What Is the Role of Molecular Techniques for Detection of Pathogen Deoxyribonucleic Acid (DNA) (Polymerase Chain Reaction [PCR] or Next-Generation Sequencing) in Patients With Infected Total Ankle Arthroplasty (TAA)?

Rationale

The culture of multiple periprosthetic tissue samples is currently considered the gold standard for microbiological diagnosis of periprosthetic joint infections (PJIs). ¹⁹ However, biofilm-associated infections are not easily detected by culture-based methods and are often resistant to conventional antimicrobial therapy. Therefore, it seems imperative to promptly investigate and subsequently integrate molecular diagnostic techniques into the clinical practice for the management of PJI. ²⁰

The most common molecular techniques that have been used to diagnose PJI are both based on polymerase chain reaction (PCR): specific PCR and broad-range PCR. ⁸ Specific PCR targets a single bacterial species (eg, Staphylococcus aureus) or a group of closely related species (eg, all staphylococcal species). These are typically considered real-time PCR assays. Specific PCRs can be used in the diagnosis of any targeted pathogen with extreme sensitivity, potentially detecting even a single copy of the target deoxyribonucleic acid (DNA). This approach provides accurate results within hours and has the advantage of singling out any organisms deemed as significant, thereby making contamination easier to control for, as well as making quantification possible. ⁸

Broad-range PCR, in contrast to specific PCR assays, provides the opportunity to detect DNA from any pathogen rather than a specific preset of expected pathogens. Almost all broad-range PCR techniques used in diagnostic microbiology laboratories are based on the gene coding for the small subunit of the bacterial ribosome (16S rDNA). The main limitations of broad-range PCR relate to inherent problems with contamination and sensitivity. Contamination arises from bacterial DNA present in PCR reagents or inadvertently introduced during the collection and handling of the sample, particularly if additional fluids are added to the culture sample during transport or laboratory processing. ⁷ Unfortunately, these “contaminant” bacteria detected with broad-range PCR are closely related to the microorganisms that cause low-grade PJI, making the distinction between true-positive versus false-positive PCR results challenging. For these reasons, broad-range PCR has not yet been integrated into the standard routine diagnostic procedure of PJI by most laboratories, but this technique is a valid option to be applied to the diagnosis of synovial fluid or periprosthetic tissue infections.^5,21

Comparing the specific and broad techniques, one study found the sensitivities of specific PCR for detection of Propionibacterium acnes and Staphylococcus spp in sonication fluid from prosthetic shoulder infections to be 89% and 97%, respectively. ¹⁵ In contrast, broad-range PCR of tissue cultures in patients with PJI has previously demonstrated a sensitivity of only 50%. ¹

The arrival of high-throughput (next-generation) sequencing techniques has enabled the generation of thousands of individual sequences from a single broad-range PCR. ⁸ This approach seems to be promising in aiding in surgical site infection and PJI detection, because it provides detailed information on the bacterial population present in prosthetic joint samples. ⁸ The next-generation technique of pyrosequencing allows for massively parallel, rapid identification of pathogens at a much lower cost per base than the traditional sequencing. The greater breadth and depth of pyrosequencing, in which hundreds of thousands of sequences can be generated in a single run, means that low-abundance species have a higher chance of being detected. ⁸

When comparing molecular and microbiological techniques on PJIs, culture and PCR have shown similar sensitivities (72.6% and 70.4%) and specificities (98.3% and 97.8%).^3,4 While using a combination of 16S rDNA PCR and lateral flow immunoassay, the 16S recombinant DNA (rDNA) test system provided a diagnostic result within 25 minutes in 97% of all patients. This can be juxtaposed to the microbiological culture of synovial fluid, which achieved a lower sensitivity than that of the 16S rDNA test with 80%. ¹¹ In terms of cost, molecular diagnosis may be a more expensive diagnostic method than bacterial culture with a cost-effectiveness that has not yet been evaluated. ¹³ The direct detection of bacterial 16S rDNA shows encouraging results and warrants further evaluation in larger patient cohorts. ¹¹

Although molecular techniques have shown to be important in diagnosing PJI in orthopedic fields other than foot and ankle, they have not been well studied in the setting of an infected total ankle arthroplasty (TAA). In one of the few studies identified studying the use of molecular techniques in the foot and ankle, Stoodley et al evaluated several techniques to ascertain the presence of a bacterial infection in an explanted TAA that had an initial negative culture. The techniques included the Ibis T5000, real time–polymerase chain reaction (RT-PCR), a direct culture of the ankle hardware, confocal microscopy, and fluorescence in situ hybridization (FISH). ¹⁶

The Ibis T5000, a research-use-only (RUO) technology based on the combination of PCR amplification of highly conserved pathogen genomes with high-performance electrospray ionization mass spectrometry and base-composition analysis, is able to tease out a variety of organisms (including bacterial and viral) down to the species level. ² Data points include number of genome copies, relative organism abundance, and antibiotic sensitivity.^9,22 Based on Ibis testing, Stoodley et al were able to identify the presence of S aureus, Staphylococcus epidermidis, and the methicillin-resistant mecA gene in tissue on the removed TAA hardware. ¹⁶ Additionally, the Ibis detected that there was close to 10 times more S aureus in comparison to the S epidermidis. Of all the techniques investigated, the authors proposed the Ibis T5000 technology to have the most potential in aiding with clinical detection of PJI with TAA. ¹⁶

In addition to the Ibis system, the authors used RT-PCR in order to detect metabolically active S aureus. ¹⁶ The authors were able to harvest ribonucleic acid (RNA) from a tissue sample, and after converting the RNA to complementary DNA via reverse transcription, they used specific PCR primers for the bacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and histidine ammonia-lyase (hutH) genes.^17,18,23 The study demonstrated the presence of S aureus messenger RNA for both the GAPDH and the hutH genes. ¹⁶

Another technique was a direct culture of the tibial metal component of the removed ankle hardware. After a detailed agar preparation protocol, the tibial component was placed in a beaker in which an agar formed. After incubation, the number of bacterial colony-forming units (CFUs) on the agar was eventually estimated. The authors reported approximately 1000 CFUs spread across the entire tibial component and composed of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant S epidermis. ¹⁶

Confocal microscopy was also implemented for viability determination after staining and using a 488-nm excitation wavelength to identify bacteria as living or dead. Fluorescence in situ hybridization (FISH) was also used with fluorophore-labeled 16S rDNA sequences specific for S aureus.^10,12,14 A red Syto59 fluorescent nucleic acid stain was used to stain all bacterial and host nuclei, allowing S aureus to be the only species stained both red and green. Bacteria that were stained with Syto59 solely were distinguished from host nuclei on the basis of size.^6,14 Confocal microscopy and FISH demonstrated a scattered distribution of biofilm formation, with clusters of bacterial colonies on tissue, the talar component edges, the polyethylene bearing surface, and the tibial component. FISH testing also indicated that bacterial growth was predominantly S aureus and S epidermidis to a lesser extent. ¹⁶

These findings presented by Stoodley et al offer to be an important diagnostic step to gauge the presence of a bacterial-infected TAA. ¹⁶ However, further research is necessary to decide the true clinical utility of these techniques.

Supplemental Material