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Virus identification at homeostasis and disease in clinical samples by unta...

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Institut Pasteur de Madagascar

Tananarive, Tananarive 101

Madagascar

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As in other medical fields, the availability of next generation sequencing (NGS) techniques is about to revolutionize diagnostics of infectious diseases and pathogen discovery. The main drawback of targeted, pathogen-specific PCRs is that they are only able to identify predefined targets, which supposes that the physician has elaborated an etiological hypothesis. Moreover, for a series of pathogens, and in particular highly variable RNA viruses like enteroviruses or DNA viruses such as papillomaviruses and adenoviruses that comprise multiple types, PCR-based tests target conserved loci that hardly discriminate between genotypes.

To bypass these difficulties, several strategies have been developed, with as main objective to broaden the range of detection. Multiplexed PCR assays for multiple loci have been and are still being developed to provide, at least in principle, simultaneous detection of several agents. The range of multiplex PCR can be considerably improved by designing primers targeting numerous pathogens and varied loci within pathogens and resolving these amplicons using electrospray ionization-mass spectrometry or NGS. Nevertheless, the design of numerous primers will have to be constantly updated along with increase in the number of sequences in databases and identification of new pathogens, in order to maintain a high range of detection.

An alternative strategy, Whole Genome-NGS takes advantage of the increasing availability and speed and decreasing cost per base of NGS. We describe the pipelines we are currently using and show comparison of analytical sensitivity with that of (RT)PCR. We also show examples of identification of pathogens not diagnosed by standard pipelines in human clinical cases with a specific focus in immunocompromised patients, and results of efforts to identify unknown persistent human viruses at homeostasis.

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Institut Pasteur de Madagascar

Tananarive, Tananarive 101

Madagascar

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