Fluconazole,Itraconazole and Ketoconazole In- Vitro Activity. A Comparative Study

Summary

We compared in-vitro activity of fluconazole, itraconazole and ketoconazole by evaluating their Minimal Inhibitory Concentrations (MICs) for 100 fungal strains isolated from different biological specimens of ARC/AIDS patients. A semisolid agar medium was used: this method is suitable for testing molds and yeasts, and can be applied to all azole antifungal drugs. Fluconazole had higher MICs than two other tested drugs, especially for Candida krusei strains; however it never had a MIC higher than 40mg/l.

Itraconazole and ketoconazole had MICs higher than 40mg/l for one Cryptococcus neoformans strain. There were no significant differences for itraconazole and ketoconazole among the tested strains.     

Mycological-diagnostic assessment of the efficacy of amphotericin B + flucytosine to control Cryptococcus neoformans in AIDS patients

Abstract: Optimal diagnostic data (microscopy, culture and serology) on 15 cases of Cr. neoformans infection in AIDS patients served as a basis for the preliminary subdivision of the stages of cryptococcosis: Primary (minor involvement of the lungs only) and secondary (hematogenous dissemination with involvement of various organs) stages. Cr. neoformans counts in body fluids and antigen titres in serum and CSF proved to be useful criteria to assess the stage of infection. The efficacy of the combination therapy with amphotericin B + flucytosine seems to be related to the stage of infection. From the mycological point of view, the standard combination of 0.3–0.5 mg/kg BW/d amphotericin B and 150 mg/kg BW/d flucytosine proved to be effective. Data on sensitivity of the agent to amphotericin B and flucytosine are presented in a summarized form. The subjects of duration of therapy, relapse and resistance to flucytosine are commented by means of examples. It should be the aim of an optimal therapy of cryptococcosis to diagnose this mycotic disease in its primary stage. Zusammenfassung: Anhand von 15 Fällen wird versucht, die Cr. neoformans-Infektion AIDS-Kranker in das Primärstadium (alleiniger und geringer Befall der Lunge) und das Sekundärstadium (hämatogene Dissemination mit Befall verschiedener Organe) einzuteilen. Die Keimzahl von Cr. neoformans in Körperflüssigkeiten und der Cr. neoformans-Antigentiter in Serum und Liquor dienten hierbei als die beiden wesentlichen Kriterien zur Einschätzung des Stadiums der Infektion und seiner Therapierbarkeit. Aus mykologischer Sicht erwies sich die Standardkombination von Amphotericin B (0,3–0,5 mg/kg KG täglich) + Flucytosin (150 mg/kg KG täglich) als wirksam. Anhand von Beispielen wird zu den Themen: Dauer der Therapie, Rezidiv und Flucytosin-Resistenz Stellung genommen. über die Empfindlichkeit der Cr. neoformans-Stämme gegen Amphotericin B und Flucytosin (Ancotil ) wird zusammenfassend berichtet. Voraussetzung für eine optimale Therapierbarkeit der Kryptokokkose scheint die Diagnostik dieser Mykose in ihrem Primärstadium zu sein.     

Susceptibility profile and epidemiological cut‐off values of Cryptococcus neoformans species complex from Argentina

Epidemiological cut‐off values (ECVs) based on minimal inhibitory concentration (MIC) distribution have been recently proposed for some antifungal drug/Cryptococcus neoformans combinations. However, these ECVs vary according to the species studied, being serotypes and the geographical origin of strains, variables to be considered. The aims were to define the wild‐type (WT) population of the C. neoformans species complex (C. neoformans) isolated from patients living in Argentina, and to propose ECVs for six antifungal drugs. A total of 707 unique C. neoformans isolates obtained from HIV patients suffering cryptococcal meningitis were studied. The MIC of amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and posaconazole was determined according to the EDef 7.2 (EUCAST) reference document. The MIC distribution, MIC₅₀, MIC₉₀ and ECV for each of these drugs were calculated. The highest ECV, which included ≥95% of the WT population modelled, was observed for flucytosine and fluconazole (32 μg ml^?1 each). For amphotericin B, itraconazole, voriconazole and posaconazole, the ECVs were: 0.5, 0.5, 0.5 and 0.06 μg ml^?1 respectively. The ECVs determined in this study may aid in identifying the C. neoformans strains circulating in Argentina with decreased susceptibility to the antifungal drugs tested.     

The Use of Intravenous Methotrexate in the Treatment of Ectopic Pregnancy

Abstract

Cryptococcosis is a disseminated fungal disease typically associated with immuno-suppression and characterized by high mortality rates. Cryptococcus neoformans has been reported to be isolated from blood cultures in around 20% of patients with cryptococcosis, and cryptococcemia has been correlated with poor prognosis.

We report a case of fatal C. neoformans fungemia in a neutropenic patient with a history of chronic lymphocytic leukemia treated with alemtuzumab. The patient presented with loss of consciousness and died after 5 days of antifungal therapy with liposomal amphotericin B. The international literature regarding opportunistic infections after immunosuppressive therapy with alemtuzumab with particular attention on fun-gal infections has also been reviewed.     

Activity of Antimicrobial Drugs Evaluated by Agar Dilution and Radiometric Methods against Strains of Nocardia asteroides Isolated in Italy from Immunocompromised Patients

Summary

Benzylpenicillin, amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cephaloridine, cefotaxime, imipenem, erythromycin, clarithromycin, azithromycin, amikacin, ciprofloxacin and trimethoprim-sulfamethoxazole were tested in vitro by the agar dilution method against eleven strains of Nocardia asteroides isolated both from AIDS and other immunocompromised patients. Imipenem, amikacin and trimethoprim-sulfamethoxazole were shown to be the most active drugs with minimum inhibitory concentrations (MIC) values nearly always lower than concentrations achievable in blood. Ciprofloxacin, cephaloridine and cefotaxime were moderately active, while the remaining drugs were totally ineffective.

When susceptibility was assessed by the radiometric method the MIC₉₀ values were uniformly lower than those in the agar method, possibly due to lower inactivation of the drugs during incubation. The two methods showed a good correlation only for imipenem, amikacin and ciprofloxacin. The results obtained by the radiometric method seem to indicate that, as for mycobacteria, this method may also give a more accurate evaluation of the antimicrobial susceptibility of Nocardiae.     

Determination of the Susceptibility In Vitro of 54 Isolates of Mycobacterium fortuitum against Three Fluoroquinolones Using Two Methods

Abstract

The In Vitro susceptibility to ofloxacin, norfloxacin and ciprofloxacin or 54 Mycobacterium fortuitum isolates originating from clinical samples (7) of patients attending the Hospital Universitario de Canarias and Hospital del Tórax, and from environmental (47) sources, were determined. For this, two methods were used: dilution in agar with Middlebrook 7H10 Agar as a base medium culture, and broth microdilution, with Mueller-Hinton Broth without supplement.

The different isolates under study revealed a uniform susceptibility by both methods against ciprofloxacin.

100% inhibition was obtained from a Minimum Inhibitory Concentration (MIC) of 0.25 μg/ml, and 2 μg/ml of ciprofloxacin, for broth microdilution and dilution in agar, respectively.

For ofloxacin and norfloxacin, all the isolates were inhibited at an MIC of 0.5 μ/ml, by the broth microdilution method, which contrasted sharply with an MIC of 32 μ/ml, in the case of dilution in agar.

In this study, we have observed the existence of differences in the In Vitro susceptibility of the isolates of M. fortuitum against the three fluoroquinolones assayed, mainly for ofloxacin and norfloxacin, by both methods. We, therefore, consider it necessary to establish a standardized, reproducible assay method, for the study of sensitivity to atypical mycobacteria.     

Molecular epidemiology and in vitro antifungal susceptibility testing of 108 clinical Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato isolates from Denmark

Cryptococcosis is mainly caused by members of the Cryptococcus gattii/Cryptococcus neoformans species complexes. Here, we report the molecular characterisation and in vitro antifungal susceptibility of Danish clinical cryptococcal isolates. Species, genotype, serotype and mating type were determined by amplified fragment length polymorphism (AFLP) fingerprinting and qPCR. EUCAST E.Def 7.2 MICs were determined for amphotericin B, flucytosine, fluconazole, voriconazole and isavuconazole. Most isolates were C. neoformans (serotype A; n = 66) and belonged to genotype AFLP1/VNI (n = 61) or AFLP1B/VNII (n = 5) followed by Cryptococcus deneoformans (serotype D; genotype AFLP2, n = 20), C. neoformans × C. deneoformans hybrids (serotype AD; genotype AFLP3, n = 13) and Cryptococcus curvatus (n = 2). Six isolates were C. gattii sensu lato, and one isolate was a C. deneoformans × C. gattii hybrid (genotype AFLP8). All isolates were amphotericin B susceptible. Flucytosine susceptibility was uniform MIC₅₀ of 4–8 mg l^?1 except for C. curvatus (MICs >32 mg l^?1). Cryptococcus gattii sensu lato isolates were somewhat less susceptible to the azoles. MICs of fluconazole (>32 mg l^?1), voriconazole (≥0.5 mg l^?1) and isavuconazole (0.06 and 0.25 mg l^?1 respectively) were elevated compared to the wild‐type population for 1/19 C. deneoformans and 1/2 C. curvatus isolates. Flucytosine MIC was elevated for 1/61 C. neoformans (>32 mg l^?1). Antifungal susceptibility revealed species‐specific differential susceptibility, but suggested acquired resistance was an infrequent phenomenon.     

Evaluation of semisolid agar screening tests for determining fluconazole and amphotericin B susceptibilities of Candida strains by using three different media

The susceptibilities of 164 Candida isolates against fluconazole and amphotericin B were determined by semisolid agar screening tests and the microdilution method according to NCCLS M27-A standards. The semisolid agar screening tests were performed with three different media containing 0.5% agar and 2, 8, and 40 microg/ml of fluconazole or 0.5 and 2.0 microg/ml of amphotericin B. These media were MOPS buffered RPMI 1640, brain-heart infusion and 1/3 diluted Sabouraud dextrose agar. The results of both methods were interpreted as susceptible, dose dependent susceptible and resistant for fluconazole and susceptible and resistant for amphotericin B. The agreement rates of semisolid agar screening tests using RPMI 1640, brain-heart infusion and Sabouraud dextrose media with the reference microdilution method were found to be 71.4%, 51.2%, and 57.3% for fluconazole and 79.3%, 53.7%, and 56.7% for amphotericin B, respectively. Overall, we conclude that semisolid agar screening tests using RPMI 1640 can be used for determining the susceptibilities of Candida isolates against fluconazole and amphotericin B in clinical microbiology laboratories.     

Skull,Patella and Thigh Cryptococcosis after a Crashing Injury of the Temporal Bone

Summary

An unusual case of triple location (skull, patella and thigh) Cryptococcus neoformans is described. The peculiarity of the case is based on the probability of direct post-traumatic bone inoculation, subsequent seeding from skull to patella, thigh and cerebral spinal fluid (CSF), patella involvement, lack of evidence of lung involvement and lack of predisposing underlying disease. The response to surgery and a combination of amphotericin B and 5-flucytosine administration was favorable without relapse at follow-up after 7-year discontinuation of therapy.     

In- Vitro Activity of Commonly Used Antifungal Agents in the Presence of Rifampin,Polymyxin B and Norfloxacin against Candida albicans

Abstract

This study assessed the in-vitro antifungal activity against Candida albicans of amphotericin B, ketocona-zole and miconazole, each in the presence of rifampin, polymyxin B and norfloxacin. Evaluation of drug interactions was estimated by the checkerboard pattern broth dilution method and by time-kill studies. Rifampin reduced the activity of the three antifungal agents used, with the reduction being more pronounced with amphotericin B. Synergy was observed when polymyxin B was combined with any of the antifungal agents used. The addition of norfloxacin resulted in minimal, if any, change in the activity.     

Flucytosine and cryptococcosis: which in vitro test is the best predictor of outcome?

The combination of flucytosine and amphotericin B is first choice treatment for active cryptococcosis. Because of innate or acquired resistance of Cryptococcus neoformans to flucytosine, in vitro testing is mandatory. Yeast nitrogen base (YNB) at pH 7.0 is the recommended medium for the broth microdilution test (NCCLS M27-A) and for the E-test. In order to verify if minimum inhibitory concentrations (MICs) were able to predict treatment outcome, the susceptibility of 24 isolates from 21 patients treated with flucytosine alone or in combination was tested by the broth microdilution, agar dilution and E-test using YNB either at pH 7.0 or at pH 5.4. Only those MICs obtained on YNB pH 5.4 proved to correlate with treatment outcome. The present study suggests that in vitro susceptibility to flucytosine of C. neoformans isolates should be evaluated on YNB pH 5.4 and the test should be standardized accordingly.     

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.     

Effects of Fluconazole Singly and in Combination with 5-Fluorocytosine or Amphotericin B in the Treatment of Cryptococcal Meningoencephalitis in an Intracranial Murine Model

Abstract

In this study we developed a highly reproducible intracranial murine model of cryptococcosis. Mice (Balb/c, 5-7 weeks old) were challenged intracranially and treated with intermediate (30 mg/kg) or high (90 mg/kg) dose fluconazole, and amphotericin B (0.75 mg/kg), administered singly or in combination with flucytosine (100 mg/kg). Survival and brain CFU analyses were performed. Effect of fluconazole prophylaxis was also determined. Our data show that the developed model mimics clinical signs of cryptococcal meningitis. In single treatment, fluconazole (30 mg/kg) was more efficacious than amphotericin B or flucytosine (P <0.0001). Combination treatment led to significantly increased anticryptococcal activity, which was highest for high dose fluconazole + flucytosine (P <0.0001). However, no significant difference was observed between high dose fluconazole treatment with and without flucytosine (P >0.05). Fluconazole prophylaxis led to a significant decrease in brain CFU. In conclusion, high dose fluconazole administered post-infection, or as prophylaxis, may be highly efficacious in the treatment and prevention of meningoencephalitis.     

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.     

Molecular typing of environmental Cryptococcus neoformans/C. gattii species complex isolates from Manaus,Amazonas, Brazil

Cryptococcus neoformans and Cryptococcus gattii are the main causative agents of cryptococcosis, a systemic fungal disease that affects internal organs and skin, and which is acquired by inhalation of spores or encapsulated yeasts. It is currently known that the C. neoformans/C. gattii species complex has a worldwide distribution, however, some molecular types seem to prevail in certain regions. Few environmental studies of Cryptococcus have been conducted in the Brazilian Amazon. This is the first ecological study of the pathogenic fungi C. neoformans/C. gattii species complex in the urban area of Manaus, Amazonas, Brazil. A total of 506 samples from pigeon droppings (n = 191), captive bird droppings (n = 60) and tree hollows (n = 255) were collected from June 2012 to January 2014 at schools and public buildings, squares, pet shops, households, the zoo and the bus station. Samples were plated on niger seed agar (NSA) medium supplemented with chloramphenicol and incubated at 25°C for 5 days. Dark‐brown colonies were isolated and tested for thermotolerance at 37°C, cycloheximide resistance and growth on canavanine‐glycine‐bromothymol blue agar. Molecular typing was done by PCR‐RFLP. Susceptibility to the antifungal drugs amphotericin B, fluconazole, itraconazole and ketoconazole was tested using Etest^® strips. In total, 13 positive samples were obtained: one tree hollow (C. gattiiVGII), nine pigeon droppings (C. neoformansVNI) and three captive bird droppings (C. neoformansVNI). The environmental cryptococcal isolates found in this study were of the same molecular types as those responsible for infections in Manaus.     

In- Vitro Comparative Activity of Fluconazole and Other Antifungal Agents Against Blastoschizomyces capitatus

Summary

Blastoschizomyces caphatus represents an emerging fungal pathogen in acute leukemia patients. The susceptibility to amphotericin B, 5-fluorocytosine, ketoconazole and fluconazole of nine clinical isolates was evaluated. A specific medium (high resolution medium) was used for testing fluconazole. This agent and 5-fluorocytosine were at least four- to eightfold more active than amphotericin B and ketoconazole against all isolates but one.     

Antifungal susceptibility testing of Malassezia yeast: comparison of two different methodologies

All Malassezia species are lipophilic; thus, modifications are required in susceptibility testing methods to ensure their growth. Antifungal susceptibility of Malassezia species using agar and broth dilution methods has been studied. Currently, few tests using disc diffusion methods are being performed. The aim was to evaluate the in vitro susceptibility of Malassezia yeast against antifungal agents using broth microdilution and disc diffusion methods, then to compare both methodologies. Fifty Malassezia isolates were studied. Microdilution method was performed as described in reference document and agar diffusion test was performed using antifungal tablets and discs. To support growth, culture media were supplemented. To correlate methods, linear regression analysis and categorical agreement was determined. The strongest linear association was observed for fluconazole and miconazole. The highest agreement between both methods was observed for itraconazole and voriconazole and the lowest for amphotericin B and fluconazole. Although modifications made to disc diffusion method allowed to obtain susceptibility data for Malassezia yeast, variables cannot be associated through a linear correlation model, indicating that inhibition zone values cannot predict MIC value. According to the results, disc diffusion assay may not represent an alternative to determine antifungal susceptibility of Malassezia yeast.     

In Vitro Susceptibility of Trichosporon beigelii to Antifungal Agents

Abstract

The minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of amphotericin B, flucytosine, miconazole, fluconazole and itraconazole against 21 isolates of Trichosporon beigelii in RPMI-1640 medium were determined using National Committee for Clinical Laboratory Standards (NCCLS) methodology in microdilution method. Most isolates were sensitive to miconazole (MIC₉₀ 0.78 μ/ml), fluconazole (MIC₉₀ 6.25 μ/ml), and itraconazole (MIC₉₀ 0.19 μ/ml), with the former being the most active agent tested (MFC₉₀ 3.12 μ/ml). Although amphotericin B inhibited most strains (MIC range, 0.78-3.12 μ/ml), poor fungicidal activity was observed (MFC range, 1.56 -12.5 μ/ml) showing a pattern of relative resistance In Vitro. Flucytosine showed generally poor activity against most isolates tested. These In Vitro findings confirm the resistance of T.beigelii to amphotericin B and suggest that azoles may be an alternative to the former for the treatment of disseminated trichosporonosis. However, in vivo studies would better validate these In Vitro findings.     

Two new media Pinus halepensis seed agar and blackberry agar for rapid identification of Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast‐like fungus that causes life‐threatening infections, particularly in immunocompromised patients. The formation of brown pigment on many media described in the literature, such as that in Niger seed (Guizotia abyssinica) agar, has been used to identify C. neoformans. The present study compares melanin production by clinical and environmental isolates of C. neoformans and other medically important yeast on two new media, Pinus halepensis seed (PHS) agar and blackberry (BlaB) agar, and the classic medium Niger seed agar. Results obtained after the culture of 46 strains of C. neoformans, for 4, 24 and 48 h at 37 °C on these three media, showed that at 24 h, 100% of strains were pigmented on BlaB agar, 91.3% on PHS agar but only 34.8% on Niger seed agar. In conclusion, PHS and BlaB agar are two interesting new media for the rapid identification of C. neoformans isolates.     

Fluconazole plus flucytosine is a good alternative therapy for non‐HIV and non‐transplant‐associated cryptococcal meningitis: A retrospective cohort study

Cryptococcal meningitis (CM) carries a high risk of mortality with increasing incidences in immune competent hosts. Current treatments are not well tolerated, and evaluation of other treatments is needed. Fluconazole and 5‐flucytosine in treating immune competent hosts have not been characterised. To evaluate the efficacy of fluconazole and 5‐flucytosine in treating non‐HIV‐ and non‐transplant‐associated CM. We performed a retrospective cohort study of the outcomes in immune competent patients with CM treated with fluconazole and 5‐flucytosine or deoxycholate‐amphotericin B and 5‐flucytosine. The primary outcome was treatment response evaluated at the 12th week after initiation of antifungal therapy. A total of 43 and 47 patients received amphotericin B deoxycholate and 5‐flucytosine or fluconazole and 5‐flucytosine, respectively. A total of 38 (88.4%) patients cannot tolerate recommended doses of amphotericin B deoxycholate and 5‐flucytosine (patients needed dose reduction during the treatment). Patients given fluconazole and 5‐flucytosine had higher baseline cryptococcal burdens (median 3632 versus 900 cryptococci/mL, P = 0.008). No significant differences were seen in cryptococcus clearance (74.4% vs 70.2%, P = 0.814), treatment time (39 days, 20‐69 days vs 21 days, 7‐63 days, P = 0.107) and successful response (including complete and partial responses) rates (69.7% vs 72.3%, P = 0.820). Fluconazole and 5‐flucytosine treatment had lower total adverse events (19.1% vs 90.7%, P < 0.001). Fluconazole and 5‐flucytosine had relatively high efficacy with few adverse events in treating CM. Fluconazole and 5‐flucytosine therapy is promising in patients that do not tolerate or are not suited for amphotericin B deoxycholate treatment.