Clinical And Laboratory Standards Institute Method Research Articles

Determination of amphotericin B antifungal susceptibility in Candida strains using an inverted microscope.

Invasive Candida infections have recently shown a significant increase in prevalence and are associated with high mortality rates. Initiating early antifungal treatment in patients with candidemia is vital. The aim of our study was to compare the antifungal susceptibility results of a new method called Flat Plate Method modified from reference "Clinical and Laboratory Standards Institute (CLSI) microdilution method by us with Sensitititre Yeast One colorimetric method and the reference CLSI method. We tested 100 Candida isolates from blood cultures. We followed the CLSI M27-A3 (reference method for broth dilution antifungal susceptibility testing of yeasts; third edition) guidelines for testing in vitro susceptibility to amphotericin B. In the Flat Plate method, 96-well plates were used for evaluation with an inverted microscope. Minimum inhibitory concentration (MIC) values in the SYO method were measured following the manufacturer's instructions. The MIC values obtained by all three methods were considered compatible if they were within ± 2 dilution limits. The SYO method detected C. albicans and C. glabrata with 100% essential agreement, whereas there was 96.29% essential agreement in the case of C. parapsilosis. In the Flat Plate method, the essential agreement with amphotericin B was 91.42%, for C. albicans isolates and 89.47%.for C. glabrata strains. When determining early antifungal susceptibility using the Flat Plate method, the results are obtained quickly, with high accuracy, and without incurring additional costs. However, there is a need for comprehensive studies comparing different antifungals.

Overestimation of Amphotericin B Resistance in Candida auris with Sensititre YeastOne Antifungal Susceptibility Testing: a Need for Adjustment for Correct Interpretation.

Significant variation in minimal inhibitory concentrations (MIC) has been reported for amphotericin B (AMB) and C. auris, depending on the antifungal susceptibility testing (AFST) method. Although the Sensititre YeastOne (SYO) is widely used in routine laboratory testing, data regarding its performance for the AFST of C. auris are scarce. We tested AMB against 65 C. auris clinical isolates with the SYO and the reference methodology by the Clinical and Laboratory Standards Institute (CLSI). The essential agreement (EA, ±1 dilution) between the two methods and the categorical agreement (CA) based on the Centers for Disease Control and Prevention (CDC)'s tentative breakpoint of MIC ≥ 2 mg/L were determined. The SYO wild type upper limit value (WT-UL) was determined using the ECOFFinder. The modal (range) CLSI growth inhibitory MIC was lower than the SYO colorimetric MIC [1(0.25-1) versus 2(1-8) mg/L, respectively]). The CLSI-colorimetric SYO EA was 29% and the CA was 11% (89% major errors; MaE). MaE were reduced when the SYO WT-UL of 8 mg/L was used (0% MaE). Alternatively, the use of SYO growth inhibition endpoints of MIC-1 (75% growth inhibition) or MIC-2 (50% growth inhibition) resulted in 88% CA with 12% MaE and 97% CA with 3% MaE, respectively. In conclusion, SYO overestimated AMB resistance in C. auris isolates when colorimetric MICs, as per SYO instructions and the CDC breakpoint of 2 mg/L, were used. This can be improved either by using the method-specific WT-UL MIC of 8 mg/L for colorimetric MICs or by determining growth inhibition MIC endpoints, regardless of the color. IMPORTANCE Candida auris is an emerging and frequently multidrug-resistant fungal pathogen that accounts for life-threatening invasive infections and nosocomial outbreaks worldwide. Reliable AF is important for the choice of the optimal treatment. Commercial methods are frequently used without prior vigorous assessment. Resistance to AMB was over-reported with the commercial colorimetric method Sensititre YeastOne (SYO). SYO produced MICs that were 1 to 2 twofold dilutions higher than those of the reference CLSI method, resulting in 89% MaE. MaE were reduced using a SYO-specific colorimetric wild type upper limit MIC value of 8 mg/L (0%) or a 50% growth inhibition endpoint (3%).

ANTIBACTERIAL AND CYTOTOXICITY STUDIES OF GOSSYPOL ISOLATED FROM FRUITS OF THESPESIA POPULNEA (L.) SOL. EX CORREA- A REMEDY FOR SKIN DISEASES

Skin infections caused by Staphylococcus aureus and Streptococcus pyogenes and dermatophytoses are becoming severe nowadays. In a previous study, gossypol from fruits of Thespesia populnea (L.) Sol. ex Correa was found to possess growth-inhibiting activity against dermatophytes, causing dermatophytoses, the major fungal disease of the skin (Anuthara et al., 2021). There may be a chance of secondary bacterial infection in the case of dermatophytoses. Hence the present study was designed to evaluate the activity of gossypol against Staphylococcus aureus and Streptococcus pyogenes, the major bacterial pathogens of the skin. Gossypol (GP) was isolated from the fruits of T. populnea and was identified and characterised by HPLC, HPTLC, FTIR, LCMS and NMR spectroscopy in a previous study (Anuthara et al., 2021). Then checked its antibacterial activity against Staphylococcus aureus and Streptococcus pyogenes. The MIC and MBC of gossypol were 31.25 µg/mL and 62.50 µg/mL for Staphylococcus aureus. The MIC of gossypol against Streptococcus pyogenes was 125 µg/mL. It was not bactericidal at the tested concentration. The antibacterial study was done by the disc diffusion method, and it showed inhibition against the growth of two pathogens tested. The minimum inhibitory concentration (MIC) was determined according to the CLSI (clinical and laboratory standards Institute) method- M07-A9 with slight modification (CLSI, 2012b). Since GP showed good inhibition activity against bacterial pathogens, the cytotoxicity of the compound was also tested towards the goal of developing a new antibacterial preparation. Cytotoxicity of gossypol has been evaluated by direct microscopic observation and MTT assay. The apoptosis was detected by acridine orange (AO) and ethidium bromide (EtBr) double staining method.

Comparison of two methods for cell count determination in the course of biocide susceptibility testing

The inoculum density is an important parameter for numerous experimental approaches in bacteriology, including antimicrobial susceptibility testing (AST), biocide susceptibility testing (BST) and biocide efficacy testing (BET). Methods to determine the inoculum density commonly refer to cell counts and have been described for BET according to the German Medical Veterinary Society (Deutsche Veterinärmedizinische Gesellschaft, DVG) and for AST according to the Clinical and Laboratory Standards Institute (CLSI). In this study, the DVG method using 1000 μL volumes of two different dilution steps and the AST method according to CLSI using a 100 μL volume of a single dilution step from the inoculum suspension were compared. For this, each of the four reference strains, Staphylococcus aureus ATCC® 6538, Enterococcus hirae ATCC® 10541, Escherichia coli ATCC® 10536 and Pseudomonas aeruginosa ATCC® 15442, was comparatively tested 28 times using the inoculum preparation according to DVG. The results were statistically analysed using Bland-Altman plots and 95 % limits of agreement (AL). Moreover, cell counts were correlated with the optical density of the bacterial suspensions used. In comparison, the CLSI method measured lower values for colony-forming units (CFU) of -0.12 log10 compared to the DVG method. Overall, both methods returned an AL of -0.52 to 0.27 log10. Since the variations observed between the two methods were within one log10 step and the measured CFUs did not differ systematically, both methods proved to be suitable for cell count determination. Therefore, the CLSI method, which is less complex and less time-consuming, is recommended.

CLSI Methods Development and Standardization Working Group Best Practices for Evaluation of Antimicrobial Susceptibility Tests.

Effective evaluations of antimicrobial susceptibility tests (ASTs) require robust study design. The Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing has recognized that many published studies reporting the performance of commercial ASTs (cASTs) suffer from major design and/or analysis flaws, rendering the results difficult or impossible to interpret. This minireview outlines the current consensus of the Methods Development and Standardization Working Group of the CLSI Subcommittee on Antimicrobial Susceptibility Testing regarding best practices for systematic evaluation of the performance of an AST, including the analysis and presentation of essential data intended for publication.

In Vitro Antibiotic Susceptibilities of Francisella tularensis Determined by Broth Microdilution following CLSI Methods.

In vitro susceptibilities for 47 antibiotics were determined in 30 genetic diverse strains of Francisella tularensis by the broth microdilution method following Clinical and Laboratory Standards Institute (CLSI) methods. The F. tularensis strains demonstrated susceptibility to aminoglycosides, fluoroquinolones, and tetracyclines. There was a distinct difference in macrolide susceptibilities between A and B type strains, as has been noted previously. The establishment and comparison of antibiotic susceptibilities of a diverse but specific set of F. tularensis strains by standardized methods and the establishment of population ranges and MIC50/90 values provide reference information for assessing new antibiotic agents and a baseline to monitor any future emergence of resistance, whether natural or intentional.

Rethinking the Antibiotic Discovery Paradigm

Rethinking the Antibiotic Discovery Paradigm

In vitro antibiotic susceptibilities of Yersinia pestis determined by broth microdilution following CLSI methods.

In vitro susceptibilities to 45 antibiotics were determined for 30 genetically and geographically diverse strains of Yersinia pestis by the broth microdilution method at two temperatures, 28°C and 35°C, following Clinical and Laboratory Standards Institute (CLSI) methods. The Y. pestis strains demonstrated susceptibility to aminoglycosides, quinolones, tetracyclines, β-lactams, cephalosporins, and carbapenems. Only a 1-well shift was observed for the majority of antibiotics between the two temperatures. Establishing and comparing antibiotic susceptibilities of a diverse but specific set of Y. pestis strains by standardized methods and establishing population ranges and MIC50 and MIC90 values provide reference information for assessing new antibiotic agents and also provide a baseline for use in monitoring any future emergence of resistance.

To be or not to be? (Resistant)

Background Our laboratory changed routine antibiotic susceptibility testing from the Clinical and Laboratory Standards Institute (CLSI) to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) method in August 2014. We have since observed a sudden increase in amoxicillin/clavulanate (Augmentin) resistant, beta-lactamase positive, Haemophilus influenzae . Aim This Quality Assurance Project compares H. influenzae susceptibility using breakpoints from EUCAST and CLSI methodology. The null hypothesis: EUCAST method is more sensitive, therefore increased detection of Augmentin resistant strains of H. influenzae is expected. Aim – to determine a gold standard for H. influenzae susceptibility testing. Method H. influenzae isolates were predominantly from respiratory specimens. Disc susceptibility and Augmentin E-tests were performed on every isolate using EUCAST and CLSI methods. Results The CLSI method provided consistent results; disc susceptibility correlated well with E-test results. The EUCAST results were variable; E-test results often did not correlate with disc susceptibility data. Discussion Using the EUCAST method, we identified more H. influenzae that were Augmentin resistant, beta-lactamase producers. Using the CLSI we would have mis-reported these isolates as susceptible to Augmentin. This study raises two important questions: which of the two susceptibility testing methods provides the accurate in vitro results, likely to correlate with clinical effectiveness; and, which method should be recommended for routine use in a diagnostic laboratory?

A rare case of breakthrough fungal pericarditis due to fluconazole‐resistant Candida auris in a patient with chronic liver disease

Introduction: Candida pericarditis is a rare clinical entity with a high fatality, primarily attributed to difficulty in diagnosis. Unfortunately, the diagnosis is made post‐mortem in more than 50% of cases, and thus a high index of clinical suspicion is crucial. Case presentation: We report a rare case of fungal pericardial effusion caused by the recently recognized multidrug‐resistant Candida auris, which was cultured from pericardial fluid, blood, bronchoalveolar lavage and urine of a chronic liver disease patient while on empiric fluconazole therapy. The yeast was misidentified as Candida haemulonii by the VITEK2 commercial identification system, and was confirmed as C. auris by internal transcribed spacer and large ribosomal subunit sequencing. In addition, the VITEK2 AST card erroneously revealed a high amphotericin B MIC (16 µg ml−1) and low caspofungin MIC (0.25 µg ml−1) that did not correlate with results from the reference Clinical and Laboratory Standards Institute (CLSI) microbroth dilution method. Based on VITEK2 MIC data, the patient was administered caspofungin. However, in vitro antifungal susceptibility data for C. auris by the CLSI method exhibited high MICs to fluconazole (64 µg ml−1) and caspofungin MIC (1 µg ml−1) but low MICs to amphotericin B (MIC range, 0.125−0.5 µg ml−1). The patient’s repeat pericardial fluid culture, despite caspofungin therapy for 12 days, grew C. auris and he died on day 13 of therapy. Conclusion: C. auris is a recently reported agent of fungaemia and deep‐seated infections and is notable for its antifungal resistance. Although early species identification and rapid antifungal susceptibility testing are needed in cases of critical infections, the reporting of rare yeast isolates exhibiting high MICs to antifungals by automated systems needs a cautionary approach.

Comparison of EUCAST and CLSI broth microdilution methods for the susceptibility testing of 10 Systemically active antifungal agents when tested against Candida spp.

The antifungal broth microdilution (BMD) method of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) was compared with Clinical and Laboratory Standards Institute (CLSI) BMD method M27-A3 for amphotericin B, flucytosine, anidulafungin, caspofungin, micafungin, fluconazole, isavuconazole, itraconazole, posaconazole, and voriconazole susceptibility testing of 357 isolates of Candida. The isolates were selected from global surveillance collections to represent both wild-type (WT) and non-WT MIC results for the azoles (12% of fluconazole and voriconazole results were non-WT) and the echinocandins (6% of anidulafungin and micafungin results were non-WT). The study collection included 114 isolates of Candida albicans, 73 of C. glabrata, 76 of C. parapsilosis, 60 of C. tropicalis, and 34 of C. krusei. The overall essential agreement (EA) between EUCAST and CLSI results ranged from 78.9% (posaconazole) to 99.6% (flucytosine). The categorical agreement (CA) between methods and species of Candida was assessed using previously determined CLSI epidemiological cutoff values. The overall CA between methods was 95.0% with 2.5% very major (VM) and major (M) discrepancies. The CA was >93% for all antifungal agents with the exception of caspofungin (84.6%), where 10% of the results were categorized as non-WT by the EUCAST method and WT by the CLSI method. Problem areas with low EA or CA include testing of amphotericin B, anidulafungin, and isavuconazole against C. glabrata, itraconazole, and posaconazole against most species, and caspofungin against C. parapsilosis, C. tropicalis, and C. krusei. We confirm high level EA and CA (>90%) between the 2 methods for testing fluconazole, voriconazole, and micafungin against all 5 species. The results indicate that the EUCAST and CLSI methods produce comparable results for testing the systemically active antifungal agents against the 5 most common species of Candida; however, there are several areas where additional steps toward harmonization are warranted.

Evaluation of Etest Performed in Mueller–Hinton Agar Supplemented with Glucose for Antifungal Susceptibility Testing of Clinical Isolates of Filamentous Fungi

Although reference broth microdilution protocol is currently available for filamentous fungi antifungal susceptibility testing (AFST), simpler alternatives as Etest(®) tend to be favoured in clinical routine, making their validation of utmost importance. In this study, Etest(®) method using 2% glucose supplemented Muller-Hinton agar was compared to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 protocol for filamentous fungi AFST. The echinocandins, caspofungin and anidulafungin, the azoles voriconazole and posaconazole, and the polyene amphotericin B were tested against 48 Aspergillus spp., seven Fusarium spp., one Beauveria bassiana and three Paecilomyces lilacinus isolates. The majority of the isolates were susceptible to the antifungals tested, and the overall level of agreement between the CLSI and Etest methods was 71.9% for one dilution and 99.7% when using two dilutions. Since interpretative breakpoints for filamentous fungi employing the CLSI or Etest methods are not available yet, the established epidemiological cut-off values for Aspergillus spp. were used to distinguish wild-type isolates from those with acquired resistance mechanisms. Forty-five Aspergillus strains did not evidence resistance mutations.

Assessment of caspofungin susceptibility of Candida glabrata by the Etest®, CLSI, and EUCAST methods, and detection of FKS1 and FKS2 mutations

Candida glabrata has emerged as a major pathogen in invasive candidiasis in recent years. Currently, guidelines for invasive candidiasis treatment recommend fluconazole or an echinocandin as the first-line therapy. Nevertheless, the resistance of Candida glabrata to echinocandin is an emerging problem and has been partly associated with mutations in the FKS1 and FKS2 genes. The Etest® is an appropriate method for determining antifungal susceptibility in emergency routine diagnosis. In this work, we evaluated the reliability of the Etest® in comparison with the two reference broth microdilution methods, Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST), to assess the caspofungin resistance of 193 isolates of Candida glabrata. The interpretation of minimum inhibitory concentration (MIC) values was also discussed according to different breakpoints. Moreover, FKS1 and FKS2 mutations were investigated for isolates with high MICs. Our results showed that the MIC50 value was similar to the MIC90 value for each method. The Etest® method showed the lowest MIC values, whereas EUCAST presented the highest. Categorical agreement between the Etest® and CLSI methods was 100 % and 36 % using the breakpoints proposed by Arendrup et al. (Antimicrob Agents Chemother 56(7):3965-3968, 2012) and Pfaller et al. (Int J Antimicrob Agents 38(1):65-69, 2011), respectively. Two isolates showed high MIC values with the three methods and both presented FKS2 mutations. A novel FKS2 mutation was also reported for one isolate. Future epidemiological studies should also evaluate the reliability of the Etest® to detect echinocandin resistance, as it remains a routine method.

Comparison of two disc diffusion methods with minimum inhibitory concentration for antimicrobial susceptibility testing of Neisseria gonorrhoeae isolates.

Background:A few studies are available comparing either minimum inhibitory concentration (MIC) values with the Clinical and Laboratory Standards Institute (CLSI) disc diffusion method or MIC with the Australian Gonococcal Surveillance Program (AGSP) method.Aim:This study was conducted with the aim to identify the most feasible and cost-effective method for antimicrobial susceptibility testing of Neisseria gonorrhoeae.Materials and Methods:Antimicrobial susceptibility testing of N. gonorrhoeae was performed using, in parallel, the E-test for MIC determination and disc diffusion by CLSI and AGSP techniques, and were compared. Susceptibility to penicillin, ciprofloxacin, tetracycline, ceftriaxone and spectinomycin and cefixime were determined by CSLI and AGSP method and Kappa statistics used to analyse the data with SPSS software.Results:All isolates were susceptible to ceftriaxone and spectinomycin by three methods. Ninety-nine (99%) strains were resistant to ciprofloxacin, while 1% showed intermediate susceptibility to ciprofloxacin by all methods. Statistically, there was a moderate level of agreement between the methods for penicillin.Conclusion:All three methods gave reproducible results. Although the media used in the disc diffusion by the AGSP method is easy and cheap to prepare, the CLSI method of disc diffusion testing is recommended for susceptibility testing of gonococcal isolates because of its feasibility and 100% accuracy, with MIC by E-test as the reference method.

In vitro activities of isavuconazole and comparator antifungal agents tested against a global collection of opportunistic yeasts and molds.

Isavuconazole is a new broad-spectrum triazole with a favorable pharmacokinetic and safety profile. We report the MIC distributions for isavuconazole and 111 isolates of Candida (42 Candida albicans, 25 Candida glabrata, 22 Candida parapsilosis, 14 Candida tropicalis, and 8 Candida krusei isolates), as determined by Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution (BMD) methods. Also, the relative activities of isavuconazole, itraconazole, fluconazole, posaconazole, voriconazole, and the three echinocandins were assessed against a recent (2011) global collection of 1,358 isolates of Candida spp., 101 of Aspergillus spp., 54 of non-Candida yeasts, and 21 of non-Aspergillus molds using CLSI BMD methods. The overall essential agreement (EA) (±2 log2 dilutions) between the CLSI and EUCAST methods was 99.1% (EA at ±1 log2 dilution, 90.1% [range, 80.0 to 100.0%]). The activities of isavuconazole against the larger collection of Candida spp. and Aspergillus spp. were comparable to those of posaconazole and voriconazole; the MIC90 values for isavuconazole, posaconazole, and voriconazole against Candida spp. were 0.5, 1, and 0.25 μg/ml and against Aspergillus spp. were 2, 1, and 1 μg/ml, respectively. Isavuconazole showed good activities against Cryptococcus neoformans (MIC90, 0.12 μg/ml) and other non-Candida yeasts (MIC90, 1 μg/ml) but was less potent against non-Aspergillus molds (MIC90, >8 μg/ml). Isavuconazole MIC values for three mucormycete isolates were 4, 1, and 2 μg/ml, whereas all three were inhibited by 1 μg/ml posaconazole. Isavuconazole demonstrates broad-spectrum activity against this global collection of opportunistic fungi, and the CLSI and EUCAST methods can be used to test this agent against Candida, with highly comparable results.

In Vitro Fluconazole and Voriconazole Susceptibilities of Candida Bloodstream Isolates in Korea: Use of the CLSI and EUCAST Epidemiological Cutoff Values

BackgroundAt present, the clinical breakpoints (CBPs) of both fluconazole and voriconazole are available only for 3 common Candida species in the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methods. Epidemiological cutoff values (ECVs) were recently applied to both methods to detect the emergence of acquired resistance (i.e., non-wild-type isolates) among 5 common Candida species.MethodsWe performed a nationwide study to determine the fluconazole and voriconazole susceptibility of Candida bloodstream isolates (BSIs) using both the CLSI and EUCAST methods. A total of 423 BSIs of 5 Candida species were collected from 8 hospitals. The azole susceptibilities were assessed on the basis of the species-specific CBPs and ECVs.ResultsOf the 341 BSIs of 3 common Candida species (i.e., C. albicans, C. tropicalis, and C. parapsilosis), 0.3% and 0.9%, 0.0% and 1.5% of isolates were categorized as fluconazole and voriconazole resistant according to the CLSI and EUCAST CBPs, respectively. Of 423 total BSIs, 1.4% and 2.6% had fluconazole minimum inhibitory concentrations (MICs) exceeding the ECVs according to the CLSI and EUCAST, respectively; 1.0% and 2.1% had voriconazole MICs exceeding the ECVs according to the CLSI and EUCAST, respectively. Categorical agreement between the methods using ECVs was 98.3% for fluconazole and 98.3% for voriconazole.ConclusionsThe EUCAST and CLSI methods using ECVs provide highly concordant results. Moreover, non-wild-type isolates with possibly acquired azole resistance were rare among the BSIs of 5 common Candida species in Korea.

Comparative Drug Resistance of Mycobacterium abscessus and M. chelonae Isolates from Patients with and without Cystic Fibrosis in the United Kingdom

The isolation of rapidly growing mycobacteria (RGM), particularly Mycobacterium abscessus, from individuals with cystic fibrosis (CF) is associated with poor clinical outcome due to broad drug resistance and the difficulty of eradicating the organisms. Susceptibility testing is recommended to guide therapy. A disc diffusion method is used in the United Kingdom, whereas in the United States, the CLSI (Clinical and Laboratory Standards Institute) recommends the broth dilution method. The purpose of this study was to investigate whether the two methods produced comparable drug resistance profiles and to test the hypotheses that the disc diffusion method overscores resistance and that isolates of M. abscessus/M. chelonae from CF patients are more likely than those from non-CF patients to show drug resistance, as a result of CF patients' greater exposure to antibiotic therapy. A total of 82 isolates (58 M. abscessus and 24 M. chelonae isolates) were tested blindly against 15 antimicrobials by broth dilution and the disc diffusion method. Isolates tested by the broth microdilution showed high levels of resistance; susceptibility to amikacin, clarithromycin, tobramycin (only in M. chelonae), and cefoxitin (only in M. abscessus) was shown. Tigecycline results varied widely depending on which breakpoint was used. Agreement between methods for a few drugs (e.g., cefoxitin and amikacin) was poor. Although there were drug resistance differences between CF and non-CF isolates, these did not reach statistical significance. The CLSI method provided more robust breakpoints, standardization, and reproducibility. An analysis of the implementation of the CLSI method demonstrated ease of use and similar drug resistance findings for the two species.

Detection of Amphotericin B Resistance in Candida haemulonii and Closely Related Species by Use of the Etest, Vitek-2 Yeast Susceptibility System, and CLSI and EUCAST Broth Microdilution Methods

The emerging fungal pathogens Candida haemulonii and Candida pseudohaemulonii often show high-level resistance to amphotericin B (AMB). We compared the utilities of five antifungal susceptibility testing methods, i.e., the Etest using Mueller-Hinton agar supplemented with glucose and methylene blue (Etest-MH), the Etest using RPMI agar supplemented with glucose (Etest-RPG), the Vitek-2 yeast susceptibility system, and the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution methods, for the detection of AMB-resistant isolates of C. haemulonii and closely related species. Thirty-eight clinical isolates (8 C. haemulonii, 10 C. pseudohaemulonii, and 20 Candida auris isolates) were analyzed. Of the 18 C. haemulonii and C. pseudohaemulonii isolates, 18, 15, 18, 10, and 9 exhibited AMB MICs of >1 μg/ml by the Etest-MH, Etest-RPG, Vitek-2, CLSI, and EUCAST methods, respectively. All 20 C. auris isolates showed AMB MICs of ≤1 μg/ml by all five methods. Of the methods, the Etest-MH generated the broadest distribution of AMB MICs for all 38 isolates and showed the best discrimination between the C. haemulonii and C. pseudohaemulonii isolates (4 to 32 μg/ml) and those of C. auris (0.125 to 0.5 μg/ml). Taking the Etest-MH as the reference method, the essential agreements (within two dilutions) for the Etest-RPG, Vitek-2, CLSI, and EUCAST methods were 84, 92, 55, and 55%, respectively; the categorical agreements were 92, 92, 79, and 76%, respectively. This study provides the first data on the efficacy of the Etest-MH and its excellent agreement with Vitek-2 for discriminating AMB-resistant from AMB-susceptible isolates of these Candida species.

Comparative assessment of CDS, CLSI disc diffusion and Etest techniques for antimicrobial susceptibility testing of Neisseria gonorrhoeae: a 6-year study

BackgroundA variety of techniques are available for antimicrobial susceptibility testing of Neisseria gonorrhoeae.ObjectiveThe aim of this study was to find a cost-effective, reliable and easily applicable microbiological method to detect...

In vitro activity of a novel broad-spectrum antifungal, E1210, tested against Aspergillus spp. determined by CLSI and EUCAST broth microdilution methods.

E1210 is a first-in-class broad-spectrum antifungal that suppresses hyphal growth by inhibiting fungal glycophosphatidylinositol (GPI) biosynthesis. In the present study, we extend these findings by examining the activity of E1210 and comparator antifungal agents against Aspergillus spp. by using the methods of the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) to test wild-type (WT) as well as amphotericin B (AMB)-resistant (-R) and azole-R strains (as determined by CLSI methods). Seventy-eight clinical isolates of Aspergillus were tested including 20 isolates of Aspergillus flavus species complex (SC), 22 of A. fumigatus SC, 13 of A. niger SC, and 23 of A. terreus SC. The collection included 15 AMB-R (MIC, ≥ 2 μg/ml) isolates of A. terreus SC and 10 itraconazole-R (MIC, ≥ 4 μg/ml) isolates of A. fumigatus SC (7 isolates), A. niger SC (2 isolates), and A. terreus SC (1 isolate). Comparator antifungal agents included anidulafungin, caspofungin, amphotericin B, itraconazole, posaconzole, and voriconazole. Both CLSI and EUCAST methods were highly concordant for E1210 and all comparators. The essential agreement (EA; ± 2 log(2) dilution steps) was 100% for all comparisons with the exception of posaconazole versus A. terreus SC (EA = 91.3%). The minimum effective concentration (MEC)/MIC(90) values (μg/ml) for E1210, anidulafungin, caspofungin, itraconazole, posaconazole, and voriconazole, respectively, were as follows for each species: for A. flavus SC, 0.03, ≤ 0.008, 0.12, 1, 1, and 1; for A. fumigatus SC, 0.06, 0.015, 0.12, >8, 1, and 4; for A. niger SC, 0.015, 0.03, 0.12, 4, 1, and 2; and for A. terreus SC, 0.06, 0.015, 0.12, 1, 0.5, and 1. E1210 was very active against AMB-R strains of A. terreus SC (MEC range, 0.015 to 0.06 μg/ml) and itraconazole-R strains of A. fumigatus SC (MEC range, 0.03 to 0.12 μg/ml), A. niger SC (MEC, 0.008 μg/ml), and A. terreus SC (MEC, 0.015 μg/ml). In conclusion, E1210 was a very potent and broad-spectrum antifungal agent regardless of in vitro method applied, with excellent activity against AMB-R and itraconazole-R strains of Aspergillus spp.