scholarly journals The Trailing End Point Phenotype in Antifungal Susceptibility Testing Is pH Dependent

1999 ◽  
Vol 43 (6) ◽  
pp. 1383-1386 ◽  
Author(s):  
Kieren A. Marr ◽  
Tige R. Rustad ◽  
John H. Rex ◽  
Theodore C. White
Keyword(s):  
Minimal Medium ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Ph Effect ◽  
Antifungal Susceptibility ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
National Committee ◽  
End Points

ABSTRACT The interpretation of end points in azole antifungal drug susceptibility testing is problematic, in part due to incomplete growth inhibition of Candida species. Such trailing growth can cause the MICs of fluconazole for some isolates to be low (<1 μg/ml) after 24 h of growth but much higher (>64 μg/ml) after 48 h. Isolates having this type of growth have been described as having a low-high phenotype. Although these isolates would be considered resistant by current National Committee of Clinical Laboratory Standards definitions, growing evidence suggests that they are susceptible in vivo. To further characterize these isolates in vitro, microdilution susceptibility testing comparing the complex defined medium RPMI 1640 to a defined minimal medium (yeast nitrogen broth) was performed. Isolates having trailing growth in MOPS (morpholinepropanesulfonic acid)-buffered RPMI 1640 (pH 7.0) were found to have clear end points in the minimal medium at its native pH of 4.5. The pH of the medium influenced the low-high phenotype, as these same isolates trailed in minimal medium adjusted to a pH of ≥6.0 but did not trail in RPMI 1640 adjusted to a pH of ≤5.0. This pH effect was independent of the medium buffering capacity, as trailing was decreased in both minimal medium and RPMI 1640 (pH 4.5) buffered in citrate. Adjustment in the pH of MOPS-buffered RPMI 1640 reduced trailing in multiple strains of Candida albicans without affecting the MICs for isolates having known susceptible (low-low) and resistant (high-high) phenotypes. Adjustment of the medium pH could be considered to eliminate trailing in azole drug susceptibility testing.

scholarly journals Comparative Evaluation of FUNGITEST and Broth Microdilution Methods for Antifungal Drug Susceptibility Testing of Candida Species and Cryptococcus neoformans

1998 ◽  
Vol 36 (4) ◽  
pp. 926-930 ◽  
Author(s):  
Kate G. Davey ◽  
Ann D. Holmes ◽  
Elizabeth M. Johnson ◽  
Adrien Szekely ◽  
David W. Warnock
Keyword(s):  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
National Committee ◽  
Broth Microdilution ◽  
Fluconazole Resistant

The FUNGITEST method (Sanofi Diagnostics Pasteur, Paris, France) is a microplate-based procedure for the breakpoint testing of six antifungal agents (amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and miconazole). We compared the FUNGITEST method with a broth microdilution test, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 180 isolates ofCandida spp. (50 C. albicans, 50C. glabrata, 10 C. kefyr, 20C. krusei, 10 C. lusitaniae, 20C. parapsilosis, and 20 C. tropicalisisolates) and 20 isolates of Cryptococcus neoformans. Overall, there was 100% agreement between the methods for amphotericin B, 95% agreement for flucytosine, 84% agreement for miconazole, 83% agreement for itraconazole, 77% agreement for ketoconazole, and 76% agreement for fluconazole. The overall agreement between the methods exceeded 80% for all species tested with the exception ofC. glabrata (71% agreement). The poorest agreement between the results for individual agents was seen with C. glabrata (38% for fluconazole, 44% for ketoconazole, and 56% for itraconazole) and C. tropicalis (50% for miconazole). The FUNGITEST method misclassified as susceptible 2 of 12 (16.6%) fluconazole-resistant isolates, 2 of 10 (20%) itraconazole-resistant isolates, and 4 of 8 (50%) ketoconazole-resistant isolates of several Candida spp. Further development of the FUNGITEST procedure will be required before it can be recommended as an alternative method for the susceptibility testing of Candida spp. or C. neoformans.

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 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 Metarrhizium anisopliae as a Cause of Sinusitis in Immunocompetent Hosts

1999 ◽  
Vol 37 (1) ◽  
pp. 195-198 ◽  
Author(s):  
Sanjay G. Revankar ◽  
Deanna A. Sutton ◽  
Stephen E. Sanche ◽  
Jyothi Rao ◽  
Marcus Zervos ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Susceptibility Testing ◽  
Chronic Sinusitis ◽  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
National Committee ◽  
Insect Populations ◽  
Clinically Significant ◽  
Control Insect

Metarrhizium anisopliae is a common pathogen of insects and has even been used to control insect populations. It is rarely isolated from human or animal sources, but recently, there have been three reported cases of disease, two in humans and one in a cat. We present our experience with five isolates from human sources, including two that were the apparent causes of two cases of sinusitis in immunocompetent hosts. The first patient was a 36-year-old male with frontal and ethmoid sinusitis, and the second was a 79-year-old female with chronic sinusitis. Both patients underwent surgery, and pathology of the surgical specimens revealed branching hyphae. Cultures grew onlyMetarrhizium species. Neither patient received antifungal therapy, and both did well postoperatively. The other three isolates were cultured from bronchoalveolar lavage specimens but were not felt to be clinically significant. Antifungal susceptibility testing using the National Committee for Clinical Laboratory Standards macrobroth method revealed that all isolates were resistant to amphotericin B, 5-flucytosine, and fluconazole. Itraconazole and newer azole compounds were more active. Metarrhizium species may cause disease in humans, even those without evidence of immunosuppression, and are apparently highly resistant to amphotericin B in vitro.

scholarly journals In Vitro Antifungal Susceptibility Of Griseofulvin, Fluconazole, Itraconazole And Terbinafine Against Clinical Isolates Of Trichophyton Rubrum And Trichophyton Mentagrophytes

2014 ◽  
Vol 2 (1) ◽  
Author(s):  
K R Reddy ◽  
S Ram Reddy
Keyword(s):  
Trichophyton Rubrum ◽  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
Trichophyton Mentagrophytes ◽  
Drug Susceptibility ◽  
Antifungal Drugs ◽  
Clinical Isolates ◽  
National Committee ◽  
In Vitro Antifungal Susceptibility

Investigations on antifungal drug susceptibility were carried out on 90 clinical isolates of Trichophyton rubrum, and Trichophyton mentagrophytes with four antifungal drugs, namely griseofulvin, fluconazole, itraconazole and terbinafine as suggested by National Committee for Clinical Laboratory Standards (NCCLS) M27–A (1997) document by broth macrodilution method to standardize in vitro antifungal susceptibility testing and to find out the Minimum Inhibitory Concentration (MIC) of the drugs. In this study, terbinafine was found to be the most efficient drug for all isolates. Terbinafine had the lowest MIC range of 0.001 g/ml to 0.09 g/ml and MIC50 was low at 0.005 g/ml and MIC90 was also low at 0.04 g/ml against T.rubrum; and MIC range of 0.001μg/ml to 0.19μg/ml with a MIC50 of 0.01μg/ml and MIC90 at 0.09μg/ml against T.mentagrophytes. Itraconazole showed antifungal activity superior to that of fluconazole, with a MIC range of 0.04g/ml to 1.56g/ml, with MIC50 at 0.19μg/ml and MIC90 at 1.56g/ml against T.rubrum; and MIC range of 0.04μg/ml to 1.56μg/ml, with MIC50 at 0.19μg/ml and MIC90 at 0.78μg/ml against T.mentagrophytes. Griseofulvin appears to be still a potent drug for management of dermatophytoses. Griseofulvin had a MIC range of 0.15g/ml to 5.07 g/ml with MIC50 at1.26 g/ml and MIC90 at 2.53 g/ml against T.rubrum; and MIC range of 0.31μg/ml to 5.07μg/ml with MIC50 at 1.26μg/ml and MIC90 at 2.53μg/ml against T.mentagrophytes. Fluconazole showed a high MIC range of 0.19 g/ml to 50 g/ml and MIC50 was high at 1.56g/ml and MIC90 was also high at 12.5 g/ml against T.rubrum; and a high MIC range of 0.09μg/ml to 25.0μg/ml, with MIC50 at 1.56μg/ml and MIC90 at 12.5μg/ml towards T.mentagrophytes. The technique was found to be easy to perform and reliable with consistent results.

scholarly journals Recommendation of an Appropriate Medium for In Vitro Drug Susceptibility Testing of the Fish Pathogen Tenacibaculum maritimum

2005 ◽  
Vol 49 (1) ◽  
pp. 82-87 ◽  
Author(s):  
Ruben Avendaño-Herrera ◽  
Rute Irgang ◽  
Soledad Núñez ◽  
Jesús L. Romalde ◽  
Alicia E. Toranzo
Keyword(s):  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Drug Susceptibility Testing ◽  
The Other ◽  
Disk Diffusion ◽  
National Committee ◽  
Good Growth ◽  
Good Correspondence ◽  
In Vitro Susceptibility

ABSTRACT In the present study, Anacker and Ordal agar, marine agar (MA), and Flexibacter maritimus medium (FMM) were compared with the dilute versions of Mueller-Hinton agar (DMHA) medium recommended by the National Committee for Clinical Laboratory Standards (NCCLS) for their use in disk diffusion tests with Tenacibaculum maritimum strains and to calculate the MICs of five drugs by the Etest method. Preliminary growth tests performed with 32 strains of this pathogen on each medium revealed that all strains failed to grow on DMHA, while the remaining media supported good growth of all isolates. In the susceptibility tests, which were carried out with the other three media, all strains were resistant to oxolinic acid and were highly susceptible to amoxicillin and trimethoprim-sulfamethoxazole, showing a good correspondence with the Etest values, which ranged from 0.064 to 0.75 and 0.006 to 1.5 μg/ml, respectively. Enrofloxacin and oxytetracycline produced significantly smaller inhibition zones and MICs on MA than on the other media assayed. However, fast, clear, and well-defined zones of inhibition were displayed for all strains at 24 h of incubation only on FMM by both the disk diffusion assay and Etest. In addition, FMM prepared with commercial sea salts instead of seawater was also suitable for bacterial isolation as well as for susceptibility testing. On the basis of these results, the use of FMM to determine the in vitro susceptibility of T. maritimum and its inclusion in a future revision of the NCCLS M42 report are recommended.

scholarly journals Comparison of a spectrophotometric microdilution method with RPMI-2% glucose with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro susceptibility testing of amphotericin B, flucytosine, and fluconazole against Candida albicans.

1996 ◽  
Vol 40 (9) ◽  
pp. 1998-2003 ◽  
Author(s):  
J L Rodríguez-Tudela ◽  
J Berenguer ◽  
J V Martínez-Suárez ◽  
R Sanchez
Keyword(s):  
Quality Control ◽  
Candida Albicans ◽  
Amphotericin B ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
National Committee ◽  
Microdilution Method ◽  
Reference Strains

The National Committee for Clinical Laboratory Standards has proposed a reference broth macrodilution method for in vitro antifungal susceptibility testing of yeasts (the M27-P method). This method is cumbersome and time-consuming and includes MIC endpoint determination by visual and subjective inspection of growth inhibition after 48 h of incubation. An alternative microdilution procedure was compared with the M27-P method for determination of the amphotericin B, flucytosine, and fluconazole susceptibilities of 8 American Type Culture Collection strains (6 of them were quality control or reference strains) and 50 clinical isolates of candida albicans. This microdilution method uses as culture medium RPMI 1640 supplemented with 18 g of glucose per liter (RPMI-2% glucose). Preparation of drugs, basal medium, and inocula was done by following the recommendations of the National Committee for Clinical Laboratory Standards. The MIC endpoint was calculated objectively from the turbidimetric data read at 24 h. Increased growth of C. albicans in RPMI-2% glucose and its spectrophotometric reading allowed for the rapid (24 h) and objective calculation of MIC endpoints compared with previous microdilution methods with standard RPMI 1640. Nevertheless, good agreement was shown between the M27-P method and this microdilution test. The MICs obtained for the quality control or reference strains by the microdilution method were in the ranges published for those strains. For clinical isolates, the percentages of agreement were 100% for amphotericin B and fluconazole and 98.1% for flucytosine. These data suggest that this microdilution method may serve as a less subjective and more rapid alternative to the M27-P method for antifungal susceptibility testing of yeasts.

In vitro antifungal susceptibility of griseofulvin, fluconazole, itraconazole and terbinafine against clinical isolates of Trichophyton rubrum and Trichophyton mentagrophytes

2014 ◽  
Vol 1 (4) ◽  
pp. 26-28
Author(s):  
KR Reddy ◽  
SR Reddy
Keyword(s):  
Trichophyton Rubrum ◽  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
Trichophyton Mentagrophytes ◽  
Drug Susceptibility ◽  
Antifungal Drugs ◽  
Clinical Isolates ◽  
National Committee ◽  
In Vitro Antifungal Susceptibility

Investigations on antifungal drug susceptibility were carried out on 90 clinical isolates of Trichophyton rubrum, and Trichophyton mentagrophytes with four antifungal drugs, namely griseofulvin, fluconazole, itraconazole and terbinafine as suggested by National Committee for Clinical Laboratory Standards (NCCLS) M27A (1997) document by broth macrodilution method to standardize in vitro antifungal susceptibility testing and to find out the Minimum Inhibitory Concentration (MIC) of the drugs. In this study, terbinafine was found to be the most efficient drug for all isolates. Terbinafine had the lowest MIC range of 0.001 g/mlto 0.09 g/ml and MIC50 was low at 0.005 g/ml and MIC90 was also low at 0.04 g/ml against T. rubrum; and MIC range of 0.001pg/ml to 0.19pg/ml with a MIC50 of 0.01pg/ml and MIC90 at 0.09 pg/ml against T. mentagrophytes. Itraconazole showed antifungal activity superior to that of fluconazole, with a MIC range of 0.04g/ml to l.56g/ml, with MIC50 at 0.19pg/m land MIC90 at l.56g/ml against T. rubrum; and MIC range of 0.04.g/ml to 1.56pg/ml, with MIC50 at 0.19pg/ml and MIC90 at 0.78pg/ml against T. mentagrophytes. Griseofulvin appears to be still a potent drug for management of dermatophytoses. Griseofulvin had a MIC range of 0.15g/ml to 5.07 g/ml with MIC50 at l.26 g/ml and MIC90 at 2.53 g/ml against T. rubrum; and MIC range of 0.31pg/ml to 5.07pg/ml with MIC50 at 1.26pg/ml and MIC90 at 2.53pg/ml against T. mentagrophytes. Fluconazole showed a high MIC range of 0.19 g/ml to 50 g/ml and MIC50 was high at 1.56g/ml and MIC90 was also high at 12.5 g/ml against T. rubrum; and a high MIC range of 0.09pg/ml to 25.0pg/ml, with MIC50 at 1.56pg/ml and MIC90 at 12.5pg/ml towards T. mentagrophytes. The technique was found to be easy to perform and reliable with consistent results.DOI: http://dx.doi.org/10.3126/jucms.v1i4.9569 Journal of Universal College of Medical Sciences (2013) Vol.1 No.04: 26-28

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