Search

Bloodstream HSV-1 and HSV-2 infections can cause devastating outcomes with high morbidity and mortality, especially in neonates or immunocompromised individuals. Proper patient management for herpes simplex virus (HSV) bloodstream infections is time-sensitive and requires a rapid, accurate, and definitive diagnosis. The absence of the U.S. Food and Drug Administration (FDA)-approved molecular assays for HSV detection in blood, coupled with a lack of consensus on the optimal sample type, underscores the unmet need for improved diagnostics. We prospectively compared the cycle threshold values in paired samples including whole blood (WB), plasma, serum, and peripheral blood mononuclear cells (PBMCs) from patients with bloodstream HSV infections. This analysis employed a modified use of the FDA-cleared Simplexa HSV-1 & 2 Direct assay. The clinical performance in serum was assessed by comparing the results of 247 remnant specimens on this sample-to-answer platform to established laboratory-developed tests in a blinded fashion. Serum samples exhibited significantly lower cycle thresholds than whole blood samples [2.6 cycle threshold (Ct) bias, P < 0.001]. The modified Simplexa assay demonstrated 100% positive percent agreement for the detection of HSV-1 and HSV-2 DNA in serum samples and yielded an overall agreement of 95% (95% CI, 0.92 to 0.97), with a κ statistic of 0.75 (95% CI, 0.62 to 0.86) compared to the composite reference method. Discordance rates were 5.20% for HSV-1 and 0.81% for HSV-2. This investigation demonstrates that serum is an optimal specimen type for HSV detection when compared to several blood compartments. Serum offers a promising sample type for rapid and accurate diagnosis of HSV bloodstream infections using the modified Simplexa assay., Importance: Rapid, accurate, and definitive diagnosis of herpes simplex virus (HSV) infections is crucial in clinical settings for patient management. The absence of FDA-authorized molecular assays for HSV-1/2 detection in blood, coupled with a lack of consensus on the optimal sample type, underscores the need for improved diagnostic methods. Furthermore, rapid diagnosis of HSV bloodstream infections enables timely administration of antiviral treatment, influences patient management decisions for those at high risk, and can contribute to shorter hospital stays, thereby reducing healthcare costs., Competing Interests: The authors declare no conflict of interest.

Results from the Solana HSV 1+2/VZV assay for the detection of herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) in cutaneous or mucocutaneous specimens were compared with that of viral culture and a commercial PCR assay (RealStar alpha herpesvirus PCR kit). Three hundred two mucocutaneous specimens, for which HSV-1, HSV-2, or VZV viral culture or PCR detection have been requested, were randomly selected and prospectively processed on the Solana assay and viral culture or the RealStar assay. Discordant results between culture and the Solana assay were further analyzed using the RealStar assay. A Bayesian latent class model was developed to estimate the performance of each method. Viral culture detected 123 positive specimens (85 HSV-1, 36 HSV-2, and 2 VZV), while the Solana assay detected 27 additional positive specimens (4 HSV-1, 11 HSV-2, and 12 VZV), in agreement with the RealStar PCR assay. The estimated sensitivity of the Solana assay according to our model was 92.7% to 98.7%, 87.1% to 97.8%, and 94.9% to 98.8% (95% confidence interval [CI]) for HSV-1 HSV-2, and VZV, respectively, while the estimated sensitivity of viral culture was 85.2% to 95.0%, 73.6% to 89.6%, and 30.9% to 45.8% (95% CI), respectively. A nonsignificant tendency toward increased sensitivity was noted for the Solana assay compared with culture for HSV-1 and HSV-2, and the Solana assay was significantly more sensitive than culture for the detection of VZV. The Solana assay performed comparably to the RealStar assay. Processing time was reduced with the Solana assay compared with viral culture., (Copyright © 2020 American Society for Microbiology.)

Herpes simplex virus (HSV) causes acute and relapsing symptoms characterized by ulcerative lesions. Laboratory diagnosis of HSV in cutaneous or mucocutaneous lesions has historically been performed with the use of viral cell culture systems; however, these tests are laborious and suffer decreased sensitivity for advanced-stage lesions. The recent availability of FDA-cleared moderately complex assays has resulted in the increased use of molecular diagnostics for the routine detection of HSV in superficial swab specimens. We performed a clinical evaluation of the recently FDA-cleared illumigene HSV 1&2 loop-mediated isothermal amplification (LAMP) assay (Meridian Bioscience, Cincinnati OH) for the detection and differentiation of HSV-1 and HSV-2 in cutaneous and mucocutaneous swab specimens. A total of 1,153 clinical swab specimens were collected and tested at 7 different clinical centers. Each specimen was tested for the presence of HSV-1 and HSV-2 using the illumigene assay, and results were compared to those of the enzyme-linked virus-inducible system (ELVIS) as the reference method. Overall, the illumigene assay demonstrated a sensitivity and specificity of 94.8% and 95.5%, respectively, for the detection of HSV-1. Detection of HSV-2 was similar, with a sensitivity of 98.9% and a specificity of 95.5%. Discrepant analysis was performed using an alternative molecular test (AmpliVue HSV1+2 assay; Quidel Molecular, San Diego, CA) on 91/99 specimens that were recorded as false positive (FP) or false negative (FN) compared to the reference method. In total, 57/78 (73%) FP and 9/13 (69%) FN illumigene results were supported by the AmpliVue result. The illumigene HSV 1&2 assay demonstrated high sensitivity and specificity to detect and differentiate HSV in clinical specimens and identified 57 additional specimens that were positive for HSV compared to culture. The use of LAMP eliminates the need for the cycling of temperatures and provides results in less than 60 min, with approximately 2 min of hands-on time per specimen., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The AmpliVue HSV 1+2 assay was compared to the ELVIS HSV ID and D(3) Typing Culture System for the qualitative detection and differentiation of herpes simplex virus 1 (HSV-1) and HSV-2 DNA in 1,351 cutaneous and mucocutaneous specimens. Compared to ELVIS, AmpliVue had sensitivities of 95.7 and 97.6% for detecting HSV-1 and HSV-2, respectively. Following arbitration of discordant results by an independent molecular method, the AmpliVue assay had a resolved sensitivity and specificity of 99.2 and 99.7%, respectively, for both HSV-1 and HSV-2, whereas ELVIS had a resolved sensitivity of 87.1% for HSV-1 and 84.5% for HSV-2., (Copyright © 2015 Granato et al.)

Detection of herpes simplex virus 1 and 2 (HSV-1 and HSV-2) in cerebrospinal fluid (CSF) is a medical emergency and requires rapid, sensitive testing. However, the volume of CSF received for microbiological studies may be limited, especially from young children. In this study, we compared three testing protocols to our routine real-time PCR method to determine the most sensitive approach for detecting HSV-1 and HSV-2 in low-volume (≤100 μl) CSF.

Central nervous system infection due to herpes simplex virus (HSV) is a medical emergency and requires rapid diagnosis and initiation of therapy. In this study, we compared a routine real-time PCR assay for HSV types 1 (HSV-1) and 2 (HSV-2) to a recently FDA-approved direct PCR assay (Simplexa HSV-1/2 Direct; Focus Diagnostics, Cypress, CA) using cerebrospinal fluid samples (n = 100). The Simplexa HSV-1/2 assays demonstrated a combined sensitivity and specificity of 96.2% (50/52) and 97.9% (47/48), respectively. In addition, the Simplexa assay does not require nucleic acid extraction, and the results are available in 60 min., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

We characterize a novel probe binding-site polymorphism detectable solely by melt curve analysis using the Roche LightCycler HSV 1/2 analyte-specific reagent real-time PCR assay. The frequencies of this novel (47°C) and previously described intermediate (60 to 62°C) melt curves were 0.016% and 4.9%, respectively.

The determination of herpes simplex virus (HSV) infection using a PCR assay is one of the most commonly requested tests for analysis of cerebrospinal fluid (CSF), although only a very low proportion of results are positive. A previously reported study showed that selecting only those CSF samples with >5 leukocytes/mm(3) or a protein level of >50 mg/dl was adequate for the diagnostic workup. The aim of the present study was to assess the reliability of alternative acceptance criteria based on elevated CSF white blood cell counts (>10 cells/mm(3)). We analyzed all requests for HSV PCR received between January 2008 and December 2011. CSF samples were accepted for analysis if they had >10 cells/mm(3) or if the sample was from an immunocompromised patient or a child aged <2 years. In order to evaluate our selection criteria, we identified those CSF samples with a leukocyte count of 5 to 10 cells/mm(3) or protein levels of >50 mg/dl in order to test them for HSV type 1 and 2 (HSV-1 and HSV-2) DNA. During the study period, 466 CSF samples were submitted to the microbiology laboratory for HSV PCR. Of these, 268 (57.5%) were rejected, and 198 (42.5%) were tested according to our routine criteria. Of the tested samples, 11 (5.5%) were positive for HSV DNA (7 for HSV-1 and 4 for HSV-2). Of the 268 rejected specimens, 74 met the criteria of >5 cells/mm(3) and/or protein levels of >50 mg/dl. Of these, 70 (94.6%) were available for analysis. None of the samples yielded a positive HSV PCR result. Acceptance criteria based on CSF leukocyte counts, host immune status, and age can help to streamline the application of HSV PCR without reducing sensitivity.

A neonate with herpes simplex virus 1 encephalitis was treated with intravenous acyclovir. During the course of therapy, the infection became intractable to the treatment and a mutation in the viral thymidine kinase gene (nucleotide G375T, amino acid Q125H) developed. This mutation was demonstrated in vitro to confer acyclovir resistance.

Herpes infections are among the most common sexually transmitted infections (STI), but diagnostic methods for genital herpes have not kept pace with the movement toward molecular testing. Here, we describe an FDA-approved molecular assay that identifies and types herpes simplex virus (HSV) infections for use in routine clinical settings. Paired samples from anogenital lesions were tested using the BD ProbeTec HSV Q(x) (HSVQ(x)) system, HSV culture and, a laboratory-developed PCR assay. Family planning, obstetrics/gynecology (OB/GYN), or sexually transmitted disease (STD) clinics in the United States served as recruitment sites. Sensitivity and specificity estimates, head-to-head comparisons, measures of agreement, and latent-class analyses were performed to provide robust estimates of performance. A total of 508 participants (174 men and 334 women) with anogenital lesions were included; 260 HSV-2 and 73 HSV-1 infections were identified. No differences in test performance based on gender, clinic type, location of the lesion, or type of lesion were observed. The sensitivity of HSV-2 detection ranged from 98.4 to 100% depending on the analytical approach, while the specificity ranged from 80.6%, compared to the less sensitive culture method, to 97.0%, compared to PCR. For HSV-1, the sensitivity and specificity ranges were 96.7 to 100% and 95.1 to 99.4%, respectively. This assay may improve our ability to accurately diagnose anogenital lesions due to herpes infection.

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.

Herpes simplex virus types 1 and 2 (HSV-1 and -2) are significant human pathogens causing clinically indistinguishable facial and genital lesions. Recently, the number of reported genital herpes cases caused by type 1 virus has increased. Identifying the HSV type is of clinical importance to determine proper treatment, as there is no licensed vaccine or cure. We assessed, by PCR, the frequency of HSV-1 and HSV-2 present in more than 60,000 clinical cervicovaginal specimens derived from samples originating from 43 continental U.S. states. Fourteen percent were positive for HSV-1 and/or HSV-2. This likely represents subclinal shedding. It was not a measurement of the prevalence of HSV infection. While the majority were HSV-2, 32% were HSV-1. The distribution of HSV types varied between the states with the largest number of specimens, New Jersey, Florida, and Texas. Specimens from women under the age of 24 had an HSV-1 positivity rate of 47 percent. Importantly, in New Jersey, an observed age effect was the disproportionately high prevalence of genital HSV-1 in young women. This represents the largest analysis of HSV types reported and has important public health implications, particularly for younger women.

We here report on the development of a novel multiplex PCR with product detection in a Luminex 100 suspension array system. The assay covers the nine most important bacterial and viral pathogens found in Danish meningitis patients. The microorganisms include Neisseria meningitidis, Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Streptococcus agalactiae, herpes simplex virus types 1 and 2, and varicella-zoster virus. The study was based on 1,187 samples, of which 55 were found to be positive by PCR. The assay was found to have an excellent sensitivity and an excellent specificity compared to the results of a "gold standard," defined by routine laboratory tests, for the two most important pathogens, S. pneumoniae (95 and 99.1%, respectively) and N. meningitidis (100 and 99.7%, respectively). The method provides a valuable supplement to the traditional microscopy and culture of cerebrospinal fluid (CSF) samples in a routine diagnostic setting, and results can be available within 1 workday. The method is suitable for use for the initial screening and identification of nine important microorganisms in CSF samples from patients with suspected meningitis. Compared to microscopy and culture of CSF, this rapid and sensitive method will support physicians with the selection of the appropriate antimicrobial agents and the initiation of timely treatment in the absence of live microorganisms in the CSF.

Thelifelong latent infection-reactivation mode of infection of herpes simplex virus type 1 (HSV-1) transmitted by close contact has allowed a diversity of restriction fragment length polymorphism (RFLP) variations to accumulate in human populations. Whether and how the variants of the HSV-1 that is ubiquitous worldwide spread to different human populations is not clear. In our previous study the geographically gradient distribution of the HSV-1 BgK(L) variant, which is a good marker for the BgK(L):SaCFJ(M):SaGH(M):SaD/E(L):KpM(S) variant, suggested that BgK(L) dispersed geographically. Southern hybridization analyses showed that in BgK(L) the BglII cleavage site between the BglII K and small "Q/#13" fragments is lost, the SalI cleavage sites between the SalI J and C and between SalI F and J fragments are lost, and the SalI E fragment is abnormally large (SaE(L) variation). The RFLP and geographic distribution of one more HSV-1 RFLP variant, BgO(L), were comparatively analyzed. The BglII cleavage site between the BglII O and Q/#13 fragments is lost in BgOL. BgO(L) clinical isolates were not associated with any of the SaCFJ(M), SaE(L), SaGH(M), or KpM(S) variations, whereas one-fourth of the non-BgO(L):non-BgK(L) isolates was associated with SaCFJ(M) and SaGH(M), indicating that BgK(L) and BgO(L) are distant in terms of diversification. BgO(L) is distributed highly in the northeastern region and the southwestern island of Kyushu but is rare between the two regions in Japan, in a remarkable contrast to BgK(L). These are the first epidemiologic data to show contrasting geographic distribution profiles of two HSV-1 variants and suggest the gradual dispersion and replacement of HSV-1 variants.

We report here on the development and validation of a prototype Invader Plus method for the qualitative detection of herpes simplex virus types 1 and 2 in cerebrospinal fluid (CSF). The method combines PCR and Invader techniques in a single, closed-tube, continuous-reaction format that gives an analytical sensitivity of approximately 10 copies per reaction. The clinical sensitivity and specificity were 100.0% and 98.6%, respectively, when the results of the method were validated against the results obtained with a PCR colorimetric microtiter plate system by use of clinical CSF specimens.

Restriction endonuclease fragment length polymorphism (RFLP) is useful for the epidemiological study of herpes simplex virus type 1 (HSV-1). We report here the identification of a major BglII RFLP variant of HSV-1, designated BgKL, found in 27.0% of 636 HSV-1 clinical isolates. We have also established its geographic distribution in Japan. BgKL has an unusually large BglII K fragment. SalI cleavage analyses showed that 97% of BgKL variant isolates lack both the SalI C-J and the F-J cleavage sites and have an unusually large SalI D or E fragment, and 91% of the BgKL variants lack both SalI G and H fragments. Furthermore, 96% of BgKL isolates have an unusually small KpnI M fragment. Therefore, BgKL is a marker for these five mutations in most HSV-1 isolates and is a useful HSV-1 RFLP marker. The BgKL variant was found in 59% of HSV-1 isolates from Shikoku Island, 44% of HSV-1 isolates from the Chugoku region of Honshu Island, 31% of HSV-1 isolates from Kyushu Island, 0% of HSV-1 isolates from Okinawa Island, 49% of HSV-1 isolates from Osaka, 27% of HSV-1 isolates from Shiga, 13% of HSV-1 isolates from the Chubu Region, and 9% of HSV-1 isolates from the Tohoku Region of Honshu Island. Differences in the frequency of BgKL between the Shikoku-Chugoku-Osaka area (49%) and Kyushu, between Kyushu and Okinawa, between the Shikoku-Chugoku-Osaka area and Shiga, and between Shiga and Tohoku are all statistically significant. The BgKL frequency decreases in a geographical gradient suggest that this HSV-1 variant was dispersed from Shikoku to the surrounding regions and then to more distant regions. The BgKL frequency in Tokyo was similar to the nationwide average. These are the first data to suggest a geographic and demographic dispersion pattern of HSV-1. Implications for the epidemiology and diversification of HSV-1 are discussed.

HSV-1 DNA was detected in 32 (30%) of 106 cerebrospinal fluid samples from patients with encephalitis. Cytomegalovirus, varicella-zoster virus, and human herpesvirus 6 (HHV-6) DNAs were each detected in three patients (3%); herpes simplex virus type 2 (HSV-2) and HHV-7 PCRs were negative. HSV detection was associated with seizure (P = 0.02), especially focal seizure (P = 0.0002), and pathological computed tomography (P = 0.02) with focal lesions (P = 0.0004).

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react rapidly and efficiently, with a high specificity, under isothermal conditions. We used a LAMP assay for the detection of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV). The virus specificities of primers were confirmed by using 50 HSV-1, 50 HSV-2, and 8 VZV strains. The assay was performed for 45 min at 65 degrees C. The LAMP assay had a 10-fold higher sensitivity than a PCR assay. An analysis of nucleotide sequence variations in the target and primer regions used for the LAMP assay indicated that 3 of 50 HSV-1 strains had single nucleotide polymorphisms. No HSV-2 or VZV strains had nucleotide polymorphisms. Regardless of the sequence variation, there were no differences in sensitivity with the HSV-1-specific LAMP assay. To evaluate the application of the LAMP assay for clinical diagnosis, we tested clinical samples from 40 genital herpes patients and 20 ocular herpes patients. With the LAMP assay, 41 samples with DNA extraction and 26 direct samples without DNA extraction were identified as positive for HSV-1 or HSV-2, although 37 samples with DNA extraction and just one without DNA extraction were positive by a PCR assay. Thus, the LAMP assay was less influenced than the PCR assay by the presence of inhibitory substances in clinical samples. These observations indicate that the LAMP assay is very useful for the diagnosis of HSV-1, HSV-2, and VZV infections.

Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are major causes of mucocutaneous lesions and severe infections of the central nervous system. Here a new semiautomated method for detecting and typing of HSV was used to analyze 479 mucocutaneous swab samples. After DNA extraction using a Magnapure LC robot, a 118-bp segment of the gB region was amplified by real-time PCR utilizing type-specific TaqMan probes to identify HSV-1 or HSV-2. HSV detection in a single well using probes labeled with carboxyfluorescein (FAM) for HSV-1 and JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein) for HSV-2 had a sensitivity similar to that seen in separate reactions. All but one of 217 samples (99.5%) that had been positive by virus culture were positive by TaqMan PCR, with a correct identification of type in all cases. Out of 262 samples negative by virus culture, 48 (18.3%) were positive by TaqMan PCR, with higher Ct values compared with culture positive samples (P < 0.0001). Overall, the Ct values for HSV-1 were lower than for HSV-2 (mean, 25.5 versus 27.9), but to some extent this could be due to weaker fluorescence by JOE. Lower C(t) values for HSV-1 were seen also in the 202 genital samples (79 HSV-1, 122 HSV-2, 1 HSV-1 and HSV-2), indicating that HSV-1 replicates as well as HSV-2 in the genital area. HSV-1 constituted 40% of genital infections and was associated with lower mean age (29.2 versus 36.4 years), probably reflecting the fact that recurrent genital HSV-1 infections are rare.

Primers for herpes simplex virus type 1 (HSV 1)-specific loop-mediated isothermal amplification (LAMP) method amplified HSV-1 DNA, while HSV-2-specific primers amplified only HSV-2 DNA; no LAMP products were produced by reactions performed with other viral DNAs. The sensitivities of the HSV-1- and HSV-2-specific LAMP methods, determined by agarose gel electrophoresis, reached 500 and 1,000 copies/tube, respectively. The turbidity assay, however, determined the sensitivity of the HSV-1- and HSV-2-specific LAMP methods to be 1,000 and 10,000 copies/tube, respectively. After initial validation studies, 18 swab samples (in sterilized water) collected from patients with either gingivostomatitis or vesicular skin eruptions were examined. HSV-1 LAMP products were detected by agarose gel electrophoresis in the 10 samples that also demonstrated viral DNA detection by real-time PCR. Nine of these 10 samples exhibited HSV-1 LAMP products by turbidity assay. Furthermore, both the agarose gel electrophoresis and the turbidity assay directly detected HSV-1 LAMP products in 9 of the 10 swab samples collected in sterilized water. Next, we examined the reliability of HSV type-specific LAMP for the detection of viral DNA in clinical specimens (culture medium) collected from genital lesions. HSV-2 was isolated from all of the samples and visualized by either agarose gel electrophoresis or turbidity assay.

Diagnostic virus isolation is still frequently used, particularly from respiratory tract secretions. Testing positive virus cultures for all possible viruses is time-consuming, and unexpected or unknown viruses may escape detection. Therefore, a novel random PCR approach was developed that allows sequence-independent amplification of viral nucleic acids from virus isolation-positive cultures. Selectivity for viral sequences is obtained by preferential isolation of nucleic acids that are particle associated and resistant to nucleases. Using primers with a degenerated 3' end, the isolated nucleic acids are amplified and the randomly amplified PCR products are cloned and sequenced. As proof of the concept, the PAN-PCR approach was applied to supernatants of coxsackievirus B3 and murine adenovirus type 1-infected cells. Enterovirus and adenovirus sequences were obtained, demonstrating that the random PCR approach allows detection of RNA and DNA viruses. As a first application of this PAN-PCR approach, we characterized a virus isolate from mouth-washing material of a patient with chronic fatigue syndrome and high antibody titers to coxsackievirus B2. The virus isolate had tested negative for enteroviruses and respiratory viruses (influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus, and adenovirus) by immunofluorescence and PCR. Particle-associated, nuclease-resistant RNA and DNA were prepared from the supernatant of infected cells. The DNA and the reverse-transcribed RNA were randomly amplified, and PCR products were cloned and sequenced. Of 25 sequences obtained from the DNA preparation, 24 contained herpes simplex virus type 1 (HSV-1) sequences from 14 different loci spread over the HSV-1 genome. This result was confirmed by using a standard diagnostic HSV-PCR, demonstrating that the PAN-PCR correctly identified the virus isolate. Although the identification of HSV-1 in mouth-washing material is not surprising in retrospect, it clearly demonstrates the applicability of the PAN-PCR approach. This method should be particularly useful for characterizing virus isolates that have tested negative for all expected viruses and for identifying unknown viruses.

Glycoprotein G-based herpes simplex virus type 2 (HSV-2) enzyme-linked immunosorbent assays from Focus and Kalon were performed with specimens from 118 patients with culture-documented genital herpes episodes, and their results were compared. Sensitivity was 52% by Kalon and 86% by Focus for first HSV-2 episodes and 100% (for each of the two tests) for recurrent HSV-2. Median times to seroconversion were 120 days by the Kalon assay, 21 days by the Focus assay, and 68 days by Western blotting assay. Values for specificity were 100% (Kalon) and 93% (Focus).

The secreted portion of glycoprotein G (sgG-2) of herpes simplex virus type 2 (HSV-2) was evaluated as a novel antigen in an enzyme-linked immunosorbent assay (ELISA) format for detection of type-specific immunoglobulin G (IgG) antibodies in HSV-2-infected patients. The results were compared with those obtained by a commercially available assay, the HerpeSelect 2 ELISA (the FOCUS2 assay). Five different panels of sera were analyzed: panel A consisted of 109 serum samples from patients with a culture-proven HSV-1 infection that were Western blotting (WB) negative for HSV-2; panel B consisted of 106 serum samples from patients with a culture-proven recurrent HSV-2 infection that were WB positive for HSV-2; panel C consisted of 100 serum samples with no detectable IgG antibodies against HSV-1 and HSV-2; panel D consisted of 70 HSV-2 negative "tricky" serum samples containing antinuclear IgG antibodies or IgM antibodies against other viruses or bacteria; and panel E consisted of consecutive serum samples from 21 patients presenting with a first episode of HSV-2-induced lesions. When sera in panels A to C were analyzed, the sgG-2 ELISA and the FOCUS2 assay both showed sensitivities and specificities of >or=98%. In total, among the samples in panel D, 13 serum samples (19%) were false positive by the FOCUS2 assay and 1 serum sample (1.4%) was false positive by the sgG-2 ELISA. When the sera in panel E were analyzed, the sgG-2 ELISA detected seroconversion somewhat later than WB or the FOCUS2 assay did. We conclude that sgG-2 induces an HSV-2 type-specific antibody response and can be used for type-discriminating serology.

Real-time PCR with melting curve analysis of PCR products is a rapid procedure for detecting and genotyping herpes simplex virus (HSV). When testing mucocutaneous samples for HSV by a real-time PCR assay targeting the DNA polymerase gene, we found that some PCR products had atypical melting curves that did not conform to the expected melting temperatures for HSV type 1 or 2. Sequence analysis showed that these strains had base-pair mismatches over the probe binding sites. An alternative assay is required to type such atypical isolates.

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

Procedures using real-time technique were developed to demonstrate the presence of herpes simplex virus type 1 (HSV-1) and HSV-2, varicella zoster virus (VZV), and cytomegalovirus (CMV) in miscellaneous clinical specimens. The assays were compared to rapid culture using centrifugation followed by detection with monoclonal antibodies. A total of 711 consecutive samples were collected from different patient groups. Throat swabs were obtained from transplant patients; dermal or oral specimens were collected from patients suspected for VZV or HSV infection. Genital specimens were taken from patients who attended the Clinic for Sexually Transmitted Diseases at the Dijkzigt Hospital Rotterdam presenting with symptoms of a primary genital ulcer. Nucleic acid extraction was carried out using a MagnaPure LC instrument. The amplification steps were performed on the ABI Prism 7700 sequence detection system. To monitor the process of extraction and amplification, a universal control consisting of seal herpesvirus type 1 (PhHV-1) was added to the clinical specimens. By culture 127 of 668 (19%) samples were positive for HSV-1, 72 of 668 (10.8%) specimens were positive for HSV-2, and 17 of 366 (4.6%) were positive for VZV. Using real-time amplification the numbers of positive specimens were 143 of 668 (21.4%), 97 of 668 (14.5%), and 27 of 366 (7.4%), respectively. Eighty-six specimens were tested for CMV, 12 (14.0%) were positive by culture, and 17 (19.8%) were positive by real-time PCR. The clinical data of the patients with discrepant results were reviewed thoroughly. In all cases the patients with only real-time PCR-positive results could be considered as truly infected. We concluded that the real-time amplification technique is suitable for the detection of human herpesvirus infection. It offers a semiquantitative and reliable assay with a quick result that is more sensitive than rapid culture, especially for the diagnosis of HSV-2 and VZV infections.

Serologic assays that utilize herpes simplex virus (HSV) type-specific glycoproteins G-1 (HSV-1) and G-2 (HSV-2) to discriminate between antibodies against HSV-1 and HSV-2 are sensitive and specific. However, the high rates of seroreversion, defined as the change in an individual's antibody status from positive to negative over time, previously reported in longitudinal evaluations of glycoprotein G type-specific tests suggests that their use in HSV acquisitional studies would be problematic. To further explore the reliability of the glycoprotein G-based serologic tests, we evaluated HSV-1 and HSV-2 enzyme immunoassays from Focus Technologies in a longitudinal cohort of 1207 young women from Pittsburgh, Pa. On enrollment of the women in the study, HSV-1 and HSV-2 antibodies were detected in 46.6 and 24.9% of the women, respectively. Among the women with at least three visits, 3.4% (15 of 447) of those who were HSV-1 antibody positive had a subsequent negative result while fewer than 1% (2 of 227) of those who were HSV-2 antibody positive seroreverted. The median of mean positive index values for women who seroreverted to HSV-1 antibody was lower than that for women who remained seropositive (1.25 versus 7.06; P < 0.001). Similarly, the median of mean positive index values for women whose HSV-2 antibody status reverted from positive to negative was lower than that for those women who did not serorevert (1.83 versus 7.46; P = 0.02). Comparative Western blot analysis demonstrated that the lower positive index values, seen more often among the HSV seroreverters, often signified false-positive immunoassay results. Overall, the seroreversion rates were low; the use of glycoprotein G-based serologic tests for the measurement of HSV-1 and HSV-2 antibodies in incidence studies therefore appears warranted.

Cryopreserved cell monolayers are a new cell culture technology intended to ensure the availability of cells in the laboratory for virus detection. Two cryopreserved cell monolayers, ELVIS for the detection of herpes simplex virus (HSV) and R-Mix for the detection of influenza virus, were evaluated. The results indicated that fresh and cryopreserved cell monolayers are comparable in sensitivity for the detection of HSV and influenza virus. The cells retain the same level of sensitivity for up to 4 months at -80 degrees C.

A multiplex PCR was designed to amplify herpes simplex virus types 1 and 2, cytomegalovirus, and varicella-zoster virus DNA present in a diverse range of clinical material. The susceptibility of these viruses to in vivo inhibition by at least one antiviral drug was an important consideration in their inclusion in the multiplex detection system. An aliquot of equine herpesvirus was introduced into each specimen prior to extraction and served as an indicator of potential inhibitors of the PCR and a detector of suboptimal PCR conditions. Compared to virus isolation and immunofluorescence-based antigen detection, the multiplex assay yielded higher detection rates for all viruses represented in the assay. The turnaround time for performance of the assay was markedly reduced compared to those for the other techniques used to identify these viruses. More than 21,000 tests have been performed using the assay. Overall, the multiplex PCR enabled the detection of substantially increased numbers of herpesviruses, in some cases in specimens or anatomical sites where previously they were rarely if ever identified using traditional detection methods.

Two companies, MRL and Meridian Diagnostics, have developed Food and Drug Administration-approved herpes simplex virus type 1 type-specific enzyme immunoassays. The sensitivity, specificity, and overall testing efficiency of these assays were 98.2, 93.8, and 96.6% for MRL and 98.8, 99.0, and 98.1% for Meridian, making both of these kits suitable for use in the clinical lab.

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.

Herpes infections are among the most common sexually transmitted diseases and are the most common cause of genital ulcer disease in the United States. This study addresses the changing distribution of herpes simplex virus type 1 (HSV-1) and HSV-2 in patients presenting for evaluation of herpetic infections. Viral culture results from the University of Kentucky Clinical Microbiology Laboratory were reviewed for a 6-year period (1994 through 1999). Data were collected on patient sex, site of culture, and culture result. These data were analyzed statistically to identify yearly trends. Of the 4,498 cultures analyzed, nearly equal proportions of HSV-1 (13.3%) and HSV-2 (12.0%) were detected for an overall culture positivity rate of 25.3%. Approximately two-thirds of all positive cultures were from women. Although HSV-2 remained the predominant type of genital herpes, over the 6-year span of this study, there was a trend toward increasing proportions of HSV-1 genitalis, with 31.8% of male patients and 44.8% of female patients demonstrating HSV-1 genitalis by 1999. The majority of patients with HSV in nongenital sites grew HSV-1. Although there was significant yearly variation, HSV-2 was isolated from only 9.4% of patients with nongenital HSV for the entire 6-year period. This study therefore concludes that HSV-2 remains primarily a genital pathogen, while HSV-1 is taking on an increasingly important role in causing genital ulcer disease in addition to being the primary nongenital HSV.

A conventional multiplex PCR assay that detects herpes simplex virus type 1 (HSV-1), HSV-2, varicella-zoster virus, and enteroviruses for the diagnosis of central nervous system infections was modified to be performed using the LightCycler system. The sensitivity of detection of each of the viruses using the LightCycler assay was compared to that of the conventional assay using external quality assessment material. The assays had equivalent sensitivities, but the LightCycler assay was more rapid, reduced the risk of contamination, and used an amplicon detection format that demonstrated greater discrimination than a gel electrophoresis method.

The most common etiologic agents of genital ulcer disease (GUD) are herpes simplex virus type 1 (HSV-1), HSV-2, Treponema pallidum, and Haemophilus ducreyi. In an outpatient clinic for sexually transmitted diseases in Amsterdam, The Netherlands, specimens from 372 patients with GUD were collected from February to November 1996. Sera were collected at the time of the symptoms and, for most patients, also during follow-up visits. Swabs in viral transport medium were used for HSV culture and for detection of DNA. The most prevalent pathogen found was HSV-2, which was detected by culture in 35% of the patients and by PCR in 48% of the patients. Also, HSV-1 infection was more often detected by PCR (7.8%) than by culture (5.6%). Evidence for an active infection with T. pallidum was found in 1.9% of the patients, using serological tests. A multiplex PCR for simultaneous T. pallidum and H. ducreyi DNA detection was positive for T. pallidum in 3.3% of the samples and for H. ducreyi in only 0.9% (3 out of 368) of the samples. The sensitivity of the PCR was superior to that of culture for HSV detection and to that of serology for T. pallidum detection. Specific H. ducreyi immunoglobulin G antibodies were detected in sera of 5.2% of the patients, with no concordance between serology and PCR. In 37% of the cases, none of the tested microorganisms was detected. Performance of PCR in addition to conventional techniques significantly improved the diagnosis of GUD.

We have introduced a time-resolved fluorometry (TRF)-based microwell hybridization assay for PCR products in detection of herpes simplex virus (HSV) in cerebrospinal fluid (CSF) specimens. TRF is a sensitive nonradioactive detection technique which involves the use of lanthanide chelates as fluorescent labels. We used PCR primers from the glycoprotein D genes of HSV type 1 (HSV-1) and HSV-2. The biotinylated PCR products were collected on streptavidin-coated microtitration wells and hybridized with short oligonucleotide probes, europium labeled for HSV-1 and samarium labeled for HSV-2. The TRF results were obtained as counts per second and as signal-to-noise (S/N) ratios. The sensitivity of the assay was 0.1 infectious units (PFU) of HSV in CSF specimens, and the S/N values increased with the virus amount, up to 68.5 for 10(3) PFU of HSV-1 and to 58.5 for 10(3) PFU of HSV-2, allowing semiquantitation of HSV in CSF. The primers and probes recognized all the studied 48 HSV wild-type samples, with S/N ratios of 12.4 to 190 (HSV-1) and 5.1 to 248 (HSV-2). We tested CSF specimens, 100 for each HSV type, which were HSV PCR negative by Southern blot and 22 CSF specimens which were HSV-1 or -2 PCR blot positive. In the TRF test, the mean S/N ratio for the HSV-1-negative CSF was 1.37 (standard deviation [SD] = 0.513) and for the HSV-2-negative CSF it was 1.03 (SD = 0.098). The HSV-1 blot-positive CSF yielded S/N ratios of 3.6 to 85.9, and the HSV-2 blot-positive CSF yielded ratios from 1.9 to 13. Using the mean S/N ratio for negative CSF specimens + 3 SD as the cutoff yielded all the previously HSV-positive specimens as TRF positive. The TRF PCR assay for HSV in CSF specimens is a rapid and sensitive method, improves interpretation of PCR results, and is well suited for automation.

Molecular detection of herpes simplex virus (HSV) DNA is recognized as the reference standard assay method for the sensitive and specific diagnosis of central nervous system infections caused by HSV. In this study, a molecular assay based on real-time PCR on the LightCycler (LC) instrument was evaluated and compared with a home-brew molecular assay. The detection limit of the LC assay was determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene and with the First European Union Concerted Action HSV Proficiency Panel. A total of 59 cerebrospinal fluid (CSF) specimens were investigated for the comparative study. With plasmid pS4, the detection limit of the LC assay was found to be 10(4) copies per ml, i.e., 12.5 copies per run. When samples of the First European Union Concerted Action HSV Proficiency Panel were tested, 2x10(3) to 5x10(3) HSV type 1 genome equivalents (GE) per ml, i.e., 2.5 to 6.3 GE per run, could consistently be detected. There was a correlation between the LC assay and the home-brew assay in 55 of 59 specimens. In conclusion, the LC assay allows very rapid detection of HSV DNA in CSF. It was found to be laborsaving and showed sufficient sensitivity.

The polymorphism of the thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1) was analyzed and was compared with the restriction fragment length polymorphism (RFLP) of the whole genome to evaluate the relative efficiency of the TK gene as a potential probe for identification and discrimination of HSV-1. The effectiveness of using the polymorphism of the TK gene in classifying HSV-1 strains was comparable to that of RFLP analysis of 66 sites, suggesting that TK gene sequencing may have important applications in epidemiological studies of HSV-1.

Using a standard plaque assay and clinical isolates of herpes simplex virus (HSV), we have tested the ability of zinc salts to inactivate HSV. Virus was treated by incubation at 37 degrees C with zinc salts in morpholinepropanesulfonic acid-buffered culture medium and was then diluted and plated onto CV-1 cells for detection and quantitation of remaining infectious virus. Of 10 randomly chosen clinical isolates (five HSV type 1 [HSV-1] isolates and five HSV-2 isolates), seven were inactivated >98% by treatment in vitro with 50 mM zinc gluconate for 2 h and nine were inactivated >97% by treatment with zinc lactate. The effect was concentration dependent. With an HSV-1 isolate, 50 mM zinc gluconate or zinc lactate caused 100% inactivation, 15 mM caused 98 to 99% inactivation, and 5 mM caused 63 to 86% inactivation. With an HSV-2 isolate, 50 and 15 mM zinc gluconate caused 30% inactivation and 5 and 1 mM caused less than 9% inactivation, whereas 50 and 15 mM zinc lactate caused greater than 92% inactivation and 5 and 1 mM caused 37 and 26% inactivation, respectively. The ability of the zinc salts to inactivate HSV was not related to pH in the pH range of 6.1 to 7.6 since inactivation by zinc gluconate or zinc lactate in that pH range was 99.7 to 100% with a 2-h treatment with 50 mM zinc salt. Short (5-min) treatments of selected isolates with zinc gluconate, zinc lactate, zinc acetate, or zinc sulfate yielded inactivation rates of 0 to 55%.

A PCR method to amplify DNA segments of the glycoprotein G gene of monkey B virus (BV) was achieved by adding betaine to the PCR mixture, in spite of the high G+C content of this gene. No product was obtained when DNA of human herpes simplex viruses (HSVs) was used as the template under the same conditions. Thus, this PCR method is useful in discriminating BV from HSVs.

The ELVIS HSV Id test kit (an enzyme-linked virus-inducible system) (Diagnostic Hybrids, Inc.) uses genetically engineered BHK cells to produce a detectable enzyme, beta-galactosidase, upon infection with either herpes simplex virus (HSV) type 1 (HSV-1) or HSV-2. Twenty six ELVIS-positive clinical specimens were selected for study by PCR and with monoclonal antibodies because they were originally low-titer HSV-positive specimens by ELVIS but HSV antibody nonreactive upon follow-up staining of the ELVIS monolayer. Twenty-one of 26 specimens were frozen, thawed, and retested with ELVIS without removing the cellular debris from the specimen; 18 were ELVIS positive and 3 were ELVIS negative on retesting. A typing result was provided upon retesting for 14 of 18 ELVIS-positive specimens (11 were HSV-1 and 3 were HSV-2) with HSV-specific monoclonal antibodies; no antibody signal was observed for 4 of 18 ELVIS-positive specimens. Sixteen of 26 specimens were subjected to blinded PCR analysis with two different primer sets, including all those that were repeat tested with ELVIS without success and those that had insufficient quantity for repeat testing. All 16 specimens analyzed were PCR positive with primer set 1; 15 of 16 were also positive with primer set 2, with the HSV type identified for all specimens (7 were HSV-1 and 8 were HSV-2). These results indicate that the original ELVIS result with these low-titer specimens was correct and further confirm the sensitivity and specificity of ELVIS HSV Id as a rapid, cell culture-based kit for the detection of HSV.

Herpes simplex virus type 1 (HSV-1)-related disease ranges from a localized, self-limiting illness to fatal disease in immunocompromised individuals. The corneal disease herpetic keratitis may develop after reactivation of a latent virus or reinfection with an exogenous herpesvirus. Molecular analysis of the virus involved may allow distinction between these two options. The HSV-1 genome contains several hypervariable regions that vary in numbers of reiterating regions (reiterations I to VIII [ReI to ReVIII]) between individual strains. Twenty-four HSV-1 clones, derived by subcloning of HSV-1 (strain F) twice in limiting dilutions, were tested in a PCR-based assay to analyze the stabilities of ReI, ReIII, ReIV, and ReVII. ReI and ReIII proved to vary in size upon subcloning, whereas ReIV and ReVII were stable. Subsequently, 37 unrelated isolates and 10 sequential isolates from five patients, all with HSV-1-induced keratitis, were genotyped for ReIV and ReVII. Of the 37 unrelated samples, 34 (92%) could be discriminated, while the genotypes of the viruses in sequential samples were identical for each individual. Conclusively, the data show that the approach presented allows the rapid and accurate discrimination of HSV-1 strains in studies that address the transmission and pathogenesis of HSV-1 infections.

The purpose of this study was to evaluate the performance of a herpes simplex virus (HSV) type 1-specific anti-glycoprotein C-1 monoclonal antibody (MAb) and a type 2-specific anti-glycoprotein G-2 MAb for typing of 2,400 clinical HSV-1 isolates and 2,400 clinical HSV-2 isolates, respectively, using an enzyme immunoassay. The anti-HSV-1 MAb showed sensitivity and specificity of 100%, and the anti-HSV-2 MAb showed a sensitivity of 99.46% and 100% specificity, indicating that these MAbs are suitable for typing of clinical HSV isolates.

We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 10(8) copies of HSV DNA/20 microl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the results obtained by assays with monoclonal antibodies against HSV-1 or HSV-2. Thus, while the real-time PCR is slightly less sensitive than the gel-based liquid hybridization system, the high throughput, the lack of contamination during processing, the better reproducibility, and the better ability to type the isolates rapidly make the real-time PCR a valuable tool for clinical investigation and diagnosis of HSV infection.

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.

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.

The purpose of the present study was to determine if the quantity of herpes simplex virus (HSV) DNA in the cerebrospinal fluid (CSF) of patients with herpes encephalitis would be useful in establishing the prognosis of the disease and to determine the effect of antiviral therapy on the clearance of viral DNA from the CSF. Quantitation of HSV DNA was done by constructing an internal standard (IS) from the glycoprotein B amplicon which had a 25-bp deletion between primer annealing sites. Each CSF specimen was coamplified with the IS and the ratio of the amount of HSV/amount of IS was compared to the ratios on a standard curve constructed with the same IS plus known amounts of HSV DNA. CSF specimens were available from 16 patients who were treated with intravenous acyclovir, and the amount of HSV DNA ranged from < 25 to 18,000 copies per microliter in CSF obtained before or within 4 days of the initiation of acyclovir therapy. Patients with > 100 copies of HSV DNA per microliter were older, were found by computed tomography to have lesions, and had poorer outcomes than patients with < 100 copies. Follow-up CSF specimens were available from seven patients. In six of these seven patients, the HSV DNA levels decreased during therapy. One patient had a twofold increase in HSV DNA levels after 1 week of therapy and died on day 8. The application of this assay may be helpful in establishing the prognosis and in the monitoring of patients with herpes simplex encephalitis.