Detection of herpes simplex virus DNA in cerebrospinal fluid samples using the polymerase chain reaction and microplate hybridization

doi:10.1016/0166-0934(95)01991-X

Journal of Virological Methods

Volume 59, Issues 1–2, May 1996, Pages 1-11

https://doi.org/10.1016/0166-0934(95)01991-X Get rights and content

Abstract

As conventional polymerase chain reaction (PCR) procedures are time-consuming and laborious, we developed and evaluated a rapid semi-automatic microplate method to detect the amplified PCR products. The use of PCR, with subsequent hybridization in microplates, is described for the detection of herpes simplex virus (HSV) DNA in cerebrospinal fluid samples. The principle of the method is based on two phases. Firstly, the amplification of the viral DNA in the sample is undertaken using a pair of primers of which one is biotinylated. Secondly, the amplified viral genomic sequences are bound to the wells of streptavidin-coated microplates and hybridized with digoxigenin-labeled oligonucleotide probes which are then detected using anti-digoxigenin antibody enzyme conjugates and either a photometric, fluorometric or luminometric substrate and microplate reader. The method is highly sensitive allowing the detection of as few as five purified DNA molecules. Compared to conventional gel electrophoresis followed by Southern blotting the established microplate hybridization is also much less time-consuming and involves less manual work. The applicability of the method is described for use as a routine diagnostic procedure for detection of early central nervous system infections caused by HSV-1 and HSV-2.

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Cited by (45)

Improved multiplex-PCR and microarray for herpesvirus detection from CSF
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Citation Excerpt :
DNA controls of HSV-1 strain MacIntyre, HSV-2 G, CMV AD169, EBV B95-8, HHV-7 H7-4, HHV-6A U1102, HHV-6B Z-29, and varicella-zoster virus (VZV) Rod (Advanced Biotechnologies, Columbia, MD) were used for dilution series. Twenty-two CSFs were collected from specimens tested with HSV-PCR (Piiparinen and Vaheri, 1991; Vesanen et al., 1996), and VZV-PCR (Echevarria et al., 1994; Koskiniemi et al., 1997) at HUSLAB (Helsinki, Finland). Ten CSFs were positive for HSV-1 or HSV-2, 10 for VZV, and two were negative.
A multiplex-PCR and microarray-based method was designed for detection of eight herpesviruses from clinical specimens.
To improve the method, especially for detection of herpes simplex (HSV) and varicella-zoster viruses (VZV), and to update and validate the method using positive cerebrospinal fluid (CSF) samples.
A new primer pair for HSV-PCR and four detection oligonucleotides for HSVs were designed. Two new detection oligonucleotides for VZV and additional oligonucleotides for CMV, EBV, HHV-6 and -7 were designed. The new and previous multiplex-PCR and microarray method were tested in parallel with dilution series of commercial herpesvirus DNAs and 20 CSF specimens positive for HSV-1, HSV-2, or VZV.
New method was more sensitive for detection of HSVs and both two new detection oligonucleotides for VZV functioned well at low levels of viral DNA.
The revised HSV-PCR and new HSV- and VZV-oligonucleotides were found to function well and be more sensitive, thereby increasing reliability of the method.
Primary Human Herpesvirus-6 Infection in the Central Nervous System Can Cause Severe Disease
2007, Pediatric Neurology
Citation Excerpt :
The sample was considered positive if the relative light unit signal was >7-fold higher than the background signal. The polymerase chain reactions to herpes simplex virus-1, herpes simplex virus-2, and varicella zoster virus from cerebrospinal fluid were performed as described [22,23]. Antibodies to human herpesvirus-6 were examined using an in-house indirect immunofluorescence test, and titers are indicated as the reciprocal of the sample dilution [24].
Human herpesvirus-6 (HHV-6) infection is common in infancy, and symptoms are usually mild. However, encephalitis and other neurologic complications have been reported. Primary HHV-6 infection has been rarely confirmed in the central nervous system. We studied 21 children with suspected HHV-6 infection, drawn from a prospective, large-scale study of neurologic infections in Finland. Human herpesvirus-6 polymerase chain reaction was performed on cerebrospinal fluid samples, and antibody tests were performed on serum and cerebrospinal fluid. We identified nine children, aged 3 to 24 months, who had HHV-6-specific nucleic acid in cerebrospinal fluid. Primary infection was confirmed by seroconversion of specific antibodies in six, whereas one had a fourfold increase, and one had a fourfold decrease, in the antibody titer supporting recent infection. Generalized and prolonged seizures appeared in six children, four had a rash, four had ataxia, and four had gastroenteritis. All but two had a high fever. At follow-up, four children had evident neurologic sequelae, ataxia, and developmental disability, and needed special education. Primary HHV-6 infection may invade the central nervous system, and can cause neurologic symptoms and potentially permanent disability in children aged ≤2 years. The possibility of HHV-6 infection must be considered when treating acutely ill children, and especially those with convulsions.
Multiplex-PCR and oligonucleotide microarray for detection of eight different herpesviruses from clinical specimens
2006, Journal of Clinical Virology
Human herpesviruses cause clinically important diseases, e.g. infections of the central nervous system. New diagnostic tools are required for rapid and reliable detection of these viruses.
A microarray-based method was designed for detection of eight human herpesviruses in cerebrospinal fluid (CSF), whole blood, plasma, serum and proficiency-testing specimens.
Herpes simplex type 1 and 2, varicella-zoster, cytomegalo-, Epstein-Barr and human herpes viruses 6A, 6B and 7 were amplified from clinical specimens by two multiplex-PCRs and transcribed to single-stranded RNAs which were hybridized to oligonucleotides on microarray. The results were compared to those from conventional PCR. In total, 227 specimens were tested including 23 CSF, 10 whole blood, 73 plasma, 10 proficiency-testing samples and 111 negative control samples.
Concordant results were obtained in 214/227 (94%). Microarray detected 10 possible double and one triple infection. Negative control samples (70 serum, 30 CSF and 11 proficiency-testing samples) were all negative.
Microarray is suitable for detection of multiple herpesviruses in clinical samples.
Clinical applications of chemiluminescence
2003, Analytica Chimica Acta
Citation Excerpt :
The LOCI-type assay described in the previous section has also been adapted for nucleic acid assays, e.g. single-nucleotide polymorphism typing [52]. Other chemiluminescent nucleic acid-based assays include infectious disease testing in combination with nucleic acid amplification techniques such as the polymerase chain reaction [e.g. telomere DNA [53], herpes simplex [54], Lassa fever virus (reverse transcriptase-polymerase chain reaction method) [55], papillomavirus in cervical scrapes [56], and Trichomonas vaginalis (detects <12 organisms) [57]. Western blotting for proteins (e.g. a Western blot for Na+/H+ exchanger isoform-1 in the study of inflammatory bowel disease [58]), Southern blotting for DNA and Northern blotting for RNA, are important techniques in clinical research and chemiluminescent end-points have been developed for each of these types of assay.
This article reviews the clinical applications of chemiluminescence in routine testing and surveys the diverse applications of chemiluminescence in clinical research. In routine clinical testing, chemiluminescent labels (acridinium ester, acridinium sulfonamide) and detection reactions for peroxidase and alkaline phosphatase enzyme labels (luminol and adamantyl 1,2-dioxetane-based reactions, respectively) are widely used in immunoassay and nucleic acid probe assays (e.g. hybridization protection assay, Hybrid Capture^® assay). In clinical research the sensitivity, dynamic range and diversity of chemiluminescent assays has led to a vast range of applications, notably in protein and nucleic acid blotting, microarray-based assays, monitoring reactive oxygen species, and as detection reactions for substances separated by HPLC, capillary electrophoresis (CE), and flow-injection analysis.
Co-detection and discrimination of six human herpesviruses by multiplex PCR-ELAHA
2003, Journal of Clinical Virology
Background: Herpesviruses are a significant cause of human morbidity. Traditional approaches to the identification of these viruses require infectious or at least antigenic virus. Multiplex PCR (mPCR) is capable of simultaneously amplifying a range of targets from a single preparation of nucleic acids and when combined with a suitable detection assay, it is capable of discriminating each of the amplicons. Objectives: Several methods have been described in the literature, however, they lack one or more significant design features required to suitably control a routinely applied nucleic acid amplification assay. We aimed to design a multiplex herpesvirus PCR that could co-amplify eight human herpesvirus targets plus an internal control (IC) molecule in a single tube. Study Design: Primers were designed to target the DNA polymerase genes of each of the human herpesviruses. Synthetic controls were developed to act as templates for the evaluation of assay sensitivity and specificity and for development of an in-house competitive quantitative PCR. Amplicon was discriminated using a simplified enzyme linked amplicon hybridisation assay (ELAHA). Results and Conclusions: For routine diagnostic use we reduced the number of herpesviral targets from 8 to 6 in order to maintain adequate clinical sensitivity. The ELAHA proved more sensitive than agarose gel electrophoresis. Additionally, 36 cytomegalovirus positive patients were examined with an in-house quantitative PCR-ELAHA which was developed to confirm that that the mPCR's co-detection limit of 10² copy of synthetic template per millitre was relevant for use in detecting virus from clinical samples. The mPCR-ELAHA was then applied to the screening of 174 patient specimens resulting in a specificity of 98% and a sensitivity of 93%. This preliminary study demonstrated that the mPCR-ELAHA was a complete approach to the detection of herpesviruses from a range of clinical samples and disease states.
Varicella zoster and Borrelia burgdorferi are the main agents associated with facial paresis, especially in children
2003, Journal of Clinical Virology
Background: The etiology of facial paresis (FP) often remains unresolved. Yet, a microbial association is frequently suspected. Objective: To evaluate the infectious etiology of FP by using sensitive tests. Study design: We studied the serum and cerebrospinal fluid of 42 patients diagnosed with idiopathic peripheral facial paresis using sensitive serological methods and nucleic acid detection and for reference, 42 patients with other neurological disorders (OND) matched for age, sex, season and geographical area. Results: Varicella zoster virus and Borrelia burgdorferi accounted for 56% of all associated agents in children with FP compared with 11% of OND (P=0.01). In adults, the respective numbers were 29 and 13%. Other treatable etiological agents, Chlamydia pneumoniae and Mycoplasma pneumoniae, accounted for 11% in children and 8% in adults and with the same prevalence between patients with FP and OND. Conclusions: Microbes, with specific therapy available accounted for 52% of all associated agents in the patients with FP when compared with 26% in controls with OND (P=0.04). Based on this, we conclude that the patients with FP may benefit from antimicrobial therapy.

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Research paperDetection of herpes simplex virus DNA in cerebrospinal fluid samples using the polymerase chain reaction and microplate hybridization

Abstract

Methods Enzymol.

Encephalitis in immunocompetent patients due to HSV type 1 or 2 as determined by type-specific polymerase chain reaction and antibody assays of CSF

J. Med. Virol.

Different patterns of neurologic involvement with herpes simplex virus types 1 and 2: isolation of herpes simplex virus 2 from the buffy coat of two adults with meningitis

J. Infect. Dis.

Acute diagnosis of herpes encephalitis

Lancet

Detection of viral DNA in neonatal herpes simplex virus infections: frequent and prolonged presence in serum and cerebrospinal fluid

J. Infect. Dis.

Diagnosis of herpes encephalitis via Southern blotting of CSF DNA amplified by polymerase chain reaction

J. Med. Virol.

Poor antibody production in fatal herpes encephalitis

J. Infect. Dis.

Polymerase chain reaction for detection of herpes simplex virus in cerebrospinal fluid. A prospective study of 1053 specimens

Ann. Neurol.

Herpes simplex virus encephalitis: laboratory evaluations and their diagnostic significance

J. Infect. Dis.

Research paper
Detection of herpes simplex virus DNA in cerebrospinal fluid samples using the polymerase chain reaction and microplate hybridization