Fluorescence In Situ Hybridization as Diagnostic Tool for Implant-associated Infections: A Pilot Study on Added Value

Abstract

Implant-associated infections are a devastating complication in surgery. Especially in infections with biofilm-forming microorganisms, the identification of the causing microorganism remains a challenge. However, the classification as biofilm is not possible with conventional polymerase chain reaction or culture-based diagnostics. The aim of this study was to evaluate the additional value of fluorescence in situ hybridization (FISH) and nucleic acid amplification technique (FISHseq) to discuss a diagnostic benefit of the culture-independent methods and to map spatial organization of pathogens and microbial biofilms in wounds.

Methods: In total, 118 tissue samples from 60 patients with clinically suspected implant-associated infections (n = 32 joint replacements, n = 24 open reduction and internal fixation, n = 4 projectiles) were analyzed using classic microbiological culture and culture-independent FISH in combination with polymerase chain reaction and sequencing (FISHseq).

Results: In 56 of 60 wounds, FISHseq achieved an added value. FISHseq confirmed the result of cultural microbiological examinations in 41 of the 60 wounds. In 12 wounds, one or more additional pathogens were detected by FISHseq. FISHseq could show that the bacteria initially detected by culture corresponded to a contamination in three wounds and could exclude that the identified commensal pathogens were a contamination in four other wounds. In five wounds, a nonplanktonic bacterial life form was detected.

Conclusions: The study revealed that FISHseq gives additional diagnostic information, including therapy-relevant findings that were missed by culture. In addition, nonplanktonic bacterial life forms could also be detected with FISHseq, albeit less frequently than previously indicated.

Fig. 1.

FISH of wound tissue from a patient, where culture was positive for Acinetobacter sp., Corynebacterium sp., and Enterococcus sp. A, Overview shows host cell nuclei, stained with DAPI (blue); the tissue background appears in green. At higher magnification (B), FISH-positive bacteria are visible within a biofilm, which are detected by the Enterococcus genus-specific FISH probe EFAEC (orange).

Fig. 2.

FISH of wound tissue culture positive for Staphylococcus aureus. A, Overview shows host cell nuclei, stained with DAPI (blue); the tissue background appears in green. At higher magnification (B and C), FISH-positive bacteria are visible in microcolonies, which are detected by the Staphylococcus genus-specific FISH probe STAPHY (green, B). Another microscopic field of the same sample shows Finegoldia magna with the species-specific FISH probe FMAG (orange, C).

Fig. 3.

FISH of negative wound tissue culture. A, Overview shows host cell nuclei, stained with DAPI (blue); the tissue background appears in green. At higher magnification (B), a FISH-positive microcolony is visible, which is detected by the Candida genus-specific FISH probe CAND10 (orange).