Respiratory Treatment and Prevention (Advances in Experimental Medicine and Biology)

fluid (CSF). Moreover, we analyzed the contamination background. We

detected 10^2 HIV copies and 10^3 HSV copies. The analysis of control

samples (two water samples and one CSF sample from an uninfected

patient) revealed the presence of human DNA in the CSF sample (91 %

of all reads), while the dominating sequences in water were qualified as

‘other’, related to plants, plant viruses, and synthetic constructs, and

constituted 31 % and 60 % of all reads. Bacterial sequences represented

5.9 % and 21.4 % of all reads in water samples and 2.3 % in the control

CSF sample. The bacterial sequences corresponded mainly to

Psychrobacter, Acinetobacter,andCorynebacterium genera. In conclu-

sion, Ribo-SPIA amplification followed by NGS metagenomic analysis is

sensitive for detection of RNA and DNA viruses. Contamination seems

common and thus the results should be confirmed by other independent

methods such as RT-PCR and PCR. Despite these reservations, NGS

seems to be a promising method for the diagnosis of viral infections.

Keywords

Bacteria • Cerebrospinal fluid • DNA • Encephalitis • Next-generation

sequencing • Metagenomics analysis • Pathogens • Viruses

1 Introduction

Next-generation sequencing (NGS) is frequently
used to analyze microbial evolution, pathogen
transmission patterns, and to identify drug-
resistant variants (Frey et al. 2014 ; Barzon
et al. 2013 ; Capobianchi et al. 2013 ; Dunne
et al. 2012 ). NGS is also employed to identify
etiologic agents in various infections (Miller
et al. 2013 ; Yozwiak et al. 2012 ; Virgin and
Todd 2011 ; Nakamura et al. 2009 ). Metagenomic
sequencing provides insight into microbial com-
position of a sample, enables the detection of
non-culturable and highly variable organisms, as
well as emerging and novel pathogens (Barzon et
al. 2013 ; Capobianchi et al. 2013 ; Frey et al.
2014 ).
Infections of the central nervous system are
caused by a variety of microorganisms including
viruses, bacteria, fungi, and parasites. Until now,
more than 100 different agents have been
identified as capable of causing encephalitis
(Granerod et al.2010a; Glaser et al. 2003 ). The
most common viral agents causing encephalitis

in Europe are herpes simplex virus (HSV),

arboviruses (tick-borne encephalitis virus), and

enteroviruses (Paradowska-Stankiewicz and

Piotrowska 2014 ; Granerod and Crowcroft

2007 ; Glaser et al. 2006 ). However, etiology

remains unknown in 40–60 % of cases, which

could be due to a wide range of factors such as

suboptimal time of sample collection, large num-

ber of potential causative microorganisms, or the

existence of yet unknown pathogens (Silva 2013 ;

Granerod et al. 2010b). Routine diagnostics,

which typically includes serological and molecu-

lar tests, enables the detection only of the most

common pathogens (Rasool et al. 2014 ; Solomon

et al. 2007 ; Steiner et al. 2005 ; Chaudhuri and

Kennedy 2002 ).

NGS could be a valuable tool in the diagnosis

of encephalitis as it could detect causative

pathogens even when the routine tests fail

(Wilson et al. 2014 ; Tan le et al. 2013 ). In the

current study we analyzed the sensitivity of NGS,

preceded by isothermal RNA amplification

(Ribo-SPIA), for the detection of HIV and HSV

in the cerebrospinal fluid (CSF).

54 I. Bukowska-Os ́ko et al.