Evaluation of Quadruple Real-Time PCR Method to Detect Enterococci Carrying Vancomycin-Resistant Genes vanA, vanB, vanM in Rectal Swabs

Abstract

Purpose: To evaluate the sensitivity and accuracy of the quadruple real-time PCR method for the detection of enterococci carrying vancomycin-resistant genes vanA, vanB, and vanM in rectal swabs. Methods: Choosing PCR-sequenced DNA extracted directly from rectal swabs as the gold standard, the results of the quadruple real-time PCR method and the traditional method (screening culture combined PCR-sequencing method whose DNA extracted from single colony) were compared with the gold standard. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the quadruple real-time PCR method and the traditional method were obtained. The time required for the three methods was calculated. Results: The results between gold standard and the quadruple real-time PCR method were similar. Compared to the traditional method, the quadruple real-time PCR method had a much higher sensitivity, specificity, PPV, NPV, and consistency. Our study found that the quadruple real-time PCR method is beneficial for detection of enterococci carrying vanM with vancomycin heteroresistance. The traditional method had high specificity and NPV, but its sensitivity and PPV were not ideal. The time needed for gold standard is a minimum of 28 h; the quadruple real-time PCR method takes 2-3 h while the traditional method consumes a minimum of 72 h. Conclusion: The quadruple real-time PCR method can provide a rapid and reliable result for the diagnosis of patients with colonized vancomycin-resistant enterococci. This new method is beneficial for the active screening, timely clinical treatment measures, epidemiological research, and hospital monitoring of the enterococci carrying vancomycin-resistant gene, especially for the enterococci with vancomycin heteroresistance carrying vanM.

Keywords: enterococci carrying vancomycin resistant gene; genotyping; quadruple real-time PCR method; rapid detection; rectal swabs; vancomycin heteroresistance enterococci carrying vanM.

Figure 1

The procedure of the three methods in this study.

Figure 2

Vancomycin heterogeneous resistant Enterococcus carrying vanM obtained from antibiotic free medium.

Figure 3

Electrophoresis results of DNA isolated from partial of rectal swabs amplified with vanB primers. Lanes 1 and 7 contain 200 bp Marker. Lanes 2, 3, and 4 represent positive control, negative control, and blank control of vanB, respectively. Lanes 5 and 6 show electrophoretic results of DNA of Enterococcus isolated from different rectal swabs amplified by vanB primers. Electrophoresis results showed that these strains do not carry vanB.