Detection of prosthetic hip infection at revision arthroplasty by immunofluorescence microscopy and PCR amplification of the bacterial 16S rRNA gene

Abstract

In this study the detection rates of bacterial infection of hip prostheses by culture and nonculture methods were compared for 120 patients with total hip revision surgery. By use of strict anaerobic bacteriological practice during the processing of samples and without enrichment, the incidence of infection by culture of material dislodged from retrieved prostheses after ultrasonication (sonicate) was 22%. Bacteria were observed by immunofluorescence microscopy in 63% of sonicate samples with a monoclonal antibody specific for Propionibacterium acnes and polyclonal antiserum specific for Staphylococcus spp. The bacteria were present either as single cells or in aggregates of up to 300 bacterial cells. These aggregates were not observed without sonication to dislodge the biofilm. Bacteria were observed in all of the culture-positive samples, and in some cases in which only one type of bacterium was identified by culture, both coccoid and coryneform bacteria were observed by immunofluorescence microscopy. Bacteria from skin-flake contamination were readily distinguishable from infecting bacteria by immunofluorescence microscopy. Examination of skin scrapings did not reveal large aggregates of bacteria but did reveal skin cells. These were not observed in the sonicates. Bacterial DNA was detected in 72% of sonicate samples by PCR amplification of a region of the bacterial 16S rRNA gene with universal primers. All of the culture-positive samples were also positive for bacterial DNA. Evidence of high-level infiltration either of neutrophils or of lymphocytes or macrophages into associated tissue was observed in 73% of patients. Our results indicate that the incidence of prosthetic joint infection is grossly underestimated by current culture detection methods. It is therefore imperative that current clinical practice with regard to the detection and subsequent treatment of prosthetic joint infection be reassessed in the light of these results.

FIG. 1

Examples of confocal laser scanning micrographs of bacteria in material removed by ultrasonication (sonicate) from culture-negative hip prostheses to illustrate coccoid cells present singly and in small groups (a) and in a large aggregate (b). Bacteria were labelled with anti-Staphylococcus spp. polyclonal antiserum and an anti-P. acnes-specific MAb, followed by labelling with suitable fluorescently conjugated secondary antibodies.

FIG. 2

Confocal laser scanning micrographs of sonicates obtained from culture-negative hip prostheses to illustrate a large number of coccoid cells associated with a smaller number of coryneform cells (a) and a large number of coryneform cells associated with a smaller number of coccoid cells (b) labelled as described in the legend to Fig. 1.

FIG. 3

Confocal laser scanning micrograph illustrating the depth of the dislodged biofilm aggregate of coccoid cells shown in Fig. 1b. The series of images were captured at 0.5-μm intervals from the top (image in the box) to the bottom (images displayed left to right) of the aggregate. The depth of this aggregate was estimated to be 3.5 μm.

FIG. 4

Immunofluorescence micrographs of material scraped from skin illustrate an isolated bacterial cell and a skin cell (a) and a small group of bacteria (b). Immunolabelling was carried out as described in the legend to Fig. 1.