Complete Blood Count

Four types of data were collected: a health survey, self-reported anthropometric measurements, a blood sample, and a medical chart review to validate selected self-reported diagnoses. In identifying clinical laboratory tests and selecting diagnoses for validation, priority was given to those with potential associations with PFC exposure as reported in the scientific literature. Clinical laboratory tests included serum lipid, immune, and inflammatory markers; liver, kidney, and thyroid function; complete blood count; serum electrolytes and protein; and endocrine function, including insulin and glucose [see Supplemental Material, Note 4 (doi:10.1289/ehp.0800379.S1)]. Validated medical diagnoses included heart disease, cancers, thyroid disease, neurologic disorders, inflammatory and autoimmune disorders, and pregnancy complications [see Supplemental Material, Note 5 (doi:10.1289/ehp.0800379.S1)].
The health survey gathered demographic data; current and historic residential and employment information, including water source and use; personal medical diagnoses, treatments including medications, and physical symptoms; family medical history; pregnancy history and pregnancy-related outcomes for women; and information about lifestyle and health behaviors. Participants also self-reported their own height, weight, and blood pressure. Brookmar, Inc. contracted with a separate company to independently pilot test the survey, and revisions were made based on pilot-test findings. The final version of the survey was accepted by the settling parties. The survey, a list of the clinical laboratory tests, and the 18 medical diagnoses verified by medical record review are publically available on The C8 Health Project WVU Data Hosting Website (C8 Health Project 2009 ).

The seven adult marmosets were inoculated endobronchially at the level of the main carina using a special narrow diameter bronchoscope with one mL of a 10⁸ CFU/mL M. intracellulare obtained from the Mycobacteria/Nocardia Research Laboratory at the UTHSCT. All procedures (bronchoscopy, blood draws and euthanasia) were conducted under ketamine anesthesia with the additional use of isoflurane anesthesia with bronchoscopy and bronchoalveolar lavage (BAL) in the presence of veterinary staff. Each animal underwent assessment of serum chemistry, and complete blood count prior to inoculation and on the day of euthanasia. Because there are no previous comparable studies with this primate, we sacrificed a group of animals at 30 days and another group at 60 days to optimize the chance of recovering M. intracelluare as well as to define the time course of an evolving inflammatory response. Cytokine analysis was obtained prior to inoculation with M. intracellualre and on a weekly basis from day 0 to day 30 for all animals and again on day 60 for the animals sacrificed at day 60. All the animals had BAL performed prior to euthanasia at either 30- or 60-days post-inoculation. The animals were then taken directly to necropsy by a primate pathologist.

Acute-phase serum or plasma samples were collected during the initial visit for study enrollment and transported to the IICS-UNA laboratory. Samples were tested for DENV NS1 antigen using the Standard Q Dengue Duo rapid immunochromatographic test (SD Biosensor, Suwon, South Korea) according to manufacturer recommendations. Qualitative antibody data acquired using this method was not evaluated in this study, see antibody section below. Primary samples were then aliquoted and stored at −80°C until later use or shipment on dry ice to Emory University for additional testing. For molecular testing, total nucleic acids were extracted from 200μL of sample on an EMAG instrument and eluted into 50μL of buffer. Samples were tested for Zika virus, chikungunya virus and DENV by real-time RT-PCR (rRT-PCR) using a validated and published multiplex assay (the ZCD assay) [60 (link)], and DENV serotype and viral load were determined with a published DENV multiplex assay [61 (link), 62 (link)]. Both rRT-PCRs were performed as previously described [60 (link)–62 (link)].
Serologic testing was performed on acute-phase samples using two different methods. First, anti-DENV IgG and IgM were analyzed using commercial ELISA kits [Dengue ELISA IgG (G1018) and Dengue ELISA IgM Capture (M1018), Vircell Microbiologists, Granada, Spain] according to manufacturer recommendations (interpretation: IgM or IgG index >11 positive, 9–11 indeterminate, <9 negative). Second, a 5μL aliquot of serum from 139 participants with sufficient sample was tested in the pGOLD assay (Nirmidas Biotech, Inc, Palo Alto, CA), which is a multiplex serological assay for IgM and IgG against DENV (DENV-2 whole virus antigen) and ZIKV (NS1 antigen). The pGOLD assay was performed as previously described [59 (link), 63 (link)]. In each well of the pGOLD slide, antigens are spotted in triplicate, and average signals are used during analysis. For IgG, the negative control signal was subtracted from the sample signal, and the difference was divided by the average signal of four IgG control spots included in each well. For IgM, a similar calculation was performed using the signal from a known anti-DENV IgM positive control sample included on each run. A positive threshold ratio of 0.1 was established for each isotype, which was ≥ 3 standard deviations above the mean of the negative control.
Chymase and LBP levels were determined using commercial ELISA kits (G-Biosciences, St. Louis, MO, USA), following the manufacturer’s instructions. Complete blood counts and chemistries were performed at the clinical site at the discretion of the care team, and results were included if the sample was obtained within ±1 day of enrollment.