蛋白质飞行质谱技术在严重急性呼吸综合征(SARS)早期诊断中的应用价值(英文)

目的探索蛋白质飞行质谱技术在严重急性呼吸综合征(SARS)早期诊断中的应用价值。方法用表面加强激光解吸电离质谱技术检测急性SARS组和对照组的血清,首先建立诊断模型,然后单盲进行模型验证。结果通过检测急性SARS组(74例)和对照组(147例)的血清,发现了早期SARS患者血清中质谱峰(M/Z)为3939.08、4137.71、8136.64、11514.20的4种特异蛋白指纹标志物,并建立诊断模型,此模型敏感性98.6%(73/74),特异性94.6%(139/147)。结论早期SARS患者有特异的蛋白指纹图谱,利用表面加强激光解吸电离质谱技术检测SARS患者或疑似SARS患者血清,可更好地协助临床进行SARS的早期诊治。     

Serum proteomic fingerprints of adult patients with severe acute respiratory syndrome

BACKGROUND: Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a new coronavirus strain, SARS-CoV. Specific proteomic patterns might be present in serum in response to the infection and could be useful for early detection of the disease. METHODS: Using surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology, we profiled and compared serum proteins of 39 patients with early-stage SARS infection and 39 non-SARS patients who were suspected cases during the SARS outbreak period. Proteomic patterns associated with SARS were identified by bioinformatic and biostatistical analyses. Features of interest were then purified and identified by tandem mass spectrometry. RESULTS: Twenty proteomic features were significantly different between the 2 groups. Fifteen were increased in the SARS group, and 5 were decreased. Their concentrations were correlated with 2 or more clinical and/or biochemical variables. Two were correlated with the SARS-CoV viral load. Hierarchical clustering analysis showed that a majority of the SARS patients (95%) had similar serum proteomic profiles and identified 2 subgroups with poor prognosis. ROC curve analysis identified individual features as potential biomarkers for SARS diagnosis (areas under ROC curves, 0.733-0.995). ROC curve areas were largest for an N-terminal fragment of complement C3c alpha chain (m/z 28 119) and an internal fragment of fibrinogen alpha-E chain (m/z 5908). Immunoglobulin kappa light chain (m/z 24 505) positively correlated with viral load. CONCLUSIONS: Specific proteomic fingerprints in the sera of adult SARS patients could be used to identify SARS cases early during onset with high specificity and sensitivity.     

Proteomics and bioinformatics approaches for identification of serum biomarkers to detect breast cancer

BACKGROUND: Surface-enhanced laser desorption/ionization (SELDI) is an affinity-based mass spectrometric method in which proteins of interest are selectively adsorbed to a chemically modified surface on a biochip, whereas impurities are removed by washing with buffer. This technology allows sensitive and high-throughput protein profiling of complex biological specimens. METHODS: We screened for potential tumor biomarkers in 169 serum samples, including samples from a cancer group of 103 breast cancer patients at different clinical stages [stage 0 (n = 4), stage I (n = 38), stage II (n = 37), and stage III (n = 24)], from a control group of 41 healthy women, and from 25 patients with benign breast diseases. Diluted serum samples were applied to immobilized metal affinity capture Ciphergen ProteinChip Arrays previously activated with Ni2+. Proteins bound to the chelated metal were analyzed on a ProteinChip Reader Model PBS II. Complex protein profiles of different diagnostic groups were compared and analyzed using the ProPeak software package. RESULTS: A panel of three biomarkers was selected based on their collective contribution to the optimal separation between stage 0-I breast cancer patients and noncancer controls. The same separation was observed using independent test data from stage II-III breast cancer patients. Bootstrap cross-validation demonstrated that a sensitivity of 93% for all cancer patients and a specificity of 91% for all controls were achieved by a composite index derived by multivariate logistic regression using the three selected biomarkers. CONCLUSIONS: Proteomics approaches such as SELDI mass spectrometry, in conjunction with bioinformatics tools, could greatly facilitate the discovery of new and better biomarkers. The high sensitivity and specificity achieved by the combined use of the selected biomarkers show great potential for the early detection of breast cancer.     

Comparative study of patients with and without SARS who fulfilled the WHO SARS case definition

To differentiate severe acute respiratory syndrome (SARS) from non-SARS illness, we retrospectively compared 53 patients with probable SARS and 31 patients with non-SARS who were admitted to Mackay Memorial Hospital from April 27 to June 16, 2003. Fever (> 38°C) was the earliest symptom (50/53 SARS vs. 5/31 non-SARS, p < 0.0001), preceding cough by a mean of 4.5 days. The initial chest X-ray study was normal in 22/53 SARS cases versus 5/31 non-SARS cases. SARS patients with an initially normal chest X-ray study developed infiltrates at a mean of 5 ± 3.44 days after onset of fever (21/22 SARS vs. 0/5 non-SARS). Rapid radiographic progression of unifocal involvement to multifocal infiltrates was seen in 22 of 24 SARS vs. 0 of 26 non-SARS patients (p < 0.0001). Pleural effusion was not present in any SARS patients but was seen in 6 of 26 non-SARS cases (p < 0.0001). Initial lymphopenia, thrombocytopenia, and elevated lactate dehydrogenase were all more common in SARS than non-SARS (p < 0.0001). They may help differentiate SARS from non-SARS if a reliable and rapid diagnostic test is not available.     

中性粒细胞碱性磷酸酶在传染性非典型肺炎诊断与鉴别诊断中的价值

目的　探讨中性粒细胞碱性磷酸酶 (alkalinephosphatase ,ALP)活性在传染性非典型肺炎 (严重急性呼吸综合征 ,SARS)患者诊断与鉴别诊断中的应用价值。方法　抽取 138例临床确诊的SARS患者、14 4例除外SARS的其他发热患者和 15 7名正常健康人 (男 80例 ,女 77例 )静脉血 ,制备血涂片。所有血涂片依改良Gomori氏法进行ALP细胞免疫组织化学染色 ,显微镜油镜观察 10 0个中性粒细胞 ,以阳性率和积分记录结果。结果　正常健康人群中性粒细胞碱性磷酸酶染色 (NAP)阳性率和积分中位数均为 10 0 0 ,差异无统计学意义 (P >0 0 5 ,P >0 0 5 ) ;非SARS发热患者组、SARS患者组中性粒细胞ALP阳性率 (中位数 )分别为 4 0 0 0、16 0 0 ,积分分别为 5 0 5 0、17 0 0 ,经统计学处理表明 ,不论NAP阳性率还是积分 ,非SARS发热患者高于SARS患者 ,SARS患者高于正常对照 (P<0 0 1)。结论　正常健康人群、非SARS发热患者、SARS患者间中性粒细胞碱性磷酸酶存在差异 ,将中性粒细胞碱性磷酸酶用于SARS的诊断与鉴别诊断有待进一步观察。     

Boosted decision tree analysis of surface-enhanced laser desorption/ionization mass spectral serum profiles discriminates prostate cancer from noncancer patients

BACKGROUND: The low specificity of the prostate-specific antigen (PSA) test makes it a poor biomarker for early detection of prostate cancer (PCA). Because single biomarkers most likely will not be found that are expressed by all genetic forms of PCA, we evaluated and developed a proteomic approach for the simultaneous detection and analysis of multiple proteins for the differentiation of PCA from noncancer patients. METHODS: Serum samples from 386 men [197 with PCA, 92 with benign prostatic hyperplasia (BPH), and 96 healthy individuals], randomly divided into training (n = 326) and test (n = 60) sets, were analyzed by surface-enhanced laser desorption/ionization (SELDI) mass spectrometry. The 124 peaks detected by computer analyses were analyzed in the training set by a boosting tree algorithm to develop a classifier for separating PCA from the noncancer groups. The classifier was then challenged with the test set (30 PCA samples, 15 BPH samples, 15 samples from healthy men) to determine the validity and accuracy of the classification system. RESULTS: Two classifiers were developed. The AdaBoost classifier completely separated the PCA from the noncancer samples, achieving 100% sensitivity and specificity. The second classifier, the Boosted Decision Stump Feature Selection classifier, was easier to interpret and used only 21 (compared with 74) peaks and a combination of 21 (vs 500) base classifiers to achieve a sensitivity and specificity of 97% for the test set. CONCLUSIONS: The high sensitivity and specificity achieved in this study provides support of the potential for SELDI, coupled with a bioinformatics learning algorithm, to improve the early detection/diagnosis of PCA.     

SARS流行期发热患者的诊治及影响因素分析

目的：对严重急性呼吸综合征(SARS)流行期发热患者的病因构成、诊治状况及影响因素进行分析，探讨SARS及发热相关疾病的早期诊断及鉴别诊断方法。方法：将2003年3月15日—5月10日北京大学附属第三医院急诊科接诊的4060例发热患者流行病学及临床资料进行总结分析，并评价其影响因素的作用。结果：4060例发热患者中，被收入隔离病房者275例，145例确诊为SARS(占3．57％)，29例为疑似病例，排除诊断101例。诊断非SARS性肺感染93例，并诊断流行性脑膜炎2例，化脓性脑膜炎8例，流行性出血热3例。结论：SARS高发期发热患者中约90％的病因为流行性感冒或其他病毒感染，非SARS性肺感染及发热相关传染病占有一定比例。应提高发热的鉴别诊断意识和水平，白细胞升高不是排除SARS的诊断依据，X线胸片异常可不与发热同时出现，必须进行胸片复查。     

Evaluation of the Wampole Laboratories ELISA-based assay for Epstein-Barr virus serology

BACKGROUND: Epstein-Barr virus (EBV) infection is associated with infectious mononucleosis, Burkitt's lymphoma, and nasopharyngeal carcinoma. Serologic diagnosis of acute EBV infections has been the method of choice, and tests are available as indirect fluorescent antibody (IFA)- and ELISA-based assays. OBJECTIVE: To evaluate the ELISA-based EBV assay from Wampole Laboratories (Cranbury, NJ). METHODS: One hundred fifty-two consecutive samples received for comprehensive EBV serology were analyzed. RESULTS: A comparison of the Wampole Laboratories' ELISA system with the Gull/Meridian Diagnostics (Cincinnati, OH) IFA, and ELISA assays showed 88% concordance for anti-viral capsid antigen (VCA) IgM (n=177); 79% concordance for anti-VCA IgG (n=177); 87% concordance for anti-NA IgG (n=172); and 48% concordance for anti-EA IgG (n=165). Using the results from all four antibody assays to identify patients with acute infection, the Wampole system had a 67% concordance with the Gull/Meridian (n=164). CONCLUSIONS: Differences in the specificity of the anti-EA IgG assays (i.e. reactivity against the D component of early antigen (EA-D) (Wampole) vs. reactivity to EA-D and the R component of early antigen (EA-R) (Gull/Meridian)) may have lead to poor concordance (48%) for this particular assay. Because the Wampole system had a <70% overall concordance with the Gull/Meridian system in diagnosis of acute infection, these data suggest that further studies are needed to determine the true clinical sensitivity and specificity of this system.     

Recurrence and persistence of fever in children who developed amantadine-resistant influenza viruses after treatment

In recent years, a dramatic increase of amantadine-resistant influenza A has occurred globally, but limited data have been available on the clinical course of patients developed amantadine-resistant viruses. We compared fever reduction between patients who developed resistance or remained sensitive in a pediatric clinic in Niigata, Japan, from 2000 to 2006. A total of 2,802 clinical samples were collected from patients who visited the pediatric outpatient clinic with influenza like illness during the seven influenza epidemic seasons. Patients were divided into 4 groups and analyzed for the fever reduction after amantadine treatment: emerged amantadine-resistant (n = 15); amantadine-sensitive (n = 35); patients administered no antiviral drugs (n = 42); and oseltamivir-treated patients (n = 320), which served as references. All 4 groups showed alleviation of fever up to day 3. The amantadine-resistant group had a significant recurrence of fever on day 4 and/or 5, and as a consequence, the course of illness was prolonged. Considering the pattern of fever, recurrent and persistent patterns were found significantly at higher rates in children with emerged resistant virus compared to other groups, and the age tended to be younger in amantadine-resistant compared to amantadine-sensitive group (3.9 +/- 3.0 vs 6.7 +/- 4.1 years old, n.s.). Therefore, we concluded that younger children were prone to develop amantadine-resistance after treatment and showed a significant recurrence of fever on day 4 and/or 5, and the course of illness was consequently prolonged.     

10例成人社区获得性病毒性肺炎分析

目的：在严重急性呼吸综合征(SARS)流行的背景下，为SARS和其它病毒性肺炎进行诊断和鉴别诊断提供依据。方法：对上海市第五人民医院2003年4月12日～6月5日收治的10例成人社区获得性病毒性肺炎流行病学史、临床特征、实验室检查、微生物学检查、影像学、治疗和预后进行回顾性分析。结果：符合SARS流行病学特点1例(10％)，符合流感流行病学特点3例(30％)。主要临床症状：发热(100％)、咳嗽(70％)。就诊时白细胞(WBC)计数减低3例(30％)，中性粒细胞百分比(NEUT％)增高5例(50％)，淋巴细胞百分比(LYMPH％)减少者3例(30％)。病原学检查：流感病毒7例(70％)，副流感病毒2例(20％)，1例SARS病原学检查结果阴性。就诊时异常的胸部X线片表现以肺实质阴影为主。进展3例(30％)，阴影在下肺9例(90％)、心缘旁3例(30％)、单侧9例(90％)、双侧1例(10％)。全部病例经治疗后痊愈出院。结论：相对SARS，成人社区获得性流感病毒性肺炎具有发热不反复、肺内病灶单发、病程短、预后好的特点，对SARS的诊断更应注意流行病学及恰当时机反复病原学检测。     

流感病毒快速检测法在北京地区2007-2008年流感季节中的应用

目的 分析评价采用流感病毒快速检测法诊治流感的应用价值.方法 前瞻性选择2007年12月-2008年3月流感季节在北京朝阳医院发热门诊就诊的符合卫生部流感样病例患者500例,留取咽部分泌物进行流感病毒培养,随机选取260例患者进行流感病毒快速检测,并调查患者性别、年龄、症状、实验室检查、症状恢复时间、治疗花费等因素.同时分析流感病毒培养阳性组与流感病毒快速检测组的敏感度、特异度、阳性预测值和阴性预测值,及对治疗方案和预后的影响.结果 2007-2008年流感季节共入选流感样病例500例,最终498例纳入分析,病毒培养结果主要为乙型流感(208例,41.8%),而甲型流感少见(51例,10.2%).498例流感样患者的平均年龄为35岁,男:女比例为1.47:1.与培养阴性组比较,培养阳性组咳嗽、咽痛、鼻塞症状比例比较高(t值分别为13.728、4.014和4.720,P均<0.01或0.05).260例经流感病毒快速检测患者中,甲型流感抗原阳性18例、乙型流感抗原阳性132例,流感病毒快速检测的敏感度77.1%、特异度70.1%、阳性预测值78.6%、阴性预测值68.2%.流感病毒快速检测抗病毒治疗的比例由0提高到26%,抗生素使用比例由63.4%降到20.7%.结论 2007-2008年流感季节主要流感类型为乙型流感,流感病毒快速检测法对临床病例检测敏感度和特异度较高,对临床抗病毒诊治有指导意义.     

医院内SARS暴发流行的临床分析

目的：总结严重急性呼吸综合征(SARS，或称传染性非典型肺炎，IAP)的临床表现、诊断和治疗，以及外界输入性病例引起暴发流行的传染链特点。方法：对2例外院输入病例的临床资料和传染链进行分析，并总结41例SARS患者的临床资料。结果：①SARS患者的传染性极强，防范不当可使大量人群感染；②SARS的临床特征主要是发热(100．0％)，有相当部分患者在发病初期(1—3d)呼吸道症状并不明显(占53．7％)，并伴有外周血白细胞(52．6％)和淋巴细胞(68．4％)比例减少；②早期给予利巴韦林、甲基氢化泼尼松和免疫增强治疗对控制病情的进一步发展有一定效果。结论：重视防范、早发现、早隔离和早治疗，对改善SARS的预后和控制其蔓延有着十分重要的意义。     

广州地区SARS流行期和非流行期献血人群SARS病毒抗体的流行病学调查

目的　分析广州地区SARS流行期和非流行期无偿献血人群中严重急性呼吸道综合征(SARS)病毒感染 情况,为制定预防输血传播SARS病毒措施提供科学依据。方法　采用酶联免疫吸附试验(ELISA)对无偿献血者血 液进行SARS病毒抗体筛查,对SARS病毒抗体阳性样本用荧光聚合酶链反应(PCR)法进一步检测SARS病毒核 酸。采用统一的个案调查表对20名SARS病毒抗体阳性无偿献血者进行电话咨询调查,同时对31名SARS康复献 浆者进行检测,分析相关数据作对照。结果　6120名无偿献血者中,共检测出SARS病毒抗体阳性56例,阳性率为 0.92%,31名SARS康复献浆者中,检测出SARS病毒抗体阳性30例,阳性率为96.77%;SARS流行期和非流行期 无偿献血者SARS病毒抗体阳性率分别为0.91%和0.92%,56名无偿献血者SARS病毒抗体阳性的平均S/CO值 (2.34)和抗体平均滴度(≤1∶2)均明显低于30名SARS康复献浆者的平均S/CO值(14.8)和抗体平均滴度(≤1∶ 32);56例SARS病毒抗体阳性无偿献血者血液样本均未检测出SARS病毒核酸;20例SARS病毒抗体阳性无偿献 血者的调查显示:献血者身体健康,无SARS患者密切接触史。结论　广州地区无偿献血人群中存在的低水平 SARS病毒抗体阳性率,是否表明SARS病毒抗体阳性献血者曾经感染过SARS病毒,尚需进一步     

35例SARS患者临床特征分析

目的 :探讨严重急性呼吸综合征 (SARS)的临床特征。方法 :回顾分析过去 5个月里我院收治的 35例SARS患者的临床特征 ,并与普通的社区获得性肺炎患者进行比较。同时用流式细胞仪检测了 13例 SARS患者和 10例健康志愿者的外周血 CD+ 4和 CD+ 8T淋巴细胞。结果 :SARS最常见的表现为发热 (10 0 .0 0 % )、咳嗽(74 .30 % )、头痛 (4 5 .70 % )、全身肌肉酸痛 (4 5 .70 % )及淋巴细胞减少 (2 0 /33)。 X线胸片表现为进行性发展的多发性肺浸润病灶。 13例 SARS患者外周血 CD+ 4和 CD+ 8明显降低〔 CD+ 4为 (16 .10± 4 .31) %比 (38.30±8.5 2 ) % ,CD+ 8为 (19.90± 5 .4 0 ) %比 (2 8.38± 4 .33) % ;t值分别为 8.174和 4 .0 5 5 ,P均 <0 .0 1〕,SARS组患者平均退热时间较肺炎组长〔(13.92± 8.35 ) d比 (3.86± 1.4 2 ) d,t=16 .4 90 ,P=0 .0 0 0〕;肺总病变吸收时间也明显延长〔(11.97± 4 .4 1) d比 (9.2 1± 4 .4 2 ) d,t=3.0 82 ,P=0 .0 0 3〕。结论 :SARS患者主要表现为发热、咳嗽、头痛、全身肌肉酸痛 ,外周血淋巴细胞计数减少 ,CD+ 4和 CD+ 8细胞明显降低 ,肺内多发性浸润病灶。     

Quality control and peak finding for proteomics data collected from nipple aspirate fluid by surface-enhanced laser desorption and ionization

BACKGROUND: Recently, researchers have been using mass spectroscopy to study cancer. For use of proteomics spectra in a clinical setting, stringent quality-control procedures will be needed. METHODS: We pooled samples of nipple aspirate fluid from healthy breasts and breasts with cancer to prepare a control sample. Aliquots of the control sample were used on two spots on each of three IMAC ProteinChip arrays (Ciphergen Biosystems, Inc.) on 4 successive days to generate 24 SELDI spectra. In 36 subsequent experiments, the control sample was applied to two spots of each ProteinChip array, and the resulting spectra were analyzed to determine how closely they agreed with the original 24 spectra. RESULTS: We describe novel algorithms that (a) locate peaks in unprocessed proteomics spectra and (b) iteratively combine peak detection with baseline correction. These algorithms detected approximately 200 peaks per spectrum, 68 of which are detected in all 24 original spectra. The peaks were highly correlated across samples. Moreover, we could explain 80% of the variance, using only six principal components. Using a criterion that rejects a chip if the Mahalanobis distance from both control spectra to the center of the six-dimensional principal component space exceeds the 95% confidence limit threshold, we rejected 5 of the 36 chips. CONCLUSIONS: Mahalanobis distance in principal component space provides a method for assessing the reproducibility of proteomics spectra that is robust, effective, easily computed, and statistically sound.     

SARS病人临床及细胞免疫特征分析

【目的】探讨严重急性呼吸综合征(SARS)的临床和细胞免疫特征。【方法】回顾分析本院收治的35例SARS病人的临床特征,并与普通肺炎病人进行比较。同时用流式细胞仪检测了13例SARS病人和10例健康志愿者的外周血CD4^ 和CD8^ T淋巴细胞。【结果】SARS病人最常见的表现为发热、咳嗽、头痛、全身肌肉酸痛,淋巴细胞减少。13例SARS病人外周血CD4^ 和CD8^ 自发病第1周至第四周均明显降低,以发病第2周最明显。胸部X线表现为进行性发展的多发性肺浸润病灶。【结论】SARS是一个严重的呼吸系疾病,主要表现为发热、咳嗽、头痛、全身肌肉酸痛。检查有外周血淋巴细胞计数减少,CD4^ 和CD8^ 明显降低,肺内多发性浸润病灶。     

自身免疫病患者血清中传染性非典型肺炎冠状病毒抗体阳性原因分析

目的 探讨传染性非典型肺炎又称严重急性呼吸综合征(SARS),冠状病毒(SARS-CoV)抗体在SARS病原学诊断中的特异性及其在系统性红斑狼疮(SLE)、类风湿性关节炎(RA)、干燥综合征(SS)和混合结缔组织病(MCTD)患者中的假阳性问题。方法 应用酶联免疫吸附试验(ELISA)和荧光定量RT-PCR技术检测了111名正常对照和58例SLE、20例RA、10例SS、16例MCTD患者做血清中SARS-CoV抗体的检测。结果 在111名正常对照中,IgM抗体均阴性,IgG抗体的阳性率为3.6％;IgG抗体诊断SARS的特异性为96.4％,两种抗体同时阳性诊断SARS的特异性为100％。58例SLE患者中,IgM抗体和IgG抗体阳性率分别为8.6％和32.8％,IgG抗体和IgM抗体同时阳性为19％;在10例SS患者中只有1例两种抗体同时阳性(10％);在16例MCTD患者中,IgG抗体阳性6例(37.5％),两种抗体同时阳性1例(6.3％);在20例RA患者中只有1例IgG抗体阳性(5％)。经RT-PCR检测,上述自身免疫病患者中的阳性病例血清SARS-CoV抗体均为阴性。结论用非纯化抗原制备的ELISA试剂盒测定自身免疫病患者的SARS-CoV抗体,可能出现假阳性,两种抗体同时测定可降低诊断的假阳性率,提高诊断的特异性。在自身免疫病患者中出现假阳性的原因可能与包被的抗原有关。     

IFA和ELISA在肿瘤患者血清SARS-CoV抗体测定中的对比分析

目的探讨酶联免疫吸附试验(ELISA)和间接免疫荧光法(IFA)检测严重急性呼吸综合征(SARS)冠状病毒(SARS-CoY)&体在SARS病原中诊断中的特异性及其在肿瘤患者血清中的假阳性问题。方法 应用ELISA和IFA检测了111例正常对照和40例肿瘤患者血清中SARS-CoV抗体的阳性率。结果在111例正常对照和40例肿瘤患者中,IgM抗体均阴性,IgG抗体在正常对照中的阳性率为3.6%(4/111),在肿瘤患者中的阳性率17.5％(7/40),IgC抗体诊断SARS的特异性为96.4％,两种抗体同时阳性诊断SARS的特异性为100％。经IFA检测,上述SARS-CoV抗体阳性者均为阴性。结论 IFA诊断SARS的特异性为100％,ELISA诊断SARS存在一定的假阳性。应用ELISA测定SARS-CoV抗体时,应同时测定两种抗体以降低诊断的假阳性率,提高诊断的特异性。