Spatiotemporal Virus Surveillance for Severe Acute Respiratory Infections in Resource-limited Settings: How Deep Need We Go?

Following the discovery of molecular cloning, nucleic acid sequencing, and polymerase chain reaction, the wonderland of molecular biology has opened its doors again to virtually unlimited possibilities, as next-generation/deep sequencing or, better, massive parallel sequencing allows for the comprehensive analysis of all genomic nucleic acids of any origin in any given sample in real time. Initially applied to the bacterial microbiome, deep sequencing has similarly detected a multitude of viral genome sequences, and changed hitherto common views on persisting viral infections [1], putting forward the concept of the “virome” in general, and of the “human viromes” in particular [2]. While the numeric summary of viral genome sequences is mind-blowing, even after careful annotation and curation through refined bioinformatic tools, the utility of massive parallel sequencing has been prominently demonstrated in epidemiologic cluster analysis, for example, leading to the identification of a novel arenavirus as the cause of donor-derived death of 3 solid organ transplant recipients [3], or of a novel human polyomavirus causing lethal multiorgan failure in a pancreas transplant recipient [4]. Given the technical and bioinformatic advances as well as the declining laboratory costs, the application of deep sequencing to identify etiologic agents in clinical samples has been approached in different pathologies, including those caused by community-acquired respiratory viruses (CARVs). Despite the general enthusiasm for metagenomics, it remains important to realize that there are caveats. In fact, multiple technical and conceptual aspects influence the results of deep sequencing, may cause significant differences in the read-outs, and hence bias their interpretation. Conceptually, it is clear that the human virome is different in different organ sites and body location (eg, the gastrointestinal, urogenital, or respiratory tract) and is subject to dynamic changes of the viral constituents, not only as a result of physiological changes (eg, dietary, hormonal, aging), but also due to complex pathological alterations such as trauma, inflammation, and immune responses [5], exemplified in inflammatory bowel disease or transplantation [6, 7]. Technically, nucleic acid extraction is presumed to be representative across all different agents, whereas in fact different sample preparations, buffers, purification, single fragment partitioning, and sequence analytic procedures may introduce an as-yet little-understood bias, which can lead to underrepresentation of certain viral genomes—for example, human coronavirus compared with paramyxovirus genomes (H. H. Hirsch, unpublished observation). Thus, every step of the wet and dry laboratory procedure is dependent on the methodology, and may yield potentially different results [8]. Independent confirmatory testing has become an essential part of the presumably hypothesis-free deep sequencing [9]. These aspects are important when discussing the results of massive parallel sequencing to either rule in or rule out the involvement of a pathogen in a given pathology. With these caveats in mind, and given the significant global burden of viral respiratory tract disease in the very young and the very old [10–12], well-established researchers from the United States and Uganda have joined forces: Cummings et al. in this issue of Clinical Infectious Diseases characterize the potential role of noninfluenza viruses in spatiotemporal clusters of severe acute respiratory infection (SARI) in a resource-limited setting. The prospectively collected epidemiological data and nasopharyngeal/ E D I T O R I A L C O M M E N T A R Y