利用即時聚合酶連鎖反應定量鮑氏不動桿菌血中菌量並監測療效

Abstract

鮑氏不動桿菌Acinetobacter baumannii(A. baumannii)所造成菌血症是一個嚴重且極重要的院內感染症，死亡率高達25-50%。對於多重抗藥性鮑氏不動桿菌，現有的抗生素都無法有效的改變預後。定性的聚合酶連鎖反應 (PCR)已被廣泛用於臨床檢體中各種致病原之偵測，不僅包括多種病毒，也包括鮑氏不動桿菌在內的數種細菌，但利用即時定量PCR定量並追蹤血中鮑氏不動桿菌尚無完整研究。 本研究的第一個目標是建立即時定量PCR的方法來定量全血中鮑氏不動桿菌DNA，本研究的第二個目標是探討鮑氏不動桿菌菌血症病人血中細菌DNA量之系列變化和臨床病程及預後的關係。 在本研究的第一個目標下，我們首先將具特異性的鮑氏不動桿菌Oxa-51 基因之增幅片段以TA 克隆植入pCRII TOPO載體，以spectrophotometry定量後以十倍系列稀釋以即時PCR建立標準曲線做陽性對照組，以健康成人全血抽取之DNA作為陰性對照組。將已知菌量的鮑氏不動桿菌(9x100-9x106colony forming units(CFU)/mL) 加入健康成人全血及生理食鹽水中，測量Oxa-51 DNA數目，分析此方法的敏感性。收集常見的非鮑氏不動桿菌院內感染菌株，測量Oxa-51 DNA數目，分析此方法的特異性。結果發現以已知量的鮑氏不動桿菌混合健康成人全血或生理食鹽水再抽取DNA以即時PCR定量，鮑氏不動桿菌之CFU菌數與Oxa-51 DNA數目成正相關，相關係數0.99。收集了85株的常見的非鮑氏不動桿菌院內感染菌株，皆測量不到Oxa-51 DNA，沒有偽陽性的情形。 在本研究的第二個目標下，我們收集台大醫院2008年4月至2009年2月間成人加護病房中鮑氏不動桿菌菌血症病人加入本研究，自菌血症起，前七天每天收集全血1mL，之後每週抽血兩次各1mL，以即時PCR測量Oxa-51 DNA數目分析和臨床的相關性。結果發現納入分析的五十一位病人中，有39位在這次住院中死亡，死亡率為76.47%。分析存活和死亡這兩組病人，在單變項分析中發現死亡與病人發生菌血症時的Pittsburgh菌血症指數(p=0.006)、SOFA指數(p=0.019)、血小板數(p=0.004)、是否為多重抗藥性(Extensively resistant)鮑氏不動桿菌感染(p=0.007)及經驗性療法期的最高菌量(p=0.001)有顯著相關。而多變數logistic回歸發現經驗性療法期的最高菌量(Odds ratio: 2.462, 95% CI: 1.236-4.905)及Pittsburgh菌血症指數(Odds ratio: 1.612, 95% CI: 1.084-2.399)為預測死亡的獨立預測因子。 進一步用多變數Cox存活分析，顯示經驗性療法期的最高菌量(Hazard ratio: 1.582, 95% CI: 1.144-2.188)及Pittsburgh菌血症指數(Hazard ratio: 1.224, 95% CI: 1.065-1.407)為死亡的獨立預測因子。而多變數線性回歸則發現Day0的菌量及病人是否有Leukemia病史和經驗性療法期的最高菌量有顯著相關。雖然死亡和存活兩組病人在Day 0的菌量沒有顯著的差異(median, 2.37 vs. 1.71 log copies/mL, p=0.0967, two-tailed Mann-Whitney U test)，但開始治療後死亡組的病人在Day 1、 Day 2有顯著較高的菌量(median, 2.73 vs. 1.49, 2.68 vs. 0 log copies/mL; p=0.0236, p=0.0031; two-tailed Mann-Whitney U test)。而根據generalized estimation equation分析血中的菌量及在經驗性治療後改變的程度，可以發現雖然起始的菌量沒有統計學上的差別(p=0.053)，但是死亡組的治療後菌量明顯較高(p=0.049)。 總結，我們在第一個目標中已建立了一個即時PCR定量法來定量血中鮑氏不動桿菌Oxa-51 DNA的數目。在第二個目標中我們利用此方法於51個來自台大醫院的血液培養證實為鮑氏不動桿菌菌血症的病人來測定血中鮑氏不動桿菌菌量及其系列變化。我們發現經驗性療法期的最高菌量及Pittsburgh菌血症指數為兩個獨立的死亡預測因子。此外，死亡組的病人在開始治療後的Day 1 及Day 2其菌量高於存活組。我們的研究顯示此Oxa-51 DNA即時定量法可能是一個有用的且有潛力的方法來定量鮑氏不動桿菌菌血症。未來實際用於臨床來監測病程或推測預後尚待更多病人數目的研究來驗證此研究的結果。

Acinetobacter baumannii (A. baumannii) bacteremia is a serious and increasingly important nosocomial infection with a high mortality rate ranging from 25% to 50%. None of the antibiotics regimens has been shown to improve the outcome of infection caused by multi-drug resistant A. baumannii. Polymerase chain reaction (PCR) has been widely employed in clinical samples to detect various pathogens covering not only many viruses but also several bacteria including A. baumannii. However, using quantitative real-time PCR to quantify A. baumannii in blood has never been explored. The long-term goal of this study is to apply a simple and convenient assay to quantify A. baumannii bacteremia in clinical practice for monitoring disease course and predicting outcome. The objective is to establish a quantitative method to measure the amounts of A. baumannii in blood and to test its potential usefulness in patients with A. baumannii bacteremia. In the first specific aim of this study, we established a quantitative real-time PCR assay to quantify the Oxa-51 DNA copy number of A. baumannii. In the second specific aim, we examined the amounts and sequential changes of A. baumannii Oxa-51 DNA in whole blood from patients with blood culture-proven A. baumannii bacteremia and investigated the relationship to clinical courses and outcomes. In the first aim, a 431-bp fragment of the A. baumannii specific Oxa-51 gene was amplified and cloned into the pCRII TOPO vector, and increasing copy numbers of the plasmid were subjected to real - time PCR to establish the standard curves. Whole blood samples from healthy adult volunteers were collected in EDTA tubes and spiked with increasing colony forming units (CFU) of A. baumannii (9x100-9x106 CFU/mL). Equal amounts of bacteria in normal saline served as positive controls and whole blood samples from healthy adult volunteers served as negative controls. DNA extraction and real-time PCR were performed using Taqman primers and probe (Oxa-51). This DNA extraction and real-time PCR assay demonstrated a good correlation between the amounts of A. baumannii DNA derived from bacteria spiked in whole blood and the CFU determined by quantitative culture, with a Pearson correlation coefficient approximating 0.99. Real-time PCR for Oxa-51 DNA were also tested for 85 common nosocomial pathogens other than A. baumannii, which were isolated in the National Taiwan University Hospital (NTUH), and all showed negative. In the second aim, we carried out a prospective observational study to include adult patients admitted to the intensive care units of NTUH between April 2008 and February 2009 with positive blood culture of A. baumannii. Quantifications of Oxa-51 DNA were performed during the clinical course. Fifty one patients were analyzed in this study. The overall in-hospital mortality rate was 76.47% (39/51). Univariate analysis showed that Pittsburgh bacteremia score (p=0.006), SOFA score (p=0.019), platelet counts (0.004), extensively resistant A.baumannii infection (p=0.007), and the maximum Oxa-51 DNA copy number during empiric treatment (p=0.001) were significant factors associated with mortality. After multivariate logistic regression analysis, the maximum Oxa-51 DNA copy number during empiric treatment (Odds ratio: 2.462, 95% CI: 1.236-4.905), and Pittsburgh bacteremia score (Odds ratio: 1.612, 95% CI: 1.084-2.399) were found to be independent risk factors for mortality. After the analysis by a multivariate Cox’s proportional hazards, the independent significant risk factors for in-hospital mortality included the maximum Oxa-51 DNA copy number during empiric treatment (Hazard ratio: 1.582, 95% CI: 1.144-2.188) and Pittsburgh bacteremia score (Hazard ratio: 1.224, 95% CI: 1.065-1.407). Although the amounts of Oxa-51 DNA of the survivors and non-survivors were not significantly different on Day 0 (median, 2.37 vs. 1.71 log copies/mL, p=0.0967, two-tailed Mann-Whitney U test), the non-survivors had significant higher Oxa-51 DNA copy number than the survivors after empiric treatment on Day 1 and Day 2 (median, 2.73 vs. 1.49, 2.68 vs. 0 log copies/mL; p=0.0236, p=0.0031; two-tailed Mann-Whitney U test). Moreover, analysis of the Oxa-51 DNA copy number between the survivors and non-survivors and their sequential change by generalized estimation equation showed that there is no significant difference in the initial DNA copy number (p=0.0533), but that the non-survivors had a significant increase in the Oxa-51 DNA copy number (p=0.049). In summary, we have established a quantitative real-time PCR assay to measure the amounts of A. baumannii Oxa-51 DNA in whole blood in the first specific aim. We employed this method to study the amounts and sequential changes of Oxa-51 DNA in whole blood from 51 patients with blood culture-proven A. baumannii bacteremia at NUTH in the second specific aim. We found that the maximum Oxa-51 DNA copy number during empiric treatment and Pittsburgh bacteremia score were two independent significant risk factors for in-hospital mortality. Moreover, the non-survivors had significant higher Oxa-51 DNA copy number than the survivors on Day 1 and Day 2 after empiric treatment. Taken together, our study suggests that the quantitative real-time PCR assay for Oxa-51 DNA is a potentially useful method for quantification of A. baumannii bacteremia. The future application of this method in clinical practice to monitor disease course and predict outcome requires further study with a larger sample size to verify the observations in this study.