Comparison of multiplex real-time PCR and PCR-reverse blot hybridization assay for the direct and rapid detection of bacteria and antibiotic resistance determinants in positive culture bottles

Abstract

The aim of this study was to evaluate the performance of a commercially available multiplex real-time PCR assay and a PCR-reverse blot hybridization assay (PCR-REBA) for the rapid detection of bacteria and identification of antibiotic resistance genes directly from blood culture bottles and to compare the results of these molecular assays with conventional culture methods. The molecular diagnostic methods were used to evaluate 593 blood culture bottles from patients with bloodstream infections. The detection positivity of multiplex real-time PCR assay for Gram-positive bacteria, Gram-negative bacteria and Candida spp. was equivalent to PCR-REBA as 99.6 %, 99.1 % and 100 %, respectively. Using conventional bacterial cultures as the gold standard, the sensitivity, specificity, positive predictive value and negative predictive value of these two molecular methods were 99.5 % [95 % confidence interval (CI), 0.980-1.000; P<0.001), 100 % (95 % CI, 0.983-1.000; P<0.001), 100 % and 99 %, respectively. However, positivity of the Real-methicillin-resistant Staphylococcus aureus multiplex real-time PCR assay targeting the mecA gene to detect methicillin resistance was lower than that of the PCR-REBA method, detecting an overall positivity of 98.4 % (n=182; 95 % CI, 0.964-1.000; P<0.009) and 99.5 % (n=184; 95 % CI, 0.985-1.000; P<0.0001), respectively. The entire two methods take about 3 h, while results from culture can take up to 48-72 h. Therefore, the use of these two molecular methods was rapid and reliable for the characterization of causative pathogens in bloodstream infections.

Development of a Rapid Reverse Blot Hybridization Assay for Detection of Clinically Relevant Antibiotic Resistance Genes in Blood Cultures Testing Positive for Gram-Negative Bacteria

“…However, as REBA-EAC assay detects one or more amino acid substitutions in targeted region, it can discriminate between ESBL TEM/SHV derivatives and non-ESBL TEM/SHV types. In general, our results are similar to previous assessments of REBA assay (sensitivity of 95–99% and specificity of 100%) (Kim et al, 2016 ; Wang et al, 2016 ) and other methods such as microarray-based check-points assay (sensitivity of 94–95% and specificity of 83–100%) (Cohen Stuart et al, 2010 ; Card et al, 2013 ) and MALDI-TOF MS-based assay (sensitivity of 97–99% and specificity of 94–99%) (Bork et al, 2015 ; Chong et al, 2015 ). Moreover, the performance of the REBA-EAC assay was better than the E -test (sensitivity of 71% and specificity of 73%) (Garrec et al, 2011 ).…”

Evaluation of multiplex tandem PCR (MT-PCR) assays for the detection of bacterial resistance genes among Enterobacteriaceae in clinical urines

“…PCR has been widely employed to seek resistance genes in clinical samples, but mostly to support infection control rather than to guide therapy. [11][12][13][14][15][16][17] We explored its potential to detect important enterobacterial resistance genes in clinical urines without culture. Two iterations of an MT-PCR assay were tested, expanding the number of resistance targets.…”

Rapid Detection of Beta-Lactamases Genes among Enterobacterales in Urine Samples by Using Real-Time PCR

“…qPCR system is a faster and more efficient technology for detection sensitivity to antibiotics compared to classical phenotypic and conventional methods [ 26 ]. It has been widely used for the research of resistance genes in clinical samples, but principally to support infection controlling rather than guiding therapy [ 21 ]. We explored its potential to detect important genes for antibioresistance to Enterobacterales in the clinic urine without culture.…”