Clinical Virology Chapter 29 PDF

CHAPTER 29 Clinical Virology Kevin M. McNabb CHAPTER OUTLINE CHARACTERISTICS OF VIRUSES Caliciviridae Structure Coronaviridae Tax...

CHAPTER 29 Clinical Virology Kevin M. McNabb CHAPTER OUTLINE CHARACTERISTICS OF VIRUSES Caliciviridae Structure Coronaviridae Taxonomy Filoviridae Viral Replication Flaviviridae LABORATORY DIAGNOSIS OF VIRAL INFECTIONS Orthomyxoviridae Specimen Selection, Collection, and Transport Paramyxoviridae Appropriate Specimens for Maximum Recovery Picornaviridae Methods in Diagnostic Virology Retroviridae DOUBLE-STRANDED DNA VIRUSES Rhabdoviridae Adenoviridae Togaviridae Herpesviridae HEPATITIS VIRUSES Papillomaviridae Hepatitis A Virus Poxviridae Hepatitis B Virus SINGLE-STRANDED DNA VIRUSES Hepatitis D Virus Parvoviridae Hepatitis C Virus DOUBLE-STRANDED RNA VIRUSES Hepatitis E Virus Reoviridae Other Hepatitis Viruses SINGLE-STRANDED RNA VIRUSES PRIONS Arenaviridae ANTIVIRAL THERAPY Bunyaviridae OBJECTIVES After reading and studying this chapter, you should be able to: 1. Describe the characteristics of viruses and how they differ from 9. Define cytopathic effect and describe how it is used to presumptively bacteria. identify viral agents. 2. Describe how viruses replicate. 10. Evaluate the vaccine program for influenza. 3. Describe the proper procedures for collection and transport of viral 11. List common opportunistic infections and other indicators of specimens. acquired immunodeficiency syndrome. 4. Name the appropriate specimen for maximum recovery of the 12. Create an algorithm for the serologic diagnosis of human suspected viral agent. immunodeficiency virus infection. 5. Compare the different methods used in the diagnosis of viral 13. Compare the genomes and modes of transmission of the human infections. hepatitis viruses. 6. Explain the advantages and limitations of conventional cell cultures 14. Develop an algorithm for the serologic diagnosis of viral hepatitis. for diagnosing viral infections. 15. Interpret the results of a hepatitis serologic profile. 7. Explain the advantages and limitations of rapid viral antigen 16. For each of the viral agents presented in this chapter, discuss how detection methods. the virus is transmitted or acquired, infection produced by the virus, 8. Discuss the indications and limitations of serologic assays in the and most effective method of laboratory diagnosis. diagnosis of viral infections. body weight, was experiencing fecal incontinence, and had been Case in Point unable to urinate for 3 days. Two years previously, the patient A 36-year-old man was admitted to the hospital after presenting had been diagnosed with human immunodeficiency virus (HIV) at the emergency department with a self-reported, 7-month history infection. A physical examination demonstrated bilateral lower of numbness and weakness in his right leg. He had lost 25 lb in extremity weakness, and his reflexes were slowed throughout 681 682 PART 2 Laboratory Identification of Significant Isolates his body. Kaposi sarcoma (KS) lesions were noted, especially on becoming much more cost-effective and much more common in the lower extremities, along with thrush and herpes lesions in the clinical microbiology laboratory and allows for much better the perianal region. The patient had no fever, and magnetic patient care. resonance imaging (MRI) ruled out spinal cord compression. The Virology is very relevant today and perhaps even more so as patient had a history of intravenous (IV) drug abuse, chronic shown by the viral threats that have literally burst into our lives, diarrhea for 1.5 years, KS for 2 years, and pancytopenia for including the following: several weeks. The patient had large right arachnoid cysts of In Bra il, outbreak of dengue fever (D ), ith over 1.5 million congenital origin. No previous laboratory reports indicated infec- reported cases tious agents in cerebrospinal fluid (CSF). Meningitis was suspected, In West frica, Ebola outbreak that started in 2014 and lasted and the patient was admitted with a diagnosis of polyradiculopathy until ell into 2015 (neuropathy of the spinal nerve roots) secondary to acquired In Bra il, the spread of ika virus in November 2015, ith a immunodeficiency syndrome (AIDS). Blood and CSF specimens link to microcephaly and subse uent spread to the southern were collected. Although numerous white blood cells (WBCs) were found, CSF produced no growth on routine bacteriologic United States culture. The blood cultures were also negative. Acyclovir was In the nited States, the spread of chikungunya virus from administered after culture results were received. the Caribbean countries and territories, first seen in late 2013 and reported in lorida, uerto Rico, and the.S. irgin Islands Introduction of West Nile virus (WN ) into North merica, Issues to Consider ith resurgence in 2012 that resulted in double the yearly After reading the patient’s case history, consider: cases seen prior to that time How the patient’s history relates to his current symptoms E plosion, spread, mortality, and then ithdra al of severe What information is obtained from the laboratory and MRI acute respiratory syndrome (S RS) results ne pected transfer of monkeypo from frica to Mid estern What information provided helps determine the most likely United States cause of the patient’s symptoms Ne variant of in uen a ( 3N2) affecting humans and s ine throughout the United States and implicated in infections in visitors to county fairs iral illness continues to be a significant problem for large Key Terms segments of people throughout the orld. or e ample, I Antigenic drift Heteroploid continues to devastate entire continents, effectively reducing large Antigenic shift Koplik spots portions of each generation. Mos uitoes continue to spread dengue Arboviruses Nucleocapsid virus throughout the orld and ika virus in Bra il, ith significant Capsid Obligate intracellular parasites impact. ver the years, there has been a rise in enterovirus 71 Cell cultures Primary cell cultures (E 71), hich has killed hundreds of children throughout parts Continuous cell cultures Prions of the sian continent. Despite in uen a surveillance programs, Cytopathic effect Syncytia reliable vaccines, and dependable antiviral medications, more than Diploid Tissue culture 30,000.S. citi ens die each year of in uen a. his chapter Envelope Vaccinia virus discusses basic virology, including the advances and challenges Hemagglutinin Virion in clinical virology in the modern clinical laboratory and how the laboratory helps diagnose viral illnesses. Characteristics of Viruses C linical virology is a challenging and e citing area of clinical microbiology. It has changed over the years from viral Structure diagnostic testing performed in only a very few, highly t a minimum, viruses contain a viral genome of ribonucleic acid speciali ed laboratories to the modern, high-comple ity laboratory (RN ) or deo yribonucleic acid (DN ) and a protein coat— of today. Many of the older, traditional diagnostic methods ere the capsid. he genome can be double stranded (ds) or single slo and cumbersome and re uired significant e pertise because stranded(ss). he genome and its protein coat together are referred they were primarily based on cell culture, serology, and microscopy to as the nucleocapsid. he entire virus particle is called the (both bright-field and electron). Results ere often too slo to virion. Some viruses also have a phospholipid labile envelope come to be clinically useful and were perhaps even irrelevant. surrounding the virion. Enveloped viruses are typically more Over the last decade, diagnostic advances have transformed the susceptible to inactivation by high temperature, e treme p , and field of virology by developing ne er methods that are many chemicals compared ith non-enveloped (naked) viruses. he times faster so that results are useful clinically. o ever, faster envelopes are of host origin but contain virus-encoded proteins. laboratory-directed diagnostics must be followed by appropriate he viruses ac uire the envelope from the host membrane as they medical interventions, or patients ill receive poor care. With bud from host cells. the emergence of molecular diagnostic testing for viral infections he morphology of virions is helical, icosahedral (a geometric over the last few years, detection is much faster, much more shape ith 20 triangular sides), or comple. he envelope masks sensitive, and much more specific, resulting in earlier interven- the shape of the virion, so most enveloped viruses are variably tion, early treatment, and better outcomes. his technology is shaped or pleomorphic. he po viruses are the largest viruses  CHAPTER 29 Clinical Virology 683 (260 × 450 nm), and the smallest human virus is the poliovirus, vacuole. Once inside the cell, the virus loses its protein coat, hich is 25 nm in diameter. releasing the genome. his process is called uncoating. RN viruses usually release the genome into the cytoplasm, whereas Taxonomy most DN viruses release their genome into the host nucleus. riginally, viruses ere classified by the diseases they caused he viral genome then directs the host cell to make viral proteins and their host range. No , viruses are classified in orders, families, and replicate the viral genome. Depending on the virus, the genera, and species based on genome type (RN or DN ), number metabolism of the host cell may be completely stopped (as ith of strands in the genome (ds or ss), morphology, and presence or polioviruses) or may continue on a restricted scale (as ith in uen a absence of an envelope. ur gro ing kno ledge of the nucleotide viruses). se uences also becomes a valuable tool for the ta onomic placement he ne t step is the assembly or maturation of the virus par- of viruses. summary of the clinically significant viruses is sho n ticles. he capsid protein subunits aggregate to form capsomers, in able 29.1. and the capsomers combine to form the capsid. he capsid and genome associate to form the nucleocapsid. he ne virions are Viral Replication then released by lysis if they are naked viruses or by budding Viruses are obligate intracellular parasites; that is, they must if they are enveloped viruses. During budding, part of the host be inside a living cell and use the host cell machinery to replicate. cell plasma membrane surrounds the viral capsid and becomes In the first step for infection of a cell to occur, virions must absorb the viral envelope. or attach to the cell surface. bsorption is specific for certain cell receptors, and receptor distribution will determine the entry point Laboratory Diagnosis of into the host. Most host cell receptors are glycoproteins, some of hich include the immunoglobulin superfamily molecules (for Viral Infections poliovirus), acetylcholine (for rabies virus), sialic acid (for in uen a Laboratories can provide different levels of services, depending virus), CD4 (for I ), and complement receptor C3d (for Epstein- on the mission, financial resources, and need. ll these must be Barr virus EB ). he virus attaches to specific receptors on the balanced to provide the most cost-effective and complete diag- surface of a susceptible cell by means of speciali ed structures nostics that ill meet the needs of the clinical staff. ull-service on its surface called adhesion molecules. virology laboratories provide viral culture and identification by he ne t step in viral replication is penetration. iruses can using different mammalian cell cultures to support the growth penetrate the cell by several different mechanisms and penetration of viruses in clinical specimens. lthough not all medical treatment is virus dependent. Naked virions can penetrate the cell membrane facilities provide full virology services, these laboratories can directly. Enveloped viruses may enter the cell by fusion ith the still obtain information about viral infections through performance cell membrane, and a third method of penetration is endocytosis, of rapid tests that detect specific viruses in clinical specimens. whereby the enveloped virus enters the cell in a cytoplasmic hese tests can involve the detection of viral antigens by using TABLE 29.1 List of Viruses Causing Human Disease, Based on Nucleic Acid Characteristics and Taxonomy Genome Strand Family (Subfamily) Genus Species dsDNA Adenoviridae Mastadenovirus Human mastadenoviruses A to G Herpesviridae Simplexvirus Human herpesviruses 1 and 2, macacine herpesvirus 1 (Alphaherpesvirinae) Varicellovirus Human herpesvirus 3 (Betaherpesvirinae) Cytomegalovirus Human herpesvirus 5 Roseolovirus Human herpesvirus 6 Human herpesvirus 7 (Gammaherpesvirinae) Lymphocryptovirus Human herpesvirus 4 Rhadinovirus Human herpesvirus 8 Papillomaviridae Papillomavirus Human papillomavirus Poxviridae Molluscipoxvirus Molluscum contagiosum virus (Chordopoxvirinae) Orthopoxvirus Cowpox virus, monkeypox virus, vaccinia virus, variola virus Parapoxvirus Orf virus Yatapoxvirus Yaba monkey tumor virus dsDNA, ssDNA Hepadnaviridae Orthohepadnavirus Hepatitis B virus ssDNA Parvoviridae Bocaparvovirus Human bocavirus (Parvovirinae) Dependoparvovirus Adeno-associated dependoparvoviruses A and B Erythroparvovirus Human parvovirus B19 dsRNA Picobirnaviridae Picobirnavirus Human picobirnavirus Reoviridae Rotavirus Rotaviruses A, B, and C (Sedoreovirinae) Orbivirus Changuinola virus, Corriparta virus, Great Island virus, Lebombo virus, Orungo virus Seadornavirus Banna virus (Spinareovirinae) Coltivirus Colorado tick fever virus Orthoreovirus Mammalian orthoreovirus Continued 684 PART 2 Laboratory Identification of Significant Isolates TABLE 29.1 List of Viruses Causing Human Disease, Based on Nucleic Acid Characteristics and Taxonomy—cont’d Genome Strand Family (Subfamily) Genus Species ssRNA Arenaviridae Arenavirus Lymphocytic choriomeningitis virus, Lassa virus, Chapare virus, Guanarito virus, Junín virus, Lujo virus, Machupo virus, Sabiá virus Astroviridae Mamastrovirus Human astroviruses 1, 6, 8, and 9 Bunyaviridae Orthobunyavirus California encephalitis virus, Bunyamwera virus, Bwamba virus, Guama virus, Madrid virus, Nyando virus, Oropouche virus, Tacaiuma virus Hantavirus Hantaan virus, Sin Nombre virus, Puumala virus, Thottapalayam virus Nairovirus Crimean-Congo hemorrhagic fever virus, Dugbe virus Phlebovirus Rift Valley fever virus, Punta Toro virus, Sandfly fever Naples virus Caliciviridae Norovirus Norwalk virus Sapovirus Sapporo virus Coronaviridae Alphacoronovirus Human coronavirus 229E, human coronavirus NL63 (Coronavirinae) Betacoronavirus Betacoronavirus 1, human coronavirus HKU1, severe acute respiratory syndrome (SARS)–related coronavirus (Torovirinae) Torovirus Human torovirus Filoviridae Marburgvirus Marburg virus Ebolavirus Zaire ebolavirus, Tai Forest ebolavirus, Sudan ebolavirus, Bundibugyo ebolavirus Flaviviridae Flavivirus Yellow fever virus, West Nile virus, dengue virus, Zika virus, Japanese encephalitis virus, Kyasanur Forest disease virus, Langat virus, louping ill virus, Murray Valley encephalitis virus, Omsk hemorrhagic fever virus, Powassan virus, St. Louis encephalitis virus, tickborne encephalitis virus, Wesselsbron virus, Yellow fever virus Hepacivirus Hepatitis C virus Hepeviridae Hepevirus Hepatitis E virus Orthomyxoviridae Influenzavirus A Influenza A virus Influenzavirus B Influenza B virus Influenzavirus C Influenza C virus Paramyxoviridae Respirovirus Human parainfluenza viruses 1 and 3 (Paramyxovirinae) Morbillivirus Measles virus Rubulavirus Human parainfluenza viruses 2 and 4, mumps virus Henipavirus Hendra virus, Nipah virus (Pneumovirinae) Pneumovirus Human respiratory syncytial virus Metapneumovirus Human metapneumovirus Picornaviridae Enterovirus Human enterovirus A (human coxsackievirus A2, human enterovirus 71), Human enterovirus B (human coxsackievirus B1, human echovirus), Human enterovirus C (human polioviruses 1 to 3, human coxsackievirus A1), Human enterovirus D (human enterovirus 68, 70 and 94), Human rhinovirus A, Human rhinovirus B, Human rhinovirus C Parechovirus Human parechovirus Hepatovirus Hepatitis A virus Rhabdoviridae Lyssavirus Rabies virus Retroviridae (Orthoretrovirinae) Lentivirus Human immunodeficiency viruses 1 and 2 Togaviridae Alphavirus Barmah Forest virus, chikungunya virus, eastern equine encephalitis virus, Mayaro virus, O’nyong-nyong virus, Ross River virus, Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis virus, western equine encephalitis virus Rubivirus Rubella virus dsDNA, Double-stranded deoxyribonucleic acid; ssDNA, single-stranded deoxyribonucleic acid; ssRNA, single-stranded ribonucleic acid. a number of methods, such as immuno uorescence (I ) or en yme laboratories, are being used more by many clinical laboratories. immunoassay (EI ). Some tests have aivers from the Clinical his technology can detect viral infections very early in infection, Laboratory Improvement ct (CLI ), and this helps bring viral and many tests are completed in less than an hour. identification services into physicians offices and clinics. ther laboratories limit their virology services to viral serology— Specimen Selection, Collection, determining the patient s immune response to viruses—rather than and Transport detecting the viruses directly. lthough this is sometimes useful, number of different clinical specimens are suitable for the it is usually takes 3 to 4 eeks after infection before these antibodies diagnosis of viral diseases. he clinical signs and symptoms of are produced, which may mean that treatment would be too late diseases often point to the target organ(s) involved, hich can or not needed. Many ne molecular methods based on nucleic help determine the most appropriate specimen(s) to collect. his, acid detection and amplification, once used in only highly comple combined with a basic understanding of the viral pathogenesis,  CHAPTER 29 Clinical Virology 685 can help in specimen selection for each specific virus. It is important respiratory mucosa are most appropriate for viral diagnosis of to ensure, however, that the specimen collected can be used to respiratory infections. spirates, or surface s abs, are usually isolate a wide range of viral pathogens because similar syndromes appropriate for lesions. If the intestinal mucosa is involved, a may overlap. stool specimen is most appropriate. o ever, if systemic, congenital, Because viral shedding is usually greatest during the early or generali ed disease is involved, specimens from multiple sites, stages of infection, the best specimens are those collected as early including blood (buffy coat), CS , and the portals of entry (oral as possible, which, in many infections, is even before symptoms or respiratory tract) or e it (urine or stool), are appropriate. occur. he sensitivity of viral culture can decrease rapidly 3 days Enteroviruses can cause respiratory infections and may be recovered after the acute onset of symptoms, so care needs to be taken from the stool after the respiratory shedding has ceased. In addition, to collect specimens appropriately to ma imi e detection and enteroviruses are a major cause of aseptic meningitis and can also identification. Specimens should be collected aseptically. Depending be isolated from urine specimens. able 29.2 lists recommended on the anatomic site and the method of collection, specimens may specimens to be collected for viral diagnosis according to the be nonsterile (i.e., contaminated ith bacteria and/or fungi) or body site affected. Incorrect or poor specimen collection can result sterile. his ill impact ho much specimen processing ill be in a false-negative diagnostic result. re uired prior to viral culture. Non culture-based test methods are typically not impacted by contamination, but that varies with the Methods in Diagnostic Virology system. Often, sterile specimens are obtained from sites that are free he clinical laboratory uses four ma or methods to diagnose viral of microorganisms, such as blood, CS , or tissue. Identification of infections: a virus in sterile sites usually means that the isolated virus is the Direct detection of the virus in clinical specimens cause of the disease. Nonsterile specimens are obtained from sites Nucleic acid based detection that contain normal ora, such as the respiratory tract, genital tract, Isolation of viruses in cell cultures skin, or stool. hese specimens may re uire processing to reduce Serologic assays to detect antibodies to virus contaminants and promote viral gro th. spirated secretions are Each laboratory must decide on the method to offer on the often preferable, but swabs are easier to use for collection. Swabs basis of the spectrum of infections encountered, population of must be made of Dacron or rayon. Calcium alginate s abs inhibit patients served, and financial resources. In most laboratories, a the replication of some viruses and can interfere with nucleic combination of several methods is used to optimi e detection and acid amplification tests. issue samples must be kept moist and reduce cost. must not be placed on media unless it is specifically designed for viral preservation. Direct Detection Viral transport medium, saline, or trypticase soy broth can be In general, direct detection methods are not as sensitive as culture added to sterile containers to keep tissues from drying. Several methods but can offer uick results to allo rapid therapy. Many viral transport systems are commercially available. Most trans- of these tests can be performed in a few minutes. Viral detec- port media consist of a buffered isotonic solution with a protein, tion allo s clinicians to make relevant decisions about therapy, such as albumin, gelatin, or serum, to protect less stable viruses. infection control measures, and hospitali ation. In many cases, Often, antibacterial and antifungal agents are added to transport virology results may be available before routine bacteriology culture systems to inhibit contamination of microorganisms. Samples results are. that can be collected with viral transport media are respiratory, Microscopy. Bright-field light microscopy is best for detecting swab, and tissue samples. Samples that should be collected po viruses because all other virus particles are too small to be ithout viral transport media include blood, bone marro , CS , seen. Electron microscopy has a greater magnification and can amniotic uid, urine, pericardial uid, and pleural uid. he be used to detect virions and is useful to detect nonculturable transport container should be unbreakable and able to ithstand viruses, such as Nor alk virus, in stool filtrates. o ever, electron free ing and tha ing. microscopy is e pensive, labor-intensive, and not a very sensitive It is optimal to process viral specimens for culture immediately. method of detecting viruses. herefore electron microscopy is Some viruses, such as respiratory syncytial virus (RS ), become rarely used in clinical laboratories and is more suited for large much more difficult to recover even a fe hours after collection. teaching or research institutions. If specimens cannot be processed immediately after collection, Many viruses produce distinctive and characteristic visual they should be stored at 4 C. Specimens should not be fro en changes in infected cells referred to as a cytopathic effect (C E). unless a significant delay (>4 days) in processing is anticipated. lthough virus particles cannot be visuali ed, the C E can be In that case, specimens should be fro en and held at 70 C. detected in cell scrapings from infected sites ith bright-field Specimens should never be stored at −20 C because this tem- microscopy. or instance, a anck smear can detect Co dry perature facilitates the formation of ice crystals that will disrupt type bodies from herpes simple virus ( S ) and varicella- the host cells and result in loss of viral viability. Repeated oster virus ( ) lesions, and apanicolaou ( ap) smears can free e tha ing cycles are to be avoided because they can also reveal human papillomavirus ( ) associated koilocytes, hich result in loss of viral viability. are s uamous cells ith an enlarged nucleus surrounded by a nonstaining halo. Rabies is sometimes diagnosed by detecting Appropriate Specimens for Negri bodies, hich are eosinophilic cytoplasmic inclusions in Maximum Recovery neurons. or optimal recovery, specimens for viral isolation should be I can be a valuable tool to detect various viral agents directly in collected from the affected site. or e ample, secretions from the clinical specimens. I -labeled antibodies allo direct visuali ation 686 PART 2 Laboratory Identification of Significant Isolates TABLE 29.2 Tests Available for Common Viral Pathogens and Specimens for Culture Body System Affected Antigen Detection Virus Isolation Serology Culture Specimens Molecular Testing Respiratory tract Adenovirus, herpes Adenovirus, coxsackie Adenovirus, coxsackie Nasal aspirate, Single marker simplex virus group A virus, group A virus, nasopharynx (NP) molecular testing (HSV), coxsackie group B coxsackie group B or throat swabs, available or panels cytomegalovirus virus, echovirus, HSV, virus, echovirus, HSV, bronchoalveolar for typical respiratory (CMV), influenza CMV, influenza virus CMV, influenza virus lavage, lung pathogens available virus types A and types A and B, types A and B, biopsy B, parainfluenza parainfluenza virus, parainfluenza virus, virus, respiratory RSV, reovirus, RSV syncytial virus rhinovirus (RSV) Gastrointestinal tract Adenoviruses 40 Adenoviruses 40 and 41, Adenoviruses 40 and Stool, rectal swab Panels for multiple and 41, rotavirus coxsackie group A 41, coxsackie group markers available virus, reovirus A virus Liver Hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), Epstein-Barr virus (EBV) Cutaneous HSV, adenovirus, HSV, adenovirus, HSV, adenovirus, Vesicle aspirate, NP Molecular testing is varicella-zoster coxsackie group A coxsackie group B aspirate and available for HSV-1 virus (VZV) virus, coxsackie group virus, dengue virus, stool, lesion and HSV-2 B virus, echovirus, echovirus, human swab enterovirus, measles herpesvirus 6 virus, VZV, reovirus, (HHV-6), measles rubella virus, vaccinia virus, VZV, parvovirus virus B19, rubella virus, vaccinia virus Central nervous HSV-1 and HSV-2, Coxsackie group A virus, Coxsackie group A Cerebrospinal fluid Molecular panel system mumps virus, coxsackie group B virus, coxsackie (CSF), brain available for CSF CMV, enterovirus, virus, echovirus, group B virus, biopsy, NP that includes CMV, HHV-6, human enterovirus, poliovirus, echovirus, poliovirus, swabs, stool enterovirus, HSV-1 parechovirus, HSV-1 and HSV-2, HSV, HHV-6, mumps and HSV-2, HHV-6, VZV mumps virus virus human parechovirus, and VZV Ocular Adenovirus, HSV Adenovirus, HSV, HSV, coxsackie group A Corneal swabs, coxsackie group A virus conjunctival virus, enterovirus scrapings Genital HSV HSV HSV Vesicle aspirate, Molecular testing vesicle swab available for HSV-1 and HSV-2 of virus infection, and some tests can amplify signals, which from respiratory specimens, hepatitis B virus ( B ) and I -1 enhance sensitivity. In direct uorescent antibody (D ) tests, cells from serum or plasma, enteric adenoviruses from the stool, and from a patient are fi ed to a microscope slide and uorescence- S from cutaneous lesions and con unctival s abs. ther tests labeled antibodies are added. If viral antigens are present in the are packaged in single-test platforms, ith positive specimens sample, the labeled antibody ill bind and uorescence ill be detected by colorimetric or optical density changes on membrane seen microscopically (see Chapter 10 for a more detailed descrip- or silicon surfaces ( ig. 29.1). hese tests can be used to detect tion). D assays are available for numerous viruses, including RS , in uen a viruses and B from respiratory specimens, adenovirus, in uen a viruses and B, measles virus, parain uen a rotavirus and enteric adenovirus from rectal s abs, and WN viruses ( I s) 1 through 4, and RS from respiratory specimens, from serum. EI is often less sensitive than cell cultures or I , S -1, S -2, and from cutaneous lesion material, and so negative results are confirmed ith cell culture or I or nucleic cytomegalovirus (CM ) from blood. acid based tests. hese assays are, by far, the most popular viral Enzyme Immunoassays. Many EI tests for viral detection testing methods in hospital-based laboratories, but as nucleic are commercially available, with most using multiwell microtiter acid based detection becomes cheaper and easier, they may be plate assays. hese tests can detect RS and in uen a virus supplanted by this newer technology.  CHAPTER 29 Clinical Virology 687 A B FIG. 29.1 A, Card format rapid immunochromatographic membrane assay, BinaxNOW (Scarborough, ME), for three common respiratory viruses—influenza A and B and respiratory syncytial virus. B, Examples of positive and negative results. Nucleic Acid–Based Detection in the United States, there has been an increased demand for n increasing interest in nucleic acid based detection assays WN testing ith CR assay. he recent in u of ika virus has compared with traditional cell culture methods has shifted the resulted in an emergency use only (E ) approval from the focus of clinical virology. Not only can the presence or absence D for this rt CR assay, hich is geared to ard patients ho of a virus be determined ith nucleic acid based analysis but, suspect they have been e posed. Detection of in uen a virus depending on the assay used, a uantitative result can also be by CR assay as sho n to be not only more sensitive than the obtained. he use of these assays has led to a better understanding traditional cell culture and shell vial methods, but it also allowed of viruses and helped develop better therapies. earlier administration of antiviral therapy to patients, resulting in dvantages of nucleic acid based detection assays include a better overall treatment. microarray assay for rapid subtyping much faster turnaround time ( ), better sensitivity compared of in uen a virus isolates has been developed and ould be ith cell culture and D , assays that can be uantitative, detection valuable in the event of an outbreak or pandemic. Lumine of viruses nonculturable by cell culture (e.g., norovirus No , assay to detect and type or subtype 20 different viral pathogens hepatitis viruses), ability to detect multiple viruses simultaneously ithin 5 hours has also been described. hese types of systems (multiple ), and potentially characteri ation of the virus geneti- will help epidemiologists, infectious disease physicians, and others cally (genotype). Disadvantages include detection of active and in the public health community by rapidly identifying viral inactivated virus, higher cost, need for speciali ed training and pathogens during an outbreak. Ne er isothermal nucleic acid more comple facilities, and lack of assays approved by the.S. amplification technology is no becoming more prevalent ( lere, ood and Drug dministration ( D ). Smaller clinical laboratories Waltham, M ) it does not re uire temperature cycling and can often rely on sending many of these tests to larger reference deliver results in as uickly as 20 minutes ith performance that laboratories at a higher cost and longer. o ever, as the is a vast improvement on slo er CR-based assays. ll of these technology for virology develops, it will get easier and cheaper molecular assays will lead to faster treatment and better patient to perform, leading to much higher specificity and sensitivity outcomes. of results. E amples of nucleic acid based assays include the hybridi ation Viral Isolation assay, traditional polymerase chain reaction ( CR) and real-time In clinical virology, isolating viruses is still the gold standard CR (rt CR) assays, branched DN assay, nucleic acid se uence against hich all other methods are compared. hree methods based amplification, and a combination of CR and o cytometry, are used for the isolation of viruses in diagnostic virology—cell such as the Lumine system (Lumine , ustin, ) for multiple culture, animal inoculation, and embryonated eggs. Of these three detection. Nucleic acid hybridi ation tests can detect viruses from methods, the most commonly used by clinical virology laboratories various clinical specimens. ssays are available to detect a number is cell culture. nimal inoculation is e tremely costly, used only of viruses, including from endocervical specimens, and as a special resource and in reference or research laboratories. classify them into types that have a high risk or a lo risk for or e ample, certain co sackie viruses re uire suckling mice cancer. ther hybridi ation tests can detect CM from blood and for isolation of the virus. Embryonated eggs are rarely used B from plasma and serum. isolation of in uen a viruses is enhanced in embryonated eggs, Numerous gene amplification techni ues are available for but this is generally accomplished more easily in cell culture. amplification and detection of viral genomes, primarily bloodborne Establishing at least a limited clinical virus isolation capability pathogens, such as I -1, B , hepatitis C virus ( C ), and in routine laboratories can be ustified, provided ualified personnel WN. With a dramatic rise in the incidence of West Nile fever and space are available. Most of the clinical orkload focuses 688 PART 2 Laboratory Identification of Significant Isolates on the detection of S in genital specimens and respiratory Optimally, several different cell lines will be used for a single viruses. significant number of common clinical viruses can specimen to recover different viruses that may be present, similar often be identified ithin 48 hours of inoculation, including S , to the strategy used ith media for the recovery of bacteria. able in uen a and B viruses, I s 1 through 4, RS , adenovirus, 29.3 lists some cell culture lines commonly used in clinical and many enteroviruses. virology. Cell Culture. he term cell culture is technically used to Mi ed or engineered cell cultures are lines of cells that contain indicate culture of cells in vitro the cells are not organi ed into a mi ture of t o different cell types or are made up of cells a tissue. he term tissue culture or organ culture is used to denote genetically modified to make identification of viral infection easier. the growth of tissues or an organ so that the architecture or function Mi ed cell lines have been developed by combining t o cell lines of the tissue or organ is preserved. Many clinical virologists use susceptible to certain types of viruses, such as respiratory or enteric these terms interchangeably; however, cell culture is the technically viruses. he mi ed line can have greater sensitivity to a ider more correct term. range of viruses and therefore reduce the number of culture vials Cell cultures can be divided into three categories—primary, that need to be incubated. Interpreting these mi ed cell cultures lo passage (or finite), and continuous. Primary cell cultures is sometimes difficult, but this is easily learned and ell orth are obtained from tissue removed from an animal. he tissue is the effort. finely minced and then treated ith an en yme, such as trypsin, Cytopathic Effect on Cell Cultures. Some viruses produce to disperse individual cells further. he cells are then seeded onto a very characteristic C E that can provide a presumptive identifica- a surface to form a monolayer, such as in a ask or a test tube. tion of a virus isolated from a clinical specimen. or e ample, With primary cell lines, only minimal cell division occurs. Cell S gro s rapidly on many different cell lines and fre uently viability is maintained by periodically removing cells from the produces a C E ithin 24 hours. predominantly cell-associated surface, diluting them, and placing them into a new container. virus, S produces a focal C E (in hich ad acent cells become his process is referred to as splitting or passaging. rimary cell infected) and pla ues, or clusters of infected cells. he combination lines can only be passaged a few times before new cells must be of rapid gro th, pla ue formation, and gro th on many different obtained. n e ample of commonly used primary cell culture is cell types, such as MRC-5 (Medical Research Council cell strain one ith primary monkey kidney ( MK) cells. 5), human fibroblasts, ero, Ep2, mink lung, and MK cells, is inite cell cultures can divide, but passage is limited to about presumptive evidence for the identification of S. S is one 50 generations. inite cell lines, like primary cell lines, are of the fe viruses that can gro on rabbit kidney cells ( ig. 29.2) diploid; that is, they contain two copies of each chromosome. therefore it is a useful cell line for S detection. Diploid is the normal genetic makeup for eukaryotic cells. s the CM produces an S -like C E ( ig. 29.3) but gro s much number of passages increases, these cells become more insensitive more slo ly and only on diploid fibroblasts. gro s on to viral infection. uman neonatal lung is an e ample of a standard several types of cells, including diploid fibroblasts, 549 cells, finite cell culture used in diagnostic virology. and ero cells. Enteroviruses characteristically produce rather Continuous cell cultures are capable of infinite passage and small, round infected cells that spread diffusely on MK cells, are heteroploid; that is, they have an abnormal and variable number diploid fibroblasts, human embryonal rhabdomyosarcoma (RD) of chromosomes that is not a multiple of the normal haploid cells, and 549 cells. denoviruses also produce cell rounding number. Ep2 (derived from a human laryngeal epithelial carci- ( ig. 29.4) on a number of cell types, including diploid fibroblasts, noma), 549 (derived from a human lung carcinoma), and ero Ep2 cells, 549 cells, and MK cells, but this is usually larger (derived from monkey kidney) are e amples of continuous cell than that caused by enteroviruses. he rounding may be diffuse lines used in diagnostic virology. Both Ep2 and 549 ere or focal, appearing like a cluster of grapes. developed from cancer tissue obtained from patients during treat- he respiratory viruses may not produce a characteristic C E. ment. Each laboratory must decide hich cell lines to use on the RS can produce classic syncytial formation in Ep2 or MKC basis of the spectrum of viral sensitivity, availability, and cost. cells. Syncytia are giant multinucleated cells resulting from cell TABLE 29.3 Cell Cultures Commonly Used in the Clinical Virology Laboratory Virus PMK HDF HEp2 RK A549 CPE Herpes simplex virus − +++ +++ +++ +++ Large, rounded cells Cytomegalovirus − +++ − − − Large, rounded cells Varicella-zoster virus − +++ − − ± Foci or rounded cells; possible syncytia Enterovirus + + ++ − + Refractile, round cells in clusters Adenovirus + ++ +++ − ++ Large, rounded cells in clusters Respiratory syncytial virus ± ± +++ − ++ Syncytia Influenza virus, parainfluenza virus +++ ± − − − Variable—none to granular appearance A549, Human lung carcinoma cell line; CPE, cytopathic effect; HDF, human diploid fibroblasts; HEp2, human laryngeal carcinoma cell line; PMK, primary monkey kidney; RK, rabbit kidney; −, negative; +, acceptable; ++, good viral recovery; +++, recommended; ±, positive or negative. Modified from Costello MJ et al: Guidelines for specimen collection, transportation, and test selection, Lab Med 24:19, 1993.  CHAPTER 29 Clinical Virology 689 A B FIG. 29.2 A, Herpes simplex virus (HSV) from the skin, showing the cytopathic effect (CPE) in less than 1 day on rabbit kidney cells. B, HSV showing the CPE in less than 1 day on HeLa cells. (Unstained, ×400.) C E, a hemagglutination or hemadsorption test is done to detect these viruses. Cells infected ith in uen a virus e press a viral hemagglutinin ( ) protein on their surface that binds red blood cells (RBCs). In the hemadsorption test, a suspension of RBCs is added to the infected cell monolayer. If in uen a virus is present, the RBCs ill adsorb or stick to the infected cells. In the hemag- glutination assay, supernatant from the infected monolayer contain- ing in uen a virus is mi ed ith a suspension of RBCs. In uen a viruses also have the protein on their surface therefore the RBCs ill visibly agglutinate. luorescent antibody stains that detect viral antigen, such as those used directly on clinical speci- mens, can also be used to screen cell cultures before a final negative result is reported. I , EI , and nucleic acid amplification assays FIG. 29.3 Cytomegalovirus from cerebrospinal fluid forming a can also be used to detect and identify viruses in cell cultures to cytopathic effect on diploid fibroblast cells (unstained, ×400). ensure true positives are not missed. Centrifugation-Enhanced Shell Vial Culture. he shell vial culture techni ue can more rapidly identify viruses than the traditional cell culture method. Cells are grown on a round coverslip in a shell vial. shell vial is a small, round, at-bottomed tube, generally ith a scre cap. he shell vial is inoculated ith the clinical sample and then centrifuged to promote viral absorption. he shell vial is incubated for 24 to 48 hours, after hich the coverslip is removed and the I techni ue performed. Based on the type of clinical specimen and suspected viruses, a variety of uorescent-labeled antibodies can be used. modification of this procedure is to use at-bottomed microtiter plates. lthough this is better than looking for a C E, in many cases it can be labor- intensive, and often cultures are done in duplicate, which results in reading at 24 hours then again at 48 hours, thus increasing the. Serologic Assays FIG. 29.4 Cytopathic effect of adenovirus on HeLa cells (unstained, ×400). Viral serology detects circulating antibodies to viruses after e posure. his method provides limited information and has certain inherent problems. irst, serologic assays measure the host response fusion as a conse uence of virus infection. I type 2, and to a rather than directly detecting the virus. Second, the antibody- lesser e tent I type 3, can also produce syncytia. In uen a producing capabilities of human hosts differ idely. or e ample, virus commonly does not e hibit a ell-defined C E. Specimens despite being actively infected, immunocompromised individuals submitted for in uen a virus cultures are usually inoculated onto may not produce enough antibodies to be detected. his is typically MK cells, LLC-MK2 (a continuous line derived from rhesus seen in I -positive individuals. hird, the antibody level does monkey kidney), or MDCK (Madin-Darby canine kidney epithelial not necessarily correlate with the acuteness or activity level of cells) cells. Because in uen a viruses typically do not produce a the infection because this is also host dependent. 690 PART 2 Laboratory Identification of Significant Isolates With fe e ceptions, paired sera (acute and convalescent) lthough half of all adenovirus infections are asymptomatic, the demonstrating seroconversion or a fourfold rise in titer are re uired virus causes about 10 of all cases of pneumonia and 5 to 15 to establish a diagnosis of recent infection. herefore serologic of all cases of gastroenteritis in children. denovirus infections studies are usually retrospective. Some assays are able to distinguish affect the respiratory tract, eye, and gastrointestinal ( I) tract, bet een immunoglobulin M (IgM) and immunoglobulin (Ig ) with lesser involvement of the urinary tract, heart, central nervous the presence of IgM indicates an acute (recent) infection. Cross- system (CNS), liver, pancreas, and genital tract. he viruses can reactions ith nonspecific antibodies can occur, hich makes also cause epidemic keratocon unctivitis, acute hemorrhagic cystitis, interpretation of results difficult. Interpretation is also difficult and pharyngocon unctival fever. denovirus infections occur because of passive transfer of antibodies, such as in transplacental throughout the year and affect every age group. denovirus serotype or transfusion transmission. he follo ing are indications for 14 is rarely reported but causes severe and sometimes fatal acute serologic testing: respiratory disease ( RD) in patients of all ages. In the nited Diagnosis of infections ith nonculturable agents, such as States, an outbreak of adenovirus 14 as reported in four states hepatitis viruses from 2006 to 2007. he outbreak included one infant in Ne Diagnosis of a past (Ig ) or acute (IgM) infection from various ork and 140 additional cases from the states of regon, e as, viral pathogens and Washington. lthough no link could be found bet een the Determination of immune status in regard to rubella virus, Ne ork case and the other cases, all isolates ere identical by measles virus, , hepatitis virus ( ), and B he on and fiber gene se uencing. Since 2007, adenovirus has Monitoring of patients ho are immunosuppressed or have been associated ith outbreaks of RD in.S. military recruits had transplantations and the general public. denovirus types 3, 4, and 7 are most Epidemiologic or prevalence studies commonly associated ith RD and can be fatal. denovirus is shed in secretions from the eyes and respiratory tract. Viral shedding in feces and urine can occur for days after Double-Stranded DNA Viruses the symptoms have disappeared. he viruses are spread by aerosols, Viruses are discussed in this chapter in groups based on nucleic fomites, the oral-fecal route, and personal contact. Most infections acid types—double-stranded DN (dsDN ), single-stranded DN are mild and re uire no specific treatment. ntil the sole manu- (ssDN ), double-stranded RN (dsRN ), and single-stranded facturer ceased production, oral vaccination was available from RN (ssRN ) viruses. epatitis viruses are the only e ception, 1971 to 1999 for types 4 and 7 and as used only for preventing and they will be discussed as one group because they do not all RD in military recruits. he development of a ne vaccine as have the same type of nucleic acid. directed by the military after outbreaks occurred among its person- nel, and this vaccine became available in ctober 2011. ood Adenoviridae infection control measures, including ade uate chlorination of denovirus as first isolated from adenoid tissue and as thus swimming pools, prevent adenovirus infections, such as adenovirus- named for the initial isolation location. uman adenoviruses belong associated conjunctivitis. to the family denoviridae and the genus Mastadenovirus. deno- denovirus types 40 and 41 are called enteric adenoviruses viruses are naked icosahedral viruses ith dsDN ( ig. 29.5). because they cause epidemics of gastroenteritis in young children, denovirus has 51 distinct serotypes (seven subgenera, through ith diarrhea being a prominent feature of the illness. here is ), and the different serotypes are associated ith numerous far less vomiting and fever than ith rotavirus infections. Enteric common clinical manifestations. he clinical manifestations seen adenoviruses have a worldwide, endemic distribution, and the are dependent on the age and immune status of the infected person. number of cases increases during the armer months. hese adeno- he most common serotypes are 1 to 8, 11, 21, 35, 37, and 40. viruses can be identified but not serotyped by EI. Commercial antigen detection kits are available, and although ine pensive, they lack sensitivity. here is a ne molecular panel ( ilm rray, Bio ire, Salt Lake City, ) that is specific for adenovirus types 40 and 41. denoviruses are uite stable and can be isolated in human embryonic kidney and many continuous epithelial cell lines. hey produce a characteristic C E, ith s ollen cells in grapelike clusters. Isolates can be identified by uorescent antibody and EI methods, along with nucleic acid tests. Serotyping is accomplished by serum neutrali ation or hemagglutination inhibition. Electron microscopy has been used in several epidemiologic studies but is not routinely used as a clinical tool. Herpesviridae he herpesviruses belong to the family erpesviridae. he her- pesviruses have a genome of linear dsDN , an icosahedral capsid, an amorphous integument surrounding the capsid, and an outer FIG. 29.5 Transmission electron micrograph of adenovirus envelope. ll herpesviruses share the property of producing latency (×60,000). (Courtesy Dr. G. William Gary, Jr., Centers for Disease and lifelong persistence in their hosts. he virus is latent bet een Control and Prevention, Atlanta, GA.) active infections. It can be activated from latency by various  CHAPTER 29 Clinical Virology 691 stimuli, including stress, caffeine, and sunlight. ctivation can involved. he symptoms are usually less severe in recurrent disease. cause lesions to reappear. enital herpes infections can as much as double the risk of se ual Eight species of human herpesviruses ( ) are currently transmission of I. kno n: Neonatal Herpes. ransmission of S from infected S -1, also kno n as -1 mothers to neonates is less common than might be e pected, but S -2, also kno n as -2 the risk of mother-to-infant transmission is 10 times higher hen , also kno n as -3 mothers have an unrecogni ed primary infection during labor EB , also kno n as -4 and delivery. o ever, mortality associated ith disseminated CM , also kno n as -5 neonatal disease is about 60 in treated neonates but e ceeds -6 70 in untreated neonates. Infection can be ac uired in utero, -7 intranatally (during birth), or postnatally (after birth). he infec- -8, also kno n as KS herpesvirus tion is usually transmitted during a vaginal delivery and is more here are other herpesviruses that infect only primates, e cept severe hen S -2 is involved. he rate of transmission is about for herpes B virus, hich has produced fatal infections in animal 50 hen the mother has a primary infection. Most ne borns handlers and researchers orking ith primates. are infected by mothers who are asymptomatically shedding the virus during a primary infection. he risk of transmission is very Herpes Simplex Viruses low when the mother has recurrent herpes. Cesarean delivery or S -1 and S -2 belong to the genus Simplexvirus. S infec- suppressive antiviral therapy at delivery significantly reduces the tions are very common. By adulthood, about 80 of mericans risk of transmission. have been infected ith S -1. ppro imately 20 of mericans Herpes Simplex Virus Encephalitis. S encephalitis is a have had S -2 infections. hese figures indicate that about one very rare but devastating disease ith a mortality rate of about 70. in si persons in the nited States has had S infection, and In the nited States, S encephalitis may account for up to 20 most infections are asymptomatic. Disease caused by S infection of all encephalitis cases. S is the leading cause of fatal sporadic is generally divided into t o categories—primary (first or initial encephalitis in the nited States. Encephalitis is usually caused by infection) and recurrent (reactivation of the latent virus). S -2 in neonates and S -1 in older children and adults. S Infections are generally spread by contact ith contaminated encephalitis is also associated with an immunocompromised status. secretions. Lesions usually occur on mucous membranes after an Survival rates and clinical outcomes are greatly improved ith I incubation period of 2 to 11 days. Infected individuals are most antiviral treatment. ne diagnostic panel for the detection of infectious during the early days of a primary infection. Virus- S -1 and S -2, as ell as several other viruses and bacteria infected cells are usually found at the edge and in the base of that cause meningitis, is available ( ilm rray, Bio ire, Salt Lake lesions; however, the virus can be transmitted from older lesions City, ), and this test is fre uently being performed to reduce as well as from asymptomatic patients. the need for antiviral therapy especially in infants. Ocular Herpes. herpes simple infection of the con unctiva Types of Infections can manifest itself as swelling of the eyelids associated with S infections can cause a ide spectrum of clinical manifesta- vesicles. Corneal involvement can result in destructive ulceration tions, including those discussed below. and perforation of the cornea, leading to blindness. S is the Oral Herpes. Oral herpes infections were thought to have most common cause of corneal infection in the United States. been caused by S -1, but it is no kno n that a number of ortunately, most infections involve only the superficial epithelial cases are caused by S -2. he incubation period ranges from layer and heal completely with treatment. 2 days to 2 eeks. rimary infections are usually asymptomatic, but when apparent, they commonly manifest themselves as rarely Diagnosis seen mucosal vesicles inside the mouth or as ulcerations that may Diagnosis of S infections is best made by antigen detection be idespread and involve the buccal mucosa, posterior pharyn , or viral isolation. he best specimens for culture are aspirates and gingival and palatal mucosae. In young adults, a primary from vesicles, open lesions, or host cells collected from infected S infection can involve the posterior pharyn and look like sites. Culture of CS is usually not productive. o obtain a culture- acute pharyngitis. Recurrent, or reactivation, S infection usually confirmed diagnosis of encephalitis, brain biopsy material is occurs on the border of the lips at the junction of the oral mucosa re uired. lternatively, CS can be assayed by CR for S. In and skin. n early symptom of burning or pain follo ed by vesicles, many studies, gene amplification for S in CS approaches ulcers, and crusted lesions is the typical pattern. 100 sensitivity. Some of the ne er nuclear assays are becoming Genital Herpes. enital herpes infections are usually caused easier to perform and less costly, so it is e pected that they ill by S -2, although S -1 can cause as many as one third of be used more fre uently in clinical laboratories. he ne est the infections. Many individuals ith antibodies to S -2 have meningitis panel ill detect S -1 and S -2 as ell as other not been diagnosed ith genital herpes. he infection manifests viruses, bacteria, and yeasts. itself in females as vesicles on the mucosa of the labia, vagina, In culture, S replicates rapidly, and the C E can be seen or both. Involvement of the cervi and vulva is not uncommon. ithin 24 hours ( ig. 29.6 also see ig. 29.2). herefore diagnosis In males, the shaft, glans, and prepuce of the penis are the most and appropriate therapy can be initiated uickly, resulting in better commonly affected sites. he urethra is commonly involved in patient outcomes. S can be isolated in numerous cell lines, both men and omen. Recurrent herpes infections involve the including human embryonic lung, rabbit kidney, Ep2, and 549 same sites as primary infections, but the urethra is less commonly cells. S is one of the most fre uently isolated viruses in the 692 PART 2 Laboratory Identification of Significant Isolates S or. CM is typically spread by close contact ith an infected person. Most adults demonstrate antibody against the virus, ith a prevalence rate in the nited States of 55 among adult omen and 32 among adult men. he seroprevalence of CM increases ith age in all populations it is highest among lo er socioeconomic groups living in cro ded conditions. ersons ho live in overcro ded conditions can ac uire CM at an early age. he virus is shed in saliva, tears, urine, stool, and breast milk. CM infection can also be transmitted se ually via semen and cervical and vaginal secretions and through blood and blood products. CM infection is the most common congenital infection in the United States. Most CM infections are asymptomatic in the immune- competent host but can manifest themselves as a self-limiting, infectious mononucleosis-like illness, ith fever and hepatitis. In immunocompromised hosts, such as transplant recipients and patients ith I infection, CM infection can become a sig- nificant, life-threatening, systemic disease involving almost any FIG. 29.6 Advanced cytopathic effect in an A549 cell line caused organ, including the lungs, liver, intestinal tract, and retina, as by herpes simplex virus infection (unstained, ×400). (Courtesy ell as the CNS. Sarah Pierson.) Congenital infections and infections in immunocompromised patients are often symptomatic and can be serious. Serious clinical manifestations can develop if the mother ac uires the primary clinical virology laboratory. Once isolated, monoclonal antibodies infection during pregnancy; congenital infection, however, is can be used to type the virus. yping genital lesion isolates can unlikely to occur if the mother as seropositive at the time of be prognostic in that S -2 reactivation occurs more readily than conception. Symptomatic congenital infection is characteri ed by S -1 reactivation. In addition, typing genital lesions from children petechiae, hepatosplenomegaly, microcephaly, and chorioretinitis. has been used to provide legal evidence supporting potential se ual ther manifestations are reduced birth eight, CNS involvement, abuse. mental impairment, deafness, and even death. CM infection is Commercially available engineered cell lines improve the one of the leading causes of mental retardation, deafness, and detection of S. In the EL IS (en yme-linked, virus-inducible intellectual impairment. system) test, a gene for the en yme β-galactosidase linked to he diagnosis of CM infection is best confirmed by isolation a virus-induced promoter has been inserted into baby hamster of the virus from normally sterile body uids, such as the buffy kidney cells. If S -1 is present in the cell line, a viral protein coat of blood or other internal uids or tissues. he virus can also will activate the promoter, resulting in β-galactosidase e pression. be cultured from urine or respiratory secretions, but because Detection is accomplished by addition of a reagent, hich is cleaved shedding of CM from these sites is common in normal hosts, by the en yme produced in virus-infected cells and results in the isolation from these sources must be interpreted ith e treme formation of a blue color, which is easily seen by light microscopy. caution. Over the last several years, a viral antigenemia test has ormerly, serology provided only limited information to aid gained ider use by clinical virology laboratories. he antigenemia in the diagnosis of S infections. Reagents that could distinguish assay is specific, sensitive, rapid, and relatively easy to perform. bet een antibodies to S -1 and to S -2 ere not previously he test is based on the immunocytochemical detection of the available. his as problematic because most adult patients have 65-kilodalton (kDa), lo er-matri phosphoprotein (pp65) in the antibodies to S -1. No , ho ever, several D -approved, nuclei of infected peripheral WBCs. he antigenemia test may type-specific assays that differentiate antibody response to S prove helpful in assessing the efficacy of antiviral therapy. o ever, are available. he tests come in a variety of formats, including there are no several ne er nucleic acid assays using CR that EI , strip immunoblot, and even simple membrane-based, point- may replace this test in smaller clinical laboratories as they become of-care assays. he difference bet een the ne er tests and those more cost-effective. Nucleic acid assay is offered at ma or.S. of the previous generation is the antigens used. he ne er tests reference laboratories and is the preferred method for determining use recombinant or affinity-purified, type-specific glycoprotein viral loads. CM produces a characteristic C E, hich can 1 or 2, giving the tests the ability to distinguish bet een S -1 sometimes be seen in clinical specimens ( ig. 29.7). and S -2. lder-generation tests used crude antigen preparations Molecular-based testing is also idely used to detect virus from lysed cell culture of the virus and have been shown to have particles in clinical samples. CR, branched DN , and hybridi a- cross-reactivity rates of as much as 82 in positive specimens. tion assays are all used for blood donor screening and diagnostic applications. ne meningitis panel, including CM isolation, Cytomegalovirus is available and is perfect for use in pediatric populations. CM is in the genus Cytomegalovirus, and the name originates congenital infection is best confirmed by isolation of CM from from the enlargement of infected cells (from Latin cyto, meaning the infant ithin the first 2 eeks of life. Isolation after the first cell, and mega, meaning large). It is a typical herpesvirus, but it 2 eeks does not confirm congenital infection. rine is the most replicates only in human cells much more slowly compared with common specimen submitted for viral detection in these patients.  CHAPTER 29 Clinical Virology 693 and sho fe signs of infection. When infection ith EB occurs in adolescence, it presents as infectious mononucleosis 35 to 50 of the time. he signs and symptoms of EB infection include sore throat, fever, lymphadenopathy, hepatomegaly, splenomegaly, and general malaise. hese usually resolve ithin a fe eeks, although malaise can be prolonged in some cases. Complications of EB infections include splenic hemorrhage and rupture, hepatitis, thrombocytopenia purpura with hemolytic anemia, Reye syndrome, encephalitis, and other neurologic syndromes. EB can be recovered from the oropharyn of symptomatic as well as healthy persons, who can transmit the virus to susceptible persons via infected saliva. he incubation period for EB infection ranges from 2 eeks to 2 months. s ith the other herpes group viruses, infection is very common and results in latency, and most adults demonstrate FIG. 29.7 Active cytomegalovirus lung infection in a patient antibody against the virus. Young children with the infection are with acquired immunodeficiency syndrome. Lung histopathol- almost al ays asymptomatic. s the age at the time of infection ogy shows cytomegalic pneumocyte containing characteristic increases to young adulthood, a corresponding increase occurs in intranuclear inclusions, hematoxylin and eosin (×1000). (Courtesy the ratio of symptomatic to asymptomatic infections. Some cancers Edwin P. Ewing, Jr., Centers for Disease Control and Prevention, have been associated ith EB , including Burkitt lymphoma, Atlanta, GA.) odgkin disease, and nasopharyngeal carcinoma (N C). Burkitt lymphoma is a malignant disease of the lymphoid tissue seen most commonly in frican children. he virus has also been increasingly recogni ed as an important infectious agent in transplant recipients. he most significant clinical effect of EB infection in these patients is the development of a B-cell lymphoproliferative disorder or lymphoma. iral culture for EB re uires human B lymphocytes, and is beyond the capabilities of most clinical virology laborato- ries. herefore laboratory diagnosis of EB infection is often accomplished ith serologic tests. EB infects circulating B lymphocytes and stimulates them to produce multiple heterophile antibodies, including antibodies to sheep and horse RBCs. he aul-Bunnell heterophile antibody test is an e cellent rapid screening test for these antibodies, although some false-positive reactions do occur. large number of rapid test kits, generally based on EI or late agglutination, are commercially available for detecting heterophile antibodies. hese tests are 80 to 85 effective. Some false-positive test results represent patients who have had infectious mononucleosis and still have low levels of antibody. Young children can have false-negative results with FIG. 29.8 Negatively stained transmission electron micrograph the heterophile test performing an EB -specific antibody test revealing the presence of numerous Epstein-Barr virus’ virions (×40,000). (Courtesy Fred Murphy, Centers for Disease Control on these individuals is appropriate. EB -specific serologic tests and Prevention, Atlanta, GA.) ( able 29.4, ig. 29.9) measure the presence or absence of the following: Anti-VCA (antibodies against the viral capsid antigen): IgM s ith S , serology is not as helpful as a culture in diagnos- to the C occurs early in the infection and disappears in ing the infection. CM can be isolated in cell culture only by about 4 eeks, so its presence indicates current infection. Ig using human diploid fibroblast cell lines, such as human embryonic often appears in the acute stage and will persist for life at lung or human foreskin fibroblasts (see ig. 29.3). he virus lower titers. replicates slo ly, so it may take up to 3 eeks for the C E to Anti-EA IgG (IgG antibody to early antigen): Ig to E can appear in culture. o ever, the use of shell vials can reduce the appear in the acute phase, and its presence indicates current time for detection to as little as 1 day. or recent infection. he antibody usually cannot be detected after 6 months. Epstein-Barr Virus Anti-EA/D (antibody to early antigen, diffuse): ntibodies to Epstein-Barr virus (EB ), in the subfamily ammaherpesvirinae E /D appear in the acute phase, and their presence indicates and the genus Lymphocryptovirus, causes infectious mononucleosis current or recent infection. he antibodies usually cannot be ( ig. 29.8). p to 95 of adults aged bet een 35 and 40 years detected after 6 months. atients ith N C often have elevated have been infected. Many children become infected ith EB levels of Ig and Ig anti-E /D antibodies. 694 PART 2 Laboratory Identification of Significant Isolates TABLE 29.4 Interpretation of Epstein-Barr Virus Serologic Markers PB Anti-VCA IgM Anti-VCA IgG Anti-EA IgG Anti-EBNA Interpretation − − − − − No previous exposure to Epstein-Barr virus + + + ± − Acute infectious mononucleosis ± ± + ± + Recent infection −− − + − + Past infection Anti-EA IgG, Immunoglobulin G antibodies to early antigen; anti-EBNA, antibodies to Epstein-Barr vius nuclear antigen; anti-VCA IgG, immunoglobulin G antibodies against the viral capsid antigen; anti-VCA IgM, immunoglobulin M antibodies against the viral capsid antigen; PB, Paul-Bunnell antibody; −, negative; +, positive; ± positive or negative. Clinical illness Antibody titer Anti-VCA IgG Anti-EBNA Anti-EA Anti-VCA IgM Heterophil (PB) antibody Weeks 1 2 3 4 5 6 12 24 FIG. 29.9 Serologic evaluation of Epstein-Barr virus infection (infectious mononucleosis) showing the rise and fall of detectable antibodies. Anti-EA, Antibody to early antigen; anti-EBNA, antibody to Epstein-Barr virus nclera antigen; anti-VCA IgG, immunoglobulin G antibody to the viral capsid antigen; anti-VCA IgM, immunoglobulin M antibody to the viral capsid antigen; PB, Paul-Bunnell. Anti-EA/R (antibody to early antigen, restricted): ntibodies to E /R appear in the acute phase and disappear soon after anti-E /D, but can persist for up to 2 years and may be lifelong in some patients. nti-E /R Ig antibody level is elevated in patients ith Burkitt lymphoma. Anti-EBNA (antibody to the EBV nuclear antigen): ntibodies appear about 1 month after infection, ith titers peaking in 6 to 12 months. here are several molecular assays coming to market that ill use rt CR to both detect and uantitate viral load that ill be key in patient treatment and also to measure the effectiveness of treatment for EB -positive patients. his ill especially critical in persons who also have other medical conditions that lower immune status, such as I infection or diabetes. Varicella-Zoster Virus is in the subfamily lphaherpesvirinae and the genus Vari- cellovirus. spreads by droplet inhalation or direct contact with infectious lesions. Cell-free virus is produced at very high levels in the skin vesicles, and thus the uid from these vesicles is highly infectious. he virus causes t o different clinical manifestations—varicella (chickenpo ) and oster (shingles). In the nited States, more than 90 of adults have antibody to. aricella is the primary infection and is highly contagious ( ig. 29.10). In contrast to infections ith the other herpesviruses that do not usually manifest symptoms, varicella is generally FIG. 29.10 Electron micrograph of a varicella virus (×100,000). clinically apparent. It commonly appears in childhood and includes (Courtesy Erskine Palmer and B.G. Partin, Centers for Disease symptoms such as a mild febrile illness, rash, and vesicular lesions. Control and Prevention, Atlanta, GA.)  CHAPTER 29 Clinical Virology 695 sually, the lesions appear first on the head and trunk and then as the fever resolves. bout 30 to 40 of infected children spread to the limbs. he lesions dry, crust over, and heal in 1 to ith symptoms e perience sei ures. s ith all members of 2 eeks. ainful oral mucosal lesions may develop, particularly the family erpesviridae, reactivation of latent infections can in adults. become clinically significant in immunocompromised individuals. erpes oster is the clinical manifestation caused by reactivation -6 has also been proposed as having some involvement in of it usually occurs in adults. ppro imately one in three the development of progressive multifocal leukoencephalopathy adults ill develop herpes oster in their lifetime. It is thought and multiple sclerosis. that the virus remains latent in the dorsal root or cranial nerve he diagnosis of -6 infection is usually made clinically. ganglia after primary infection. In a small proportion of patients, Isolation of the virus is most sensitive ith lymphocyte cell culture, the virus becomes reactivated, travels down the nerve, and causes which is not practical for routine diagnosis. Serology may not be oster. he most common presentation is rash, follo ed by vesicular helpful unless paired sera are available. atients do not usually lesions in a unilateral dermatome pattern. hese lesions may be have a positive IgM result until about 5 days after infection Ig associated with prolonged disabling pain that can remain for appears several days later. CR and viral load testing offer the months, long after the vesicular lesions disappear. most sensitive and specific means of diagnosing primary -6 infection is usually diagnosed on the basis of characteristic infection. clinical findings. In atypical cases, such as in immunosuppressed patients, the diagnosis may be more difficult or uestionable. In Human Herpesvirus 7 such patients, culture of fresh lesions (vesicles) or the use of -7 is in the genus Roseolovirus ith -6. he CD4 uorescent-labeled monoclonal antibodies against confirms molecule serves as a receptor for -7 to infect lymphocytes. the diagnosis. can be cultured on human embryonic lung or It also uses other receptors and has a broad range of host cells. ero cells. Cytopathic changes may not be evident for 3 to 7 days. Like -6, -7 is e tremely common and is shed in the ver the last fe years amplified nucleic assays, such as CR saliva of 75 of adults. he virus causes roseola, hich is clinically assays, have become the standard for the diagnosis of disease. identical to that caused by -6. -7 causes latent infections hese assays have revolutioni ed the diagnosis of disease in lymphocytes. Despite the similarities bet een -6 and of the CNS and of disseminated infection, especially in -7, their antigenic diversity is such that antibodies to one immunocompromised patients ( I infection, diabetes), and the virus do not protect against infection from the other. In addition, identification of herpes oster in patients ho do not develop the e posure to -7 seems to occur later in life than e posure to rash associated ith. he advantages of these molecular -6. Most 2-year-olds are seronegative for -7, but most assays are that they re uire small specimen volumes and are hig