Targeted Metagenomic Sequencing-based Approach Applied to 2146 Tissue and Body Fluid Samples in Routine Clinical Practice

Abstract

Background: The yield of next-generation sequencing (NGS) added to a Sanger sequencing-based 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR) assay was evaluated in clinical practice for diagnosis of bacterial infection.

Methods: PCR targeting the V1 to V3 regions of the 16S rRNA gene was performed, with amplified DNA submitted to Sanger sequencing and/or NGS (Illumina MiSeq) or reported as negative, depending on the cycle threshold value. A total of 2146 normally sterile tissues or body fluids were tested between August 2020 and March 2021. Clinical sensitivity was assessed in 579 patients from whom clinical data were available.

Results: Compared with Sanger sequencing alone (400 positive tests), positivity increased by 87% by adding NGS (347 added positive tests). Clinical sensitivity of the assay that incorporated NGS was 53%, which was higher than culture (42%, P < .001), with an impact on clinical decision-making in 14% of infected cases. Clinical sensitivity in the subgroup that received antibiotics at sampling was 41% for culture and 63% for the sequencing assay (P < .001).

Conclusions: Adding NGS to Sanger sequencing of the PCR-amplified 16S rRNA gene substantially improved test positivity. In the patient population studied, the assay was more sensitive than culture, especially in patients who had received antibiotic therapy.

Keywords: 16S ribosomal RNA gene PCR; clinical metagenomics; targeted metagenomics; tissue and body fluids.

Figure 1.

Laboratory workflow of the targeted metagenomics approach (tNGS). (1) A 2-mm³ piece of tissue or 100 µL of fluid was placed into a lysis tube with 20 µL of 0.1-mm silica/zirconium beads, 160 µL proteinase K buffer (PKB), and 20 µL of proteinase K. For FFPE tissues, a 10-µm section was put into a 1.5-mL centrifuge tube with 160 µL of PKB and 20 µL of proteinase K and heated to 60°C for 2 minutes to melt the wax. After centrifugation, liquid and tissue below the wax were transferred to a lysis tube with 20 µL of 0.1-mm beads. (2) Lysis tubes were incubated/spun, cooled for 5 minutes at room temperature, and then centrifuged. (3) Then, 2 mL of prewarmed (40°C) NUCLISENS easyMAG lysis buffer was added to the disposable NUCLISENS easyMAG cartridge with a maximum of 270 µL of the lysed sample and incubated at room temperature for 10 minutes followed by extraction loading. (4) Next, the 16S rRNA gene polymerase chain reaction targeted the V1–V3 region of the bacterial 16S rRNA gene using dual priming oligonucleotides. (5) Depending on the Ct value, samples were sent to Sanger or next-generation sequencing or reported negative. Abbreviations: AQV, average quality value; Ct, cycle threshold; FFPE, formalin-fixed paraffin-embedded; F,R, forward, reverse; PCR, polymerase chain reaction. Created with Biorender.com.

Figure 2.

Sample flowchart. Skin and nasal biopsies were excluded as they are not normally sterile sources. Implants, screws, mesh, swabs, bone marrow, and samples of unknown source were excluded. Abbreviations: Ct, cycle threshold; NGS, next-generation sequencing.

Figure 3.

Additional value of targeted metagenomics (tNGS). A, The x-axis is the positivity rate of the test (calculated as the number positive by the tNGS assay divided by the total number of samples in each group on the y-axis). Incremental yield of the tNGS assay offered by NGS was calculated as the number of positive results by NGS divided by the number of samples positive by Sanger sequencing and displayed as + XX% next to each bar group. The “other tissue” group includes biopsies of serous membranes, muscle, organs (brain, liver, spleen, kidney, ovary, breast), digestive tract, masses or tumors not identified as abscesses, thrombi, and deep tissues from pacemaker pockets. The “other fluid” group includes mediastinal fluid, abdominal fluid (including bile), and any fluid not identified as being from an abscess. B, Descriptive microbiology of the microorganisms sequenced by tNGS in the 747 positive samples. Of the 607 monomicrobial samples, 529 (87%) were identified to the species level and 78 (13%) to the genus level only. For formatting reasons, some bacteria are grouped by genus in the figure, even if identified to the species level. Abbreviations: Ct, cycle threshold; NGS, next-generation sequencing.

Figure 4.

Clinical sensitivity by sample type and antimicrobial exposure. A, Clinical sensitivity (determined as the percentage of positive identifications in the infected group) of cultures, tNGS, and the combination of the 2 for tissue samples by source. B, Clinical sensitivity (percentage of positive identifications in the infected group) of cultures, tNGS, and the combination of the 2 for fluid samples by source. C, Sensitivity (percentage of positive identifications in the infected group) by numbers of days without antimicrobial therapy prior to sampling. Sensitivities of cultures and tNGS were compared using the McNemar test of paired proportions. *P value < .05. D, Kaplan-Meier curve showing probability of positivity for cultures, tNGS, and both based on duration of antimicrobial therapy before sampling in the infected group (n = 214). Patients who had received more than 100 days of antimicrobial therapy prior to sampling were included in the analysis but were censored at 100 days on the graphic representation. The probability of culture positivity was compared with tNGS and the combination of the 2 using a log-rank test (Mantel-Cox). Abbreviations: CI, confidence interval; ns = nonsignificant; tNGS, targeted metagenomics.