Molecular Diagnosis of Orthopedic-Device-Related Infection Directly from Sonication Fluid by Metagenomic Sequencing

Abstract

Culture of multiple periprosthetic tissue samples is the current gold standard for microbiological diagnosis of prosthetic joint infections (PJI). Additional diagnostic information may be obtained through culture of sonication fluid from explants. However, current techniques can have relatively low sensitivity, with prior antimicrobial therapy and infection by fastidious organisms influencing results. We assessed if metagenomic sequencing of total DNA extracts obtained direct from sonication fluid can provide an alternative rapid and sensitive tool for diagnosis of PJI. We compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other orthopedic device infections. Reads from Illumina MiSeq sequencing were taxonomically classified using Kraken. Using 50 derivation samples, we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and confirmed our findings in 47 independent validation samples. Compared to results from sonication fluid culture, the species-level sensitivity of metagenomic sequencing was 61/69 (88%; 95% confidence interval [CI], 77 to 94%; for derivation samples 35/38 [92%; 95% CI, 79 to 98%]; for validation samples, 26/31 [84%; 95% CI, 66 to 95%]), and genus-level sensitivity was 64/69 (93%; 95% CI, 84 to 98%). Species-level specificity, adjusting for plausible fastidious causes of infection, species found in concurrently obtained tissue samples, and prior antibiotics, was 85/97 (88%; 95% CI, 79 to 93%; for derivation samples, 43/50 [86%; 95% CI, 73 to 94%]; for validation samples, 42/47 [89%; 95% CI, 77 to 96%]). High levels of human DNA contamination were seen despite the use of laboratory methods to remove it. Rigorous laboratory good practice was required to minimize bacterial DNA contamination. We demonstrate that metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings, combined with the increasing availability of portable, random-access sequencing technology, offer the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI.

Keywords: diagnosis; metagenomic sequencing; orthopedic device infection; prosthetic joint infection.

Copyright © 2017 Street et al.

Figures

FIG 1

Study samples and quality control. Sequences with

FIG 2

Sequencing data filtering calibration heat maps. Two thresholds (threshold 1 and threshold 2) and three parameters (parameter a, parameter b, and parameter c) were used to determine true infection. Samples meeting either threshold were determined to be true infection. The final parameter values were chosen by maximizing the Youden index, calculated as follows: (sensitivity + specificity) − 1. For threshold 1, samples with more reads from a given species than an upper-read cutoff (parameter a; plotted on each x axis) were included. For threshold 2, samples with more species-specific reads than a lower-read cutoff (parameter b; the six panels show six different values for parameter b: 50, 100, 125, 150, 200, and 250, which are indicated within each y-axis title) and with the percentage of species-specific reads as a proportion of all bacterial reads present above a percentage cutoff (parameter c, plotted on each y axis) were included.

FIG 3

Sonication culture and sequencing comparison. The proportion of sequencing reads classified as bacterial is shown on the y axis on a log scale, and the number of CFU from sonication fluid culture is shown on the x axis. Markers are colored by the concordance of sonication fluid culture and sequencing. A single marker is shown per patient sample. Where only one of several species isolated was found by sequencing, this is shown as a false-negative. Similarly, any sample with one or more false-positive species identified by sequencing is shown as false positive. False-negative results where a coagulase-negative Staphylococcus was cultured from sonication fluid but not found in tissue samples or on sequencing are shown separately, as are samples identified only to the genus level by sequencing. Results were very similar if absolute numbers of bacterial reads were plotted on the y axis instead.