Editorial commentary: Unbiased next-generation sequencing and new pathogen discovery: undeniable advantages and still-existing drawbacks.

Traditional tools for microbial diagnosis might be unsuccessful in identifying the causative agent of an infection. Since the advent of polymerase chain reaction (PCR) technology, the nucleic acid– based detection of pathogens in clinical samples has significantly improved the percentage of successful identifications. The most commonly used PCR-based methodologies require the knowledge of the microorganism’s genome sequences; however, this knowledge is not always available. A typical case is represented by the outbreaks of emerging pathogens, where the causative agent was never before identified, at least in the human population. Even more important for the daily practice is the case of diseases for which the diagnosis of the etiological determinant still poses a challenge in a high percentage of cases. A paradigmatic example is represented by encephalitis, where, in up to 60%–75% of cases, the identification of the causing pathogen remains unmet, even after broad-range PCR-based screenings [1–3]. Different reasons might account for this failure. For instance, the causative agent might be represented by a microorganism that was never before associated with encephalitis (unexpected) or that is completely unknown. The discovery of novel pathogens through the analysis of their genome requires in these cases a random, sequence-independent, and thus “universal” amplification strategy [4]. Unbiased next-generation sequencing (NGS) is emerging as an attractive approach for the detection of pathogens in different types of samples, including clinical specimens. NGS applied to microbial diagnosis ideally should allow the amplification and analysis of any type of genetic material present in the clinical sample with a dose-independent efficiency, without the need to design specific primers to preamplify target sequences. Because random/unbiased amplification amplifies the host nucleic acids along with the microbial ones, searching for the microbial nucleic acids is like looking for a needle in a haystack. Indeed, even though different strategies have been developed to either enrich the samples with microorganism nucleic acids or to decrease the amount of the host nucleic acids [5], the level of host background still limits the sensitivity of NGS in pathogen discovery. Moreover, the choice of the NGS platform is crucial, with read length and number of sequences generated per runs as key parameters guiding the selection [6]. Taking into account these aspects, the potentiality of unbiased NGS in recovering genetic material of unknown pathogens, especially viruses, has been already proven [5]. In the present issue of Clinical Infectious Diseases, 2 different articles, one by Brown et al [7] and one by Naccache et al [8], address the problem of the diagnosis of unexplained encephalitis in 2 immunocompromised patients, an 18-month-old boy and a 42-year-old man, respectively, by employing an unbiased NGS approach. In both cases, high-throughput RNA sequencing (RNA-Seq, Illumina Miseq) allowed the identification of a novel astrovirus, HAstV-VA1/HMO-C-UK1, as the potential causative agent of the fatal encephalitis. Astroviruses are nonenveloped, singlestranded RNA viruses, commonly associated with gastroenteritis in humans and only recently reported as possible causative agents of encephalitis [9, 10]. In one case, the patient was found to be infected by HAstV-SG, an astrovirus of the VA/ HMO clade [9]. In the second case, the Received 6 November 2014; accepted 7 November 2014; electronically published 7 January 2015. Correspondence: Giorgio Palù, MD, Department of Molecular Medicine, University of Padova, via Gabelli 63, 35121 Padova, Italy ([email protected]). Clinical Infectious Diseases 2015;60(6):889–91 © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals. [email protected]. DOI: 10.1093/cid/ciu913