Development of a multiplex nested consensus PCR for detection and identification of major human herpesviruses in CNS infections.

BACKGROUND: Rapid, sensitive and economical detection and identification of human herpesviruses as causative agents of central nervous system (CNS) infections are of clinical importance. The traditional methods for the detection of herpesviruses in CNS infections all suffer from limitations. PCR has a potential to overcome each of them. OBJECTIVES: The aims of this study were reducing the number of primers in multiplex PCR and increasing the sensitivity of the assay by nested PCR. STUDY DESIGN: A multiplex nested consensus PCR (MNC-PCR) was developed for the simultaneous detection of major human herpesviruses. A pair of conserved primers was designed for detection of HSV-1, HSV-2, CMV and EBV and another pair of conserved primers for nested PCR. For VZV, a different pair of primers was designed and another pair of primers for nested PCR. A reduction in the number of designed primer pairs (from five pairs to two in both stages of PCR) is an advantage in this assay. One hundred forty-seven cerebral spinal fluid (CSF) samples from patients that showed clinical manifestation of CNS infections were tested. Results of MNC-PCR in CSF samples were compared with those of single PCR assay for each individual DNA virus. Sensitivity of the assay was determined with a plasmid containing VZV DNA binding protein gene and another plasmid for HSV-1 DNA polymerase gene. False negative results (due to the presence of inhibitor of DNA amplification in CSF samples) were avoided by the inclusion of beta2-microglobulin primers in the MNC-PCR assay as an internal control. RESULTS: Positive results were obtained in 20 CSF samples (8 HSV-1, 2 HSV-2, 4 CMV, 3 VZV, 3 HSV-1/CMV, CMV/VZV and HSV-1/EBV coinfections). The comparison between single PCR and MNC-PCR showed a marked increase in sensitivity of MNC-PCR test, since six negative samples in single PCR proved positive in MNC-PCR (P<0.005). Sensitivity was determined 1-5 plasmid copies for VZV and 50-100 plasmid copies for HSV-1. CONCLUSIONS: The MNC-PCR assay presented in this study can provide a rapid, sensitive and economical method for detection of viral infections and is applicable to small volumes of CSF samples.     

Laboratory Diagnosis of Common Herpesvirus Infections of the Central Nervous System by a Multiplex PCR Assay

A sensitive multiplex PCR assay for single-tube amplification that detects simultaneous herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (CMV), and Epstein-Barr virus (EBV) is reported with particular emphasis on how the method was optimized and carried out and its sensitivity was compared to previously described assays. The assay has been used on a limited number of clinical samples and must be thoroughly evaluated in the clinical context. A total of 86 cerebrospinal fluid (CSF) specimens from patients which had the clinical symptoms of encephalitis, meningitis or meningoencephalitis were included in this study. The sensitivity of the multiplex PCR was determined to be 0.01 and 0.03 50% tissue culture infective doses/the reciprocal of the highest dilution positive by PCR for HSV-1 and HSV-2 respectively, whereas for VZV, CMV and EBV, 14, 18, and 160 ag of genomic DNA were detected corresponding to 48, 66, and 840 genome copies respectively. Overall, 9 (10.3%) of the CSF samples tested were positive in the multiplex PCR. HSV-1 was detected in three patients (3.5%) with encephalitis, VZV was detected in four patients (4.6%) with meningitis, HSV-2 was detected in one neonate (1.16%), and CMV was also detected in one neonate (1.16%). None of the samples tested was positive for the EBV genome. None of the nine positive CSF samples presented herpesvirus coinfection in the central nervous system. Failure of DNA extraction or failure to remove any inhibitors of DNA amplification from CSF samples was avoided by the inclusion in the present multiplex PCR assay of alpha-tubulin primers. The present multiplex PCR assay detects simultaneously five different herpesviruses and sample suitability for PCR in a single amplification round of 40 cycles with an excellent sensitivity and can, therefore, provide an early, rapid, reliable noninvasive diagnostic tool allowing the application of antiviral therapy on the basis of a specific viral diagnosis. The results of this preliminary study should prompt a more exhaustive analysis of the clinical value of the present multiplex PCR assay.     

DNA microarray technology for simultaneous detection and species identification of seven human herpes viruses

The aim of the study was to develop a multiplex PCR-based DNA microarray technology for simultaneous detection and species identification of seven human herpes viruses, namely herpes simplex virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpes virus 6 (HHV-6A, HHV-6B), and to apply this technology to accurate diagnosis of herpesvirus-associated diseases. Primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase gene in human herpes viruses. DNA microarrays were made by printing the oligonucleotide probes onto special glass slides. After amplification and labeling with CY5, the PCR products were hybridized with the DNA microarrays and species identified. Sixty-one cerebrospinal fluid (CSF) and 132 blood specimens were analyzed by this technique, and the results were compared with those of TaqMan PCR. Several specimens were sequenced further after cloning. The PCR products of the seven human herpes viruses ranged from 224 to 252 bp, and could be species identified with DNA microarrays. The detection limits were 10(1) copies/microl for each virus. And the test showed no cross-reaction to DNA extracted from S. aureus, E. coli, hepatitis B virus, Cryptococcus neoformans, Candida albicans and human genome. Among 132 blood and 61 CSF specimens, 55 were tested positive for human herpes virus DNA. Compared with the results of TaqMan PCR, the sensitivity and specificity of the DNA microarray technology was 96.2% and 99.3%, respectively. This multiplex PCR-based DNA microarray technology, which is rapid, specific and sensitive, serves as an effective technique for simultaneous detection and species identification of seven human herpes viruses.     

Herpesvirus (HSV-1, EBV and CMV) infections in atherosclerotic compared with non-atherosclerotic aortic tissue

The viral nucleic acid of herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) was studied by polymerase chain reaction (PCR), Southern blotting and in situ hybridization (ISH) in aortic tissues from 33 autopsies. In 23 cases involving persons who ranged from 23 weeks to 75 years of age at the time of death, the tissue was histologically non-atherosclerotic. Of these 23, aortic tissues tested positive for HSV-1 in 13%, for EBV in 13% and for CMV in 4%. In the other 10 cases involving persons who were 53-75 years old at death, atherosclerotic aortic tissue tested positive for HSV-1 in 80%, for EBV in 80% and for CMV in 40%. Neither double nor triple infections occurred in the non-atherosclerotic group, whereas six of 10 were positive for two viruses, and two of 10 were positive for three viruses in the atherosclerotic group. By in situ hybridization, the viruses were localized in cells morphologically consistent with endothelial cells and smooth muscle cells. We detected HSV-1, EBV and CMV DNA in cells in the upper portion of the non-atherosclerotic aortic wall, whereas viral DNA was detected more extensively in atherosclerotic lesions than in non-atherosclerotic tissue. We also are the first to show the existence of EBV DNA in the human aortic wall. In conclusion, we suggest that the high incidence and kinds of herpesviruses are related to the high incidence of atherosclerosis.     

Multiplex PCR-based DNA array for simultaneous detection of three human herpesviruses, EVB, CMV and KSHV

Human lymphotropic herpesviruses, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are responsible for a wide variety of human diseases. Due to an increase in diseased states associated with immunosuppression, more instances of co-morbid infections with these herpesviruses have resulted in viral reactivations that have caused numerous fatalities. Therefore, the development of rapid and accurate method to detect these viruses in immunocompromised patients is vital for immediate treatment with antiviral prophylactic drugs. In this study, we developed a new multiplex PCR method coupled to DNA array hybridization, which can simultaneously detect all three human herpesviruses in one single cell sample. Multiplex PCR primers were designed to amplify specific regions of the EBV (EBER1), CMV (IE) and KSHV (LANA) viral genomes. Pre-clinical application of this method revealed that this approach is capable of detecting as few as 1 copy of the viral genomes for KSHV and CMV and 100 copies of the genome for EBV. Furthermore, this highly sensitive test showed no cross-reactivity among the three viruses and is capable of detecting both KSHV and EBV viral genomes simultaneously in the lymphoblastoid cells that have been double infected with both viruses. Thus, this array-based approach serves as a rapid and reliable diagnostic tool for clinical applications.     

Epidemiology of Epstein-Barr virus, cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus infections in peripheral blood leukocytes revealed by a multiplex PCR assay

A multiplex polymerase chain reaction (PCR) has been developed for the simultaneous detection of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and Kaposi's sarcoma-associated herpesvirus (KSHV) in a clinical sample. Primers of multiplex PCR were designed to amplify specific regions of the EBV EBNA1, CMV IE2, and KSHV LANA genes. This multiplex PCR assay was found to have detection sensitivities of 1-10 copies of purified viral DNA cloned into the plasmid. To assess diagnostic and pre-clinical applications with this method, we utilized KSHV-positive primary effusion lymphoma (PEL) cells, EBV-positive Burkitt's lymphoma cells, CMV-infected fibroblast cells, and clinically prepared peripheral blood leukocytes (PBLs) that had been infected with viruses. We found that this multiplex PCR assay has high sensitivity and specificity for simultaneous detection of EBV, CMV, and KSHV genomes in a single amplification from a clinical material. Using this multiplex PCR assay, we investigated the prevalence of EBV, CMV, and KSHV in PBL samples from normal Japanese randomly selected. KSHV, EBV, and CMV genomes were detected in samples from 2 (0.2%), 377 (39.5%), and 27 (2.8%) of the 953 blood donors, respectively. Interestingly, both EBV and CMV genomes were detected in samples from all KSHV-positive donors.     

Parallel detection of five human herpes virus DNAs by a set of real-time polymerase chain reactions in a single run.

BACKGROUND: Human herpes viruses cause a spectrum of diseases that are usually self-limiting but can be reactive during immuno-suppression and may then lead to severe or even life-threatening diseases. The LightCycler technology allows rapid polymerase chain reaction (PCR) including product analysis within a closed system. This approach has been demonstrated to be suitable for routine diagnostic virus detection. Several LightCycler PCR assays have been established to the detection of human herpes viruses. The assays vary in their detection formats and PCR cycling protocols. So, they cannot be performed within a single LightCycler run. OBJECTIVES: Development of four LightCycler PCR assays for parallel detection of DNA derived from human cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV) in a single run. STUDY DESIGN: Primers and hybridization probes were tailored to suit one LightCycler PCR program. LightCycler PCRs were established, detection limits were determined, and clinical samples were evaluated. RESULTS: With quantified herpes virus type specific DNA spiked into cerebrospinal fluid, serum or EDTA plasma the detection limits were found either at 500 or 250 viral DNA copies per ml depending on the virus DNA specific PCR and on the specimen type used. The applicability of the new LightCycler assays for routine molecular testing was evaluated by testing 96 clinical samples. CONCLUSION: The developed set of LightCycler PCRs permits parallel detection of CMV, EBV, HSV-1, HSV-2, and VZV in a single LightCycler run. The new molecular assays can easily be used to the rapid, simple, and convenient detection of herpes virus DNA in cerebrospinal fluid, serum and EDTA plasma in the routine diagnostic laboratory.     

Loop-mediated isothermal amplification assay for the diagnosis of retinitis caused by herpes simplex virus-1

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of herpes simplex virus 1 (HSV-1). The specificity of the assay was tested using DNA extracted from HSV-1-infected rabbit corneal epithelium cultures, HSV-2 grown on Vero cell line, cytomegalovirus (CMV) (AD-169), varicella zoster virus (VZV) (Oka-vaccine), adenovirus, Aspergillus flavus and Staphylococcus aureus. The specificity of LAMP was confirmed by bidirectional sequencing of the amplicons. The sensitivity of the LAMP assay was tested using different concentrations of HSV-1 DNA. To evaluate the application of the LAMP assay in clinical diagnosis, we tested vitreous samples from 20 patients with suspected viral retinitis using LAMP and real-time PCR for HSV-1. The LAMP primers amplified only HSV-1 DNA; no LAMP products were detected with the DNAs of HSV-2, CMV, VZV, adenovirus A. flavus and S. aureus. The sequences of the positive HSV-1 LAMP products perfectly (99–100%) matched the HSV-1 sequences deposited in the GenBank database. LAMP is as sensitive as real-time PCR, with the lowest detection limit being 10 copies/μL of HSV-1 DNA. Of the 20 patients with suspected viral retinitis, four tested positive for HSV-1 using real- time PCR and LAMP. A 100% concordance was observed across the two methods. The LAMP assay is a rapid, highly specific and sensitive method for the diagnosis of retinitis caused by HSV-1.     

Simultaneous quantification of Epstein-Barr virus, cytomegalovirus, and human herpesvirus 6 DNA in samples from transplant recipients by multiplex real-time PCR assay

We developed a multiplex real-time PCR assay using 6-carboxyfluorescein, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, and carbocyanine 5-labeled probes to simultaneously quantify Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) DNA. When previously tested and stored DNA samples were examined, results of the multiplex real-time PCR assay were as sensitive and specific as those of a single real-time PCR assay. The multiplex assay was used to quantify the EBV, CMV, and HHV-6 DNA in 46 transplant recipients. A total of 303 whole-blood and plasma specimens were collected and analyzed. According to the results of the multiplex assay, the detection rates for viral DNA in whole blood and plasma were 23.8% and 5.9% for EBV, 11.2% and 5.3% for CMV, and 12.5% and 2.0% for HHV-6, respectively. All forms of viral DNA were detected more frequently in whole blood than in plasma. During the symptomatic period, EBV DNA was detected in all whole-blood specimens but not in all plasma specimens. Furthermore, the EBV DNA load in whole blood was higher during the symptomatic period than during the asymptomatic period, whereas the EBV DNA load in plasma was similar for both periods. These results demonstrate that whole blood is more suitable for the quantification of EBV DNA in transplant patients. However, a cutoff value with clinical relevance still needs to be determined.     

Vitreous fluid sampling and viral genome detection for the diagnosis of viral retinitis in patients with AIDS

Cytomegalovirus (CMV) causes severe necrotizing retinitis in patients with the acquired immune deficiency syndrome (AIDS) and other herpesviruses have been implicated in the acute retinal necrosis syndrome (ARN), seen in both the immunocompetent and the immunosuppressed. At present the diagnosis of viral retinitis relies solely on clinical appearances. In order to assess whether the detection of herpesvirus-specific DNA in cell-free vitreous biopsy samples could be useful in the early diagnosis of viral retinitis, vitreous fluid samples were taken from 100 patients. Fifty patients had AIDS as defined by the Centers for Disease Control, (MMWR 36 (suppl 1S):1S–15S, 1987) and retinal disease. The remainder were not known to be HIV infected and had no clinical evidence of retinal infection. Each sample was tested for the presence of CMV, herpes simplex virus 1 (HSV-1), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV6), by amplification of viral DNA using a sensitive and specific nested poly-merase chain reaction (PCR). The presence of detectable CMV or VZV DNA was clearly associated with clinical disease whereas the presence of HSV-1, EBV, and HHV6 sequences were not. Clinical discrimination between CMV- and VZV-associated retinitis was greatly enhanced when the PCR results were taken into consideration. © 1994 Wiley-Liss, Inc.     

Enhancing sensitivity of human herpes virus diagnosis with DNA microarrays using dendrimers

DNA microarray technology has become a promising new tool for the detection and identification of viral pathogens in human plasma and cell cultures. For exploration of this technology, we have developed DNA microarrays that encode capture oligonucleotide probes for different human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The on-chip hybridization is accomplished with the PCR amplicons of the respective human herpes virus types. In this original article, we attached multiple Cy3-fluorophores to the branched 5' ends of the labeling oligonucleotide primers. For the first time, we experimentally demonstrated that the self-designed, knowledge-based, and focused microarrays specifically hybridized to fluorophore-labeled pathogenic DNAs using dendrimer technology. The fluorescence signal enhancement via the dendrimers was up to 30 times compared with the quenched single Cy3-fluorophore-labeled HSV-1 DNA. The on-chip signal-amplifying effect depended upon the number of branches and the concentration of fluorophore-labeled pathogenic DNAs. Treblers were superior to doublers, as trebler-labeled nucleic acids had fluorescence-signal-enhancing effects over a broad range of labeled DNA concentrations exemplified for the quenched single Cy3-fluorophore-labeled HSV-1 and non-quenched single Cy3-fluorophore-labeled CMV DNAs.     

Comparison of a Transplant Multiplex Viral Panel on the ICEPlex System with Real-Time PCR for Detection of Cytomegalovirus,Epstein-Barr Virus,and BK Virus in Clinical Specimens

We compared a multiplex viral transplant panel on the ICEPlex system to real-time PCR for the detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and BK virus (BKV). The sensitivities of the ICEPlex were 83.3%, 95.5%, and 65.5% for the detection of CMV, EBV, and BKV, respectively. Interestingly, the multiplex assay detected dual infections in 16/280 (5.7%) samples tested.     

Association of viral factors with non-familial breast cancer in Taiwan by comparison with non-cancerous, fibroadenoma, and thyroid tumor tissues

To study the etiologic factors of non-familial breast cancer, the polymerase chain reaction (PCR) and Southern hybridization were used to detect six viruses including human papillomavirus (HPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV)-1, HSV-2, and human herpesvirus (HHV)-8 DNA in 69 patients with breast cancer and 60 specimens from non-cancerous or other individuals with thyroid tumors or fibroadenoma (non-breast cancer controls). Two specimens from patients with a familial history of breast cancer and five breast cancer specimens with negative results for beta-globin, which was used as internal control, were excluded from this study. Eight (12.9%) HSV-1, 28 (45.2%) EBV, 47 (75.8%) CMV, 8 (12.9%) HPV, and 28 (45.2%) HHV-8 positive samples out of the 62 breast cancer specimens were detected; no HSV-2 DNA was detected in any group. Among the viral gene-positive breast cancer samples, 12 (23.1%) were positive for 1 virus, 16 (30.8%) were positive for 2 viruses, 21 (40.4%) were positive for 3 viruses, and 3 (5.8%) were positive for 4 viruses. Among the viral gene-positive specimens of the control groups, only one virus, CMV, was found in the non-cancerous and thyroid tumor specimens, while multiple viruses were found in the fibroadenoma specimens. The viruses associated with breast cancer were HHV-8 > EBV (P <0.01). The viruses associated with fibroadenoma were HSV-1 and HHV-8 > EBV (P <0.01). The presence of more than one virus was found predominantly in breast cancer and exclusively found in fibroadenoma. CMV was the only virus associated with thyroid tumors.     

DETECTION OF OPPORTUNISTIC DNA VIRAL INFECTIONS BY MULTIPLEX PCR AMONG HIV INFECTED INDIVIDUALS RECEIVING CARE AT A TERTIARY CARE HOSPITAL IN SOUTH INDIA

Purpose: Opportunistic viral infections cause increased morbidity and mortality among human immunodeficiency virus (HIV) infected individuals, especially those who are not on antiretroviral treatment. Early diagnosis of these opportunistic viruses will be able to reduce the risk of disease progression with appropriate intervention. Materials and Methods: Multiplex PCR was attempted to detect the opportunistic herpes viruses (HSV-1, HSV-2, VZV, EBV, and CMV), adenovirus and polyoma viruses (JC and BK) in three cocktails of PCR reactions. Subsequently, all the viruses detected were quantitated by testing using monoplex real time PCR. Whole blood samples collected between 2006 and 2007 from 68 treatment naïve HIV-1 infected and 30 normal healthy individuals were tested for these eight viruses. Among the 68 HIV -1 infected individuals 35 had CD4+ T cell count less than or equal to 200 while the other 33 had greater than 200 CD4+ T cells. Results: Among the 68 HIV-1 infected individuals, 49 (72%) were positive for EBV, 5 (7%) samples were positive for CMV. All the five CMV positive individuals had CD4+ T cell count of less than or equal to 200 cells/µL. The mean EBV load among the individuals with a CD4+ T cells of less than or equal to 200 cells/µL was 3.88 log₁₀ while among those with greater than 200 CD4+ T cells it was 3.75 log₁₀. The mean CMV load was 6.98 log_10. Three samples were positive for both CMV & EBV. None of the samples was positive for HSV-1, HSV-2, VZV, Adenovirus, JC and BK viruses. Conclusions: In our study, multiplex PCR based detection system was found useful in detecting opportunistic viruses in HIV infected individuals. Though EBV is the most prevalent opportunistic viral infection among HIV infected individuals, there was no significant association between EBV load, CD4+ T cell counts and HIV-1 virus load. CMV was seen in HIV infected individuals with low CD4+ T cell counts (less than 200 cells/μL).     

Rapid quantitative PCR assays for the simultaneous detection of herpes simplex virus, varicella zoster virus, cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6 DNA in blood and other clinical specimens

Rapid diagnosis of human herpesvirus primary infections or reactivations is facilitated by quantitative PCRs. Quantitative PCR assays with a standard thermal cycling profile permitting simultaneous detection of herpes simplex virus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV6) DNA were developed and validated for diagnostic use. High specificity and sensitivity were achieved and the new PCR assays correlated well with commercial PCR assays. Twenty two thousand eight hundred sixty eight PCR tests were undertaken on specimens obtained from immunosuppressed patients. DNAemia was frequent with EBV (43.5%), HHV6 (32.4%), CMV (12.8%), and VZV (12.9%). As already described for EBV and CMV, high virus loads of HHV6 and VZV were associated with clinical symptoms and poor clinical outcome, for example, three of four patients with VZV virus loads >10(5) copies/ml died. A high proportion of lower respiratory specimens was positive for EBV- (38.8%), HHV6- (29.4%), and CMV-DNA (18.2%). For CMV, infection was confirmed in 66.7% of patients by virus isolation or positive pp65 antigenaemia. Differentiation of HHV6A, -B and HSV-1, -2 by melting curve analysis revealed that HHV6A and HSV-2 represented only 1.8% and 3.3% of all positive specimens, respectively. In conclusion, these results indicate significant improvements for the early diagnosis of primary infections or reactivations of five human herpesviruses especially in immunosuppressed patients. Detection of coinfections with multiple herpesviruses is facilitated. Quantitative results enable monitoring of virus load during antiviral therapy. A standard thermal cycling profile permits time and cost effective use in a routine diagnostic setting.     

Evaluation of a new general primer pair for rapid detection and differentiation of HSV-1, HSV-2, and VZV by polymerase chain reaction

The polymerase chain reaction (PCR) enables rapid and sensitive detection of VZV and HSV DNA and its efficiency depends mainly on the choice of the primers. Primers should hybridize to conserved DNA sequences within the viral genomes in order to avoid unreliable amplification due to DNA sequence variation between different strains. The aim of the study was to design and to evaluate a general primer pair which permits fast and reliable detection of HSV and VZV. The genes UL 15 of HSV and UL 42 of VZV share the highest degree of homology within the two genomes. We designed a primer pair (GPHV-RU) which hybridizes to these genes. The genetic variability of amplified sequences from clinical specimens was analyzed by restriction enzyme cleavage analysis and by temperature gradient SSCP analysis (TG-SSCP). PCR with GPHV-RU amplified viral sequences from all analyzed specimens (25×VZV, 10×HSV-1, 5×HSV-2) obtained from patients with clinical evidence of HSV or VZV infection. Restriction enzyme cleavage analysis with Hpa II further permitted reliable distinction between VZV, HSV-1, and HSV-2. Analysis of the heterogeneity of the amplified sequences by restriction enzyme cleavage and by TG-SSCP demonstrated no variability between the analyzed clinical specimens of VZV and of HSV-2 and only one differing TG-SSCP-pattern within the HSV-1 isolates. The results suggest that detection of HSV and VZV using the new primer pair GPHV-RU should give reliable results as the amplified sequences show little genetic variability within clinical isolates of HSV-1/2 and VZV. © 1996 Wiley-Liss, Inc.     

Amplification of the six major human herpesviruses from cerebrospinal fluid by a single PCR

We used a novel type of primer system, a system that uses stair primers, in which the primer sequences are based on consensus sequences in the DNA polymerase gene of herpesvirus to detect herpesviruses by PCR. A single PCR in a single tube detected the six major herpesviruses that infect the central nervous system: herpes simplex virus type 1 (HSV-1), and type 2 (HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and human herpesvirus 6 (HHV-6). We used the technique to analyze 142 cerebrospinal fluid (CSF) samples that had been stored at -80 degrees C and compared the results with those obtained previously for the same samples by standard, targeted PCR. Four hundred one targeted PCR tests had been run with the 142 samples to detect HSV-1, HSV-2, CMV, and VZV; screening for EBV and HHV-6 was not prescribed when the samples were initially taken. Eighteen CSF samples tested positive by classic targeted PCR. The herpesvirus consensus PCR detected herpesviruses in 37 samples, including 3 samples with coinfections and 17 viral isolates which were not targeted. Two samples identified as infected by the targeted PCR tested negative by the consensus PCR, and eight samples that tested positive by the consensus PCR were negative by the targeted PCR. One hundred three samples scored negative by both the targeted and the consensus PCRs. This preliminary study demonstrates the value of testing for six different herpesviruses simultaneously by a sensitive and straightforward technique rather than screening only for those viruses that are causing infections as suggested by clinical signs.     

Diagnostic utility of a multiplex herpesvirus PCR assay performed with cerebrospinal fluid from human immunodeficiency virus-infected patients with neurological disorders

We used a multiplex nested-PCR assay for the simultaneous detection in cerebrospinal fluid (CSF) of five human herpesviruses (HVs) (cytomegalovirus [CMV], Epstein-Barr virus [EBV], varicella-zoster virus [VZV], herpes simplex virus [HSV], and human herpesvirus 6 [HHV-6]) in a clinical evaluation of human immunodeficiency virus (HIV)-infected patients with neurological disorders. This method, which has the advantages of being rapid and economical, would be of particular interest for the diagnosis of neurological syndromes caused by more than one HV. We studied 251 CSF samples from 219 patients. HV DNA was demonstrated in 93 (37%) of the CSF samples (34% of the patients). CMV was the HV most frequently detected in our patients (25%), while EBV, VZV, HSV, and HHV-6 DNAs were present in significantly fewer cases (7, 4, 3, and 1%, respectively). When results were compared with the final etiological diagnoses of the patients, the multiplex HV PCR showed high specificity for the diagnosis of CMV and VZV neurological diseases and for cerebral lymphoma (0.95, 0.97, and 0.99, respectively). The sensitivity of the assay was high for CMV disease (0.87), was low for cerebral lymphoma (0.33), and was not evaluable for VZV disease due to the small number of patients with this diagnosis. Nevertheless, detection of VZV DNA had possible diagnostic value in four of the nine cases, and EBV DNA amplification always predicted the diagnosis of cerebral lymphoma in patients with cerebral masses. Detection of HSV DNA was frequently associated with CMV amplification and fatal encephalitis. HHV-6 was not considered to have a pathogenetic role in the three cases in which it was detected. This multiplex HV PCR assay is a specific and clinically useful method for the evaluation of HIV-infected patients with neurological disorders related to HV.     

Automated detection of five human herpes virus DNAs by a set of LightCycler PCRs complemented with a single multiple internal control.

BACKGROUND: Herpes viruses represent important causes of morbidity and mortality especially in immuno-compromised patients. To assist in rapid diagnosis real-time PCR assays have been developed for the detection of herpes virus DNA in patient specimens. A recently described set of real-time PCR assays using LightCycler technology enabled parallel detection of DNA from cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus type 1 and 2 (HSV-1/-2), and varicella-zoster virus (VZV) by using a single LightCycler program [J. Clin. Virol. 26 (2003) 85]. The set of assays lacked automation of DNA purification and of PCR mixture preparation, and was not furnished with measures to monitor for sample adequacy. OBJECTIVES: Development of a set of automated LightCycler-PCR assays for the detection CMV-, EBV-, HSV-1/-2- and VZV-DNA in plasma samples and complementation of the assays with internal amplification controls (ICs). STUDY DESIGN: The MagNA Pure LC instrument was used for automated DNA purification and automated preparation of PCR mixtures. A single multiple IC-DNA specific for all four herpes virus type-specific PCRs was generated and used in all four LightCycler assays. Detection limits were determined and clinical samples were evaluated. RESULTS: With quantified herpes virus type-specific reference DNA spiked into EDTA plasma, the detection limits were found at 250 copies/ml of CMV-, EBV-, HSV-1/-2-DNA and at 500 copies/ml of VZV-DNA. The novel set of assays was evaluated by testing 112 EDTA plasma samples. The use of the IC led to the detection of PCR-inhibited samples. CONCLUSION: The set of automated LightCycler assays was found rapid, markedly labour saving and suitable for the routine diagnostic laboratory. The use of the one internal control molecule simplified the assay protocol and allowed monitoring for sample adequacy.