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Review
. 2003 Jan;26(1):1-28.
doi: 10.1016/s1386-6532(02)00173-7.

Molecular analysis of cerebrospinal fluid in viral diseases of the central nervous system

Paola Cinque  1 , Simona Bossolasco, Ake Lundkvist
Affiliations
Review

Molecular analysis of cerebrospinal fluid in viral diseases of the central nervous system

Paola Cinque et al. J Clin Virol. 2003 Jan.

Abstract

The use of nucleic acid (NA) amplification techniques has transformed the diagnosis of viral infections of the central nervous system (CNS). Because of their enhanced sensitivity, these methods enable detection of even low amounts of viral genomes in cerebrospinal fluid. Following more than 10 years of experience, the polymerase chain reaction or other NA-based amplification techniques are nowadays performed in most diagnostic laboratories and have become the test of choice for the diagnosis of several viral CNS infections, such as herpes encephalitis, enterovirus meningitis and other viral infections occurring in human immunodeficiency virus-infected persons. Furthermore, they have been useful to establish a viral etiology in neurological syndromes of dubious origin and to recognise unusual or poorly characterised CNS diseases. Quantitative methods have provided a valuable additional tool for clinical management of these diseases, whereas post-amplification techniques have enabled precise genome characterisation. Current efforts are aiming at further improvement of the diagnostic efficiency of molecular techniques, their speed and standardisation, and to reduce the costs. The most relevant NA amplification strategies and clinical applications of to date will be the object of this review.

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Figures

Fig. 1
Fig. 1
Example of multiplex PCR. Three unrelated sequences of HSV-1, HSV-2 and VZV, are amplified simultaneously in the same test tube by using three different primer pairs, specific for each virus. The amplification products can be differentiated on agarose gel if the amplified fragments yield bands of different size (M: 100 bp DNA ladder marker). Alternatively, amplification products can be identified through an additional step, by hybridization with specific probes, restrictions enzyme analysis, nested PCR with specific internal primers, or DNA sequencing.
Fig. 2
Fig. 2
Example of PCR assay with consensus primers (adapted from Rozenberg and Lebon, 1991). Conserved DNA sequence from the polymerase genes of HSV-1, HSV-2, EBV and CMV are amplified simultaneously in the same tube by a consensus primer pair targeting regions in common to these viruses. Following agarose gel electrophoresis, the amplified products have similar length (top) but each virus can be distinguished by using restriction enzymes (bottom) (a, SmaI and b, BamHI; M: DNA marker). Alternatively, viruses can be differentiated following hybridization with virus-specific probes or DNA sequencing.
Fig. 3
Fig. 3
Example of a standard curve consisting of 5–50 000 EBV DNA genomes/reaction generated by automated real-time PCR using the TaqMan technology. The fluorescence, proportional to the amount of amplified products, is acquired at each PCR cycle by an automated fluorometer. A threshold cycle (CT) defines the cycle number at which the fluorescence passes a fixed threshold. Quantification of the amount of target in unknown samples is accomplished by measuring the CT and comparing this value to the CT values of the standard curve.
Fig. 4
Fig. 4
DNA sequencing from paired CSF and plasma specimens. An example of nucleotide sequencing from paired CSF and plasma samples using cycle-sequencing with dye-labeled oligonucleotides. Amplified products are obtained from paired CSF and plasma specimens following nucleic acid extraction, RNA retrotranscription and PCR amplification of a fragment from the HIV-1 reverse transcriptase (RT) gene. The amplified DNA is purified from unincorporated primers and nucleotides and added to the sequencing reaction mixture. The products of the sequencing reaction are subected to automated electrophoresis and recognized by a laser scanner. A four-color electropherogram is produced, which is translated into a linear nucleotide sequence by a computer software. The final sequence is compared to reference sequences, e.g., HXB2 for HIV-1. Three nucleotide mutations, resulting in two aminoacid substitutions at codons 215 (treonin→phenylalanin) and 219 (lysin→glutamin) are found in plasma but not in SCF (arrows). Such mutations are associated with resistance to the RT inhibitor drug zidovudine.
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