Evaluation of Etest Method for Determining Posaconazole MICs for 314 Clinical Isolates of Candida Species

The performance of the Etest for posaconazole (SCH 56592) susceptibility testing of 314 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 314 isolates with RPMI agar containing 2% glucose (RPG agar) and were read after incubation for 48 h at 35 degrees C. The Candida isolates included C. albicans (n = 174), C. glabrata (n = 57), C. tropicalis (n = 31), C. parapsilosis (n = 39), C. krusei (n = 5), C. guilliermondii (n = 6), and C. lusitaniae (n = 2). The Etest results correlated well with reference MICs. Overall agreement was 95%, and agreements for individual species were as follows: C. krusei, 100%; C. albicans, 98%; C. tropicalis, 97%; C. glabrata, 93%; C. parapsilosis, 85%; C. guilliermondii, 83%; and C. lusitaniae, 50%. The problem of trailing end points was minimized with RPG agar, and good agreement with broth dilution MICs was obtained when discernible growth within an established ellipse was ignored. The Etest method using RPG agar appears to be a useful method for determining posaconazole 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.     

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.     

Evaluation of broth microdilution testing parameters and agar diffusion Etest procedure for testing susceptibilities of Aspergillus spp. to caspofungin acetate (MK-0991)

The NCCLS M38-A document does not describe guidelines for testing caspofungin acetate (MK-0991) and other echinocandins against molds. This study evaluated the susceptibilities of 200 isolates of Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus to caspofungin (MICs and minimum effective concentrations [MECs]) by using standard RPMI 1640 (RPMI) and antibiotic medium 3 (M3), two inoculum sizes (10(3) and 10(4) CFU/ml), and two MIC determination criteria (complete [MICs-0] and prominent growth inhibition [MICs-2]) at 24 and 48 h. Etest MICs were also determined. In general, caspofungin MIC-2 and MEC pairs were comparable with both media and inocula (geometric mean ranges of MECs and MICs, respectively, with larger inoculum: 0.12 to 0.64 microg/ml and 0.12 to 0.44 microg/ml with RPMI versus 0.04 to 0.51 microg/ml and 0.03 to 0.21 microg/ml with M3); however, MEC results were less influenced by testing conditions than MICs, especially with the larger inoculum. Overall, the agreement between caspofungin Etest MICs and broth dilution values was higher with MECs obtained with M3 (>90%) and the large inoculum than under the other testing conditions. Because RPMI is a more stable and chemically defined medium than M3, the determination at 24 h of the easier visual MECs with RPMI and the inoculum recommended in the M38-A document appears to be a suitable procedure at present for in vitro testing of caspofungin against Aspergillus spp. Future in vitro correlations with in vivo outcome of both microdilution and Etest procedures may detect more-relevant testing conditions.     

Disk diffusion method for determining susceptibilities of Candida spp. to MK-0991

We have developed an agar-based methodology for testing susceptibilities of Candida spp. to the new antifungal agent MK-0991, a glucan synthase inhibitor. Results obtained with this method correlated well with the results obtained by the National Committee for Clinical Laboratory Standards M27-A broth microdilution reference method. However, as noted with prior comparisons of broth- and agar-based systems, some isolates yielded inhibition zones which were not consistent with the MICs obtained for them. Understanding the implications of these differences will require testing in an in vivo system.     

In vitro activity of caspofungin (MK-0991) against Candida albicans clinical isolates displaying different mechanisms of azole resistance

Caspofungin inhibits the synthesis of 1,3-beta-D-glucan, a key step in fungal cell wall biosynthesis. Here we report on its potent in vitro activity (MIC at which 90% of the isolates tested are inhibited = 1 microg per ml of RPMI medium) against 32 Candida albicans fluconazole-susceptible and -resistant clinical isolates irrespective of the underlying resistance mechanism (alterations in ERG11 and/or upregulation of MDR and CDR genes encoding efflux pumps) and provide further evidence that caspofungin is not a substrate for multidrug transporters.     

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

Trends in the Prevalence of Amphotericin B-Resistance (AmBR) among Clinical Isolates of Aspergillus Species

The challenges of the invasive infections caused by the resistant Aspergillus species include the limited access to antifungals for treatment and high mortality. This study aimed to provide a global perspective of the prevalence of amphotericin B resistance (AmBR), geographic distribution, and the trend of AmBR from 2010 to 2020. To analyze the prevalence of in vitro AmBR in clinical Aspergillus species, we reviewed the literature and identified a total of 72 articles. AmBR was observed in 1128 out of 3061 Aspergillus terreus (36.8%), 538 out of 3663 Aspergillus flavus (14.9%), 141 out of 2691 Aspergillus niger (5.2%), and 353 out of 17,494 Aspergillus fumigatus isolates (2.01%). An increasing trend in AmB-resistant isolates of A. fumigatus and a decreasing trend in AmB-resistant A. terreus and A. flavus isolates were observed between 2016 and 2020. AmB-resistant A. terreus and A. niger isolates, accounting for 40.4% and 20.9%, respectively, were the common AmB-resistant Aspergillus species in Asian studies. However, common AmB-resistant Aspergillus species reported by European and American studies were A. terreus and A. flavus isolates, accounting for 40.1% and 14.3% in 31 studies from Europe and 25.1% and 11.7% in 14 studies from America, respectively. The prevalence of AmB-resistant A. niger in Asian isolates was higher than in American and European. We found a low prevalence of A. terreus in American isolates (25.1%) compared to Asian (40.4%) and European (40.1%). Future studies should focus on analyzing the trend of AmBR on a regional basis and using the same methodologies.     

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.     

Use of Anidulafungin as a Surrogate Marker To Predict Susceptibility and Resistance to Caspofungin among 4,290 Clinical Isolates of Candida by Using CLSI Methods and Interpretive Criteria

This study addressed the application of anidulafungin as a surrogate marker to predict the susceptibility of Candida to caspofungin due to unacceptably high interlaboratory variation of caspofungin MIC values. CLSI reference broth microdilution methods and species-specific interpretive criteria were used to test 4,290 strains of Candida (eight species), including 71 strains with documented fks mutations. Caspofungin MIC values were compared with those of anidulafungin to determine the percentage of categorical agreement (CA) and very major (VME), major (ME), and minor error rates, as well as the ability to detect fks mutants. For all 4,290 isolates the CA was 97.1% (0.2% VME and ME, 2.5% minor errors) using anidulafungin as the surrogate. Among the 62 isolates of Candida albicans (4 isolates), C. tropicalis (5 isolates), C. krusei (4 isolates), C. kefyr (2 isolates), and C. glabrata (47 isolates) that were nonsusceptible (NS; either intermediate [I] or resistant [R]) to both caspofungin and anidulafungin, 52 (83.8%) contained a mutation in fks1 or fks2. Eight mutants of C. glabrata, two of C. albicans, and one each of C. tropicalis and C. krusei were classified as susceptible (S) to both antifungal agents. The remaining 7 mutants (2 C. albicans and 5 C. glabrata) were susceptible to one of the agents and either intermediate or resistant to the other. Using the epidemiological cutoff value (ECV) of 0.12 μg/ml for both caspofungin and anidulafungin to differentiate wild-type (WT) from non-WT strains of C. glabrata, 42 of the 55 (76.4%) C. glabrata mutants were non-WT and 8 of the 55 (14.5%) were WT for both agents (90.9% concordance). Anidulafungin can accurately serve as a surrogate marker to predict S and R of Candida to caspofungin.     

Evaluation of the ESP Culture System II for Testing Susceptibilities of Mycobacterium tuberculosis Isolates to Four Primary Antituberculous Drugs

The reliability of the ESP Culture System II (herein referred to as ESP II) for testing susceptibilities of Mycobacterium tuberculosis isolates to isoniazid, rifampin, ethambutol, and streptomycin was evaluated by comparing results to those of the method of proportion (MOP), which was considered the reference method, for 20 clinical isolates and 30 challenge strains provided by the Centers for Disease Control and Prevention (CDC). Clinical isolates also were tested with the BACTEC TB 460 system; these results agreed with those obtained by the MOP for all isolates and all drugs, except the high concentration of isoniazid, for which agreement was 95%. After resolution of discrepancies, levels of agreement between ESP II and MOP for the clinical isolates were 95 and 100%, respectively, for the low and high concentrations of isoniazid, 100% for rifampin and ethambutol, and 95% for streptomycin. For the 30 challenge isolates, ESP II results for both concentrations of isoniazid agreed with the expected results in all cases, whereas agreement was 93% for both rifampin and streptomycin and 90% for ethambutol. All discrepancies with the CDC isolates were due to failure of ESP II to correctly classify resistant strains. By testing isolates yielding discrepant ethambutol and streptomycin results with a lower concentration of both drugs in the ESP II system, agreement increased to 93% for ethambutol and 100% for streptomycin. For the clinical isolates, the times to an ESP II result of susceptible (means ± standard errors of the means) were 8.47 ± 0.12 days (range, 7 to 10 days) and 8.73 ± 0.29 days (range, 5 to 11 days) when the inoculum was prepared from a McFarland equivalent and from a seed bottle, respectively. The time to an ESP II result of resistant varied by drug and method of inoculum preparation, ranging from 5.50 ± 0.22 days for ethambutol with the inoculum prepared from a McFarland standard to 8.0 days for ethambutol with the inoculum prepared from a seed bottle. These data suggest that the ESP II system is a rapid and reliable method for testing susceptibilities of M. tuberculosis isolates to isoniazid and rifampin. Performance, however, may be suboptimal for ethambutol and streptomycin. Testing additional ethambutol-resistant and streptomycin-resistant strains with two concentrations of both drugs is necessary.

Tuberculosis remains an important public health problem in the United States today, despite its declining incidence over the past several years. One aspect of tuberculosis control is treatment with effective antituberculous drugs. To help ensure appropriate therapy early in the course of the disease, experts at the Centers for Disease Control and Prevention (CDC) recommend that for isolates of Mycobacterium tuberculosis complex (MTBC) susceptibility test results be available an average of 28 to 30 days from receipt of a specimen in the laboratory (7). Currently in the United States, susceptibility testing of MTBC is performed by using either the method of proportion, which is considered the reference method, or the BACTEC TB 460 system (Becton Dickinson, Sparks, Md.). Of these two methods only the BACTEC TB 460 system has the potential to consistently meet the suggested turnaround time.The ESP Culture System II (AccuMed International, Westlake, Ohio [formerly Difco]), a fully automated continuously monitoring system for growth and detection of mycobacteria, has been available for commercial use for a few years (9). As for the ESP blood culture system, the technology of the ESP II is based on detection of pressure changes (i.e., either gas production or gas consumption due to microbial growth) within the headspace above the broth culture medium in a sealed bottle. A special detection algorithm has been developed for the very slowly growing mycobacteria, in addition to the detection algorithm used with the ESP blood culture system. Recently, a method for testing susceptibilities of isolates of MTBC to isoniazid, rifampin, ethambutol, and streptomycin was developed for the ESP II system (1, 3, 5, 6). The purpose of this study was to evaluate the reliability of the ESP II system for testing susceptibilities of MTBC isolates to these four drugs.     

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.     

Effects of caspofungin (MK-0991) and anidulafungin (LY303366) on phagocytosis,oxidative burst and killing of <Emphasis Type="Italic">Candida albicans</Emphasis> by human phagocytes

The objective of the present study was to investigate the influence of the new echinocandins caspofungin (MK-0991) and anidulafungin (LY303366) on human phagocytes. Phagocytosis, oxidative burst and intracellular killing of Candida albicans were analyzed by flow cytometry. Neither caspofungin nor anidulafungin significantly influenced phagocytosis. Only caspofungin significantly influenced oxidative burst after 15 min of incubation (P<0.05). Both caspofungin and anidulafungin improved intracellular killing rates of C. albicans after 2 h of incubation (42.4% and 43.2%, respectively, compared to 37.9% in controls; P<0.05). In conclusion, caspofungin significantly improves oxidative burst and intracellular killing, which may be advantageous for clinical therapy.     

Use of Micafungin as a Surrogate Marker To Predict Susceptibility and Resistance to Caspofungin among 3,764 Clinical Isolates of Candida by Use of CLSI Methods and Interpretive Criteria

Due to unacceptably high interlaboratory variation in caspofungin MIC values, we evaluated the use of micafungin as a surrogate marker to predict the susceptibility of Candida spp. to caspofungin using reference methods and species-specific interpretive criteria. The MIC results for 3,764 strains of Candida (eight species), including 73 strains with fks mutations, were used. Caspofungin MIC values and species-specific interpretive criteria were compared with those of micafungin to determine the percent categorical agreement (%CA) and very major error (VME), major error (ME), and minor error rates as well as their ability to detect fks mutant strains of Candida albicans (11 mutants), Candida tropicalis (4 mutants), Candida krusei (3 mutants), and Candida glabrata (55 mutants). Overall, the %CA was 98.8% (0.2% VMEs and MEs, 0.8% minor errors) using micafungin as the surrogate marker. Among the 60 isolates of C. albicans (9 isolates), C. tropicalis (5 isolates), C. krusei (2 isolates), and C. glabrata (44 isolates) that were nonsusceptible (either intermediate or resistant) to both caspofungin and micafungin, 54 (90.0%) contained a mutation in fks1 or fks2. An additional 10 C. glabrata mutants, two C. albicans mutants, and one mutant each of C. tropicalis and C. krusei were classified as susceptible to both antifungal agents. Using the epidemiological cutoff values (ECVs) of 0.12 μg/ml for caspofungin and 0.03 μg/ml for micafungin to differentiate wild-type (WT) from non-WT strains of C. glabrata, 80% of the C. glabrata mutants were non-WT for both agents (96% concordance). Micafungin may serve as an acceptable surrogate marker for the prediction of susceptibility and resistance of Candida to caspofungin.     

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.     

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.     

International Surveillance of Bloodstream Infections Due to Candida Species: Frequency of Occurrence and Antifungal Susceptibilities of Isolates Collected in 1997 in the United States, Canada, and South America for the SENTRY Program

An international program of surveillance of bloodstream infections (BSIs) in the United States, Canada, and South America between January and December 1997 detected 306 episodes of candidemia in 34 medical centers (22 in the United States, 6 in Canada, and 6 in South America). Eighty percent of the BSIs were nosocomial and 50% occurred in patients hospitalized in an intensive care unit. Overall, 53.3% of the BSIs were due to Candida albicans, 15.7% were due to C. parapsilosis, 15.0% were due to C. glabrata, 7.8% were due to C. tropicalis, 2.0% were due to C. krusei, 0.7% were due to C. guilliermondii, and 5.8% were due to Candida spp. However, the distribution of species varied markedly by country. In the United States, 43.8% of BSIs were due to non-C. albicans species. C. glabrata was the most common non-C. albicans species in the United States. The proportion of non-C. albicans BSIs was slightly higher in Canada (47.5%), where C. parapsilosis, not C. glabrata, was the most common non-C. albicans species. C. albicans accounted for 40.5% of all BSIs in South America, followed by C. parapsilosis (38.1%) and C. tropicalis (11.9%). Only one BSI due to C. glabrata was observed in South American hospitals. Among the different species of Candida, resistance to fluconazole (MIC, ≥64 μg/ml) and itraconazole (MIC, ≥1.0 μg/ml) was observed with C. glabrata and C. krusei and was observed more rarely among other species. Isolates of C. albicans, C. parapsilosis, C. tropicalis, and C. guilliermondii were all highly susceptible to both fluconazole (99.4 to 100% susceptibility) and itraconazole (95.8 to 100% susceptibility). In contrast, 8.7% of C. glabrata isolates (MIC at which 90% of isolates are inhibited [MIC₉₀], 32 μg/ml) and 100% of C. krusei isolates were resistant to fluconazole, and 36.9% of C. glabrata isolates (MIC₉₀, 2.0 μg/ml) and 66.6% of C. krusei isolates were resistant to itraconazole. Within each species there were no geographic differences in susceptibility to fluconazole or itraconazole.     

Incidence of Acinetobacter Species Other than A. baumannii among Clinical Isolates of Acinetobacter: Evidence for Emerging Species

Six hundred ninety nonduplicate isolates of Acinetobacter species were identified using a combination of detection of bla_OXA-51-like and rpoB sequence cluster analysis. Although most isolates were identified as A. baumannii (78%), significant numbers of other species, particularly A. lwoffii/genomic species 9 (8.8%), A. ursingii (4%), genomic species 3 (1.7%), and A. johnsonii (1.7%), were received, often associated with bacteremias.The Acinetobacter genus consists of more than 30 species, of which A. baumannii, and to a lesser extent genomic species 3 and 13TU, are most associated with the clinical environment and nosocomial infections. Identification within the genus is difficult and requires molecular methods, and these organisms are rarely identified to the species level using appropriate methods (3, 4, 6, 24). While A. baumannii can relatively readily be identified by detection of bla_OXA-51-like, the intrinsic carbapenemase gene in this species (22), the use of rpoB sequencing has facilitated identification across the genus (5, 8), and it is becoming clear that other species, such as A. ursingii (which has also been called A. septicus [13]) and A. haemolyticus, are also important nosocomial pathogens in some cases (3, 4, 5, 6, 9, 24). rpoB sequencing has advantages over such techniques as amplified ribosomal DNA restriction analysis (ARDRA), based on 16S rRNA gene sequences, and those based on the 16S-23S intergenic spacer region, since there is a relatively high degree of polymorphism in this gene among the Acinetobacter species, and sequences are available for all the currently described species (8), including those more recently described (11-14).Our laboratory provides a typing and identification service for hospitals in the United Kingdom and Republic of Ireland for this organism, and here we describe the species found among 690 isolates of Acinetobacter, each from a different patient, submitted over a 20-month period during 2008/2009, from some 135 hospitals. While A. baumannii, which is frequently associated with outbreaks, is still by far the most common of the Acinetobacter species among clinical isolates, it is clear that lesser-known species, such as A. lwoffii, A. ursingii, and A. parvus, are regularly encountered, have been associated with serious infections, and may represent emerging pathogens.The majority of isolates were received as Acinetobacter species for typing, identification, and/or susceptibility determinations and were subjected to a multiplex PCR for detection of bla_OXA-58-like, bla_OXA-23-like, bla_OXA-51-like, bla_OXA-40-like, and class 1 integrase genes, as described by Turton et al. (22), with the addition of primers for bla_OXA-58-like (27). Detection of bla_OXA-51-like was regarded as a positive identification of A. baumannii; the identity of a proportion of such isolates was also checked by rpoB sequence cluster analysis; in addition, many were shown by pulsed-field gel electrophoresis (PFGE) to be further representatives of strains previously identified as A. baumannii, and all gave amplicons in a PCR to amplify variable-number tandem repeat loci found in A. baumannii (23). The remaining isolates were identified by rpoB sequence cluster analysis using primers described by La Scola et al. (8). Briefly, a 903-bp portion of the rpoB gene covering two variable regions was amplified using the primers Ac696F and Ac1598R. Amplicons were treated with Exo-SAP-IT (USB Corporation, Cleveland, OH) according to the manufacturer''s instructions, and four sequencing reactions were carried out, using the primers Ac696F, Ac1055F, Ac1093R, and Ac1598R, respectively. The resulting fragments were separated on a Beckman-Coulter CEQ8000 genetic analysis system or an Applied Biosystems 3730 DNA analyzer and aligned, and sequences of a 765-bp fragment corresponding to nucleotides (nt) 2964 to 3728 of the coding sequence were compared using the BioNumerics software program; a phylogenetic tree was constructed using the MEGA software program (http://www.megasoftware.net/mega41.html) (19) (Fig. ​(Fig.1).1). Sequences of reference isolates were also included, and isolates were identified both by BLAST searches and according to which species they clustered most closely with; as more isolates were added to the database, a measure of the extent of sequence diversity associated with each species was obtained, allowing determination of whether isolates clustered closely enough to be assigned to that species. Susceptibilities to at least 16 antibiotics were determined by agar dilution and interpreted using British Society of Antimicrobial Chemotherapy (BSAC) breakpoints (http://www.bsac.org.uk). Pulsed-field gel electrophoresis (PFGE) of ApaI-digested genomic DNA was carried out as described previously (21).Open in a separate windowFIG. 1.Phylogenetic tree of sequences corresponding to nt 2964 to 3728 of the rpoB coding sequence of isolates of Acinetobacter species. Clinical and reference isolates were included, with GenBank accession numbers being included with the latter. Phylogenetic analyses were conducted in MEGA4 (19) using the neighbor-joining method. One thousand replicates were used for bootstrap analysis.Using this method, we were able to identify most isolates to the species level, although isolates of A. lwoffii and genomic species 9 clustered too closely to be distinguished from one another, as did those of A. baylyi and genomic species 11 (A. guillouiae). Similarly, as has been observed by others (26), A. grimontii and A. junii could not be distinguished and are likely to be a single species. Three isolates (UA1 to -3), two of which had highly similar rpoB sequences, did not cluster closely enough with any of the described species and may represent new species. For all three, the closest currently described species is A. towneri. Isolates that were identified as A. radioresistens were PCR positive for bla_OXA-23-like, the naturally occurring carbapenemase gene in this species (17), consistent with the identification. Detection of OXA carbapenemase genes among species other than A. baumannii was rare, with the only other examples being two isolates, one of genomic species 3 and the other of genomic species 16, with bla_OXA-58-like.As expected, the majority (78.0%) of isolates were identified as A. baumannii; with A. lwoffii/genomic species 9 (8.8%), A. ursingii (4.0%), genomic species 3 (1.7%), A. johnsonii (1.7%), and A. parvus (1.3%) accounting for most of the rest (Table ​(Table1).1). In most cases, these non-A. baumannii isolates were from blood and were associated with bacteremia or septicemia. Of note is that some isolates (of A. johnsonii, genomic species 13, and A. beijerinckii) were implicated in endocarditis; this has been described for other Acinetobacter species (7, 18, 28) but not these and provides further evidence that these organisms can cause life-threatening infections. The relatively high incidence of A. ursingii, which exceeded those of both genomic species 3 and 13TU, was unexpected but agrees with observations from hospitals in the Netherlands and Northern Ireland (2, 24). It has previously been documented that this organism has the capacity to cause bloodstream infections in hospitalized patients (4, 9, 11), and it has been associated with a nosocomial outbreak of bloodstream infections in a neonatal intensive care unit, in which two babies died (6). The isolates in the present study were from 28 patients in 24 centers, suggesting that they were not epidemiologically related; nevertheless, three isolates, each from different centers, formed a cluster by PFGE (see Fig. S1 in the supplemental material). Similarly, A. lwoffii has previously been linked with catheter-related bloodstream infections (20), as has A. parvus (12).

TABLE 1.

Submissions of Acinetobacter sp. other than A. baumannii received during the study period and associated clinical information^a