MALDI-TOF MS-based drug susceptibility testing of pathogens: The example of Candida albicans and fluconazole (original) (raw)
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Application of MALDI-TOF MS for requalification of a Candida clinical isolates culture collection
Brazilian Journal of Microbiology, 2014
Microbial culture collections underpin biotechnology applications and are important resources for clinical microbiology by supplying reference strains and/or performing microbial identifications as a service. Proteomic profiles by MALDI-TOF MS have been used for Candida spp. identification in clinical laboratories and demonstrated to be a fast and reliable technique for the routine identification of pathogenic yeasts. The main aim of this study was to apply MALDI-TOF MS combined with classical phenotypic and molecular approaches to identify Candida clinical isolates preserved from 1 up to 52 years in a Brazilian culture collection and assess its value for the identification of yeasts preserved in this type of collections. Forty Candida spp. clinical isolates were identified by morphological and biochemical analyses. Identifications were also performed by the new proteomic approach based on MALDI-TOF MS. Results demonstrated 15% discordance when compared with morphological and biochemical analyses. Discordant isolates were analysed by ITS sequencing, which confirmed the MALDI-TOF MS identifications and these strains were renamed in the culture collection catalogue. In conclusion, proteomic profiles by MALDI-TOF MS represents a rapid and reliable method for identifying clinical Candida species preserved in culture collections and may present clear benefits when compared with the performance of existing daily routine methods applied at health centres and hospitals.
Rapid detection of fluconazole resistance in Candida tropicalis by MALDI-TOF MS
Medical Mycology
With the changing epidemiology and emergence of antifungal resistance among Candida species, rapid antifungal susceptibility testing (AFST) is crucial for optimization of antifungal therapy. This study was conducted to standardize a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) based AFST method (ms-AFST) for susceptibility of Candida tropicalis isolates. Clinical isolates of C. tropicalis were confirmed for fluconazole resistance by the CLSI (M27-A3) method. The incubation period and drug concentration were optimized to determine the minimal profile change concentration (MPCC) by MALDI-TOF MS. The data were analyzed first by direct visual observation of the spectra followed by composite correlation index (CCI) matrix analysis, virtual gel analysis, and cluster analysis for confirmation. Finally, the correlation between minimum inhibitory concentrations (MICs) and MPCCs was evaluated. A total of 15 fluconazole resistant (MICs ranging from 16 to 128 μg/ml) and 19 fluconazole susceptible C. tropicalis isolates (MIC ≤1 μg/ml) were included in this study. All C. tropicalis isolates had significant spectral changes after 4h incubation with fluconazole. Of 34 isolates, MPCCs and MICs were equivalent for 16 isolates, and the MPCC was one dilution lower than the respective MIC in the remaining 18 isolates. This finding was further supported by visual analysis, CCI matrix analysis, virtual gel and principal component analysis dendrogram analysis. The correlation between MPCC and MIC was significant (P < .05). Therefore, a MALDI-TOF MS based AFST assay may be used as a rapid screening technique for fluconazole resistance in C. tropicalis.
2021
Background C. albicans is the most important yeast that caused the infection in humans; the trend of resistance to fluconazole (FLC) was also increased, while the FLC susceptibility by conventional method takes time causing the treatment failure. To investigate FLC susceptibility to C. albicans using MALDI-TOF MS and Real-time PCR for CDR1, CDR2, MDR1 and ERG11, overall, 32 C. albicans strains included 4 reference strains (3 FLC susceptible (S) and 1 FLC resistant (R), 1 spontaneous mutant strain (FLC susceptible-dose dependent, SDD) and 27 clinical strains obtained from 2 Thai University Hospitals were performed FLC susceptibility testing by Sensititre YeastOne and broth microdilution method, FLC resistant expression mechanism by Real-time PCR and the major peak determination by MALDI-TOF MS.Results The change of CDR1 and CDR2 mRNA expression were only significantly observed in SDD and R strains. Using MALDI-TOF MS, the change of mass spectral intensity at range 3376-3382 m/z (majo...
Journal of Antimicrobial Chemotherapy, 2019
Background: With the rise of fluconazole resistance in Candida tropicalis in Asian countries, the rapid detection of resistance is required to optimize therapy. Objectives: We evaluated a stable isotope labelling by/with amino acids in cell culture (SILAC) approach for rapid detection of fluconazole resistance in C. tropicalis by MALDI-TOF MS. Methods: Twenty-six fluconazole-resistant and 19-susceptible C. tropicalis isolates were used. Isolates were grown in media containing normal lysine (NL), isotopically labelled ('heavy') lysine (HL) and fluconazole (FLC) with labelled lysine (HL ! FLC). MALDI-TOF MS was performed, acquired spectra were visually compared and composite correlation index (CCI) values were calculated. The results were analysed by CCI matrix, virtual gel and principal component analysis (PCA). Results: The MICs of fluconazole in 26 resistant and 19 susceptible isolates were 16-256 mg/L and 0.5-1 mg/L, respectively. The m/z values of 15 peaks specific for media containing NL and their corresponding peaks specific for media with HL were detected to differentiate resistant and susceptible isolates. The CCI cutoff values for susceptible and resistant isolates were significant (P , 0.05). The CCI matrix, virtual gel and PCA dendrogram confirmed the results. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of this method for detection of fluconazole resistance were 92.31%, 100%, 100%, 90.48% and 95.56%, respectively. Conclusions: SILAC, a promising approach for rapid resistance detection in C. tropicalis using MALDI-TOF MS, may be used in the routine laboratory in the near future.
Establishing Surrogate Markers for Fluconazole Resistance in Candida albicans
Microbial Drug Resistance, 2005
Azole-resistant Candida can be a confounding factor for clinical management of opportunistic infections in immunocompromised patients, but rapid identification of such resistant organisms can improve patient outcome. New target-based molecular diagnostic strategies have the potential to identify resistant organisms faster than current culture-based assays. It was the objective of this study to determine whether target site mutations and/or drug pump over-expression are suitable surrogate markers of drug resistance that could aid new molecular-based diagnostic assays. A collection of 59 clinical isolates displaying a range of azole susceptibilities were assayed for mutations within the target gene Erg11 and for over-expression of drug-efflux pumps Cdr1, Cdr2, Flu1, and Mdr1, as well as drug target gene Erg11 by quantitative real-time PCR with molecular beacons. A fluconazole-resistant (MIC Ն 64 g/ml) phenotype was closely associated with over-expression of Cdr1 (p ؍ 0.005), Cdr2 (p ؍ 0.01), and Mdr1 (p ؍ 0.03) along with four mutations in Erg11 (T229A, Y132F, S405F, G464S). Changes in expression levels for Erg11 and Flu1 were not statistically correlated with resistance (p ؍ 0.27 and p ؍ 0.86, respectively). Overall, these findings provide a statistical basis to establish Erg11 mutations and drug pump over-expression as surrogate markers for phenotypic fluconazole resistance.
Retrospective Re- Identification of Candida Strains by Maldi-Tof in Seek of Candida Auris
PROBLEMS of Infectious and Parasitic Diseases
The yeast C. auris was first described as a new species in 2009. Since then, this species was recognized as an emerging multi-drug-resistant (MDR) yeast that can cause a wide spectrum of infections, ranging from fungemia to deep-seated infections, especially in intensive care settings. It can be not- or misidentified by commercial identification systems. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently been considered as a convenient, rapid and accurate technology in the identification of yeast isolates to species level. A total of 132 clinical isolates, collected from 2015 to 2021 in the National Reference Laboratory of Mycology and Sexually Transmitted Infections were included in the study. The isolates were mainly from patients with candidemia and other specimens from invasive Candida infections. The isolates were identified by standard mycological procedures, the assimilation profile was done by commercially available strip...
Perspectives on antifungal susceptibility testing in Candida
International Journal of Infectious and Tropical Diseases, 2014
Background: Along with the increase in the incidence of candidiasis, there has been an important shift away from Candida albicans towards non albicans spp. Many species from non albicans Candida group exhibit varying degrees of resistance, either intrinsic or acquired or both, to commonly used antifungal drugs. The emergence of antifungal resistance and a gradual increase in the number of new and broad spectrum antifungal drugs has complicated the choice of antifungal drug for treatment of candidiasis. Accurate, reproducible and reliable antifungal susceptibility testing is necessary for the selection of an appropriate and accurate antifungal therapeutic agent. The standard reference method of in vitro antifungal susceptibility testing is complex and labor intensive. Aim: This paper reviews various methodologies commonly used for antifungal susceptibility testing of Candida spp is reviewed. Method: Relevant research articles on Pubmed and Google Scholar were collected and reviewed. Results: A total of 51 research articles from various journals were reviewed for various methodologies commonly used for antifungal susceptibility testing of Candida spp. Conclusion: The in vitro antifungal susceptibility of an infecting species of Candida is one of the several factors that influence the successful therapy. In developed countries, antifungal susceptibility testing is now increasingly used to supplement the treatment of mycotic infection. In resource-constrained laboratories of developing countries where determination of antifungal susceptibility testing by the Clinical and Laboratory Standards Institute (CLSI) standardized broth method is not always feasible, simple, user friendly methods like disc diffusion, Etest can be easily incorporated as alternative reliable techniques.
Antimicrobial Agents and Chemotherapy, 1992
In vitro antifungal susceptibility testing generally remains unstandardized and unreliable for directing therapy. When azoles are tested, this problem is further compounded by the lack of definite reading end points. We determined the in vitro susceptibility of 50 Candida albicans isolates (including 10 reference strains) to fluconazole by using a microbroth dilution method in which microtiter plates were agitated immediately before reading. Six fungal inoculum sizes (ranging from 2 x 102 to 4 x 105 CFU/ml), three different times of reading (24, 48, and 72 h), and two temperatures (30 and 35°C) were tested. We also compared visual and spectrophotometric determinations of MIC end points. This agitation method resulted in clear-cut visual end points that were reproducible for different observers within the same laboratory, that were independent of inoculum size, temperature of incubation, and time of reading, and that correlated well with the degree of fungal inhibition as determined by spectrophotometry. Median MICs also correlated with usually achievable levels of fluconazole in serum and tissue of humans and experimental animals.
International Journal of Analytical Chemistry, 2015
Candida albicans (strains NCTC-885-653 and ATCC-10231) long-term cultivated in the presence of antifungal agent fluconazole (FLC) and classical microbiological methods for determination of minimal inhibitory concentration (MIC) were used in this study. A simple and sensitive method based on reverse-phase high-performance liquid chromatography (RP-HPLC) has been developed for the determination of FLC intracellular concentration in C. albicans using tinidazole as an internal standard. Following extraction with dichloromethane, the chromatographic separation was achieved on a Machery-Nagel EC250/2 Nucleodur-100-3 C18 column by gradient elution using the mobile phase consisting of (A) 0.01 M ammonium acetate buffer, pH = 5.00, and (B) acetonitrile. Different analytical performance parameters such as linearity, precision, accuracy, limit of quantification (LOQ), and robustness were determined according to US DHHS FDA and EMEA guidelines. The method was linear for FLC (= 0.9999) ranging from 100 to 10000 ng/mL. The intraday and interday precisions (relative standard deviation) were within 2.79 and 2.64%, respectively, and the accuracy (relative error) was less than 2.82%. The extraction recovery ranged from 79.3 to 85.5%. The reliable method was successfully applied to C. albicans azole-resistance study and it was shown that intracellular concentration of FLC correlated with a yeast drug susceptibility profile and MIC values.