We herein explored the value of NGS for diagnosing PJI. NGS is widely used to perform metagenetic analyses, and it can simultaneously detect sequences from millions of bases in a single biochemical reaction. In 2005, Margulies et al. performed genome-wide sequencing of Mycoplasma genitalium and Streptococcus pneumoniae, marking the beginning of the “NGS revolution.” [19] Further, in 2014, Wilson et al. used NGS for the first time to diagnose infectious diseases [16]. NGS could reportedly diagnose clinically uncommon pathogenic bacterial infections and markedly improve the detection of clinically important pathogens, revolutionizing microbial diagnosis. In 2018, Prof. Tarabichi (Rothman Institute, US) et al. reported the use of NGS for diagnosing PJI for the first time. Based on the MSIS criteria, they found that the positive diagnostic rate of NGS was 89.3% for infected cases and 25% for non-infected and culture-negative cases [5]. In the present study, the positive diagnostic rate of NGS was 93.3% for infected cases and 5.3% for non-infected and culture-negative cases. This disparity in results can be attributed to differences in threshold standards. Different NGS platforms, reagents, and databases tend to lead to variances, and NGS detection results also greatly vary. NGS can be not only qualitative but also quantitative, and it can detect the sequence number of bacterial fragments in a specimen. Further studies are warranted to explore the diagnostic significance of the sequence number.
At present, bacterial culture remains the standard method for the definitive diagnosis of PJI; however, the sensitivity of this technique is highly inconsistent (58–95%), which can be attributed to the use of antibiotics before sampling, inconsistency in standardization of sampling and specimen transmission, variances in cultivation time, and presence of unusual bacteria [20, 21]. Berbari et al. found that 53% patients with culture-negative PJI had been treated using antibiotics before sampling. On using large quantities of broad-spectrum antibiotics before sampling, bacterial count reduces or secretions contain antibiotics, consequently affecting bacterial growth and propagation [22]. Moreover, the current bacterial culture time may not be sufficiently long, typically ranging from 3 to 5 days, rather than 2 weeks [23]. In addition, other medical institutions use conventional culture media, which makes the cultivation of microorganisms such as fungi and mycobacteria challenging. Studies have reported that 46% culture-negative PJIs are caused by fungal infections and 43% by mycobacteria [24]. Diagnosing culture-negative PJI is very difficult and may involve a comprehensive evaluation with clinical methods, radiology, serology (inflammatory markers), histopathology, and microbiology. We herein found that the sensitivity of the bacterial culture method to diagnose PJI was 47%, which is lower than the data reported in the literature. This could be because among the eight culture-negative patients with PJI, NGS results indicated that one patient was infected with Candida albicans. Further, two patients had a history of treatment using broad-spectrum antibiotics. Our findings indicate that bacterial culture and NGS results were generally consistent. To summarize, the sensitivity of NGS to diagnose PJI (93.3%) was significantly higher than that of bacterial culture (47%); moreover, NGS showed a higher positive diagnostic rate for culture-negative cases (87.5%).
We herein also compared the diagnostic value of NGS and commonly used serological biomarkers to detect PJI. To the best of our knowledge, such an investigation has not been conducted as yet. We found that the accuracy and sensitivity of NGS for diagnosing PJI was significantly higher than those of serological biomarkers. The ideal method to screen PJI should be highly sensitive so as to minimize false negatives. Serological markers are still the most commonly used method for the clinical diagnosis of PJI. Our results showed that the AUC of ESR and D-dimer was 0.667 and 0.572, respectively, but the results showed no statistical significance. The sensitivity of CRP to diagnose PJI is 67%, which was lower than that reported by the most recent study (85.1%) [25]. The main reason for this is that in this study, the diagnostic cut-off value calculated by the Youden index for CRP was > 11.2 mg/L, which is higher than the cut-off value recommended by the MSIS (> 10 mg/L). A higher cut-off value can lead to a decrease in sensitivity. IL-6 was initially considered to be a highly sensitive and specific marker of PJI [26]. However, because pertinent studies did not consider the confounding effects of previous antibiotic use and related inflammatory conditions on IL-6 and other inflammatory markers, there may have been a selection bias [27]. PCT is often used as a marker of systemic infection, but its role in the diagnosis of local infections (such as PJI) is limited because the threshold of PCT in patients with local infections substantially overlaps with its normal range [28]. We herein found that with regard to PJI diagnosis, in comparison with CRP, there was no significant difference in the overall test results of IL-6 and PCT. This result is the same as that previously reported in the literature, i.e., studies have not confirmed that IL-6 and PCT are superior to traditional blood biomarkers for diagnosing PJI [29].
As with other methods to diagnose PJI, NGS also has both advantages and disadvantages. The main advantage of NGS is unbiased sampling, leading to the identification of known as well as novel organisms [30]. Further, NGS can provide auxiliary genomic information needed to predict drug resistance [31]. It can also generate quantitative or semiquantitative data pertaining to bioconcentration by counting sequencing reads, which is very useful for samples containing multiple microorganisms or in case of disease processes involving more than one pathogen [32]. The main inherent disadvantage of NGS is that microbial nucleic acids in a sample can be affected by the presence of human nucleic acids. The vast majority of sequences (usually > 99%) come from human hosts, consequently limiting the overall analytical sensitivity of pathogen detection methods [33]. Another potential challenge is that testing samples, reagents, or contaminating microorganisms in the laboratory environment can affect the accuracy of results [34, 35]. In addition, while metagenes can be used to detect pathogenic bacteria, they cannot be used for drug sensitivity tests at the same time, which remains a persistent issue.
This study had some limitations: the sample size was relatively small, choosing the MSIS criteria as the gold standard for diagnosing PJI may cause grouping errors, and benefit analysis was not performed for NGS. Our results nevertheless clearly indicated that NGS had higher accuracy and sensitivity than the bacterial culture method and commonly used serological biomarkers, and thus, we report that its diagnostic value to detect PJI is relatively much higher.