Grys, Thomas E.

Abstract Background Coccidioidomycosis is often diagnosed with a collection of tests that measure a patient’s ability to mount an immune response to the fungus (antibody-based diagnostics) utilizing fungal protein preparations. Here we present an antigen-based assay that detects and quantifies coccidioidal chitinase-1 (CTS1) in diagnostic antigen preparations with potential for use in human serum. Methods An inhibition-based enzyme-linked immunoassay (ELISA) was developed that utilizes a monoclonal antibody specific for coccidioidal CTS1. CTS1 was quantified in commercial antigen preparations using recombinant CTS1 as a standard. Sera from 192 individuals from an endemic area were tested which included 78 patients (40.6%) with proven or probable coccidioidomycosis. Results The quantity of CTS1 in diagnostic commercial antigen preparations from different suppliers varied. Temporal constraints of availability of different lots of commercial antigens does not allow for immediate comparison of lot-to-lot variability. Assay results from patient serum samples correlated with low- and high-titer serology from patients with a coccidioidomycosis diagnosis. Further analysis suggested that patient derived anti-CTS1 antibodies may overlap with the mouse monoclonal antibody used in the assay. This unexpected overlap in CTS1 binding suggests the assay can detect antigen, antibody, or both, which contributes to its high level of clinical sensitivity of 89.74% and specificity of 94.90%. Conclusions The CTS1 inhibition ELISA described in this report is a promising tool to aid in quality control of antigens used in the diagnosis of coccidioidomycosis. Further optimization is needed to harness its utility as a diagnostic tool to aid in diagnosis and disease monitoring of coccidioidomycosis.

Coccidioidomycosis, or Valley Fever (VF), is a potentially lethal fungal infection that results in more than 200 deaths/year in the United States. Differential diagnosis and timely treatment of VF is made difficult due to presentation of vague symptoms that often mimic other pulmonary conditions. Accurate diagnosis therefore relies on a combination of clinical presentation, serology, radiography, histology and culture. These diagnostic methods are either costly, time‐consuming, invasive, or indeterminate. Previous studies have used MS‐based methods for the accurate diagnosis of similar fungal diseases as well as characterization of host‐fungal interactions. Herein, we present the first targeted plasma and urine liquid chromatography tandem mass spectrometry (LC‐MS/MS) profiling approach for the relatively non‐invasive, rapid and accurate detection of VF. In this targeted approach, 207 plasma metabolites and 231 urine metabolites were reliably detected using LC‐MS/MS. These metabolites, which are representative of more than 35 metabolic pathways of potential biological relevance, were monitored in 59 plasma samples and 88 urine samples acquired from excess clinical specimens taken from two groups of patients (48 VF patients and 99 non‐VF controls) at Mayo Clinic Arizona. Univariate and multivariate statistical analyses were performed to develop predictive plasma and urine biomarker panels. Pathway and enrichment analyses were performed to elucidate potential pathogenic mechanisms and therapeutic targets. Results of our univariate significance testing and multivariate model estimation informed the construction of a 3‐metabolite panel of plasma biomarkers ( q < 0.05 and VIP > 2; Fig 1) and a 9‐metabolite panel of urinary biomarkers ( q < 0.05 and VIP > 1; Fig 2). Receiver operating characteristic (ROC) curves (Fig 3) generated based on enhanced orthogonal partial least squares‐discriminant analysis (OPLS‐DA) models (Fig 4) showed excellent overall classification performance (99.5%), with 94.4% sensitivity and 97.6% specificity for plasma metabolites. Although urine metabolites were less accurate (92.5%), demonstrating 89.7% sensitivity and 88.1% specificity, they represent a substantial increase in diagnostic accuracy over currently available urinary antigen tests, which only report sensitivity of around 70%. Pathway analysis (Fig 5) and enrichment analysis (Fig 6) ubiquitously showed alterations in metabolic pathways related to fungal virulence, DNA/RNA synthesis and energy utilization, and host immune escape mechanisms. These results potentially reveal pathways or markers that could be targeted therapeutically. This study is the first‐ever MS‐based metabolomics approach for rapid and highly accurate diagnosis of VF. Support or Funding Information Support from the College of Health Solutions at Arizona State University, Arizona Biomedical Research Council (ABRC) grant# 16–162513 and American Achievement Rewards for College Scientists (ARCS) Foundation Award is gratefully acknowledged. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Laser capture microdissection (LCM) coupled to label-free quantitative mass spectrometry is a viable strategy to identify biomarkers from infected tissues. In this study, LCM was employed to take a “snapshot” of proteins produced in vivo during Coccidiodies spp. infection in human lungs. Proteomic analysis of LCM lung sections revealed hundreds of hosts and Coccidioidal proteins. Twenty-seven highly abundant Coccidioides spp. proteins were identified which do not share significant sequence orthology with human proteins. Three of the 27 Coccidioidal proteins are also potential Coccidoides-specific biomarkers, as they also do not share sequence homology to any other pathogenic fungus or microbe. Gene ontology analysis of the 27 biomarker candidate proteins revealed enriched hydrolase activity and increased purine and carbohydrate metabolism functions. Finally, we provide proteomic evidence that all 27 biomarker candidates are produced by the fungus when grown in vitro in a media- and growth-phase dependent manner.

Abstract With the increasing prevalence of antibiotic resistance, the need to develop antimicrobial susceptibility testing (AST) technologies is urgent. The current challenge has been to perform the antibiotic susceptibility testing in short time, directly with clinical samples, and with antibiotics over a broad dynamic range of clinically relevant concentrations. Here, a technology for point‐of‐care diagnosis of antimicrobial‐resistant bacteria in urinary tract infections, by imaging the clinical urine samples directly with an innovative large volume solution scattering imaging (LVSi) system and analyzing the image sequences with a single‐cell division tracking method is developed. The high sensitivity of single‐cell division tracking associated with large volume imaging enables rapid antibiotic susceptibility testing directly on the clinical urine samples. The results demonstrate direct detection of bacterial infections in 60 clinical urine samples with a 60 min LVSi video, and digital AST of 30 positive clinical samples with 100% categorical agreement with both the clinical culture results and the on‐site agar plating validation results. This technology provides opportunities for precise antibiotic prescription and proper treatment of the patient within a single clinic visit.

Abstract Coccidioides spp. are important pneumonia-causing pathogens of the American southwest, but little is known about their glycobiology and how their glycosylations differ from other pneumonia-causing fungi. There is mounting preliminary evidence to suggest genus or even species-specific glycosylations in the fungal kingdom due to the presence of unique Carbohydrate-Active Enzymes (CAZymes) in fungal genomes (Deshpande and others 2008; Karkowska-Kuleta and Kozik 2015). If Coccidioides spp.-specific glycans can be identified, it may be possible to exploit these differences to develop more specific diagnostic approaches and more effective therapeutics. Herein we i) mined Coccidioides spp. and other pathogenic fungal genomes to identify CAZymes specific for Coccidiodes spp., ii) proteomically determined the Coccidioides spp. “CAZome” produced in vivo and in vitro, and iii) utilized glycomics to differentiate Coccidioides genus-specific N-glycans from other pathogenic fungi. As far as we are aware, this is the first proteomic and glycomic comparison of the N-glycomes and CAZomes of different fungal genera during infection in human hosts.