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好医生医师节答案1.重症肺炎时期，哪一项不是可能引发的疾病(B)A.急性呼吸窘迫综合征B.急性心肌梗死c.急性肾功能衰竭D.多脏器功能衰竭2.下列关于H7N9禽流感病毒对外界抵抗力的描述准确的是：A.较低温度粪便中可存活4周B.在4℃水中只可存活1个月c.加热至65℃15分钟或煮沸(100℃)2分钟以上可灭活D.加热至65℃30分钟或煮沸(100℃)2分钟以上可灭活5.禽流感患者体温大多持续在(B)℃以上A.38 B.39 C.40 D.426.禽流感患者使用Zanamivir适用于(C)岁以上人群A.3 B.5 C.7D.9D7.截至2016年1月，至少在非洲、亚洲、美洲的(C)个国家有寨卡病毒传播的证据A.30B.35 C.45 D.508.主要分布于海南省、广东省雷州半岛以及云南省的西双版纳州、德宏州、临沧市等地区是(A)伊蚊A.埃及伊蚊B.白纹伊蚊C.非洲伊蚊D.黄头伊蚊9.寨卡病毒病通常症状较轻，不需要做出特别处理，以(D)为主A.抗炎治疗B.抗菌治疗C.解热镇痛治疗D.对症治疗10.以下关于埃博拉出血热错误的是(C)A.病毒分离应采集发病一周内患者血清标本，用Vero细胞进行病毒分离B.发病前21天内，在没有恰当个人防护的情况下，接触过埃博拉患者的血液、体液、分泌物、排泄物或尸体等符合其流行病学c.疑似病例应及时采集标本，并送至所在医院检验科进行病原学检测D.尚无特异性治疗措施，主要是对症和支持治疗11.未解除隔离的患者死亡后，应当及时对尸体进行处理。
处理方法为：用双层布单包裹尸体，装入双层尸体袋中，由(D)直接送至指定地点火化。
A.救护车B.冷冻车c.普通车辆D.专用车辆12.(C)应当符合上级卫生计生行政部门的设置条件及《医院隔离技术规范》等有关要求。
A.建筑布局B.工作流程C.建筑布局和工作流程D.建筑布局、医疗器械的使用规范和工作流程13.截止到(D)年，韩国卫生福利部通报该国确诊病例达108例。
高二英语科技词汇单选题40题(带答案)1.The new smartphone has a large _____.A.screenB.keyboardC.mouseD.printer答案：A。
“screen”是屏幕，新智能手机有一个大屏幕，符合常理。
“keyboard”是键盘，“mouse”是鼠标，“printer”是打印机，都与智能手机不匹配。
2.We can use a _____ to take pictures.puterB.cameraC.televisionD.radio答案：B。
“camera”是相机，可以用来拍照。
“computer”是电脑，“television”是电视，“radio”是收音机，都不能用来拍照。
3.The _____ can play music and videos.ptopB.speakerC.projectorD.scanner答案：A。
“laptop”是笔记本电脑，可以播放音乐和视频。
“speaker”是扬声器，“projector”是投影仪，“scanner”是扫描仪，都不能播放音乐和视频。
4.My father bought a new _____.A.tabletB.bookC.penD.pencil答案：A。
“tablet”是平板电脑。
“book”是书，“pen”是钢笔，“pencil”是铅笔，只有平板电脑是科技设备。
5.The _____ is very useful for online meetings.A.headphoneB.microphoneC.speakerD.camera答案：D。
“camera”摄像头在在线会议中很有用。
“headphone”是耳机，“microphone”是麦克风，“speaker”是扬声器，都不如摄像头在在线会议中的作用直接。
6.We can store a lot of data in a _____.A.flash driveB.penC.pencilD.book答案：A。
Codevelopment of Two or More Unmarketed Investigational Drugs for Use in CombinationDRAFT GUIDANCEThis guidance document is being distributed for comment purposes only. Comments and suggestions regarding this draft document should be submitted within 60 days of publication in the Federal Register of the notice announcing the availability of the draft guidance. Submit comments to the Division of Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, rm. 1061, Rockville, MD 20852. All comments should be identified with the docket number listed in the notice of availability that publishes in the Federal Register.For questions regarding this draft document contact (CDER) Colleen Locicero 301-796-1114.U.S. Department of Health and Human ServicesFood and Drug AdministrationCenter for Drug Evaluation and Research (CDER)December 2010Clinical MedicalCodevelopment of Two or More Unmarketed Investigational Drugs for Use in CombinationAdditional copies are available from:Office of CommunicationsDivision of Drug Information, WO51, Room 220110903 New Hampshire Ave.Silver Spring, MD 20993Phone: 301-796-3400; Fax: 301-847-8714druginfo@/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htmU.S. Department of Health and Human ServicesFood and Drug AdministrationCenter for Drug Evaluation and Research (CDER)December 2010Clinical MedicalTable of Contents 1234 5 6 7 8 910111213141516171819202122 I.INTRODUCTION (1)II.BACKGROUND (2)III.DETERMINING WHETHER CODEVELOPMENT IS AN APPROPRIATEDEVELOPMENTOPTION (2)IV.NONCLINICAL CODEVELOPMENT (3)A.Demonstrating the Biological Rationale for the Combination (3)B.Nonclinical Safety Characterization (3)V.CLINICAL CODEVELOPMENT (4)A.Early Human Studies (Phase 1) (4)1.Safety of the Individual Components (4)2.Safety and Dosing of the Combination (5)B.Clinical Pharmacology (5)C.Proof of Concept Studies (Phase 2) (6)D. Confirmatory Studies (Phase 3) (8)VI.REGULATORY PROCESS ISSUES IN CODEVELOPMENT (8)A. Early Interaction with FDA (8)B. IND Submissions and Marketing Applications (9)beling Issues (9)D.Pharmacovigilance (9)232425262728Guidance for Industry1Codevelopment of Two or More Unmarketed Investigational Drugsfor Use in Combination2930 This draft guidance, when finalized, will represent the Food and Drug Administration’s (FDA’s) current31 thinking on this topic. It does not create or confer any rights for or on any person and does not operate to32 bind FDA or the public. You can use an alternative approach if the approach satisfies the requirements of33 the applicable statutes and regulations. If you want to discuss an alternative approach, contact the FDA34 staff responsible for implementing this guidance. If you cannot identify the appropriate FDA staff, call35 the appropriate number listed on the title page of this guidance.36373839404142434445464748495051525354555657 I. INTRODUCTIONThis guidance is intended to assist sponsors in the codevelopment2 of two or more novel (not previously marketed) drugs to be used in combination to treat a disease or condition. The guidance provides recommendations and advice on how to address certain scientific and regulatory issues that will arise during codevelopment. It is not intended to apply to development of fixed-dose combinations of already marketed drugs or to development of a single new investigational drug to be used in combination with an approved drug or drugs. The guidance is also not intended to apply to vaccines, gene or cellular therapies, blood products, or medical devices.3FDA’s guidance documents, including this guidance, do not establish legally enforceable responsibilities. Instead, guidances describe the Agency’s current thinking on a topic and should be viewed only as recommendations, unless specific regulatory or statutory requirements are cited. The use of the word should in Agency guidances means that something is suggested or recommended, but not required.1 This guidance has been prepared by the Office of Medical Policy in the Center for Drug Evaluation and Research (CDER) at the Food and Drug Administration.2Codevelopment herein refers to the concurrent development of two or more drug products with the intent that the products be used in combination to treat a disease or condition.3 For purposes of this guidance, the term drug includes therapeutic biological products that are regulated by CDER. Consult the Therapeutic Biologics web page for further information on the types of biological products to which this guidance applies:/Drugs/DevelopmentApprovalProcess/HowDrugsareDevelopedandApproved/ApprovalApplications/T herapeuticBiologicApplications/default.htmII. BACKGROUND585960616263646566676869707172737475767778798081828384858687888990919293949596979899 100 101 102 103 Combination therapy is an important treatment modality in many disease settings, including cancer, cardio-vascular disease, and infectious diseases. Recent scientific advances have increased our understanding of the pathophysiological processes that underlie these and other complex diseases. This increased understanding has provided further impetus for new therapeutic approaches using combinations of drugs directed at multiple therapeutic targets to improve treatment response or minimize development of resistance. In settings in which combination therapy provides significant therapeutic advantages, there is growing interest in the development of combinations of investigational drugs not previously developed for any purpose.Because the existing developmental and regulatory paradigm focuses primarily on assessment of the effectiveness and safety of a single new investigational drug acting alone, or in combination with an approved drug, FDA believes guidance is needed to assist sponsors in the codevelopment of two or more unmarketed drugs. Although interest in codevelopment has been most prominent in oncology and infectious disease settings, codevelopment also has potential application in other therapeutic settings. Therefore, this guidance is intended to describe a high-level, generally applicable approach to codevelopment of two or more unmarketed drugs. It describes the criteria for determining when codevelopment is an appropriate option, makes recommendations about nonclinical and clinical development strategies, and addresses certain regulatory process issues. III. DETERMININGWHETHERCODEVELOPMENT IS AN APPROPRIATE DEVELOPMENT OPTIONConcurrent development of two or more novel drugs for use in combination generally will provide less information about the safety and effectiveness of the individual drugs than would be obtained if the individual drugs were developed alone. How much less will vary depending on a variety of factors, including the stage of development at which the individual drug components cease to be studied independently. For example, in codevelopment scenarios in which rapid development of resistance to monotherapy is a major concern, it may not be possible or appropriate to obtain clinical data for the individual components of the combination beyond phase 1 testing. Because codevelopment will generally provide less information about the safety and effectiveness of the individual drugs, it will present greater risk compared to development of an individual drug. Therefore, FDA believes that codevelopment should ordinarily be reserved for situations that meet the following criteria:•The combination is intended to treat a serious disease or condition.•There is a compelling biological rationale for use of the combination (e.g., the agents inhibit distinct targets in the same molecular pathway, provide inhibition of both aprimary and compensatory pathway, or inhibit the same target at different binding sites to decrease resistance or allow use of lower doses to minimize toxicity).• A preclinical model (in vivo or in vitro) or short-term clinical study on an established biomarker suggests that the combination has substantial activity and provides greater thanadditive activity or a more durable response (e.g., delayed resistance) compared to the individual agents alone. 104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146 • There is a compelling reason for why the agents cannot be developed individually (e.g., monotherapy for the disease of interest leads to resistance and/or one or both of the agents would be expected to have very limited activity when used as monotherapy). FDA recommends that sponsors consult with FDA on the appropriateness of codevelopment before initiation of clinical development of the combination. IV. NONCLINICAL CODEVELOPMENT A. Demonstrating the Biological Rationale for the Combination The biology of the disease, pathogen, or tumor type should be sufficiently understood to provide a plausible biological rationale for the use of combination therapy to treat the disease or condition. For example, in an oncology setting the biological rationale may be to intervene at different steps in the cell proliferation pathway. The biological rationale for a combination anti-infective therapy may be to target different metabolic pathways or different steps in the replication cycle of the pathogen to reduce the chance of developing resistance to the therapy or increase efficacy in treating disease caused by resistant organisms (e.g., multidrug-resistant atypical tuberculosis). Sponsors should develop evidence to support the biological rationale for the combination in an in vivo (preferable) or in vitro model. The model should compare the activity of the combination to the activity of the individual components. Ordinarily, the model should demonstrate that, compared to the individual components, the combination has substantial activity and provides greater than additive activity or a more durable response in a pathophysiological process considered pertinent to the drug’s intended use in humans. An animal model of activity generally would not be necessary. However, if there is an animal model relevant to the human disease, valuable activity data, as well as information about the relative doses of the drugs, might be obtained from evaluating the combination in that model. B. Nonclinical Safety Characterization For detailed recommendations regarding nonclinical safety characterization for two or more investigational drugs to be used in combination, sponsors should consult the recently revised International Conference on Harmonisation (ICH) Guidance on Nonclinical Safety Studies.4 Section XVII of that guidance (Combination Drug Toxicity Testing) includes a discussion of nonclinical safety studies appropriate in a combination drug development setting involving two early stage entities. The ICH guidance defines early stage entities as compounds with limited clinical experience (i.e., phase 2 studies or less), so the discussion is specifically applicable to the4 Guidance for Industry: M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials andMarketing Authorization, January 2010 (this guidance is a revision of 1997 ICH guidance M3: Nonclinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals).147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 type of development described in this guidance. In situations in which it is possible to obtain only limited clinical data for the individual drugs, additional nonclinical data for the individual drugs or combination may be needed before beginning human studies with the combination. (e.g., see section V.A.1).V. CLINICAL CODEVELOPMENTThis section provides a general roadmap and guiding principles for concurrent clinical development of two or more investigational drugs to be used in combination. It includes recommendations for characterizing the clinical safety and effectiveness of the combination and, to the extent needed or possible, the individual components of the combination.Note: The appropriate review division should always be consulted on the specifics of a given clinical development program.A. Early Human Studies (Phase 1)The main objectives of early studies in humans are to characterize the safety and pharmacokinetics of the individual components and then the combination and to provide data to support appropriate dosing for the combination in phase 2 testing.1. Safety of the Individual ComponentsWhenever possible, the safety profile of each individual drug should be characterized in phase 1 studies in healthy volunteers in the same manner as would be done fordevelopment of a single drug, including determination of the maximum tolerated dose(MTD), the nature of the dose limiting toxicity (DLT), and pharmacokinetic parameters.If there is a useful measure (e.g., biomarker) of pharmacologic activity, it will also beimportant to determine dose-response for that measure. If testing in healthy volunteers is not possible (e.g., if nonclinical data suggest a drug may be genotoxic or otherwiseunacceptable for studies in healthy volunteers), the safety profile of the individual drugs should be evaluated in patients with the disease of interest. These safety data will guide decisions in later studies about starting doses, dose escalation increments, and final dose selection.If it is not possible to characterize the safety of the individual drugs in humans (e.g.,where drug toxicity prevents use of healthy volunteers and monotherapy would beunethical in patients with the disease of interest), the sponsor should conduct nonclinical studies of the combination to support initial dosing of the combination in humans.The nonclinical data for the combination should include pharmacokinetic (absorption,distribution, metabolism, and excretion) and toxicokinetic data and appropriatebiomarker/target inhibition, if relevant.2. Safety and Dosing of the Combination 193194195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237For initial human effectiveness studies of the combination, the combination starting dose, dosing escalation intervals, and doses to be used in dose-response studies should bedetermined based on phase 1 safety data for the individual components, if available. Ifphase 1 safety data for the components are unavailable, nonclinical data for thecombination will be needed to determine the initial combination dose in humans (seeprevious paragraph). Phase 1 safety studies of the combination could also be conducted — for example, sequential testing in which subjects get drug A, then drug B, then AB — to support dosing in subsequent studies.B. ClinicalPharmacologyThe sponsor should conduct the same clinical pharmacology studies for each of the individual drugs in the combination as would be done if the drugs were being developed separately. In general, such studies include the assessment of bioavailability, characterization of pharmacokinetics, mass balance, the evaluation of effects of intrinsic (such as renal impairment and hepatic impairment) and extrinsic (such as food effect and drug interactions) factors on pharmacokinetics or pharmacodynamics, and exposure-response. Studies to address intrinsic and extrinsic factors could be conducted with the combination instead of the individual drugs.The evaluation of drug interaction potential follows the same sequence as in other development programs; results of in vitro drug metabolism and drug transporter studies inform the need for in vivo drug interaction studies. The role of pharmacogenomics should be investigated and incorporated into the combination drug development plan to identify potential sources of pharmacokinetic or pharmacodynamic variability.Dose-response should be evaluated for each drug of the combination. The results of such studies should be used to determine doses to further explore for the combination. If the drug products cannot be administered alone, various doses of each drug administered as the combination should be assessed.If one drug has no activity or minimal activity by itself, dose-response should be assessed when the drug products are administered in combination using a number of doses of the active drug and the inactive drug. The same approach should be used in evaluating dose-response for the combination of drugs where each drug has minimal activity when used alone.In addition to evaluating dose-response, response should be evaluated with respect to systemic drug concentration to provide insight into efficacy and safety as a function of drug exposure. Concentration-response assessments should be done in both phase 2 and phase 3 trials. To increase exposure ranges in phase 3 and to further assess dose-response, the incorporation of more than one dose of each of the drugs used in the combination in the phase 3 trials should be considered.C. Proof of Concept Studies (Phase 2) 238239240 241 242 243 244 245 246247 248 249 250 251 252 253 254 255 256 257 258 In general, phase 2 testing should accomplish the following to the extent needed for a given combination (e.g., to the extent not sufficiently established by existing data): •Demonstrate the contribution of each component of the combination to the extent possible and needed (given available nonclinical and pharmacologic data);•Provide evidence of the effectiveness of the combination; and•Optimize the dose or doses of the combination for phase 3 trials.The amount and types of clinical data needed and appropriate study designs will vary depending on the nature of the combination being developed, the disease, and other factors. For the types of combinations contemplated by this guidance, it will often be inappropriate to use monotherapy treatment arms in studies of the disease of interest, or it will be possible to administer the components of the combination as monotherapy only for short durations. In these circumstances, the study design typically employed to determine the contributions of the components to the combination — a four-arm factorial design comparing the combination to individual components and placebo or standard of care (SOC) therapy (AB v. A. v. B v. placebo or SOC) — will have limited utility. The following scenarios illustrate possible phase 2 study designs for combinations of two investigational drugs in different situations.Scenario 1: The components of the combination cannot be administered individually259 260261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278If in vivo or in vitro models, or phase 1 or other early clinical studies make clear that the components of the combination cannot be administered individually in clinical trials inthe disease of interest (e.g., because such testing would involve administering treatment known to be ineffective as monotherapy), or can’t be administered as monotherapy forthe duration needed to evaluate effectiveness (e.g., because of rapid development ofresistance), proof-of-concept evidence for the combination ordinarily should come froma study directly comparing the combination (AB) to SOC. Alternatively, if SOC isknown to be an effective therapy (not solely palliative), an add-on design could be used comparing the combination plus SOC to SOC alone.In some resistance scenarios, it may be possible to administer the individual drugs in acombination as monotherapy for a short duration, but long enough to establish proof ofconcept in humans. For example, direct-acting antivirals (DAAs) to treat chronichepatitis C virus infection can be administered as monotherapy for three days to establish antiviral activity and for initial dose exploration. For DAA studies of longer duration, the combination should be used or the individual components should be added to an activecontrol.55 See draft guidance for industry: Chronic Hepatitis C Virus Infection: Developing Direct-Acting Antiviral Agents for Treatment (section III. 4. b. – Phase 1b (proof-of-concept) trials) or consult the Division of Antiviral Drug Products in CDER for more specific recommendations.279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 Scenario 2: Each drug alone has activity and can be administered individuallyIf in vivo or in vitro models, or phase 1 or other early clinical studies indicate that each drug has some activity, but the combination appears to have greater than additive activity, and rapid development of resistance is not a concern, a four-arm, phase 2 trial comparing the combination to each drug alone and to placebo or SOC (AB v. A v. B v. SOC or placebo6) should be used to demonstrate the contribution of the components to the combination and proof of concept. As noted above, if SOC is a known effective therapy, a study design in which each of the arms is added to SOC could be used (AB + SOC v. A + SOC v. B + SOC v. placebo + SOC).An adaptive trial design with the same four treatment arms might also be used where appropriate, initially using the treatment arms described above. The single-drug arms could be terminated early if it became clear that they had much less activity than the combination. These designs could demonstrate the activity of each component of (i.e., the contribution of each component to the combination) without exposing the large numbers of patients typically required for phase 3 trials to therapeutic products with inadequate activity. For these trials, it may not be necessary to use a clinical endpoint as a primary efficacy measurement. A credible pharmacodynamic or other biomarker, such as tumor response, may be adequate.300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 Scenario 3: One drug is active alone and one is inactiveIf in vivo or in vitro models, or phase 1 or other early clinical studies suggest that one of the drugs is inactive or minimally active and one drug is modestly active, but the combination has substantial activity, the more active drug generally will require greater scrutiny and should ordinarily be studied as a single drug in a phase 2 study. The minimally active drug generally would not require study as a single drug beyond initial phase 1 safety studies. In this scenario, proof of concept and the contribution of each component could be demonstrated using a three-arm comparison of the active drug alone, SOC, and the combination (AB v. A v. SOC), or the combination and the individual drug added to SOC where SOC is a known effective therapy (AB + SOC v. A + SOC v. SOC). If the inactive drug in a combination is a pharmacokinetic or metabolic enhancer that contributes to the activity of the combination only by increasing the therapeutic concentrations of the active drug, human pharmacokinetic data may provide adequate evidence to support the enhanced activity of the combination and demonstrate the contribution of the inactive drug. A confirmatory study of the combination would usually be needed to provide evidence of effectiveness for the combination (see section V.D). Dose Finding319 320321 322Dose-finding studies could be very important to refine the combination dose or doses and select doses for phase 3 trials. Depending on the role of each component, it may be6 Note that the placebo arm is intended to show the effect size compared to non-treatment, not to show the contribution of each component.323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367useful to test multiple doses of both components to establish a best dose in terms of risks and benefits. If one component in a two-drug combination is more active than the other, it may be more important to study multiple doses of the more active drug (as part of thecombination). For the same reason, it may be more important to study multiple doses ofa drug that is significantly more toxic than the other component of the combination.Other study designs and types of studies also may be appropriate.D. Confirmatory Studies (Phase 3)If findings from in vivo or in vitro models and/or phase 2 trials adequately demonstrate the contribution of each component to the combination, phase 3 trials comparing the combination to SOC or placebo generally will be sufficient to establish effectiveness. If the contribution of the individual components is not clear and it is ethically feasible to use a component or components of the combination as monotherapy in a study arm, it may be necessary to demonstrate the contribution of the components in phase 3 studies (e.g., by use of a factorial design). For example, if phase 2 data do not provide sufficient evidence of the contribution of each component of a two drug combination, but provide strong evidence that the combination is superior to one of the components, a phase 3 trial comparing the combination to the more active component alone and SOC may be needed to demonstrate that the less active component contributes to the activity of the combination. In this and other situations, it will often be useful to study more than one dose of the more active drug in phase 3 studies.Unexpected toxicity (e.g., serious adverse events observed at higher than expected rates) in phase 2 trials is a potential complication for development of a combination and progressing to phase 3 trials. If the toxicity can be attributed to one component of the combination, it may be possible to conduct phase 3 trials with the combination using a lower dose or doses of the more toxic component. If the toxicity cannot be attributed to an individual component of the combination, additional studies may be needed to identify the more toxic component and appropriate dosing for the combination before initiating phase 3 trials. The specifics of any phase 3 design should be discussed with the appropriate FDA review division at an End-of-Phase 2 meeting.VI. REGULATORY PROCESS ISSUES IN CODEVELOPMENTSponsors should consider a number of regulatory issues when planning the codevelopment of two or more novel drugs for use in combination. Key issues are outlined below.A. Early Interaction with FDASponsors are encouraged to communicate as early as possible (e.g., pre-IND meeting) with the appropriate FDA review division when considering codevelopment of innovative combination therapy. Sponsors also are encouraged to consult FDA frequently throughout the development process. We believe such communication will help facilitate development of the combination therapy.368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398B. IND Submissions and Marketing ApplicationsDecisions about the type of IND submission(s) and marketing application(s) needed (e.g., individual component submissions, combination submission) will depend on the sponsor's overall codevelopment and marketing strategy. Until FDA has more experience with codevelopment, FDA recommends that these decisions be made on a case-by-case basis in consultation with the appropriate review division.C. Labeling IssuesFDA also anticipates that the content of labeling for the combination and/or the components will be case specific, depending on the nature of the combination, the intended uses of the individual components, the marketing strategy, and other factors. Therefore, FDA does not believe it can provide generally applicable labeling guidance at this time. Again, we recommend consultation with the appropriate review division.D. PharmacovigilanceApplicants should develop a pharmacovigilance plan that takes into account the additional postmarket risks presented by initial marketing of two or more previously unapproved drugs for use in combination (compared to risks associated with marketing of a single drug). Risk will vary, depending on the nature of the combination and how the combination is marketed. The risk assessment should consider, among other things:•Potential for use of each drug individually;•Potential for use of any of the components of the combination in combinations with other drugs; and•Drugs likely to be co-administered with the combination.Applicants should discuss their pharmacovigilance plans with the appropriate review division and the Office of Surveillance and Epidemiology.。
GMPMay 2011EMA/CHMP/ICH/425213/2011ICH/ Committee for medicinal products for human use (CHMP)ICH guideline Q11 on development and manufacture of drug substances (chemical entitiesand biotechnological/biological entities)ICH 指导原则 Q11 原料药的开发和生产(化学实体和生物技术/生物实体)Step 3翻译/审核：谢永/ChankTransmission to CHMP May 2011 Comments Should be provided using this template. The Completed comments form7 Westferry Circus ● Canary Wharf ● London E14 4HB ● United KingdomTelephone +44 (0)20 7418 8400 Facsimile +44 (0)20 7418 8416E-mail ich@ema.europa.eu Website www.ema.europa.eu An agency of the European Union© European Medicines Agency, 2011. Reproduction is authorised provided the source is acknowledged.T ABLE OF CONTENTS目录1.I NTRODUCTION 介绍 (4)2.S COPE 范围 (4)3.M ANUFACTURING P ROCESS D EVELOPMENT 制造工艺开发 (5)3.1. General Principles 总则 (5)3.1.1. Drug Substance Quality Link to Drug Product将原料药质量与制剂药品联系起来 (5)3.1.2. Process Development Tools 工艺开发工具 (5)3.1.3. Approaches to Development 开发的方法 (6)3.1.4. Drug Substance Critical Quality Attributes 原料药的关键质量属性（CQA） (7)3.1.5. Linking Material Attributes and Process Parameters to Drug Substance CQAs 将物料属性和工艺参数与原料药的关键质量属性相关联 (8)3.1.6. Design Space 设计空间 (9)3.2. Submission of Manufacturing Process Development Information 制造工艺开发信息的注册递交 (10)3.2.1. Overall Process Development Summary 全面的工艺开发总结 (10)3.2.2. Drug Substance CQAs 原料药的CQAs (11)3.2.3. Manufacturing Process History 制造工艺历史 (11)3.2.4. Manufacturing Developmental Studies 制造开发研究 (12)4.D ESCRIPTION OF M ANUFACTURING P ROCESS AND P ROCESS C ONTROLS 制造工艺描述和工艺控制. 125.S ELECTION OF S TARTING M ATERIALS AND S OURCE M ATERIALS 起始物料和源物料的选择 (13)5.1. General Principles 通则 (13)5.1.1. Selection of Starting Materials for Synthetic Drug Substances 化学合成原料药的起始物料的选择 (13)5.1.2. Selection of Starting Materials for Semi-synthetic Drug Substances 半合成原料药的起始物料的选择 (14)5.1.3. Selection of Source Materials for Biotechnological/Biological Products生物产品的起始物料的选择 (15)5.2. Submission of Information for Starting Material or Source Material 起始物料或源物料的信息申报 (15)5.2.1. Justification of Starting Material Selection for Synthetic Drug Substances 合成原料药的起始物料的选择的合理解释 (15)5.2.2. Justification of Starting Material Selection for Semi-Synthetic Drug Substances 半合成原料药的起2 / 37始原料选择的合理解释 (16)5.2.3. Qualification of Source Materials for Biotechnological/Biological Products 生物产品源物料的确认 (16)6. C ONTROL S TRATEGY控制策略 (16)6.1. General Principles 通则 (16)6.1.1. Approaches to Developing a Control Strategy 开发控制策略的方法 (17)6.1.2. Considerations in Developing a Control Strategy 开发控制策略中的考虑 (17)6.2. Submission of Control Strategy Information 控制策略信息的注册申报 (18)7. P ROCESS V ALIDATION/E VALUATION工艺验证/评估 (19)7.1. General Principles 一般原则 (19)7.2. Principles Specific to Biotechnological/Biological Products 生物制品的特殊原则 (20)8. S UBMISSION OF M ANUFACTURING P ROCESS D EVELOPMENT AND R ELATED I NFORMATION I N C OMMONT ECHNICAL D OCUMENTS (CTD)F ORMAT生产工艺开发及相关信息在CTD格式的递交 (21)8.1. Quality Risk Management and Process Development 质量风险管理和工艺开发 (21)8.2. Critical Quality Attributes (CQAs) 关键质量属性（CQAs） (21)8.3. Design Space 设计空间 (21)8.4. Control Strategy 控制策略 (22)9. L IFECYCLE M ANAGEMENT 生命周期管理 (22)10. Illustrative Examples 实例 (23)10.1. Example 1: Linking Material Attributes and Process Parameters to Drug Substance CQAs - ChemicalEntity 将物料属性和工艺参数与原料药的关键质量属性（CQA）相关联—化学药部分 (23)10.2. Example 2: Use of Quality Risk Management to Support Lifecycle Management of Process Parameters使用质量风险管理支持工艺参数的生命周期管理 (27)10.3. Example 3: Presentation of a Design Space for a Biotechnological Product Unit Operation 例3：生物产品单元操作设计空间的介绍 (28)10.4. Example 4: Selecting an Appropriate Starting Material 例4：选择一个恰当的起始物料 (30)10.5. Example 5: Summary of Control Elements for select CQAs 选择CQA 的控制要素的小结 (31)11.G LOSSARY术语 (35)3 / 371.I NTRODUCTION 介绍This guideline describes approaches to developing process and drug substance understanding and also provides guidance on what information should be provided in CTD sections 3.2.S.2.2 ¨C 3.2.S.2.6.It provides further clarification on the principles and concepts described in ICH guidelines on Pharmaceutical Development (Q8), Quality Risk Management (Q9) and Pharmaceutical Quality Systems (Q10) as they pertain to the development and manufacture of drug substance.此指南描述了开发原料药工艺及理解的方法，也提供了那些信息需要在CTD 章节 3.2.S.2.2 和3.2.S.2.6 中提供的指南。
非典型病原体社区获得性肺炎的进展范志强朱惠莉摘要：非典型病原体肺炎在社区获得性肺炎中的地位逐年升高，国内研究发现肺炎支原体的感染率已超过肺炎链球菌，成为我国成人社区获得性肺炎首要致病菌，非典型病原体肺炎有其相对特征的临床和影像表现，确诊有赖于实验室检测，不能明确病原体的情况下，治疗应该覆盖非典型病原体，选用β内酰胺类联合大环内酯类抗生素或者喹诺酮单药治疗，其中大环内酯类药物的治疗效应不仅限于对非典型病原体的杀菌作用，而且具有调节炎症反应和抑制炎症瀑布效应，降低死亡率；当明确非典型病原体感染并排除混合感染后可选用针对性药物如大环内酯类、多西环素或者呼吸喹诺酮类。
社区获得性肺炎( community-acquired pneumonia，CAP)是指在医院外罹患的肺实质感染性( 含肺泡壁，即广义上的肺间质) 炎症，包括具有明确潜伏期的病原体感染，在入院后潜伏期内发病的肺炎。
CAP是严重感染性疾病，占所有住院患者的1%。
近年来发发病率显著增长，英国1997年至2005年间，CAP的发病率增加了34%[1]。
非典型病原体如肺炎支原体（Mycoplasma pneumoniae）、军团菌（Legionella pneumophilia）和肺炎衣原体（Chlamydia pneumoniae）是引起CAP的常见的病原体，是临床工作中不可回避的重要问题，本文将对非典型病原体CAP的现状进行综述。
一、非典型病原体CAP的流行病学与流感病毒型肺炎的治病机理相似，肺炎衣原体或者支原体也通过吸入感染性飞沫传播，支原体肺炎好发于生活封闭的人群，如大学生、监狱囚犯和部队，也有家庭聚集特点，发病高峰在秋冬季。
嗜肺军团菌是引起呼吸道感染的主要非典型病原体之一，在1976 年美国费城退伍军人中首次被发现并命名。
LP 广泛存在于自然界和空调、冷却塔、淋浴器等供水系统中，机体可因吸入被LP 污染的水源产生的气溶胶感染。
CAP的病原学分布有显著的地区差异，引起CAP的首要病原体仍是肺炎链球菌，以往的研究提示肺炎链球菌引起的肺炎的比例为50%, 近期的证据显示随着肺炎多糖疫苗在成人中的广泛应用、普遍的儿童肺炎球菌疫苗接种，以及吸烟率的下降，社区获得性肺炎的肺炎链球菌的感染率下降，美国住院的CAP患者中仅10-15%的患者检测到肺炎链球菌[2-4]。
pcr反应的名词解释PCR（聚合酶链式反应）是一种分子生物学技术，用于在体外扩增DNA片段。
PCR技术可以在较短的时间内复制和扩增DNA片段，因此在医学、生物学、犯罪学和进化生物学等领域被广泛应用。
以下是27个双语例句：1. PCR amplifies specific DNA fragments in vitro.PCR在体外扩增特定的DNA片段。
2. The use of PCR has revolutionized molecular biology research.PCR的应用彻底改变了分子生物学研究。
3. PCR technology allows for quick and efficient DNA amplification.PCR技术可以快速高效地扩增DNA。
4. The PCR reaction requires a DNA template, primers, nucleotides, and DNA polymerase.PCR反应需要DNA模板、引物、核苷酸和DNA聚合酶。
5. The denaturation step in PCR involves heating the DNA to separate the strands.PCR中的变性步骤是通过加热DNA来使两股DNA分离。
6. Annealing is the process of binding the primers to their complementary DNA sequences.等温是指引物与其互补的DNA序列结合的过程。
7. Extension is the step in PCR where DNA polymerase synthesizes new DNA strands.延伸是PCR中DNA聚合酶合成新的DNA链的步骤。
8. PCR amplification can be done using a thermal cycler machine.可以使用热循环机进行PCR扩增。
埃博拉出血热病程（）进入极期，可出现神志的改变，如谵妄、嗜睡等A、1-2天B、2-3天C、4-5天D、5-7天E、7-10天2、以下不能对寨卡病毒灭活的是（）A、70%乙醇B、1%次氯酸钠C、红外线照射D、过氧乙酸E、脂溶剂3、人感染H7N9禽流感重症患者胸部影像常呈双肺多发（）及肺实变影像A、空洞状影B、磨玻璃影C、新月样影D、粟粒样结节影E、蜂窝状影4、人感染H7N9禽流感治疗时，扎那米韦适用于（）以上人群A、3岁B、5岁C、7岁D、10岁E、18岁5、埃博拉出血热病程5-7日可出现麻疹样皮疹，以（）多见A、面部、手心和脚掌B、肩部、胸部和脚掌C、肩部、四肢和脚掌D、肩部、手心和脚掌E、面部、胸部和脚掌6、以下不属于人感染H7N9禽流感重症病例次要标准的是（）A、呼吸频率≥30次/分B、氧合指数≥250mmHgC、多肺叶浸润D、血尿素氮≥7.14mmol/LE、收缩压＜90mmHg需要积极的液体复苏7、以下不能确诊中东呼吸综合征的是（）A、至少双靶标PCR检测阳性B、单个靶标PCR阳性产物，经基因测序确认C、从呼吸道标本中分离出MERS-CoVD、恢复期血清中MERS-CoV抗体较急性期血清抗体水平呈2倍以上升高E、恢复期血清中MERS-CoV抗体较急性期血清抗体水平阳转8、埃博拉出血热的潜伏期为（）A、2-14天B、7-14天C、2-21天D、7-21天E、14-21天9、中东呼吸综合征病理主要表现不包括（）A、肺充血B、小叶性肺炎C、双肺散在分布结节D、间质性肺炎E、肺炎性渗出10、以下关于黄热病有误的是（）A、潜伏期通常为3-6天，也可长达10天B、典型病例临床过程可分为感染期、缓解期、中毒期和恢复期C、血常规：外周血白细胞减少，中性粒细胞比例降低，血小板下降D、肾功能检查：血肌酐水平下降E、流行病学史：发病前14天内有在黄热病流行地区居住或旅行史1、中东呼吸综合征的临床诊断病例为（）A、符合流行病学史和临床表现，但尚无实验室确认依据B、满足疑似病例标准，仅有实验室阳性筛查结果C、单个靶标PCR阳性产物，经基因测序确认D、从呼吸道标本中分离出MERS-CoVE、恢复期血清中MERS-CoV抗体较急性期血清抗体水平阳转2、中东呼吸综合征的病毒核酸检测最好采用（）A、multiplex PCRB、nested PCRC、RT-PCRD、real-time RT-PCRE、real-time PCR3、以下关于黄热病流行病学有误的是（）A、主要经蚊叮咬传播B、城市型黄热病传播媒介主要是非洲伊蚊C、人对黄热病毒普遍易感。
Journal of Clinical Virology32(2005)318–324Development of a multiplex nested consensus PCR for detection and identification of major human herpesviruses in CNS infectionsNarges Kharazani Tafreshi a,Majid Sadeghizadeh a,∗,Samad Amini-Bavil-Olyaee b,Ali Mohammad Ahadi a,Issa Jahanzad c,Mohammad Hassan Roostaee da Department of Genetics,Faculty of Basic Sciences,Tarbiat Modarres University,P.O.Box14155-4843,Tehran,Iranb Biotechnology Department,Pasteur Institute of Iran,Tehran,Iranc Pouya Zistech Ltd.,P.O.Box16315-769,Tehran,Irand Department of Virology,Faculty of Medical Sciences,Tarbiat Modarres University,Tehran,IranReceived2December2003;received in revised form1May2004;accepted11May2004AbstractBackground:Rapid,sensitive and economical detection and identification of human herpesviruses as causative agents of central nervous system(CNS)infections are of clinical importance.The traditional methods for the detection of herpesviruses in CNS infections all suffer from limitations.PCR has a potential to overcome each of them.Objectives:The aims of this study were reducing the number of primers in multiplex PCR and increasing the sensitivity of the assay by nested PCR.Study design:A multiplex nested consensus PCR(MNC-PCR)was developed for the simultaneous detection of major human herpesviruses.A pair of conserved primers was designed for detection of HSV-1,HSV-2,CMV and EBV and another pair of conserved primers for nested PCR.For VZV,a different pair of primers was designed and another pair of primers for nested PCR.A reduction in the number of designed primer pairs(fromfive pairs to two in both stages of PCR)is an advantage in this assay.One hundred forty-seven cerebral spinalfluid(CSF) samples from patients that showed clinical manifestation of CNS infections were tested.Results of MNC-PCR in CSF samples were compared with those of single PCR assay for each individual DNA virus.Sensitivity of the assay was determined with a plasmid containing VZV DNA binding protein gene and another plasmid for HSV-1DNA polymerase gene.False negative results(due to the presence of inhibitor of DNA amplification in CSF samples)were avoided by the inclusion of2-microglobulin primers in the MNC-PCR assay as an internal control. Results:Positive results were obtained in20CSF samples(8HSV-1,2HSV-2,4CMV,3VZV,3HSV-1/CMV,CMV/VZV and HSV-1/EBV coinfections).The comparison between single PCR and MNC-PCR showed a marked increase in sensitivity of MNC-PCR test,since six negative samples in single PCR proved positive in MNC-PCR(P<0.005).Sensitivity was determined1–5plasmid copies for VZV and 50–100plasmid copies for HSV-1.Conclusions:The MNC-PCR assay presented in this study can provide a rapid,sensitive and economical method for detection of viral infections and is applicable to small volumes of CSF samples.©2004Elsevier B.V.All rights reserved.Keywords:Human herpesviruses;CNS infection;Multiplex nested consensus-PCR assay(MNC-PCR)Abbreviations:CNS,central nervous system;CMV,cytomegalovirus; CSF,cerebrospinalfluid;DNA,deoxyribonucleic acid;EBV,Epstein-Barr virus;HHV-8,human herpesvirus8;HSV,herpes simplex virus;MNC-PCR, multiplex nested consensus polymerase chain reaction;VZV,varicella-zoster virus∗Corresponding author.Tel.:+98218011001x4409;fax:+98218009730.E-mail address:sadeghma@modares.ac.ir(M.Sadeghizadeh).1.IntroductionHuman herpesviruses,herpes simplex virus type1(HSV-1),herpes simplex virus type2(HSV-2),varicella-zoster virus (VZV),human cytomegalovirus(HCMV)and Epstein-Barr virus(EBV)are agents causing a wide range of acute cen-tral nervous system(CNS)infections in human(Markoulatos et al.,2001).Although infections caused by different her-1386-6532/$–see front matter©2004Elsevier B.V.All rights reserved. doi:10.1016/j.jcv.2004.05.018N.K.Tafreshi et al./Journal of Clinical Virology32(2005)318–324319pesviruses may have specific clinical manifestations,an ac-curate etiological diagnosis of herpes viral infection is often complicated.Quite frequently,clinical and laboratoryfind-ings are non-specific and often similar not only in patients with different herpesviral infections but with non-herpesviral infections as well(Demkin et al.,2002).Diagnosis on basis of clinical symptoms is often unreli-able,mainly due to the fact that a wide variety of viruses must be considered as etiological agents of CNS infections.A rapid and reliable diagnosis for CNS infection is important (Sauerbrei and Wutzler,2002).Polymerase chain reaction(PCR)has the potential to over-come traditional methods and can be used to amplify any vi-ral DNA present in cerebral spinalfluid(CSF)from patients, even in thefirst few days of infections(Debiasi et al.,2002).In the past,a series of individual PCR tests had to be performed for each single virus.This was both time-consuming and ex-pensive(Elnifro et al.,2000);hence,several approaches have been developed for simultaneous detection of herpesviruses in a single assay.Multiplex-PCR is a variant of PCR in which two or more target sequences are simultaneously amplified in the same reaction(Markoulatos et al.,2003)and utilizes a set of primers targeting multiple specific sites.This approach requires the use of many primers that impair the specificity and cost-effectiveness of the assay(Demkin et al.,2002).In another approach,which makes use of consensus primers for conserved regions,the number of primers has been reduced (Rozenberg and Lebon,1991;Johnson et al.,2000;Demkin et al.,2002).The aims of the present study were two folds:reducing the use of primers in multiplex PCR approach and increasing the sensitivity of the assay by utilizing multiplex nested PCR in CNS infections.2.Materials and methods2.1.Patients and clinical specimensA total of147CSF samples were analyzed(82females and65males).One hundred and two samples from patients, suspected of CNS infection(exhibiting symptoms such as fever,headache,dizziness,malaise,sezure,vomiting,neck stiffness,disorientation,etc.)and hospitalized in the neurol-ogy and emergency ward of Imam Khomeini and Baghiatal-lah Hospitals(Tehran,Iran),were collected.In addition,45 CSF samples that had previously been referred to Diagno-sis Viral Laboratory(Keyvan Laboratory,Tehran,Iran)were tested.All CSF samples were tested for biochemical anal-ysis(the concentration of protein and glucose and lympho-cyte count)and bacterial infection(by culturing)in hospitals. Samples obtained from the virology laboratory showed high protein concentration,normal glucose level and high lympho-cyte count.These observations led the attending physicians to believe that the patients may have viral infections of CSF. Moreover,all147samples proved negative when cultured for bacterial infections.Two CSF samples,collected from patients with clinical manifestations unrelated to herpesvirus infections,were used as negative controls.All CSF samples were obtained within10days after the onset of symptoms and patients were not receiving any anti-herpetic drug at the time of lumber puncture.Samples were stored at−70◦C.2.2.Viral isolates and positive controlsIranian HSV-1(Soleimanjahi et al.,2003),CMV(Amini Bavil-Olyaee et al.,2003),EBV(Keyvan Viral Laboratory) viral isolates(that were obtained from Iranian patients)as well as VZV(Ling et al.,1992)genomic DNA,were used as positive controls.For the propagation of HSV-1,Vero cell line;for CMV,HFF cell line and for EBV,B95-8cell line were infected inflasks at a concentration that gave a50% cytopathic affect(CPE).The culture medium was removed and the cells were harvested(all cells were obtained from Cell Culture Bank,Pasteur Institute,Tehran,Iran).HSV-2 was isolated from an Iranian patient in this research;Vero cells were used to isolate the virus.Genomic DNA was ex-tracted from all viruses and CSF samples by a commercial viral DNA extraction kit as described by manufacturers(Nu-cleospin Blood,MN,Germany).2.3.Multiplex nested consensus-PCR(MNC-PCR)2.3.1.Design of MNC-PCR primersThe GenBank accession number for the DNA sequences used,are as follows:HSV-1M10792;HSV-2AY038367; CMV AF133627;EBV V01555and VZV NC001348.Highly conserved regions within DNA polymerase gene of HSV-1,HSV-2,CMV and EBV were determined by utility of multiple alignment DNA sequences from different viruses (/clustalw).Primers assigned as HHV-F1,HHV-R1within highly conserved regions of HSV-1, HSV-2,CMV and EBV and primers HHV-F2and HHV-R2 were also designed for nested PCR within highly conserved region.Low homology between VZV DNA polymerase gene and other viruses was the reason for designing a different primer for this virus.Therefore,VZV DNA binding protein gene was selected to design the primers(assigned as VZV-F1, VZV-R1and VZV-F2,VZV-R2as nested primers).All VZV primers were designed from regions with least homology to other herpesviruses.Design of primers was performed with Gene Runner Software for Windows version3.05.Primer sequences used are illustrated in Table1.First round primers have identical T m and so do the sec-ond round primers.All primer sequences were checked for hairpin loop and dimer formation.The accuracy of designed primers was confirmed by Blast program (/BLAST/).2.3.2.MNC-PCR amplification conditionsAfter optimization of PCR,first round PCR reactions con-sisting of3l of extracted DNA was added to22l PCR mix320N.K.Tafreshi et al./Journal of Clinical Virology32(2005)318–324 Table1List of primers and their propertiesPrimers Primer function Primer sequences(5 to3 )Type ofvirusesdetection Size ofamplicons(bp)Fragments sizeafter Taq Idigestion(bp)HHV-F1First round-PCR(outer sense)GTCGTGTTTGACTTTGCCAGC HSV-1742HHV-R1First round-PCR(outer antisense)GTCTTGCGCACCAGATCCAC HSV-2742HCMV817EBV748VZV-F1First round-PCR(outer sense)TGATTTGGCTTTATTACCTTTGATGCGVZV-R1Fisrt round-PCR(outer antisense)CGACGGCAGTTACATTTCTGACG VZV650HHV-F2Second round-PCR(inner sense)GCATCATCCTGGCTCACAACC HSV-149388,93and312 HHV-R2Second round-PCR(inner antisense)GTCCGTGTCCCCGTAGATG HSV-249388,99,144and167HCMV56567,237and261EBV49921,229and249 VZV-F2Second round-PCR(inner sense)GCCTACTGTTGACGGTGCTGVZV-R2Second round-PCR(inner antisense)CCATGGGAGCAATAACACAGT VZV356152and204containing:0.5M of each primers HHV-F1,HHV-R1and0.125M of each primers VZV-F1,VZV-R1,1×PCR buffer,1.5mM MgCl2,0.2mM dNTPs,2.5U Taq DNA polymerase (Cinnagen,Iran),0.07%BSA as enhancer(Gibco/BRL, Germany)of PCR assay was performed.First round PCR was carried out using the following programs:94◦C for 3min(predenaturation),35cycles at94◦C for45s,65.5◦C for1min,72◦C for1min,with afinal extension at72◦C for7min.For the nested PCR,0.5l offirst-round prod-uct was transferred to24.5l of an identical PCR mix but containing second-round primers with the same concen-tration as thefirst round.PCR conditions were the same as forfirst-round PCR,except that the annealing temper-ature was changed to63◦C.Positve and negative controls were included in each run.To monitor the presence of in-hibitors,2-microglobulin primers(F:GGTGTCTTGAG-GCTCAGGGAG,R:CAACTTCAATGTCGGATGGATG) were included in each test sample as internal control.DNA extraction was repeated when samples in thefirst round PCR were not positive forβ2-microglobulin gene.Also other negative controls from patients with uninfected CNS were used.Electrophoretic separation of amplified products was per-formed on a1.5%agarose gel(GibcoBRL,Germany)in 1×TBE buffer pH8.0.Agarose gels were stained with ethid-ium bromide,bands visualized under UV–light and pho-tographed for documentation.2.3.3.Precautions against PCR contaminationThe preparation of master mix,the extraction of DNA and addition of template to the PCR mixture and specially the addition offirst round PCR reaction to second round PCR were performed in four different rooms.Each stage was performed with separate micropipettes and all PCR proce-dures were carried out using aerosolfilter tips.Other precau-tions such as a frequent change of gloves,use of UV–light and sodium hypochlorate solution to decontaminate sur-faces were strictly adhered to in order to prevent contami-nation.2.4.Restriction enzyme digestion analysisTo identify HSV-1,HSV-2and EBV and also to recon-firm the identity of VZV and CMV,the nested PCR products for each viral sample was digested with Taq I restriction en-zyme(Cinnagen,Iran).The digestion mixture consisted of 8l of second round PCR product,2l of appropriate en-zyme buffer,five units of enzyme and deionized water to give afinal volume of20and20l mineral oil also was added to prevent evaporation.The reaction mixture was incubated for 4h at65◦C.Digested products were separated by2%agarose gel electrophoresis.2.5.Single target PCRAll samples were tested with single PCR for HSV-1,HSV-2,CMV,VZV and EBV viruses.The primers used were spe-cific for each virus as described by Markoulatos et al.(2001).2.6.MNC-PCR sensitivityTo test MNC-PCR sensitivity,two plasmids,containing HSV-1DNA polymerase gene(pNNI plasmid,Challberg, 1986)and VZV DNA binding protein gene(pMS29plasmid, Arvin et al.,2002)were used.Defined copy numbers of VZV and HSV-1cloned fragments were prepared for sensitivity assay.3.Results3.1.Analysis of PCR productsThe size offirst round PCR products using HHV-F1,HHV-R1primers as well as nested PCR products and other features are indicated in Table1.Size of PCR products of internal control(by2-microglobulin primers)was315bp(data not shown).322N.K.Tafreshi et al./Journal of Clinical Virology 32(2005)318–324Fig.1.The specific Taq I digestion pattern of MNC-PCR products clearly distinguish HSV-1,HSV-2and EBV from each other (Table 1):Lanes 1,3,5corresponding to undigested HSV-1,HSV-2and EBV PCR products,respectively and lanes 2,4,6corresponding to restriction enzyme digestion of HSV-1,HSV-2and EBV PCR products,respectively.CMV and VZV amplified products were accurately iden-tified by their molecular weight,but those of HSV-1,HSV-2and EBV were identified by single restriction analysis of the amplicons.The specific Taq I digestion pattern of these ampli-fied PCR products clearly distinguished HSV-1,HSV-2and EBV from each other (Table 1and Fig.1).3.2.Clinical samplesA total of 147CSF samples from patients were tested by optimized MNC-PCR as well as single PCR assay.Of these,eight were positive for HSV-1,two were positive for HSV-2,three were positive for VZV ,four were positive for CMV ,three of them showed EBV/HSV-1,HSV-1/CMV and CMV/VZV coinfections (Figs.2and 3).None of the sam-ples were positive for EBV .Clinical manifestation ofpa-Fig.2.MNC-PCR of selected positive CSF samples and controls after agarose gel electrophoresis,lane 1control mixture containing HSV-1,CMV ,VZV PCR products,lane 2negative control,lanes 3,7,13CSF samples con-taining HSV-1DNA and lanes 4,8HSV-1digestion of PCR product,lane 5CSF samples containing HSV-2DNA and lane 6HSV-2PCR product digestion,lanes 11,12CSF samples containing CMV DNA,lanes 9,10,14CSF samples containing VZV .M;100bp DNA size marker (Fermentase AB,Lithuania).Fig.3.Agarose gel electrophoresis of MNC-PCR products from CSF sam-ples and ne 1:control mixture containing HSV-1,CMV ,VZV .Lane 2:negative ne 3:patient CSF sample containing CMV ne 5:CSF containing coinfection of CMV/VZV ne 6:CSF con-taining coinfection of HSV-1/CMV DNA and lane 7,digestion of lane 6by Taq ne 10:patient CSF sample HSV-1DNA and lane 11,digestion of lane nes 4,8,9:CSF samples with no herpes viral infection in this assay.M:100–1000bp molecular ruler.tients with positive CSF samples in MNC-PCR is shown in Table 2.3.3.Single target PCRAll 147CSF samples were also tested by single PCR for each virus (HSV-1,HSV-2,CMV ,VZV and EBV).All neg-ative CSF samples that were tested by MNC-PCR,were also negative by single PCR,but six samples (cases 5,9,12,16,18,19)that were negative in single PCR,tested positive in MNC-PCR.The number of positive samples by using both assays were significantly different (P <0.005).3.4.Sensitivity of MNC-PCRThe sensitivity was determined to be between 50and 100genomic DNA copies for HSV-1(this sensitivity was applied to HSV-2,CMV and EBV as well,since PCR was performed with same primers to HSV-1)and 1–5DNA copies for VZV .4.DiscussionMultiplex PCR is a variant of PCR in which two or more target sequences are simultaneously amplified in the same re-action (Markoulatos et al.,2003).The presence of more than one primer pair in the multiplex PCR increases the chance of obtaining spurious amplification products,due to the for-mation of dimers,hairpin loops,internal loops between two primers (Elnifro et al.,2000).To circumvent these problems,another approach has been developed in which consensus primers for conserved regions are used thus reducing the number of primers.N.K.Tafreshi et al./Journal of Clinical Virology32(2005)318–324323Rozenberg and Lebon(1991)designed a single pair of primers within a highly conserved region of the DNA poly-merase gene of HSV-1,HSV-2,CMV and EBV.Individual viruses were then identified by double digestion of ampli-cons.Minjolle et al.(1999)designed stair primers in con-served region of DNA polymerse gene for detection of ma-jor human herpesviruses.However,11oligonucleotides were used in each reaction and subsequently,PCR products were analyzed by hybridization to specific probes in microtiter plates.Johnson et al.(2000)designed two primer pairs target-ing well-conserved regions of the genome of herpesviruses. Individual viruses were identified using the same approach. However,separate PCR assays had to be preformed to detect viruses.Demkin et al.(2002)proposed a semi-nested PCR for HSV-1,HSV-2,CMV and EBV.In this study,a multiplex nested consensus PCR assay to detect herpesviral DNA in a single tube was established in which total of two pairs of primers were used in thefirst PCR reaction and two pairs of primers in the second round PCR reactions.Since a conserved region of DNA polymerase gene of HSV-1,HSV-2,CMV and EBV is used to design the primers,the total number of primers in both rounds of PCR is less than other multiplex PCR.CMV and VZV am-plified products were accurately identified by their molecular weight,but a simple digestion of PCR products by Taq I was sufficient to distinguish among HSV-1,HSV-2and EBV.The selected restriction endonuclease proved efficient without the need to purify amplicons from PCR mixture.To avoid false negative results,an internal control was de-signed(2-microglubolin).Detection of a315bp PCR prod-uct in either positive or negative CSF samples in thefirst round indicated that DNA extraction from CSF was performed cor-rectly.All samples were also tested by single PCR for each virus with the same initial concentration of DNA as in MNC-PCR. Six CSF samples shown to be negative by single PCR,were demonstrated positive with MNC-PCR,out of which two proved to be coinfections with different herpesviruses(cases 18and19,Table2).These results were confirmed by repeat-ing both PCR reactions.Negative and positive controls were included for each series of PCR.In addition,the follow up of clinical manifestation of these patients assured the MNC-PCR test validity.The results obtained from the MNC-PCR assay demonstrate that it is a much more sensitive method for detection of viral DNA in CSF specially for low num-ber of virus particles(1–5genomic copy number for VZV and∼50–100genomic copy number for other viruses as de-termined by the sensitivity assay).It is generally believed using nested primers in the second round of PCR reaction in-creases the sensitivity of the assay.It is hence proposed that higher sensitivity of MNC-PCR is the major advantage of the assay.The drawback,however,is the increased risk of con-tamination compared to single PCR.Therefore,laboratories involved in PCR should take extra precautionary measures to avoid contamination.Another important advantage of this assay is its economi-cal aspect.The required number of PCR assays for detection offive major herpesviruses by single PCR(not nested)is 2.5-fold higher than the required number of multiplex nested PCR for detection of same viruses in same number of CSF samples.Hence,all the reagents including dNTP,Taq DNA polymerase,the number of primers and the amount of CSF and as well as the required time are reduced in the MNC-PCR assay.Also,multiple infections may be particularly problem-atic for single PCR assays,since coinfections will not be de-tected unless the laboratory is specifically instructed to look for multiple organisms(Tang et al.,1999).In addition,anal-ysis of the corresponding nucleotide sequences showed that HHV-F1,HHV-F2,HHV-R2can also detect HHV-8virus.It is therefore proposed that if the three primers are used in a semi-nested PCR,HHV-8may also be detected.In conclusion,the MNC-PCR assay presented in this study can provide a rapid,sensitive and economical method to de-tect viral infection and is applicable to small volumes of CSF samples.This assay proved particularly valuable for diagno-sis of viral CNS infection in immunocompromised or im-munocompetent patients(when there is a potential for a mul-tiplicity of possible pathogens as the cause of a patient’s neu-rological disorder)and it is applicable for diagnosis of these viruses in neonates.The assay offers the potential for very rapid and sensitive detection of human herpesviruses,within a single assay,single clinical sample,thereby allowing earlier application of specific antiviral therapy and avoiding the use of redundant and potentially toxic pharmaceuticals. AcknowledgmentsWe thank Dr.S.Gharavi and Dr.F.Sabahi for critical reading of the manuscript and Dr.H.Keyvani,E.Sabouri and H.Mashi for kindly providing us CSF samples.This research was supported by a grant from Tarbiat Modarres University and Pouya Zistech Ltd,Tehran,Iran.ReferencesAmini Bavil-Olyaee S,Sabahi F,Karimi M.PCR optimization:improving of human cytomegalovirus(HCMV)PCR to achieve a highly sensitive detection method.Iranian J Biotechnol2003;4(1):59–64.Arvin AM,Sharp M,Moir M,Kinchington PR,Sadeghi-Zadeh M, Ruyechan WT,et al.Memory cytotoxic T cell responses to vi-ral tegument and regulatory proteins encoded by open reading frames4,10,29,and62of varicella-zoster virus.Viral Immunol 2002;15(3):507–16.Challberg MD.A method for identifying the viral genes required for her-pesvirus DNA replication.Proc Natl Acad Sci USA1986;83:9094–8. Debiasi RL,Kleinscmidt BK,Weinberg A,Tyler e of PCR for the diagnosis of herpesviruses infections of the central nervous system.J Clin Virol2002;25:S5–11.Demkin V,Kruglova AI,Nikolaeva NP,Yurchenko JV.Detection and species identification of herpesviruses using polymerase chain reac-tion coupled with restriction endonuclease analysis.J Virol Methods 2002;103:121–8.324N.K.Tafreshi et al./Journal of Clinical Virology32(2005)318–324Elnifro M,Ashsli MA,Cooper RJ,Klapper PE.Multiplex PCR:Opti-mization and application in diagnostic virology.Clin Microbiol Rev 2000;13(4):559–70.Johnson G,Nelson S,Martin P,Fellier prehensive PCR-based assay for detection and species identification of human herpesviruses.J Clin Microbiol2000;38(9):3272–9.Ling P,Kinchingon R,Sadeghi-Zadeh M,Ruychan W,Hay J.Transcrip-tion from Varicella-Zoster virus gene67.J Virol1992;66:3690–8. Markoulatos P,Georgopoulou A,Siafakas N,Plakokefalos E,Tzanakaki G,Kourea-Kremastinou boratory diagnosis of common her-pesviruses infections of the central nervous system by a multiplex PCR assay.J Clin Microbiol2001;39(12):4426–32.Markoulatos P,Siafakas N,Katsorchis T,Moncany M.Multiplex PCR: rapid DNA cycling in a conventional thermal cycler.J Clin Lab Anal 2003:17108–12.Minjolle S,Michelet C,Jusselin I,Joannes M,Cartier F,Colimon R.Am-plification of the six major human herpesviruses from cerebrospinal fluid by a single PCR.J Clin Microbiol1999;37(4):950–3. Rozenberg F,Lebon P.Amplification and characterization of herpesviruses DNA in cerebrospinalfluid from patients with acute encephalitis.J Clin Microbiol1991;29(11):2412–7.Sauerbrei A,Wutzler boratory diagnosis of central nervous sys-tem infections caused by herpesviruses.J Clin Virol2002;25: S45–51.Soleimanjahi H,Roostaee MH,Rasaee MJ,Mahboodi F,Bamdad T.Immunogenecity and efficacy of baculovirus-derived glycoprotein d of herpes simplex virus type-1in mice.Arch Razi Ins2003;55:19–28. Tang YW,Shawn Mitchell P,Epsy J,Smith F,Persing H.Molecular diagnosis of herpes simplex virus infection in the central nervous system.J Clin Microbiol1999;37(7):2127–36.。
