Sample acquisition and preparation.

Oropharyngeal swabs and stool samples were collected from a cohort of patients with human papillomavirus-positive (HPV⁺) locoregionally advanced oropharyngeal squamous cell carcinoma (LA-OPSCC) treated with chemoradiotherapy (CRT) (13). Oropharyngeal swabs over the tumor site and stool samples were collected up to 3 weeks prior to the start of radiotherapy and again at completion of CRT (up to 3 weeks following last day of radiotherapy) (13). Oropharyngeal swabs over the tumor site and stool samples were collected using the ZymoBIOMICS DNA/RNA mini prep kits (Zymo Research, Irvine, CA). DNA was extracted using ZymoBIOMICS DNA micro kit (13).

Vaginal secretions were collected from a cohort of patients 10 to 18 days after the last day of bleeding from their previous menstrual period (11). Instead SoftCups (Evofem, San Diego, CA) were self-inserted to collect undiluted cervicovaginal secretions for 1 min (14). The SoftCup was placed into a 50-ml conical tube and transported on ice to the lab within 2 h (14). DNA was extracted using the DNeasy PowerSoil kit (Qiagen) (14).

Rectal swabs (FLOQSwab; Copan) were collected from a cohort of HIV-positive, antiretroviral therapy (ART)-treated men who have sex with men in Toronto, Canada (15). Rectal swabs were inserted and rotated 360° inside the anal canal (15). All rectal swabs were stored at −80°C in 300 μl of ultrapure-grade phosphate-buffered saline (VWR, Radnor, USA) prior to DNA extraction (15). DNA was extracted using the DNeasy PowerSoil kit (Qiagen) per the manufacturer’s instructions with one exception: 30 μl of solution C1 was used to treat both the supernatant and the swab for the first step before removing the swab and adding another 30 μl of C1 to complete the process (15).

All studies were approved by the institutional research ethics board. All patients provided written, signed, informed consent to participate. In total, 46 oropharyngeal swabs, 46 stool samples, 7 vaginal samples, and 13 rectal swabs were included in this study. Two oropharyngeal swabs and 1 stool sample were not taken into consideration in model derivation due to insufficient amounts of sample for qPCR. Oropharyngeal and stool samples were grouped into a sample set used to derive models, while vaginal and rectal samples comprised the sample set used to validate.