流行性乙型脑炎PCR检测病毒和中医证候特征研究
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摘要
研究目的：对2011年重庆医科大学附属儿童医院采集流行性乙型脑炎患者（乙脑）的标本进行病毒分离与鉴定；2011年和2012年收集的乙脑病例，从证候分型和中医证候方面分析流行性乙型脑炎的临床特点，进一步明确流行性乙型脑炎的证侯特点和中医命名、病因、病机、演变规律及中医药疗效优势，探索解决流行性乙型脑炎中的三大主证问题，形成中医药防治乙脑的理论体系，为救治乙脑提供循证医学证据，促进中医药防治乙脑的技术方法和方案的制定。
研究方法：
（1）采集重庆医科大学附属儿童医院流行性乙型脑炎患者血液及脑脊液标本共145份，通过巢式PCR，实时荧光PCR，BHK‐21细胞培养以及接种2‐3日龄乳鼠等方法，检测并分离乙脑病毒。
（2）研究现场：研究现场主要是武汉市医疗救治中心、重庆科大学附属儿童医院、杭州市第六人民医院、成都市传染病医院、贵阳市第五人民医院。因为上述的几家医院分别是各省的传染病医院，一旦上述省份有乙脑流行，都会把患者转入这几家医院治疗，所以保证了患者的集中性和全面性。另外这几家医院有一定的研究基础，在研究周期内，具备相对固定的数据管理人员。
（3）研究对象
武汉市医疗救治中心、重庆科大学附属儿童医院、杭州市第六人民医院、成都市传染病医院、贵阳市第五人民医院在2011年‐2012年收治的确诊为流行性乙型脑炎患者。
入选标准：①符合卫生部《流行性乙型脑炎诊断标准》（2008年版）临床诊断。②血清或脑脊液检测乙脑病毒IgM抗体阳性的病例。
排除标准：①血清或脑脊液检测乙脑病毒IgM抗体阴性的病例。②未进行血清或脑脊液检测的病例。
（4）实验室检测
主要是血清学检测，血清学检测1个月内未接种过乙脑疫苗者，用免疫荧光法或E LISA法检测患者发病初期的血液或脑脊液中的病毒抗原，有助于早期诊断。用免疫荧光法或E LISA检测患者血清或脑脊液中乙脑特异性抗体，具有早期诊断意义。取患者急性期及恢复期双份血清，抗体效价呈4倍以上升高有诊断意义。
由各医院检验科分别检测。
（5）研究方法
现场考查2011年和2012年武汉市医疗救治中心、重庆科大学附属儿童医院、杭州市第六人民医院、成都市传染病医院、贵阳市第五人民医院收治的确诊为乙脑患者。根据患者的病例和CRF表，填写“流行性乙型脑炎病例调查表”，内容有：患者基本情况、诊断、入院和出院时间、典型症状、体征、中医辨证、出院时恢复情况等。根据调查表对乙脑发病临床症状和中医辨证进行分析。
研究结果
（1）本实验仅从儿童乙脑患者脑脊液标本中分离到1株乙脑病毒，命名为CQ11-66，该病毒能致2-3日龄乳鼠死亡，也能使BHK-21细胞发生细胞病变效应。
（2）2011年和2012年共收集病例472例，发病大都集中在7、8、9三个月。其中男性患者271例，女性患者201例，男女之间的比例为1.05:1。5岁以下的儿童225例，6-10岁的儿童173例，大于10岁的患者74例。有450例患者来自农村，居住环境简陋，附近有养猪场或自家养猪，没有防蚊措施。
（3）临床症候：2011年和2012年乙脑患者主要观察的症候分别是发热445例（94.30%）、头痛108例（22.90%）、呕吐43例（9.10%）、抽搐157例（33.30%）、意识障碍360例（76.30%）、呼吸异常20例（4.20%）、纳差375例（79.40%）、乏力418例（88.60%）。
（4）病因证候特点：2011年流行性乙型脑炎病因证候特点是：毒邪195例，占100%；暑热之邪148例，占75.9%，夹湿邪152例，占77.9%；内陷者146例，占74.9%；轻型、普通型表示卫表轻证，出现发热，烦躁不安，意识障碍较少。重型毒陷心包出现抽搐、嗜睡及烦躁不安等症状。2012年流行性乙型脑炎病因证候特点是：毒邪277例，占100%；暑邪256例，占92.4%；热邪258例，占93.1%；夹湿邪213例，占76.9%；内陷者216例，占78.0%；轻型、普通型表示卫表轻证，出现发热，烦躁不安，意识障碍较少。重型毒陷心包出现抽搐、嗜睡及烦躁不安等症状。亡阴1例，占0.4%；亡阳6例，占1.4%，转到危重病房治疗。
（5）辨证分型：2011年收治流行性乙型脑炎195例，证型分布：毒蕴肺胃35例，占17.9%；毒损脑络98例，占50.3%；毒陷心包62例，占31.8%，未见阴阳衰竭、气阴两伤证型。2012年收治流行性乙型脑炎277例，证型分布：毒蕴肺胃61例，占22.0%；毒损脑络115例，占41.5%；毒陷心包90例，占32.5%，阴阳衰竭11例，占4.0%，未见气阴两伤证型。
（6）临床疗效：2011年随机对照组疗效：治疗组共54例，其中痊愈51例，好转3例，治愈率,94.40%，好转率5.60%；对照组共43例，痊愈38例，好转5例，治愈率88.40%，好转率11.60%。队列研究组疗效：治疗组共51例，其中痊愈45例，好转6例，治愈率88.20%，好转率11.80%；对照组共47例，痊愈35例，好转11例，死亡1例，治愈率74.50%，好转率23.40%，死亡率2.10%。2012年随机对照组疗效：治疗组共119例，其中痊愈99例，好转20例，治愈率83.20%，好转率16.80%；对照组共57例，痊愈42例，好转13例，治愈率73.70%，好转率26.30%。队列研究组疗效：治疗组共69例，其中痊愈50例，好转18例，死亡1例，治愈率72.50%，好转率26.10%，死亡率1.40%；对照组共32例，痊愈22例，好转6例，死亡4例，治愈率68.80%，好转率18.80%，死亡率12.50%。
（7）三大主症：
2011年随机对照组：治疗组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（5.12±2.20）天、（3.35±1.42）天、（3.8±2.20）天、（20±2.50）天。对照组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（8.26±1.56）天、（7.58±1.39）天、（7.4±1.30）天、（26±1.86）天。
2011年队列研究组：治疗组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（5.50±1.30）天、（5.84±1.03）天、（2.23±1.19）天、(23±2.19)天。对照组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（8.74±1.42）天、（9.72±1.61）天、（5.47±1.39）天、（29±1.87）天。
2012年随机对照组：治疗组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（5.68±1.98）天、（4.76±1.39）天、（4.19±2.18）天、（19±3.10）天。对照组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（9.26±2.29）天、（8.43±1.26）天、（8.26±1.27）天、（24±1.29）天。
2012年队列研究组：治疗组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（5.50±1.30）天、（6.72±1.17）天、（2.69±1.23）天、（28±2.76）天。对照组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（7.98±1.69）天、（10.12±1.79）天、（6.47±1.29）天、（30±1.29）天。
结论
1.本研究是重庆市首次从儿童乙脑患者脑脊液标本中检测并分离到流行性乙型脑炎病毒，命名为CQ11-66。
2.从近3年的临床观察，根据叶天士“夏暑发自阳明”，暑温病，以暑邪为患，暑性炎热，暑邪致病，多见火热之证；暑季潮湿，常有暑中夹湿之候；暑热耗气伤阴，故后期又有气阴两亏之状。乙脑病机是暑热毒邪侵入人体，迅速里传，气营两燔为主，既有温病传变的共性，又有自身演变的特殊性。初步提炼出流行性乙型脑炎的核心病机。
3.流行性乙型脑炎中医证候规律，毒蕴肺胃型主要表现为微恶寒,发热，头痛，神志清楚,伴有恶心，口渴，喜饮，少抽搐等证候；毒损脑络型主要表现为高热，头痛，嗜睡昏蒙，偶有抽搐发作等证候；毒陷心包型主要表现为发病急骤，剧烈头痛，躁动或狂躁，昏迷，反复抽搐等症状；阴阳衰竭型病势险恶，主要表现高热，体若燔炭，迅速陷入深昏迷，顽固、持续的抽搐，呼吸气粗或急促无力，呼吸不规则，出现急性亡阴亡阳症状，如颜面苍白晦暗、口唇紫绀，汗多如油，手足厥冷等证候。
4.临床分型中，根据2009年中医药行业专项《中医药防治流行性乙型脑炎临床规律与诊疗方案的研究》课题组制定中医预案，基本确立乙脑的有毒蕴肺胃、毒损脑络、毒陷心包和阴阳衰竭。从近2年临床观察和分析，毒陷心包所占比例较大，其次为毒损脑络、毒蕴肺胃。高热、抽搐和意识障碍仍是流行性乙型脑炎的三大主证，以毒损脑络、毒陷心包型多见；发热是流行性乙型脑炎常见症状，抽搐在毒蕴肺胃和毒损脑络发作较少，在毒陷心包证型中比较常见。患者体温越高，抽搐发作次数就越多，意识障碍持续时间就越长。
5.流行性乙型脑炎病机和传变特点，主要体现在暑热毒、常夹湿、喜内陷、易动风、伤气阴、亡阴阳。毒邪贯穿整个流行性乙型脑炎发病过程，这为确立流行性乙型脑炎治疗原则奠定基础。进一步认识乙脑病的传变符合卫气营血及三焦演变规律，但不拘泥卫气营血和三焦传变的规律。
6.在治疗上，在常规治疗的基础上，积极加强中医辨证治疗，轻型（毒壅肺胃）治疗“宜清不宜下，重用石膏”，故宜辛寒清气，应用白虎汤和银翘散加减；普通型（毒损脑络）清热解毒，凉营醒脑，应用清营汤加减。重型（毒陷心包）清热解毒、凉血熄风，应用清瘟败毒饮和止痉散加减。极重型（阴阳衰竭）要分亡阴应用救逆汤加生脉注射液；亡阳应用通脉四逆汤加参附注射液；同时安宫牛黄丸、至宝丹、紫雪丹在高热、神昏、抽搐治疗过程中起到了重要作用。
Objective:
Of the2011Chongqing Medical University Children's Hospital,Children's Hospital Affiliated collecting specimens for virusisolation and identification of patients with Japaneseencephalitis (JE);JE cases collected in2011and2012, from theevidence, climate the typing and TCM syndromes analysis of theclinical features of JE, to further clarify the Japaneseencephalitis card Hou characteristics and Chinese medicine named,etiology, pathogenesis, evolutionregularity and efficacy ofChinese medicine advantages, explore solutions to the problem ofJapanese encephalitis in the three main card, the formation of thetheoretical system of Chinese medicine prevention and control ofJE, evidence based medicine for the treatment of JE JE promote theprevention and treatment of Traditional Chinese Medicinetechnicalmethods and program development.
Methods:
(1)The acquisition of a total of145copies of Chongqing Medical University Children's Hospital, Japanese encephalitis patientsblood and cerebrospinal fluid specimens by nested PCR, real-timefluorescence PCR, BHK-21cell cultures as well as vaccination2-3day-old suckling mice and other methods detection and separationof the Japanese encephalitis virus.
(2)Study the scene: The study site medical care center inWuhanCity the Chongqing University Children's Hospital, the SixthPeople's Hospital of Hangzhou, ChengduInfectiousDiseasesHospital,GuiyangCity, and the Fifth People's Hospital. Above severalhospitals is provincial infectious disease hospital, once theprovince of JE epidemic will the patient transferred to severalhospitals treatment, so to ensure the patient centralized andcomprehensiveness. Another research base in several hospitals inthe study period, with a relatively fixed data management.
(3)study： Wuhan City medical treatment center, ChongqingUniversity Children's Hospital, the Sixth People's Hospital ofHangzhou, Chengdu Infectious Diseases Hospital, Fifth People'sHospital of Guiyang City, admitted in2011-2012diagnosed patientswith Japanese encephalitis.
Inclusion criteria:(1) comply with the Ministry of Health,"Japanese encephalitis diagnostic criteria"(2009) clinicaldiagnosis.②serum or cerebrospinal fluid detected cases of JEvirus IgM antibodies.
Exclusion criteria:①serum or cerebrospinal fluid to detectcases of JE virus IgM antibody negative.②not detected cases ofserum or cerebrospinal fluid.
(4)Laboratory testing Serological tests, serological tests JEvaccine unvaccinated months, with viral antigen in the blood or cerebrospinal fluid of patients with early onset of immuneofluorescence or E LISA assay useful for early diagnosis. JEspecific antibodies with immunofluorescence or E LISA detection ofserum or cerebrospinal fluid, with early diagnosis. Take patientswith acute and convalescent serum antibody titer was more than fourtimes higher diagnostic significance.By the hospital laboratorywere detected.
(5)Research Methods Site visits to medical treatment center inWuhan City in2011and2012, the Children's Hospital of ChongqingUniversity, Hangzhou Sixth People's Hospital, Chengdu InfectiousDiseases Hospital, Guiyang City Fifth People's Hospital of Japaneseencephalitis patients. Patient cases and CRF table, fill in thecases of Japanese encephalitis questionnaire content: basicsituation of the patients, the diagnosis, admission and dischargetime, the typical symptoms, signs, TCM syndrome discharge recovery.JE onset of clinical symptoms and TCM analysis on census forms.
Results:
(1)In this study, CSF samples only from children JE patientsisolated from a Japanese encephalitis virus, named CQ11-66, thevirus can cause2-3day-old suckling mice died, BHK-21cells canoccur Cellcytopathic effect.
(2)In2011and2012,472cases were collected cases, the onsetmostly concentrated in the three months7,8,9. Which271casesof male patients, female patients with201cases, between men andwomen, the proportion was1.05:1.225cases of children under theage of5,6-10-year-old children in173cases, more than10-year-oldpatients74cases.450patients from rural areas, the poor livingenvironment, near a pig farm or their own pig, there is no anti-mosquito measures.
(3)Clinical symptoms: The JE patients the main symptom observedin2011and2012were fever in445cases (94.30%), headache in108cases (22.90%), vomiting in43cases (9.10%), seizures in157cases(33.30%) and360cases of disturbance of consciousness,(76.30%),abnormal breathing20cases (4.20%) and anorexia375cases (79.40%),fatigue in418cases (88.60%).
(4)Cause of syndrome characteristics: Japanese encephalitiscause of syndromes characterized by2011: toxic evil195cases,accounting for100%; the heat evil148cases, accounting for75.9%clip dampness152cases, accounting for77.9%; hiinvagination of146cases, accounting for74.9%; Light, ordinary type indicatesGuardian table light card, fever, irritability, less disturbanceof consciousness. The heavy-duty the poison trap pericardialconvulsions, drowsiness, and irritability symptoms. Japaneseencephalitis cause of syndromes characterized by2012: the toxin277cases (100%); damp256cases, accounting for92.4%; the heat evil258cases, accounting for93.1%; dampness evil213cases,accounting for76.9%; hi invagination of the216cases, accountingfor78.0%; lightweight, ordinary type of representation Guardiantable light card, fever, irritability, and less disturbance ofconsciousness.The heavy-duty the poison trap pericardialconvulsions, drowsiness,and irritability symptoms. Death overcastcases, accounting for0.4%; death yang6cases (1.4%) go to the ICUtreatment.
(5)Differentiation:2011Japanese encephalitis195casestreated, the syndrome distribution: drug Yun lung and stomach in35cases, accounting for17.9%;98cases of poison damage of brain collateral, accounting for50.3%of the62cases; poison trappericardial accounted for31.8%, and no yin and yang failure, Qiand Yin injury syndromes.2012treated277cases of Japaneseencephalitis syndrome distribution: drug Yun lung and stomach in61cases, accounting for22.0%; poison damage of brain collateral115cases, accounting for41.5%; the poison trap the pericardium90cases,32.5%, yin and yang failure11cases, accounting for4.0%,and no Qi and Yin injury syndrome type.
(6)Clinical efficacy:2011random control group efficacy:treatment group of54cases, of which51cases were cured, improvedin3cases, the cure rate,94.40%, improvement rate of5.60%;control group of43cases,38cases cured, improved five cases, thecure rate was88.40%,11.60%improvement rate. Cohort study grouptherapeutic effect: a total of51cases, of which45cases were cured,improved in six cases, the cure rate was88.20%, the improvementrate of11.80%; control group of47cases,35cases cured, improvedin11cases,1patient died, the cure rate74.50%, improvement ratewas23.40%, and the mortality rate was2.10%.2012randomizedcontrol group therapeutic effect: a total of119cases, of which99cases were cured,20cases improved, the cure rate was83.20%,the improvement rate of16.80%; control group of57cases,42casescured, improved in13cases, the cure rate73.70%improvement ratewas26.30%. Cohort study group efficacy: treatment group of69cases,50cases were cured,18cases improved, and1died, the cure ratewas72.50%, the improvement rate of26.10%,1.40%mortality;control group of32cases,22cases cured, improved six cases, fourcases of death, the cure rate of68.80%, the improvement rate of18.80%,12.50%mortality.
(7)The three main symptoms:
2011randomized control groups: the treatment group, bodytemperature dropped to normal time conscious average, averagerelieve convulsions time, average length of stay (5.12±2.20) days,(3.35±1.42) days,(3.8±2.20) days,(20±2.50) days. Controlgroup, the temperature dropped to normal time conscious average,average relieve convulsions time, the average length of stay (8.26±1.56) days,(7.58±1.39) days,(7.4±1.30) days,(26±1.86)days.
2011cohort study groups: the treatment group body temperaturedropped to normal time conscious average, average relieveconvulsions time, average length of stay (5.50±1.30) days,(5.84±1.03) days,(2.23±1.19) days,(23±2.19) days. Control group,the temperature dropped to normal time, the average conscious,average relieve convulsions time, the average length of stay (8.74±1.42) days,(9.72±1.61) days,(5.47±1.39) days,(29±1.87)days.
2012randomized control group: the treatment group, bodytemperature dropped to normal time conscious average, averagerelieve convulsions time, the average length of stay (5.68±1.98)days,(4.76±1.39) days,(4.19±2.18) days,(19±3.10) days.Control group, the temperature dropped to normal time, the averageconscious, average relieve convulsions time, the average length ofstay (9.26±2.29) days,(8.43±1.26) days,(8.26±1.27) days,(24±1.29) days.
2012cohort study groups: the treatment group body temperaturedropped to normal time conscious average, average relieveconvulsions time, the average length of stay (5.50±1.30) days, (6.72±1.17) days,(2.69±1.23) days,(28±2.76) days. Controlgroup, the temperature dropped to normal time, the averageconscious, average relieve convulsions time, the average length ofstay (7.98±1.69) days,(10.12±1.79) days,(6.47±1.29) days,(30±1.29) days.
Conclusion:
1This study Chongqing for the first time detected in CSFsamples from children JE patients and isolated Japaneseencephalitis virus, named CQ11-66.
2From nearly three years of clinical observation, accordingto Ye Tina shin of summer from the bottom of Yang Ming, summerfebrile disease to summer evil infestation sex hot summer, dampdiseases more common hot the card; humid summer season, often thereis summer in the folder the wet of waiting; summer heat gasconsumption Shang in therefore late there Qiyin the two losses andthe like. JE pathogenesis heat the toxin invades the human body,quickly years pass gas camp two burnt the of both febrile diseasetransmission becomes common, has its own evolution particularity.Preliminary refining the Japanese encephalitis core pathogenesis.
3Japanese encephalitis TCM Syndromes, Drug Yun lung andstomach mainly micro chills, fever, headache, conscious,accompanied by nausea, thirst hi drink less and convulsionssyndrome; Poison damaged brain collaterals mainly high fever,headache, lethargy darkened, occasional seizure syndromes; Thepoison trap pericardial mainly for rapid-onset, severe headache,restlessness or mania, coma, repeated convulsions and othersymptoms; Yin and Yang exhausted disease potential dangerous,mainly fever, body burnt charcoal, quickly fell into a deep coma, stubborn, continuous convulsions, rough breathing or shortness ofbreath, weakness, irregular breathing, acute death overcast deathyang symptoms, such as facial pale dull lips The cyanosis, sweatmore, such as oil, hand-foot Jueleng syndrome.
4. Clinical classification, medicine, prevention and treatmentof epidemic encephalitis laws of clinical and treatment programsin the pharmaceutical industry, special in2009,"the researchgroup to develop the Chinese plan, basically established the JEtoxic Yun lung and stomach poison brain collateral damage, poisontrap pericardium and yin and yang failure. From nearly2years ofclinical observation and analysis, the poison trap pericardialproportion, followed by drug damage of brain collateral, Yun-lungand stomach poison. High fever, convulsions and unconsciousness arestill three Japanese encephalitis master card, Yidu damage of braincollateral poison trap pericardial more common type; Fever arecommon symptoms of epidemic encephalitis, convulsions in drug Yunlung and stomach poison damage brain collateral attack less, isrelatively common in the poison trap pericardial card type. Thehigher the patient's temperature, the more the number of seizure,and the longer the duration of the disturbance of consciousness.
5.Japanese encephalitis pathogenesis and mass changecharacteristics, the poison in the summer heat, constant dampnessand retraction, and easy to move wind hurt Qiyin death of yin andyang. Toxic evil throughout the process of the incidence of Japaneseencephalitis, which lay the foundation for the establishment ofJapanese encephalitis treatment principle. The furtherunderstanding JE disease mass change in line with the Wei Qi YingXue the triple burner EVOLUTION, but not stickle Wei Qi Ying Xue and the triple burner Biography change the law.
6Treatment on the basis of conventional treatment, andactively strengthen TCM treatment, light treatment (poisonobstruct the lung and stomach) Yawing should not reuse gypsum,therefore should Xinhua Qing gas, the white tiger soup andYinqiaosan plus less; Common type (toxic damage of brain collateral)detoxify, cool camp refreshing Qingyingtang addition andsubtraction. The heavy(the poison trap pericardium) detoxification,cooling Fifing, application Wingmen Baidu Decoction ZhejiangDecoction. Heavy (yin and yang failure) to death of yin applicationfrom collapse Tonga Shengmai the injection; death of Appliedtongmai Sini Tonga Shenfu injection;While Angongniuhuang, thetreasure of Dan, Dixie high fever, coma, played an important rolein the process of convulsive therapy. Alpha
研究方法：
（1）采集重庆医科大学附属儿童医院流行性乙型脑炎患者血液及脑脊液标本共145份，通过巢式PCR，实时荧光PCR，BHK‐21细胞培养以及接种2‐3日龄乳鼠等方法，检测并分离乙脑病毒。
（2）研究现场：研究现场主要是武汉市医疗救治中心、重庆科大学附属儿童医院、杭州市第六人民医院、成都市传染病医院、贵阳市第五人民医院。因为上述的几家医院分别是各省的传染病医院，一旦上述省份有乙脑流行，都会把患者转入这几家医院治疗，所以保证了患者的集中性和全面性。另外这几家医院有一定的研究基础，在研究周期内，具备相对固定的数据管理人员。
（3）研究对象
武汉市医疗救治中心、重庆科大学附属儿童医院、杭州市第六人民医院、成都市传染病医院、贵阳市第五人民医院在2011年‐2012年收治的确诊为流行性乙型脑炎患者。
入选标准：①符合卫生部《流行性乙型脑炎诊断标准》（2008年版）临床诊断。②血清或脑脊液检测乙脑病毒IgM抗体阳性的病例。
排除标准：①血清或脑脊液检测乙脑病毒IgM抗体阴性的病例。②未进行血清或脑脊液检测的病例。
（4）实验室检测
主要是血清学检测，血清学检测1个月内未接种过乙脑疫苗者，用免疫荧光法或E LISA法检测患者发病初期的血液或脑脊液中的病毒抗原，有助于早期诊断。用免疫荧光法或E LISA检测患者血清或脑脊液中乙脑特异性抗体，具有早期诊断意义。取患者急性期及恢复期双份血清，抗体效价呈4倍以上升高有诊断意义。
由各医院检验科分别检测。
（5）研究方法
现场考查2011年和2012年武汉市医疗救治中心、重庆科大学附属儿童医院、杭州市第六人民医院、成都市传染病医院、贵阳市第五人民医院收治的确诊为乙脑患者。根据患者的病例和CRF表，填写“流行性乙型脑炎病例调查表”，内容有：患者基本情况、诊断、入院和出院时间、典型症状、体征、中医辨证、出院时恢复情况等。根据调查表对乙脑发病临床症状和中医辨证进行分析。
研究结果
（1）本实验仅从儿童乙脑患者脑脊液标本中分离到1株乙脑病毒，命名为CQ11-66，该病毒能致2-3日龄乳鼠死亡，也能使BHK-21细胞发生细胞病变效应。
（2）2011年和2012年共收集病例472例，发病大都集中在7、8、9三个月。其中男性患者271例，女性患者201例，男女之间的比例为1.05:1。5岁以下的儿童225例，6-10岁的儿童173例，大于10岁的患者74例。有450例患者来自农村，居住环境简陋，附近有养猪场或自家养猪，没有防蚊措施。
（3）临床症候：2011年和2012年乙脑患者主要观察的症候分别是发热445例（94.30%）、头痛108例（22.90%）、呕吐43例（9.10%）、抽搐157例（33.30%）、意识障碍360例（76.30%）、呼吸异常20例（4.20%）、纳差375例（79.40%）、乏力418例（88.60%）。
（4）病因证候特点：2011年流行性乙型脑炎病因证候特点是：毒邪195例，占100%；暑热之邪148例，占75.9%，夹湿邪152例，占77.9%；内陷者146例，占74.9%；轻型、普通型表示卫表轻证，出现发热，烦躁不安，意识障碍较少。重型毒陷心包出现抽搐、嗜睡及烦躁不安等症状。2012年流行性乙型脑炎病因证候特点是：毒邪277例，占100%；暑邪256例，占92.4%；热邪258例，占93.1%；夹湿邪213例，占76.9%；内陷者216例，占78.0%；轻型、普通型表示卫表轻证，出现发热，烦躁不安，意识障碍较少。重型毒陷心包出现抽搐、嗜睡及烦躁不安等症状。亡阴1例，占0.4%；亡阳6例，占1.4%，转到危重病房治疗。
（5）辨证分型：2011年收治流行性乙型脑炎195例，证型分布：毒蕴肺胃35例，占17.9%；毒损脑络98例，占50.3%；毒陷心包62例，占31.8%，未见阴阳衰竭、气阴两伤证型。2012年收治流行性乙型脑炎277例，证型分布：毒蕴肺胃61例，占22.0%；毒损脑络115例，占41.5%；毒陷心包90例，占32.5%，阴阳衰竭11例，占4.0%，未见气阴两伤证型。
（6）临床疗效：2011年随机对照组疗效：治疗组共54例，其中痊愈51例，好转3例，治愈率,94.40%，好转率5.60%；对照组共43例，痊愈38例，好转5例，治愈率88.40%，好转率11.60%。队列研究组疗效：治疗组共51例，其中痊愈45例，好转6例，治愈率88.20%，好转率11.80%；对照组共47例，痊愈35例，好转11例，死亡1例，治愈率74.50%，好转率23.40%，死亡率2.10%。2012年随机对照组疗效：治疗组共119例，其中痊愈99例，好转20例，治愈率83.20%，好转率16.80%；对照组共57例，痊愈42例，好转13例，治愈率73.70%，好转率26.30%。队列研究组疗效：治疗组共69例，其中痊愈50例，好转18例，死亡1例，治愈率72.50%，好转率26.10%，死亡率1.40%；对照组共32例，痊愈22例，好转6例，死亡4例，治愈率68.80%，好转率18.80%，死亡率12.50%。
（7）三大主症：
2011年随机对照组：治疗组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（5.12±2.20）天、（3.35±1.42）天、（3.8±2.20）天、（20±2.50）天。对照组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（8.26±1.56）天、（7.58±1.39）天、（7.4±1.30）天、（26±1.86）天。
2011年队列研究组：治疗组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（5.50±1.30）天、（5.84±1.03）天、（2.23±1.19）天、(23±2.19)天。对照组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（8.74±1.42）天、（9.72±1.61）天、（5.47±1.39）天、（29±1.87）天。
2012年随机对照组：治疗组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（5.68±1.98）天、（4.76±1.39）天、（4.19±2.18）天、（19±3.10）天。对照组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（9.26±2.29）天、（8.43±1.26）天、（8.26±1.27）天、（24±1.29）天。
2012年队列研究组：治疗组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（5.50±1.30）天、（6.72±1.17）天、（2.69±1.23）天、（28±2.76）天。对照组体温降至正常时间、平均意识清醒、平均缓解抽搐时间、平均住院时间分别是（7.98±1.69）天、（10.12±1.79）天、（6.47±1.29）天、（30±1.29）天。
结论
1.本研究是重庆市首次从儿童乙脑患者脑脊液标本中检测并分离到流行性乙型脑炎病毒，命名为CQ11-66。
2.从近3年的临床观察，根据叶天士“夏暑发自阳明”，暑温病，以暑邪为患，暑性炎热，暑邪致病，多见火热之证；暑季潮湿，常有暑中夹湿之候；暑热耗气伤阴，故后期又有气阴两亏之状。乙脑病机是暑热毒邪侵入人体，迅速里传，气营两燔为主，既有温病传变的共性，又有自身演变的特殊性。初步提炼出流行性乙型脑炎的核心病机。
3.流行性乙型脑炎中医证候规律，毒蕴肺胃型主要表现为微恶寒,发热，头痛，神志清楚,伴有恶心，口渴，喜饮，少抽搐等证候；毒损脑络型主要表现为高热，头痛，嗜睡昏蒙，偶有抽搐发作等证候；毒陷心包型主要表现为发病急骤，剧烈头痛，躁动或狂躁，昏迷，反复抽搐等症状；阴阳衰竭型病势险恶，主要表现高热，体若燔炭，迅速陷入深昏迷，顽固、持续的抽搐，呼吸气粗或急促无力，呼吸不规则，出现急性亡阴亡阳症状，如颜面苍白晦暗、口唇紫绀，汗多如油，手足厥冷等证候。
4.临床分型中，根据2009年中医药行业专项《中医药防治流行性乙型脑炎临床规律与诊疗方案的研究》课题组制定中医预案，基本确立乙脑的有毒蕴肺胃、毒损脑络、毒陷心包和阴阳衰竭。从近2年临床观察和分析，毒陷心包所占比例较大，其次为毒损脑络、毒蕴肺胃。高热、抽搐和意识障碍仍是流行性乙型脑炎的三大主证，以毒损脑络、毒陷心包型多见；发热是流行性乙型脑炎常见症状，抽搐在毒蕴肺胃和毒损脑络发作较少，在毒陷心包证型中比较常见。患者体温越高，抽搐发作次数就越多，意识障碍持续时间就越长。
5.流行性乙型脑炎病机和传变特点，主要体现在暑热毒、常夹湿、喜内陷、易动风、伤气阴、亡阴阳。毒邪贯穿整个流行性乙型脑炎发病过程，这为确立流行性乙型脑炎治疗原则奠定基础。进一步认识乙脑病的传变符合卫气营血及三焦演变规律，但不拘泥卫气营血和三焦传变的规律。
6.在治疗上，在常规治疗的基础上，积极加强中医辨证治疗，轻型（毒壅肺胃）治疗“宜清不宜下，重用石膏”，故宜辛寒清气，应用白虎汤和银翘散加减；普通型（毒损脑络）清热解毒，凉营醒脑，应用清营汤加减。重型（毒陷心包）清热解毒、凉血熄风，应用清瘟败毒饮和止痉散加减。极重型（阴阳衰竭）要分亡阴应用救逆汤加生脉注射液；亡阳应用通脉四逆汤加参附注射液；同时安宫牛黄丸、至宝丹、紫雪丹在高热、神昏、抽搐治疗过程中起到了重要作用。
Objective:
Of the2011Chongqing Medical University Children's Hospital,Children's Hospital Affiliated collecting specimens for virusisolation and identification of patients with Japaneseencephalitis (JE);JE cases collected in2011and2012, from theevidence, climate the typing and TCM syndromes analysis of theclinical features of JE, to further clarify the Japaneseencephalitis card Hou characteristics and Chinese medicine named,etiology, pathogenesis, evolutionregularity and efficacy ofChinese medicine advantages, explore solutions to the problem ofJapanese encephalitis in the three main card, the formation of thetheoretical system of Chinese medicine prevention and control ofJE, evidence based medicine for the treatment of JE JE promote theprevention and treatment of Traditional Chinese Medicinetechnicalmethods and program development.
Methods:
(1)The acquisition of a total of145copies of Chongqing Medical University Children's Hospital, Japanese encephalitis patientsblood and cerebrospinal fluid specimens by nested PCR, real-timefluorescence PCR, BHK-21cell cultures as well as vaccination2-3day-old suckling mice and other methods detection and separationof the Japanese encephalitis virus.
(2)Study the scene: The study site medical care center inWuhanCity the Chongqing University Children's Hospital, the SixthPeople's Hospital of Hangzhou, ChengduInfectiousDiseasesHospital,GuiyangCity, and the Fifth People's Hospital. Above severalhospitals is provincial infectious disease hospital, once theprovince of JE epidemic will the patient transferred to severalhospitals treatment, so to ensure the patient centralized andcomprehensiveness. Another research base in several hospitals inthe study period, with a relatively fixed data management.
(3)study： Wuhan City medical treatment center, ChongqingUniversity Children's Hospital, the Sixth People's Hospital ofHangzhou, Chengdu Infectious Diseases Hospital, Fifth People'sHospital of Guiyang City, admitted in2011-2012diagnosed patientswith Japanese encephalitis.
Inclusion criteria:(1) comply with the Ministry of Health,"Japanese encephalitis diagnostic criteria"(2009) clinicaldiagnosis.②serum or cerebrospinal fluid detected cases of JEvirus IgM antibodies.
Exclusion criteria:①serum or cerebrospinal fluid to detectcases of JE virus IgM antibody negative.②not detected cases ofserum or cerebrospinal fluid.
(4)Laboratory testing Serological tests, serological tests JEvaccine unvaccinated months, with viral antigen in the blood or cerebrospinal fluid of patients with early onset of immuneofluorescence or E LISA assay useful for early diagnosis. JEspecific antibodies with immunofluorescence or E LISA detection ofserum or cerebrospinal fluid, with early diagnosis. Take patientswith acute and convalescent serum antibody titer was more than fourtimes higher diagnostic significance.By the hospital laboratorywere detected.
(5)Research Methods Site visits to medical treatment center inWuhan City in2011and2012, the Children's Hospital of ChongqingUniversity, Hangzhou Sixth People's Hospital, Chengdu InfectiousDiseases Hospital, Guiyang City Fifth People's Hospital of Japaneseencephalitis patients. Patient cases and CRF table, fill in thecases of Japanese encephalitis questionnaire content: basicsituation of the patients, the diagnosis, admission and dischargetime, the typical symptoms, signs, TCM syndrome discharge recovery.JE onset of clinical symptoms and TCM analysis on census forms.
Results:
(1)In this study, CSF samples only from children JE patientsisolated from a Japanese encephalitis virus, named CQ11-66, thevirus can cause2-3day-old suckling mice died, BHK-21cells canoccur Cellcytopathic effect.
(2)In2011and2012,472cases were collected cases, the onsetmostly concentrated in the three months7,8,9. Which271casesof male patients, female patients with201cases, between men andwomen, the proportion was1.05:1.225cases of children under theage of5,6-10-year-old children in173cases, more than10-year-oldpatients74cases.450patients from rural areas, the poor livingenvironment, near a pig farm or their own pig, there is no anti-mosquito measures.
(3)Clinical symptoms: The JE patients the main symptom observedin2011and2012were fever in445cases (94.30%), headache in108cases (22.90%), vomiting in43cases (9.10%), seizures in157cases(33.30%) and360cases of disturbance of consciousness,(76.30%),abnormal breathing20cases (4.20%) and anorexia375cases (79.40%),fatigue in418cases (88.60%).
(4)Cause of syndrome characteristics: Japanese encephalitiscause of syndromes characterized by2011: toxic evil195cases,accounting for100%; the heat evil148cases, accounting for75.9%clip dampness152cases, accounting for77.9%; hiinvagination of146cases, accounting for74.9%; Light, ordinary type indicatesGuardian table light card, fever, irritability, less disturbanceof consciousness. The heavy-duty the poison trap pericardialconvulsions, drowsiness, and irritability symptoms. Japaneseencephalitis cause of syndromes characterized by2012: the toxin277cases (100%); damp256cases, accounting for92.4%; the heat evil258cases, accounting for93.1%; dampness evil213cases,accounting for76.9%; hi invagination of the216cases, accountingfor78.0%; lightweight, ordinary type of representation Guardiantable light card, fever, irritability, and less disturbance ofconsciousness.The heavy-duty the poison trap pericardialconvulsions, drowsiness,and irritability symptoms. Death overcastcases, accounting for0.4%; death yang6cases (1.4%) go to the ICUtreatment.
(5)Differentiation:2011Japanese encephalitis195casestreated, the syndrome distribution: drug Yun lung and stomach in35cases, accounting for17.9%;98cases of poison damage of brain collateral, accounting for50.3%of the62cases; poison trappericardial accounted for31.8%, and no yin and yang failure, Qiand Yin injury syndromes.2012treated277cases of Japaneseencephalitis syndrome distribution: drug Yun lung and stomach in61cases, accounting for22.0%; poison damage of brain collateral115cases, accounting for41.5%; the poison trap the pericardium90cases,32.5%, yin and yang failure11cases, accounting for4.0%,and no Qi and Yin injury syndrome type.
(6)Clinical efficacy:2011random control group efficacy:treatment group of54cases, of which51cases were cured, improvedin3cases, the cure rate,94.40%, improvement rate of5.60%;control group of43cases,38cases cured, improved five cases, thecure rate was88.40%,11.60%improvement rate. Cohort study grouptherapeutic effect: a total of51cases, of which45cases were cured,improved in six cases, the cure rate was88.20%, the improvementrate of11.80%; control group of47cases,35cases cured, improvedin11cases,1patient died, the cure rate74.50%, improvement ratewas23.40%, and the mortality rate was2.10%.2012randomizedcontrol group therapeutic effect: a total of119cases, of which99cases were cured,20cases improved, the cure rate was83.20%,the improvement rate of16.80%; control group of57cases,42casescured, improved in13cases, the cure rate73.70%improvement ratewas26.30%. Cohort study group efficacy: treatment group of69cases,50cases were cured,18cases improved, and1died, the cure ratewas72.50%, the improvement rate of26.10%,1.40%mortality;control group of32cases,22cases cured, improved six cases, fourcases of death, the cure rate of68.80%, the improvement rate of18.80%,12.50%mortality.
(7)The three main symptoms:
2011randomized control groups: the treatment group, bodytemperature dropped to normal time conscious average, averagerelieve convulsions time, average length of stay (5.12±2.20) days,(3.35±1.42) days,(3.8±2.20) days,(20±2.50) days. Controlgroup, the temperature dropped to normal time conscious average,average relieve convulsions time, the average length of stay (8.26±1.56) days,(7.58±1.39) days,(7.4±1.30) days,(26±1.86)days.
2011cohort study groups: the treatment group body temperaturedropped to normal time conscious average, average relieveconvulsions time, average length of stay (5.50±1.30) days,(5.84±1.03) days,(2.23±1.19) days,(23±2.19) days. Control group,the temperature dropped to normal time, the average conscious,average relieve convulsions time, the average length of stay (8.74±1.42) days,(9.72±1.61) days,(5.47±1.39) days,(29±1.87)days.
2012randomized control group: the treatment group, bodytemperature dropped to normal time conscious average, averagerelieve convulsions time, the average length of stay (5.68±1.98)days,(4.76±1.39) days,(4.19±2.18) days,(19±3.10) days.Control group, the temperature dropped to normal time, the averageconscious, average relieve convulsions time, the average length ofstay (9.26±2.29) days,(8.43±1.26) days,(8.26±1.27) days,(24±1.29) days.
2012cohort study groups: the treatment group body temperaturedropped to normal time conscious average, average relieveconvulsions time, the average length of stay (5.50±1.30) days, (6.72±1.17) days,(2.69±1.23) days,(28±2.76) days. Controlgroup, the temperature dropped to normal time, the averageconscious, average relieve convulsions time, the average length ofstay (7.98±1.69) days,(10.12±1.79) days,(6.47±1.29) days,(30±1.29) days.
Conclusion:
1This study Chongqing for the first time detected in CSFsamples from children JE patients and isolated Japaneseencephalitis virus, named CQ11-66.
2From nearly three years of clinical observation, accordingto Ye Tina shin of summer from the bottom of Yang Ming, summerfebrile disease to summer evil infestation sex hot summer, dampdiseases more common hot the card; humid summer season, often thereis summer in the folder the wet of waiting; summer heat gasconsumption Shang in therefore late there Qiyin the two losses andthe like. JE pathogenesis heat the toxin invades the human body,quickly years pass gas camp two burnt the of both febrile diseasetransmission becomes common, has its own evolution particularity.Preliminary refining the Japanese encephalitis core pathogenesis.
3Japanese encephalitis TCM Syndromes, Drug Yun lung andstomach mainly micro chills, fever, headache, conscious,accompanied by nausea, thirst hi drink less and convulsionssyndrome; Poison damaged brain collaterals mainly high fever,headache, lethargy darkened, occasional seizure syndromes; Thepoison trap pericardial mainly for rapid-onset, severe headache,restlessness or mania, coma, repeated convulsions and othersymptoms; Yin and Yang exhausted disease potential dangerous,mainly fever, body burnt charcoal, quickly fell into a deep coma, stubborn, continuous convulsions, rough breathing or shortness ofbreath, weakness, irregular breathing, acute death overcast deathyang symptoms, such as facial pale dull lips The cyanosis, sweatmore, such as oil, hand-foot Jueleng syndrome.
4. Clinical classification, medicine, prevention and treatmentof epidemic encephalitis laws of clinical and treatment programsin the pharmaceutical industry, special in2009,"the researchgroup to develop the Chinese plan, basically established the JEtoxic Yun lung and stomach poison brain collateral damage, poisontrap pericardium and yin and yang failure. From nearly2years ofclinical observation and analysis, the poison trap pericardialproportion, followed by drug damage of brain collateral, Yun-lungand stomach poison. High fever, convulsions and unconsciousness arestill three Japanese encephalitis master card, Yidu damage of braincollateral poison trap pericardial more common type; Fever arecommon symptoms of epidemic encephalitis, convulsions in drug Yunlung and stomach poison damage brain collateral attack less, isrelatively common in the poison trap pericardial card type. Thehigher the patient's temperature, the more the number of seizure,and the longer the duration of the disturbance of consciousness.
5.Japanese encephalitis pathogenesis and mass changecharacteristics, the poison in the summer heat, constant dampnessand retraction, and easy to move wind hurt Qiyin death of yin andyang. Toxic evil throughout the process of the incidence of Japaneseencephalitis, which lay the foundation for the establishment ofJapanese encephalitis treatment principle. The furtherunderstanding JE disease mass change in line with the Wei Qi YingXue the triple burner EVOLUTION, but not stickle Wei Qi Ying Xue and the triple burner Biography change the law.
6Treatment on the basis of conventional treatment, andactively strengthen TCM treatment, light treatment (poisonobstruct the lung and stomach) Yawing should not reuse gypsum,therefore should Xinhua Qing gas, the white tiger soup andYinqiaosan plus less; Common type (toxic damage of brain collateral)detoxify, cool camp refreshing Qingyingtang addition andsubtraction. The heavy(the poison trap pericardium) detoxification,cooling Fifing, application Wingmen Baidu Decoction ZhejiangDecoction. Heavy (yin and yang failure) to death of yin applicationfrom collapse Tonga Shengmai the injection; death of Appliedtongmai Sini Tonga Shenfu injection;While Angongniuhuang, thetreasure of Dan, Dixie high fever, coma, played an important rolein the process of convulsive therapy. Alpha
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