scholarly journals Method for Measuring Postantifungal Effect in Aspergillus Species

2002 ◽  
Vol 46 (6) ◽  
pp. 1960-1965 ◽  
Author(s):  
Roxana G. Vitale ◽  
Johan W. Mouton ◽  
Javier Afeltra ◽  
Jacques F. G. M. Meis ◽  
Paul E. Verweij
Keyword(s):  
Amphotericin B ◽  
Growth Curve ◽  
Clinical Laboratory ◽  
Growth Curves ◽  
National Committee ◽  
Control Growth ◽  
And Control ◽  
Postantifungal Effect

ABSTRACT An in vitro method for determination of postantifungal effect (PAFE) in molds was developed by using three isolates each of Aspergillus fumigatus, A. flavus, A. terreus, A. nidulans, and A. ustus. MICs of amphotericin B and itraconazole were determined by using National Committee for Clinical Laboratory Standards guidelines (M38-P). The inoculum was prepared in RPMI 1640 broth buffered with MOPS (morpholinepropanesulfonic acid) at pH 7.0, and conidia were exposed to amphotericin B and itraconazole at concentrations of 4, 1, and 0.25 times the MIC, each for 4, 2, and 1 h at 37°C. The same procedure was followed for controls with drug-free medium. Following exposure, the conidia were washed three times in saline and the numbers of CFU per milliliter were determined. Exposed and control conidia were then inoculated into microtitration plates and incubated at 37°C for 48 h in a spectrophotometer reader. The optical density (OD) was measured automatically at 10-min intervals, resulting in growth curves. PAFE was quantified by comparing three arbitrary points in the control growth curve, the first increase of OD and the points when 20 and 50% of the maximal growth were reached, with the growth curve of drug-exposed conidia. Amphotericin B induced PAFE in A. fumigatus at four times the MIC after 2 and 4 h of exposure ranging from 1.83 to 6.00 h and 9.33 to 10.80 h, respectively. Significantly shorter PAFEs or lack of PAFE was observed for A. terreus, A. ustus, and A. nidulans. Itraconazole did not induce measurable PAFE in the Aspergillus isolates at any concentration or exposure time tested. Further studies are warranted to investigate the implications of PAFE in relation to clinical efficacy and dosing frequency.

scholarly journals Evaluation of a flow cytofluorometric method for rapid determination of amphotericin B susceptibility of yeast isolates.

1997 ◽  
Vol 41 (7) ◽  
pp. 1537-1540 ◽  
Author(s):  
F Peyron ◽  
A Favel ◽  
H Guiraud-Dauriac ◽  
M El Mzibri ◽  
C Chastin ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Fluorescence Intensity ◽  
Clinical Laboratory ◽  
Rapid Determination ◽  
Reference Method ◽  
National Committee ◽  
Candida Spp ◽  
Broth Macrodilution

A rapid-flow cytofluorometric susceptibility test for in vitro amphotericin B testing of yeasts was evaluated and compared to the National Committee for Clinical Laboratory Standards (NCCLS) M27-T reference broth macrodilution method. The flow cytofluorometric method is based on the detection of decreased green fluorescence intensity of cells stained with DiOC5(3), a membrane potential-sensitive cationic dye, after drug treatment. Testing was performed on 134 clinical isolates (Candida spp. and Torulopsis glabrata). From the dose-response curve obtained for each isolate, three endpoints were calculated by computer analysis (the concentrations at which the fluorescence intensity was reduced by 50, 80, and 90%, i.e., 50% inhibitory concentration [IC50], IC80, and IC90, respectively). A regression analysis correlating these endpoints with the M27-T MICs showed that the best agreement was obtained with IC80. The flow cytofluorometric method showed good reproducibility with control strains. These initial results suggest that the flow cytofluorometric method is a valid alternative to the NCCLS reference method.

Use of spectrophotometric reading for in vitro antifungal susceptibility testing ofAspergillusspp.

1999 ◽  
Vol 45 (10) ◽  
pp. 871-874 ◽  
Author(s):  
Eric Dannaoui ◽  
Florence Persat ◽  
Marie-France Monier ◽  
Elisabeth Borel ◽  
Marie-Antoinette Piens ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
Reference Method ◽  
Antifungal Susceptibility Testing ◽  
Aspergillus Species ◽  
National Committee ◽  
Broth Microdilution Method

A comparative study of visual and spectrophotometric MIC endpoint determinations for antifungal susceptibility testing of Aspergillus species was performed. A broth microdilution method adapted from the National Committee for Clinical Laboratory Standards (NCCLS) was used for susceptibility testing of 180 clinical isolates of Aspergillus species against amphotericin B and itraconazole. MICs were determined visually and spectrophotometrically at 490 nm after 24, 48, and 72h of incubation, and MIC pairs were compared. The agreement between the two methods was 99% for amphotericin B and ranged from 95 to 98% for itraconazole. It is concluded that spectrophotometric MIC endpoint determination is a valuable alternative to the visual reference method for susceptibility testing of Aspergillus species.Key words: antifungal, susceptibility testing, Aspergillus, spectrophotometric reading.

scholarly journals Comparison of the Echinocandin Caspofungin with Amphotericin B for Treatment of Histoplasmosis following Pulmonary Challenge in a Murine Model

2000 ◽  
Vol 44 (7) ◽  
pp. 1850-1854 ◽  
Author(s):  
Steve Kohler ◽  
L. Joseph Wheat ◽  
Patricia Connolly ◽  
Carol Schnizlein-Bick ◽  
Michelle Durkin ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Histoplasma Capsulatum ◽  
Clinical Laboratory ◽  
Antigen Detection ◽  
National Committee ◽  
Slight Effect ◽  
Fungal Burden ◽  
Detection Techniques ◽  
The Mean

ABSTRACT Twenty clinical isolates of Histoplasma capsulatum were tested for their in vitro susceptibilities to caspofungin in comparison to those to amphotericin B by following National Committee for Clinical Laboratory Standards guidelines for yeasts. The mean MICs were 16.6 μg/ml (range, 8 to 32 μg/ml) for caspofungin and 0.56 μg/ml (range, 0.5 to 1.0 μg/ml) for amphotericin B. Survival experiments used a 105 dose in a pulmonary challenge model with B6C3F1 mice. All mice that received amphotericin B at 2 mg/kg of body weight every other day (q.o.d.), 30% of mice that received caspofungin at 8 mg/kg/day, and 20% of mice that received caspofungin at 4 mg/kg/day survived to day 15, while mice that received caspofungin at 2 mg/kg/day and all control mice that received the vehicle died by day 14. Amphotericin B at 2 mg/kg q.o.d. markedly reduced the fungal burden in the lungs and spleens, as measured byHistoplasma antigen detection techniques and quantitative cultures, for each comparison. Caspofungin at 10 mg/kg twice a day (b.i.d.) did not reduce the fungal burden, as measured by antigen detection techniques, but slightly reduced the levels of fungi in both the lungs and spleens, as determined by quantitative cultures. Caspofungin at 5 mg/kg b.i.d. did not affect fungal burden. Overall, caspofungin had only a slight effect on survival or fungal burden.

scholarly journals In Vitro Activities of Voriconazole, Itraconazole, and Amphotericin B against Blastomyces dermatitidis,Coccidioides immitis, and Histoplasma capsulatum

2000 ◽  
Vol 44 (6) ◽  
pp. 1734-1736 ◽  
Author(s):  
Ren-Kai Li ◽  
Meral A. Ciblak ◽  
Nicole Nordoff ◽  
Lester Pasarell ◽  
David W. Warnock ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Histoplasma Capsulatum ◽  
Clinical Laboratory ◽  
Lethal Effect ◽  
Blastomyces Dermatitidis ◽  
National Committee ◽  
Human Pathogens ◽  
Coccidioides Immitis ◽  
Dimorphic Fungi

ABSTRACT The in vitro activity of voriconazole was compared to those of itraconazole and amphotericin B against the mold forms of 304 isolates of three dimorphic fungi, Blastomyces dermatitidis,Coccidioides immitis, and Histoplasma capsulatum. MICs were determined by a broth microdilution adaptation of the National Committee for Clinical Laboratory Standards M27-A procedure. RPMI 1640 medium was used for tests with voriconazole and itraconazole, whereas Antibiotic Medium 3 with 2% glucose was used for amphotericin B. Minimum fungicidal concentrations (MFCs) were also determined. Amphotericin B was active against all three dimorphic fungi, with MICs at which 90% of the isolates tested are inhibited (MIC90s) of 0.5 to 1 μg/ml. Itraconazole had MIC90s of 0.06 μg/ml for H. capsulatum, 0.125 μg/ml for B. dermatitidis, and 1 μg/ml for C. immitis. The MIC90s of voriconazole were 0.25 μg/ml for all three fungi. Amphotericin B was fungicidal for B. dermatitidis and H. capsulatum with MFCs at which 90% of strains tested are killed (MFC90s) of 0.5 and 2 μg/ml, respectively. It was less active against C. immitis, with MFCs ranging from 0.5 to >16 μg/ml. Voriconazole and itraconazole were lethal for most isolates of B. dermatitidis, with MFC50s and MFC90s of 0.125 and 4 μg/ml, respectively. Both azoles were fungicidal for some isolates of H. capsulatum, with MFC50s of 2 and 8 μg/ml for itraconazole and voriconazole, respectively; neither had a lethal effect upon C. immitis. Our results suggest that voriconazole possesses promising activity against these important human pathogens.

Amphotericin B in Lipid Emulsion: Stability, Compatibility, and In Vitro Antifungal Activity

1998 ◽  
Vol 42 (4) ◽  
pp. 762-766 ◽  
Author(s):  
Scott Walker ◽  
Sandra A. N. Tailor ◽  
Mark Lee ◽  
Lisa Louie ◽  
Marie Louie ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Amphotericin B ◽  
Clinical Laboratory ◽  
National Committee ◽  
Compatibility Study ◽  
The Stability ◽  
Few Data ◽  
Reference Strains ◽  
In Vitro Antifungal Activity

ABSTRACT Newer formulations of amphotericin B (AmB) complexed with liposomes or lipid suspensions have been developed. Preliminary studies have suggested that AmB in Intralipid (IL) may be as effective as, but less toxic than, conventional formulations of AmB, but few data are available regarding its stability, compatibility, or in vitro antifungal activity. A compatibility study was done to evaluate the effects of AmB concentrations in IL containing either 10 or 20% soybean oil. The effects of temperature, shaking, and AmB and IL concentrations on the stability of AmB-IL suspensions were analyzed by visual inspection and liquid chromatography. The in vitro antifungal activity of AmB-IL, compared to that of AmB alone against reference strains of Candida species was determined by using a broth macrodilution method in accordance with National Committee for Clinical Laboratory Standards guidelines (M27-T). Samples of AmB-IL which were lightly shaken retained more than 90% of the AmB concentration over 21 days when stored at either 4 or 23°C. Varying the AmB concentration did not appear to affect the stability of AmB-IL. However, a precipitate was formed when mixtures with more than 30% lipid as a proportion of the total volume were centrifuged. AmB-IL and AmB alone had similar in vitro antifungal activities against reference strains of yeasts. Further pharmacologic and clinical studies with AmB-IL are warranted, although AmB should not be combined with IL in concentrations capable of producing a precipitate.

scholarly journals In Vitro Amphotericin B Resistance in Clinical Isolates of Aspergillus terreus, with a Head-to-Head Comparison to Voriconazole

1999 ◽  
Vol 37 (7) ◽  
pp. 2343-2345 ◽  
Author(s):  
Deanna A. Sutton ◽  
Stephen E. Sanche ◽  
Sanjay G. Revankar ◽  
Annette W. Fothergill ◽  
Michael G. Rinaldi
Keyword(s):  
Amphotericin B ◽  
Aspergillus Terreus ◽  
Clinical Laboratory ◽  
Immunocompromised Host ◽  
Clinical Isolates ◽  
National Committee ◽  
High Dose ◽  
Etiologic Agents ◽  
Disseminated Disease

Amphotericin B therapy continues to be the “gold standard” in the treatment of invasive aspergillosis in the immunocompromised host. Although Aspergillus fumigatus and Aspergillus flavus constitute the major species, several reports have described invasive pulmonary or disseminated disease due to the less common Aspergillus terreus and dismal clinical outcomes with high-dose amphotericin B. We therefore evaluated 101 clinical isolates of A. terreus for their susceptibility to amphotericin B and the investigational triazole voriconazole by using the National Committee for Clinical Laboratory Standards M27-A method modified for mould testing. Forty-eight-hour MICs indicated 98 and 0% resistance to amphotericin B and voriconazole, respectively. We conclude that A. terreus should be added to the list of etiologic agents refractory to conventional amphotericin B therapy and suggest the potential clinical utility of voriconazole in aspergillosis due to this species.

scholarly journals The Calgary Biofilm Device: New Technology for Rapid Determination of Antibiotic Susceptibilities of Bacterial Biofilms

1999 ◽  
Vol 37 (6) ◽  
pp. 1771-1776 ◽  
Author(s):  
H. Ceri ◽  
M. E. Olson ◽  
C. Stremick ◽  
R. R. Read ◽  
D. Morck ◽  
...  
Keyword(s):  
Antibiotic Susceptibility ◽  
Clinical Laboratory ◽  
Rapid Determination ◽  
New Technology ◽  
Growth Curves ◽  
National Committee ◽  
Standard Group ◽  
Antibiotic Susceptibilities ◽  
Significant Difference

Determination of the MIC, based on the activities of antibiotics against planktonic bacteria, is the standard assay for antibiotic susceptibility testing. Adherent bacterial populations (biofilms) present with an innate lack of antibiotic susceptibility not seen in the same bacteria grown as planktonic populations. The Calgary Biofilm Device (CBD) is described as a new technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. The CBD produces 96 equivalent biofilms for the assay of antibiotic susceptibilities by the standard 96-well technology. Biofilm formation was followed by quantitative microbiology and scanning electron microscopy. Susceptibility to a standard group of antibiotics was determined for National Committee for Clinical Laboratory Standards (NCCLS) reference strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, andStaphylococcus aureus ATCC 29213. Growth curves demonstrated that biofilms of a predetermined size could be formed on the CBD at specific time points and, furthermore, that no significant difference (P > 0.1) was seen between biofilms formed on each of the 96 pegs. The antibiotic susceptibilities for planktonic populations obtained by the NCCLS method or from the CBD were similar. Minimal biofilm eradication concentrations, derived by using the CBD, demonstrated that for biofilms of the same organisms, 100 to 1,000 times the concentration of a certain antibiotic were often required for the antibiotic to be effective, while other antibiotics were found to be effective at the MICs. The CBD offers a new technology for the rational selection of antibiotics effective against microbial biofilms and for the screening of new effective antibiotic compounds.

scholarly journals Interactions between Triazoles and Amphotericin B against Cryptococcus neoformans

2000 ◽  
Vol 44 (9) ◽  
pp. 2435-2441 ◽  
Author(s):  
Francesco Barchiesi ◽  
Anna M. Schimizzi ◽  
Francesca Caselli ◽  
Andrea Novelli ◽  
Stefania Fallani ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Clinical Laboratory ◽  
Fungal Infections ◽  
National Committee ◽  
Positive Interaction ◽  
Pronounced Increase

ABSTRACT The interaction of amphotericin B (AmB) and azole antifungal agents in the treatment of fungal infections is still a controversial issue. A checkerboard titration broth microdilution-based method that adhered to the recommendations of the National Committee for Clinical Laboratory Standards was applied to study the in vitro interactions of AmB with fluconazole (FLC), itraconazole (ITC), and the new investigational triazole SCH 56592 (SCH) against 15 clinical isolates ofCryptococcus neoformans. Synergy, defined as a fractional inhibitory concentration (FIC) index of ≤0.50, was observed for 7% of the isolates in studies of the interactions of both FLC-AmB and ITC-AmB and for 33% of the isolates in studies of the SCH-AmB interactions; additivism (FICs, >0.50 to 1.0) was observed for 67, 73, and 53% of the isolates in studies of the FLC-AmB, ITC-AmB, and SCH-AmB interactions, respectively; indifference (FICs, >1.0 to ≤2.0) was observed for 26, 20, and 14% of the isolates in studies of the FLC-AmB, ITC-AmB, and SCH-AmB interactions, respectively. Antagonism (FIC >2.0) was not observed. When synergy was not achieved, there was still a decrease, although not as dramatic, in the MIC of one or both drugs when they were used in combination. To investigate the effects of FLC-AmB combination therapy in vivo, we established an experimental model of systemic cryptococcosis in BALB/c mice by intravenous injection of cells of C. neoformans 2337, a clinical isolate belonging to serotype D against which the combination of FLC and AmB yielded an additive interaction in vitro. Both survival and tissue burden studies showed that combination therapy was more effective than FLC alone and that combination therapy was at least as effective as AmB given as a single drug. On the other hand, when cells of C. neoformans 2337 were grown in FLC-containing medium, a pronounced increase in resistance to subsequent exposures to AmB was observed. In particular, killing experiments conducted with nonreplicating cells showed that preexposure to FLC abolished the fungicidal activity of the polyene. However, this apparent antagonism was not observed in vivo. Rather, when the two drugs were used sequentially for the treatment of systemic murine cryptococcosis, a reciprocal potentiation was often observed. Our study shows that (i) the combination of triazoles and AmB is significantly more active than either drug alone against C. neoformans in vitro and (ii) the concomitant or sequential use of FLC and AmB for the treatment of systemic murine cryptococcosis results in a positive interaction.

scholarly journals In Vitro Activities of Free and Lipid Formulations of Amphotericin B and Nystatin against Clinical Isolates of Coccidioides immitis at Various Saprobic Stages

2002 ◽  
Vol 46 (5) ◽  
pp. 1583-1585 ◽  
Author(s):  
Gloria M. González ◽  
Rolando Tijerina ◽  
Deanna A. Sutton ◽  
John R. Graybill ◽  
Michael G. Rinaldi
Keyword(s):  
Amphotericin B ◽  
Clinical Laboratory ◽  
National Committee ◽  
Growth Stages ◽  
Coccidioides Immitis ◽  
In Vitro Susceptibility ◽  
Broth Macrodilution ◽  
Lipid Formulations ◽  
Different Growth Stages

ABSTRACT We investigated the susceptibilities of hyphal, mixed hyphal, ungerminated arthroconidial, and germinated arthroconidial populations of Coccidioides immitis to lipid formulations of amphotericin B and nystatin and their conventional preparations, utilizing the National Committee for Clinical Laboratory Standards M38-P broth macrodilution method. The differences in effects of the three different growth stages of the saprobic phase of C. immitis on the MIC/minimum lethal concentration (MLC) ratio were not statistically significant for any of the antifungal agents tested. These results suggest that either inocula could be used for in vitro susceptibility studies with C. immitis.

scholarly journals Evaluation of Amphotericin B Interpretive Breakpoints for Candida Bloodstream Isolates by Correlation with Therapeutic Outcome

2006 ◽  
Vol 50 (4) ◽  
pp. 1287-1292 ◽  
Author(s):  
Benjamin J. Park ◽  
Beth A. Arthington-Skaggs ◽  
Rana A. Hajjeh ◽  
Naureen Iqbal ◽  
Meral A. Ciblak ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
National Committee ◽  
Restricted Range ◽  
Broth Microdilution ◽  
Therapeutic Success ◽  
In Vitro Susceptibility ◽  
In Vitro Susceptibility Testing

ABSTRACT One hundred seven Candida bloodstream isolates (51 C. albicans, 24 C. glabrata, 13 C. parapsilosis, 13 C. tropicalis, 2 C. dubliniensis, 2 C. krusei, and 2 C. lusitaniae strains) from patients treated with amphotericin B alone underwent in vitro susceptibility testing against amphotericin B using five different methods. Fifty-four isolates were from patients who failed treatment, defined as death 7 to 14 days after the incident candidemia episode, having persistent fever of ≥5 days' duration after the date of the incident candidemia, or the recurrence of fever after two consecutive afebrile days while on antifungal treatment. MICs were determined by using the Clinical Laboratory Standards Institute (formally National Committee for Clinical Laboratory Standards) broth microdilution procedure with two media and by using Etest. Minimum fungicidal concentrations (MFCs) were also measured in two media. Broth microdilution tests with RPMI 1640 medium generated a restricted range of MICs (0.125 to 1 μg/ml); the corresponding MFC values ranged from 0.5 to 4 μg/ml. Broth microdilution tests with antibiotic medium 3 produced a broader distribution of MIC and MFC results (0.015 to 0.25 μg/ml and 0.06 to 2 μg/ml, respectively). Etest produced the widest distribution of MICs (0.094 to 2 μg/ml). However, none of the test formats studied generated results that significantly correlated with therapeutic success or failure.

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