Rapid Detection of Infectious Agents INFECTIOUS AGENTS AND PATHOGENESIS Series Editors: Mauro Bendinelli, University of Pisa Herman Friedman, University of South Florida College of Medicine Recentvolumes in theseries: DNATUMORVIRUSES Oncogenic Mechanisms Edited by Giuseppe Barbanti-Brodano, Mauro Bendinelli, and Herman Friedman ENTERIC INFECTIONS AND IMMUNITY Edited by Lois J. Paradise, Mauro Bendinelli, and Herman Friedman FUNGALINFECTIONSANDIMMUNERESPONSES Edited by Juneann W. Murphy, Herman Friedman, and Mauro Bendinelli HERPESVIRUSES AND IMMUNITY Edited by Peter G. Medveczky, Herman Friedman, and Mauro Bendinelli MICROORGANISMSANDAUTOIMMUNEDISEASES Edited by Herman Friedman, Noel R. Rose, and Mauro Bendinelli NEUROPATHOGENIC VIRUSES AND IMMUNITY Edited by Steven Specter, Mauro Bendinelli, and Herman Friedman PSEUDOMONAS AERUGINOSA AS AN OPPORTUNISTIC PATHOGEN Edited by Mario Campa, Mauro Bendinelli, and Herman Friedman PULMONARY INFECTIONS AND IMMUNITY Edited by Herman Chmel, Mauro Bendinelli, and Herman Friedman RAPID DETECTION OF INFECTIOUS AGENTS Edited by Steven Specter, Mauro Bendinelli, and Herman Friedman RICKETTSIALINFECTIONAND IMMUNITY Edited by Burt Anderson, Herman Friedman, and Mauro Bendinelli VIRUS-INDUCEDIMMUNOSUPPRESSION Edited by Steven Specter, Mauro Bendinelli, and Herman Friedman A Continuation Order Plan is available for this series. A continuation order will bring delivery of each new volume immediately upon publication. Volumes are billed only upon actual shipment. For further information please contact the publisher. Rapid Detection of Infectious Agents Edited by StevenSpecter University of South Florida College ofMedicine Tampa, Florida Mauro Bendinelli University of Pisa Pisa, Italy and Herman Friedman University of South Florida College ofMedicine Tampa, Florida (cid:46)(cid:79)(cid:88)(cid:90)(cid:72)(cid:85) (cid:36)(cid:70)(cid:68)(cid:71)(cid:72)(cid:80)(cid:76)(cid:70)(cid:51)(cid:88)(cid:69)(cid:79)(cid:76)(cid:86)(cid:75)(cid:72)(cid:85)(cid:86) (cid:49)(cid:72)(cid:90)(cid:60)(cid:82)(cid:85)(cid:78)(cid:15) (cid:37)(cid:82)(cid:86)(cid:87)(cid:82)(cid:81)(cid:15) (cid:39)(cid:82)(cid:85)(cid:71)(cid:85)(cid:72)(cid:70)(cid:75)(cid:87)(cid:15) (cid:47)(cid:82)(cid:81)(cid:71)(cid:82)(cid:81)(cid:15) (cid:48)(cid:82)(cid:86)(cid:70)(cid:82)(cid:90) eBookISBN: 0-306-46807-7 Print ISBN: 0-306-45848-9 ©2002 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow All rights reserved No part of this eBook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher Created in the United States of America Visit Kluwer Online at: http://www.kluweronline.com and Kluwer's eBookstore at: http://www.ebooks.kluweronline.com Contributors BURT ANDERSON • College ofMedicine, Department ofMedical Micro- biology and Immunology, University of South Florida, Tampa, Florida 33612-4799 M. N. BOBROW • Dupont-NEN Life Science Products, Medical Products Department,Boston,Massachusetts02118 CHARLOTTE A. GAYDOS • Division of Infectious Disease, TheJohns HopkinsUniversitySchoolof Medicine,Baltimore, Maryland 21205 RICHARD V. GOERING • Department of Medical Microbiology and Im- munology, Creighton University School of Medicine, Omaha, Nebraska 68178 SPENCER R. HEDGES • Department ofMicrobiology, University ofAla- bama at Birmingham, Birmingham, Alabama 35294-2170 RICHARDL.HODINKA • DepartmentsofPathologyandPediatrics,Clini- cal Virology Laboratory, Children’s Hospital ofPhiladelphia, and Univer- sity ofPennsylvania School ofMedicine, Philadelphia, Pennsylvania 19104 SUSAN B. HUNTER • Foodborne and Diarrheal Diseases Branch, Division ofBacterial andMycoticDiseases, National CenterforInfectiousDiseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333 G.J.LITT • Dupont-NENLife ScienceProducts, MedicalProductsDepart- ment,Boston,Massachusetts02118 JAMES J. McSHARRY • Department ofMicrobiology, Immunology, and Molecular Genetics, AlbanyMedical College,Albany, NewYork 12208 JIRI MESTECKY • Department ofMicrobiology, University ofAlabama at Birmingham,Birmingham,Alabama35294-2170 V vi CONTRIBUTORS SUSANNE MODROW • Institute for Medical Microbiology and Hygiene, University ofRegensburg D-93053 Regensburg, Germany GERARDJ. NUOVO • MGNMedicalResearchLaboratory,Setauket,New York11733 PAUL D. OLIVO • Department of Molecular Microbiology, Washington UniversitySchoolofMedicine, St. Louis, Missouri63110 DAVID PERSING • Division ofClinical Microbiology, Mayo Clinic Foun- dation, Rochester, Minnesota 55905 THOMAS C. QUINN • Divison ofInfectious Disease, TheJohns Hopkins University School ofMedicine, Baltimore, Maryland 21205; and National Institute ofAllergy and Infectious Diseases, National Institutes ofHealth, Bethesda, Maryland 20892-2520 MICHAELW. RUSSELL • DepartmentofMicrobiology, UniversityofAla- bamaatBirmingham, Birmingham, Alabama 35294-2170 BALA SWAMINATHAN • Foodborne and Diarrheal Diseases Branch, Divi- sionofBacterialandMycoticDiseases, NationalCenterfor InfectiousDis- eases, CentersforDisease ControlandPrevention,Atlanta, Georgia 30333 DANNY L. WIEDBRAUK • Departments ofClinical Pathology and Pedi- atrics, WilliamBeaumontHospital, RoyalOak, Michigan 48073 HANSWOLF • Institute forMedicalMicrobiologyandHygiene, University ofRegensburg, D-93053 Regensburg, Germany XIAOTIAN ZHENG • Division of Clinical Microbiology, Mayo Clinic Foundation, Rochester, Minnesota 55905 Preface to the Series Themechanismsofdiseaseproductionbyinfectiousagentsarepresentlythefocus of an unprecedented flowering of studies. The field has undoubtedly received impetus from the considerable advances recently made in the understanding of the structure, biochemistry, and biology of viruses, bacteria, fungi, and other parasites. Another contributing factor is our improved knowledge of immune responses andotheradaptive orconstitutive mechanisms bywhich hosts reactto infection. Furthermore, recombinant DNAtechnology, monoclonal antibodies, andother newer methodologies have provided the technical tools for examining questionspreviouslyconsideredtoocomplextobesuccessfullytackled. Themost importantincentive ofall is probablytheregeneratedideathatinfectionmightbe theinitiatingeventin manyclinical entitiespresentlyclassified as idiopathicorof uncertainorigin. Infectiouspathogenesisresearchholdsgreatpromise.Asmoreinformationis uncovered, itisbecomingincreasinglyapparentthatourpresentknowledgeofthe pathogenic potential ofinfectious agents is often limited to the most noticeable effects, which sometimes represent only the tip ofthe iceberg. For example, it is nowwell appreciated that pathologic processes caused by infectious agents may emerge clinically after an incubation of decades and may result from genetic, immunologic, and other indirect routes more than from the infecting agent in itself.Thus,thereisageneralexpectationthatcontinuedinvestigationwillleadto the isolationofnewagents of infection, the identificationofhithertounsuspected etiologic correlations, and, eventually, more effective approaches to prevention andtherapy. Studies onthemechanisms ofdisease causedbyinfectiousagentsdemanda breadthofunderstandingacrossmanyspecializedareas,aswellasmuchcoopera- tionbetweencliniciansandexperimentalists. The seriesInfectiousAgentsandPatho- genesisisintendednotonlytodocumentthestateofthe artinthisfascinatingand challengingfield but also to help lay bridges amongdiverse areas andpeople. M.Bendinelli H. Friedman vii Preface 1. INTRODUCTION Theevolution ofdiagnosticmicrobiologybeganwith thedevelopment ofthe mi- croscope and has progressed through a variety of tools that have allowed us initiallytovisualizemicroorganismsandthentotakeadvantageofvariousproper- ties ofthe bacteria, viruses, fungi, orparasites to aid in their diagnosis. Earliest techniques reliedon describinggross ormicroscopic morphologyandprogressed to observing growth on differential and selective media. Both ofthese methods dependedonvisualizingtheorganismsandprovidedfairlyrapiddetection,usually within24hours.Morepowerfultools,liketheelectronmicroscope,allowedvisual identification of microbes including viruses within 8-24 hours. Additionally, the development of cell culture techniques facilitated detecting viruses and other obligateintracellularparasites,yetdetectionrequiredfrom 1 daytoafewweeksin some cases. However, although effective forgross identification, these tools still were often too crude to distinguish microorganisms with like morphologies. Subsequently,immunologicandbiochemicaltoolsthatdifferentiatedspecies andstrainsweredeveloped,anddiagnosticmicrobiologybecameahighlyspecific science, sometimes providing diagnosis within one or two days. Furthermore, automationallowedfordiagnosisinsomecasesinlessthan24hours. Serological profiles that depend on detecting multiple antigens and/or antibodies or the presence of IgM antibodies, as an indication of primary infection, also aided detectionandidentificationwithinhoursofspecimen submission. Moresophisti- cated immunologic tools also are being applied in the clinical lab, such as new uses for flow cytometry, development ofsynthetic peptides as reagents for diag- nostic testing, and measuring antibodies in mucosal secretions. The development ofmolecular biology has expanded exponentially in the past decade and has begun to be accorded rapidly increasing importance in the diagnostic laboratory. This has tremendously improved our ability to recognize ix x PREFACE even small differences among isolates, has greatly refined our diagnostic skills, and has truly made almost all aspects ofdiagnostic microbiology rapid. Many ofthe methods developed for detecting microorganisms depend on the viability of the organisms. Many conventional serological techniques, al- thoughnotsufferingthisdisadvantage, areoftenlimitedbytheneedformatched, timely collected, acute and convalescent sera, which delays diagnosis, often by weeks to months. Thus, there is a tremendous advantage in using direct detection methods for rapid diagnosis ofinfectious diseases, This has been achieved by a varietyofmethodsandtools. Ourincreasingsophisticationhasalloweddetecting small amounts ofinfectious material, achieving even more rapid identification and oftenprovidingpositive identification in afewhours. Additionally, we have expandedourcapabilitiesnowto distinguishstrainswithinaspeciesandspecific isolates. This has resulted in great improvements in epidemiological studies. Our expandingrepertoireoftechniqueshasalso increasedthediversityofinformation thatwe can obtain and therefore the contribution ofthe diagnostic laboratoryto guidepatientmanagementeffectively. Nowtechniques allowquantitationofviral loads, which has been linked to pathogenic activity for the human immunodefi- ciencyvirus(HIV),hepatitisCvirus(HCV),andcytomegalovirus(CMV),among others, chronically present once infection is established. Genotyping has been most important for relating to therapeutic protocols, especially for HCV. Anti- viralsusceptibility testing hasbecome arealitywiththeadventofnewtechniques thatrapidlydetectandquantitatevirusandalsoprobeforresistancemutationsin the viral genome. 2. IMMUNOLOGIC ADVANCES Serological andimmunologic assays have been utilized extensively in diag- nosinginfectious diseases, in many cases for rapid diagnosis. Yet many ofthese assaysdependonpairedsamples,whichsignificantlydelaystesting.Furthermore, serologicaltestsoftendependondevelopingmeasurablelevelsofantibody,which takesanywherefrom 10daystoseveralmonths, dependingonthepathogenand the individual patient response. The development and use ofmonoclonal anti- bodies has greatly expanded our ability to perform direct antigen detection test- ing, one ofthe singularly most important steps in developing rapid diagnostic services. Parallel tothis isthemore recentdevelopmentofsyntheticpeptides that can be used to dissect and more specifically measure immune responses. These allowrapid detection andgreater definition ofthe antigenic epitopes responsible forinducingimmunity.Althoughsyntheticpeptidesarealsolikelytohaveagreat impact on vaccine development and understanding of immunodominant epi- topes,thereareadditionalusesforthesepeptidesinidentifyingepitopesresponsi- ble for generating immunity duringinfections. Recent developments anduse of PREFACE xi syntheticpeptidesin infectiousdisease diagnosisaredescribedby Susanne Mod- row and Hans Wolfofthe Institute for Medical Microbiology and Hygiene of Regensburg,Germany. A most interesting development in the use of immunologic methods for rapidly diagnosing viral infections is the adaptation offlow cytometry for such use.AsdescribedbyJimMcSharry,AlbanyMedicalCollege,Albany,NewYork, thismethodisreproducible, rapid, andhas avarietyofapplicationsthatcanhelp indiagnosingseveralviral diseases. Furthermore, ithasbeenutilizedto assistin determiningdrugefficacyin HIV infections. Detectingantibodies influids other than serum has become amore impor- tant diagnostic approach in recentyears. In this regardmeasuringmucosal anti- bodies is gaining in significance, most notably secretory IgA. The manner of detection and utility of this form of testing is detailed in the chapter byJiri Mestecky and co-workers, University ofAlabama at Birmingham, Birmingham, Alabama. 3. CELL CULTURE AND GENETIC ENGINEERING Cell culture has been the gold standard for viral isolation for the past 50 years. More recently the development ofthe shellvialandenhanced cell culture using labeled antibodies for detecting virus has brought this into the realm of rapid detection. The newest advance in cell culture is the use of genetically engineered cell lines that express a gene, such as the beat-galactosidase gene, when infected only by a particular virus. This results in a colorimetric reaction (cells turnblue when infected) that self-identifies an infectedcellwithout adding antibodies. Thismode ofrapid detectionis describedbyPaul Olivo, Washington University, St. Louis, Missouri, who adapted this method for detecting herpes simplex virus in clinical specimens. This approach is being adapted for other viruses and it might be possible in the future to have engineered cells that have several such reporter geneseach ofwhich expresses a different colorin response toaparticularvirus. Thus,byexpressingcellcolor,wemightisolateandidentify viruses rapidly in one cell type in a very futuristic technique. Dr. Olivo also describes alternative uses for genetic engineering ofcells, such as transgenic cell linesorcelllinesdesignedtoresistonevirusbut not another,toaidindifferentiat- ingviruses in mixed infections or other uses. 4. MOLECULARBIOLOGYAND RAPID DETECTION The remainder of the work presented in this volume utilizes molecular techniqueswhichareapplied forrapidlydetectingandcharacterizing avarietyof