PCR-based Sepsis@Quick test is superior in comparison with blood culture for identification of sepsis-causative pathogens

Abstract

Sepsis is an acute, often fatal syndrome that requires early diagnosis and proper treatment. Blood culture (BC) is the gold standard for the identification of pathogens, however it has marked limitations, including that it is time-consuming (delaying treatment) and can only detect microbes that readily grow under culture conditions. Alternatively, non-culture-based methodologies like polymerase chain reaction (PCR) are faster but also have limitations; e.g., the reaction is often inhibited by the abundance of human DNA and thus can only detect limited known target pathogens. In our previous publication, we have demonstrated a proof-of-concept of a simple pre-analytical tool to remove human DNA from patients' blood specimens, hence allowing downstream PCRs to detect rare bacterial genetic materials. In the current study, we reported a better performance of a novel prototype diagnosis kit named Sepsis@Quick that combines human DNA removal step with real-time PCRs compared to blood-culture for identifying sepsis causative bacteria. Our data showed that Sepsis@Quick is superior to blood culture in which the novel diagnostic kit could identify more pathogens and even polymicrobial infection, faster and less influenced by the empirical administration of broad spectrum antibiotic therapy (single administration or combination of cephalosporin III and fluoroquinolon). Additionally, for the first time, we demonstrated that positive results achieved by Sepsis@Quick are significantly associated with a reduction of sepsis-related mortality.

Figure 1

Scheme of study design and workflow. (A) Former MCLB-1 based stepwise realtime PCR protocol including Group-specific screening by PCR reactions targeting bacterial 16SrRNA gene to differentiate Gram-positive, Gram-negative and Enterobacteriaceae groups. Samples positive in the screening assay were subjected to genus-specific real-time PCR reactions to detect 13 most common sepsis causative pathogens. (B) the current protocol of Sepsis@quick diagnostics kit was subjected to individual to genus-specific real-time PCR of E. coli, K. pneumoniae, P. auriginosa, A. baumannii, N. meningitidis, Staphylococus sp, S aureus, Streptococus sp. S. suis, S. pneumonia, Enterococcocus sp, Fusobacterium sp and Bacteriodes sp.

Figure 2

Numbers of individual pathogens detected by blood culture and Sepsis@quick: Upper panel shows the numbers positive cases or number of polymicrobial infection detected by blood culture or sepsis@quick, lower panel shows number of individual microbial pathogens detected by blood culture or sepsis@quick.

Figure 3

Diagnostic performance and concordance portion between the two methods: Total 144 patient blood samples were recruited into this current study, of which, Sepsis@Quick identified 83/144 (57.64%) positive. Blood culture approach detected 49 cases (34%) positive and 43 out of 49 culture-positive cases (87.8%) were co-detected by Sepsis@Quick and only 40 cases (27.78) were not diagnosed.

Figure 4

Relationship between sepsis-related mortality rate and diagnostic performance of Sepsis@quick: Two left bars show comparisons of mortality rate between patient group carrying sespsis@quick positive result versus patient group with sespsis@quick negative diagnosis. Two right bars show comparisons of mortality rate between patient group of blood culture negative but sepsis@quick positive versus patient group with blood culture negative and sepsis@quick negative (double nengative). Both comparison were performed by Chi-square test.