Development and Clinical Application of a PCR-UV Assay for Detection of Carbapenem Resistant Acinetobacter baumannii in Bloodstream infections

Objective: Bloodstream infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) are a significant public health concern, with high morbidity and mortality rates. The detection and identification of CRAB is essential for early diagnosis and treatment. Hence, a rapid and economical CRAB-associated Bloodstream infections(BSIs) detection method is of urgent need. Methods: A tripling PCR-UV reaction system was designed for detecting the antibiotic resistance gene OXA23, OXA51 and AB-specific gene. The specificity of the primers , limit of detection (LOD), reproducibility, and accuracy of the assay were evaluated. The PCR products were analyzed using UV and ImageJ analysis directly, which provided a quickly interpretation of the results. Furthermore, the established assay was validated on clinical isolates and compared with blood culture and drug sensitivity tests. Results: The tripling PCR-UV method established in this study demonstrated strong primer specificity, distinguishing CRAB among 23 common clinical pathogens. The results of this PCR method have been validated by electrophoresis for good accuracy and reproducibility, with a Limit of Detection (LOD) of 3.0 x 10 ^-1 ng/uL. Meanwhile, the optimal annealing temperature for the triple method was 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 batch. 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 sensitivity tests. Conclusion: The tripling 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 grassroots units where expensive molecular instruments are not readily available and could help in the diagnosis and treatment of CRAB infections in BSIs.