Qiasymphony virus bacteria mini kit

Total nucleic acid was extracted from 200 μL of 10% fecal suspension prepared in normal saline using the QIAsymphony Virus/Bacteria Mini kit (Qiagen, United Kingdom) according to the QIAsymphony SP instrument (Qiagen, United Kingdom). Genomic DNA of the isolates was extracted using the TIANamp bacteria DNA Kit (Tiangen Biotech Beijing Co., Ltd, Beijing, China). The extract was eluted in 50 μL DNase-free and RNase-free water and stored at -80°C. The quality of the DNA extract was assessed using a NanoDrop spectrophotometer (ND-1000; Thermo Fisher Scientific Inc., Waltham, MA, United States). All procedures were conducted following the manufacturers’ instructions. PCR products were purified using the high pure PCR product purification kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. The sequencing was performed by Sanger method using the ABI 3730XL automated DNA analyzer (Applied Biosystems Inc., Foster City, CA, United States). The information of primers for sequencing was listed in the Supplementary Table 1. The DNA sequence analysis was performed using the DNAStar Lasergene software (DNAstar Inc., Madison, WI, United States).

For detection of respiratory viral and bacterial organisms, nucleic acid was extracted from swab samples using the Qiasymphony Virus/Bacteria Mini Kit (Qiagen) and quantified using a multiplex real-time quantitative PCR
assay kit (Fast Track Diagnostics Respiratory Pathogens 33 Kit), according to the manufacturer’s instructions, on the CFX96 Touch System lightcycler platform (Bio-Rad). This kit is designed to detect a panel of respiratory tract bacteria, viruses, and fungi (Table E4).

RNA was extracted using the QIASymphony Virus/Bacteria Mini Kit (Qiagen, Hilden, Germany) by the CellFree200 Default IC protocol with a 60 μL extract elution volume.

The Respiratory Pathobionts cohort, a subset of participants screened for eligibility for the CORTIS-01 trial at the Worcester site, has previously been described^{24 (link)}. Briefly, HIV-uninfected participants were consecutively enroled in this sub-study irrespective of CORTIS-01 enrolment, or signs and symptoms of upper respiratory tract infections. Paired nasopharyngeal and oropharyngeal flocked swabs (FLOQSwabs, Copan Diagnostics, Murrieta, CA, USA) were collected and stored in Primestore buffer (Longhorn Vaccines and Diagnostics, San Antonio, TX, USA) at −80 °C, and viral and bacterial nucleic acid was later extracted (Qiasymphony Virus/Bacteria Mini Kit, Qiagen, Hilden, Germany) and quantified using a multiplex RT-qPCR assay kit (Respiratory Pathogens 33 Kit, Fast Track Diagnostics, Luxembourg) on the CFX96 Touch System lightcycler platform (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions. Participants co-enroled into the CORTIS-01 trial were investigated for TB at baseline; those only enroled into the Respiratory Pathobionts cohort were not investigated for TB (Supplementary Fig. S1c). All participants provided written, informed consent and the protocols were approved by the University of Cape Town Faculty of Health Sciences Human Research Ethics Committee (HREC 327/2017).

Viral RNA was extracted directly from 200 μL of clinical sample with either the Qiagen EZ1 Virus mini kit v2.0 or the QIAsymphony Virus/Bacteria mini kit, using their respective proprietary Bio Robot EZ1 and QIAsymphony automated platforms (Qiagen, Valencia, CA), according to the manufacturer’s instructions. All extracted RNAs were eluted into a final volume of 60 μL of elution buffer.