double combination of polymyxin B and gatifloxacin and the triple combination of meropenem, polymyxin B and gatifloxacin were indifferent for the majority of the strains tested, that is, 90 and 80% respectively.     

In vitro antifungal activity of amphotericin B and 11 comparators against Aspergillus terreus species complex

Aspergillus terreus infections are difficult to treat because of the intrinsic resistance to amphotericin B, and higher mortality compared to infections caused by other Aspergillus species. The aim of the present study was to determine the in vitro antifungal activity of amphotericin B and 11 comparators against clinical (n = 36) and environmental (n = 45) A. terreus isolates. In vitro antifungal susceptibility was performed using the CLSI M38‐A2 procedure. Amphotericin B exhibited the highest MICs (MIC range, 0.125‐4 μg/mL; MIC₉₀, 2 μg/mL), followed by terbinafine (MIC range, 0.002‐1 μg/mL; MIC₉₀, 1 μg/mL). Only one isolate (1/81) showed amphotericin B MIC above the epidemiologic cut‐off value (ECV; 4 μg/mL). None of the isolates had a MIC of ≥ ECV for voriconazole, itraconazole and posaconazole. The reasons for the difference in amphotericin B susceptibility patterns between studies remain unknown. The genetic and species diversity, clinical, environmental and ecological factors in Terrei section on various amphotericin B susceptibility profiles in different countries should be considered more as the main reasons associated with these differences.     

In vitro combination between antifungals and diphenyl diselenide against Cryptococcus species

Cryptococcus species are an encapsulated fungal pathogen that cause cryptococcal meningitis. There are limited therapeutic options for this infection. The management includes the use of different antifungals such as amphotericin B, flucytosine, or fluconazole, either alone or in combination. However, numerous therapeutic failures, as well as the limited effectiveness of such therapeutics, have been described. Diphenyl diselenide is a chemically synthesised molecule with was found to have antimicrobial activity. In this study, we evaluated the antifungal activities of fluconazole, amphotericin B and flucytosine, in combination with diphenyl diselenide against 30 clinical isolates of Cryptococcus spp. using CLSI M27‐A3 method and the checkerboard microdilution technique. Our results show that the combination of flucytosine and diphenyl diselenide displayed 100% of synergism. However, when we analysed (PhSe)₂ plus AMB or FLZ we observed around 70% of indifference. Our results suggest that the combination of diphenyl diselenide with other antifungal agents deserves attention as a new option for the development of alternative therapies for cryptococcosis.     

In vitro antifungal susceptibility of clinical isolates of Arthrographis kalrae,a poorly known opportunistic fungus

The in vitro antifungal activity of amphotericin B (AMB), itraconazole, voriconazole, posaconazole, terbinafine (TRB), caspofungin, anidulafungin and micafungin were evaluated by a broth microdilution technique against 22 isolates of Arthrographis kalrae of clinical origin. TRB showed the highest activity, followed by the azoles, particularly posaconazole. AMB exerted low activity whereas the echinocandins showed almost no antifungal activity.     

Emergence of Resistance to Amphotericin B and Triazoles in Candida glabrata Vaginal Isolates in a Case of Recurrent Vaginitis

Abstract

Emergence of resistance to triazoles and amphotericin B in Candida glabrata vaginal isolates is documented by Etest. During the 18-month follow-up of a case of vaginitis, 14 consecutive isolates of C. glabrata were examined. The isolates exhibited development of in vitro resistance beginning with itraconazole (>32 μg/ml), followed by fluconazole (>256 μg/ml), amphotericin B (>32 μg/ml), and voriconazole (>32 μg/ml). The DNA sequence analyses and finger printing of the isolates strongly suggest that our patient remained colonized with a single strain. The report underscores the propensity of C. glabrata to acquire resistance during antifungal therapy and the importance of susceptibility testing in the management of infections caused by this species.     

Therapy for fungal infections in leukemia

Invasive fungal infections remain a common cause of morbidity and mortality among patients with leukemia who become further compromised by neutropenia. Candida and Aspergillus spp account for the vast majority of these infections, but other, less commonly recognized fungi can cause life-threatening infection in these hosts as well. The earlier, more limited antifungal armamentarium of ketoconazole, flucytosine, and amphotericin B has been substantially augmented by the availability of fluconazole, itraconazole, and the lipid-associated amphotericin formulations. Intense clinical study has focused on the use of these agents in empiric treatment, treatment of suspected or proven infection, and prophylaxis. Recognition of the limitations of antifungal therapy in the neutropenic host has led to evaluation of the adjunctive role of immunotherapy.     

In-Vitro Activities of Terbinafine,Itraconazole and Amphotericin B Against Aspergillus and Cladosporium species

Abstract

The in vitro fungicidal and fungistatic activities of terbinafine were compared with those of itraconazole and amphotericin B against Aspergillus (n=63) and Cladosporium (n=21) isolates. The broth macrodilution modification of NCCLS reference method for filamentous fungi (M38-P) was used to assess the minimum inhibitory concentrations (MICs). Our data show that the in vitro activities of terbinafine were comparable to those of itraconazole and amphotericin B against the Aspergillus spp and Cladosporium strains tested. Despite strain-dependent variabilities, in general, itraconazole's activity was similar to that of amphotericin B against strains of Aspergillus and Cladosporium. Our data suggest that terbinafine may be useful in the treatment of Aspergillus and Cladosporium infections.     

In Vitro Activity of Ampicillin-Sulbactam Against Clinical Multiresistant Acinetobacter baumannii Isolates

Abstract

We evaluated the in vitroactivity of ampicillin-sul-bactam in comparison with that of broad-spectrum antimicrobial agents against Acinetobacter baumannii isolates. Two hundred and twelve clinical isolates collected between January 1993 and March 1995 from two tertiary hospitals located in São Paulo, Brazil were tested for susceptibility by the disk diffusion method against several broad-spectrum antimicrobial agents, including imipenem, ciprofloxacin, ceftazidime, aztreonam, amikacin, and polymyxin B. All strains were susceptible to polymyxin B. The second most active compound was the combination ampicillin-sulbactam (88% susceptibility). Only 79% of the isolates were susceptible to imipenem. Ciprofloxacin was active against 60 (28%) and amikacin against 34 (16%) isolates. Ceftazidime was the most active cephalosporin; however, only 9% of the isolates were susceptible to this compound. Both aztreonam and ampicillin alone showed very poor activity against this species (1% susceptibility). The prevalence of severe infections due to A. baumannii is increasing very rapidly in the tertiary hospitals of São Paulo and there are very few options for the treatment of these infections. Polymyxin B is invariably in vitro active against this species; however, this compound can cause severe side effects and is not commercially available for intravenous use in Brazil and in several other countries. Our results indicated that the combination ampicillin-sulbactam may be an alternative drug for the treatment of infections due to multiresistant A. baumannii; however, further studies are necessary to evaluate the clinical role of this compound for the treatment of severe infections.     

Pulmonary Aspergillosis with Possible Cerebral Involvement in a Previously Healthy Pregnant Woman

Abstract

Invasive aspergillosis is observed mainly in immunodepressed patients. Here we report a case of pulmonary aspergillosis with CNS involvement in a pregnant woman without other known causes of immunodeficiency.

Case report: A 23-years old pregnant woman underwent a caesarean because of unexplained seizures. During the subsequent days worsening headache and a dete-riorating neurological status were reported suggesting meningitis. Stiffness, right sided hemiparesis and cranial nerve palsies were observed at admission. Radiological findings revealed lesions involving the right pulmonary apex, the right cerebellar hemisphere and the Pterygopalatina fossa. Microbiological studies revealed large colonies of Aspergillus fumigatus. A favorable outcome was observed after administration of liposomal amphotericin B and 5-fluorocytosine and, at improved conditions, when oral uptake of itraconazole was given.

The authors conclude that risk of infections sustained by fungal opportunistic agents during pregnancy must be considered. Sequential antifungal administration may be an efficient therapy able to shorten hospitalization.     

Influence of New Quinolones on Normal Immune Capabilities

Summary

Ciprofloxacin, pefloxacin, norfloxacin, and ofloxacin were investigated for immunomodulatory activity on humoral and cell-mediated immune responses. Ciprofloxacin and pefloxacin altered the humoral immune responses of mice to sheep red blood cells. This effect was not exhibited by norfloxacin or ofloxacin. All four quinolones did not alter cell-mediated responses. When these antimicrobial agents were tested for their interaction with human polymorphonuclear phagocytic activity, all agents suppressed this activity. In addition, all except norfloxacin showed anti-inflammatory activity.     

Persistence of Candida albicans Candidemia in Non-Neutropenic Surgical Patients: Management of a Representative Patient in the Absence of Second-Line Treatment Guidelines

Abstract

Primary treatment failure and mortality in non-neutropenic patients with candidemia is high according to clinical trial experience. Current guidelines are mainly useful only for first line treatment strategies.

We describe treatment failure and persistent protracted Candida albicans candidemia without clinically evident ocular involvement nor catheter recolonization in a malnourished non-neutropenic surgical patient with peritonitis. Primary antifungal treatment failure with fluconazole and secondary treatment failure with caspofungin occurred in the absence of evident Candida seeding the eye, valvular endocardium, or the intravascular catheter. Switch to liposomal amphotericin B was followed by clinical and microbiological cure.

In patients with multiple risk factors for the acquisition of candidemia and life-threatening clinical conditions, the possibility of primary/secondary failure of new potent antifungal regimens may be initially neglected.

Additional multicenter controlled clinical data are needed to guide the timing and choice of secondary antifungal treatment regimens in non-neutropenic candidemia patients.     

In vitro susceptibility of chromoblastomycosis agents to five antifungal drugs and to the combination of terbinafine and amphotericin B

Chromoblastomycosis is a chronic mycosis that affects the skin and subcutaneous tissues caused by several genera of dematiaceous fungi. There is not a treatment of choice. Thus, tools that help guide clinical practice are fundamental. In this sense, antifungal activity tests in vitro could be useful. However, trials with chromoblastomycosis agents are scarce. The aim of this study was to evaluate both the in vitro susceptibility of 60 chromoblastomycosis agents to five antifungals and the combination of amphotericin B (AMB) and terbinafine (TRB). TRB, itraconazole (ITZ) and ketoconazole (KTZ) were, in this order, the drugs which showed better activity against the chromoblastomycosis agents. The less active drugs were voriconazole (VRZ) and AMB. The more differentiated group was Exophiala spinifera. Cladophialophora carrionii and Fonsecaea spp. are significantly more susceptible to KTZ than Phialophora verrucosa, whereas C. carrionii is significantly more sensitive to VRZ than P. verrucosa and E. spinifera. Assays in this direction allow the knowledge of the susceptibility of the causative agents which may help the management of patients with this disease. This study includes the largest number of these agents and of genera found in the literature.     

Identification and characterisation of human pathogenic filamentous fungi and susceptibility to Thymus schimperi essential oil

Twenty‐eight clinical fungal isolates were characterised by morphological (macro‐ and micro‐features and growth response at 25, 30 and 37 °C) and molecular (nuclear rDNA‐internal transcriber spacer, calmodulin, cytochrome c oxidase 1 and the largest subunit of RNA polymerase II) analyses. The clinical fungal isolates were ascribed to the following taxa: Penicillium chrysogenum, Verticillium sp., Aspergillus tubingensis, Aspergillus minutus, Beauveria bassiana and Microsporum gypseum. In addition, in vitro susceptibility testing of the isolates to conventional antifungal agents and to two chemically well‐defined chemotypes of Thymus schimperi essential oil was performed. Most of the isolates were resistant to amphotericin B (except A. minutus), and itraconazole, while terbinafine was quite active on these fungi. T. schimperi essential oil showed antifungal activity against all of the tested fungal isolates with minimal inhibitory concentration values similar or lower than those of terbinafine. Transmission electron microscopy analyses revealed that fungal growth inhibition by essential oil was accompanied by marked morphological and cytological changes.     

In Vitro Testing of Aspergillus fumigatus Clinical Isolates for Susceptibility to Voriconazole,Amphotericin B and Itraconazole: Comparison of Sensititre versus NCCLS M38-A Using Two Different Inocula

Abstract

Voriconazole, amphotericin B and itraconazole were tested In Vitro against 18 strains of Aspergillus fumigatus isolated from cystic fibrosis patients. Susceptibility was tested with the broth microdilution method (M38-A protocol- NCCLS). Results of this reference method were compared with those of an experimental commercial microdilution broth method (Sensititre). Two different inocula, prepared from 2- and 7-day cultures, were used. Minimum inhibitory concentrations (MICs) of the reference method ranged from 0.25 to 2 μg/ml for voriconazole, 0.06 to 1 μg/ml for amphotericin B, 0.016 to >16 μg/ml for itraconazole. There were no significant differences in the MIC ranges or MIC₉₀ values obtained with the two testing methods or with the two types of inocula. These findings confirm the good In Vitro activity of voriconazole, itraconazole and amphotericin B against A. fumigatus. They also indicate that reliable susceptibility data can be generated more rapidly by commercial systems and use of 2-day cultures for inoculum preparation.     

In vitro activity of colistin as single agent and in combination with antifungals against filamentous fungi occurring in patients with cystic fibrosis

Because published reports indicate that the antibiotic colistin (COL) has antifungal properties, this study investigated the antifungal in vitro activity of COL as single agent and in combination with the antifungal compounds voriconazole (VRC), caspofungin (CAS) and amphotericin B (AMB) against Scedosporium/Pseudallescheria spp., Exophiala dermatitidis and Geosmithia argillacea. In total, susceptibility was determined for 77 Scedosporium/Pseudallescheria spp., 82 E. dermatitidis and 17 G. argillacea isolates. The minimal inhibitory concentrations (MICs) of COL and the antifungals as single compound and in combination were determined with MIC test strips. Drug interactions were detected by crossing the MIC test strips at a 90º angle. The fractional inhibitory concentration index was used to categorise the drugs’ interaction. The MIC₅₀ value of COL was 12 μg ml^?1 for S. prolificans, 16 μg ml^?1 for P. apiosperma, 16 μg ml^?1 for P. boydii, 12 μg ml^?1 for E. dermatiditis and 6 μg ml^?1 for G. argillacea. VRC was the most active drug in combination without any antagonism with the exception of few P. boydii isolates. COL as single agent and in most combinations with antifungals exhibits in vitro antifungal activity against filamentous ascomycetes occurring in cystic fibrosis patients and may offer a novel therapeutic option, especially for multidrug‐resistant S. prolificans.