Biology subjects

Chaturvedi, V.

Publications and source records attributed to Chaturvedi, V..

10 recordsLinked to original sources

bioRxiv2024

What makes Candida auris pan-drug resistant? Integrative insights from genomic, transcriptomic, and phenomic analysis of clinical strains resistant to all four major classes of antifungal drugs

The global epidemic of drug-resistant Candida auris continues unabated. We do not know what caused the unprecedented appearance of pan-drug resistant (PDR) Candida auris strains in a hospitalized patient in New York; the initial report highlighted both known and unique mutations in the prominent gene targets of azoles, amphotericin B, echinocandins, and flucytosine antifungal drugs. However, the factors that allow C. auris to acquire multi-drug resistance and pan-drug resistance are not known. Therefore, we conducted a comprehensive genomic, transcriptomic, and phenomic analysis to better understand PDR C. auris. Among 1,570 genetic variants in drug-resistant C. auris, 299 were unique to PDR strains. The whole genome sequencing results suggested perturbations in genes associated with nucleotide biosynthesis, mRNA processing, and nuclear export of mRNA. Whole transcriptome sequencing of PDR C. auris revealed two genes to be significantly differentially expressed - a DNA repair protein and DNA replication-dependent chromatin assembly factor 1. Of 59 novel transcripts, 12 candidate transcripts had no known homology among expressed transcripts found in other organisms. We observed no fitness defects among multi-drug resistant (MDR) and PDR C. auris strains grown in nutrient-deficient or - enriched media at different temperatures. Phenotypic profiling revealed wider adaptability to nitrogenous nutrients with an uptick in the utilization of substrates critical in upper glycolysis and tricarboxylic acid cycle. Structural modelling of 33-amino acid deletion in the gene for uracil phosphoribosyl transferase suggested an alternate route in C. auris to generate uracil monophosphate that does not accommodate 5-fluorouracil as a substrate. Overall, we find evidence of metabolic adaptations in MDR and PDR C. auris in response to antifungal drug lethality without deleterious fitness costs.

microbiology↗

bioRxiv2021

Reverse Transcriptase Real-Time PCR Assay for the Rapid Enumeration of Live Candida auris from the Healthcare Environment

Ongoing healthcare-associated outbreaks of multidrug-resistant yeast Candida auris have prompted development of several rapid DNA-based molecular diagnostic tests. These tests do not distinguish between live and dead C. auris cells, limiting their use for environmental surveillance and containment efforts. We addressed this critical gap by developing a reverse transcriptase (RT) real-time PCR assay for rapid detection of live C. auris in healthcare environments. The assay targets the internal transcribed spacer 2 (ITS2) cDNA of C. auris, amplified by obtaining pure RNA followed by reverse transcription and real-time PCR assay. The assay was highly sensitive, with the detection limit of ten colony-forming units (CFU) per RT real-time PCR reaction. In C. auris viability studies, ITS2 cDNA was detectable in heat- and ethanol-killed C. auris cells, but not bleach-killed cells. Validation studies showed no cycle threshold (Ct) values were obtained from samples on a sponge matrix spiked with either dead C. auris (105/ml) or other Candida species (105/ml), while most environmental samples spiked with 102 to 105 CFU of live C. auris yielded positive Ct values. Finally, 33 environmental samples positive for C. auris DNA but negative by culture were all negative by RT real-time PCR assay, confirming concordance between culture and the assay. The RT real-time PCR assay appears highly reproducible, robust, and specific for detecting live C. auris from environmental samples, and could be an invaluable tool in surveillance efforts to control the spread of live C. auris in healthcare environments.

molecular biology↗

bioRxiv2020

Pan-resistant Candida auris: New York Sub-cluster Susceptible to Antifungal Combinations

Four pan-resistant Candia auris strains from New York outbreak were 100% inhibited in vitro by combinations of two antifungal drugs using fixed concentrations achievable in vivo. Pan-resistant C. auris strains have mutations in eleven gene targets associated with major antifungal drugs, and constituted a distinct sub-cluster among NY strains.

microbiology↗

bioRxiv2020

Evaluation of in vitro activity of manogepix against multidrug-resistant and pan-resistant Candida auris from the New York Outbreak

An ongoing Candida auris outbreak in the New York metropolitan area is the largest recorded to date in North America. Laboratory surveillance revealed NY C. auris isolates are resistant to fluconazole, with variable resistance to other currently used broad-spectrum antifungal drugs, and that several isolates are pan-resistant. Thus, there is an urgent need for new drugs with a novel mechanism of action to combat the resistance challenge. Manogepix (MGX) is a first-in-class agent that targets the fungal Gwt1 enzyme. The prodrug, fosmanogepix, is currently in Phase 2 clinical development for the treatment of fungal infections. We evaluated the susceptibility of 200 New York C. auris isolates to MGX and 10 comparator drugs using CLSI methodology. MGX demonstrated lower MICs than comparators (MIC50 and MIC90 0.03 mg/L; range 0.004-0.06 mg/L). The MGX epidemiological cutoff value (ECV, 99% cutoff) for the tested C. auris isolates was 0.06 mg/L. MGX was 8-32-fold more active than the echinocandins, 16-64-fold more active than the azoles, and 64-fold more active than amphotericin B. No differences were found in the MGX or comparators MIC50, MIC90, or GEOMEAN values when subsets of clinical, surveillance, and environmental isolates were evaluated. The range of MGX MIC values for six C. auris pan-resistant isolates was 0.008-0.015 mg/L, and the median and mode MIC values were 0.015 mg/L, demonstrating that MGX retains activity against these isolates. These data support further clinical evaluation of fosmanogepix for the treatment of C. auris infections, including highly resistant isolates.

microbiology↗

bioRxiv2019

In vitro Efficacy of Novel Glucan Synthase Inhibitor, Ibrexafungerp (SCY-078), Against Multidrug- and Pan-resistant Candida auris Isolates from the Outbreak in New York

We report low MIC50 of Ibrexafungerp (SCY-078) for 102 Candida auris clinical and surveillance isolates from outbreak in New York. The group included C. auris with a variable resistance to antifungal drugs. Five pan-resistant C. auris isolates were susceptible to Ibrexafungerp with low MIC50 range of 0.12-1 {micro}g/ml.

microbiology↗

bioRxiv2019

Laboratory Analysis of an Outbreak of Candida auris in New York from 2016 to 2018-Impact and Lessons Learned

Candida auris is a multidrug-resistant yeast which has emerged in healthcare facilities worldwide, however little is known about identification methods, patient colonization, spread, environmental survival, and drug resistance. Colonization on both biotic and abiotic surfaces, along with travel, appear to be the major factors for the spread of this pathogen across the globe. In this investigation, we present laboratory findings from an ongoing C. auris outbreak in NY from August 2016 through 2018. A total of 540 clinical isolates, 11,035 patient surveillance specimens, and 3,672 environmental surveillance samples were analyzed. Laboratory methods included matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for yeast isolate identification, real-time PCR for rapid surveillance sample screening, culture on selective/non-selective media for recovery of C. auris and other yeasts from surveillance samples, antifungal susceptibility testing to determine the C. auris resistance profile, and Sanger sequencing of ribosomal genes for C. auris genotyping. Results included: a) identification and confirmation of C. auris in 413 clinical isolates and 931 patient surveillance isolates, as well as identification of 277 clinical cases and 350 colonized cases from 151 healthcare facilities including 59 hospitals, 92 nursing homes, 1 long-term acute care hospital (LTACH), and 2 hospices, b) successful utilization of an in-house developed C. auris real-time PCR assay for the rapid screening of patient and environmental surveillance samples, c) demonstration of relatively heavier colonization of C. auris in nares compared to the axilla/groin, and d) predominance of the South Asia Clade I with intrinsic resistance to fluconazole and elevated minimum inhibitory concentration (MIC) to voriconazole (81%), amphotericin B (61%), 5-FC (3%) and echinocandins (1%). These findings reflect greater regional prevalence and incidence of C. auris and the deployment of better detection tools in an unprecedented outbreak.

microbiology↗

bioRxiv2019

A rapid and automated sample-to-result Candida auris real-time PCR assay for high throughput testing of surveillance samples with BD MAX™ open system

The multidrug-resistant yeast pathogen Candida auris continues to cause outbreaks and clusters of clinical cases worldwide. Previously, we developed a real-time PCR assay for the detection of C. auris from surveillance samples (Leach et al. JCM. 2018: 56, e01223-17). The assay played a crucial role in the ongoing investigations of C. auris outbreak in New York City. To ease the implementation of the assay in other laboratories, we developed an automated sample-to-result real-time C. auris PCR assay using BD MAX open system. We optimized sample extraction at three different temperatures and four incubation periods. Sensitivity was determined using eight pools of patient samples, and specificity was calculated using four clades of C. auris, and closely and distantly related yeasts. Three independent extractions and testing of two patient sample pools in the quadruplicate yielded assay precision. BD MAX optimum assay conditions were: DNA extraction at 75{degrees}C for 20 min, and the use of PerfeCTa Multiplex qPCR ToughMix. The limit of detection (LOD) of the assay was one C. auris CFU/PCR reaction. We detected all four clades of C. auris without cross-reactivity to other yeasts. Of the 110 patient surveillance samples tested, 50 were positive for C. auris using the BD MAX System with 96% clinical sensitivity and 94% accuracy compared to the manual assay. BD MAX assay allows high-throughput C. auris screening of 180 surveillance samples in a 12-hour workday.

microbiology↗

bioRxiv2019

Efficacy of antifungal drug combinations against widespread primary resistance, cross-resistance, and multidrug-resistance in Candida auris from New York Hospitals and Healthcare Facilities

Since 2016, New York hospitals and healthcare facilities have faced an unprecedented outbreak of pathogenic yeast Candida auris. We tested over one thousand C. auris isolates from affected facilities and found high-resistance to fluconazole (FLC, MIC50>256 mg/L), and variable resistance to other antifungal drugs. Therefore, we evaluated if two-drug combinations are effective in vitro against multidrug-resistant C. auris. Broth micro-dilution (BMD) plates were custom-designed, and quality controlled by TREK Diagnostic System. We used MIC100 endpoints for the drug combination readings as reported earlier for the intra- and inter-laboratory agreements against Candida species and Aspergillus fumigatus. The study results were derived from 12,960 MIC100 readings, for fifteen C. auris isolates tested against 864 possible two-drug antifungal combinations for nine antifungal drugs. Flucytosine (5FC) at 1.0 mg/L potentiated the most successful combinations with other drugs. Micafungin (MFG), Anidulafungin (AFG), Caspofungin (CAS) at individual concentrations of 0.25 mg/L in combination with 5FC (1.0 mg/L) yielded MIC100 for 14, 13, and 12 of 15 C. auris test isolates. AMB / 5FC (0.25/1.0 mg/L) yielded MIC100 for 13 isolates. None of the combinations were effective for C. auris 18-1, which tested resistant against FLC and 5FC, except POS/5FC (0.12/1.0 mg/L). The simplified two-drug combination susceptibility test format would permit laboratories to provide clinicians and public health experts with additional data to deal with multidrug-resistant C. auris.

microbiology↗

bioRxiv2019

Characterization of novel Chrysosporium morrisgordonii sp. nov., from bat white-nose syndrome (WNS) affected mines, northeastern United States

Psychrotolerant hyphomycetes including unrecognized taxon are commonly found in bat hibernation sites in Upstate New York. During a mycobiome survey, a new fungal species, Chrysosporium morrisgordonii sp. nov., was isolated from bat White-nose syndrome (WNS) afflicted Graphite mine in Warren County, New York. This taxon was distinguished by its ability to grow at low temperature spectra from 6{degrees}C to 25{degrees}C. Conidia were tuberculate and thick-walled, globose to subglobose, unicellular, 3.5-4.6 {micro}m x3.5-4.6 {micro}m, sessile or borne on narrow stalks. The stalks occurred at right angles to the hypha. Intercalary aleuroconidia were also observed; the hyphae were septate, hyaline, smooth-walled, 1-3 {micro}m wide, straight or branched. The ITS1-5.8S-ITS2 sequence of C. morrisgordonii differed from the closest match to C. speluncarum by 37 nucleotides (94% identity); the Neighbor-joining phylogenetic tree confirmed that C. morrisgordonii was placed in a well-supported monophyletic group with C. speluncarum. LSU and ITS phylogenetic trees further indicated that C. morrisgordonii, C. speluncarum, Renispora flavissima, Neogymnomyces virgineus and Gymnoascus demonbreunii formed a distinct group in the Onygenales.

microbiology↗

bioRxiv2017

Validation and Implementation of CLIA-Compliant Whole Genome Sequencing (WGS) in Public Health Laboratory

BackgroundPublic health microbiology laboratories (PHL) are at the cusp of unprecedented improvements in pathogen identification, antibiotic resistance detection, and outbreak investigation by using whole genome sequencing (WGS). However, considerable challenges remain due to the lack of common standards.\n\nObjectives1) Establish the performance specifications of WGS applications used in PHL to conform with CLIA (Clinical Laboratory Improvements Act) guidelines for laboratory developed tests (LDT), 2) Develop quality assurance (QA) and quality control (QC) measures, 3) Establish reporting language for end users with or without WGS expertise, 4) Create a validation set of microorganisms to be used for future validations of WGS platforms and multi-laboratory comparisons and, 5) Create modular templates for the validation of different sequencing platforms.\n\nMethodsMiSeq Sequencer and Illumina chemistry (Illumina, Inc.) were used to generate genomes for 34 bacterial isolates with genome sizes from 1.8 to 4.7 Mb and wide range of GC content (32.1%-66.1%). A customized CLCbio Genomics Workbench - shell script bioinformatics pipeline was used for the data analysis.\n\nResultsWe developed a validation panel comprising ten Enterobacteriaceae isolates, five gram-positive cocci, five gram-negative non-fermenting species, nine Mycobacterium tuberculosis, and five miscellaneous bacteria; the set represented typical workflow in the PHL. The accuracy of MiSeq platform for individual base calling was >99.9% with similar results shown for reproducibility/repeatability of genome-wide base calling. The accuracy of phylogenetic analysis was 100%. The specificity and sensitivity inferred from MLST and genotyping tests were 100%. A test report format was developed for the end users with and without WGS knowledge.\n\nConclusionWGS was validated for routine use in PHL according to CLIA guidelines for LDTs. The validation panel, sequencing analytics, and raw sequences will be available for future multi-laboratory comparisons of WGS in PHL. Additionally, the WGS performance specifications and modular validation template are likely to be adaptable for the validation of other platforms and reagents kits.

microbiology↗