Epidemiologic analysis and antifungal susceptibility of Candida blood isolates in southern Taiwan.

Candidemia is a clinically important disease which has increased in incidence worldwide in recent decades. In order to identify the risk factors for mortality in candidemic patients and to elucidate the role of antifungal susceptibility testing, a retrospective cohort study was performed of 56 episodes of candidemia in 1998 at a medical center in southern Taiwan. The minimal inhibitory concentration (MIC) of these isolates was determined by E-test. Malignancy and alimentary diseases (42.9%) were the most common underlying conditions of these patients. There was no difference of Candida spp. distribution among patients treated in medical or surgical departments, except that all 5 isolates of C. intermedia were found in patients treated in medical departments (p=0.02) and 50% of candidemic infants had C. parapsilosis isolates (p=0.046). Among all Candida isolates, 3 (5.4%) were fluconazole non-susceptible. C. tropicalis had a significantly higher rate of amphotericin B resistance than the other species (p=0.007). Thirty four patients died and 70.6% of these deaths were attributable to candidemia. Thrombocytopenia, septic shock at the date of candidemia onset, C-reactive protein > 100 mg/L, blood urea nitrogen > 20 mg/dL, length of stay < 60 days, and Acute Physiology and Chronic Health Evaluation II score > or = 10 points were significantly associated with the death attributable to candidemia. Thrombocytopenia was the only independent predictor for mortality in the multivariate analysis. When the breakpoint of fluconazole was set at 2 microg/mL, as opposed to 8 microg/mL as in the National Committee for Clinical Laboratory Standards (NCCLS) criteria, the clinical outcome of death was significantly correlated to the MICs of the blood isolates. The correlation between MIC of fluconazole determined by E-test data, which is more easily obtainable than with NCCLS methods, and outcome requires larger scale investigation.     

Correlation between microdilution, E-test, and disk diffusion methods for antifungal susceptibility testing of posaconazole against Candida spp

Agar-based antifungal susceptibility testing is an attractive alternative to the microdilution method. We examined the correlation between the microdilution, E-test, and disk diffusion methods for posaconazole against Candida spp. A total of 270 bloodstream isolates of Candida spp. with a broad range of posaconazole MICs were tested using the CLSI M27-A2 method for microdilution, as well as the M-44A method and E-test methods for agar-based testing on Mueller-Hinton agar supplemented with 2% glucose and 0.5 microg of methylene blue. MICs and inhibitory zone diameters at the prominent growth reduction endpoint were recorded at 24 and 48 h. The Candida isolates included Candida albicans (n = 124), C. parapsilosis (n = 44), C. tropicalis (n = 41), C. glabrata (n = 36), C. krusei (n = 20), C. lusitaniae (n = 3), and C. dubliniensis (n = 2). The overall concordance (i.e., the percentage of isolates within two dilutions) between the E-test and microdilution was 64.8% at 24 h and 82.6% at 48 h. When we considered an arbitrary breakpoint of < or = 1 microg/ml, the agreement between the E-test and microdilution methods was 87.8% at 24 h and 93.0% at 48 h. The correlation of MICs with disk diffusion zone diameters was better for the E-test than the microdilution method. Zone correlation for diameters produced by the disks of two manufacturers was high, with a Pearson test value of 0.941 at 24 h. The E-test and microdilution MICs show good concordance and interpretative agreement. The disk diffusion zone diameters are highly reproducible and correlate well with both the E-test and the microdilution method, making agar-based methods a viable alternative to microdilution for posaconazole susceptibility testing.     

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.     

In vitro susceptibility testing and DNA typing of Saccharomyces cerevisiae clinical isolates.

Saccharomyces spp. are widely distributed in nature and may colonize the normal human gastrointestinal tract. Although Saccharomyces cerevisiae isolates have been previously considered nonpathogenic, they appear to be increasingly associated with infections in immunocompromised or otherwise debilitated patients. The antifungal susceptibility and epidemiology of S. cerevisiae are poorly defined at present. A series of 76 isolates (mostly stool surveillance and throat swab isolates) from 70 bone marrow transplant patients hospitalized at two different medical centers were characterized by antifungal susceptibility testing and restriction endonuclease analysis of chromosomal DNA. For DNA typing, digestion with NotI followed by pulsed-field gel electrophoresis was applied. Typing results revealed 62 distinct DNA types among the 76 clinical isolates. Despite this genomic diversity, clusters of identical isolates were identified among different patients hospitalized concurrently in the same unit, indicating possible nosocomial transmission. The MICs of amphotericin B, 5-fluorocytosine, fluconazole, and itraconazole were determined by a broth microdilution method, as recommended by the National Committee for Clinical Laboratory Standards. The MICs at which 90% of the strains were inhibited were as follows: amphotericin B, 1.0 micrograms/ml; 5-fluorocytosine, 0.25 micrograms/ml; fluconazole, 8.0 micrograms/ml; and itraconazole, 1.0 micrograms/ml. The relative resistance of S. cerevisiae to fluconazole and itraconazole may promote the emergence of this species as a pathogen among immunosuppressed patients.     

Comparison of E-test and broth microdilution methods for antifungal drug susceptibility testing of molds

We compared the E test with a broth microdilution method, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 90 isolates of pathogenic molds (10 Absidia corymbifera, 10 Aspergillus flavus, 10 Aspergillus fumigatus, 10 Aspergillus niger, 10 Aspergillus terreus, 10 Exophiala dermatitidis, 10 Fusarium solani, 10 Scedosporium apiospermum, 5 Scedosporium prolificans, and 5 Scopulariopsis brevicaulis). Overall, there was 71% agreement between the results of the two methods for amphotericin B (E-test MICs within +/-2 log2 dilutions of broth microdilution MICs) and 88% agreement with the results for itraconazole. The overall levels of agreement (within +/-2 log2 dilutions) were >/=80% for 5 of the 10 species tested against amphotericin B and 8 of the 10 species tested against itraconazole. The best agreement between the results was seen with A. fumigatus and A. terreus (100% of results for both agents within +/-2 log2 dilutions). The poorest agreement was seen with S. apiospermum, S. prolificans, and S. brevicaulis tested against amphotericin B (20% of results within +/-2 log2 dilutions). In every instance, this low level of agreement was due to isolates for which the broth microdilution MICs were low but for which the E-test MICs were much higher. The E test appears to be a suitable alternative procedure for testing the susceptibility of Aspergillus spp. and some other molds to amphotericin B or itraconazole.     

Multisite reproducibility of colorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates.

MICs of fluconazole and amphotericin B were determined independently for 100 coded yeast isolates by each of six laboratories to determine reproducibility of results by using a colorimetric oxidation-reduction-based broth microdilution test. In addition, each site tested five quality control isolates on at least four different occasions during the study. Results agreed within a three-dilution range (mode +/- 1 log2 dilution) for 96.2% of fluconazole tests and 92.7% of amphotericin B tests. Agreement among tests with the quality control isolates was 99.4% with fluconazole and 98.6% with amphotericin B. These results indicate that the colorimetric microdilution method is reproducible among laboratories.     

In vitro method to study antifungal perfusion in Candida biofilms

Antimycotic perfusion through Candida biofilms was demonstrated by a modification of a simple in vitro diffusion cell bioassay system. Using this model, the perfusion of three commonly used antifungal agents, amphotericin B, fluconazole, and flucytosine, was investigated in biofilms of three different Candida species (i.e., Candida albicans, Candida parapsilosis, and Candida krusei) that were developed on microporous filters. Scanning electron microscopy revealed that C. albicans formed a contiguous biofilm with tightly packed blastospores and occasional hyphae compared with C. parapsilosis and C. krusei, which developed confluent biofilms displaying structural heterogeneity and a lesser cell density, after 48 h of incubation on nutrient agar. Minor structural changes were also perceptible on the superficial layers of the biofilm after antifungal perfusion. The transport of antifungals to the distal biofilm-substratum interface was most impeded by C. albicans biofilms in comparison to C. parapsilosis and C. krusei. Fluconazole and flucytosine demonstrated similar levels of perfusion, while amphotericin B was the least penetrant through all three biofilms, although the latter appeared to cause the most structural damage to the superficial cells of the biofilm compared with the other antifungals. These results suggest that the antifungal perfusion through biofilm mode of growth in Candida is dependent both on the antimycotic and the Candida species in question, and in clinical terms, these phenomena could contribute to the failure of Candida biofilm-associated infections. Finally, the in vitro model we have described should serve as a useful system to investigate the complex interactions that appear to operate in vivo within the biofilm-antifungal interphase.     

Improved method for azole antifungal susceptibility testing.

A reproducible method is described for the determination of the MICs of ketoconazole, miconazole, fluconazole, and itraconazole with sharp endpoints when employed with either yeasts or molds. A semisolid medium is used with controlled pH and standardized inoculum. The time of reading results is a critical factor in the conduct of this test. The medium is simple to prepare and has a relatively long refrigerator shelf life in a user-ready state, requiring only the addition of a freshly prepared inoculum after restoration to room temperature.     

Molecular identification and antifungal susceptibility testing of clinical isolates of the Candida rugosa species complex and proposal of the new species Candida neorugosa

Candida rugosa is a poorly known fungal species occasionally involved in human infections. A molecular analysis of the sequences of the D1/D2 domains and the internal transcribed spacer (ITS) region of the ribosomal genes of 24 clinical isolates phenotypically identified as C. rugosa demonstrated that only 10 (41.6%) isolates belonged to that species. The other isolates were identified as Candida pararugosa (41.6%) and Candida pseudorugosa (8.3%). The remaining two isolates, from human and equine infections, respectively, were clearly different from the others and represent a new species proposed here as Candida neorugosa. The closest species by D1/D2 sequences was the type strain of C. rugosa, with only 92.3% similarity. C. neorugosa can also be differentiated from all other species of the C. rugosa complex by phenotypic features. The eight antifungal drugs tested showed high in vitro activity against the 24 isolates included in the study.     

In vitro Azole antifungals susceptibility of Candida spp. isolates from HIV-infected patients with periodontitis

ObjectiveThe objective of the present study was to determine the in vitro Azole antifungals susceptibility of Candida spp. strains isolated from HIV-positive patients with periodontitis.MethodsOral examination was performed in 500 HIV-positive patients, of which 228 were included in the study for having periodontitis which and separated in two groups based on their TCD4⁺ T-cells: (A) n = 110 (≤200 CD4⁺); (B) n = 118 (>200 CD4⁺). Candida spp. were isolated from the subgingival biofilm and crevicular fluid by seeding on CHROMagar plates and confirmed by endpoint PCR and MALDI-TOF. The susceptibility test in vitro for five antifungals was performed using the disc diffusion method.ResultsFrom the 228 HIV-positive patients with periodontitis, 174 were positive to Candida spp., and 204 isolations were obtained. 138 (67.64%) were C. albicans, and 66 (32.35%) were Candida non-albicans species. The most frequent Candida non-albicans species in order of frequency were C. glabrata with 48 (23.52%), C. tropicalis with 10 (4.9%), C. krusei with 7 (3.43%), and C. dubliniensis with 1 (0.49%). All species presented resistance to any antifungal: 149 to 5-fluorocytosine (73.0%), 149 to fluconazole (73.0%), and 144 to voriconazole (70.7%). Miconazole and econazole presented the highest susceptibility rates with 129 (63.2%) and 130 (63.7%) isolations, respectively.ConclusionThe Candida spp. involved in periodontitis of HIV-positive patients have a multi-resistant feature. It is necessary to implement recurrent research regarding the antifungal resistance of the Candida spp. that take part in periodontitis pathogenesis to promote an effective treatment in HIV patients.     

Evaluation of a novel colorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates.

A comparative evaluation of two broth microdilution methods for antifungal susceptibility testing of 600 clinical yeast isolates (Candida spp., Torulopsis glabrata, and Cryptococcus neoformans) against amphotericin B, fluconazole, and flucytosine (5FC) was conducted. Microdilution testing was performed according to National Committee for Clinical Laboratory Standards (NCCLS) recommendations (NCCLS document M27-P). By using the growth control for comparison, reference microdilution MIC endpoints for amphotericin B were scored as the lowest concentration at which a score of 0 (complete absence of growth) was observed, and those for 5FC and fluconazole were scored at the lowest concentration at which a score of 2 (prominent decrease in turbidity) (MIC-2) was observed. The second microdilution method employed a colorimetric endpoint using an oxidation-reduction indicator (Alamar Biosciences, Inc., Sacramento, Calif.) and was assessed independently of the reference microdilution MICs. The MICs for the two microdilution test systems were read after 24 and 48 h of incubation. Excellent agreement between the reference and colorimetric microdilution MICs was observed. Overall agreement was > or = 95% for all three drugs at 24 h. At 48 h, agreement was > or = 98% for amphotericin B and 5FC but dropped to 84% for fluconazole. Given these results it appears that the colorimetric microdilution approach to antifungal susceptibility testing may be viable alternative to the NCCLS reference method for testing yeasts.     

Trends in antifungal susceptibility among Candida sp. Urinary isolates from 1994 and 1998

Antifungal susceptibilities were determined from 80 urinary isolates of Candida species collected in 1994 and 1998. Our findings demonstrate increasing geometric means of fluconazole MICs and fluconazole resistance in Candida albicans and Candida tropicalis (those for Candida glabrata were unchanged) within the 4-year span. Amphotericin B and voriconazole MICs remained constant.     

Comparative evaluation of macrodilution and alamar colorimetric microdilution broth methods for antifungal susceptibility testing of yeast isolates.

A comparative evaluation of the macrodilution method and the Alamar colorimetric method for the susceptibility testing of amphotericin B, fluconazole, and flucytosine was conducted with 134 pathogenic yeasts. The clinical isolates included 28 Candida albicans, 17 Candida tropicalis, 15 Candida parapsilosis, 12 Candida krusei, 10 Candida lusitaniae, 9 Candida guilliermondii, 18 Torulopsis glabrata, and 25 Cryptococcus neoformans isolates. The macrodilution method was performed and interpreted according to the recommendations of the National Committee for Clinical Laboratory Standards (document M27-P), and the Alamar colorimetric method was performed according to the manufacturer's instructions. For the Alamar colorimetric method, MICs were determined at 24 and 48 h of incubation for Candida species and T. glabrata and at 48 and 72 h of incubation for C. neoformans. The overall agreement within +/- 1 dilution for Candida species and T. glabrata against the three antifungal agents was generally good, with the values for amphotericin B, fluconazole, and flucytosine being 85.3, 77.9, and 86.2%, respectively, at the 24-h readings and 69.3, 65.2, and 97.2%, respectively, at the 48-h readings. Most disagreement was noted with fluconazole against C. tropicalis and T. glabrata. Our studies indicate that determination of MICs at 24 h by the Alamar colorimetric method is a valid alternate method for testing amphotericin B, fluconazole, and flucytosine against Candida species but not for testing fluconazole against C. tropicalis and T. glabrata. For flucytosine, much better agreement can be demonstrated against Candida species and T. glabrata at the 48-h readings by the Alamar method.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro antifungal susceptibility of Cryptococcus gattii

We have determined the in vitro susceptibilities of 57 strains of Cryptococcus gattii to nine antifungal agents and have compared the MICs for these strains with those for C. neoformans. MICs were determined by a microdilution reference method. Albaconazole and ravuconazole (MICs of 0.04 and 0.05 microg/ml, respectively) showed the best activities. Micafungin showed no activity (MIC of >128 microg/ml). In general, C. gattii was less susceptible than C. neoformans to all drugs tested, with the exception of amphotericin B and flucytosine.     

Comparison of the E-test with the NCCLS M38-P method for antifungal susceptibility testing of common and emerging pathogenic filamentous fungi

The National Committee for Clinical Laboratory Standards (NCCLS) M38-P method describes standard parameters for testing the fungistatic antifungal activities (MICs) of established agents against filamentous fungi (molds). The present study evaluated the in vitro fungistatic activities of itraconazole and amphotericin B by the E-test and the NCCLS M38-P microdilution method against 186 common and emerging pathogenic molds (123 isolates of Aspergillus spp. [five species], 16 isolates of Fusarium spp. [two species], 4 Paecilomyces lilacinus isolates, 5 Rhizopus arrhizus isolates, 15 Scedosporium spp., 18 dematiaceous fungi, and 5 Trichoderma longibrachiatum isolates). The agreement between the methods for amphotericin B MICs ranged from 70% for Fusarium solani to > or =90% for most of the other species after the first reading; agreement was dependent on both the incubation time and the species being evaluated. Major discrepancies between the amphotericin B MICs determined by the E-test and the NCCLS M38-P method were demonstrated for three of the five species of Aspergillus tested and the two species of Fusarium tested. This discrepancy was more marked after 48 h of incubation; the geometric mean MICs determined by the E-test increased between 24 and 48 h from between 1.39 and 3.3 microg/ml to between 5.2 and >8 microg/ml for Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans. The agreement between the itraconazole MICs determined by the E-test and the NCCLS M38-P method ranged from 83.3% for A. nidulans to > or =90% for all the other species tested; the overall agreement was higher (92.7%) than that for amphotericin B (87.9%). The agreement was less dependent on the incubation time. Clinical trials need to be conducted to establish the role of the results of either the E-test or the NCCLS M38-P method in vitro for molds with the two agents as predictors of clinical outcome.     

In vitro susceptibility of Candida species to lactoferrin.

Lactoferrin is an antimicrobial protein present in human mucosal secretions as well as saliva. As there is no information on the relative fungicidal activity of human and bovine lactoferrin, an oral isolate of Candida albicans was studied for its susceptibility to these two proteins. Exposure to a concentration of 20 micrograms ml-1 of either HLF or BLF at 37 degrees C inactivated the yeast to the same degree irrespective of the incubation time of 45, 90 or 150 min. A similar study, using 20 micrograms ml-1 BLF and an incubation time of 150 min, elicited varying anticandidal activity against 35 isolates belonging to six different Candida species. Thus, BLF was fungicidal for the six Candida species in the following decreasing order, C. tropicalis > C. krusei > C. albicans > C. guilliermondii > C. parapsilosis > C. glabrata; the latter being the most resistant. These Candida species also demonstrated significant intra-species variation in susceptibility to the protein (P < 0.05). When the yeast cells exposed to BLF were examined by cryo-scanning electron microscopy, profound cell wall changes such as cell surface blebs, swelling and cell collapse were noted. These findings suggest that lactoferrin, a constituent of saliva, may differentially modulate the carriage of Candida species in the oral cavity.     

Interlaboratory evaluation of the Etest for antifungal susceptibility testing of dermatophytes.

A three-site interlaboratory reproducibility evaluation of the Etest concentration gradient strip method for testing antifungal susceptibilities was conducted using 30 strains of dermatophytes exposed to strips loaded with ketoconazole (KTZ), itraconazole (ITZ), amphotericin B (AMB) and fluconazole (FCZ). Etest minimal inhibitory concentrations were compared with those obtained using a broth microdilution method. All isolates produced clearly detectable growth at 28 degrees C within 72-96 h for reading with the Etest method. The highest interlaboratory agreement between Etest and the microdilution method was shown with FCZ (94%), and the lowest was seen with KTZ (60%). Overall, agreement between the Etest and microdilution method was variable. It was excellent for AMB (97%), good for ITZ (80%) and KTZ (77%), and low for fluconazole (27%).     

Species identification and antifungal susceptibility of uncommon blood yeast isolates

Background/PurposeAccurate identification of Candida species is increasingly important in the era of emergence of Candida auris. We aimed to compare the identification performance of two matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems (Vitek MS and Bruker biotyper MS) and an oligonucleotide array for uncommon blood yeast isolates and demonstrate the susceptibilities among those isolates.MethodCandida species isolates from blood culture other than Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, and Candida krusei identified by biochemical methods were collected from multiple hospitals and further identified by an oligonucleotide array based on the internal transcribed spacer-1 (ITS-1) and ITS-2 sequences of the rRNA genes, Vitek MS and Bruker biotyper MS. The minimal inhibitory concentrations (MICs) of these clinical isolates were determined by the Sensititre YeastOne (SYO) system.ResultsAmong 136 isolates, Candida guilliermondii was most common (52, 38.2%), followed by C. lusitaniae (13, 9.6%) and C. haemulonii (12, 8.8%). The oligonucleotide array, Vitek MS and Bruker biotyper MS correctly identified 89.7% (122), 90.4% (123), and 92.6% (126) of these isolates, respectively. Elevated minimal inhibitory concentrations (MICs) of fluconazole were observed for C. haemulonii (MIC₉₀: 256 mg/L), and C. guilliermondii (MIC₉₀: 16 mg/L) with 28.4% of uncommon Candida isolates with MIC ≧ 8 mg/L.ConclusionsFor uncommon Candida species, the unmet need for current databases of two commercial MALDI-TOF MS systems is highlighted, and the oligonucleotide array may serve as a supplement.