Beth A. Arthington-Skaggs

Incidence of Bloodstream Infections Due to Candida Species and In Vitro Susceptibilities of Isolates Collected from 1998 to 2000 in a Population-Based Active Surveillance Program

ABSTRACT To determine the incidence of Candida bloodstream infections (BSI) and antifungal drug resistance, population-based active laboratory surveillance was conducted from October 1998 through September 2000 in two areas of the United States (Baltimore, Md., and the state of Connecticut; combined population, 4.7 million). A total of 1,143 cases were detected, for an average adjusted annual incidence of 10 per 100,000 population or 1.5 per 10,000 hospital days. In 28% of patients, Candida BSI developed prior to or on the day of admission; only 36% of patients were in an intensive care unit at the time of diagnosis. No fewer than 78% of patients had a central catheter in place at the time of diagnosis, and 50% had undergone surgery within the previous 3 months. Candida albicans comprised 45% of the isolates, followed by C. glabrata (24%), C. parapsilosis (13%), and C. tropicalis (12%). Only 1.2% of C. albicans isolates were resistant to fluconazole (MIC, ≥64 μg/ml), compared to 7% of C. glabrata isolates and 6% of C. tropicalis isolates. Only 0.9% of C. albicans isolates were resistant to itraconazole (MIC, ≥1 μg/ml), compared to 19.5% of C. glabrata isolates and 6% of C. tropicalis isolates. Only 4.3% of C. albicans isolates were resistant to flucytosine (MIC, ≥32 μg/ml), compared to <1% of C. parapsilosis and C. tropicalis isolates and no C. glabrata isolates. As determined by E-test, the MICs of amphotericin B were ≥0.38 μg/ml for 10% of Candida isolates, ≥1 μg/ml for 1.7% of isolates, and ≥2 μg/ml for 0.4% of isolates. Our findings highlight changes in the epidemiology of Candida BSI in the 1990s and provide a basis upon which to conduct further studies of selected high-risk subpopulations.

Epidemiology of Candidemia in Brazil: a Nationwide Sentinel Surveillance of Candidemia in Eleven Medical Centers

ABSTRACT Candidemia studies have documented geographic differences in rates and epidemiology, underscoring the need for surveillance to monitor trends. We conducted prospective candidemia surveillance in Brazil to assess the incidence, species distribution, frequency of antifungal resistance, and risk factors for fluconazole-resistant Candida species. Prospective laboratory-based surveillance was conducted from March 2003 to December 2004 in 11 medical centers located in 9 major Brazilian cities. A case of candidemia was defined as the isolation of Candida spp. from a blood culture. Incidence rates were calculated per 1,000 admissions and 1,000 patient-days. Antifungal susceptibility tests were performed by using the broth microdilution assay, according to the Clinical and Laboratory Standards Institute guidelines. We detected 712 cases, for an overall incidence of 2.49 cases per 1,000 admissions and 0.37 cases per 1,000 patient-days. The 30-day crude mortality was 54%. C. albicans was the most common species (40.9%), followed by C. tropicalis (20.9%) and C. parapsilosis (20.5%). Overall, decreased susceptibility to fluconazole occurred in 33 (5%) of incident isolates, 6 (1%) of which were resistant. There was a linear correlation between fluconazole and voriconazole MICs (r = 0.54 and P < 0.001 [Spearmans rho]). This is the largest multicenter candidemia study conducted in Latin America and shows the substantial morbidity and mortality of candidemia in Brazil. Antifungal resistance was rare, but correlation between fluconazole and voriconazole MICs suggests cross-resistance may occur.

Epidemiologic and Molecular Characterization of an Outbreak of Candida parapsilosis Bloodstream Infections in a Community Hospital

ABSTRACT Candida parapsilosis is an important cause of bloodstream infections in the health care setting. We investigated a large C. parapsilosis outbreak occurring in a community hospital and conducted a case-control study to determine the risk factors for infection. We identified 22 cases of bloodstream infection with C. parapsilosis: 15 confirmed and 7 possible. The factors associated with an increased risk of infection included hospitalization in the intensive care unit (adjusted odds ratio, 16.4; 95% confidence interval, 1.8 to 148.1) and receipt of total parenteral nutrition (adjusted odds ratio, 9.2; 95% confidence interval, 0.9 to 98.1). Samples for surveillance cultures were obtained from health care worker hands, central venous catheter insertion sites, and medical devices. Twenty-six percent of the health care workers surveyed demonstrated hand colonization with C. parapsilosis, and one hand isolate was highly related to all case-patient isolates by tests with the DNA probe Cp3-13. Outbreak strain isolates also demonstrated reduced susceptibilities to fluconazole and voriconazole. This largest known reported outbreak of C. parapsilosis bloodstream infections in adults resulted from an interplay of host, environment, and pathogen factors. Recommendations for control measures focused on improving hand hygiene compliance.

Comparison of Visual and Spectrophotometric Methods of Broth Microdilution MIC End Point Determination and Evaluation of a Sterol Quantitation Method for In Vitro Susceptibility Testing of Fluconazole and Itraconazole against Trailing and Nontrailing Candida Isolates

ABSTRACT Visual determination of MIC end points for azole antifungal agents can be complicated by the trailing growth phenomenon. To determine the incidence of trailing growth, we performed testing of in vitro susceptibility to fluconazole and itraconazole using the National Committee for Clinical Laboratory Standards broth microdilution M27-A reference procedure and 944 bloodstream isolates of seven Candida spp., obtained through active population-based surveillance between 1998 and 2000. Of 429 C. albicans isolates, 78 (18.2%) showed trailing growth at 48 h in tests with fluconazole, and 70 (16.3%) showed trailing in tests with itraconazole. Of 118 C. tropicalis isolates, 70 (59.3%) showed trailing growth in tests with fluconazole, and 35 (29.7%) showed trailing in tests with itraconazole. Trailing growth was not observed with any of the other five Candida spp. tested (C. dubliniensis, C. glabrata, C. krusei, C. lusitaniae, and C. parapsilosis). To confirm whether or not isolates that showed trailing growth in fluconazole and/or itraconazole were resistant in vitro to these agents, all isolates that showed trailing growth were retested by the sterol quantitation method, which measures cellular ergosterol content rather than growth inhibition after exposure to azoles. By this method, none of the trailing isolates was resistant in vitro to fluconazole or itraconazole. For both agents, a 24-h visual end point or a spectrophotometric end point of 50% reduction in growth relative to the growth control after 24 or 48 h of incubation correlated most closely with the result of sterol quantitation. Our results indicate that MIC results determined by either of these end point rules may be more predictive of in vivo outcome for isolates that give unclear visual end points at 48 h due to trailing growth.

Comparative Evaluation of NCCLS M27-A and EUCAST Broth Microdilution Procedures for Antifungal Susceptibility Testing of Candida Species

ABSTRACT A two-laboratory study was performed to evaluate the correlation between the NCCLS M27-A and EUCAST microdilution procedures for antifungal testing of Candida spp. A panel of 109 bloodstream isolates was tested against amphotericin B, flucytosine, fluconazole, and itraconazole. Overall, the agreement was 92% and the intraclass correlation coefficient was 0.90 (P < 0.05).

In Vitro Evolution of Itraconazole Resistance in Aspergillus fumigatus Involves Multiple Mechanisms of Resistance

ABSTRACT We investigated the evolution of resistance to the antifungal drug itraconazole in replicate populations of Aspergillus fumigatus that were founded from a strain with a genotype of sensitivity to a single drug and then propagated under uniform conditions. For each population, conidia were serially transferred 10 times to agar medium either with or without itraconazole. After 10 transfers in medium supplemented with itraconazole, 10 itraconazole-resistant mutant strains were isolated from two populations. These mutant strains had different growth rates and different levels of itraconazole resistance. Analysis of the ergosterol contents of these mutants showed that they accumulate ergosterol when they are grown in the presence of itraconazole. The replacement of the CYP51A gene of the wild-type strain changed the susceptibility pattern of this strain to one of itraconazole resistance only when CYP51A genes with N22D and M220I mutations were used as selectable marker genes. Real-time quantitative reverse transcription-PCR was used to assess the levels of expression of the Afumdr1, Afumdr2, Afumdr3, Afumdr4, AtrF transporter, CYP51A, and CYP51B genes in these mutant strains. Most mutants showed either constitutive high-level expression or induction upon exposure of Afumdr3, Afumdr4, and AtrF to itraconazole. Our results suggest that overexpression of drug efflux pumps and/or selection of drug target site mutations are at least partially responsible for itraconazole resistance and could be considered mechanisms for the emergence of clinical resistance to this drug.

Quantitation of Candida albicans Ergosterol Content Improves the Correlation between In Vitro Antifungal Susceptibility Test Results and In Vivo Outcome after Fluconazole Treatment in a Murine Model of Invasive Candidiasis

ABSTRACT MIC end point determination for the most commonly prescribed azole antifungal drug, fluconazole, can be complicated by “trailing” growth of the organism during susceptibility testing by the National Committee for Clinical Laboratory Standards approved M27-A broth macrodilution method and its modified broth microdilution format. To address this problem, we previously developed the sterol quantitation method (SQM) for in vitro determination of fluconazole susceptibility, which measures cellular ergosterol content rather than growth inhibition after exposure to fluconazole. To determine if SQM MICs of fluconazole correlated better with in vivo outcome than M27-A MICs, we used a murine model of invasive candidiasis and analyzed the capacity of fluconazole to treat infections caused by C. albicansisolates which were trailers (M27-A MICs at 24 and 48 h, ≤1.0 and ≥64 μg/ml, respectively; SQM MIC, ≤1.0 μg/ml), as well as those which were fluconazole sensitive (M27-A and SQM MIC, ≤1.0 μg/ml) and fluconazole resistant (M27-A MIC, ≥64 μg/ml; SQM MIC, 54 μg/ml). Compared with the untreated controls, fluconazole therapy increased the survival of mice infected with a sensitive isolate and both trailing isolates but did not increase the survival of mice infected with a resistant isolate. These results indicate that the SQM is more predictive of in vivo outcome than the M27-A method for isolates that give unclear MIC end points due to trailing growth in fluconazole.

Paradoxical growth effect of caspofungin observed on biofilms and planktonic cells of five different Candida species

ABSTRACT The paradoxical growth (PG) of Candida sp. biofilms in the presence of high caspofungin (CAS) concentrations was previously unknown. We sought to characterize the PG at supra-MICs of CAS among clinical Candida sp. isolates grown as biofilms in 96-well polystyrene microtiter plates. The MICs of CAS were determined for 30 clinical Candida sp. isolates (4 Candida albicans, 6 C. tropicalis, 7 C. parapsilosis, 8 C. orthopsilosis, and 5 C. metapsilosis isolates) when they were grown as planktonic cells and biofilms and were defined as the lowest drug concentrations that resulted in a prominent decrease in growth and a 50% reduction in metabolic activity, respectively. PG was defined as a resurgence of growth (>50% of that in the drug-free growth control well) at drug concentrations above the MIC. With the exception of C. tropicalis, all isolates displayed PG more frequently when they were grown as biofilms than when they grown as planktonic cells. PG was undetectable among C. metapsilosis isolates in planktonic cell MIC tests but was present in 100% of the isolates in biofilm MIC tests. The drug concentration and the number of drug dilutions supporting PG were higher for biofilms than for planktonic cells. Microscopic changes in cell morphology were observed among both planktonic and biofilm cells with PG. Specifically, the accumulation of enlarged, globose cells was associated with PG, and we hypothesize that CAS-induced changes in the cell wall composition may be the explanation.

Multicenter Evaluation of a New Disk Agar Diffusion Method for Susceptibility Testing of Filamentous Fungi with Voriconazole, Posaconazole, Itraconazole, Amphotericin B, and Caspofungin

ABSTRACT The purpose of this study was to correlate inhibition zone diameters, in millimeters (agar diffusion disk method), with the broth dilution MICs or minimum effective concentrations (MECs) (CLSI M38-A method) of five antifungal agents to identify optimal testing guidelines for disk mold testing. The following disk diffusion testing parameters were evaluated for 555 isolates of the molds Absidia corymbifera, Aspergillus sp. (five species), Alternaria sp., Bipolaris spicifera, Fusarium sp. (three species), Mucor sp. (two species), Paecilomyces lilacinus, Rhizopus sp. (two species), and Scedosporium sp. (two species): (i) two media (supplemented Mueller-Hinton agar [2% dextrose and 0.5 μg/ml methylene blue] and plain Mueller-Hinton [MH] agar), (ii) three incubation times (16 to 24, 48, and 72 h), and (iii) seven disks (amphotericin B and itraconazole 10-μg disks, voriconazole 1- and 10-μg disks, two sources of caspofungin 5-μg disks [BBL and Oxoid], and posaconazole 5-μg disks). MH agar supported better growth of all of the species tested (24 to 48 h). The reproducibility of zone diameters and their correlation with either MICs or MECs (caspofungin) were superior on MH agar (91 to 100% versus 82 to 100%; R, 0.71 to 0.93 versus 0.53 to 0.96 for four of the five agents). Based on these results, the optimal testing conditions for mold disk diffusion testing were (i) plain MH agar; (ii) incubation times of 16 to 24 h (zygomycetes), 24 h (Aspergillus fumigatus, A. flavus, and A. niger), and 48 h (other species); and (iii) the posaconazole 5-μg disk, voriconazole 1-μg disk, itraconazole 10-μg disk (for all except zygomycetes), BBL caspofungin 5-μg disk, and amphotericin B 10-μg (zygomycetes only).

Biofilm production and evaluation of antifungal susceptibility amongst clinical Candida spp. isolates, including strains of the Candida parapsilosis complex

Candida cells can form biofilms that frequently are sources of infections and are less susceptible to antifungal drugs. Some authors have reported that Candida orthopsilosis and Candida metapsilosis isolates are not able to produce biofilms in vitro and there are no studies available on biofilm susceptibility for these species to antifungals. The aims of this study were to (i) quantify Candida spp. biofilms in vitro, and (ii) test the in vitro susceptibilities of Candida spp. biofilms to fluconazole (FLC) and amphotericin B (AMB). Isolates studied included four Candida albicans, six C. tropicalis, seven C. parapsilosis, eight C. orthopsilosis, and five C. metapsilosis. We compared two different methods to evaluate biofilm production, i.e., crystal violet (CV) staining and XTT-reduction assays (XTT). Scanning electron microscopy (SEM) was used to observe high, medium and low biofilm producing isolates screened by these two methods. To determine the minimum biofilm eradication concentration (MBEC) for FLC and AMB, XTT-reduction assay was used to measure cell metabolic activity. Biofilm quantification by CV and XTT showed that C. tropicalis isolates were the highest biofilm producer, followed by C. albicans, C. parapsilosis, C. orthopsilosis and C. metapsilosis. Examination of SEM images revealed that the extent of biofilms formed by high, medium, and low producers was highly correlated to the results generated by CV assay. Biofilm of all the isolates evaluated were resistant to FLC (MBEC(80) ≥ 256 ug/ml) but, in general, susceptible to AMB, except for six C. parapsilosis strains (MBEC(80) ≥ 8 ug/ml).