不同腸道病毒感染手足口病患兒病毒載量及臨床特征對(duì)比分析

陳 蘇，杜潘艷，鄭 紅，王寶林，高翠紅，張 雙

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不同腸道病毒感染手足口病患兒病毒載量及臨床特征對(duì)比分析

陳 蘇，杜潘艷，鄭 紅，王寶林，高翠紅，張 雙

063000河北省唐山市第四醫(yī)院檢驗(yàn)科(陳蘇)；唐山市婦幼保健院檢驗(yàn)科(杜潘艷，鄭紅，王寶林，高翠紅，張雙)

【摘要】目的對(duì)比分析腸道病毒71型(EV71)與柯薩奇A組16型(CoxA16)感染手足口病(HFMD)患兒的病毒載量及臨床特征。方法收集2015年5—9月唐山市婦幼保健院兒科確診的HFMD患兒430例，根據(jù)臨床癥狀、體征和病毒種類(lèi)將患兒分為EV71輕度組162例、EV71重度組87例、CoxA16輕度組158例、CoxA16重度組23例。采用實(shí)時(shí)熒光定量反轉(zhuǎn)錄PCR技術(shù)檢測(cè)患兒咽拭子EV71、CoxA16 RNA，計(jì)算病毒載量；記錄患兒的病程、體溫、熱程、口腔潰瘍、流涎、咳嗽、手部出疹、足部出疹、嗜睡、驚厥、嘔吐、意識(shí)改變、肢體抖動(dòng)、肌痙攣情況。結(jié)果標(biāo)準(zhǔn)曲線顯示循環(huán)閾值(Ct值)與病毒載量的對(duì)數(shù)呈高度負(fù)相關(guān)(r=-1.000，P<0.01)。Ct值(X)與病毒載量的對(duì)數(shù)(Y)的關(guān)系為Y=-0.29X+13.03。CoxA16輕度組、CoxA16重度組HFMD患兒Ct值較EV71輕度組、EV71重度組降低(P<0.05)；EV71輕度組與EV71重度組、CoxA16輕度組與CoxA16重度組HFMD患兒Ct值比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。EV71重度組、CoxA16重度組HFMD患兒病程較EV71輕度組、CoxA16輕度組延長(zhǎng)(P<0.05)；EV71重度組HFMD患兒病程較CoxA16重度組延長(zhǎng)(P<0.05)。4組HFMD患兒體溫≥38.5 ℃、口腔潰瘍、流涎、手部出疹、足部出疹發(fā)生率比較，差異均無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。EV71重度組、CoxA16重度組HFMD患兒熱程≥3 d發(fā)生率較EV71輕度組、CoxA16輕度組升高(P<0.007)；EV71重度組、CoxA16重度組HFMD患兒體溫≥38.5 ℃+熱程≥3 d發(fā)生率較EV71輕度組、CoxA16輕度組升高，EV71輕度組HFMD患兒體溫≥38.5 ℃+熱程≥3 d發(fā)生率較CoxA16輕度組升高，EV71重度組HFMD患兒體溫≥38.5 ℃+熱程≥3 d發(fā)生率較CoxA16重度組升高(P<0.007)；CoxA16輕度組、CoxA16重度組HFMD患兒咳嗽、手部出疹數(shù)≥15個(gè)(雙手)、足部出疹數(shù)≥15個(gè)(雙足)發(fā)生率較EV71輕度組、EV71重度組升高(P<0.007)。EV71重度組、CoxA16重度組HFMD患兒嗜睡、嘔吐發(fā)生率較EV71輕度組升高，EV71重度組HFMD患兒嗜睡發(fā)生率較CoxA16重度組升高(P<0.01)。結(jié)論HFMD患兒病毒載量與感染病毒相關(guān)，但不同病情程度間病毒載量無(wú)差別。不同病毒感染及病情程度患兒間熱程≥3 d、體溫≥38.5 ℃+熱程≥3 d、咳嗽、手部出疹數(shù)≥15個(gè)(雙手)、足部出疹數(shù)≥15個(gè)(雙足)、嗜睡、驚厥、嘔吐、意識(shí)改變、肢體抖動(dòng)、肌陣攣發(fā)生率有差異。

【關(guān)鍵詞】手足口病；腸道病毒屬；柯薩奇病毒感染；病毒載量；體征和癥狀

陳蘇，杜潘艷，鄭紅，等.不同腸道病毒感染手足口病患兒病毒載量及臨床特征對(duì)比分析[J].中國(guó)全科醫(yī)學(xué)，2016，19(18)：2211-2215.[www.chinagp.net]

Chen S,Du PY,Zheng H,et al.Analysis of the viral load and clinical features of children with hand,foot and mouth disease by different enteroviruses[J].Chinese General Practice，2016，19(18)：2211-2215.

手足口病(hand，foot and mouse disease，HFMD)是一種易感染低齡兒童的急性傳染病，病原體以腸道病毒71型(enterovirus 71，EV71)和柯薩奇A組16型(coxsackievirus，CoxA16)最為常見(jiàn)，而EV71感染常會(huì)引起嚴(yán)重的神經(jīng)系統(tǒng)癥狀[1]。兩種病原體感染與病毒載量及引起的臨床特征是否有關(guān)，目前關(guān)于此方面的研究較少。因此，本研究分析EV71和CoxA16感染HFMD患兒病毒載量及臨床特征的差異，旨在為臨床診療和致病機(jī)制的研究提供依據(jù)。

1資料與方法

1.2方法

1.2.1標(biāo)本采集及處理規(guī)范采集患兒入院當(dāng)日咽拭子標(biāo)本，在裝有3～5 ml 0.9%氯化鈉溶液采樣管中充分?jǐn)噭?dòng)，得液態(tài)標(biāo)本，密封，用于EV71、CoxA16 RNA檢測(cè)。

1.2.2EV71、CoxA16 RNA檢測(cè)EV71、CoxA16 RNA檢測(cè)引物和試劑盒均采用上海科華生物工程股份有限公司產(chǎn)品。嚴(yán)格按說(shuō)明書(shū)分別對(duì)液態(tài)標(biāo)本進(jìn)行預(yù)處理，EV71、CoxA16 RNA提取及PCR擴(kuò)增檢測(cè)。設(shè)置循環(huán)條件為：反轉(zhuǎn)錄50 ℃ 25 min，1個(gè)循環(huán)；預(yù)變性94 ℃ 2 min，1個(gè)循環(huán)； 預(yù)擴(kuò)增95 ℃ 10 s→55 ℃ 15 s→72 ℃ 15 s，5個(gè)循環(huán)；擴(kuò)增、檢測(cè)95 ℃ 10 s→60 ℃ 40 s，40個(gè)循環(huán)。實(shí)時(shí)熒光定量反轉(zhuǎn)錄PCR(RT-qPCR)法擴(kuò)增曲線陽(yáng)性結(jié)果判定標(biāo)準(zhǔn)：在陰性、陽(yáng)性質(zhì)控均滿(mǎn)足要求的條件下，陽(yáng)性標(biāo)本擴(kuò)增曲線呈典型S型且循環(huán)閾值(Ct值)<36.0，陰性標(biāo)本無(wú)典型的S型擴(kuò)增曲線或無(wú)Ct值。

1.2.3病毒載量檢測(cè)檢測(cè)已知濃度標(biāo)準(zhǔn)品的Ct值，在對(duì)數(shù)坐標(biāo)紙上以標(biāo)準(zhǔn)品的濃度為縱坐標(biāo)，標(biāo)準(zhǔn)品的Ct值為橫坐標(biāo)，繪制標(biāo)準(zhǔn)曲線。通過(guò)標(biāo)本的Ct值在標(biāo)準(zhǔn)曲線上得出各待測(cè)標(biāo)本的濃度即為標(biāo)本的病毒載量。

1.2.4臨床資料采用回顧性分析方法，記錄患兒的病程、體溫、熱程、口腔潰瘍、流涎、咳嗽、手部出疹、足部出疹、嗜睡、驚厥、嘔吐、意識(shí)改變、肢體抖動(dòng)、肌痙攣情況。

2結(jié)果

2.1EV71、CoxA16 RNA檢測(cè)結(jié)果HFMD患兒RT-qPCR法擴(kuò)增曲線及標(biāo)準(zhǔn)曲線見(jiàn)圖1。標(biāo)準(zhǔn)曲線顯示，Ct值與病毒載量的對(duì)數(shù)呈高度負(fù)相關(guān)(r=-1.000，P<0.01)。Ct值(X)與病毒載量的對(duì)數(shù)(Y)的關(guān)系為：Y=-0.29X+13.03。陽(yáng)性臨界對(duì)照循環(huán)曲線呈S型，Ct值為34.6，病毒載量為1.02×103copies/ml；陽(yáng)性標(biāo)本Ct值為20.8，病毒載量為1.01×107copies/ml；陰性標(biāo)本未檢出病毒，Ct值>40.0。

注：Ct值=循環(huán)閾值；A為擴(kuò)增曲線，B為標(biāo)準(zhǔn)曲線

圖1HFMD患兒RT-qPCR法擴(kuò)增曲線及標(biāo)準(zhǔn)曲線

Figure 1Amplification and standard curve of real-time fluorescent quantitative RT-PCR on children with HFMD

2.2不同組間Ct值比較EV71輕度組、EV71重度組、CoxA16輕度組、CoxA16重度組HFMD患兒Ct值比較，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；其中CoxA16輕度組、CoxA16重度組HFMD患兒Ct值較EV71輕度組、EV71重度組降低，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；EV71輕度組與EV71重度組、CoxA16輕度組與CoxA16重度組HFMD患兒Ct值比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05，見(jiàn)表1)。

2.3不同組間患兒臨床特征比較EV71輕度組、EV71重度組、CoxA16輕度組、CoxA16重度組HFMD患兒病程分別為(9.0±3.1)d、(14.6±3.2)d、(8.7±2.2)d、(12.6±3.0)d，差異有統(tǒng)計(jì)學(xué)意義(F=101.59，P<0.001)；其中EV71重度組、CoxA16重度組HFMD患兒病程較EV71輕度組、CoxA16輕度組延長(zhǎng)，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；EV71重度組HFMD患兒病程較CoxA16重度組延長(zhǎng)，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。4組HFMD患兒體溫≥38.5 ℃、口腔潰瘍、流涎、手部出疹、足部出疹發(fā)生率比較，差異均無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。EV71重度組、CoxA16重度組HFMD患兒熱程≥3 d發(fā)生率較EV71輕度組、CoxA16輕度組升高，差異有統(tǒng)計(jì)學(xué)意義(P<0.007)；EV71重度組、CoxA16重度組HFMD患兒體溫≥38.5 ℃+熱程≥3 d發(fā)生率較EV71輕度組、CoxA16輕度組升高，EV71輕度組HFMD患兒體溫≥38.5 ℃+熱程≥3 d發(fā)生率較CoxA16輕度組升高，EV71重度組HFMD患兒體溫≥38.5 ℃+熱程≥3 d發(fā)生率較CoxA16重度組升高，差異有統(tǒng)計(jì)學(xué)意義(P<0.007)；CoxA16輕度組、CoxA16重度組HFMD患兒咳嗽、手部出疹數(shù)≥15個(gè)(雙手)、足部出疹數(shù)≥15個(gè)(雙足)發(fā)生率較EV71輕度組、EV71重度組升高，差異有統(tǒng)計(jì)學(xué)意義(P<0.007)。EV71重度組、CoxA16重度組HFMD患兒嗜睡、嘔吐發(fā)生率較EV71輕度組升高，EV71重度組HFMD患兒嗜睡發(fā)生率較CoxA16重度組升高，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)。EV71重度組HFMD患兒驚厥、意識(shí)改變、肢體抖動(dòng)、肌陣攣發(fā)生率與CoxA16重度組比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05，見(jiàn)表2)。

3討論

HFMD一年四季均可發(fā)病，以夏季及秋季高發(fā)。傳染源為患者和病毒攜帶者，病毒主要通過(guò)呼吸道和消化道傳播。迄今為止發(fā)現(xiàn)能夠引發(fā)HFMD的腸道病毒有20余種，而實(shí)驗(yàn)室多以檢測(cè)EV71和CoxA16兩種病毒為主。目前，病毒核酸檢測(cè)多采用PCR技術(shù)，而本研究所采用的反轉(zhuǎn)錄PCR(RT-qPCR)方法較普通RT-PCR方法更靈敏、快捷，且能夠直觀地顯示病毒載量[3]。本研究根據(jù)臨床癥狀、體征和病毒種類(lèi)將患兒分為EV71輕度組162例、EV71重度組87例、CoxA16輕度組158例、CoxA16重度組23例，CoxA16輕度組、CoxA16重度組HFMD患兒Ct值較EV71輕度組、EV71重度組降低，EV71輕度組與EV71重度組、CoxA16輕度組與CoxA16重度組HFMD患兒Ct值無(wú)差異，提示病毒載量與感染病毒相關(guān)，而與病情程度無(wú)關(guān)，與已有研究相符[4-5]。

表1　不同組間HFMD患兒Ct值比較

注：EV71=腸道病毒71型，CoxA16=柯薩奇A組16型，Ct值=循環(huán)閾值；與EV71輕度組比較，aP<0.05；與EV71重度組比較，bP<0.05

表2　不同組間HFMD患兒臨床特征比較〔n(%)〕

注：與EV71輕度組比較，aP<0.007；與EV71重度組比較，bP<0.007；與CoxA16重度組比較，cP<0.007

HFMD起病急、病情發(fā)展迅速、臨床表現(xiàn)多樣，重度患兒可合并無(wú)菌性腦膜炎、腦炎、脊髓灰質(zhì)炎性麻痹、神經(jīng)源性肺水腫等中樞神經(jīng)系統(tǒng)疾病，如不及時(shí)治療，極易危及生命[6]。國(guó)內(nèi)外學(xué)者將臨床特征作為判斷重度患兒的危險(xiǎn)因素,以及時(shí)發(fā)現(xiàn)和控制重癥的發(fā)生發(fā)展[7-8]。本研究通過(guò)病例資料整理分析發(fā)現(xiàn)，兩種病毒感染均有發(fā)熱、口腔潰瘍、流涎、手部出疹、足部出疹等臨床癥狀。而重度患兒可出現(xiàn)嗜睡、驚厥、嘔吐、意識(shí)改變、肢體抖動(dòng)、肌陣攣等早期神經(jīng)系統(tǒng)受累表現(xiàn)[9]，病程和熱程均較長(zhǎng)，而體溫≥38.5 ℃+熱程≥3 d在EV71重度患兒表現(xiàn)尤為明顯，可作為重度患兒的危險(xiǎn)因素之一；CoxA16感染患兒的臨床表現(xiàn)主要是咳嗽，手、足部出疹數(shù)多，提示CoxA16感染對(duì)皮膚黏膜的損傷較明顯，少數(shù)CoxA16感染患兒也可出現(xiàn)神經(jīng)系統(tǒng)受累表現(xiàn)。既往研究發(fā)現(xiàn)，病毒載量與臨床特征無(wú)關(guān)，臨床表現(xiàn)的復(fù)雜性與所感染病毒的種類(lèi)存在一定的關(guān)聯(lián)性[9-10]。

綜上所述，盡管病毒載量與病情程度無(wú)關(guān)，但病毒載量的監(jiān)測(cè)有助于臨床及時(shí)掌握患兒的病毒復(fù)制情況，本研究發(fā)現(xiàn)不同的病毒種類(lèi)引起宿主不同的臨床表現(xiàn)，因此對(duì)病毒種類(lèi)及病毒載量的及時(shí)檢測(cè)對(duì)臨床診療、病情監(jiān)測(cè)和重度患兒的預(yù)防控制有重要意義。有研究表明,HFMD患兒復(fù)雜的臨床表現(xiàn)可能與宿主的免疫防御功能和病毒的生物學(xué)特性有關(guān)[11]，而同一腸道病毒中包括不同基因型，本課題組將進(jìn)一步擴(kuò)大樣本量，分析HFMD患兒不同的臨床特征與腸道病毒基因型間的關(guān)系。

作者貢獻(xiàn)：陳蘇、杜潘艷進(jìn)行試驗(yàn)設(shè)計(jì)與實(shí)施、資料收集整理、撰寫(xiě)論文、成文并對(duì)文章負(fù)責(zé)；鄭紅、王寶林、高翠紅進(jìn)行試驗(yàn)實(shí)施、評(píng)估、資料收集；張雙進(jìn)行質(zhì)量控制及審校。

本文無(wú)利益沖突。

參考文獻(xiàn)

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[2]衛(wèi)生部辦公廳關(guān)于印發(fā)《手足口病診療指南(2010年版)》的通知[EB/OL].(2010-04-06)[2010-06-01].http://www.moh.gov.cn/publicfiles/business/htmlfiles/mohyzs/s3586/202004/46884.htm.

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(本文編輯：陳素芳)

Analysis of the Viral Load and Clinical Features of Children With Hand,Foot and Mouth Disease by Different Enteroviruses

CHENSu,DUPan-yan,ZHENGHong,etal.

DepartmentofLaboratory，theFourthHospitalofTangshan,Tangshan063000,China

【Abstract】ObjectiveTo analyze the viral load and clinical features of children with hand,foot and mouth disease(HFMD) by EV71 and CoxA16.MethodsA total of 430 children who were definitely diagnosed with HFMD in the Department of Pediatrics in Tangshan Women and Children Health-care Hospital from May to September in 2015 were enrolled.According to clinical symptoms,physical signs and species of virus,the children were divided into EV71 mild group(162 cases),EV71 severe group(87 cases),CoxA16 mild group(158 cases)and CoxA16 severe group(23 cases).Throat swabs of the children were collected and the RNA of EV71 and CoxA16 was detected by real-time fluorescent quantitative RT-qPCR method,and viral load was calculated.Length of disease,temperature,length of fever,dental ulcer,salivation,cough,hand rash,foot rash,somnolence,convulsion,vomit,change of consciousness,limb jitter,and myospasm of the children were recorded.ResultsCycle threshold(Ct value) shown by standard curve had highly negative correlation with the viral load(r=-1.000，P<0.01).The relation between the Ct value(X) and the logarithm of viral load(Y) was Y=-0.29X+13.03.CoxA16 mild group and CoxA16 severe group were lower than EV71 mild group and EV71 severe group in Ct value (P<0.05).There was no significant difference in the Ct value of HFMD children between EV71 mild group and EV71 severe group and between CoxA16 mild group and CoxA16 severe group(P>0.05).EV71 severe group and CoxA16 severe group had longer length of disease than EV71 mild group and CoxA16 mild group (P<0.05).EV71 severe group had longer length of disease than CoxA16 severe group (P<0.05).The four groups were not significantly different in the incidence rates of temperature ≥38.5 ℃,dental ulcer,salivation,hand rash and foot rash(P>0.05).EV71 severe group and CoxA16 severe group had higher incidence rate of the length of fever ≥3 d than EV71 mild group and CoxA16 mild group (P<0.007).EV71 severe group and CoxA16 severe group had higher incidence rate of temperature ≥38.5 ℃ plus length of fever ≥3 d than EV71 mild group and CoxA16 mild group,EV71 mild group was higher than CoxA16 mild group in the incidence rate of temperature ≥38.5 ℃ plus length of fever ≥3 d,and EV71 severe group was higher than CoxA16 severe group in the incidence rate of temperature ≥38.5 ℃ plus length of fever ≥3 d(P<0.007).CoxA16 mild group and CoxA16 severe group had higher incidence rates of cough,the number of hand rashes ≥15(both hands) and the number of foot rashes ≥15(both feet) than EV71 mild group and EV71 severe group(P<0.007).EV71 severe group and CoxA16 severe group had higher incidence rates of somnolence and vomit than EV71 mild group, and EV71 severe group was higher than CoxA16 severe group in the incidence rates of somnolence(P<0.01).ConclusionThe viral load of HFMD children varies with different types of enterovirus but is not different among different disease severity levels.HFMD children with different types of enterovirus and disease severity levels are different in the incidence rates of length of fever ≥3 d,temperature ≥38.5 ℃ plus length of fever ≥3 d,cough,the number of hand rashes ≥15(both hands) and the number of foot rashes ≥15(both feet),somnolence,convulsion,vomit,change of consciousness,limb jitter and myospasm.

【Key words】Hand,foot and mouth disease；Enterovirus；Coxsackievirus infections；Viral load；Signs and symptoms

通信作者：杜潘艷，063000河北省唐山市婦幼保健院檢驗(yàn)科；E-mail：dpy405@sohu.com

【中圖分類(lèi)號(hào)】R 725.1

【文獻(xiàn)標(biāo)識(shí)碼】B

doi：10.3969/j.issn.1007-9572.2016.18.020

(收稿日期：2015-11-12；修回日期：2016-02-22)

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