Evaluation of Etest Method for Determining Caspofungin (MK-0991) Susceptibilities of 726 Clinical Isolates of Candida Species

The performance of the Etest for testing the susceptibilities to caspofungin (MK-0991) of 726 isolates of Candida spp. was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. MICs were determined by Etest for all 726 isolates with RPMI agar containing 2% glucose (RPG) and were read after incubation for 48 h at 35 degrees C. The Candida isolates included Candida albicans (n = 486), Candida glabrata (n = 96), Candida tropicalis (n = 51), Candida parapsilosis (n = 47), Candida krusei (n = 11), Candida lusitaniae (n = 2), and Candida guilliermondii (n = 33). In addition, a subset of 314 isolates were also tested by Etest using Casitone agar (CAS) and antibiotic medium 3 agar (AM3). The Etest results obtained using RPG correlated well with reference MICs. Overall agreement was 94% with RPG, 82% with CAS, and 79% with AM3. When RPG was used, agreement ranged from 79% for C. parapsilosis to 100% for C. krusei, C. lusitaniae, and C. guilliermondii. When CAS was used, agreement ranged from 0% for C. lusitaniae to 100% for C. glabrata. With AM3, agreement ranged from 0% for C. lusitaniae to 100% for C. guilliermondii. All three media supported growth of each of the Candida species. Etest results were easy to read, with sharp zones of inhibition. In most instances (75%) where a discrepancy was observed between the Etest and the reference method, the Etest MIC was lower. The Etest method using RPG appears to be useful for determining caspofungin susceptibilities of Candida species.     

Evaluation of the Etest Method for Determining Fluconazole Susceptibilities of 402 Clinical Yeast Isolates by Using Three Different Agar Media

The performance of the Etest for fluconazole susceptibility testing of 402 yeast isolates was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI agar containing 2% glucose (RPG), Casitone agar (CAS), and Mueller-Hinton agar (MHA) and were read after incubation for 48 h at 35°C. The yeast isolates included Candida albicans (n = 161), Candida glabrata (n = 41), Candida tropicalis (n = 35), Candida parapsilosis (n = 29), Candida krusei (n = 32), Candida lusitaniae (n = 31), Candida species (n = 19), Cryptococcus neoformans (n = 40), and miscellaneous yeast species (n = 14). The Etest results correlated well with reference MICs. Overall agreement was 94% with RPG, 97% with CAS, and 53% with MHA. When RPG was used, agreement ranged from 89% for Candida spp. to 100% for C. krusei. When CAS was utilized, agreement ranged from 93% for Cryptococcus neoformans to 100% for C. tropicalis, C. parapsilosis, C. lusitaniae, Candida spp., and miscellaneous yeast species. With MHA, agreement ranged from 17% for C. parapsilosis to 90% for C. krusei. Both RPG and CAS supported growth of all yeast species, whereas growth on MHA was comparatively weaker. Etest results were somewhat easier to read on CAS. The Etest method using either RPG or CAS, but not MHA, appears to be a viable alternative to the NCCLS reference method for determining fluconazole susceptibilities of yeasts.     

Evaluation of Etest method for determining fluconazole and voriconazole MICs for 279 clinical isolates of Candida species infrequently isolated from blood

The performance of Etest in fluconazole and voriconazole testing of 279 isolates of uncommon Candida spp. was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS)-approved standard broth microdilution (BMD) method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48 h at 35 degrees C. The isolates include Candida krusei, C. lusitaniae, C. guilliermondii, C. kefyr, C. rugosa, C. lipolytica, C. pelliculosa, C. dubliniensis, C. famata, C. zeylanoides, C. inconspicua, and C. norvegensis. Overall agreement between Etest and BMD MICs was 96% for fluconazole and 95% for voriconazole. Where a discrepancy was observed between Etest and the reference method, the Etest tended to give lower values with both fluconazole and voriconazole. The Etest method using RPMI agar appears to be a useful method for determining fluconazole and voriconazole susceptibilities of uncommon species of Candida.     

In Vitro Activity of Posaconazole against Clinical Isolates of Dermatophytes

A broth macrodilution method following the recommendations established by the National Committee for Clinical Laboratory Standards was used to compare the in vitro activity of posaconazole (PCZ) with that of itraconazole (ITC) against 30 clinical isolates of dermatophytes belonging to six different species. In terms of MICs, PCZ showed an activity equal to that of ITC. MICs of PCZ at which 50% (MIC(50)) and 90% (MIC(90)) of the isolates were inhibited were 0.5 and > 4.0 microg/ml, respectively. The MIC(50) and MIC(90) of ITC were 1.0 and > 4.0 microg/ml, respectively. However, PCZ showed a more potent fungicidal activity than that of ITC against isolates belonging to the genus Microsporum (P = 0.03). PCZ merits further investigation as a potentially useful agent for treatment of dermatophytosis.     

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.     

Vancomycin MIC for Methicillin-Resistant Coagulase-Negative Staphylococcus Isolates: Evaluation of the Broth Microdilution and Etest Methods

Vancomycin MIC results were determined by the broth microdilution (BMD) method and by Etest using 130 methicillin-resistant coagulase-negative staphylococcus bloodstream isolates obtained from a tertiary hospital. The majority (98.5%) of MIC results determined by BMD were ≤1 μg/ml, in contrast to MIC results determined by Etest (72.3% were ≥1.5 μg/ml). The MICs obtained by Etest were, in general, 1- to 2-fold higher than the MICs obtained by BMD.Coagulase-negative staphylococci (CoNS) have emerged as important nosocomial pathogens during the last decade, particularly in nosocomial bloodstream infections (4). Resistance to methicillin in CoNS is very common among isolates recovered from hospitalized individuals (4, 19). For this reason, vancomycin is usually the drug of choice for treatment of infections by methicillin-resistant CoNS (MRCoNS) (17).A reduction in the efficacy of vancomycin has been described in studies of methicillin-resistant Staphylococcus aureus (MRSA) infections treated with this antibiotic, and it has been suggested that slight increases in vancomycin MICs of between 1 and 2 μg/ml, which are within the susceptible range, may be related to suboptimal clinical outcomes (12, 16). Therefore, the determination of the MIC of vancomycin has been recommended for these pathogens (3). Some of these studies have used the broth microdilution (BMD) method for determining vancomycin MICs, while others have used the commercial Etest technique (AB Biodisk, Solna, Sweden). It has been reported that the Etest provides higher MICs than those obtained with BMD in S. aureus, mainly MRSA (14, 15). Nevertheless, there is no study comparing both methodologies for determination of the MIC of vancomycin in CoNS.Considering the increasing incidence of MRCoNS, the need for MIC determination of vancomycin, and the absence of studies assessing the performance of the Etest with these organisms, we aimed to compare the Etest and BMD methods for determination of the MICs of vancomycin in MRCoNS isolates.A total of 130 clinical isolates of CoNS recovered from blood samples obtained from patients who were hospitalized from May 2004 to August 2005 at the Hospital de Clínicas de Porto Alegre were analyzed. Only one isolate per patient was included. Blood cultures were performed using BacT/Alert (bioMérieux, Marcy l''Etoile, France). The colony morphology, Gram stain reaction, catalase testing, and absence of the coagulase enzyme were used to identify CoNS. Isolates were identified as Staphylococcus epidermidis by PCR using primers for tuf (10). The isolates that were not identified as S. epidermidis by PCR were identified using the API ID 32 Staph (bioMérieux, Marcy l''Etoile, France) semiautomated system, according to the instructions of the manufacturer. Results yielding a quality of identification of 85% or higher were accepted. The presence of the mec A gene was assessed by PCR using specific primers (13). The MICs of vancomycin were determined in duplicate by reference BMD, as recommended by CLSI, using in-house-prepared panels. The following dilutions of vancomycin were tested: 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg/ml. The standard Etest procedure was performed using Mueller-Hinton agar (Becton Dickinson, Sparks, MD), with an inoculum density equivalent to a 0.5 McFarland standard. Vancomycin Etest strips were placed onto the agar with sterile forceps. The cultures were incubated for 24 h at 35°C. S. aureus ATCC 29213 was used for quality control (3).The Wilcoxon test was used to compare the MICs obtained by Etest and BMD. Two sets of comparisons were done, one with the exact MIC values determined by Etest and the other with the MICs determined by Etest rounded up to the next dilution.All 130 isolates of CoNS proved to be mec A positive and were identified as follows: 87 (66.9%) were S. epidermidis isolates, 13 (10.0%) were S. haemolyticus isolates, 12 (9.2%) were S. hominis isolates, and 11 (8.5%) were S. capitis isolates. Seven isolates (5.4%) were not identified to the species level. The MICs of vancomycin ranged from 0.25 to 2 μg/ml by BMD and from 0.38 to 3 μg/ml by Etest. No discrepancies were observed in duplicates performed by BMD. The MIC₅₀s and MIC₉₀s for vancomycin were both 1 μg/ml by BMD and 1.5 and 2 μg/ml, respectively, by Etest (P < 0.001 for both comparisons). Most MICs determined by Etest were ≥1.5 μg/ml (94 isolates, 72.3%), while most MICs determined by BMD were ≤1 μg/ml (128 isolates, 98.5%). A total of 113 (86.9%) and 5 (3.8%) isolates presented MICs determined by Etest that were 1- and 2-fold dilutions higher than those determined by BMD, respectively. Only 10 isolates (7.7%) presented the same MIC using the two methods (Fig. ​(Fig.1).1). When the Etest MICs of 0.38, 0.75, 1.5, and 3 μg/ml were converted to 0.5, 1, 2, and 4 μg/ml, respectively, there was an even higher discrepancy between the two methods. In this case, almost all MRCoNS had MICs of ≥1 μg/ml (124 isolates, 95.4%) (Fig. ​(Fig.2).2). MICs determined by Etest were higher than those determined by BMD for all MRCoNS species, when analyzed separately (Table ​(Table1).1). No isolate had a MIC higher than 4 μg/ml, regardless of the method used.Open in a separate windowFIG. 1.Scattergram of correlation between vancomycin MICs obtained by the broth microdilution and Etest methods.Open in a separate windowFIG. 2.Scattergram of correlation between vancomycin MICs obtained by the broth microdilution method and the Etest method after rounding up.

TABLE 1.

Distribution of vancomycin MICs according to CoNS species

Epidemiological Cutoff Values for Fluconazole,Itraconazole, Posaconazole,and Voriconazole for Six Candida Species as Determined by the Colorimetric Sensititre YeastOne Method

In the absence of clinical breakpoints (CBP), epidemiological cutoff values (ECVs) are useful to separate wild-type (WT) isolates (without mechanisms of resistance) from non-WT isolates (those that can harbor some resistance mechanisms), which is the goal of susceptibility tests. Sensititre YeastOne (SYO) is a widely used method to determine susceptibility of Candida spp. to antifungal agents. The CLSI CBP have been established, but not for the SYO method. The ECVs for four azoles, obtained using MIC distributions determined by the SYO method, were calculated via five methods (three statistical methods and based on the MIC₅₀ and modal MIC). Respectively, the median ECVs (in mg/liter) of the five methods for fluconazole, itraconazole, posaconazole, and voriconazole (in parentheses: the percentage of isolates inhibited by MICs equal to or less than the ECVs; the number of isolates tested) were as follows: 2 (94.4%; 944), 0.5 (96.7%; 942), 0.25 (97.6%; 673), and 0.06 (96.7%; 849) for Candida albicans; 4 (86.1%; 642), 0.5 (99.4%; 642), 0.12 (93.9%; 392), and 0.06 (86.9%; 559) for C. parapsilosis; 8 (94.9%; 175), 1 (93.7%; 175), 2 (93.6%; 125), and 0.25 (90.4%; 167) for C. tropicalis; 128 (98.6%; 212), 4 (95.8%; 212), 4 (96.0%; 173), and 2 (98.5; 205) for C. glabrata; 256 (100%; 53), 1 (98.1%; 53), 1 (100%; 33), and 1 (97.9%; 48) for C. krusei; 4 (89.2%; 93), 0.5 (100%; 93), 0.25 (100%; 33), and 0.06 (87.7%; 73) for C. orthopsilosis. All methods included ≥94% of isolates and yielded similar ECVs (within 1 dilution). These ECVs would be suitable for monitoring emergence of isolates with reduced susceptibility by using the SYO method.     

Increased Adherence of Fluconazole-Resistant Isolates of Candida Species to Explanted Esophageal Mucosa

 The adherence of fluconazole-resistant and fluconazole-susceptible isolates of Candida albicans to explanted rabbit esophageal mucosa was examined in vivo. Among six Candida albicans isolates collected from HIV-infected patients, three fluconazole-resistant (MIC>64 μg/ml) isolates attached more avidly than three fluconazole-susceptible strains (MIC≤0.5 μg/ml) to esophageal mucosa (P≤0.05). When three strains each of six different Candida spp. were compared, the more inherently fluconazole-resistant isolates adhered more avidly in the following order: Candida glabrata>Candida krusei>Candida albicans fluconazole-sensitive >Candida tropicalis>Candida parapsilosis. Nonetheless, fluconazole-resistant Candida albicans demonstrated the greatest degree of adherence in comparison to all fluconazole-susceptible Candida albicans (P<0.001) and to all Candida spp. tested (P<0.001). Thus, the refractoriness of esophageal candidiasis in patients infected with fluconazole-resistant isolates may be related to both in vitro drug resistance and increased mucosal adherence.     

Comparative in Vitro Activity of Voriconazole and Itraconazole Against Fluconazole-Susceptible and Fluconazole-Resistant Clinical Isolates of Candida Species from Spain

 The in vitro activity of voriconazole was compared with that of itraconazole against 299 fluconazole-susceptible (MIC≤8 μg/ml) and 130 fluconazole-resistant (MIC≥16 μg/ml) clinical isolates of Candida spp. An adaptation of the National Committee for Clinical Laboratory Standards reference method was employed for determination of MICs. Voriconazole showed more potent activity than either fluconazole and itraconazole, even against some Candida albicans, Candida glabrata, and Candida krusei isolates resistant to fluconazole. However, for fluconazole-resistant isolates, the MICs of itraconazole and voriconazole were proportionally higher than for fluconazole-susceptible isolates. These data may indicate cross-resistance.     

Electrophoretic Karyotyping and Triazole Susceptibility of Candida glabrata Clinical Isolates

 A series of 35 strains of Candida glabrata isolated from 29 subjects (5 AIDS patients and 24 HIV-seronegative individuals) were typed by electrophoretic karyotyping and tested for their susceptibilities to both fluconazole and itraconazole. Almost every individual harboured his/her own specific isolate (DNA type). Neither the source of isolation nor the patient's HIV status was associated with a given DNA type. Recurrences were generally due to the persistence of the same DNA type over time. Only 9% of the isolates showed reduced susceptibility to fluconazole (MIC≥8.0 μg/ml), while 43% of the isolates showed reduced susceptibility to itraconazole (MIC≥0.25 μg/ml) (P=0.02). These data show that electrophoretic karyotyping is a useful technique for DNA typing of isolates of Candida glabrata. Care must be taken prior to inititation of antifungal therapy with either of these drugs.     

Determination of MICs of aminocandin for Candida spp. and filamentous fungi

Candida and Aspergillus spp., as well as other filamentous molds, have increasingly been reported as the causes of severe invasive fungal infections. We evaluated the new echinocandin aminocandin (AMN) for its antifungal activities against a range of fungal pathogens by determination of the MICs for the organisms. The MICs of the comparator drugs amphotericin B, caspofungin, micafungin, and voriconazole were also determined. The MICs of AMN for 25 strains each of non-Candida albicans Candida spp. (including Candida parapsilosis, Candida krusei, Candida guilliermondii, and Candida tropicalis), Aspergillus fumigatus, Scedosporium spp., Fusarium spp., and zygomycetes (including Absidia, Mucor, and Rhizopus spp.) were determined by using the Clinical and Laboratory Standards Institute M27-A2 and M38-A methodologies for yeasts and filamentous molds, respectively. The MIC ranges of AMN for all yeasts were similar (0.03 to 4.0 microg/ml), while the MIC ranges of AMN for filamentous fungi were species specific. AMN demonstrated potent antifungal activity against A. fumigatus, limited activity against Scedosporium spp., and no activity against zygomycetes or Fusarium spp. Our data showed that AMN demonstrated potent antifungal activities against all of the yeasts and Aspergillus isolates tested, suggesting that AMN could be an important addition to our arsenal of antifungals for the treatment of invasive fungal disease.     

Multicenter Evaluation of Candida QuickFISH BC for Identification of Candida Species Directly from Blood Culture Bottles

Candida species are common causes of bloodstream infections (BSI), with high mortality. Four species cause >90% of Candida BSI: C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis. Differentiation of Candida spp. is important because of differences in virulence and antimicrobial susceptibility. Candida QuickFISH BC, a multicolor, qualitative nucleic acid hybridization assay for the identification of C. albicans (green fluorescence), C. glabrata (red fluorescence), and C. parapsilosis (yellow fluorescence), was tested on Bactec and BacT/Alert blood culture bottles which signaled positive on automated blood culture devices and were positive for yeast by Gram stain at seven study sites. The results were compared to conventional identification. A total of 419 yeast-positive blood culture bottles were studied, consisting of 258 clinical samples (89 C. glabrata, 79 C. albicans, 23 C. parapsilosis, 18 C. tropicalis, and 49 other species) and 161 contrived samples inoculated with clinical isolates (40 C. glabrata, 46 C. albicans, 36 C. parapsilosis, 19 C. tropicalis, and 20 other species). A total of 415 samples contained a single fungal species, with C. glabrata (n = 129; 30.8%) being the most common isolate, followed by C. albicans (n = 125; 29.8%), C. parapsilosis (n = 59; 14.1%), C. tropicalis (n = 37; 8.8%), and C. krusei (n = 17; 4.1%). The overall agreement (with range for the three major Candida species) between the two methods was 99.3% (98.3 to 100%), with a sensitivity of 99.7% (98.3 to 100%) and a specificity of 98.0% (99.4 to 100%). This study showed that Candida QuickFISH BC is a rapid and accurate method for identifying C. albicans, C. glabrata, and C. parapsilosis, the three most common Candida species causing BSI, directly from blood culture bottles.     

Rapid Screening Tests for Determining In Vitro Susceptibility of Herpes Simplex Virus Clinical Isolates

The susceptibility of human herpes simplex virus (HSV) to acyclovir (ACV) was determined with the use of a single dose of the drug (1 and 2 μg of ACV per ml for HSV-1 and HSV-2, respectively) in two rapid assays: a rapid cytopathic effect inhibitory assay (Rapid CIA) and a rapid dye uptake assay (Rapid DUA). These tests allow the simultaneous determination of virus titer and susceptibility to ACV at a determined viral concentration (100 50% tissue culture infective doses and 100 50% dye uptake units). These tests were compared with a conventional susceptibility assay (dye uptake assay) and showed similar results. Indeterminate results with the Rapid CIA appeared in 3 of 30 samples. With the use of both Rapid CIA and Rapid DUA, we were able to determine the susceptibility of 100% of the isolates. The rapid tests, unlike conventional assays, are able to provide susceptibility results within 3 days after the virus has been isolated from a clinical specimen and could thus play a direct role in therapeutic decisions.     

Identification and Susceptibility Profile of Candida fermentati from a Worldwide Collection of Candida guilliermondii Clinical Isolates

Candida fermentati isolates make up a small percentage of the clinical isolates of the Candida guilliermondii complex and have a global distribution pattern. With the exception that the MICs of micafungin were significantly lower, the calculated average MICs for C. fermentati were not significantly different from those for C. guilliermondii.     

In Vitro Activity of the New Echinocandin Antifungal, MK-0991, against Common and Uncommon Clinical Isolates of Candida Species

 A broth macrodilution method, performed as recommended by the National Committee for Clinical Laboratory Standards, was used for comparative testing of the new echinocandin antifungal agent MK-0991 and fluconazole against 50 yeast isolates belonging to 12 species of Candida. MK-0991 was shown to be highly effective against both fluconazole-susceptible and -resistant Candida spp., yielding minimum inhibitory concentrations ranging from ≤0.19 to 0.78 μg/ml. Fungicidal activity was exerted at ≤1.5 μg/ml for 73% of the isolates tested. This study suggests that MK-0991 has significant potential for clinical development.     

In vitro susceptibility testing of filamentous fungi: comparison of Etest and reference microdilution methods for determining itraconazole MICs

The performance of the Etest for itraconazole susceptibility testing of 50 isolates of filamentous fungi was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS) proposed standard microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. Etest MICs were determined with RPMI agar containing 2% glucose and with Casitone agar and were read after incubation for 24 h (Aspergillus spp. and Rhizopus spp.) and 48 h (all species except Rhizopus spp.) at 35 degrees C. The isolates included Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Fusarium spp., Pseudallescheria boydii, Rhizopus spp., Paecilomyces variotii, and an Acremonium sp. Overall agreement between Etest and microdilution MICs was 96% with RPMI agar and 80% with Casitone agar. The agreement was 100% for all species except Rhizopus spp. (83%) and Paecilomyces varioti (0%) with RPMI agar. When Casitone agar was used, the agreement ranged from 50% with Rhizopus spp. to 100% with Fusarium spp., P. boydii, P. varioti, and an Acremonium sp. Notably, for Aspergillus spp., the agreement between itraconazole Etest MICs read at 24 h and reference microdilution MICs read at 48 h was 100% with both RPMI and Casitone agar. Both media supported the growth of all filamentous fungi tested. Where a discrepancy was observed between Etest and the reference method, the Etest MIC was generally higher. The Etest method using RPMI agar appears to be a useful method for determining itraconazole susceptibilities of Aspergillus spp. and other filamentous fungi.     

Evaluation of Etest for susceptibility testing of Mycobacterium tuberculosis

The Etest method for susceptibility testing of Mycobacterium tuberculosis was compared to the agar proportion method using four first-line agents and two fluoroquinolones. Catergorical agreement between the methods was 100% for rifampin, ethambutol, streptomycin, and ofloxacin and 98% for isoniazid. Results were obtained in 6 to 10 days by Etest. The Etest method is suitable for testing the agents evaluated against M. tuberculosis.     

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.     

3144株临床分离细菌和念珠菌的分布及耐药性分析

目的了解本院临床分离细菌和念珠菌的分布特征和耐药性,为合理使用抗生素提供依据。方法大多数分离细菌的鉴定和药敏试验利用BD Phoenix仪,少数利用手工鉴定和K-B法。念珠菌利用显色平板进行分离和鉴定,K-B法测药敏。结果3144株细菌和念珠菌中前6位的种类及百分比为白色念珠菌12.02%、铜绿假单胞菌11.10%、大肠埃希菌9.64%、金黄色葡萄球菌7.09%、表皮葡萄球菌6.36%和肺炎型肺炎克雷伯菌6.14%。G-杆菌中耐药率较低的为亚安培南、哌拉西林/他唑巴坦、头孢哌酮/舒巴坦和阿米卡星。大肠埃希菌和肺炎克雷伯菌产超广谱β-内酰胺酶（ESBLs）的检出率为44.2%和43.5%。G＋球菌耐药率较低的为万古霉素、替考拉宁、呋喃妥因、阿米卡星、氯霉素、头孢哌酮/舒巴坦和哌拉西林/他唑巴坦。金黄色葡萄球菌和凝固酶阴性葡萄球菌对甲氧西林耐药率分别为52.1%和65.1%。念珠菌对两性霉素B和制菌霉素的耐药率均为0。结论本院临床分离肠杆菌科各种细菌产ESBLs水平、非发酵菌耐药率和葡萄球菌对甲氧西林耐药率均较高,应加强抗生素的合理使用。     

Determination of Echinocandin MICs for Candida Species in Less than 8 Hours: Comparison of the Rapid Susceptibility Assay with the Clinical and Laboratory Standards Institute's Broth Microdilution Assay

The echinocandins prevent fungal cell wall synthesis by inhibiting β-1,3-glucan synthesis, a significant glucose-consuming process. Previous studies suggested that echinocandin inhibitory activity is evident within 1 h of exposure. We hypothesized that a susceptibility assay based on glucose consumption may provide clinically useful MICs rapidly. The rapid susceptibility assay (RSA), which provides MICs in less than 8 h, was compared with the standard broth microdilution susceptibility assay (Clinical and Laboratory Standards Institute, document M27-A3, 2008) for 56 Candida species strains. Variables which are known to influence MICs determined by the M27-A3 method were also assessed for their effects on the RSA results. Excellent agreement (>90%) between the results of the RSA and M27-A3 methods was achieved for all three FDA-approved echinocandins (micafungin, caspofungin, and anidulafungin). Candida lusitaniae strains were responsible for most of the discordant results. Assay variables such as the test medium, the age of the inoculum culture, and the presence of human serum affected MIC results from the RSA and the M27-A3 method similarly. The RSA is equivalent to the standard M27-A3 method for determining echinocandin MICs for Candida species. The RSA provides MIC results in less than 8 h and can be applied to old and young yeast colonies. The assay could potentially provide clinically useful MICs on the same day that yeast growth from a specimen is first detected on solid medium.Echinocandins are the newest class of FDA-approved drugs for the treatment of systemic fungal infections and the first approved class of antifungals that target the cell wall, specifically the synthesis of β-1,3-glucan. The spectrum of fungi which are susceptible to echinocandins is narrower than the spectrum of fungi susceptible to the polyenes, despite the presence of β-1,3-glucan in the cell walls of many nonzygomycetous fungi. However, the echinocandins have proven to be excellent agents for the treatment of candidiasis. In 2004, the Infectious Diseases Society of America published a practice guideline in which echinocandins are listed as one of the first-line options for the treatment of candidemia (8).Similar to the actions of β-lactam antibacterial agents, the inhibition of cell wall polysaccharide synthesis by echinocandins potentially results in cell death. Echinocandins inhibit β-1,3-glucan synthase (GS) in the cell membrane by targeting the Fks1p and Fks2p components of GS complexes. As a consequence, β-1,3-glucan, which is the predominant polysaccharide in the cell walls of Candida species, is not produced while cell wall remodeling and cell expansion continue to occur, resulting in weakness in the wall and susceptibility to cell lysis due to overwhelming turgor pressure (6). A potential outcome of the events prior to cell death is diminution of glucose uptake or consumption.Clancy et al. (2) have shown that cells exposed to echinocandins for only 1 h at levels in excess of the known MIC for the strain exhibit growth inhibition, as demonstrated by decreased numbers of CFU compared to those of untreated controls. This observation suggested that it may be possible to determine MICs of echinocandins for different yeast strains on the same day of testing by using the rapid susceptibility assay (RSA), especially because the RSA is based on levels of glucose consumption by cells exposed to the drug relative to those by control cells (4, 11). The RSA is similar to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 broth microdilution assay except that the RSA inoculum concentration is higher, the incubation period is shorter, and the end point is based on glucose uptake measured spectrophotometrically (3, 4, 11).The RSA was originally used by Cutler et al. (4, 11) to provide MICs of fluconazole and amphotericin in less than 24 h. Their results showed that MICs of the rapidly acting antifungal agent amphotericin B can be obtained in 6 h but that the slower-acting triazole fluconazole requires at least 8 to 18 h. The RSA has since been applied to Aspergillus fumigatus to obtain MICs of voriconazole, itraconazole, and amphotericin B (12, 13).Here, we evaluate the ability of the RSA to provide echinocandin MICs equivalent to those obtained by the “gold standard” CLSI M27-A3 method, which requires an incubation period of 24 h, for Candida species on the same day of testing. Further, the RSA and M27-A3 method were compared to determine if the MICs provided are similarly affected by variations in inoculum growth conditions, the test medium, and the presence of human serum. The results demonstrate that the RSA method is comparable to the M27-A3 method but provides MICs in less than 8 h regardless of whether inoculum cells were prepared from 4- or 1-day-old cultures.