Patients presenting to the ED with suspected sepsis can follow a spectrum of clinical courses, ranging from prompt resolution to substantial morbidity and death. Many factors that help determine which trajectory a patient takes have been identified. Vitally important to clinical recovery is the prompt administration of appropriate antibiotics (4
). Currently, the initial choice of antibiotics is empirical and broad and covers the most likely etiologic agents. The major limitation to a more targeted antibiotic strategy is the lack of an available diagnostic method for the rapid and reliable identification of the etiologic agent. Blood culture is the most widely utilized technique for the diagnosis of bloodstream infections. However, despite its high degree of specificity, its sensitivity remains low and the mean time to positivity in our study was approximately 24 h and reached nearly 5 days in some cases. Antigen and serological testing is highly specific but has great variations in sensitivity, depending on the targeted organism. Furthermore, it is generally limited to a particular organism, and consequently, the suspected etiologic agent must be specified a priori. This study aimed to evaluate a real-time multiplex PCR assay as an adjunct to blood culture for the rapid identification of bloodstream infections.
The findings of this study suggest a high degree of concordance between the results of blood culture and PCR, with the two methods having similar operating characteristics (sensitivities, 0.25 and 0.20, respectively). This is most evident among those patients without infection and is manifested by high specificities: 95% for blood culture and 100% for PCR (when contaminants are included). Interestingly, of 127 subjects with definite infections, 55 had concordant negative results, whereas 40 had concordant positive results, for overall agreement in 74.8% of cases. The remaining 32 discordant cases are represented by 26 blood culture-positive and PCR-negative results and 6 blood culture-negative and PCR-positive results. Due to the significant number of infections that would be missed, it does not appear that this multiplex PCR assay could replace blood culture for the identification of bloodstream infections in patients presenting to the ED with suspected sepsis. However, the time to the identification of the etiologic agent is clearly much shorter with PCR than with blood culture. PCR also added diagnostic yield, with PCR detecting an additional 24 isolates that were missed by blood culture. The clinical relevance of such PCR-positive but blood culture-negative situations is an important question. Our results suggest that the organisms identified by PCR alone were clinically relevant on the basis of chart review and confirmation of the result by other microbiological diagnostic methods, such as urine culture and antigen testing. Potential reasons for the discordant results are numerous. We were unable to ensure that an adequate volume of blood for culture was collected, which could have contributed to the discordant blood culture-negative and PCR-positive results. Furthermore, the ability of PCR to detect DNA in sterilized blood, such as after antibiotic administration, could also have yielded these discordant results. However, an analysis of the PCR and blood culture results stratified by prior antibiotic use did not support this hypothesis. In contrast, discordant blood culture-positive and PCR-negative results could arise from the intrinsic variability of PCR, inhibiting factors present in the patient sample or other unidentified factors.
The cases in which PCR identified an infection are those in which the benefits of PCR can be most clearly realized. When there is concordance between PCR and blood culture results, the primary advantage to a PCR-based assay is the timeliness with which those results are available compared to the time to the availability of conventional blood culture results: 6 to 7 h and 24 to 72 h, respectively. This may allow the treating physician to narrow antibiotic coverage early in the course of treatment, potentially avoiding the toxicity and costs associated with the use of broad and empirical antimicrobial therapy. PCR can also identify an infection that was not being adequately treated. For example, empirical antibiotic choices typically do not treat fungal infections. However, the identification of fungemia by PCR can allow the physician to appropriately expand the antimicrobial regimen early in the course of disease. The potential clinical utility of the SeptiFast PCR to effect the clinical treatment and outcome was not addressed in this study, which used banked blood samples. Nevertheless, our results affirm the hypothesis that PCR and blood culture can identify a partially overlapping set of bloodstream infections, making PCR a useful adjunct to blood culture. Prospective clinical trials for the assessment of treatment and patient outcomes will be important to the further advancement of a multiplex PCR approach.
This study has several strengths in comparison to some previous evaluations of this multiplex PCR platform. First, patients were recruited from the ED on the basis of a suspicion of community-acquired sepsis. This relatively undifferentiated patient group is in contrast to the patient populations used in other studies, which enriched for patients with confirmed infection or hospital inpatients, who are at particularly high risk of developing bloodstream infections. Extrapolation of the findings from those studies could otherwise lead to context and spectrum biases. Furthermore, our use of blinded clinical adjudicators to determine infection status resulted in a better classification system. As a result, 14% of patients in this study were ultimately determined to have a noninfectious disease process, serving as a valuable internal control.
Second, another significant strength of this study is the way in which this new diagnostic tool was evaluated. Most published reports evaluating the SeptiFast
PCR platform have tested multiple isolates from a small number of subjects, leading to spectrum bias, such that multiple samples are more likely to be drawn for sicker patients and these patients are subsequently more likely to have positive assay results (7
). In contrast, our study is the largest to date in which subjects were identified prospectively and samples were collected from any given subject only once (21
There are several potential limitations to this study. First, patients were recruited from the EDs of two tertiary-care centers, and consequently, the results may not be applicable to other clinical settings with different patient characteristics, resources, and laboratory procedures. A second potential limitation in interpreting the results of this study is the heterogeneity in the methods by which blood samples for culture were drawn. Although there are specific guidelines for how blood samples for culture should be drawn, including guidelines on skin cleansing, the volume per bottle, and the number of bottles, there is no way to ensure that all samples were collected according to those guidelines. This can have implications both for the sensitivity and for the specificity of blood culture, leading to an inadequate criterion bias, although it is more representative of actual clinical practice. Furthermore, although infection status is based on a multitude of factors, blood culture results are part of the criterion standard. As a result, incorporation bias likely plays a role in our observation that blood culture was statistically significantly better than PCR for those patients with definite infections. In addition, our clinical adjudication of infection includes viral, rickettsial, and other infectious etiologies that are expected to yield negative blood culture and PCR results, such as herpes simplex virus, influenza virus, Rickettsia rickettsii, and Clostridium difficile. This would tend to underestimate the performance characteristics of blood culture and PCR when the clinical infection criterion is used as the gold standard.
Current unanswered questions include the optimal assay conditions needed to minimize PCR inhibition by substances such as the heme in blood, the ethanol introduced during the extraction process, and other unidentified inhibitors found in blood (8
). In addition, the patient population most likely to benefit from this diagnostic tool needs to be better defined. Clinical utility may vary depending on the population prevalence of septicemia, with potentially lower levels being detected in the ED setting than in the ICU setting. Finally, the assays were performed with frozen samples and did not inform actual clinical practice. To that end, a recent study by Dierkes et al. reported on the effects of performing the SeptiFast
-based PCR in parallel with blood culture with samples from a cohort of hospitalized patients (7
). PCR was performed at the discretion of treating physicians without any subject enrollment or randomization, and the analysis was retrospective. Despite the biases that this might introduce, their findings are largely consistent with our own and serve as an important step toward assessing the directed clinical application of this technology.
In summary, PCR is a promising tool for the rapid identification of bloodstream infections as an adjunct to blood culture. Overall, the operating characteristics of PCR are similar to those of blood culture for the diagnosis of infection, but a significant number of additional isolates are identified by PCR. High-quality research into the real-clinical-time application of these results is a critical next step in the development of this technology.