Human Herpesviruses HHV-6A, HHV-6B and HHV-7 (eBook)

Diagnosis and Clinical Management

Dharam Ablashi, Louis Flamand, Gerhard Krueger, Irmeli Lautenschlager (Herausgeber)

eBook Download: PDF | EPUB

2014 | 3. Auflage
362 Seiten
Elsevier Science (Verlag)
978-0-444-62716-2 (ISBN)

Infos

Completely revised and updated, Human Herpesviruses HHV-6A, HHV-6B & HHV-7, Third Edition, delivers a contemporary and thorough review of HHV-6, beginning with foundational explorations of classification, molecular biology, and chromosomal integration of the virus, through to clinical details, including pathologic features of infection, and clinical effects on various organ systems. The work now includes coverage of HHV-7, a virus that remains underestimated in transplant reactivation, seizures, and encephalitis. The book examines the role of HHV-6 in myriad diseases, including cognitive disorders following bone marrow transplant, mesial temporal lobe epilepsy, multiple sclerosis, autoimmune disease, encephalitis, Hodgkin's disease lymphoma (HHV-6B), and glioma (HHV-6A). Descriptions of current and novel molecular and serological diagnostic assays, along with detailed protocols, are included to aid researchers and clinicians in discriminating potential false positives originating from chromosomally integrated HHV-6, and in differentiating between active and latent infection. Human Herpesviruses HHV-6A, HHV-6B & HHV-7, Third Edition is a valuable reference for both clinical and basic scientists, including epidemiologists, virologists, practicing clinicians, infectious disease specialists, pathologists, and all scientists entering the field of herpes virus research. The work is a practical and thorough resource for a foundational understanding of HHV-6 and HHV-7, while providing a cutting-edge translational and clinical reference to those looking to understand and diagnose associated viral diseases. - Delivers the latest molecular diagnostic tests, the present-day gold standard for monitoring HHV-6 - Provides expert analysis of T-cell immune response against HHV-6 - Presents tools for differential diagnosis in HHV-6 viral infections - Includes new information on the previously-largely-unknown biological consequences of ciHHV-6 - Offers coverage of new discoveries relating to HHV-7

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Chapter 2

Practical Diagnostic Procedures for HHV-6A, HHV-6B, and HHV-7

Agnès Gautheret-Dejeana,b,c and Henri Aguta,b,    aAP-HP Groupe hospitalier Pitié-Salpêtrière, Paris, France,    bUniversité Pierre-et-Marie-Curie, Paris, France,    cUniversité Paris Descartes, Paris, France

The diagnosis of HHV-6A, HHV-6B, and HHV-7 is based on both serologic and direct approaches. Serology is a classical convenient tool for the diagnosis of primary infection and epidemiologic studies; however, its use is limited by some difficulties in the interpretation of antibody serum titers and positive IgM detection in the context of a lifelong persisting infection, inability to differentiate the two HHV-6 viruses, and cross-reactivity between betaherpesviruses. Direct diagnosis was historically based on virus isolation in primary cell cultures. Molecular diagnosis, based on quantitative PCR and RT-PCR, is more sensitive and accessible than virus culture and antigen detection. It allows us to detect infection in a wide range of human specimens, to distinguish between active and latent infections as well as between HHV-6A and HHV-6B, to assess the efficacy of antiviral therapies, and to readily recognize HHV-6 chromosomal integration. Combined with PCR, nucleotide sequencing allows epidemiologic investigations as well as the recognition of resistance to antivirals. The most recent developments in molecular techniques—namely, digital PCR and next-generation sequencing—offer novel opportunities for the accurate investigation of infection pathophysiology.

Keywords

antigen detection; chromosomal integration; diagnosis; follow-up; immunofluorescence assay; quantitative PCR; real-time PCR; serology; transcripts

An Overview of Diagnostic Strategies

Objectives

The goal of diagnostic approaches is to provide the proof of a human herpesvirus 6A (HHV-6A), HHV-6B, or HHV-7 infection and to precisely define the status of this infection in terms of distinction between latent and active form, quantification of spread within the infected organism, and causative relationship with the concomitant clinical symptoms. A specific concern is that of chromosomal integration of HHV-6 (ciHHV-6), which raises numerous novel questions regarding both pathophysiology and diagnosis. Diagnostic procedures are undertaken either in the context of medical management of a given patient or for a scientific purpose within the general framework of a planned study involving a cohort of human subjects. Beyond the recognition of viral infection, the objective is to use the results of virological diagnosis to decide whether or not the current infection has to be treated, using the limited means at our disposal. In addition, it may be necessary to characterize the causative virus strain more precisely by means of molecular studies if its interhuman transmission or phylogeny have to be investigated. This is also necessary if the infection exhibits an unusual level of complexity such as in the case of a mixed infection involving distinct viral variants or viruses. Of note, the fact that HHV-6A and HHV-6B are now defined as distinct viruses (see Chapter 1) makes their formal differentiation relevant early in any HHV-6 infection diagnosis procedure and strictly necessary for the publication of HHV-6-related scientific articles. Finally, the diagnosis procedures are not dispensable for the follow-up of infection, once diagnosed, and specific treatment, if initiated. This implies not only the serial quantification of virus replication but also the detection of a putative resistance to antivirals in case of therapeutic failure.

Direct and Indirect Approaches

Two general complementary approaches can be used.1 Direct diagnosis is based on the detection and characterization of whole virions or some of their components, the most convenient ones currently being nucleic acids. The detected viral components may come from either cell-free virions or infected cells, the virus factories, which provide an even larger set of viral targets than virus particles. Indeed, infected cells synthesize not only the components of released viral particles but also numerous virus-encoded proteins and nucleic acids, not structural, which are necessary for the sequence of intracellular viral life cycle but are absent from virions. A wide set of methods can be used for direct diagnosis depending on the target selected for detection: infectious viral particles, genomic viral DNA, viral messenger RNAs, and viral proteins. As a consequence, the constraints of getting appropriate human specimens, the time necessary for obtaining a contributive result, and the cost of reagents, as well as the relevance of information provided, may vary significantly according to the method applied for diagnosis. The rapidity, accessibility, sensitivity, and specificity of these techniques thus remain crucial factors that will essentially determine the final choice of strategy.

The indirect approach, also known as serology, is based on the detection and characterization of virus-specific antibodies in a body fluid, mainly serum or plasma (Fig. 2.1). The presence of antibodies is established through their reactivity against reference viral antigens. The antigen–antibody complexes are detected and, when necessary, quantified thanks to a limited number of methods, some of them automated. Antibodies are stable proteins that can keep their immunologic properties after prolonged storage of body fluids, which confers a good reproducibility to serologic assays and provides convenient opportunities for retrospective studies. Apparently, the specimens and techniques used in serology are much easier to manage than those needed for direct diagnosis; however, the interpretation of serologic results may be equivocal, for many different reasons. The most basic one is that HHV-6A, HHV-6B, and HHV-7 infections are widespread in the general population; they are acquired primarily within the first years of life and definitely persist as lifelong latent infections. For this reason, seropositivity to any of these three viruses is a highly frequent finding of limited medical interest. In addition, the kinetics of antibody response and the presence of immunoglobulin M (IgM) antibodies that are useful to identify a primary infection often fail to recognize acute infections related to virus reactivations. The serologic profile is even more complex in case of immune suppression or ciHHV-6, two situations in which the capacity to mount or maintain a humoral immune response may be deeply altered. Ultimately, a cross-reactivity of antibodies directed against the four human betaherpesviruses has been reported, and currently available serologic assays cannot discriminate HHV-6A from HHV-6B infections.2,3

Figure 2.1 Overview on the current approaches for HHV-6A, HHV-6B, and HHV-7 serologic testing.
Serum (or another body fluid) is assayed for HHV-6-specific antibodies according to a theoretical two-step procedure: first-line analysis is dedicated to the detection of infection, and second-line analysis is dedicated to the detailed characterization of the current stage of infection.
Abbreviations: ELISA, enzyme-linked immunosorbent assay; IBA, immunoblot assay; IFA, immunofluorescence antibody assay (includes anticomplement immunofluorescence assay, ACIF); NTA, neutralization assay.

Specimens

A wide range of human specimens can support virological diagnosis procedures. The most common ones are whole blood, plasma, and serum. The relevance of saliva samples for diagnosis has to be established but this body fluid has the advantages of being readily obtained and stored and containing virions (and/or viral DNA), infected cells, and antibodies.4 Cerebrospinal fluid is mandatory for the diagnosis of central nervous system infections. Bronchoalveolar lavages allow exploration of lung infections. Finally, any cell fraction obtained from a body fluid, as exemplified by peripheral blood mononuclear cells (PBMCs), cell smear, or tissue biopsy, can be tested for the presence of virus components by means of direct diagnosis assays. Of particular interest in this domain are the molecular biology techniques, particularly the most recent refinements of PCR, such as digital PCR, which allows clonal characterization of infecting viruses in human samples and will be a prominent tool in future pathophysiology investigations. Whatever the sample and assay used, the quality of sample collection and appropriate conditions for their transport and storage remain key factors for the success of diagnostic procedures.

Methods of Direct Diagnosis

Cell Culture

The isolation of HHV-6A, HHV-6B, and HHV-7 in cell cultures is the method that enabled their discovery.5,6 This is also the only way to prove the presence of infectious viral particles in a sample, and it may be used for the diagnosis of an active infection. However, these methods are time consuming, laborious, and expensive, and they cannot be used for routine work in a clinical setting. All clinical HHV-6 isolates may grow on PBMCs or cord blood lymphocytes activated by phytohemagglutinin, in the presence of interleukin-2 and polybren. A cytopathic effect may appear within about 10 days, consisting of enlarged and refractive cells, but some strains do not exhibit any cytopathic effect. Isolation on other cell types such as fibroblasts is also possible,...

Produkt-Details

• Erscheint lt. Verlag: 11.2.2014
• Sprache: englisch
• Themenwelt: Medizin / Pharmazie ► Medizinische Fachgebiete
• Themenwelt: Studium ► Querschnittsbereiche ► Infektiologie / Immunologie
• Themenwelt: Naturwissenschaften ► Biologie ► Mikrobiologie / Immunologie
• ISBN-10: 0-444-62716-2 / 0444627162
• ISBN-13: 978-0-444-62716-2 / 9780444627162