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

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.

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,...