Molecular methods for diagnosis of viral encephalitis

Abstract

Hundreds of viruses cause central nervous system (CNS) disease, including meningoencephalitis and postinfectious encephalomyelitis, in humans. The cerebrospinal fluid (CSF) is abnormal in >90% of cases; however, routine CSF studies only rarely lead to identification of a specific etiologic agent. Diagnosis of viral infections of the CNS has been revolutionized by the advent of new molecular diagnostic technologies to amplify viral nucleic acid from CSF, including PCR, nucleic acid sequence-based amplification, and branched-DNA assay. PCR is ideally suited for identifying fastidious organisms that may be difficult or impossible to culture and has been widely applied for detection of both DNA and RNA viruses in CSF. The technique can be performed rapidly and inexpensively and has become an integral component of diagnostic medical practice in the United States and other developed countries. In addition to its use for identification of etiologic agents of CNS disease in the clinical setting, PCR has also been used to quantitate viral load and monitor duration and adequacy of antiviral drug therapy. PCR has also been applied in the research setting to help discriminate active versus postinfectious immune-mediate disease, identify determinants of drug resistance, and investigate the etiology of neurologic disease of uncertain cause. This review discusses general principles of PCR and reverse transcription-PCR, including qualitative, quantitative, and multiplex techniques, with comment on issues of sensitivity, specificity, and positive and negative predictive values. The application of molecular diagnostic methods for diagnosis of specific infectious entities is reviewed in detail, including viruses for which PCR is of proven efficacy and is widely available, viruses for which PCR is less widely available or for which PCR has unproven sensitivity and specificity, and nonviral entities which can mimic viral CNS disease.

FIG. 1.

General PCR schema.

FIG. 2.

Probes for probe-based quantitative PCR. In probe-based quantitative PCR, reagents which are capable of generating a fluorescent signal within the PCR can be monitored and quantified to reflect the starting amount of nucleic acid template at each cycle of the PCR. Probe-based quantitative PCR methods rely on the use of a fluorescent reporter molecule that increases as the PCR product accumulates, which is composed of a sequence-specific oligonucleotide with an integrated fluorophore and quencher, as in molecular beacon probes (Scorpion) and TaqMan probes. FRET-based PCR (not shown) makes use of dual sequence-specific oligonucleotide probes with either integrated 3′ donor or 5′ acceptor fluorophores; the PCR product is quantified as a function of measured energy transfer between adjacent donor and acceptor fluorophores.

FIG. 3.

SYBR Green-based quantitative PCR. SYBR Green-based quantitative PCR utilizes a sensitive DNA binding dye that binds any double-stranded product generated by the PCR. Thus, the same dye can be used in different quantitative PCR approaches, but the specificity must be verified by other methods (see the text).

FIG. 4.

Quantitative real-time PCR with melting curve analysis. (A) Detection of HSV DNA in a serially diluted suspension by LightCycler PCR with the FRET assay. The sequential numbers indicated at the base of each signal designation refer to the corresponding sample number and dilution (two left-hand columns). (B) Melting curve analysis of HSV-1 and HSV-2 genotypes determined by LightCycler PCR with the FRET assay. Reprinted from reference with permission.