Establishment of Multiplex qPCR for Identification of Pathogens in Fracture-Related Infection (FRI)

Abstract

Background: Fracture-related infection (FRI) is one of the most challenging complications after fracture surgery. Tissue culture is the most commonly used but time-consuming method for identifying FRI pathogens in clinical. Here we aimed to use the five common pathogenic bacteria in FRI to establish a simple and fast multiplex qPCR method for clinical FRI detection. 

Objectives: Here we aimed to use the five common pathogenic bacteria in FRI to establish a simple and fast multiplex qPCR method for clinical FRI detection. 

Methods: A total of 66 patients with FRI and 24 non-infectious volunteers were enrolled in this study. Results from tissue culture and multiplex qPCR were analysed and compared. Then the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), Youden Index and area under the ROC curve (AUC) of the two methods were calculated respectively.

Results: For the detection of FRI, all results can be obtained by multiplex qPCR within 5 hours while 3-7 days through tissue culture. For 66 FRI cases, tissue culture detected 63 cases (95.5%) and multiplex qPCR detected 56 cases (84.8%). Among the 24 control cases, 3 cases (12.5%) and 4 cases (16.7%) were positive by tissue culture and multiplex qPCR, respectively. The sensitivity and specificity of multiplex qPCR were 93.3% and 66.7%, while those of tissue culture were 95.4% and 87.5%, respectively. If cases within the detection range were compared, which were especially for the five common pathogens, the specificity of multiplex qPCR changed from 66.7% to 90.9%.

Conclusions: The performance of multiplex qPCR is comparable to tissue cultures, having the advantage of being automated and providing results within 5 h. Improvement of the primer setup, especially for additional species may increase the performance of the PCR. The results in this study demonstrate that TaqMan-probe based multiplex qPCR assays have the advantages of accurate quantification, rapidity, simplicity and reproducibility, promising for a wide range of applications in the initial detection of pathogenic bacteria.