Development and clinical application of a PCR-UV assay for detection of carbapenem resistant Acinetobacter baumannii in bloodstream infections.

Abstract

Objective: Bloodstream infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) are a significant public health problem, with high morbidity and mortality. Detection and identification of CRAB is essential for early diagnosis and treatment. Therefore, a rapid and economical method for the detection of CRAB-associated Bloodstream infections (BSIs) is urgently needed.

Methods: A triple PCR-UV reaction system has been developed for the detection of the antibiotic resistance genes OXA23, OXA51 and AB-specific gene. Primer specificity, limit of detection (LOD), reproducibility, and accuracy of the assay were evaluated. The PCR products were directly analyzed using UV and ImageJ analysis, which provided a quickly interpretation of the results. Furthermore, the established assay was validated on clinical isolates and compared with blood culture and drug susceptibility testing.

Results: The triple PCR-UV method established in this study demonstrated strong primer specificity and discriminated CRAB among 23 common clinical pathogens. The results of this PCR method were validated by electrophoresis and showed good accuracy and reproducibility, with a limit of detection (LOD) of 3.0 × 10^-1 ng/uL. Meanwhile, the optimal annealing temperature for the triple method has been optimized to 56.4 ℃. The result of PCR amplification could be judged by the result of the gray value of the tube to be tested / the gray value of the blank control of the same lot. The ratio > 1.3 is CRAB, the ratio between 1.1-1.3 is carbapenem-sensitive Acinetobacter baumannii (CSAB), the ratio < 1.1 is negative result. When applied to detect 30 patients with BSIs of AB, the results were consistent with clinical blood culture identification and drug susceptibility testing.

Conclusion: The triple PCR-UV assay developed in this study is a UV-visual, rapid, and cost-effective method for the detection of Acinetobacter baumannii (AB) and identification of CRAB in bloodstream infections. The assay could be particularly useful in community settings where expensive molecular instrumentation is not readily available and could help in the diagnosis and management of CRAB infections in BSIs.