Nested multiplex polymerase chain reaction for the diagnosis of cutaneous herpes simplex and herpes zoster infections and a comparison with electronmicroscopy

Herpes simplex virus (HSV) and varicella zoster virus (VZV) are common causes of cutaneous and mucocutaneous vesicular eruptions. Laboratory diagnostic techniques include Tzanck smears, electronmicroscopy, antigen detection and viral culture. This paper describes a nested multiplex polymerase chain reaction with respective sensitivities of 0.0001, 0.01 and 0.1 TCID50 for VZV, HSV-1 and HSV-2. The assay was used in (a) a salvage capacity for slides already processed for electronmicroscopy, and (b) as a front-line assay for prospectively processed specimens. Sixty-two glass slides with vesicle lymph/scrapings from 58 patients with suspected cutaneous herpetic lesions were examined. The clinical presentations were described as atypical/not specified (24), VZV (20) or HSV (18), and involved eruptions from diverse anatomical sites, including the genitalia. Of the 62 specimens, 6 and 38 were positive by electronmicroscopy and multiplex PCR respectively, giving a comparative sensitivity of 16% for electronmicroscopy. Nested multiplex PCR identified 15 VZV and 20 HSV-1 infections. Where the clinical details indicated either HSV or VZV (38/62), nested multiplex PCR was statistically likely to be reactive (26/38 vs. 9/24) (chi2 P = 0.000004) whereas electronmicroscopy was not (4/38 vs. 2/24) (chi2 P= 0.77). Where the clinical details indicated VZV (20/62) or HSV (18/62), nested multiplex PCR was statistically more likely to confirm VZV (10/20 vs. 5/42) (chi2 P= 0.001) or HSV (9/18 vs. 11/44) (chi2 P = 0.05) respectively. Two suspected HSV and 6 suspected VZV infections were shown to be VZV and HSV respectively by nested multiplex PCR.     

Detection of herpes simplex virus type 1, herpes simplex virus type 2 and varicella-zoster virus in skin lesions. Comparison of real-time PCR, nested PCR and virus isolation.

BACKGROUND: Herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella-zoster virus (VZV) cause a wide range of signs and symptoms, varying from trivial mucocutaneous lesions to life-threatening infections, especially in immuno-suppressed patients. Since antiviral drugs are available, rapid and sensitive laboratory diagnosis of these virus infections is important. OBJECTIVE: To set up and evaluate HSV-1, HSV-2 and VZV qualitative real-time PCR on the Lightcycler system and to compare the results with those of the 'in-house' nested PCR and virus isolation. STUDY DESIGN: 110 consecutive samples from dermal or genital lesions from patients suspected of having HSV infections and another 110 samples from patients with suspected VZV infections were tested with real-time PCR, nested PCR and virus isolation. RESULTS: 24 samples (22%) were positive for HSV-1 by virus isolation and nested PCR, whereas 26 (24%) were positive by real-time PCR. HSV-2 was detected in 28 samples (25%) by virus isolation, in 41 (37%) by nested PCR and in 40 (36%) by real-time PCR. VZV was isolated in 15 samples (14%) and VZV DNA was detected in 51 samples (46%) by nested PCR as well as by real-time PCR. Nucleic acid amplification increased the detection rate of HSV-2 and VZV DNA in particular compared to virus isolation. No significant difference in sensitivity was found between real-time PCR and nested PCR. CONCLUSION: Real-time PCR has the advantage of rapid amplification, a reduced risk for contamination and it is a suitable method for diagnosis of VZV and HSV in specimens from skin lesions.     

Clinical Validation of the Lyra Direct HSV 1+2/VZV Assay for Simultaneous Detection and Differentiation of Three Herpesviruses in Cutaneous and Mucocutaneous Lesions

We evaluated the Lyra Direct HSV 1+2/VZV multiplex real-time PCR assay for the detection and differentiation of herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) on 695 consecutive cutaneous and mucocutaneous lesion specimens. The intra-assay and interassay coefficient of variation values for the Lyra assay were 0.29 to 1.30% and 2.33 to 2.61%, respectively. The sensitivities, specificities, and positive and negative predictive values were 93.4 to 95.0%, 96.1 to 96.8%, 78.0 to 80.3%, and 99.0 to 99.1%, respectively, in comparison to those of viral culture. The values were further improved when a resolution analysis was performed with a laboratory-developed PCR assay.     

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.     

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.     

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.     

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.     

Routine use of duplex real-time PCR assays including a commercial internal control for molecular diagnosis of opportunistic DNA virus infections

The aim of this work was to improve the validity of laboratory-developed real-time PCR protocols implemented in the laboratory for molecular diagnosis of opportunistic DNA virus infections using the Simplexa? extraction and amplification control (SEAC) which allows the monitoring of the whole extraction and amplification process. Herpes simplex virus (HSV), varicella-zoster virus (VZV), human cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK virus (BKV), and adenovirus (AdV) genomes were investigated in 152 different clinical specimens. The use of the SEAC did not influence the results of the different virus-specific PCRs. The SEAC results showed high reproducibility with a mean Cp value of 31.08±1.44, and were not influenced by the virus-specific PCR performed or the type of clinical specimen tested. The SEAC in the DNA extracts showed high stability during storage at both +4°C and -20°C. These data allowed establishing a new procedure for the validation of viral PCR results. In conclusion, the SEAC provides a reliable option for improving the diagnosis of opportunistic viral infections by laboratory-developed real-time PCR assays in quality assurance programs.     

Oral bromovinyldeoxyuridine therapy for herpes simplex and varicella-zoster virus infections in severely immunosuppressed patients: a preliminary clinical trial

Twenty-five patients with haematological diseases were treated orally with the highly potent and selective anti-herpes agent, bromovinyldeoxyuridine (BVDU), in a dosage of 7.5 mg/kg/day (divided over three or four doses a day) for 5 days for an intercurrent mucocutaneous herpesvirus infection. Of these 25 patients, 8 were severely granulocytopenic at the time of the viral infection, and 12 recently had undergone bone-marrow transplantation; 5 were under cytotoxic therapy for a lymphoproliferative disorder; 13 had herpes simplex virus type 1 (HSV-1); 1 had herpes simplex virus type 2 (HSV-2); and 11 had varicella-zoster virus (VZV) infection. In all but two patients, BVDU arrested progression of the HSV or VZV infection within 1-2 days after treatment was started. One of the two patients who failed to respond to BVDU had an HSV-2 infection. The other had an HSV-1 infection, which was highly sensitive to BVDU in vitro; BVDU may have failed in this patient because of incomplete drug intake or profuse diarrhoea, or both. The results of this preliminary uncontrolled clinical trial suggest that BVDU may be an effective and safe drug for the oral treatment of HSV-1 and VZV infections in severely immunosuppressed patients.     

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.     

Genital Zoster Infection: the Great Imposter

Herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), and varicella-zoster virus (VZV) are taxonomically closely related viruses that produce clinically distinct diseases but share common features of infection. HSV-1 and HSV-2 both produce cutaneous and mucocutaneous primary infections followed by reactivation disease occurring at the same or a nearby anatomic site. Primary VZV infection, also known as chickenpox, is usually a childhood disease that produces pox-like cutaneous lesions distributed over the entire body. Reactivation VZV infection, often called herpes zoster, generally occurs later in life but, unlike HSV disease, produces pox-like lesions at a different anatomic location, typically along dermatomes. As such, the clinical differential diagnosis of reactivation HSV infection and zoster is generally based upon the anatomic location and the appearance of the lesion(s). The recent availability of molecular assays has resulted in the unexpected diagnosis of zoster infections in male and female patients who presented with genital lesions. The purpose of this article is to present a brief overview of HSV and VZV infections and to relate a laboratory experience that resulted in the diagnosis of zoster infections using a molecular assay in which almost 10% of the 282 positive VZV specimens were from genital sites.     

Laboratory diagnosis of common viral infections of the central nervous system by using a single multiplex PCR screening assay

A multiplex PCR assay that detects the four commonest causes of viral meningitis and encephalitis in the United Kingdom (herpes simplex virus [HSV] type 1 [HSV-1], HSV type 2 [HSV-2], varicella-zoster virus [VZV], and enteroviruses) was developed, and its sensitivity was compared with those of similar assays described previously for this application. Compared to the previous assays, this single multiplex PCR assay had higher molecular sensitivities for the detection for each of the viruses and improved utility for routine use in a diagnostic laboratory. The assay was used to test a series of 1,683 consecutive cerebrospinal fluid (CSF) samples between June 1997 and March 1998 inclusively. Viral nucleic acid was detected in 138 (8.2%) of the CSF samples, including enteroviruses in 51 samples, HSV-2 in 33 samples, VZV in 28 samples, and HSV-1 in 25 samples. Compared to the accepted relative incidence of viral etiologies, aseptic meningitis due to HSV-2 infection was high, and in adult female patients with symptoms of aseptic meningitis, HSV-2 was the virus most commonly detected in the CSF.     

Suitability and clinical application of a multiplex nested PCR assay for the diagnosis of herpes simplex virus infections

A novel multiplex nested polymerase chain reaction (PCR) assay was designed and evaluated for routine diagnosis of herpes simplex virus (HSV) infections in patients with either putative HSV infection of the central nervous system or suspected HSV keratitis. Single-tube amplification of HSV type 1 (HSV-1) or type 2 (HSV-2) DNA extracted from cerebrospinal fluid (CSF) or from keratectomy specimens was followed by differentiation of the virus type-specific PCR products either by agarose gel analysis or by DNA enzyme immunoassay. Among 417 CSF specimens obtained from 395 consecutive patients with clinically suspected HSV infection, 11 (2.6%) were positive for HSV-1 DNA and four (1.0%) probes were positive for HSV-2 DNA. None of the specimens was positive for both HSV-1 and HSV-2 DNA. The genome of HSV-2 was detected in a CSF sample obtained from a woman with meningoencephalitis and genital herpes. The presence of PCR inhibitors was detected in six of 111 (5.4%) reconstructed CSF samples. Inhibition could be removed following extraction with a commercial kit. HSV-1 DNA, but no HSV-2 DNA, was detected in corneal buttons obtained from patients with suspected herpetic keratitis. No contamination has been recorded during the 2-year routine use of this test, which has met the specific requirements of a diagnostic laboratory. © 1996 Wiley-Liss, Inc.     

Investigation of vesicular rashes for HSV and VZV by PCR

Vesicular fluid from rashes of 132 patients was tested by a multiplex PCR shown to be specific for herpes simplex virus (HSV) type 1 and 2, and varicella zoster virus (VZV) genomic DNA. The results were compared with those obtained by examination by electron microscopy and virus isolation by cell culture. The PCR did not differentiate between HSV 1 and 2. By PCR, 64 HSV infections and 53 VZV infections were identified, with presumed 100% sensitivity and specificity. Fifteen specimens tested negative by PCR, electron microscopy, and virus isolation for herpes viruses. The sensitivities of virus isolation and electron microscopy for detection of herpes simplex virus were 56% and 80%. For varicella zoster virus, the sensitivities of virus isolation and electron microscopy were 47% and 60%. These data illustrate the advantage of rapid PCR diagnosis of herpes simplex virus and varicella zoster virus in vesicle fluids. J. Med. Virol. 54:155–157, 1998. © 1998 Wiley-Liss, Inc.     

Are false negative direct immnufluorescence assays caused by varicella zoster virus gE mutant strains?

Strains of Varicella zoster virus (VZV) have been described recently in which a single base mutation in the gE epitope abrogates binding of the 3B3 monoclonal antibody, which is widely used for virus detection in diagnostic laboratories. These strains, named VZV-MSP, are associated with a distinct phenotype in both in vitro culture and in SCID-hu mice. We investigated the possibility that negative direct immunofluorescence results, using the 3B3 antibody, where the presence of virus was confirmed by polymerase chain reaction (PCR) or tissue culture are due in some cases to the MSP strain of VZV. A total of 249 vesicle fluid specimens from people with suspected shingles were examined using direct immunofluorescence, tissue culture and a nested multiplex PCR for VZV, herpes simplex virus type 1 (HSV-1) and 2 (HSV-2). VZV was detected in 218 of 249 (87.6%) cases. Forty-five confirmed VZV specimens, but with negative (30) or indeterminate (15) immunofluorescence results, were analysed further. PCR was used to amplify a fragment in ORF 68 that encodes the VZV gE ectodmain recognised by 3B3 antibody. The fragments were sequenced and analysed for the single base change G448A (D150N), which is present in VZV-MSP as compared with the reference Dumas strain. No VZV gE mutant (MSP/MSP-like) was detected. Overall, PCR was found to be the most sensitive method of confirming VZV infection. False negative VZV immunofluorescence results are unlikely to be due to virus variants.     

Routine use of a highly automated and internally controlled real-time PCR assay for the diagnosis of herpes simplex and varicella-zoster virus infections.

BACKGROUND: Detection of herpes viruses can be significantly improved by PCR. The development of real-time PCR, which has overcome several limitations of conventional PCR, improved the prospects for implementation of PCR-based assays in diagnostic laboratory. OBJECTIVES: To compare the diagnostic performance of an automated sample extraction procedure in combination with an internally controlled real-time PCR assay for detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) to conventional shell vial culture. STUDY DESIGN: One hundred eighty-two consecutive specimens from patients suspected of HSV or VZV infection were examined by internally controlled PCR and shell vial culture. An internal control consisting of phocine herpes virus was processed along with the specimens during the entire procedure and permitted to monitor extraction and amplification efficiency, including inhibition. RESULTS: A total of 48 (26.4%) specimens were positive for HSV or VZV by culture, and 77 (42.3%) by real-time PCR. Thus, overall sensitivity increased by 60.4%. All culture-positive specimens were detected and typed correctly by PCR, except for a single specimen that contained PCR inhibitors. Specifically, the real-time PCR assay increased the detection rate for HSV-1 and HSV-2 by 43.9% and 62.5%, respectively. In PCR-positive specimens, lower levels of viral DNA were found in culture-negative than in culture-positive specimens. The increase of HSV detection rates by PCR varied with the origin of specimen and was particularly significant for skin specimens (7/14 versus 3/14 detected by culture) and bronchoalveolar lavages (8/8 versus 1/8). In addition, real-time PCR significantly increased the detection rate for VZV. CONCLUSIONS: Compared to shell vial culture, our real-time PCR assay demonstrated a superior sensitivity and an added value of using internal control for checking the quality of examination of each specimen. These results provide a solid basis for implementation of real-time PCR in the routine diagnosis of HSV and VZV infections in various clinical specimens.     

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.     

Virological diagnosis of herpes simplex virus 1 esophagitis by quantitative real-time PCR assay

Herpes simplex virus 1 (HSV-1) esophagitis diagnosis is routinely based on the endoscopic findings confirmed by histopathological examination of the esophagitis lesions. Virological diagnosis is not systematically performed and restricted to viral culture or to qualitative PCR assay from esophagitis biopsy specimens. The aim of this study was to assess the interest of quantitative real-time PCR assay in HSV-1 esophagitis diagnosis by comparing the results obtained to those of histological examination associated with immunohistochemical staining, which is considered the "gold standard." From 53 esophagitis biopsy specimens, the PCR assay detected HSV-1 in 18 of 19 histologically proven to have herpetic esophagitis and in 9 of 34 that had esophagitis related to other causes, demonstrating sensitivity, specificity, positive predictive value, and negative predictive value of 94.7%, 73%, 66.7%, and 96%, respectively. Interestingly, HSV-1 was not detected in 16 specimens without the histological aspect of esophagitis. The viral loads normalized per μg of total extracted DNA in each biopsy specimen detected positive by HSV PCR were then compared and appeared to be significantly higher in histopathologically positive herpetic esophagitis (median = 2.9 × 10(6) ± 1.1 × 10(8)) than in histopathologically negative herpetic esophagitis (median = 3.1 × 10(3) ± 6.2 × 10(3)) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 10(4) copies would allow an HSV-1 esophagitis diagnosis with a sensitivity and specificity of 83.3% and 100%, respectively. In conclusion, this work demonstrated that HSV quantitative PCR results for paraffin-embedded esophageal tissue was well correlated to histopathological findings for an HSV-1 esophagitis diagnosis and could be diagnostic through viral load assessment when histopathological results are missing or uncertain.     

Challenges in designing a Taqman-based multiplex assay for the simultaneous detection of herpes simplex virus types 1 and 2 and Varicella-zoster virus

To optimise molecular detection of herpesviruses an internally controlled multiplex Taqman-PCR for the detection of Herpes simplex virus 1 (HSV1), Herpes simplex virus 2 (HSV2) and Varicella-zoster virus (VZV) was developed. The selection of the dye combination working on the ABI 7700 cycler for this multiplex PCR revealed crosstalk phenomena between several combinations of reference dyes and reporter dyes. A final dye combination with CY5 as reference dye and FAM/JOE/TXR as reporter dyes was selected. The influence of the concentration of the internal positive control (IPC) concentration on the quantitative results of HSV1, HSV2 and VZV positive patient samples was analysed. The results indicate that high IPC concentrations are detrimental for the sensitivity of the multiplex assay and that the presence of the IPC molecule narrows the dynamic range of the duplex PCRs between any of the virus PCRs and the IPC-PCR. The optimised multiplex assay detecting HSV1, HSV2 and VZV using 10(3) IPC molecules showed a performance and sensitivity comparable to that of the individual assays.     

Rapid detection of herpes simplex virus and varicella-zoster virus infections by real-time PCR

The herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2) and varicella-zoster virus (VZV) can cause life-threatening infections of the central nervous system and lead to severe infections in immunocompromised subjects and newborns. In these cases, rapid diagnosis is crucial. We developed three different real-time PCR assays based on TaqMan chemistry for the LightCycler instrument to detect HSV-1, HSV-2, and VZV. When the TaqMan assays were compared to our in-house nested PCR assays, the test systems had equal sensitivities of 相似文献