반코마이신 내성 장구균 검출을 위한 다중 실시간 중합효소연쇄반응 검사시약 평가
Evaluation of a Newly Developed Multiplex Real-time PCR Assay for the Detection of Vancomycin-Resistant Enterococci from Rectal Swabs
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배경: 반코마이신 내성 장구균(vancomycin-resistant enterococci, VRE) 감염 환자는 장내 집락화가 선행된 후 감염 증상없이 다른 환자로 VRE를 전파하는 보균자의 역할을 한다. VRE의 전파 방지를 위하여 장내 집락화된 환자의 신속한 검출이 중요하지만 배양에 기초한 VRE 검색 방법은 시간이 오래 걸리는 문제점이 있다. 이번 연구는 VRE 검출을 위해 최근 개발된 다중 실시간 중합효소연쇄반응(multiplex real-time PCR)법의 진단 수행능을 평가하고자 하였다. 방법: VRE 감시배양을 위하여 의뢰된 직장도말 105검체를 대상으로, 반코마이신 6 μg/mL이 첨가된 enterococcosel broth(EB)에 24시간 배양한 후 AnyplexTM VanR Real-time Detection (VanR) (Seegene, Inc., Seoul, Korea) 검사시약으로 다중 실시간 중합효소연쇄반응을 시행하였고 기존 배양검사 결과와 비교하였다. 또한 VanR 검사시약의 VRE 내성 유전자 검출 특이도와 검출 한계를 조사하였다. 결과: 다중 실시간 중합효소연쇄반응을 시행한 105검체 중 96 (91.4%)건에서 VRE 양성이었으며, 배양검사에서는 85 (80.9%)건에서 VRE가 배양되었다. 두 검사방법 간에 11 (10.4%)건의 결과가 일치하지 않았으며, 모두 다중 실시간 중합효소연쇄반응 양성, 배양 음성이었다. 반코마이신 감수성 장구균을 포함한 그람 음성과 양성 표준균주를 대상으로 시행한 다중 실시간 중합효소연쇄반응결과는 모두 VRE 음성이었으며, 10배씩 단계 희석한 반코마이신 내성 vanA Enterococcus faecium 균주의 DNA는 0.035 pg/reaction (3 μL), vanB Enterococcus faecalis 균주의 DNA는 0.35 pg의 농도까지 증폭반응이 관찰되었다. 결론: 직장도말 검체를 EB에 배양한 후 시행한 다중 실시간 중합효소연쇄반응 검사법은 26-28시간에 VRE 내성 유전자를 신속하고 민감하게 검출할 수 있었다. 이 방법은 시기적절한 격리시행에 도움을 주어 VRE 내성 유전자의 전파를 방지할 수 있을 것으로 기대된다.
Abstract
Background: Asymptomatic vancomycin-resistant enterococci (VRE) colonization precedes infection. VREcolonized patients serve as silent reservoirs of enterococci that go on to colonize other patients. Rapidly identifying colonized patients is crucial to prevent the spread of VRE. The culture-based method of VRE screening is time-consuming. We evaluated the diagnostic performance of a recently developed multiplex real-time PCR for the detection of VRE. Methods: We obtained 105 rectal swabs from patients who were being monitored for carriage of VRE. After 24 hour incubation of swabs in enterococcosel broth (EB) supplemented with 6 μg/mL vancomycin, multiplex real-time PCR was performed using the AnyplexTM VanR Real-time Detection (VanR) kit (Seegene,Inc., Seoul, Korea). The results of multiplex real-time PCR were compared to those of culture. We evaluated the specificity and detection limits of multiplex real-time PCR using VanR for VRE. Results: A total of 96/105 (91.4%) samples were VRE positive according to multiplex real-time PCR with EB while 85/105 (80.9%) samples were positive in culture. Eleven discordant results (10.4%) (multiplex real-time PCR positive, culture negative) were noted. All non-enterococcal bacteria and vancomycin-susceptible enterococci were negative. The DNA detection limits of VanR were 0.035 pg per reaction (3 μL) for Enterococcus faecium and 0.35 pg for Enterococcus faecalis. Conclusion: The application of multiplex real-time PCR after EB incubation allows rapid and sensitive detection in 26-28 hours for VRE screening from rectal swabs. This method could facilitate the timely implementation of contact isolation to prevent the spread of VRE.
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