Improved detection of amphotericin B-resistant isolates of Candida lusitaniae by Etest

Both intrinsic and acquired resistance to amphotericin B have been documented for Candida lusitaniae. Amphotericin B remains the drug of choice for many critical fungal infections, and the detection of resistance is essential to monitor treatment effectively. The limitations of the National Committee for Clinical Laboratory Standards (NCCLS) reference methodology for detection of amphotericin B resistance are well documented, and several alternative methods have been proposed. Etest assays with RPMI and antibiotic medium 3 (AM3) agar were compared to the NCCLS M27-A broth macrodilution method using AM3 for amphotericin B resistance testing with 49 clinical isolates of C. lusitaniae. The panel included nine isolates with known or presumed resistance to amphotericin B on the basis of in vivo and/or in vitro data. The distribution of amphotericin B MICs by Etest with RPMI ranged from 0. 032 to 16 microg/ml and was bimodal. All of the putatively resistant isolates were inhibited by amphotericin B at >/=0.38 microg/ml and could be categorized as resistant using this breakpoint. Etest with AM3 yielded a broader amphotericin B MIC range (0.047 to 32 microg/ml), and there were six putatively resistant isolates for which MICs were >1 microg/ml. The separation of putatively susceptible and resistant isolates was less obvious. Broth macrodilution with AM3 generated a unimodal distribution of MICs (ranging from 0.032 to 2 microg/ml) and failed to discriminate most of the putatively resistant isolates at both 24 and 48 h. Etest using RPMI and, to a lesser extent, using AM3 provided better discrimination between amphotericin B-resistant and -susceptible isolates of C. lusitaniae.     

Lot-to-lot variability of antibiotic medium 3 used for testing susceptibility of Candida isolates to amphotericin B.

We have previously shown that use of antibiotic medium 3 in the microdilution variant of the M27-T test method permits detection of amphotericin B-resistant Candida isolates. As this medium is not standardized and our initial work used only a single lot of antibiotic medium 3, we studied the lot-to-lot variability of three commercial lots of antibiotic medium 3 obtained from two sources. The MICs obtained with the new lots were lower than those of with the original lot, but the new lots still consistently separated putatively resistant and putatively susceptible isolates and this permits proposal of possible breakpoints for this assay system.     

Evaluation of Etest method for determining voriconazole and amphotericin B MICs for 162 clinical isolates of Cryptococcus neoformans

The performance of the Etest for voriconazole and amphotericin B susceptibility testing of 162 isolates of Cryptococcus neoformans was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 72 h at 35 degrees C. MICs were determined by Etest for all 162 isolates with RPMI 1640 agar containing 2% glucose (RPG agar) and were read after incubation for 72 h at 35 degrees C. The Etest results for both voriconazole and amphotericin B correlated well with reference MICs. Agreement was 94% for voriconazole and 99% for amphotericin B. When discrepancy was noted between the results obtained by Etest and broth microdilution for voriconazole, the Etest generally provided a higher MIC. The Etest method using RPG agar appears to be a useful method for determining the susceptibility of C. neoformans to voriconazole and amphotericin B.     

Application of the Etest to the antimicrobial susceptibility testing of Mycobacterium marinum clinical isolates.

Mycobacterium marinum, a well-recognized cutaneous pathogen, is usually treated by chemotherapy without available standardized in vitro susceptibility testing information. In this study, we have attempted to apply the stable-gradient method (Etest; AB Biodisk, Solna, Sweden) to susceptibility testing of M. marinum in order to assess the activities of eight antimicrobial agents against 60 recent clinical strains of M. marinum collected from 10 geographic sites within the United States. Two plated media (5% sheep blood Mueller-Hinton agar and Middlebrook 7H11 agar) were compared, and 7H11 agar was found to be superior in supporting the growth of all strains. Four reference strains of M. marinum were tested on five occasions with eight drugs (160 tests) in order to evaluate Etest reproducibility. Results were observed to be within 1 log2 dilution of the all-test median MIC for 97.5% of the Etests. Our MIC results for the 60 strains clearly demonstrate the best in vitro potency against M. marinum isolates to be as follows (rank order): trimethoprim-sulfamethoxazole (MIC at which 90% of the isolates are inhibited [MIC90], 0.25 and 4.25 microg/ml, respectively) = ethambutol > clarithromycin (MIC90, 1 microg/ml) > minocycline = doycycline (MIC90, 4 microg/ml) > amikacin (MIC90, 8 microg/ml). Rifampin was only marginally active against the M. marinum strains tested (MIC90, at the National Committee for Clinical Laboratory Standards) breakpoint of 1 microg/ml), and ciprofloxacin was not active (MIC90, 8 microg/ml). These data should enhance the empiric drug selection for contemporary M. marinum infections and also provide evidence that the Etest can be utilized to guide chemotherapy with alternative agents.     

Evaluation of Etest and disk diffusion methods compared with broth microdilution antifungal susceptibility testing of clinical isolates of Candida spp. against posaconazole

We performed Etest, disk diffusion, and broth microdilution susceptibility testing of 2,171 clinical isolates of Candida spp. against posaconazole. By using provisional breakpoints for comparison purposes only, the categorical agreement between the agar-based methods and broth microdilution results ranged from 93 to 98%, with <1% very major errors. The essential agreement (within 2 well dilutions) between the Etest and broth microdilution methods was 94%. These agar-based methods hold promise as simple and reliable methods for determination of the posaconzole susceptibilities of Candida spp.     

Interlaboratory reproducibility of Etest amphotericin B and caspofungin yeast susceptibility testing and comparison with the CLSI method

This study aimed to assess the interlaboratory reproducibility at four university hospital laboratories in the southeast region of France of the Etest technique for the determination of caspofungin (CAS) and amphotericin B (AMB) MICs and to compare it to the CLSI broth microdilution reference method. Consecutive clinical yeast isolates (n = 198) were included in the study. AMB and CAS MICs were read at 24 and 48 h. Interlaboratory reproducibility was estimated by using (i) an intraclass correlation coefficient (ICC), (ii) essential agreement (EA), and (iii) categorical agreement (CA). For Etest interlaboratory reproducibility for CAS, ICCs were 0.80 (95% confidence interval [CI], 0.76 to 0.84) and 0.81 (95% CI, 0.77 to 0.85) at 24 and 48 h, respectively. For AMB, the ICCs were 0.51 (95% CI, 0.43 to 0.58) and 0.69 (95% CI, 0.63 to 0.74) at 24 and 48 h, respectively. At 48 h, the between-center EAs ranged from 94.4 to 99.0% for both antifungals. For the comparison of the CLSI method and the Etest, the between-technique ICCs were 0.69 (95% CI, 0.63 to 0.74) and 0.62 (95% CI, 0.55 to 0.68) for CAS and AMB, respectively. The EAs ranged from 76.5 to 98.5% for CAS and from 90.3 to 97.4% for AMB according to the centers. CAs ranged from 87.9% to 91.4%, with four very major errors for 2 strains (1 Candida albicans strain and 1 Candida krusei strain), for CAS and from 97.5 to 99.5%, with four major errors, for AMB. In conclusion, the Etest showed a good interlaboratory reproducibility and a good correlation with the CLSI technique. It is well suited for the routine clinical laboratory and can thus be used to monitor clinical yeast isolates' in vitro susceptibilities in this setting.     

Biofilm production by clinical isolates of Candida species.

Candida species are a leading cause of infections especially in immunocompromised hosts. Usage of bio-prostheses such as IV lines and urinary catheters provide ample opportunity for Candida biofilms to set up a nidus for disease that is not easily amenable to conventional antifungal therapy. To understand the prevalence of biofilm producers among clinical isolates, 58 Candida isolates from immunocompromised patients were examined. Thirty of the 36 (83.3%) blood stream' isolates and 18 of the 22 (81.8%) oral isolates were biofilm producers. Biofilm producing blood stream isolates were significantly more among non-C. albicans Candida (93.1%) in comparison to C. albicans (42.9%).     

Speciation and amphotericin B sensitivity studies on blood isolates of Candida from burned patients

Methods of speciating Candida isolates from clinical specimens are described and the necessity of speciation is emphasized. Differences in susceptibility of C. albicans and C. tropicalis to amphotericin B were observed and the implications of this in relation to treatment with amphotericin B and the development of resistance are discussed.     

Microdilution susceptibility testing of amphotericin B, itraconazole, and voriconazole against clinical isolates of Aspergillus and Fusarium species

We compared the activities of amphotericin B, itraconazole, and voriconazole against clinical Aspergillus (n = 82) and Fusarium (n = 22) isolates by a microdilution method adopted from the National Committee for Clinical Laboratory Standards (NCCLS-M27A). RPMI 1640 (RPMI), RPMI 1640 supplemented to 2% glucose (RPMI-2), and antibiotic medium 3 supplemented to 2% glucose (AM3) were used as test media. MICs were determined after 24, 48, and 72 h. A narrow range of amphotericin B MICs was observed for Aspergillus isolates, with minor variations among species. MICs for Fusarium isolates were higher than those for Aspergillus isolates. MICs of itraconazole were prominently high for two previously defined itraconazole-resistant Aspergillus fumigatus isolates and Fusarium solani. Voriconazole showed good in vitro activity against itraconazole-resistant isolates, but the MICs of voriconazole for F. solani were high. RPMI was the most efficient medium for detection of itraconazole-resistant isolates, followed by RPMI-2. While the significance remains unclear, AM3 lowered the MICs, particularly those of amphotericin B.     

Determination of antimicrobial susceptibility patterns of Nocardia spp. from clinical specimens by Etest

Susceptibilities to 11 antimicrobial agents were determined by Etest for 93 Nocardia isolates from clinical specimens and 15 type strains belonging to different Nocardia spp. All isolates were susceptible to trimethoprim-sulphamethoxazole, amikacin and linezolid, but susceptibilities of the various Nocardia spp. to beta-lactams, aminoglycosides, ciprofloxacin and clarithromycin varied markedly. Overall, there was a good correlation between the drug resistance patterns and the species identification established by conventional phenotypic tests and 16S rDNA sequencing. Among the different species encountered, Nocardia farcinica and Nocardia brasiliensis displayed the most multiresistant profiles, with resistance to imipenem occurring mainly among isolates of N. brasiliensis and Nocardia abscessus. The species variability in susceptibility profiles and the numerous recent taxonomic changes means that in-vitro susceptibility tests may be a complementary tool for the identification of Nocardia isolates from human clinical specimens. Further studies on a larger number of species from more diverse geographical sources, including species that are found less commonly among clinical isolates, are required to validate and extend the results.     

Virulence and thrombocyte affectation of two Aspergillus terreus isolates differing in amphotericin B susceptibility

Aspergillus terreus-induced invasive infections exhibit high lethality, partly due to the intrinsic resistance for amphotericin B (AmB). We compared the virulence and pathogenesis of an AmB-resistant isolate of A. terreus (ATR) with that of a rare variant showing enhanced sensitivity for AMB (ATS). The modifications that result in enhanced AmB sensitivity of isolates are not associated with reduced virulence in vivo; instead, the ATS-infected mice died even faster than the ATR-infected animals. Since A. terreus enters the blood stream in most patients and frequently induces thrombosis, we studied a putative correlation between virulence of the two A. terreus isolates and their effect on thrombocytes. Those mice infected with the more virulent ATS isolate had lower thrombocyte numbers and more phosphatidylserine exposure on platelets than ATR-infected mice. In vitro experiments confirmed that ATS and ATR differ in their effect on thrombocytes. Conidia, aleurioconidia and hyphae of ATS were more potent than ATR to trigger thrombocyte stimulation, and thrombocytes adhered better to ATS than to ATR fungal structures. Furthermore, ATS secreted more soluble factors that triggered platelet stimulation than ATR. Thus, it might be suggested that the capacity of a fungal isolate to modulate thrombocyte parameters contributes to its virulence in vivo.     

Detection of caspofungin resistance in Candida spp. by Etest

The caspofungin susceptibilities of 28 Candida sp. clinical isolates, including 8 caspofungin-resistant isolates characterized by mutations in the Fks1 protein, were determined by the Etest in RPMI and AM3 media. Good discrimination between wild-type and mutant isolates was obtained. These results suggest that the Etest is valuable for the detection of caspofungin resistance in Candida spp.     

Detection of amphotericin B resistance in Candida haemulonii and closely related species by use of the Etest, Vitek-2 yeast susceptibility system, and CLSI and EUCAST broth microdilution methods

The emerging fungal pathogens Candida haemulonii and Candida pseudohaemulonii often show high-level resistance to amphotericin B (AMB). We compared the utilities of five antifungal susceptibility testing methods, i.e., the Etest using Mueller-Hinton agar supplemented with glucose and methylene blue (Etest-MH), the Etest using RPMI agar supplemented with glucose (Etest-RPG), the Vitek-2 yeast susceptibility system, and the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution methods, for the detection of AMB-resistant isolates of C. haemulonii and closely related species. Thirty-eight clinical isolates (8 C. haemulonii, 10 C. pseudohaemulonii, and 20 Candida auris isolates) were analyzed. Of the 18 C. haemulonii and C. pseudohaemulonii isolates, 18, 15, 18, 10, and 9 exhibited AMB MICs of >1 μg/ml by the Etest-MH, Etest-RPG, Vitek-2, CLSI, and EUCAST methods, respectively. All 20 C. auris isolates showed AMB MICs of ≤1 μg/ml by all five methods. Of the methods, the Etest-MH generated the broadest distribution of AMB MICs for all 38 isolates and showed the best discrimination between the C. haemulonii and C. pseudohaemulonii isolates (4 to 32 μg/ml) and those of C. auris (0.125 to 0.5 μg/ml). Taking the Etest-MH as the reference method, the essential agreements (within two dilutions) for the Etest-RPG, Vitek-2, CLSI, and EUCAST methods were 84, 92, 55, and 55%, respectively; the categorical agreements were 92, 92, 79, and 76%, respectively. This study provides the first data on the efficacy of the Etest-MH and its excellent agreement with Vitek-2 for discriminating AMB-resistant from AMB-susceptible isolates of these Candida species.     

In vitro activity of nystatin compared with those of liposomal nystatin, amphotericin B, and fluconazole against clinical Candida isolates

We investigated the in vitro activity of nystatin and liposomal nystatin against 103 Candida isolates to determine the effect of both time and medium on MICs. We also compared the nystatin MICs with those of amphotericin B and fluconazole. Testing was performed in accordance with the National Committee for Clinical Laboratory Standards M27-A microdilution methodology with RPMI 1640, RPMI 1640 supplemented with glucose to 2% (RPMI-2), and antibiotic medium 3 supplemented with glucose to 2% (AM3). While nystatin MICs were similar to or slightly lower than liposomal nystatin MICs in RPMI 1640 and RPMI-2, they were markedly higher than liposomal nystatin MICs in AM3. Use of AM3 and determination of the MIC after 24 h of incubation provided a slightly wider range of liposomal nystatin MICs (0.06 to >16 microg/ml). Under these conditions, the MICs at which 90% of isolates were inhibited of nystatin and liposomal nystatin were 2 and 1 microg/ml, respectively. Nystatin and liposomal nystatin in general showed good activity against all Candida spp. tested. Although the MICs of nystatin and liposomal nystatin tended to rise in parallel with the amphotericin B MICs, nystatin and liposomal nystatin MICs of 1 to 2 and 0.5 to 1 microg/ml, respectively, were obtained for seven and six, respectively, of nine isolates for which amphotericin B MICs were >or=0.25 microg/ml. No correlation between fluconazole and nystatin or liposomal nystatin MICs was observed. As amphotericin B MICs of >or=0.25 microg/ml correlate with in vitro resistance, these results suggest that liposomal nystatin might have activity against some amphotericin B-resistant isolates. In vivo testing in animal models is required for clarification of this issue.     

Voriconazole and fluconazole susceptibility of Candida isolates

An adapted NCCLS M27-A method was used to evaluate the activity of voriconazole (VRC) and fluconazole (FLC) against 295 Candida isolates collected from 189 patients (including isolates from deep sites). Isolates included 186 C. albicans, 54 C. glabrata, 27 C. tropicalis, 14 C. parapsilosis, 6 C. krusei, 6 C. lusitaniae, 1 C. lypolytica and 1 C. sake. Forty-two isolates had reduced susceptibility to FLC (MIC >8 mg/L); 83.3% of these had VRC MICs < or =2 mg/L (9 of 11 C. albicans, 18 of 19 C glabrata, 6 of 6 C. krusei, 2 of 2 C. lusitaniae and 0 of 4 C. tropicalis), including 60% of isolates collected from deep-seated infections. These results suggested that in the era of azole resistance, VRC has a promising antifungal activity for serious infections with Candida spp., including most species with low susceptibility to FLC and uncommonly isolated species.     

Evaluation of Etest for susceptibility testing of multidrug-resistant isolates of Mycobacterium tuberculosis

To prescribe effective treatment schemes for patients with tuberculosis, more-efficient susceptibility testing techniques for Mycobacterium tuberculosis are needed, especially in regions with multidrug resistance. Etest (AB BIODISK, Solna, Sweden) is a simple technique that provides quantitative drug susceptibility results for M. tuberculosis in 5 to 10 days from a culture grown at low cost. The performance of Etest was compared to that of the reference proportion method, using 95 M. tuberculosis clinical isolates of which 42.1% (40 of 95) were resistant to at least one antibiotic by the reference method. Overall agreement between Etest and the reference method was 98.9% (94 of 95) for detection of multidrug resistance; for resistance to individual drugs, agreement was 97.9% (93 of 95) for rifampin, 96.0% (92 of 95) for ethambutol, 94.7% (90 of 95) for isoniazid, and 85.3% (81 of 95) for streptomycin. This study supports the utility of Etest for timely detection of drug resistance in M. tuberculosis and for use in tuberculosis control programs.     

Comparison of Etest and agar dilution method for antimicrobial susceptibility testing of Flavobacterium isolates.

The Etest was evaluated as a possible alternative to the standard agar dilution method for susceptibility testing of nine antimicrobial agents against Flavobacterium species. In studies of 100 clinical isolates, the agreement between the MICs (+/-1 log2 dilution) obtained by the two methods was acceptable for cefotaxime, ceftazidime, amikacin, minocycline, ofloxacin, and ciprofloxacin (> 90%). Conversely, the agreement between the results obtained for piperacillin was limited (84%). The overall agreement was 92.5%.     

Multisite reproducibility of the Etest MIC method for antifungal susceptibility testing of yeast isolates.

A multicenter study was performed to establish the interlaboratory reproducibility of Etest, to provide an additional comparison of Etest MICs with reference broth macrodilution MICs, and to develop some tentative quality control (QC) guidelines for using Etest for antifungal susceptibility testing of Candida spp. Two QC strains, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, were tested by Etest against amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole in each of four laboratories. The QC strains were tested 20 times each against the five antifungal agents by using a common lot of RPMI agar. A total of 80 MICs per drug per strain were generated during the study. Overall, 98 to 100% of the MICs fell within a 3 log2 dilution range for the respective yeast-antifungal agent combinations. The level of agreement of Etest MICs with broth macrodilution MICs was 86 to 100% with amphotericin B (C. krusei and C. parapsilosis), itraconazole (C. krusei and C. parapsilosis), flucytosine (C. parapsilosis), and fluconazole (C. parapsilosis). A lower level of agreement was observed with ketoconazole (C. krusei and C. parapsilosis). Although all participants reported identical Etest MICs, the MICs of flucytosine and fluconazole when tested against C. krusei fell well above the upper limits of the reference range for this strain. The tentative QC limits for the two QC strains and five antifungal agents when tested by the Etest methodology are the same as the QC limits when tested by the reference broth macrodilution method for amphotericin B and C. krusei, itraconazole and C. krusei, flucytosine and C. parapsilosis, fluconazole and C. parapsilosis, and itraconazole and C. parapsilosis. The Etest QC ranges are 1 dilution broader (4-dilution range) than the reference macrodilution method QC ranges for ketoconazole and C. krusei, amphotericin B and C. parapsilosis, and ketoconazole and C. parapsilosis.