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AAOS Now

Published 5/1/2017
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Terry Stanton

Study Demonstrates Rapid Method for Detecting P Acnes

A study presented at the AAOS Annual Meeting reported on an assay and technique for detecting Propionibacterium acnes (P acnes). This technique was shown to identify the organism in tissue obtained from a shoulder biopsy within 24 hours.

The study evaluated the effectiveness of using a sensitive and robust polymerase chain reaction (PCR) technique coupled with specific restriction fragment length polymorphism (RFLP) analyses to achieve a clinically relevant assay that can identify P acnes.

George S. Athwal, MD, FRCSC, one of the investigators, explained that the study was undertaken because "presently, P acnes is a common infecting organism after shoulder surgery. Unfortunately, our ability to definitively diagnose an infection is limited, and common diagnostic test results are frequently normal. Additionally, to culture P acnes from a tissue sample may take greater than 7 to 10 days. Our goal was to develop a technique to rapidly determine the presence of P acnes in a biopsy sample."

In the study, primers were designed to amplify a unique region of the 16S rRNA gene in P acnes that contained a unique HaeIII restriction enzyme site. PCR and RFLP analyses were optimized to detect P acnes DNA in in vitro cultures and in arthroscopic surgical biopsy specimens from patients with P acnes infections.

Specimens from five surgical biopsies of rotator cuff tendon and five biopsies of glenohumeral joint capsule were obtained during arthroscopy. These normal human tissues were used as a source of human chromosomal DNA to simulate the "background" DNA that would be present in any shoulder tissue lysate sample being assessed for the presence of P acnes. Tissues were dissected to 1.5 g portions and homogenized in 4 mL of reverse osmosis (RO) purified water to enhance cellular lysis under hypo-osmotic conditions.

Tissues were homogenized using a polytron at 9,000 rpm, and aliquoted into 1.5 mL tubes. Two chromosomal DNA extraction methods were compared during assay development to identify the optimal method for cellular lysis: 1) aliquots were boiled in a water bath for 20 minutes, or 2) aliquots were homogenized with 100 µm glass beads in a bead beater at 4,500 rpm for 30 seconds.

In parallel, aliquots of a verified P acnes isolate were added to shoulder tissue lysates to simulate tissue samples infected with P acnes. An initial culture of 2 × 108 bacterial cells/mL was serially diluted in 10-fold increments to 2 × 103 cells/mL in shoulder tissue lysates and underwent the DNA extraction methods to isolate total (human and bacterial) DNA; 5 µL aliquots containing between 1 × 106 and 1 × 101 cells underwent PCR and RFLP analyses.

P acnes-specific amplicons were detected in as few as 10 bacterial cells and in clinical biopsy specimens of infected shoulder tissues.

The authors commented: "A major strength of PCR-RFLP assay described in this report is its specificity for P acnes. This specificity is predicted to be greater than routine culture-based approaches and previously published PCR approaches, because it has two levels of verification. The first level is PCR amplification with P acnes-specific primers, and the second is a P acnes-specific RFLP pattern. Previous reports had the first (albeit less specific than ours), but not the second. The strength of this extremely sensitive technology is the ability to detect fewer than 10 P acnes cells in the sample. The weakness is that this level of sensitivity makes aseptic sample collection very important. PCR and RFLP mapping are techniques that are routinely performed in many clinical microbiology and pathology laboratories, and so this approach is not restricted to highly specialized research laboratories."

They concluded: "The PCR-RFLP assay is simple, robust, distinguishes between P acnes and other Propionibacterium sp, and is (theoretically) cost effective. In its current form, the assay provides proof of concept for using a PCR-RFLP approach to rapidly and consistently identify P acnes in tissues from shoulder biopsies within 24 hours."

Dr. Athwal said that although this study provides the foundation for further work in diagnostics and P acnes pathogenicity, "further research is absolutely needed in this area." He continued, "Several P acnes virulence factors have been identified that can predict the pathogenicity of a strain. Presently, we are investigating the potential to detect these virulence factors in a multiplex PCR. Our hope is to develop a method that will differentiate P acnes contamination from true infection. Finally, an end goal would be to develop a point-of-care diagnostic test that would immediately identify the presence of a P acnes infection."

Dr. Athwal's coauthors of "A Rapid Method for Detecting Propionibacterium Acnes in Surgical Biopsy Specimens from the Shoulder," are Scott Holmes, MSc; Ana M. Pena Diaz, MSc; Kenneth J. Faber, MD, FRCSC; and David B. O'Gorman, PhD.

The authors' disclosure information can be accessed at www.aaos.org/disclosure

Terry Stanton is the senior science writer for AAOS Now. He can be reached at tstanton@aaos.org