曹燕 劉易林 李莉 李曉勇 孫燁 鐘利民 謝恒 劉科成
【摘要】目的探究高通量測序技術(shù)(HTS)對(duì)重癥膿毒癥患者的病原學(xué)診斷價(jià)值。方法選取122例重癥膿毒癥患者,隨機(jī)分為研究組(62例)和對(duì)照組(60例)。研究組患者采用血培養(yǎng)(BC)聯(lián)合HTS檢測,對(duì)照組患者采用BC檢測。兩組患者均接受經(jīng)驗(yàn)性抗感染治療。統(tǒng)計(jì)病原學(xué)診斷結(jié)果及類型,并比較兩組病原學(xué)診斷陽性率、病毒診斷陽性率;比較兩組接受經(jīng)驗(yàn)性抗感染治療后診斷陽性率、重癥監(jiān)護(hù)病房(ICU)住院時(shí)間、28d生存率、并發(fā)癥發(fā)生情況。結(jié)果研究組62例患者中58例患者病原學(xué)診斷結(jié)果陽性,對(duì)照組60例患者中24例患者病原學(xué)診斷結(jié)果陽性。研究組患者病原學(xué)診斷陽性率93.55%(58/62)高于對(duì)照組的40.00%(24/60),病毒診斷陽性率20.97%(13/62)高于對(duì)照組的0(0/60),差異均有統(tǒng)計(jì)學(xué)意義(X2=39.675、14.081,P<0.05)。接受經(jīng)驗(yàn)性抗感染治療后,研究組診斷陽性率為32.26%,高于對(duì)照組的15.OO%,差異有統(tǒng)計(jì)學(xué)意義(x2=5.012,P<0.05);兩組并發(fā)癥發(fā)生率比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。研究組ICU住院時(shí)間(10.04±2.06)d短于對(duì)照組的(18.25±2.87)d,28(1生存率85.48%高于對(duì)照組的56.67%,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論HTS檢測范圍廣,能夠提高對(duì)重癥膿毒癥患者病原微生物的診斷陽性率,為重癥膿毒癥患者抗感染方案的選擇提供方向,以避免盲目使用抗生素,縮短ICU住院時(shí)間,提高生存率。
【關(guān)鍵詞】膿毒癥;重癥;高通量測序技術(shù);病原學(xué);診斷價(jià)值
DOl: 10.14163/j.cnki.ll-5547/r.2020.19.002
Value analysis of high-throughput sequencing technology in pathogenic diagnosis of patients withsevere sepsis CAO Yan.LLU Yi-Lin,et at Department of Critical Care Medicine,Yuebei Peoples Ho.spital,Shaoguan 512026,China
【Abstract】Objective To investigate the value of high-throughput sequencing (HTS)technology inpathogenic diagnosis of patients with severe sepsis. Methods A total of 122 patients with severe sepsis wererandomlv divided into research group 62 (cases) and control group (60 cases). Patients in the research groupreceived blood culture (BC) combined HTS test, and patients in the control group received BC test. Both groupsof patients received empirical anti-infective treatment. The results and types of pathogen diagnosis was collectedand the positive rate of pathogen diagnosis and virus diagnosis were compared between the two groups. Thediagnostic positive rate after empirical anti-infective treatment, hospitalization time in intensive care unit (ICU).28 d survival rate and occurrence of complications were compared between the two groups. Results There were58 patients with positive pathogen diagnosis in 62 patients of the research group, and 24 patients with positivepathogen diagnosis in 60 patients of the control group. The positive rate of pathogen diagnosis 93.55%(58/62) ofthe research group was higher than than of the control group 40.OO%(24/60), and positive rate of virus diagnosis20.97%(13/62) was higher than that of the control group 0(0/60), and the difference was statistically significant(X2=39.675, 14.081. P<0.05). After empirical anti-infective treatment, the diagnosis-positive rate of the researchgroup was 32.26%. which was higher than that of the control group 15.00%. and the difference was statisticallysignificant (X2=5.012, P<0.05). There was no statistically significant difference in incidence of complicationsbetween the two groups (P>0.05). The hospitalization time in ICU (10.04 ± 2.06) d of the research group wasshorter than that of the control group (18.25 ± 2.87) d. and 28 d survival rate 85.48% was higher than that of thecontrol group 56.67%, and the difference was statistically significant (P<0.05). Conclusion HTS has a widerange of detection. and can improve the diagnosis-positive rate of pathogenic micrrganisms in patients withsevere sepsis, and provide direction for the selection of anti-infective programs for severe sepsis to avoid blind useof antibiotics. shorten the hospitalization time in ICU, and improve survival rate.
【Key words】 Sepsis; Severe; High-throughput sequencing technology; Pathogenic; Diagnlstic value
膿毒癥是由感染引起的全身炎癥反應(yīng)綜合征,可導(dǎo)致嚴(yán)重膿毒癥和膿毒性休克,是ICU患者死亡的主要原因。據(jù)文獻(xiàn)報(bào)道,全球膿毒癥發(fā)病率約0.3%,病死率可達(dá)25%,并發(fā)膿毒性休克患者病死率高達(dá)80%[1]。高通量測序技術(shù)(high-throughput sequencing,HTS),即二代測序技術(shù),近年來應(yīng)用廣泛,被證實(shí)可用于腫瘤的診治、感染性疾病診斷、遺傳疾病篩查[2]。國外研究顯示,HTS常用于病毒感染診斷,相比血培養(yǎng)(bacterialculture,BC)具有明顯優(yōu)勢[3]。但目前國內(nèi)外HTS應(yīng)用于膿毒癥病原學(xué)診斷的前瞻性研究報(bào)道罕見。鑒于本院重癥膿毒癥約占膿毒癥患者的2/3,本研究探析HTS對(duì)重癥膿毒癥患者病原學(xué)的診斷價(jià)值,以期為臨床應(yīng)用提供新的方向和依據(jù)?,F(xiàn)報(bào)告如下。
1資料與方法
1.1 一般資料選取2016年7月-2019年10月本院收治的122例重癥膿毒癥患者為研究對(duì)象,隨機(jī)分為研究組(62例)和對(duì)照組(60例)。研究組:男37例,女25例;年齡22-79歲,平均年齡(59.86±9.27)歲;急性生理學(xué)及慢性健康狀況評(píng)分系統(tǒng)Ⅱ(acute physiologyand chronic health evaluationⅡ.APACHEⅡ)評(píng)分[4]11-17分,平均APACHEⅡ評(píng)分(14.26±3.24)分;序貫器官衰竭評(píng)估(sequential organ failure assessment. SOFA)[5]4-7分,平均SOFA評(píng)分(5.78±1.41)分。對(duì)照組:男35例,女25例;年齡24-77歲,平均年齡(59.29±9.18)歲;APACHEⅡ評(píng)分10-17分,平均APACHEⅡ評(píng)分(14.15±3.19)分;SOFA評(píng)分3-7分,平均SOFA評(píng)分(5.59±1.37)分。兩組一般資料比較,差異無統(tǒng)計(jì)學(xué)意義(P>0.05),具有可比性。納入標(biāo)準(zhǔn):①重癥膿毒癥符合《中國嚴(yán)重膿毒癥/膿毒性休克治療指南》[6]中診斷標(biāo)準(zhǔn);②年齡>18歲;③患者家屬知情同意。排除標(biāo)準(zhǔn):①血液系統(tǒng)、自身免疫性疾病;②合并惡性腫瘤;③器官功能不全終末期;④入院24h內(nèi)死亡;⑤妊娠、哺乳期婦女;⑥長期服用抗生素或其他影響本研究結(jié)果的藥物;⑦嚴(yán)重創(chuàng)傷、甲狀旁腺功能異常。所有患者及其家屬知情同意本研究并簽署知情同意書。本研究所涉及的研究方法均遵循《赫爾辛基宣言》,符合醫(yī)學(xué)倫理學(xué)原則,并獲得粵北人民醫(yī)院醫(yī)學(xué)倫理委員會(huì)批準(zhǔn)。
1.2方法研究組患者采用BC聯(lián)合HTS檢測,對(duì)照組患者采用BC檢測。具體如下。
1.2.1 BC檢測采用Bact/ALERT 3D血培養(yǎng)儀及配套血培養(yǎng)瓶進(jìn)行BC;通過VITEK 2 Compact全白動(dòng)微生物分析系統(tǒng)(法國生物梅里埃公司)進(jìn)行病原微生物鑒定。
1.2.2 HTS檢測 ①構(gòu)建DNA文庫:對(duì)血樣離心
10 min/1600g,分離血漿,轉(zhuǎn)移血漿至無菌試管;通過微量樣本基因組DNA提取試劑盒(DP316,天根生化科技北京有限公司)提取血漿中DNA,用于DNA文庫構(gòu)建。②測序:先行PCR擴(kuò)增f美國ABI公司,7500FAST型實(shí)時(shí)熒光定量PCR儀),并使用IonTorrent基因組測序儀(Life TeChnologies公司)分析,結(jié)合Agilent 2100生物分析儀(Agilent Technologies公司)分析,評(píng)定DNA文庫質(zhì)量,而后進(jìn)行測序。采用IonTorrent Proton測序儀(Life TeChnologies公司)對(duì)合格DNA文庫展開測序,分析得出報(bào)告。③預(yù)處理及參考數(shù)據(jù)庫:去除低質(zhì)量序列、短序列(長度<35 hp),完成預(yù)處理;采用Burrows-Wheeler比對(duì)工具將測序數(shù)據(jù)與人類參考基因組(GRCh37)進(jìn)行比對(duì),并將數(shù)據(jù)與細(xì)菌、真菌、病毒數(shù)據(jù)庫進(jìn)行比對(duì),讀取計(jì)數(shù)及每種物種的基因組覆蓋率。所有病原體序列參考數(shù)據(jù)庫均來白NCBI。④測定病原體:根據(jù)以往對(duì)正常者血漿樣品的測序計(jì)算細(xì)菌、真菌、病毒的參考范圍,確定通過干凈序列讀數(shù)標(biāo)準(zhǔn)化的每種生物的檢測值,采用箱線圖計(jì)算正常樣本范圍,以Q3+3IQR最大、Q1-3IQR為最?。≦1、Q3分別為較低四分位數(shù)和較高四分位數(shù),IQR為四分位數(shù)間距),獲取細(xì)菌、真菌、病毒的讀碼數(shù)。根據(jù)以下確定患者是否存在病原生物:一是必須根據(jù)每種生物的覆蓋率對(duì)一個(gè)樣品中鑒定出的所有生物進(jìn)行分類,獲得了前十個(gè)清單則進(jìn)一步分析;二是在前十個(gè)生物中,嚴(yán)格映射的讀數(shù)必須為03,并且超過參考范圍的上限,覆蓋率也必須超過參考范圍的上限。⑤Sanger測序驗(yàn)證:對(duì)于HTS和BC陽性結(jié)果,采用特異性PCR驗(yàn)證。將提取的DNA作為PCR反應(yīng)模板,反應(yīng)條件為:94。C5 min(94。C 30s;55℃50s;72℃30s),40循環(huán),72℃5 min。瓊脂糖凝膠電泳檢測PCR產(chǎn)物并純化;以ABI 3730基因分析儀進(jìn)行Sanger測序,驗(yàn)證測序結(jié)果。
兩組患者均接受經(jīng)驗(yàn)性抗感染治療。
1.3觀察指標(biāo)統(tǒng)計(jì)病原學(xué)診斷結(jié)果及類型,并比較兩組病原學(xué)診斷陽性率、病毒診斷陽性率;比較兩組接受經(jīng)驗(yàn)性抗感染治療后診斷陽性率、ICU住院時(shí)間、28d生存率、并發(fā)癥發(fā)生情況。
1.4統(tǒng)計(jì)學(xué)方法采用SPSS22.0統(tǒng)計(jì)學(xué)軟件對(duì)研究數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,采用i檢驗(yàn);計(jì)數(shù)資料以率(%)表示,采用X2檢驗(yàn)。P<0.05表示差異具有統(tǒng)計(jì)學(xué)意義。
2結(jié)果
2.1病原學(xué)診斷結(jié)果及類型研究組62例患者中58例患者病原學(xué)診斷結(jié)果陽性,對(duì)照組60例患者中24例患者病原學(xué)診斷結(jié)果陽性。研究組患者病原學(xué)診斷陽性率93.55%(58/62)高于對(duì)照組的40.OO%(24/60),病毒診斷陽性率20.97%(13/62)高于對(duì)照組的0(0/60),差異均有統(tǒng)計(jì)學(xué)意義(X2=39.675、14.081,P<0.05)。見表1。
2.2兩組接受經(jīng)驗(yàn)性抗感染治療后的診斷陽性率及并發(fā)癥發(fā)生率比較接受經(jīng)驗(yàn)性抗感染治療后,研究組診斷陽性率為32.26%,高于對(duì)照組的15.OOo%,差異有統(tǒng)計(jì)學(xué)意義(X2=5.012, P<0.05);兩組并發(fā)癥發(fā)生率比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見表2。
2.3兩組ICU住院時(shí)間、28 d生存率比較研究組ICU住院時(shí)間短于對(duì)照組,28 d生存率高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見表3。
3討論
目前ICU感染的病原學(xué)診斷仍然依賴于BC和分子技術(shù),而BC被認(rèn)為是診斷血流感染的金標(biāo)準(zhǔn),但它陽性率較低,一般只能進(jìn)行細(xì)菌/真菌培養(yǎng),且并非所有的病原體都能被培養(yǎng)出,同時(shí)培養(yǎng)周期較長(3-7d),雖然反復(fù)培養(yǎng)可增加陽性概率,但同時(shí)將增加患者經(jīng)濟(jì)負(fù)擔(dān),且可能延誤最佳治療時(shí)機(jī),因此它具有一定局限性[7,8]。國外研究報(bào)道,延遲th的有效抗菌及支持治療可導(dǎo)致患者死亡率增加5%-10%[9]。而由于臨床診斷技術(shù)層面的局限性,30%-50%膿毒癥患者難以明確病原微生物,造成初治延遲或失敗,增加病死率[10]。因此,探尋準(zhǔn)確、快速的病原學(xué)診斷方法,以促進(jìn)臨床實(shí)施早期精準(zhǔn)的抗感染治療方案,提高重癥膿毒癥患者生存率,成為研究的重點(diǎn)。
自2016年7月開始,本研究對(duì)傳統(tǒng)的膿毒癥病原學(xué)診斷方法進(jìn)行了改良,在常規(guī)BC的基礎(chǔ)上聯(lián)合應(yīng)用HTS對(duì)重癥膿毒癥患者進(jìn)行病原體檢測,取得了良好效果。研究組病原體診斷陽性率為93.55%,明顯高于對(duì)照組(40.OO%),同時(shí)對(duì)于已經(jīng)接受了經(jīng)驗(yàn)性抗感染治療的患者,研究組診斷陽性率(32.260%)明顯高于對(duì)照組(15.OO%)。本研究進(jìn)一步發(fā)現(xiàn),研究組ICU住院時(shí)間明顯短于對(duì)照組,28d生存率明顯高于對(duì)照組,差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。另近年Gumaz等[11]研究發(fā)現(xiàn),與BC相比,HTS檢測膿毒癥的病原菌有較高的敏感性和特異性。HTS應(yīng)用于ICU重癥膿毒癥患者病原微生物檢測可提高診斷價(jià)值,這可能與HTS可篩選出標(biāo)本中核酸所含種類,可全面分析物種轉(zhuǎn)錄組及基因組,還能夠?qū)λ拗鱩iRNAs進(jìn)行檢測,也可獲得含量低的病原序列,并通過未知序列鑒定新物種,若預(yù)先定義每種病原微生物的特征序列譜,在測序結(jié)果中逐一比對(duì),可以迅速識(shí)別各類病原微生物,使診斷時(shí)間縮短,診斷結(jié)果更加精確[12-14]。目前HTS能檢測出約2700余種病原體,種類包括細(xì)菌、病毒、真菌、寄生蟲、支原體、衣原體、立克次氏體,48-96h得出報(bào)告,具有檢測速度快、準(zhǔn)確率高、成本低、覆蓋度廣及產(chǎn)出巨大等特點(diǎn)[15-16],特別是病毒、支原體、衣原體等少見病原體類型的病原學(xué)診斷,HTS較BC具有更加獨(dú)特和明顯的優(yōu)勢[ 17,18]。在重癥膿毒癥患者的治療中,BC聯(lián)合HTS可明顯縮短ICU住院時(shí)間,同時(shí)可明顯提高28d生存率,這可能與HTS能更快速和精準(zhǔn)地檢測出病原菌,促進(jìn)臨床早期選擇合理的治療方案,避免或減少抗生素的不合理應(yīng)用,從而提高重癥膿毒癥患者的存活率,改善患者的治療預(yù)后,同時(shí)也降低了患者的治療費(fèi)用。
綜上所述,HTS檢測范圍廣且時(shí)間短,能夠提高對(duì)重癥膿毒癥患者病原微生物的診斷陽性率,可為重癥膿毒癥患者抗感染方案的選擇提供方向,以避免盲目使用抗生素,縮短ICU住院時(shí)間,提高生存率,具有重要應(yīng)用價(jià)值。但本研究樣本量較少,且全為本院患者,可能影響其可靠性,今后還有待進(jìn)行大樣本、多中心研究檢驗(yàn)。
參考文獻(xiàn)
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[收稿日期 : 2020-04-16]
基金項(xiàng)目:韶關(guān)市科技汁劃項(xiàng)目(項(xiàng)目編號(hào):2018sn109);韶關(guān)市衛(wèi)生計(jì)生科研項(xiàng)目(項(xiàng)目編號(hào):Y18064)作者單位:512026粵北人民醫(yī)院重癥醫(yī)學(xué)科