scholarly journals Pentamidine Is Active In Vitro against Fusarium Species

2003 ◽  
Vol 47 (10) ◽  
pp. 3252-3259 ◽  
Author(s):  
Michail S. Lionakis ◽  
Russell E. Lewis ◽  
George Samonis ◽  
Dimitrios P. Kontoyiannis
Keyword(s):  
Clinical Laboratory ◽  
Fusarium Species ◽  
Tissue Perfusion ◽  
In Vivo Studies ◽  
National Committee ◽  
Yeast Nitrogen Base ◽  
Nitrogen Base ◽  
Minimum Fungicidal Concentration

ABSTRACT Fusariosis is an emerging opportunistic mycosis against which currently used antifungals have limited activity. Here, we investigated the in vitro activities of pentamidine (PNT) against 10 clinical isolates of Fusarium species (five Fusarium solani isolates and five non-F. solani isolates) by using the National Committee for Clinical Laboratory Standards microdilution method in three different media (RPMI, RPMI-2, and a yeast nitrogen base medium), disk diffusion testing, and viability dye staining. PNT had significant activities against all 10 Fusarium isolates. Non-F. solani isolates were more susceptible than F. solani isolates (P < 0.05). Additionally, PNT was fungicidal against all non-F. solani isolates, whereas it had fungistatic effects against four of the five F. solani isolates. PNT also exhibited greater activity against conidial than against hyphal development of the fungus. This fungicidal activity against non-F. solani Fusarium isolates was confirmed microscopically after staining of PNT-treated Fusarium oxysporum hyphae with the fluorescent viability dyes 5,(6)-carboxyfluorescein diacetate (CFDA) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC). The MICs at which 50% of the isolates were inhibited (2 μg/ml for non-F. solani isolates and 4 μg/ml for F. solani isolates) and the minimum fungicidal concentration at which 50% of the isolates were killed (8 μg/ml for non-F. solani isolates) were much lower than the PNT tissue concentrations previously reported in humans using conventional daily intravenous PNT dosing. Finally, PNT was more active against Fusarium isolates in a hypoxic environment of in vitro growth (P < 0.05). This finding may be clinically significant, because Fusarium, an angiotropic mold, causes tissue infarcts with resultant low tissue perfusion. Our findings suggest that PNT may have a role in the management of Fusarium infections. Future in vivo studies are needed to verify these in vitro findings.

scholarly journals Detection of Resistance to Amphotericin B amongCryptococcus neoformans Clinical Isolates: Performances of Three Different Media Assessed by Using E-Test and National Committee for Clinical Laboratory Standards M27-A Methodologies

1998 ◽  
Vol 36 (10) ◽  
pp. 2817-2822 ◽  
Author(s):  
M. Lozano-Chiu ◽  
V. L. Paetznick ◽  
M. A. Ghannoum ◽  
J. H. Rex
Keyword(s):  
Amphotericin B ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Diffusion Method ◽  
National Committee ◽  
Yeast Nitrogen Base ◽  
Nitrogen Base ◽  
Agar Diffusion ◽  
Agar Diffusion Method

Although reliable detection of resistance in vitro is critical to the overall performance of any susceptibility testing method, the recently released National Committee for Clinical Laboratory Standards M27-A methodology for susceptibility testing of yeasts discriminates poorly between resistant and susceptible isolates ofCandida spp. We have previously shown that both substitution of antibiotic medium 3 for RPMI 1640 medium in the microdilution variant of the M27-A method and use of the E-test agar diffusion methodology permit detection of amphotericin B-resistantCandida isolates. To determine the relevance of these observations to Cryptococcus neoformans, we have evaluated the performances of both the M27-A and the E-test methodologies with this yeast using three different media (RPMI 1640 medium, antibiotic medium 3, and yeast nitrogen base). As with Candida, we found that only antibiotic medium 3 permitted consistent detection of resistant isolates when testing was performed in broth by the M27-A method. When testing was performed by the E-test agar diffusion method, both RPMI 1640 medium and antibiotic medium 3 agar permitted ready detection of the resistant isolates. Reading of the results after 48 h of incubation was required for testing in broth by the M27-A method, while the MIC could be determined after either 48 or 72 h when the agar diffusion method was used.

scholarly journals Quantitation of Candida albicans Ergosterol Content Improves the Correlation between In Vitro Antifungal Susceptibility Test Results and In Vivo Outcome after Fluconazole Treatment in a Murine Model of Invasive Candidiasis

2000 ◽  
Vol 44 (8) ◽  
pp. 2081-2085 ◽  
Author(s):  
Beth A. Arthington-Skaggs ◽  
David W. Warnock ◽  
Christine J. Morrison
Keyword(s):  
Murine Model ◽  
Invasive Candidiasis ◽  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
Resistant Isolate ◽  
National Committee ◽  
Ergosterol Content ◽  
In Vitro Antifungal Susceptibility

ABSTRACT MIC end point determination for the most commonly prescribed azole antifungal drug, fluconazole, can be complicated by “trailing” growth of the organism during susceptibility testing by the National Committee for Clinical Laboratory Standards approved M27-A broth macrodilution method and its modified broth microdilution format. To address this problem, we previously developed the sterol quantitation method (SQM) for in vitro determination of fluconazole susceptibility, which measures cellular ergosterol content rather than growth inhibition after exposure to fluconazole. To determine if SQM MICs of fluconazole correlated better with in vivo outcome than M27-A MICs, we used a murine model of invasive candidiasis and analyzed the capacity of fluconazole to treat infections caused by C. albicansisolates which were trailers (M27-A MICs at 24 and 48 h, ≤1.0 and ≥64 μg/ml, respectively; SQM MIC, ≤1.0 μg/ml), as well as those which were fluconazole sensitive (M27-A and SQM MIC, ≤1.0 μg/ml) and fluconazole resistant (M27-A MIC, ≥64 μg/ml; SQM MIC, 54 μg/ml). Compared with the untreated controls, fluconazole therapy increased the survival of mice infected with a sensitive isolate and both trailing isolates but did not increase the survival of mice infected with a resistant isolate. These results indicate that the SQM is more predictive of in vivo outcome than the M27-A method for isolates that give unclear MIC end points due to trailing growth in fluconazole.

scholarly journals Correlation of Fluconazole MICs with Clinical Outcome in Cryptococcal Infection

2000 ◽  
Vol 44 (6) ◽  
pp. 1544-1548 ◽  
Author(s):  
A. I. Aller ◽  
E. Martin-Mazuelos ◽  
F. Lozano ◽  
J. Gomez-Mateos ◽  
L. Steele-Moore ◽  
...  
Keyword(s):  
Clinical Outcome ◽  
Maintenance Therapy ◽  
National Committee ◽  
Yeast Nitrogen Base ◽  
Nitrogen Base ◽  
Cryptococcal Infection ◽  
Microdilution Method ◽  
Cryptococcal Disease ◽  
Fluconazole Therapy

ABSTRACT We have correlated the in vitro results of testing the susceptibility of Cryptococcus neoformans to fluconazole with the clinical outcome after fluconazole maintenance therapy in patients with AIDS-associated cryptococcal disease. A total of 28 isolates of C. neoformans from 25 patients (24 AIDS patients) were tested. The MICs were determined by the broth microdilution technique by following the modified guidelines described in National Committee for Clinical Standards (NCCLS) document M27-A, e.g., use of yeast nitrogen base medium and a final inoculum of 104 CFU/ml. The fluconazole MIC at which 50% of isolates are inhibited (MIC50) and MIC90, obtained spectrophotometrically after 48 h of incubation, were 4 and 16 μg/ml, respectively. Of the 25 patients studied, 4 died of active cryptococcal disease and 2 died of other causes. Therapeutic failure was observed in five patients who were infected with isolates for which fluconazole MICs were ≥16 μg/ml. Four of these patients had previously had oropharyngeal candidiasis (OPC); three had previously had episodes of cryptococcal infection, and all five treatment failure patients had high cryptococcal antigen titers in either serum or cerebrospinal fluid (titers, >1:4,000). Although 14 of the 18 patients who responded to fluconazole therapy had previously had OPC infections, they each had only a single episode of cryptococcal infection. It appears that the clinical outcome after fluconazole maintenance therapy may be better when the infecting C. neoformans strain is inhibited by lower concentrations of fluconazole for eradication (MICs, <16 μg/ml) than when the patients are infected with strains that require higher fluconazole concentrations (MICs, ≥16 μg/ml). These findings also suggest that the MICs determined by the modified NCCLS microdilution method can be potential predictors of the clinical response to fluconazole therapy and may aid in the identification of patients who will not respond to fluconazole therapy.

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 Interpretive Breakpoints for Fluconazole and Candida Revisited: a Blueprint for the Future of Antifungal Susceptibility Testing

2006 ◽  
Vol 19 (2) ◽  
pp. 435-447 ◽  
Author(s):  
M. A. Pfaller ◽  
D. J. Diekema ◽  
D. J. Sheehan
Keyword(s):  
Clinical Studies ◽  
Randomized Clinical Trials ◽  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
Strong Relationship ◽  
National Committee ◽  
Disk Diffusion Testing ◽  
Invasive Infections

SUMMARY Developing interpretive breakpoints for any given organism-drug combination requires integration of the MIC distribution, pharmacokinetic and pharmacodynamic parameters, and the relationship between in vitro activity and outcome from both in vivo and clinical studies. Previously, the Subcommittee for Antifungal Testing of the Clinical and Laboratory Standards Institute (CLSI [formerly National Committee for Clinical Laboratory Standards]) proposed MIC interpretive breakpoints for fluconazole and Candida spp. These breakpoints were considered to be somewhat weak, because the clinical data supporting them came largely from mucosal infections and there were very few infections involving strains with elevated fluconazole MICs. We readdress the issue of fluconazole breakpoints for Candida by using published clinical and microbiologic data to provide further validation of the breakpoints proposed by the CLSI in 1997. We also address interpretive breakpoints for agar disk diffusion testing of fluconazole. The MIC distribution for fluconazole was determined with a collection of 13,338 clinical isolates. The overall MIC at which 90% of the isolates were inhibited was 8 μg/ml: 91% were susceptible (S) at a MIC of ≤8 μg/ml and 3% were resistant (R) (MIC ≥ 64 μg/ml). Similar results were obtained for 2,190 isolates from randomized clinical trials. Analysis of available data for 1,295 patient-episode-isolate events (692 represented mucosal infections and 603 represented invasive infections) from 12 published clinical studies demonstrated an overall success rate of 77%, including 85% for those episodes in which the fluconazole MIC was ≤8 μg/ml, 67% for those episodes in which the MIC was 16 to 32 μg/ml, and 42% for those episodes with resistant (MIC ≥ 64 μg/ml) isolates. Pharmacodynamic analysis demonstrated a strong relationship between MIC, fluconazole dose, and outcome. A dose/MIC ratio of ∼25 was supportive of the following susceptibility breakpoints for fluconazole and Candida spp.: S, MIC ≤ 8 μg/ml; susceptible-dose dependent (SDD), MIC = 16 to 32 μg/ml; R, MIC ≥ 64 μg/ml. The corresponding disk test breakpoints are as follows: S, ≥19 mm; SDD, 15 to 18 mm; R, ≤14 mm.

scholarly journals Growth Media for Mixed Multispecies Oropharyngeal Biofilm Compositions on Silicone

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Matthias Leonhard ◽  
Beata Zatorska ◽  
Doris Moser ◽  
Berit Schneider-Stickler
Keyword(s):  
Culture Conditions ◽  
Microbial Colonization ◽  
Future Research ◽  
Growth Media ◽  
Yeast Nitrogen Base ◽  
Microbial Composition ◽  
Nitrogen Base ◽  
Voice Prostheses

Aims. Microbial colonization of silicone voice prostheses by bacteria and Candida species limits the device lifetime of modern voice prostheses in laryngectomized patients. Thus, research focuses on biofilm inhibitive properties of novel materials, coatings, and surface enhancements. Goal of this in vitro study was the evaluation of seven commonly used growth media to simulate growth of mixed oropharyngeal species as mesoscale biofilms on prosthetic silicone for future research purposes. Methods and Results. Yeast Peptone Dextrose medium (YPD), Yeast Nitrogen Base medium (YNB), M199 medium, Spider medium, RPMI 1640 medium, Tryptic Soy Broth (TSB), and Fetal Bovine Serum (FBS) were used to culture combined mixed Candida strains and mixed bacterial-fungal compositions on silicone over the period of 22 days. The biofilm surface spread and the microscopic growth showed variations from in vivo biofilms depending on the microbial composition and growth medium. Conclusion. YPD and FBS prove to support continuous in vitro growth of mixed bacterial-fungal oropharyngeal biofilms deposits over weeks as needed for longterm in vitro testing with oropharyngeal biofilm compositions. Significance and Impact of Study. The study provides data on culture conditions for mixed multispecies biofilm compositions that can be used for future prosthesis designs.

scholarly journals Melanization of Cryptococcus neoformans and Histoplasma capsulatum Reduces Their Susceptibilities to Amphotericin B and Caspofungin

2002 ◽  
Vol 46 (11) ◽  
pp. 3394-3400 ◽  
Author(s):  
David van Duin ◽  
Arturo Casadevall ◽  
Joshua D. Nosanchuk
Keyword(s):  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Fungal Pathogens ◽  
Histoplasma Capsulatum ◽  
Clinical Laboratory ◽  
National Committee ◽  
Broth Macrodilution ◽  
Mammalian Infection

ABSTRACT The fungal pathogens Cryptococcus neoformans and Histoplasma capsulatum produce melanin-like pigments in the presence of l-dopa in vitro and during mammalian infection. We investigated whether melanization affected the susceptibilities of the fungi to amphotericin B, caspofungin, fluconazole, itraconazole, or flucytosine (5FC). Using the standard macrodilution MIC protocol (the M27A protocol) of the National Committee for Clinical Laboratory Standards for yeast, we found no difference in the susceptibilities of melanized and nonmelanized C. neoformans and H. capsulatum isolates. Killing assays demonstrated that melanization reduced the susceptibilities of both fungi to amphotericin B and caspofungin. Laccase-deficient C. neoformans cells grown with l-dopa were significantly more susceptible than congenic melanin-producing yeast to killing by amphotericin B or caspofungin. Preincubation of amphotericin B or caspofungin with melanins decreased their antifungal activities. Elemental analysis of melanins incubated with amphotericin B or caspofungin revealed an alteration in the C:N ratios of the melanins, which indicated binding of these drugs by the melanins. In contrast, incubation of fluconazole, itraconazole, or 5FC with melanins did not significantly affect the antifungal efficacies of the drugs or the chemical composition of the melanins. The results suggest a potential explanation for the inefficacy of caspofungin against C. neoformans in vivo, despite activity in vitro. Furthermore, the results indicate that fungal melanins protect C. neoformans and H. capsulatum from the activities of amphotericin B and caspofungin and that this protection is not demonstrable by standard broth macrodilution assays.

scholarly journals Correlation between Antifungal Susceptibilities ofCoccidioides immitis In Vitro and Antifungal Treatment with Caspofungin in a Mouse Model

2001 ◽  
Vol 45 (6) ◽  
pp. 1854-1859 ◽  
Author(s):  
Gloria M. González ◽  
Rolando Tijerina ◽  
Laura K. Najvar ◽  
Rosie Bocanegra ◽  
Michael Luther ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
National Committee ◽  
In Vitro Susceptibility ◽  
Minimum Effective Concentration ◽  
Study Results ◽  
Significant Difference

ABSTRACT Caspofungin (Merck Pharmaceuticals) was tested in vitro against 25 clinical isolates of Coccidoides immitis. In vitro susceptibility testing was performed in accordance with the National Committee for Clinical Laboratory Standards document M38-P guidelines. Two C. immitis isolates for which the caspofungin MICs were different were selected for determination of the minimum effective concentration (MEC), and these same strains were used for animal studies. Survival and tissue burdens of the spleens, livers, and lungs were used as antifungal response markers. Mice infected with strain 98-449 (48-h MIC, 8 μg/ml; 48-h MEC, 0.125 μg/ml) showed 100% survival to day 50 when treated with caspofungin at ≥1 mg/kg. Mice infected with strain 98-571 (48-h MIC, 64 μg/ml; 48-h MEC, 0.125 μg/ml) displayed ≥80% survival when the treatment was caspofungin at ≥5 mg/kg. Treatment with caspofungin at 0.5, 1, 5, or 10 mg/kg was effective in reducing the tissue fungal burdens of mice infected with either isolate. When tissue fungal burden study results were compared between strains, caspofungin showed no statistically significant difference in efficacy in the organs of the mice treated with both strains. A better in vitro-in vivo correlation was noted when we used the MEC instead of the MIC as the endpoint for antifungal susceptibility testing. Caspofungin may have a role in the treatment of coccidioidomycosis.

scholarly journals Attenuation of Itraconazole Fungicidal Activity following Preexposure of Aspergillus fumigatus to Fluconazole

2003 ◽  
Vol 47 (11) ◽  
pp. 3592-3597 ◽  
Author(s):  
Wei Liu ◽  
Michail S. Lionakis ◽  
Russell E. Lewis ◽  
Nathan Wiederhold ◽  
Gregory S. May ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Fungicidal Activity ◽  
Clinical Laboratory ◽  
Marrow Transplant ◽  
National Committee ◽  
In Vitro Susceptibility ◽  
Fourfold Increase ◽  
Microdilution Method

ABSTRACT Fluconazole (FLC), a triazole with limited activity against Aspergillus species, is frequently used as prophylaxis in leukemia patients and bone marrow transplant recipients. Prior FLC use has been associated with an increasing incidence of invasive aspergillosis in these patients. We hypothesized that prior exposure of Aspergillus fumigatus to FLC could result in altered in vitro susceptibility of this fungus to other, more active triazoles. Thus, we performed serial passages of conidia of 10 clinical isolates of A. fumigatus (all itraconazole [ITC] susceptible) on FLC-containing yeast agar glucose plates. The MICs and minimal fungicidal concentrations (MFCs) of amphotericin B, FLC, ITC, and voriconazole (VRC) for A. fumigatus conidia were measured following four passages on FLC-containing medium according to the National Committee for Clinical Laboratory Standards microdilution method. Serial passages on FLC-containing plates resulted in a fourfold increase in the MFCs (but not the MICs) of ITC for nine isolates. The attenuated ITC fungicidal activity against A. fumigatus following FLC preexposure was medium independent and was also observed against FLC-preexposed A. fumigatus hyphae with the viability staining FUN-1 dye. Moreover, FLC preexposure of A. fumigatus conidia resulted in an analogous increase in the MFCs (but not the MICs) of VRC. Our findings suggest that preexposure of A. fumigatus to FLC attenuates the in vitro fungicidal activity of subsequent ITC use against it. This phenotypic adaptation is not captured by a routine MIC determination but requires MFC measurement. The in vivo significance of this in vitro phenomenon requires further investigation.

scholarly journals AACC Guidance Document on Biotin Interference in Laboratory Tests

2020 ◽  
Vol 5 (3) ◽  
pp. 575-587 ◽  
Author(s):  
Danni Li ◽  
Angela Ferguson ◽  
Mark A Cervinski ◽  
Kara L Lynch ◽  
Patrick B Kyle
Keyword(s):  
Laboratory Tests ◽  
Clinical Laboratory ◽  
Case Reports ◽  
In Vivo Studies ◽  
High Dose ◽  
Guidance Document ◽  
Health Related ◽  
Biotin Binding

Abstract Background Laboratory tests that use streptavidin–biotin binding mechanisms have the potential to be affected by high circulating biotin concentrations, which would produce positive and negative interference in biotinylated competitive and noncompetitive (sandwich) immunoassays, respectively. Consumption of high-dose biotin supplements for cosmetic or health-related reasons has drawn attention to biotin interference in clinical laboratory tests. Case reports and in vivo studies show that ingestion of supplemental biotin can cause clinically significant errors in select biotinylated immunoassays. Content This AACC Academy document is intended to provide guidance to laboratorians and clinicians for preventing, identifying, and dealing with biotin interference. In vivo and in vitro spiking studies have demonstrated that biotin concentrations required to cause interference vary by test and by manufacturer. This document includes discussion of biotin’s mechanisms for interference in immunoassays, pharmacokinetics, and results of in vitro and in vivo studies and cites examples of assays known to be affected by high biotin concentrations. This document also provides guidance recommendations intended to assist laboratories and clinicians in identifying and addressing biotin interference in laboratory testing. Summary The recent increase in the use of high-dose biotin supplements requires laboratorians and clinicians to be mindful of the potential for biotin interference in biotinylated immunoassay-based laboratory tests. Laboratories, clinicians, regulators, and patients should work together to ensure accurate laboratory results. Laboratories have several options for identifying suspected biotin interference in specimens. Alternatively, the relatively fast elimination of biotin allows the potential for rapid follow-up specimen analysis if necessary.

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