Abstract
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This work represents a continuation of a career-long effort to develop and/or apply ever better (and broader) MDx methodologies to help characterize human infectious disease in general (Kwok et al., 1987; Ehrlich and Greenberg, 1994; Mañez et al., 1996; Marshall et al., 1997; Aul et al., 1998; Boase et al., 2013). More recently we have focused on orthopedic infectious diseases, wherein multiple studies have suggested that patients with osteoarthritis (Stoodley et al., 2011; Ehrlich et al., 2012, 2014; Jacovides et al., 2012; Maixner et al., 2014) may have bacteria present within their joints. Thus, this study was designed to look specifically at this issue using two groups of patients with arthritis of the knee. The first group were patients suspected of having an acute episode of septic arthritis based on standard clinical diagnostic criteria and the second group were patients with osteoarthritis who were undergoing primary arthroplasty of the knee, that is, replacement of their native joint with an artificial joint. In the septic arthritis group, we expected to find bacteria present in the synovial fluid of a significant fraction of the patients, but according to traditional etiologies of osteoarthritis, we would not expect to find bacteria present in the synovial fluid of the latter group.
Because we did not want to bias our studies, a pan-domain diagnostic was chosen that has multiple advantages over earlier simplex MDx. Most simplex MDx testing is characterized by the development of a highly sensitive and specific PCR-based assay that is used to assess the presence of a specific pathogen, but it is useful only if the organism that the tester hypothesized was present is actually present. If the clinical symptoms were caused by any other organism, a false-negative result would be reported with regard to infection in general. Thus, the positive predictive values of such simplex tests are very high, assuming appropriate controls are in place to control for contamination, but the negative predictive values are essentially worthless except for ruling out a single pathogen. This problem of a lack of breadth has bedeviled DNA-based diagnostics since the development of PCR some 30 years ago (Saiki et al., 1985). Twenty years ago, I published a plea for panel-based tests that would include multiple pathogens that were known to be associated with specific clinical syndromes (Ehrlich, 1996), and there have been some tests developed along these lines. However, these tests have by-and-large been developed by combining a series of simplex tests into a single test tube producing a multiplex test (Gluestein et al., 1998).
In this study, we utilized a next-generation pan-domain MDx that employs both PCR and electrospray ionization mass spectrometric analyses to simultaneously detect and speciate essentially any and all bacterial or fungal infectious agents that are present (Eckert et al., 2008). The results of this pan-domain MDx were then compared with microbial culture results, and discrepencies were adjudicated using fluorescent in situ hybridization (FISH). FISH provides for generic or species-specific verification of bacterial/fungal biofilms that are invested into the tissues. Because of the physical harshness of the FISH procedure, which includes multiple washing and agitation steps, any unaffixed bacteria or bacterial contaminants introduced through the collection method would not be retained or observed. Thus, FISH now serves as the “gold standard” among infectious disease confirmatory diagnostics—as it is both specific and spatially precise (Hall-Stoodley et al., 2006; Nistico et al., 2009).
The results of the study indicated that the vast majority of the septic arthritis cases from which an organism was detected were associated with Staphylococcus aureus, and culture only detected S. aureus. Interestingly, however, culture misidentified a septic arthritis case as S. aureus that was demonstrated by both the MDx and FISH to be Streptococcus agalactiae. More importantly culture only detected a pathogen in ∼30% of the suspected cases of septic arthritis that were demonstrated by the MDx to be infected. Examination of the osteoarthritis cases was even more telling; ∼30% were MDx positive and FISH positive for fastidious or slow-growing bacteria as we have seen before, but culture did not detect any of these nontraditional pathogens. This study supports our contention that microbial culture, although an important means to obtain isolates for study, should never be used as the sole clinical diagnostic methodology for infectious agents.