Division of Healthcare Quality Promotion, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton RD NE, Atlanta, GA, 30333, USA.
Division of Healthcare Quality Promotion, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton RD NE, Atlanta, GA, 30333, USA.
Anaerobe. 2020 Feb;61:102077. doi: 10.1016/j.anaerobe.2019.102077. Epub 2019 Jul 19.
Clostridioides difficile is a common pathogen that is well known to survive for extended periods of time on environmental healthcare surfaces from fecal contamination. During epidemiological investigations of healthcare-associated infections, it is important to be able to detect whether or not there are viable spores of C. difficile on surfaces. Current methods to detect C. difficile can take up to 7 days for culture and in the case of detection by PCR, viability of the spores cannot be ascertained. Prevention of C. difficile infection in healthcare settings includes adequate cleaning and disinfection of environmental surfaces which increases the likelihood of detecting dead organisms from an environmental sample during an investigation. In this study, we were able to adapt a rapid-viability PCR (RV-PCR) method, first developed for detection of viable Bacillus anthracis spores, for the detection of viable C. difficile spores. RV-PCR uses the change in cycle threshold after incubation to confirm the presence of live organisms. Using this modified method we were able to detect viable C. difficile after 22 h of anaerobic incubation in Cycloserine Cefoxitin Fructose Broth (CCFB). This method also used bead beating combined with the Maxwell 16 Casework kit for DNA extraction and purification and a real-time duplex PCR assay for toxin B and cdd3 genes to confirm the identity of the C. difficile spores. Spiked environmental sponge-wipes with and without added organic load were tested to determine the limit of detection (LOD). The LOD from spiked environmental sponge-wipe samples was 10 spores/mL but after incubation initial spore levels of 10 spores/mL were detected. Use of this method would greatly decrease the amount of time required to detect viable C. difficile spores; incubation of samples is only required for germination (22 h or less) instead of colony formation, which can take up to 7 days. In addition, PCR can then quickly confirm or deny the identity of the organism at the same time it would confirm viability. The presence of viable C. difficile spores could be detected at very low levels within 28 h total compared to the 2 to 10-day process that would be needed for culture, identification and toxin detection.
艰难梭菌是一种常见的病原体，它可以从粪便污染中在环境卫生保健表面存活很长时间。在与卫生保健相关的感染的流行病学调查中，重要的是能够检测到表面是否存在艰难梭菌的活孢子。目前用于检测艰难梭菌的方法需要培养长达 7 天，而在通过 PCR 检测的情况下，无法确定孢子的活力。在卫生保健环境中预防艰难梭菌感染包括充分清洁和消毒环境卫生表面，这增加了在调查期间从环境样本中检测到死生物体的可能性。在这项研究中，我们能够改编一种快速活力 PCR（RV-PCR）方法，该方法最初是为检测活炭疽芽孢而开发的，用于检测活艰难梭菌孢子。RV-PCR 使用孵育后循环阈值的变化来确认活生物体的存在。使用这种改良方法，我们能够在环丝氨酸头孢西丁果糖肉汤（CCFB）中厌氧孵育 22 小时后检测到活的艰难梭菌。该方法还使用珠击与 Maxwell 16 案例工作包相结合进行 DNA 提取和纯化，以及实时双 PCR 检测毒素 B 和 cdd3 基因，以确认艰难梭菌孢子的身份。测试了添加和未添加有机负荷的污染环境海绵拭子，以确定检测限（LOD）。从污染环境海绵拭子样本中检测到的 LOD 为 10 个孢子/mL，但孵育后最初检测到 10 个孢子/mL 的初始孢子水平。使用这种方法将大大减少检测活艰难梭菌孢子所需的时间；样品仅需进行萌发（22 小时或更短）而无需进行菌落形成，这可能需要长达 7 天的时间。此外，PCR 可以同时快速确认或否认生物体的身份，同时确认其活力。与培养、鉴定和毒素检测所需的 2 至 10 天相比，在总共 28 小时内可以以非常低的水平检测到活的艰难梭菌孢子。
