Rapid detection of fluconazole resistance in Candida tropicalis by MALDI-TOF MS

Paul, Saikat; Singh, Meenu; Shamanth, A.S.; Rudramurthy, Shivaprakash M.; Chakrabarti, Arunaloke; Ghosh, Anup

doi:10.1093/mmy/myx042

Cited by 33 publications

(33 citation statements)

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“…Our findings provide further insights into the clinical diagnostic possibilities of MALDI–TOF MS shown for Candida species (including C. parapsilosis ). These possibilities encompass the in vitro assays for fungal identification, susceptibility/resistance detection, and typing, particularly for emerging Candida species (De Carolis et al, 2014a; Dhieb et al, 2015; Vella et al, 2017; Bao et al, 2018; Paul et al, 2018; Vatanshenassan et al, 2018, 2019). Very few studies, to date, tempted to apply the MALDI–TOF MS technology to the direct detection of biofilm-forming isolates of Candida species.…”

Section: Resultsmentioning

confidence: 99%

BIOF–HILO Assay: A New MALDI–TOF Mass Spectrometry Based Method for Discriminating Between High- and Low-Biofilm-Producing Candida parapsilosis Isolates

et al. 2019

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Candida parapsilosis is the most frequent cause of catheter-related candidemia among non- Candida albicans species. This may be related to intrinsic capabilities as adhering and forming a biofilm on abiotic surfaces such as on medical devices. As previously demonstrated, patients infected with high biofilm-producing C. parapsilosis isolates had a greater mortality risk compared to patients infected with low biofilm-producing C. parapsilosis isolates. We developed the BIOF–HILO assay, a MALDI–TOF mass spectrometry (MS)-based assay, which compares mass spectra obtained from attached and suspended isolate cells during the early (i.e., 3-h) adhesion phase of in vitro biofilm formation. The composite correlation index (CCI) analysis was used to discriminate between mass spectra differences of the two cell types, classifying all 50 C. parapsilosis clinical isolates, included in the study, after only 3-h of testing, in high or low biofilm producers. All high ( n = 25) or low ( n = 25) biofilm producers had, according to CCI mass spectra comparison values, higher or lower than one CCI ratios, which were obtained by dividing the CCI suspended cells by the CCI attached cells . In conclusion, the BIOF–HILO assay allows a rapid categorization of C. parapsilosis clinical isolates in high or low biofilm producers. This information, if timely provided to physicians, may improve treatment outcomes in patients with C. parapsilosis candidemia.

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Section: Resultsmentioning

confidence: 99%

BIOF–HILO Assay: A New MALDI–TOF Mass Spectrometry Based Method for Discriminating Between High- and Low-Biofilm-Producing Candida parapsilosis Isolates

et al. 2019

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“…All the experiments used in this study was performed as per the guidelines and regulations approved by the institutional ethics committee of the Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. All these isolates were also used in our previous studies and their details MICs are presented in Supplementary Table S1 43,44 . A total of 60 samples (30 fluconazole treated and 30 untreated controls) were used for stability analysis.…”

Section: Resultsmentioning

confidence: 99%

Selection and evaluation of appropriate reference genes for RT-qPCR based expression analysis in Candida tropicalis following azole treatment

Paul

Chakrabarti

Rudramurthy

et al. 2020

Sci Rep

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Candida tropicalis arises as one of the predominant non-Candida albicans Candida (ncAc) species causing invasive candidiasis in Asian countries. A rise in reports of C. tropicalis with a parallel increase in fluconazole resistance has also been observed. The genes and underlying pathways associated with azole antifungal resistance in C. tropicalis is still not properly understood. the Rt-qpcR is the most promising approach for expression analysis of target genes to understand the mechanisms of resistance. the reliability and reproducibility of this technique depend on the selection of suitable reference genes for the normalization in expression study. the present study investigated the expression stability levels of ten genes including ACT1, EF1, GAPDH, PGK1, RDN5.8, RDN18, RDN28, SDHA, TUB1, and UBC13 for their suitability in fluconazole treated/untreated C. tropicalis. the stability levels of these genes were examined by the ∆∆ct, ΔCT, Pfaffl methods and five independent software including hkgfinder, genorm, normfinder, BestKeeper, and Reffinder software. We report, the EF1 and ACT1 were the most stable reference genes for normalization and can be used for the gene expression analysis in C. tropicalis. To the best of our knowledge, our study is the first to select and validate the reference genes in C. tropicalis for Rt-qpcR based expression analysis. Candida tropicalis, a non-Candida albicans Candida (NCAC) resides in human skin, genitourinary, respiratory, and gastrointestinal tracts as a part of the normal microbiota 1-3. In Asian countries, C. tropicalis has emerged as the predominant NCAC species causing invasive candidiasis (IC), particularly candidemia 4-6. Fluconazole is the most common antifungal drug used to treat candidemia due to C. tropicalis. The rise in IC due to C. tropicalis has been paralleled with an increase in fluconazole resistance, especially in Asian countries 4-6. The differential expression of ergosterol biosynthesis pathway genes, ATP-binding cassette (ABC), and major facilitator superfamily (MFS) drug transporters are directly linked with the azole resistance in C. tropicalis 7-9. Although various studies demonstrated the role of these mechanisms in azole resistant C. tropicalis, the principle pathways and regulatory circuits implicated are yet to be elucidated. Profiling of gene expression is a powerful approach to determine the pattern response to various stimuli including drugs and gives a holistic impression of cellular function in any living cell 10. Usually, gene expression can be estimated by multiple methods, including RNase protection assay, Northern blotting, real-time quantitative PCR (RT-qPCR), and semi-quantitative reverse-transcription PCR 11. RT-qPCR has received special attention due to its significantly higher accuracy, sensitivity, and rapidity allowing high throughput results, detection of mRNAs with low-abundance 12 and mRNA copy number measurement 13. As a result, RT-qPCR platform has been utilized for diverse applications including gene expression analysis 11,14...

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“…In CCI matrix, closely related spectra appeared as a color contrast between yellow and dark red, whereas distantly related spectra produce a contrast between green and dark blue. The color intensity of the CCI matrix determined the relationship between individual melanized fungal isolates …”

Section: Methodsmentioning

confidence: 99%

“…MSPs of all 59 isolates were used to make the dendrogram. The distance between isolates was represented by different clades in dendrogram and it was normalized up to a value of 1000 …”

Section: Methodsmentioning

confidence: 99%

MALDI‐TOF MS‐Based Identification of Melanized Fungi is Faster and Reliable After the Expansion of In‐House Database

Paul

Singh

Sharma

et al. 2018

Proteomics Clinical Apps

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Rapid detection of fluconazole resistance in Candida tropicalis by MALDI-TOF MS

Cited by 33 publications

References 20 publications

BIOF–HILO Assay: A New MALDI–TOF Mass Spectrometry Based Method for Discriminating Between High- and Low-Biofilm-Producing Candida parapsilosis Isolates

BIOF–HILO Assay: A New MALDI–TOF Mass Spectrometry Based Method for Discriminating Between High- and Low-Biofilm-Producing Candida parapsilosis Isolates

Selection and evaluation of appropriate reference genes for RT-qPCR based expression analysis in Candida tropicalis following azole treatment

MALDI‐TOF MS‐Based Identification of Melanized Fungi is Faster and Reliable After the Expansion of In‐House Database

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