#METABOLOMICS WORKBENCH ehiggins_20220930_132014 DATATRACK_ID:3482 STUDY_ID:ST002350 ANALYSIS_ID:AN003836 PROJECT_ID:PR001064 VERSION 1 CREATED_ON November 17, 2022, 10:43 am #PROJECT PR:PROJECT_TITLE Volatile Biomarkers for a Valley Fever Breath Test PR:PROJECT_TYPE GCxGC-TOFMS metabolomics PR:PROJECT_SUMMARY Coccidioidomycosis, or valley fever, is prevalent in AZ, with more than 12,000 PR:PROJECT_SUMMARY new human infections diagnosed every year. In highly endemic areas, e.g., PR:PROJECT_SUMMARY Phoenix and Tucson, up to 30% of community-acquired pneumonia may be caused by PR:PROJECT_SUMMARY Valley fever, and cases are on the rise. The current diagnostics for Valley PR:PROJECT_SUMMARY fever are severely lacking due to invasiveness (biopsy) and poor sensitivity PR:PROJECT_SUMMARY (serology), strongly contributing to an unacceptable 23-day median PR:PROJECT_SUMMARY time-to-diagnosis. There is a critical need for sensitive and non-invasive PR:PROJECT_SUMMARY diagnostics for identifying Valley fever lung infections. Our long-term goal is PR:PROJECT_SUMMARY to substantially shorten the time-to-diagnosis for Valley fever through the PR:PROJECT_SUMMARY development of sensitive and specific breath-based diagnostics for PR:PROJECT_SUMMARY coccidioidomycosis lung infections. The overall objective of this application is PR:PROJECT_SUMMARY to identify and validate putative volatile biomarkers of Coccidioides infections PR:PROJECT_SUMMARY via metabolomics analyses of in vitro cultures, mouse model lung infections, and PR:PROJECT_SUMMARY lung specimens from humans with Valley fever. At the completion of the proposed PR:PROJECT_SUMMARY study, we expect to have identified and validated a panel of 10-15 volatile PR:PROJECT_SUMMARY biomarkers for the sensitive and specific detection of valley fever in lung PR:PROJECT_SUMMARY specimens. PR:INSTITUTE Arizona State University PR:DEPARTMENT School of Life Sciences PR:LABORATORY Bean Laboratory PR:LAST_NAME Bean PR:FIRST_NAME Heather PR:ADDRESS PO Box 874501, Tempe, AZ, 85287, USA PR:EMAIL Heather.D.Bean@asu.edu PR:PHONE 480-727-3395 PR:FUNDING_SOURCE Arizona Biomedical Research Centre New Investigator Award to HDB PR:PROJECT_COMMENTS Add to Project ID: PR001064 #STUDY ST:STUDY_TITLE Identify putative volatile biomarkers of Valley fever using a murine lung ST:STUDY_TITLE infection model ST:STUDY_TYPE Untargeted metabolomics ST:STUDY_SUMMARY Coccidioides immitis and Coccidioides posadasii are soil-dwelling fungi of arid ST:STUDY_SUMMARY regions in North and South America that are responsible for Valley fever ST:STUDY_SUMMARY (coccidioidomycosis). Forty percent of patients with Valley fever exhibit ST:STUDY_SUMMARY symptoms ranging from mild, self-limiting respiratory infections, to severe, ST:STUDY_SUMMARY life-threatening pneumonia that requires treatment. Misdiagnosis as bacterial ST:STUDY_SUMMARY pneumonia commonly occurs in symptomatic Valley fever cases, resulting in ST:STUDY_SUMMARY inappropriate treatment with antibiotics, increased medical costs, and delay in ST:STUDY_SUMMARY diagnosis. In this study, we explored the feasibility of developing breath-based ST:STUDY_SUMMARY diagnostics for Valley fever using a murine lung infection model. To investigate ST:STUDY_SUMMARY potential volatile biomarkers of Valley fever that arise from host-pathogen ST:STUDY_SUMMARY interactions, we infected C57BL/6J mice with C. immitis RS and C. posadasii ST:STUDY_SUMMARY Silveira via intranasal inoculation. We collected bronchoalveolar lavage fluid ST:STUDY_SUMMARY (BALF) for cytokine profiling and for untargeted volatile metabolomics via solid ST:STUDY_SUMMARY phase microextraction (SPME) and two-dimensional gas chromatography coupled to ST:STUDY_SUMMARY time-of-flight mass spectrometry (GC×GC-TOFMS). We identified 36 volatile ST:STUDY_SUMMARY organic compounds (VOCs) that were significantly correlated to cytokine ST:STUDY_SUMMARY abundances and clustered mice by disease severity. These 36 VOCs were also able ST:STUDY_SUMMARY to separate mice with a moderate to high disease severity by infection strain. ST:STUDY_SUMMARY The data presented here show that Coccidioides and/or the host produce volatile ST:STUDY_SUMMARY metabolites that may yield biomarkers for a Valley fever breath test that can ST:STUDY_SUMMARY detect Coccidioidal infection and provide clinically relevant information on ST:STUDY_SUMMARY disease severity. ST:INSTITUTE Arizona State University ST:DEPARTMENT School of Life Sciences ST:LABORATORY Bean Laboratory ST:LAST_NAME Bean ST:FIRST_NAME Heather ST:ADDRESS PO Box 874501 ST:EMAIL Heather.D.Bean@asu.edu ST:PHONE 4807273395 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J SU:AGE_OR_AGE_RANGE 6-8 weeks SU:GENDER Female SU:ANIMAL_ANIMAL_SUPPLIER The Jackson Laboratory, Bar Harbor, ME #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Blank_14 Subject_ID:Empty run | treatment:NA RAW_FILE_NAME=Blank_14.SMP SUBJECT_SAMPLE_FACTORS - Blank_20 Subject_ID:Empty run | treatment:NA RAW_FILE_NAME=Blank_20.SMP SUBJECT_SAMPLE_FACTORS - Blank_8 Subject_ID:Empty run | treatment:NA RAW_FILE_NAME=Blank_8.SMP SUBJECT_SAMPLE_FACTORS - Grob_1 Subject_ID:grob mix | treatment:NA RAW_FILE_NAME=Grob_1.SMP SUBJECT_SAMPLE_FACTORS - Grob_2 Subject_ID:grob mix | treatment:NA RAW_FILE_NAME=Grob_2.SMP SUBJECT_SAMPLE_FACTORS - Grob_3 Subject_ID:grob mix | treatment:NA RAW_FILE_NAME=Grob_3.SMP SUBJECT_SAMPLE_FACTORS - KI mix_1 Subject_ID:alkane standard | treatment:NA RAW_FILE_NAME=KI mix_1.SMP SUBJECT_SAMPLE_FACTORS - KI_1 Subject_ID:alkane standard | treatment:NA RAW_FILE_NAME=KI_1.SMP SUBJECT_SAMPLE_FACTORS - KI_2 Subject_ID:alkane standard | treatment:NA RAW_FILE_NAME=KI_2.SMP SUBJECT_SAMPLE_FACTORS - PBS_M1-1_1 Subject_ID:PBS Mouse | treatment:NA RAW_FILE_NAME=PBS_M1-1_1.SMP SUBJECT_SAMPLE_FACTORS - PBS_M1-2_1 Subject_ID:PBS Mouse | treatment:NA RAW_FILE_NAME=PBS_M1-2_1.SMP SUBJECT_SAMPLE_FACTORS - PBS_M1-3_1 Subject_ID:PBS Mouse | treatment:NA RAW_FILE_NAME=PBS_M1-3_1.SMP SUBJECT_SAMPLE_FACTORS - PBS_M2-1_1 Subject_ID:PBS Mouse | treatment:PBS RAW_FILE_NAME=PBS_M2-1_1.SMP SUBJECT_SAMPLE_FACTORS - PBS_M2-2_1 Subject_ID:PBS Mouse | treatment:PBS RAW_FILE_NAME=PBS_M2-2_1.SMP SUBJECT_SAMPLE_FACTORS - PBS_M2-3_1 Subject_ID:PBS Mouse | treatment:PBS RAW_FILE_NAME=PBS_M2-3_1.SMP SUBJECT_SAMPLE_FACTORS - PBS_M3-1_1 Subject_ID:PBS Mouse | treatment:PBS RAW_FILE_NAME=PBS_M3-1_1.SMP SUBJECT_SAMPLE_FACTORS - PBS_M3-2_1 Subject_ID:PBS Mouse | treatment:PBS RAW_FILE_NAME=PBS_M3-2_1.SMP SUBJECT_SAMPLE_FACTORS - PBS_M3-3_1 Subject_ID:PBS Mouse | treatment:PBS RAW_FILE_NAME=PBS_M3-3_1.SMP SUBJECT_SAMPLE_FACTORS - PBS_M4-1_1 Subject_ID:PBS Mouse | treatment:PBS RAW_FILE_NAME=PBS_M4-1_1.SMP SUBJECT_SAMPLE_FACTORS - PBS_M4-2_1 Subject_ID:PBS Mouse | treatment:PBS RAW_FILE_NAME=PBS_M4-2_1.SMP SUBJECT_SAMPLE_FACTORS - PBS_M4-3_1 Subject_ID:PBS Mouse | treatment:PBS RAW_FILE_NAME=PBS_M4-3_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M1-1_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M1-1_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M1-2_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M1-2_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M1-3_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M1-3_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M2-1_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M2-1_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M2-2_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M2-2_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M2-3_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M2-3_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M3-1_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M3-1_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M3-2_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M3-2_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M3-3_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M3-3_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M4-1_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M4-1_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M4-2_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M4-2_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M4-3_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M4-3_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M5-1_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M5-1_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M5-2_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M5-2_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M6-1_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M6-1_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M6-2_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M6-2_1.SMP SUBJECT_SAMPLE_FACTORS - RS_M6-3_1 Subject_ID:C. immitis | treatment:Fungi RAW_FILE_NAME=RS_M6-3_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M1-1_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M1-1_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M1-2_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M1-2_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M1-3_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M1-3_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M2-1_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M2-1_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M2-2_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M2-2_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M2-3_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M2-3_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M3-1_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M3-1_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M3-2_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M3-2_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M3-3_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M3-3_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M4-1_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M4-1_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M4-2_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M4-2_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M5-1_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M5-1_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M5-2_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M5-2_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M5-3_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M5-3_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M6-1_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M6-1_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M6-2_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M6-2_1.SMP SUBJECT_SAMPLE_FACTORS - SIL_M6-3_1 Subject_ID:C. posadasii | treatment:Fungi RAW_FILE_NAME=SIL_M6-3_1.SMP #COLLECTION CO:COLLECTION_SUMMARY Female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) 6-8 weeks of age CO:COLLECTION_SUMMARY were used for these studies. Mice were housed according to NIH guidelines for CO:COLLECTION_SUMMARY housing and care in a biosafety level 3 animal laboratory. All procedures were CO:COLLECTION_SUMMARY approved by the Institutional Animal Care and Use Committee (protocol number CO:COLLECTION_SUMMARY 16-011) of Northern Arizona University. The Coccidioides isolates used in this CO:COLLECTION_SUMMARY study were the type strains C. immitis strain RS (ATCC® catalog no. NR-48942; CO:COLLECTION_SUMMARY NCBI accession no. AAEC00000000.3) and C. posadasii strain Silveira (ATCC® CO:COLLECTION_SUMMARY catalog no. NR-48944; NCBI accession no. ABAI00000000.2). Mice were anesthetized CO:COLLECTION_SUMMARY with ketamine/xylene (80/8 mg/kg) and intranasally inoculated with 100 CO:COLLECTION_SUMMARY arthroconidia of C. immitis strain RS (n=6) or C. posadasii strain Silveira CO:COLLECTION_SUMMARY (n=6) suspended in 30 μL phosphate-buffered saline (PBS), as described CO:COLLECTION_SUMMARY previously (22, 58). Control mice were inoculated with PBS alone (n=4). The mice CO:COLLECTION_SUMMARY were sacrificed at day 10 post-inoculation. The lungs were rinsed with 2 mL of CO:COLLECTION_SUMMARY PBS to collect bronchoalveolar lavage fluid (BALF), which were filtered with CO:COLLECTION_SUMMARY 0.22 μm Ultrafree® - MC centrifugal filter devices with Durapore® membrane CO:COLLECTION_SUMMARY (MilliporeSigma, Burlington, MA). One milliliter of each BALF sample was stored CO:COLLECTION_SUMMARY at –80°C for volatilomics analysis. Halt™ Protease Inhibitor Cocktail (10 CO:COLLECTION_SUMMARY μL/mL) was added to the remainder of each BALF sample for cytokine analysis. CO:COLLECTION_SUMMARY Spleen and brain were homogenized in 1 ml of sterile PBS followed by culture of CO:COLLECTION_SUMMARY 10-fold dilutions of each tissue on 2X GYE agar (2% glucose (VWR™, USA), 1% CO:COLLECTION_SUMMARY yeast extract (BD™, Franklin Lakes, New Jersey, USA, and 1.5% bacteriological CO:COLLECTION_SUMMARY agar (Difco, USA)) to assess fungal dissemination. CO:SAMPLE_TYPE Bronchoalveolar lavage #TREATMENT TR:TREATMENT_SUMMARY Mice were anesthetized with ketamine/xylene (80/8 mg/kg) and intranasally TR:TREATMENT_SUMMARY inoculated with 100 arthroconidia of C. immitis strain RS (n=6) or C. posadasii TR:TREATMENT_SUMMARY strain Silveira (n=6) suspended in 30 μL phosphate-buffered saline (PBS), as TR:TREATMENT_SUMMARY described previously (22, 58). Control mice were inoculated with PBS alone TR:TREATMENT_SUMMARY (n=4). The mice were sacrificed at day 10 post-inoculation. The lungs were TR:TREATMENT_SUMMARY rinsed with 2 mL of PBS to collect bronchoalveolar lavage fluid (BALF), which TR:TREATMENT_SUMMARY were filtered with 0.22 μm Ultrafree® - MC centrifugal filter devices with TR:TREATMENT_SUMMARY Durapore® membrane (MilliporeSigma, Burlington, MA). One milliliter of each TR:TREATMENT_SUMMARY BALF sample was stored at –80°C for volatilomics analysis. Halt™ Protease TR:TREATMENT_SUMMARY Inhibitor Cocktail (10 μL/mL) was added to the remainder of each BALF sample TR:TREATMENT_SUMMARY for cytokine analysis. Spleen and brain were homogenized in 1 ml of sterile PBS TR:TREATMENT_SUMMARY followed by culture of 10-fold dilutions of each tissue on 2X GYE agar (2% TR:TREATMENT_SUMMARY glucose (VWR™, USA), 1% yeast extract (BD™, Franklin Lakes, New Jersey, USA, TR:TREATMENT_SUMMARY and 1.5% bacteriological agar (Difco, USA)) to assess fungal dissemination. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The BALF samples were allowed to thaw at 4°C overnight, and then split into SP:SAMPLEPREP_SUMMARY technical triplicates of 200 μL that were transferred and sealed into SP:SAMPLEPREP_SUMMARY sterilized 2mL GC headspace vials with Supelco® PTFE/silicone septum magnetic SP:SAMPLEPREP_SUMMARY screw caps (Sigma-Aldrich®, St. Louis, MO). Samples were randomized for SP:SAMPLEPREP_SUMMARY analysis. Volatile metabolites sampling was performed by solid phase SP:SAMPLEPREP_SUMMARY microextraction (SPME) using a Gerstel® MPS Robotic Pro MultiPurpose SP:SAMPLEPREP_SUMMARY autosampler directed by Maestro® software (Gerstel®, Inc., Linthicum, MD). SP:SAMPLEPREP_SUMMARY Sample extraction and injection parameters are provided in Table S3 (see SP:SAMPLEPREP_SUMMARY Autosampler Method). Volatile metabolite analysis was performed by SP:SAMPLEPREP_SUMMARY two-dimensional gas chromatography−time-of-flight mass spectrometry SP:SAMPLEPREP_SUMMARY (GC×GC–TOFMS) using a LECO® Pegasus® 4D and Agilent® 7890B GC (LECO® SP:SAMPLEPREP_SUMMARY Corp., St. Joseph, MI). Chromatographic, mass spectrometric, and peak detection SP:SAMPLEPREP_SUMMARY parameters are provided in Table S3 (see GC×GC Method and Mass Spectrometry SP:SAMPLEPREP_SUMMARY Method). An external alkane standards mixture (C8 – C20; Sigma-Aldrich®) was SP:SAMPLEPREP_SUMMARY sampled multiple times for calculating retention indices (RI). The injection, SP:SAMPLEPREP_SUMMARY chromatographic, and mass spectrometric methods for analyzing the alkane SP:SAMPLEPREP_SUMMARY standards were the same as for the samples. SP:EXTRACTION_METHOD Solid-phase microextraction (SPME) #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GCxGC CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Column 1: Rxi-624Sil MS, (60m × 0.25mm × 1.4um); Column 2: Stabilwax, (1m × CH:COLUMN_NAME 0.25mm × 0.5um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Leco Pegasus 4D GCxGC TOF MS:INSTRUMENT_TYPE GC x GC-TOF MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS See attached protocol MS:MS_RESULTS_FILE ST002350_AN003836_Results.txt UNITS:Peak areas Has m/z:No Has RT:Yes RT units:Seconds #END