評估兩套質譜分析儀於草綠色鏈球菌的鑑定效果

Abstract

草綠色鏈球菌 (viridans group streptococci) 是人體的重要正常菌群之一，分佈於上呼吸道、胃腸道和女性生殖道，以口腔分佈為最多。但其也可導致亞急性心內膜炎、腦膜炎、敗血症與其他深部組織，亦為嚴重感染的重要致病菌。草綠色鏈球菌是一群構成相當不同的細菌，可依照細菌菌落生長特徵、在血液培養基上溶血型式、細胞壁抗原的組成、生化反應和最近發展的分子生物學方法等，依據核醣體核醣核酸 (16S rRNA基因) 序列將其分為五群。分別為：Streptococcus bovis group、Streptococcus anginosus group、Streptococcus mitis group、Streptococcus mutans group、Streptococcus salivarius group。目前臨床實驗室對於草綠色鏈球菌的鑑定方法多以快速鑑定或自動化鑑定系統，例如：API 20S、API Rapid ID 32 Strep、Vitek GPI card、MicroScan GP、Phoenix SMIC等。臺大醫院細菌檢驗組於臨床草綠色鏈球菌之鑑定方針為若為無菌部位或血液培養陽性之檢體須提供臨床正確之菌名，鑑定流程為使用BD Phoenix 100 SMIC panel，提供臨床草綠色鏈球菌之菌名。然而此方法之失敗率偏高，常常發生無法鑑定出菌名之狀況，需重複上機或使用API Rapid ID 32 Strep進行臨床菌株鑑定。鑑定草綠色鏈球菌確切菌種的正確性與時效性是臨床實驗室目前所遭遇到的一大難題。近年來質譜鑑定大量運用於臨床實驗室之細菌鑑定，其具有大幅縮短檢驗時間、降低實驗成本，提高菌名正確率之特色。本研究為評估自動化細菌鑑定儀與質譜儀於草綠色鏈球菌鑑定之表現。先使用27株已知草綠色鏈球菌參考菌株，以不同的MALDI-TOF MS之方式進行菌名鑑定。確認質譜鑑定儀的菌種庫資料。之後收集台大醫院2015年1月到6月之血液培養陽性之草綠色鏈球菌臨床分離菌株，共110株。分別使用自動化儀器 (BD Phoenix 100和VITEK 2)與MALDI-TOF MS(MALDI Biotyper和VITEK MS) 鑑定菌種。在S. anginosus group 31株菌株中，有3株於Phoenix鑑定錯誤，5株於VITEK 2鑑定錯誤，但於質譜鑑定全部皆為正確。在S. salivarius group 7株菌株中，有1株於VITEK 2鑑定錯誤，有1株需要再進一步鑑定。在S. bovis group 22株菌株中，有2株於Phoenix無法鑑定，有2株需要進一步鑑定。在S. mitis group 46 株菌株中，於Phoenix中4株無法鑑定和6株鑑定錯誤；於VITEK 2中有2株無法鑑定。在MALDI Biotyper質譜鑑定中有24株鑑定錯誤；在VITEK MS中有2株無法鑑定。最後利用multiplex PCR偵測gyrB gene以區分S. mitis與S. oralis。使用質譜儀鑑定草綠色鏈球菌，可大幅縮短檢驗時間；但須解決MALDI Biotyper於S.mitis group中面臨錯誤率偏高之情況。

Viridans group streptococci (VGS) are important flora in human. Most species reside as normal flora in the human respiratory, urogenital, and gastrointestinal tracts. Despite the low virulence, several studies have shown that many species in this group are associated with infections like endocarditis, meningitis, abscesses, and septicemia, especially in patients with different underlying conditions can cause serious infection. The nomenclature and classification have been under constant renewal, mainly due to new molecular typing methods. At present, the species of VGS can be divided into following five major groups according to their 16S rRNA sequences, Streptococcus bovis group, Streptococcus anginosus group, Streptococcus mitis group, Streptococcus mutans group and Streptococcus salivarius group. The API tests (bioMerieux, API 20S, API Rapid ID 32 Strep) or the automated commercial kits (Vitek GPI card, MicroScan GP and Phoenix SMIC) are usually used in the clinical microbiology laboratories to identify VGS. In National Taiwan University Hospital, the Phoenix 100 panel (BD) is used as an identification method for VGS when the blood culture was positive or the sterile site was positive. Recently, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) fingerprinting has been introduced as a routine microbial identification tool and found widespread acceptance thanks to its broad species coverage and superior turn around time. In this study, the commercial biochemical panels and MALDI-TOF MS systems were evaluated for identification of VGS. The 27 ATCC strains were selected to check MALDI-TOF MS database and 110 clinical blood culture positive isolates from Jan 2015 to Jun 2015 in the NTUH were collected to evaluate the two identification systems. Among the 31 S. anginosus group isolates, 3 isolates were misidentified in Phoenix system and 5 isolates were misidentified in VITEK 2 system but all the isolates were identified correctly in MALDI-TOF MS system. In the 7 S. salivarius group isolates, 1 isolate was misidentified in VITEK 2 system and another isolate needed more tests to confirm. In the 22 S. bovis group isolates, 2 isolates were misidentified in Phoenix system and 2 isolates needed more tests to confirm. Among the 46 S. mitis group isolates, 4 isolates had no result and 6 isolates were misidentified in Phoenix system and 2 isolates had no result in VITEK 2 system; 24 isolates were misidentified in MALDI Biotyper and 2 isolates had no result in VITEK MS system. These results suggested that the MALDI-TOF systems at present can offer a good alternative for the identification of VGS and perform as well as or better than the commercial biochemical methods. However, there are still some challenges in the identification of Streptococcus oralis and Streptococcus mitis.