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Fig. 1 The percentage of cases in which NGS had detected at least one pathogen.
Courtesy of Javad Parvizi, MD, FRCS

AAOS Now

Published 11/1/2017

Next-generation Sequencing: The Next Step in Identifying Infections?

A promising technology for diagnosing potential PJI
Next-generation sequencing (NGS) is a DNA-sequencing technology that has revolutionized genomic research, reducing both the time and costs involved. "The cost to sequence one gigabase of DNA in 2000 was $230; to sequence the same amount of DNA today costs just $1," said Javad Parvizi, MD, FRCS, during his presentation at the 2017 annual meeting of the Musculoskeletal Infection Society.

Dr. Parvizi had previously experimented with using a polymerase chain reaction (PCR) method of DNA sequencing to identify infectious organisms. However, PCR, which rapidly replicates a small section of DNA, requires that researchers identify organisms in advance. As a result, Dr. Parvizi and the other researchers concluded that it was not the right test to use in total joint replacement (TJR).

Dr. Parvizi and his coauthors wondered whether NGS could be used to help identify organisms in synovial fluid. Cultures from synovial fluid or deep tissues are frequently used to help identify organisms in patients with suspected periprosthetic joint infections (PJI). But the limitations involved in growing cultures from synovial fluid mean that the technique is not applied prior to surgery. Additionally, said Dr. Parvizi, "Up to 55 percent of PJIs are caused by culture-negative infections."

He also noted that outcomes after treatment for culture-negative PJI are worse than those achieved when the organism has been isolated and targeted antibiotic therapy has been administered. If NGS could identify specific organisms in synovial fluid in advance of surgery, enabling a targeted approach, results could improve.

Study parameters
Dr. Parvizi and his colleagues conducted a prospective, blinded study using 86 anonymous fluid samples from patients undergoing hip or knee aspiration as part of a routine evaluation for PJI. Each sample was subjected to a panel of synovial fluid tests, including the following:

  • C-reactive protein
  • human neutrophil elastase (HNE)
  • total neutrophil count
  • alpha (α) defensin
  • culture

Aliquots obtained from each sample also underwent NGS. The results were stratified according to their α-defensin and culture result (Fig. 1). Of the 86 samples, 30 were α-defensin positive and culture positive (definite infection); 24 were α-defensin positive and culture negative; and 32 were α-defensin negative and culture negative (definitely not infected). "It's the middle group that we're really interested in," said Dr. Parvizi.

In the first group (definite infection), NGS was concordant with culture in 25 of the 30 samples. In four of the five discordant samples, NGS detected antibiotic-resistant bacteria, whereas the culture analysis did not. In one sample with relatively low levels of synovial inflammatory biomarkers, the culture was positive but NGS was negative.

Overall, 10 samples had a positive result with NGS and a negative result from cultures; five of these samples were α-defensin positive and had high levels of inflammatory biomarkers. In the other five samples, the levels of α-defensin and inflammatory biomarkers were low. In addition, NGS identified several organisms in each sample. In samples with a higher probability of infection, a single organism was predominant; in samples presumed not infected, many organisms were prevalent, but none was predominant. "We are approaching an era when we may rethink the notion that synovial fluid in our joints is sterile," said Dr. Parvizi. "We have three organisms for every cell in our body, so we are more microbe than we are mammals."

Value of molecular markers
"I believe this is the first report of NGS being used to isolate organisms in synovial fluid," Dr. Parvizi continued. "Bear in mind that any molecular technique used to detect an organism depends on the number of organisms present in the tissue provided to the lab. The more of this fluid or the deeper the tissues that have the organisms, the better these techniques are at detecting organisms. Unfortunately, most of the samples we had were of low quantity, which may have affected the outcome.

"I think it's important for us to keep an eye on molecular markers," he continued, "in cases of metal-on-metal implant failures, adverse local issue reactions, and maybe even crystalline deposition diseases, because the molecular markers can be abnormally elevated."

In conclusion, Dr. Parvizi noted, "At this point, the gold standard for diagnosing PJI remains to be determined. Overall, it does appear NGS isolated organisms in at least 20 percent of culture-negative cases, from low-volume synovial fluid that was provided to the lab. NGS can identify known pathogens causing PJI in synovial fluid in both culture-positive and culture-negative cases. It was also able to detect numerous organisms in a given sample."

Dr. Javad Parvizi's coauthors for "Next-generation Sequencing Is a Valid Tool for Detecting Pathogens in Synovial Fluid" are Majd Tarabichi, MD, and Noam Shohat, MD.

Bottom Line

  • NGS is a DNA-sequencing technique that has reduced both the time and the costs associated with genomic research.
  • In this study, NGS was able to identify known pathogens causing PJIs in synovial fluid in both culture-positive and culture-negative samples.
  • In samples with a higher probability of infection, a single organism was predominant; in samples presumed not infected, many organisms were prevalent, but none was predominant, raising the possibility that there is a normal microbiome in the synovial fluid.
  • Using NGS to identify specific organisms in synovial fluid in advance of surgery could enable use of targeted antibiotic treatment, thus improving results.