評估以delta hemolysis assay 及Etest GRD方式偵測異質性萬古黴素抗藥性金黃色葡萄球菌

Detection of Heterogeneous Vancomycin-intermediate Staphylococcus aureus Isolates: Comparison of Delta-hemolysis Assay and Etest GRD Method

目前針對異質性萬古黴素抗藥性金黃色葡萄球菌(heterogeneous vancomycin- intermediate S. aureus; hVISA)並沒有準確且快速的篩選方法，而確認hVISA的PAP/AUC (population analysis profile/area under the curve)試驗，不但步驟繁複且耗時，並不適合臨床實驗室操作。為了找出一個適合微生物實驗室可常規執行的hVISA篩選方法，我們比較Etest glycopeptide resistant detection (GRD)及delta-hemolysis assay用於篩選hVISA。GRD Etest是使用雙頭的GRD Etest strips，將菌液接種於含5%羊血的Mueller-Hinton agar，並於24及48小時觀察結果的方式測定，delta-hemolysis assay是將待測菌株與S. aureus (RN4220)以相互垂直方式，接種在血液培養基上，於24小時觀察兩菌株交叉處是否產生δ-hemolysis現象來決定結果。在本研究中收集臨床萬古黴素的最小抑菌濃度≤2 μg/ ml的對2,6-二甲氧基苯青黴素抗藥性金黃色葡萄球菌(methicillin-resistant S. aureus; MRSA)菌株138株，同時以GRD Etest及delta -hemolysis assay二種方式篩選hVISA，並以PAP/AUC方式確認篩選結果。以PAP/AUC、delta-hemolysis assay及GRD Etest可分別偵測出36，41及47株hVISA，相較於GRD Etest方式，delta-hemolysis assay結果顯示出較佳的敏感性、特異性、陽性及陰性預測率，分別為88.9%(32/36)，91.2%(93/102)，78%(32/41)及95.9%(93/97)，而以GRD Etest方式的篩選結果則為41.7% (15/36) ， 94.1% (96/102) ， 71.4%(15/21) ， 82%(96/117) 於24 小時判讀；80.6%(29/36)，82.3%(84/102)，61.7%(29/47)及92.3%(84/91)於48小時判讀。在耗材人力成本及結果判讀時間考量上，delta-hemolysis assay分析每株菌株成本為0.6美元，結果判讀時間為24小時，而以GRD Etest方式篩選hVISA，每株菌株成本為5.3美元，結果判讀時間為48小時。上述結果顯示出delta-hemolysis assay是一種快速又經濟的hVISA篩選方法，並且適合常規地執行於臨床微生物實驗室。

The best screening method for detecting heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) remains unclear. Population analysis profile/area under the curve (PAP/AUC) is the gold standard method for detection of hVISA, but is time-consuming and labor-intensive. In order to find a suitable hVISA screening method for clinical microbiology laboratory, we compared Etest glycopeptide resistant detection (GRD) with delta-hemolysis assay for detecting hVISA. The GRD Etest was conducted by using vancomycin-teicoplanin double-sided gradient test strips on Mueller- Hinton agar with 5% sheep blood. The delta-hemolysis assay was determined by cross-streaking perpendicularly to S. aureus RN4220. Delta-hemolysis produced by a test strain resulted in a zone of synergistic hemolysis in an area where the hemolysis zone overlapped the alpha-hemolysis zone of RN4220. A total of 138 methicillin-resistant S. aureus isolates with vancomycin MICs ≤ 2 μg/ ml were screened for hVISA by GRD Etest method and delta-hemolysis assay using PAP/AUC as the reference method. There were 36, 41, and 47 hVISA isolates detected by PAP/AUC, delta-hemolysis assay and GRD Etest method (48 hours data), respectively. Delta-hemolysis assay displayed a superior sensitivity, specificity, positive predictive value, and negative predictive value of 88.9% (32/36), 91.2% (93/102), 78% (32/41), and 95.9% (93/97), GRD Etest gave corresponding values of 41.7% (15/36)，94.1% (96/102)，71.4%(15/21)，82%(96/117) at 24 hours read data, 80.6% (29/36), 82.3% (84/102), 61.7% (29/47), and 92.3% (84/91)at 48hours read data, respectively. The cost and detecting time of delta-hemolysis assay were 0.6 USD per isolate and 24 hours; GRD Etest were 5.3 USD/ isolate and 48 hours. These findings suggest that delta-hemolysis assay was a fast and cost-effective method for hVISA detection in clinical microbiology laboratory.