{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002350","ANALYSIS_ID":"AN003836","VERSION":"1","CREATED_ON":"November 17, 2022, 10:43 am"},

"PROJECT":{"PROJECT_TITLE":"Volatile Biomarkers for a Valley Fever Breath Test","PROJECT_TYPE":"GCxGC-TOFMS metabolomics","PROJECT_SUMMARY":"Coccidioidomycosis, or valley fever, is prevalent in AZ, with more than 12,000 new human infections diagnosed every year. In highly endemic areas, e.g., Phoenix and Tucson, up to 30% of community-acquired pneumonia may be caused by Valley fever, and cases are on the rise. The current diagnostics for Valley fever are severely lacking due to invasiveness (biopsy) and poor sensitivity (serology), strongly contributing to an unacceptable 23-day median time-to-diagnosis. There is a critical need for sensitive and non-invasive diagnostics for identifying Valley fever lung infections. Our long-term goal is to substantially shorten the time-to-diagnosis for Valley fever through the development of sensitive and specific breath-based diagnostics for coccidioidomycosis lung infections. The overall objective of this application is to identify and validate putative volatile biomarkers of Coccidioides infections via metabolomics analyses of in vitro cultures, mouse model lung infections, and lung specimens from humans with Valley fever. At the completion of the proposed study, we expect to have identified and validated a panel of 10-15 volatile biomarkers for the sensitive and specific detection of valley fever in lung specimens.","INSTITUTE":"Arizona State University","DEPARTMENT":"School of Life Sciences","LABORATORY":"Bean Laboratory","LAST_NAME":"Bean","FIRST_NAME":"Heather","ADDRESS":"PO Box 874501, Tempe, AZ, 85287, USA","EMAIL":"Heather.D.Bean@asu.edu","PHONE":"480-727-3395","FUNDING_SOURCE":"Arizona Biomedical Research Centre New Investigator Award to HDB","PROJECT_COMMENTS":"Add to Project ID: PR001064"},

"STUDY":{"STUDY_TITLE":"Identify putative volatile biomarkers of Valley fever using a murine lung infection model","STUDY_TYPE":"Untargeted metabolomics","STUDY_SUMMARY":"Coccidioides immitis and Coccidioides posadasii are soil-dwelling fungi of arid regions in North and South America that are responsible for Valley fever (coccidioidomycosis). Forty percent of patients with Valley fever exhibit symptoms ranging from mild, self-limiting respiratory infections, to severe, life-threatening pneumonia that requires treatment. Misdiagnosis as bacterial pneumonia commonly occurs in symptomatic Valley fever cases, resulting in inappropriate treatment with antibiotics, increased medical costs, and delay in diagnosis. In this study, we explored the feasibility of developing breath-based diagnostics for Valley fever using a murine lung infection model. To investigate potential volatile biomarkers of Valley fever that arise from host-pathogen interactions, we infected C57BL/6J mice with C. immitis RS and C. posadasii Silveira via intranasal inoculation. We collected bronchoalveolar lavage fluid (BALF) for cytokine profiling and for untargeted volatile metabolomics via solid phase microextraction (SPME) and two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS). We identified 36 volatile organic compounds (VOCs) that were significantly correlated to cytokine abundances and clustered mice by disease severity. These 36 VOCs were also able to separate mice with a moderate to high disease severity by infection strain. The data presented here show that Coccidioides and/or the host produce volatile metabolites that may yield biomarkers for a Valley fever breath test that can detect Coccidioidal infection and provide clinically relevant information on disease severity.","INSTITUTE":"Arizona State University","DEPARTMENT":"School of Life Sciences","LABORATORY":"Bean Laboratory","LAST_NAME":"Bean","FIRST_NAME":"Heather","ADDRESS":"PO Box 874501","EMAIL":"Heather.D.Bean@asu.edu","PHONE":"4807273395"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENOTYPE_STRAIN":"C57BL/6J","AGE_OR_AGE_RANGE":"6-8 weeks","GENDER":"Female","ANIMAL_ANIMAL_SUPPLIER":"The Jackson Laboratory, Bar Harbor, ME"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Blank_14",
"Factors":{"Subject_ID":"Empty run","treatment":"NA"},
"Additional sample data":{"RAW_FILE_NAME":"Blank_14.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"Blank_20",
"Factors":{"Subject_ID":"Empty run","treatment":"NA"},
"Additional sample data":{"RAW_FILE_NAME":"Blank_20.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"Blank_8",
"Factors":{"Subject_ID":"Empty run","treatment":"NA"},
"Additional sample data":{"RAW_FILE_NAME":"Blank_8.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"Grob_1",
"Factors":{"Subject_ID":"grob mix","treatment":"NA"},
"Additional sample data":{"RAW_FILE_NAME":"Grob_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"Grob_2",
"Factors":{"Subject_ID":"grob mix","treatment":"NA"},
"Additional sample data":{"RAW_FILE_NAME":"Grob_2.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"Grob_3",
"Factors":{"Subject_ID":"grob mix","treatment":"NA"},
"Additional sample data":{"RAW_FILE_NAME":"Grob_3.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"KI mix_1",
"Factors":{"Subject_ID":"alkane standard","treatment":"NA"},
"Additional sample data":{"RAW_FILE_NAME":"KI mix_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"KI_1",
"Factors":{"Subject_ID":"alkane standard","treatment":"NA"},
"Additional sample data":{"RAW_FILE_NAME":"KI_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"KI_2",
"Factors":{"Subject_ID":"alkane standard","treatment":"NA"},
"Additional sample data":{"RAW_FILE_NAME":"KI_2.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"PBS_M1-1_1",
"Factors":{"Subject_ID":"PBS Mouse","treatment":"NA"},
"Additional sample data":{"RAW_FILE_NAME":"PBS_M1-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"PBS_M1-2_1",
"Factors":{"Subject_ID":"PBS Mouse","treatment":"NA"},
"Additional sample data":{"RAW_FILE_NAME":"PBS_M1-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"PBS_M1-3_1",
"Factors":{"Subject_ID":"PBS Mouse","treatment":"NA"},
"Additional sample data":{"RAW_FILE_NAME":"PBS_M1-3_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"PBS_M2-1_1",
"Factors":{"Subject_ID":"PBS Mouse","treatment":"PBS"},
"Additional sample data":{"RAW_FILE_NAME":"PBS_M2-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"PBS_M2-2_1",
"Factors":{"Subject_ID":"PBS Mouse","treatment":"PBS"},
"Additional sample data":{"RAW_FILE_NAME":"PBS_M2-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"PBS_M2-3_1",
"Factors":{"Subject_ID":"PBS Mouse","treatment":"PBS"},
"Additional sample data":{"RAW_FILE_NAME":"PBS_M2-3_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"PBS_M3-1_1",
"Factors":{"Subject_ID":"PBS Mouse","treatment":"PBS"},
"Additional sample data":{"RAW_FILE_NAME":"PBS_M3-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"PBS_M3-2_1",
"Factors":{"Subject_ID":"PBS Mouse","treatment":"PBS"},
"Additional sample data":{"RAW_FILE_NAME":"PBS_M3-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"PBS_M3-3_1",
"Factors":{"Subject_ID":"PBS Mouse","treatment":"PBS"},
"Additional sample data":{"RAW_FILE_NAME":"PBS_M3-3_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"PBS_M4-1_1",
"Factors":{"Subject_ID":"PBS Mouse","treatment":"PBS"},
"Additional sample data":{"RAW_FILE_NAME":"PBS_M4-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"PBS_M4-2_1",
"Factors":{"Subject_ID":"PBS Mouse","treatment":"PBS"},
"Additional sample data":{"RAW_FILE_NAME":"PBS_M4-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"PBS_M4-3_1",
"Factors":{"Subject_ID":"PBS Mouse","treatment":"PBS"},
"Additional sample data":{"RAW_FILE_NAME":"PBS_M4-3_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M1-1_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M1-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M1-2_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M1-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M1-3_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M1-3_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M2-1_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M2-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M2-2_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M2-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M2-3_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M2-3_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M3-1_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M3-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M3-2_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M3-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M3-3_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M3-3_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M4-1_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M4-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M4-2_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M4-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M4-3_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M4-3_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M5-1_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M5-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M5-2_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M5-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M6-1_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M6-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M6-2_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M6-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"RS_M6-3_1",
"Factors":{"Subject_ID":"C. immitis","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"RS_M6-3_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M1-1_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M1-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M1-2_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M1-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M1-3_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M1-3_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M2-1_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M2-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M2-2_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M2-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M2-3_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M2-3_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M3-1_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M3-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M3-2_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M3-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M3-3_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M3-3_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M4-1_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M4-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M4-2_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M4-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M5-1_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M5-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M5-2_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M5-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M5-3_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M5-3_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M6-1_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M6-1_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M6-2_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M6-2_1.SMP"}
},
{
"Subject ID":"-",
"Sample ID":"SIL_M6-3_1",
"Factors":{"Subject_ID":"C. posadasii","treatment":"Fungi"},
"Additional sample data":{"RAW_FILE_NAME":"SIL_M6-3_1.SMP"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) 6-8 weeks of age were used for these studies. Mice were housed according to NIH guidelines for housing and care in a biosafety level 3 animal laboratory. All procedures were approved by the Institutional Animal Care and Use Committee (protocol number 16-011) of Northern Arizona University. The Coccidioides isolates used in this study were the type strains C. immitis strain RS (ATCC® catalog no. NR-48942; NCBI accession no. AAEC00000000.3) and C. posadasii strain Silveira (ATCC® catalog no. NR-48944; NCBI accession no. ABAI00000000.2). Mice were anesthetized with ketamine/xylene (80/8 mg/kg) and intranasally inoculated with 100 arthroconidia of C. immitis strain RS (n=6) or C. posadasii strain Silveira (n=6) suspended in 30 μL phosphate-buffered saline (PBS), as described previously (22, 58). Control mice were inoculated with PBS alone (n=4). The mice were sacrificed at day 10 post-inoculation. The lungs were rinsed with 2 mL of PBS to collect bronchoalveolar lavage fluid (BALF), which were filtered with 0.22 μm Ultrafree® - MC centrifugal filter devices with Durapore® membrane (MilliporeSigma, Burlington, MA). One milliliter of each BALF sample was stored at –80°C for volatilomics analysis. Halt™ Protease Inhibitor Cocktail (10 μL/mL) was added to the remainder of each BALF sample for cytokine analysis. Spleen and brain were homogenized in 1 ml of sterile PBS followed by culture of 10-fold dilutions of each tissue on 2X GYE agar (2% glucose (VWR™, USA), 1% yeast extract (BD™, Franklin Lakes, New Jersey, USA, and 1.5% bacteriological agar (Difco, USA)) to assess fungal dissemination.","SAMPLE_TYPE":"Bronchoalveolar lavage"},

"TREATMENT":{"TREATMENT_SUMMARY":"Mice were anesthetized with ketamine/xylene (80/8 mg/kg) and intranasally inoculated with 100 arthroconidia of C. immitis strain RS (n=6) or C. posadasii strain Silveira (n=6) suspended in 30 μL phosphate-buffered saline (PBS), as described previously (22, 58). Control mice were inoculated with PBS alone (n=4). The mice were sacrificed at day 10 post-inoculation. The lungs were rinsed with 2 mL of PBS to collect bronchoalveolar lavage fluid (BALF), which were filtered with 0.22 μm Ultrafree® - MC centrifugal filter devices with Durapore® membrane (MilliporeSigma, Burlington, MA). One milliliter of each BALF sample was stored at –80°C for volatilomics analysis. Halt™ Protease Inhibitor Cocktail (10 μL/mL) was added to the remainder of each BALF sample for cytokine analysis. Spleen and brain were homogenized in 1 ml of sterile PBS followed by culture of 10-fold dilutions of each tissue on 2X GYE agar (2% glucose (VWR™, USA), 1% yeast extract (BD™, Franklin Lakes, New Jersey, USA, and 1.5% bacteriological agar (Difco, USA)) to assess fungal dissemination."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The BALF samples were allowed to thaw at 4°C overnight, and then split into technical triplicates of 200 μL that were transferred and sealed into sterilized 2mL GC headspace vials with Supelco® PTFE/silicone septum magnetic screw caps (Sigma-Aldrich®, St. Louis, MO). Samples were randomized for analysis. Volatile metabolites sampling was performed by solid phase microextraction (SPME) using a Gerstel® MPS Robotic Pro MultiPurpose autosampler directed by Maestro® software (Gerstel®, Inc., Linthicum, MD). Sample extraction and injection parameters are provided in Table S3 (see Autosampler Method). Volatile metabolite analysis was performed by two-dimensional gas chromatography−time-of-flight mass spectrometry (GC×GC–TOFMS) using a LECO® Pegasus® 4D and Agilent® 7890B GC (LECO® Corp., St. Joseph, MI). Chromatographic, mass spectrometric, and peak detection parameters are provided in Table S3 (see GC×GC Method and Mass Spectrometry Method). An external alkane standards mixture (C8 – C20; Sigma-Aldrich®) was sampled multiple times for calculating retention indices (RI). The injection, chromatographic, and mass spectrometric methods for analyzing the alkane standards were the same as for the samples.","EXTRACTION_METHOD":"Solid-phase microextraction (SPME)"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"GCxGC","INSTRUMENT_NAME":"Agilent 7890B","COLUMN_NAME":"Column 1: Rxi-624Sil MS, (60m × 0.25mm × 1.4um); Column 2: Stabilwax, (1m × 0.25mm × 0.5um)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Leco Pegasus 4D GCxGC TOF","INSTRUMENT_TYPE":"GC x GC-TOF","MS_TYPE":"EI","ION_MODE":"POSITIVE","MS_COMMENTS":"See attached protocol","MS_RESULTS_FILE":"ST002350_AN003836_Results.txt UNITS:Peak areas Has m/z:No Has RT:Yes RT units:Seconds"}

}