Diagnosis of orthopedic device-related infection is challenging, and causative pathogens may be difficult to culture. Metagenomic sequencing can diagnose infections without culture, but attempts to detect antimicrobial resistance (AMR) determinants using metagenomic data have been less successful.

The human airways were once thought sterile in health. Now metagenomic techniques suggest bacteria may be present, but their role in asthma is not understood.

Background Diagnosis of orthopaedic device-related infection is challenging, and causative pathogens may be difficult to culture. Metagenomic sequencing can diagnose infections without culture, but attempts to detect antimicrobial resistance (AMR) determinants using metagenomic data have been less successful. Human DNA depletion may maximise the amount of microbial DNA sequence data available for analysis. Methods Human DNA depletion by saponin was tested in 115 sonication fluid samples generated following revision arthroplasty surgery, comprising 67 where pathogens were detected by culture and 48 culture-negative samples. Metagenomic sequencing was performed on the Oxford Nanopore Technologies GridION platform. Filtering thresholds for detection of true species versus contamination or taxonomic misclassification were determined. Mobile and chromosomal genetic AMR determinants were identified in Staphylococcus aureus-positive samples. Results Of 114 samples generating sequence data, species-level sensitivity of metagenomic sequencing was 49/65 (75%; 95%CI 63-85%) and specificity 103/114 (90%; 95%CI 83-95%) compared with culture. Saponin treatment reduced the proportion of human bases sequenced in comparison to 5um filtration from a median (IQR) 98.1% (87.0%-99.9%) to 11.9% (0.4%-67.0%), improving reference genome coverage at 10-fold depth from 18.7% (0.30%-85.7%) to 84.3% (12.9%-93.8%). Metagenomic sequencing predicted 13/15 (87%) resistant and 74/74 (100%) susceptible phenotypes where sufficient data were available for analysis. Conclusions Metagenomic nanopore sequencing coupled with human DNA depletion has the potential to detect AMR in addition to species detection in orthopaedic device-related infection. Further work is required to develop pathogen-agnostic human DNA depletion methods, improving AMR determinant detection and allowing its application to other infection types.

Metagenomics has the potential to revolutionise infectious diseases diagnostics, from rapid species and antimicrobial resistance prediction, to finding unrecognised and sometimes untreated infections. Our aim was to summarise all literature on culture-independent metagenomic sequencing to describe the accuracy of species and antimicrobial resistance prediction and, describe the challenges and progress in the field. We conducted a systematic review with meta-analysis from eligible studies retrieved from PubMed, Google Scholar and bioRxiv and, assessed risk of bias and quality using the QUADAS-2 tool. This study is registered with PROSPERO, number CRD42020163777. We identified 36 studies, 22 of which used a species-agnostic approach to identify all possible pathogens. In these studies, the overall sensitivity and specificity of pathogen species detection were 88% (95%CI 81-92%) and 86% (95%CI 70-94%) respectively. Antimicrobial resistance prediction and comparison to phenotypic results was undertaken in six studies. Categorical agreement was 83% (95%CI 68-92%), very major (prediction sensitive, phenotype resistant) and major error (prediction resistant, phenotype sensitive) rates were 9% (95%CI 2-27%) and 1% (95%CI 0-20%) respectively. We report limited use of negative controls in studies 61% (22/36) which contribute to a major challenge of discriminating true pathogens from contamination, where there is no convergence on methodology. More efficient human DNA depletion methods are required as a median of 79% (IQR 62-96) [Range 7-98] of sequences were classified as human despite laboratory depletion techniques. The median time from sample to result was 23.5 hours (7-31) [4-144], with sequencing time accounting 10 hours (4.8-16) [1-16]. The average reported consumables cost per sample ranged from $128 to $685. The science and regulatory environment are rapidly developing, and its role as a routine test or test of last resort still needs to be determined, however it is likely that clinical metagenomics will be an increasing part of the clinician′s armamentarium to diagnose infectious diseases in the near future.