Broth Microdilution Antifungal Susceptibility Testing Research Articles

Detection and Characterization of Two Phenotypes of Candida parapsilosis in South Korea: Clinical Features and Microbiological Findings.

We newly detected two (sinking and floating) phenotypes of Candida parapsilosis among bloodstream infection (BSI) isolates from Korean hospitals and assessed their microbiological and clinical characteristics. During the performance of a Clinical and Laboratory Standards Institute (CLSI) broth microdilution antifungal susceptibility testing, the sinking phenotype had a characteristic smaller button-like appearance because all yeast cells sank to the bottoms of the CLSI U-shaped round-bottom wells, whereas the floating phenotype comprised dispersed cells. Phenotypic analysis, antifungal susceptibility testing, ERG11 sequencing, microsatellite genotyping, and clinical analysis were performed on C. parapsilosis isolates from 197 patients with BSI at a university hospital during 2006 to 2018. The sinking phenotype was detected in 86.7% (65/75) of the fluconazole-nonsusceptible (FNS) isolates, 92.9% (65/70) of the isolates harboring the Y132F ERG11 gene substitution, and 49.7% (98/197) of all isolates. Clonality was more frequently observed for the Y132F-sinking isolates (84.6% [55/65]) than for all other isolates (26.5% [35/132]; P < 0.0001). Annual incidence of Y132F-sinking isolates increased 4.5-fold after 2014, and two dominant genotypes, persistently recovered for 6 and 10 years, accounted for 69.2% of all Y132F-sinking isolates. Azole breakthrough fungemia (odds ratio [OR], 6.540), admission to the intensive care unit (OR, 5.044), and urinary catheter placement (OR, 6.918) were independent risk factors for BSIs with Y132F-sinking isolates. The Y132F-sinking isolates exhibited fewer pseudohyphae, a higher chitin content, and lower virulence in the Galleria mellonella model than the floating isolates. These long-term results illustrate the increasing BSIs caused by clonal transmission of the Y132F-sinking isolates of C. parapsilosis. IMPORTANCE We believe that this is the first study describe the microbiological and molecular characteristics of bloodstream isolates of C. parapsilosis in Korea exhibiting two phenotypes (sinking and floating). An important aspect of our findings is that the sinking phenotype was observed predominantly in isolates harboring a Y132F substitution in the ERG11 gene (92.9%), fluconazole-nonsusceptible (FNS) isolates (86.7%), and clonal BSI isolates (74.4%) of C. parapsilosis. Although the increase in the prevalence of FNS C. parapsilosis isolates has been a major threat in developing countries, in which the vast majority of candidemia cases are treated with fluconazole, our long-term results show increasing numbers of BSIs caused by clonal transmission of Y132F-sinking isolates of C. parapsilosis in the period with an increased echinocandin use for candidemia treatment in Korea, which suggests that C. parapsilosis isolates with the sinking phenotype continue to be a nosocomial threat in the era of echinocandin therapy.

In Vitro Activity of Disinfectants against Mold Fungi Isolated from Different Environments of the Children’s Medical Center Hospital, Tehran, Iran

Introduction: Fungal aerosols cause life-threatening infections in patients hospitalized in critical wards. Antiseptics and disinfectants have broad-spectrum antimicrobial activity against the living tissue and inert surfaces microorganisms; hence, they have an essential role in controlling and preventing nosocomial infections. This study aimed to evaluate in vitro antifungal activity of benzalkonium chloride (BAC), chlorhexidine digluconate (CHX), and sodium hypochlorite (SH) against isolated fungal aerosols from the hospital environment.&#x0D; Materials and Methods: The susceptibility tests were performed on fungal aerosols isolated from various wards of Children’s Medical Center, based on broth microdilution antifungal susceptibility testing of filamentous fungi approved by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 document. The isolates included Aspergillus (Aspergillus flavus (n = 14), Aspergillus niger complex (n = 12), Penicillium spp. (n = 14), and Cladosporium spp. (n = 14).&#x0D; Results: The geometric means (GM) of the Minimum Inhibitory Concentrations (MICs) of the biocides across all isolates were as follows: BAC, 3.56 µg/ml, CHX, 9.45 µg/ml, and SH, 810.35 µg/ml. The highest range of MICs was found for SH (50-12800 µg/ml), while the lowest range was for BAC (1-16 µg/ml) against all fungal isolates. Generally, BAC showed the highest in vitro activity among disinfectants tested. The lowest MIC50 and MIC90 values were 4 and 8 µg/ml for BAC, followed by 16 and 32 µg/ml for CHX, and 800 and 6400 µg/ml for SH, respectively.&#x0D; Conclusion: The findings showed that BAC was an effective disinfectant, which can prevent resistant species and fungal pathogens and be used an alternative to other disinfectants and antiseptics.

Case of Candida auris Identified From the External Ear Canal of a Healthy Minnesota Outpatient With Travel to South Korea

Background:Candida auris is a globally emerging, multidrug-resistant fungal pathogen that causes healthcare-associated outbreaks and can be misidentified in clinical laboratories. Most US C. auris cases occur in hospitalized or long-term care patients with underlying medical conditions. Also, 4 global phylogenetic C. auris clades largely cluster geographically. Receiving health care abroad is a risk factor for US C. auris cases. In December 2019, the Minnesota Department of Health (MDH) confirmed Minnesota’s first C. auris case, isolated from the external ear canal of a healthy young adult outpatient with right-sided otitis externa. We describe the investigation and response for this uncommon US presentation of C. auris. Methods: The MDH initiated mandatory reporting and submission of confirmed or possible C. auris isolates in August 2019. The MDH Public Health Laboratory (MDH-PHL) confirmed C. auris by MALDI-TOF (Bruker) from an isolate submitted by a hospital laboratory as C. duobushaemulonii to rule out C. auris. The MDH-PHL performed broth microdilution antifungal susceptibility testing (AFST). The CDC Mycotics Diseases Branch laboratory performed whole-genome sequencing (WGS). The MDH epidemiologists obtained a patient history through interviews with healthcare staff and the patient, and they collected environmental samples from otoscopes. The MDH-PHL tested environmental samples by C. auris RT-PCR and culture. The MDH recommended disinfection of examination rooms and otoscopes and 3 months of C. auris surveillance for patients evaluated with otoscopes who later returned with otic inflammation. Swabs from the patient’s axilla, groin, and external ear canals were tested for C. auris by PCR at the MDH-PHL. Results: The patient reported recurrent right ear infections in 2016 during a 16-month visit to South Korea, with treatment in multiple ENT clinics. December 2019 otitis resolved after treatment with oral amoxicillin/clavulanate and otic ciprofloxacin/dexamethasone. AFST showed resistance to fluconozale and susceptibility to 8 antifungals, including echinocandins. WGS placed the isolate in the East Asian clade, indicating similarity to isolates from South Korea and Japan. Environmental cultures were negative. The asymptomatic left ear was colonized with C. auris; other sites were negative. As of January29, 2020, no additional cases were detected. Conclusions: We identified prolonged colonization of C. auris in the external ear canals of a healthy patient. WGS and travel in South Korea, including ENT clinic exposure, provide strong evidence of C. auris acquisition in South Korea. No spread has been reported in Minnesota. Deliberate communication with clinical laboratories regarding ruling out C. auris was key to case discovery. Clinicians should be aware of C. auris epidemiology, including healthcare exposure abroad, particularly in young, healthy patients.Funding: NoneDisclosures: None

Identification and broth-microdilution antifungal susceptibility testing of yeast directly from automated blood cultures.

Aim: To standardize MALDI-TOF-MS based identification and antifungal susceptibility (AFST) for yeasts directly from automated blood cultures to reduce turnaround time. Materials & methods: Direct-ID after lysis-centrifugation (0.5% SDS) standardized in 40 and validated in 250 yeast positive samples. Direct-AFST was standardized with fluconazole (28 samples) and evaluated (70 samples) for seven antifungals. Results: Direct-ID had a high sensitivity (97.2%) and specificity (94.3%). Correct species-level identification showed 100% in C. tropicalis, C. krusei, C. parapsilosis. Direct-AFST had a 100% categorical agreementwith culture-AFST for posaconazole, anidulafungin and >90% categorical agreement for amphotericin B, voriconazole and fluconazole. Conclusion: Direct-ID and subsequent direct-AFST is a rapid and robust method to reduce the turnaround time for the diagnosis of invasive candidiasis.

Epidemiology and Risk Factors for Echinocandin Nonsusceptible Candida glabrata Bloodstream Infections: Data From a Large Multisite Population-Based Candidemia Surveillance Program, 2008-2014.

Background. Echinocandins are first-line treatment for Candida glabrata candidemia. Echinocandin resistance is concerning due to limited remaining treatment options. We used data from a multisite, population-based surveillance program to describe the epidemiology and risk factors for echinocandin nonsusceptible (NS) C glabrata candidemia.Methods. The Centers for Disease Control and Prevention's Emerging Infections Program conducts population-based laboratory surveillance for candidemia in 4 metropolitan areas (7.9 million persons; 80 hospitals). We identified C glabrata cases occurring during 2008–2014; medical records of cases were reviewed, and C glabrata isolates underwent broth microdilution antifungal susceptibility testing. We defined echinocandin-NS C glabrata (intermediate or resistant) based on 2012 Clinical and Laboratory Standards Institute minimum inhibitory concentration breakpoints. Independent risk factors for NS C glabrata were determined by stepwise logistic regression.Results. Of 1385 C glabrata cases, 83 (6.0%) had NS isolates (19 intermediate and 64 resistant); the proportion of NS isolates rose from 4.2% in 2008 to 7.8% in 2014 (P < .001). The proportion of NS isolates at each hospital ranged from 0% to 25.8%; 3 large, academic hospitals accounted for almost half of all NS isolates. In multivariate analysis, prior echinocandin exposure (adjusted odds ratio [aOR], 5.3; 95% CI, 2.6–1.2), previous candidemia episode (aOR, 2.5; 95% CI, 1.2–5.1), hospitalization in the last 90 days (aOR, 1.9; 95% CI, 1.0–3.5, and fluconazole resistance [aOR, 3.6; 95% CI, 2.0–6.4]) were significantly associated with NS C glabrata. Fifty-nine percent of NS C glabrata cases had no known prior echinocandin exposure.Conclusion. The proportion of NS C glabrata isolates rose significantly during 2008–2014, and NS C glabrata frequency differed across hospitals. In addition to acquired resistance resulting from prior drug exposure, occurrence of NS C glabrata without prior echinocandin exposure suggests possible transmission of resistant organisms.

In vitro fungicidal activity of biocides against pharmaceutical environmental fungal isolates.

To determine the minimum inhibitory concentrations (MIC) of a range of cleanroom fungi against three disinfectants common to the pharmaceutical and healthcare sectors: biguanide (chlorhexidine) and two quaternary ammonium compounds (benzalkonium chloride and cetrimide). The in vitro fungicidal activities of the three biocides were studied against 112 cleanroom fungal isolates using broth microdilution technique (CLSI M38-A2 standard). Minimum inhibitory concentration (MIC) for all three biocides against hyaline fungi showed results of not more than 16 μg ml(-1). Alternaria showed <32 μg ml(-1) and other dematiaceous fungi reported that 8-16 μg ml(-1) for biguanides and QACs. This study clearly demonstrates that the most frequently isolated micro-organisms from an environmental monitoring programme may be periodically subjected to broth microdilution testing with cleanroom disinfectant agents used in the disinfection programme confirm their sensitivity profile. No large collection of data exists on the activity of biocides on pharmaceutical cleanroom fungal isolates. This is the first study report with large collection of cleanroom fungal isolates tested against common biocides using the broth microdilution antifungal susceptibility testing to determine the MIC value. The data presented support a quality control procedure for cleanroom disinfection.

Antifungal and cytotoxicity activities of the fresh xylem sap of Hymenaea courbaril L. and its major constituent fisetin.

BackgroundThe great potential of plants as Hymenaea courbaril L (jatoba) has not yet been throughly explored scientifically and therefore it is very important to investigate their pharmacological and toxicological activities to establish their real efficacy and safety. This study investigated the cytotoxicity of xylem sap of Hymenaea courbaril L and its bioactivity against the fungi Cryptococcus neoformans species complex and dermatophytes.MethodsThe fresh xylem sap of H. courbaril was filtered resulting in an insoluble brown color precipitate and was identified as fisetin. In the filtrate was identified the mixture of fisetinediol, fustin, 3-O-methyl-2,3-trans-fustin and taxifolin, which were evaluated by broth microdilution antifungal susceptibility testing against C. neoformans species complex and dermatophytes. The fresh xylem sap and fisetin were screened for cytotoxicity against the 3T3-A31 cells of Balb/c using neutral red uptake (NRU) assay.ResultsThe fresh xylem sap and the fisetin showed higher in vitro activity than the filtrate. The xylem sap of H. courbaril inhibited the growth of dermatophytes and of C. neoformans with minimal inhibition concentration (MIC) < 256 μg/mL, while the fisetin showed MIC < 128 μg/mL for these fungi. Fisetin showed lower toxicity (IC50 = 158 μg/mL) than the fresh xylem sap (IC50 = 109 μg/mL).ConclusionNaturally occurring fisetin can provide excellent starting points for clinical application and can certainly represent a therapeutic potential against fungal infections, because it showed in vitro antifungal activity and low toxicity on animal cells.

In vitro Antifungal Efficacy of Biguanides and Quaternary Ammonium Compounds against Cleanroom Fungal Isolates

In vitro antifungal activities of three biocides including one biguanide (chlorhexidine) and two quaternary ammonium compounds (benzalkonium chloride and cetrimide) were studied against eight cleanroom fungal isolates by using a microbiological broth dilution technique as per Clinical and Laboratory Standards Institute (CLSI) M38-A guidelines. No data exists on the activity of biocides on pharmaceutical cleanroom fungal isolates. Minimum inhibitory concentrations (MICs) for all three biocides against species of Aspergillus and Penicillium species ranged between 4 and 16 μg/mL. MICs of Curvularia, Cladosporium, and Alternaria species also showed less than 16 μg/mL. To date, susceptibility breakpoints have not been established for biocides, and this is the first study using the CLSI broth microdilution antifungal susceptibility testing to determine the MIC value of biocides. This study clearly demonstrates that the most frequently isolated micro-organisms from an environmental monitoring program may be periodically subjected to microbroth dilution testing with cleanroom disinfectant agents used in the disinfection program to confirm their sensitivity profile. Further work is needed in this field to increase our understanding of biocides against different fungal isolates and to enable to the design of more efficient disinfection and contamination control programs. Increased trend of fungal growth is observed in pharmaceutical and medical device cleanrooms. It is essential to have knowledge of choosing effective disinfectants for minimizing fungal occurrence in cleanrooms. The present study establish minimum cut-offs for specific fungal isolates that are problematic in cleanrooms against commonly used disinfectants (quaternary ammonium compounds and biguanides). Further studies based on this minimum inhibitory concentration of disinfectants, effective time, and cleaning methods could prevent fungal occurrence.

The Optimal Wavelength of Spectrophotometric Broth Microdilution Antifungal Susceptibility Test for Flucytosine and Three Azoles

There is no guideline for the appropriate wavelength at which to measure the optical density (OD) value in broth microdilution antifungal susceptibility testing, although a spectrophotometric reading method is commonly used. The present study aimed to analyze the difference in the OD values over the range of visible light and to ascertain the optimal wavelength for the spectrophotometric method of microdilution testing. We measured the OD of background blank controls of broth medium, antifungal agents, and inocula of five type strains using a Synergy HT multi-detection microplate reader at 5-nm intervals from 380 nm to 760 nm. We also estimated the OD differences between the 50% of growth control and blank control. The OD of the blank control showed a parabola shape with two peaks and steadily decreased at longer wavelengths. The curves of the antifungal agent were similar to those of blank controls, and the influence of each antifungal agent on the OD was minimal. For the difference in OD between 50% of growth control and the blank control, the curve was the opposite of the blank control, and the OD increased steadily at the wavelengths above 600 nm. The range between 600 nm and 700 nm was the optimal wavelength for broth microdilution antifungal susceptibility testing, although any wavelength within the visible light spectrum can be used.

Catheter-related fungemia caused by Candida intermedia

Candida intermedia is rarely reported as a human pathogen. We report two cases of catheter-related fungemia caused by C. intermedia which were treated successfully with intravenous fluconazole and catheter removal. The isolates were identified by commercial biochemical methods, oligonucleotide array, and partial sequencing analysis of rRNA genes.

Selection of a Surrogate Agent (Fluconazole or Voriconazole) for Initial Susceptibility Testing of Posaconazole against Candida spp.: Results from a Global Antifungal Surveillance Program

There are currently no FDA-approved broth microdilution antifungal susceptibility testing products or interpretive breakpoints for susceptibility testing of the new triazole posaconazole. Fluconazole and voriconazole are in the same triazole class as posaconazole, have CLSI-approved interpretive MIC breakpoints, and are available on some commercially available MIC panels. We investigated whether one or both of these agents may be useful as a surrogate marker for posaconazole susceptibility. Fluconazole, voriconazole, and posaconazole MIC results for 10,807 isolates of Candida spp. were analyzed to validate a potential surrogate marker for posaconazole activity against indicated species. For illustrative purposes, we applied the voriconazole MIC breakpoints to posaconazole (susceptible, < or =1 microg/ml; susceptible dose dependent, 2 microg/ml; resistant, > or =4 microg/ml) and compared these MIC results and categorical interpretations with those of fluconazole and voriconazole by using regression statistics and categorical agreement. For all 10,807 isolates, the absolute categorical agreement was 91.1% (0.1% very major errors [VME], 1.2% major errors [ME], and 7.6% minor errors [M]) using fluconazole as the surrogate marker and 97.7% (0.3% VME 0.1% ME, and 1.9% M) using voriconazole as the surrogate. The results with fluconazole improved to a categorical agreement of 93.7% (0.1% VME, 0.2% ME, and 6.0% M) when results for Candida krusei (not indicated for fluconazole testing) were omitted. Either fluconazole or voriconazole MIC results may serve as surrogate markers to predict the susceptibility of Candida spp. to posaconazole.

P1676 Evaluation of isavuconazole (BAL8557/BAL4815) Etest compared to broth microdilution antifungal susceptibility testing against quality control strains and fluconazole-susceptible clinical Candida isolates

P1676 Evaluation of isavuconazole (BAL8557/BAL4815) Etest compared to broth microdilution antifungal susceptibility testing against quality control strains and fluconazole-susceptible clinical Candida isolates

Evaluation of Etest and Disk Diffusion Methods Compared with Broth Microdilution Antifungal Susceptibility Testing of Clinical Isolates of Candida spp. against Posaconazole

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

Interactions of posaconazole and flucytosine against Cryptococcus neoformans.

A checkerboard methodology, based on standardized methods proposed by the National Committee for Clinical Laboratory Standards for broth microdilution antifungal susceptibility testing, was applied to study the in vitro interactions of flucytosine (FC) and posaconazole (SCH 56592) (FC-SCH) against 15 isolates of Cryptococcus neoformans. Synergy, defined as a fractional inhibitory concentration (FIC) index of <0.50, was observed for 33% of the isolates tested. When synergy was not achieved, there was still a decrease in the MIC of one or both drugs when they were used in combination. Antagonism, defined as a FIC of >4.0, was not observed. The in vitro efficacy of combined therapy was confirmed by quantitative determination of the CFU of C. neoformans 486, an isolate against which the FC-SCH association yielded a synergistic interaction. To investigate the potential beneficial effects of this combination therapy in vivo, we established two experimental murine models of cryptococcosis by intracranial or intravenous injection of cells of C. neoformans 486. At 1 day postinfection, the mice were randomized into different treatment groups. One group each received each drug alone, and one group received the drugs in combination. While combination therapy was not found to be significantly more effective than each single drug in terms of survival, tissue burden experiments confirmed the potentiation of antifungal activity with the combination. Our study demonstrates that SCH and FC combined are significantly more active than either drug alone against C. neoformans in vitro as well in vivo. These findings suggest that this therapeutic approach could be useful in the treatment of cryptococcal infections.

Development of reference procedures for broth microdilution antifungal susceptibility testing of yeasts with standardized endpoint determination.

Standard guidelines for the broth microdilution antifungal susceptibility testing of amphotericin B, flucytosine, fluconazole, miconazole and itraconazole are reported. These are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) on the following two points: standardization of the means of endpoint determination and the inclusion of miconazole and itraconazole in the testing. MIC was determined to be when the positive control had a turbidity of 0.2 at the 630 nm wavelength. The endpoint was 80% inhibition for azoles and 100% inhibition for other drugs. The method provided good reproducibility, and a wide range of MIC distribution was observed in all antifungal agents except amphotericin B.

Factors Affecting the Results of a Broth Microdilution Antifungal Susceptibility Testing In Vitro

In experiments involving 10 antifungal drugs and 46 strains of potentially pathogenic fungi, the factors affecting the results of in vitro susceptibility testing were studied. The composition of the test medium, inoculum size, temperature and length of incubation were the most pronounced effects influencing the results of testing in vitro. Minimal inhibitory concentrations (MIC) of the antimycotics tested were lowest in complex media (Brain Heart Infusion, Antibiotic Medium 3, Sabouraud broth) except for 5-fluorocytosine which was most effective in Yeast Nitrogen Base medium. Inoculum sizes of 10(3) to 10(4) cfu* mL(-1) had no marked effect on MIC but starting from a final concentration of 10(5) cfu*mL(-1), an abrupt increase in MIC in azole derivatives and 5-fluorocytosine was observed. There was a direct relationship between the duration of incubation and MIC of fungistatic antimycotics. The influence of the incubation temperature became generally manifest primarily in fungi with retarded growth at elevated temperature (>35 degrees C). In these fungal species, a tendency towards a decrease in MIC with increasing temperature was apparent. The other factors studied (medium pH, buffer, solvent) had no substantial influence on the antifungal activity of the drugs tested.