- 期刊
抗酸性染色價數、含菌量以及肺結核菌Real-Time PCR檢出率之相關性
The Relevance between Acid-Fast Stain Titer, Bacteria Quantity and ＂Mycobacterium tuberculosis＂ Real-Time PCR Positive Rate
摘要
結核分枝桿菌（Mycobacterium tuberculosis）的臨床診斷上，除了首先利用抗酸菌染色進行快速篩選之外，分枝桿菌的培養結果仍是黃金標準。目前，分子生物技術快速偵測結核分枝桿菌已經是一種趨勢，旨在希望能快速與準確地診斷病原菌，以達到有效治療以及避免群聚傳染的危險性。本文主要以回朔方式探討，能培養出結核分枝桿菌的檢體，將其先前結合分枝桿菌real-time PCR（即時定量聚合□連鎖反應）的結果與同一套檢體的抗酸菌染色價數來做相關性比對，並統計real-time PCR的肺結核菌陽性檢出率。另外也根據抗酸菌染色的操作方法以及價數的判斷方法，計算出在不同價數中相對應的菌數含量，並以此與實驗室的real-time PCR偵測極限做比對。由結果可以發現，當檢體含有結核分枝桿菌時，即抗酸菌染色價數越高，檢體的菌數含量越高，相對的real-time PCR肺結核菌陽性檢出率也越高，兩者結果呈現正相關性。數據顯示當抗酸菌染色為positive3+以上的時候，肺結核菌real-time PCR的陽性檢出率為100%（檢體含菌量＞6,900 CFU/mL，每一個PCR反應的含菌數＞400CFU以上）。同樣地，抗酸菌染色價數低的檢體，real-time PCR的結果容易呈現偽陰性。數據顯示當抗酸菌染色價數為positive+/-時，real-time PCR陽性率為73.7%（檢體含菌量8~17 CFU/mL，每一個PCR反應的含菌數為0.5~1 CFU），抗酸菌染色價數為positive1+時，real-time PCR陽性率為78.3%（檢體含菌量69~625 CFU/mL，每一個PCR反應的含菌數為4~39 CFU），而抗酸菌染色為positive 2+時，陽性率為89.9%（檢體含菌量690~6,250 CFU/mL，每一個PCR反應的含菌數為40~390 CFU）。而我們計算出了抗酸菌染色不同的價數中的含菌量，發現到只要抗酸菌染色為陽性，其含菌量均落在實驗室的real-time PCR操作檢體檢測極限內，但是低價數仍然有偽陰性的情況。其可能是因為剩餘檢體量不足、檢體本身菌數太少、檢體中含有抑制物或是人為的誤差因素。綜合以上結果分析，雖然本實驗室之結核分枝桿菌real-time PCR的操作檢體檢測極限為10 CFU/mL，但是在此範圍內仍然無法百分之百偵測到檢體中的結核分枝桿菌，因此並不能完全取代傳統的抗酸菌染色以及培養。
並列摘要
Acid-fast stain (AFS) is the primary step to screen Mycobecterium tuberculosis (M. tuberculosis; TB) infection in clinics. The golden standard for TB diagnosis is bacterium culture. Recently, many molecular biology techniques have been developed for rapid detection and identification of TB complex to give early and effective treatments and avoid the risk of outbreak infection. This study tries to figure out the accuracy and detection limitation of real-time PCR to detect the AFS positive specimen when the culture has been identified TB. TB culture positive cases have been selected and compared the real-time PCR results and the AFS titer to get the real-time PCR positive rate and the limit of detection. We also calculated the quantity of bacteria in different AFS titers. The data showed that higher AFS titers corresponded to more bacteria and higher real-time PCR positive rate. When the AFS titer is more than positive 3+, the real-time PCR positive rate is 100%(there are more than 6,900 CFU/mL T B in one specimen and 400 CFU of TB in one PCR reaction). Samples which were real- time PCR falsenegative results also had low titer of AFS. When the AFS titer is positive +/-, the real-time PCR positive rate is 73.7% (there are 8~17 CFU/mL TB in one specimen and 0.5~1 CFU of TB in one PCR reaction). When the AFS titer is positive 1+, the real-time PCR positive rate is 78.3% (there are 69~625 CFU/mL TB in one specimen and 4~39 CFU of TB in one PCR reaction). When the AFS titer is positive 2+, the real-time PCR positive rate is 89.9% (there are 690~6,250 CFU/mL TB in one specimen and 40~390 CFU of TB in one PCR reaction).We also found that although the quantity of bacteria in different AFS titers are always more than the limitation of TB real-time PCR, there were still some realtime PCR false negative results. It might be due to the insufficient specimen volume, insufficient bacteria, interference substances in specimen and operator error. In conclusion, although the limitation of TB real-time PCR in our laboratory is 10 CFU / mL, we can not detect Mycobacterium tuberculosis complex in specimens for 100%. The molecular biology techniques can not completely substitute for the traditional acidfast stain and culture now.