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Identification of pathogens causing viral encephalitis remains challenging, and in over 50% of cases the etiologic factor remains undetermined. Next-generation sequencing (NGS) based metagenomics has been successfully used to detect novel and rare infections, but its value for routine diagnosis of encephalitis remains unclear. The aim of the present study was to determine the sensitivity of shotgun metagenomic sequencing protocols, which include preamplification, and testing it against cerebrospinal fluid (CSF) samples from encephalitis patients. For sensitivity testing HIV and HBV positive sera were serially diluted in CSF from an uninfected patient. NGS repeatedly detected HIV and HBV sequences present at concentrations from 10 5 to 10 2 and from 10 5 to 10 viral copies/reaction, respectively. However, when the same protocols were applied to RT-PCR/PCR positive CSF samples from 6 patients with enteroviral encephalitis (median viral load 47 copies/ml) and 15 patients with HSV, CMV or VZV encephalitis (median viral load 148 copies/ml), only 7 (28.6%) were identified as positive. In conclusions, while NGS has the advantage of being able to identify a wide range of potential pathogens it seems to be less sensitive compared to the standard amplification-based assays in the diagnosis of encephalitis, where low viral loads are common.

Hepatitis C virus (HCV) is characterized by high genetic variability, which is manifested both at the inter-host and intra-host levels. However, its role in the clinical course of infection is less obvious. The aim of the present study was to determine the genetic variability of HCV HVR1 (hypervariable region 1) of genotype 1b and 3 in plasma of blood donors in the early seronegative stage of infection (HCV-RNA+, anti-HCV-) and in samples from chronically infected patients using next-generation sequencing. Sequencing errors were corrected, and haplotypes inferred using the ShoRAH software. Genetic diversity parameters (intra-host number of variants, number of nucleotide substitutions and diversity per site) were assessed by DNA SP and MEGA. During the early infection, the number of variants were significantly lower in subjects infected with genotype 3 than with genotype 1b (p < 0.02). Similarly, intra-host number of variants, number of nucleotide substitutions and diversity per site were lower in genotype 3 chronic infection (p < 0.0005). In addition, early infection was characterized by significantly lower HVR1 variability values (p < 0.04) when compared to chronic infection for both genotypes. It seems that the observed differences in HVR1 variability represent an inherent property of particular viral genotypes.