Reverse blot hybridization assay Sepsis-ID test for simultaneous identification of pathogens and antimicrobial resistance genes of mecA, vanA and vanB from blood culture bottles

Abstract

The rapid and accurate identification of pathogens in blood with sepsis is crucial for prompt initiation of appropriate therapy to decrease related morbidity and mortality rates. The aim of this study was to evaluate diagnostic performance of newly designed Reverse blot hybridization assay Sepsis -Identification test (REBA Sepsis-ID test) for rapid diagnosis of bacteremia and for detection of antimicrobial resistance genes. The 45 reference strains and 118 clinical isolates from various specimens for optimization of the REBA Sepsis-ID test were used. To evaluate the REBA Sepsis-ID test, a total of 1,400 consecutive blood culture bottles were selected from March to July 2013. By continuous monitoring blood culture system (CMBCS), a total of 300 positive and 1,100 negative blood culture bottles from patients with a delta neutrophil index (DNI) of > 2.7% were included in the study. The positive and negative bottles were tested by the conventional PCR-REBA and real-time PCR-REBA, respectively. All 45 reference strains and 118 clinical isolates showed strong specific hybridization signals at the positions of the corresponding probes derived from their respective sequences. Of the blood culture positive bottles, 67.0% (201/300) were positive for Gram positive bacteria (GPB), 24.3% (73/300) for Gram negative bacteria (GNB), 4.7% (14/300) for Candida species, and 4.0% (12/300) for polymicrobials. The agreement rates between conventional identification test and REBA Sepsis-ID test for GPB, GNB, fungi, and polymicrobials were 94.5% (190/201), 97.3% (71/73), 100% (14/14) and 91.7% (11/12), respectively. Among the 92 methicillin-resistant Staphylococcus isolates, the detection rate of mecA gene was 97.8% (90/92). The vanA gene was detected in one blood culture sample from which vancomycin-resistant Enterococcus was isolated. When the cycle threshold for real-time PCR was defined

as 30.0, 2.4% (26/1,100) of blood culture negative samples tested were positive by real-time PCR. The results of 26 samples showed discordance by conventional test, real-time PCR-REBA, and 16S rRNA sequence analysis. The REBA Sepsis-ID test could be useful to simultaneously and rapidly detect causative agents and antimicrobial resistance genes, such as mecA and vanA or vanB, in blood culture positive samples. The application of REBA Sepsis-ID test with culture methods in clinical microbiology laboratories would reduce the time for detection of pathogens responsible for sepsis, presumably resulting in reduction of patient mortality rates.