Detecting bacteria in revision shoulder arthroplasty: culture and next generation sequencing

Comparative study of cultures and next-generation sequencing in the diagnosis of shoulder prosthetic joint infections

In 44 patients undergoing revision shoulder arthroplasty, these authors compared bacterial identification by (1) culturing and (2) next-generation sequencing (NGS). They included patients with and without preoperative clinical signs of infection. Tissue samples were obtained from the anterior capsule, inferior capsule, glenoid, humeral canal, and underneath the prosthetic humeral head was obtained using “fresh” instruments. Culture media included anaerobic sheep blood agar and anaerobically prereduced hemin-thioglycolate broth. Aerobic media were apparently not used.

The total genomic DNA was isolated from tissue samples. These were then amplified for pyrosequencing. Amplification products were visualized, pooled, and subjected to size selection. Size-selected pools were then quantified and hybridized to generate single-stranded DNA. Single-stranded DNA was diluted and analyzed by emulsion-based polymerase chain reaction. The resulting amplification products were subsequently enriched and sequenced. Trimmed sequences were then run through USEARCH to cluster the sequences and mapped using the USEARCH UPARSE operational taxonomic unit (OTU) selection algorithm. Mapped sequences were then grouped by OTU; quality scoring-based sequence correction was then performed. Corrected sequences were then run through the Research and Testing Laboratory Genomics taxonomic analysis pipeline to determine the taxonomic classifications and abundance for each sample. Selected OTUs were then aligned using MUSCLE and a phylogenetic tree generated using FastTree. The selected OTU sequences were then globally aligned using USEARCH against a database of classified 16S sequences. Confidence values were assigned to each OTU classification, and the lowest common ancestor was determined based on these confidence values. The top hit and lowest common ancestor was then reported for each OTU.

Positive cultures were present in more than 50%, and positive NGS results were present in almost 40% of revision arthroplasty cases.

Cutibacterium (formerly Propionibacterium) acnes was the most common bacterial species cultured (8 of 13 [61.5%]) and identified by NGS (12 of 17 [70.1%]) in cases of definite and probable infection. The concordance (κ) between the 2 diagnostic criteria for defining infection that included culture or NGS was 0.333 (fair). Data from the 44 cases is shown below.

They concluded that culture data from revision shoulder arthroplasty cases commonly yields monomicrobial results; whereas, NGS data suggests that bacterial loads in revision arthroplasty are most commonly polymicrobial.

Comment: These results are interesting for several reasons. 

First, they indicate that, like culturing, NGS cannot provide information of use to the surgeon while the patient is undergoing revision arthroplasty; thus the surgeon must make decisions regarding prosthesis removal and immediate antibiotic therapy without knowledge of the results. 

Second, as can be seen from the chart above, in some cases Cutibacterium and Coagulase Negative Staph we cultured but not detected by NGS. These are the two most commonly recovered organisms in revision of failed shoulder arthroplasty. It is unclear whether NGS was insensitive to these bacteria.

Third, in many of the other cases, NGS commonly suggested the presence of organisms that have not been previously reported in the culture results from cases of revision arthroplasty: A junii, A tetradius, A radioresistens, A calcoaceticus, A rhizogenes, A ferrireducens, B cepacia, B fungorum, B nordic, B doer, B cepacia, B fragilis, C diphtheria, C tuberculostearicum, C hominis,  C chromoreductans, C kroppenstedtii, C quinii, C hveragerdense, C paradoxus, C acidisoli, C vibrioides, C circulars, E hormaechei, G ruanii, K palustris, K rosea, L crispatus, L agilis, L albida, M catarrhalis, M Luteus, R picettii, R insidiosa, P saccharophilia, R gnavus, S maltophilia, S agalactiae, and others. Thus it is uncertain whether live forms of these bacteria are actually present in the wounds of these patients.

NGS is a very, very sensitive test! The question is whether it is TOO sensitive for clinical use, i.e. if these microbes do not grow in culture are they truly pathogenic and of deep shoulder origin? Or could they be present in the skin itself and killed during the prep process but leaving their DNA behind? We suspect the latter, mostly due to the fact that NGS can (in theory) detect a single genetic copy in the specimen.

The paper does not provide information on the cost and time necessary to complete NGS in comparison to standard culturing.

NGS is a powerful tool being put to increasingly broad use. We look forward to further work demonstrating its value in the management of failed arthroplasty
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