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HSV-1 epidemiology in the Middle East and North Africa (MENA) remains poorly understood. Our study aimed to measure HSV-1 antibody prevalence (seroprevalence) and its age-distribution among select MENA populations residing in Qatar. Sera were collected from male blood donors attending Hamad Medical Corporation 2013-2015. A total of 2,077 sera were tested for anti-HSV-1 antibodies using HerpeSelect ® 1 ELISA IgG kits (Focus Diagnostics, Cypress, CA). Robust Poisson regression was conducted to estimate adjusted infection prevalence ratios. Country-specific HSV-1 seroprevalence was estimated for 10 national populations: 97.5% among Egyptians, 92.6% among Yemenis, 90.7% among Sudanese, 88.5% among Syrians, 86.5% among Jordanians, 82.3% among Qataris, 81.4% among Iranians, 81.4% among Lebanese, 80.5% among Palestinians, and 77.0% among Pakistanis. Age-specific HSV-1 seroprevalence was estimated for Egypt, the Fertile Crescent (Iraq, Jordan, Lebanon, Palestine, and Syria), and Qatar. Seroprevalence increased with age among Fertile Crescent and Qatari nationals. Seroprevalence increased from 70.0% among those aged ≤ 24 years up to 98.0% among those aged ≥55 years among Fertile Crescent nationals. Seroprevalence was consistently above 90% for all ages among Egyptians. HSV-1 seroprevalence is high in MENA, though with some variation across countries. The seroprevalence appears to have declined among current young age cohorts compared to its levels a few decades ago., (© 2017 The Authors. Journal of Medical Virology Published by Wiley Periodicals, Inc.)

Corneal transplants have one of the highest success rates among all transplantological procedures. Corneas intended for transplantation are stored in a preservation fluid, which is then tested for bacterial and fungal infections. Among all analyses of infectious complications following corneal transplants, infections caused by bacteria or fungi are the most prominent. Surprisingly, however, apart from a few publications, there is a lack of data regarding the occurrence of viruses in donor corneas and the risk of transmitting these to their recipients. The intention of this research was therefore to determine the frequency with which human herpesvirus 1 (HHV-1), human herpesvirus 2 (HHV-2), and human adenovirus (HAdV) occur in transplanted corneal tissue, as well as in samples of preservation fluid. The study comprised 57 paired samples, with each pair consisting of a fragment of the corneal tissue remaining after its trepanation for transplantation surgery and a sample of corneal preservation fluid. Sample pairs were all tested for the presence of the DNA of three viruses (HHV-1, HHV-2, and HAdV) using real time PCR technique. Viral DNA was found in three of the tested corneas-HHV-1 DNA in one paired sample (1.8%) and adenovirus DNA in two single samples (3.5%). We postulate that virological testing of corneas for transplantation should be considered, particularly in the case of donors with increased risk factors for herpesvirus and adenovirus reactivation. J. Med. Virol. 89:732-736, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The relationship between viral infections and first-trimester spontaneous abortions is not well-understood. The study aim was to investigate the prevalence of cytomegalovirus (CMV), human parvovirus B19 (B19V), and herpes simplex virus-1/2 (HSV-1/2) infection by molecular and serological techniques in women experiencing spontaneous miscarriage in the first trimester of pregnancy. Plasma samples were examined for CMV, B19V, and HSV-1/2 DNA using real-time quantitative polymerase chain reaction (Real-time qPCR), and for specific IgG antibodies against B19V, CMV, and HSV-1/2 using serological assays. The abortion group consisted of women (n = 1,716) with a history of two or more first-trimester spontaneous abortions. Women younger than 30 years possess higher portion to experience spontaneous abortion. No specimens were positive for B19V or CMV DNA. Seven out of the 1,716 specimens were positive for HSV-1/2 DNA. By serology, 47.24% of patients were positive for B19V IgG, 39.66% for HSV IgG, 79.31% for CMV IgG, and 9.31% for B19V IgM. The high rate of positivity for CMV IgG suggests that the majority of women with first-trimester spontaneous abortions are not susceptible to primary CMV infection. The lack of virus DNA in the majority of cases indicates that B19V, CMV, and HSV-1/2 infection is not commonly associated with first-trimester spontaneous abortion., (© 2015 Wiley Periodicals, Inc.)

Nucleic acid extraction of clinical samples is accepted as a key requirement in molecular diagnostics. At Barts Health NHS Trust, swabs taken from patients with clinical suspicion of HSV infection were routinely extracted on the Qiagen MDx BioRobot prior to testing with a real-time triplex PCR for HSV1, HSV2, and VZV. The aim of this study was to adapt an existing HSV1/HSV2/VZV real-time PCR by replacing VZV with phocine herpesvirus 1 (PhHV) as an internal control (IC) and evaluate whether this adapted assay required the nucleic acid extraction step for predominantly genital swabs. First 313 non-extracted and extracted swabs were tested in parallel with the existing triplex HSV1/HSV2/VZV real-time PCR. The second stage involved testing 176 non-extracted swabs using a triplex real-time PCR for HSV1, HSV2, and PhHV and comparing the results with the samples extracted and tested by the original triplex assay. The results correlated well when the existing assay was used, with only three non-extracted samples that would have been reported as negative compared to the extracted sample result (Cq s 33, 39, 35-two samples HSV1, one sample HSV2). In the evaluation using the adapted assay containing the IC, two of 176 samples were discordant, where a HSV negative non-extracted sample result would have been reported differently to the extracted sample result (Cq s 32, 33-both HSV1). This study demonstrated that it is feasible to test non-extracted swabs for HSV in a real-time PCR that includes an IC. J. Med. Virol. 87: 125-129, 2015. © 2014 Wiley Periodicals, Inc., (© 2014 Wiley Periodicals, Inc.)

In previous reports on the viral causes of central nervous system (CNS) infections, it has been generally recognized that HSV-1 is a major cause of encephalitis, while HSV-2 is the predominant cause of aseptic meningitis in adults. To examine this matter, the clinical characteristics in the two types of HSV CNS infections were investigated. In a retrospective cohort study which included all adult patients (≥16 years) between January 1999 and December 2013 in a 2,700-bed tertiary care hospital, all the patients in whom PCR of the CSF for HSV was positive were identified. Ninety-five patients with positive CSF PCR results for HSV were included, 21 with HSV-1 and 74 with HSV-2. Many patients with HSV-1 had encephalitis (13/21, 61.9%), whereas most patients with HSV-2 had meningitis (62/74, 83.8%). However, HSV-1 and HSV-2 accounted for similar proportion of patients with HSV encephalitis (13/25, 52.0% vs. 12/25, 48.0%). Neurological sequelae were more frequent among patients with HSV-1 (9/21, 42.9% vs. 6/74, 8.1%; P = 0.001). The present study suggests that HSV-2 is not only a major cause of aseptic meningitis, but also it may cause serious manifestation as HSV-1 encephalitis in adults., (© 2014 Wiley Periodicals, Inc.)

Bell's palsy is the most common cause of facial paralysis. Although it has been associated with diabetes mellitus, hypertension, pregnancy, and preeclampsia, the etiology of Bell's palsy remains unknown. The reactivation of latent herpes simplex virus (HSV) or varicella-zoster virus (VZV) with subsequent inflammation and entrapment of the facial nerve in the narrow labyrinthine segment has been implicated as a cause of facial paralysis, but the active role of these viruses in Bell's palsy is still discussed. This study quantified HSV-1 DNA, VZV DNA, and HHV-6 DNA in 95 saliva samples collected from patients within 48 hr from the onset of paralysis. HSV-1, VZV, and HHV-6 were detected in 13%, 3%, and 61% of patients, respectively. The detection rate did not differ significantly between patients and a control group of healthy donors. Interestingly, however, the value of HHV-6 DNA copies was significantly higher than that detected in healthy donors. In addition, the mean value of HHV-6 DNA recorded in patients who had at least a one grade improvement of palsy at the first visit was significantly lower than that detected in patients who showed no change in facial palsy grade or an increase of at least one grade. These findings call into question the role of HSV-1 and VZV in the etiology of Bell's palsy, and suggest that HHV-6 may be involved in the development of the disease or that the underlying disease mechanism might predispose patients to HHV-6 reactivation., (© 2014 Wiley Periodicals, Inc.)

Primary herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) infection leads to a life-long latent infection of ganglia innervating the oral mucosa. HSV-1 and VZV reactivation is more common in immunocompromised individuals and may result in viral shedding in saliva. We determined the kinetics and quantity of oral HSV-1 and VZV shedding in HSV-1 and VZV seropositive individuals infected with HIV and to assess whether HSV-1 shedding involves reactivation of the same strain intra-individually. HSV-1 and VZV shedding was determined by real-time PCR of sequential daily oral swabs (n = 715) collected for a median period of 31 days from 22 individuals infected with HIV. HSV-1 was genotyped by sequencing the viral thymidine kinase gene. Herpesvirus shedding was detected in 18 of 22 participants. Shedding of HSV-1 occurred frequently, on 14.3% of days, whereas solely VZV shedding was very rare. Two participants shed VZV. The median HSV-1 load was higher compared to VZV. HSV-1 DNA positive swabs clustered into 34 shedding episodes with a median duration of 2 days. The prevalence, duration and viral load of herpesvirus shedding did not correlate with CD4 counts and HIV load. The genotypes of the HSV-1 viruses shed were identical between and within shedding episodes of the same person, but were different between individuals. One-third of the individuals shed an HSV-1 strain potentially refractory to acyclovir therapy. Compared to HSV-1, oral VZV shedding is rare in individuals infected with HIV. Recurrent oral HSV-1 shedding is likely due to reactivation of the same latent HSV-1 strain., (Copyright © 2013 Wiley Periodicals, Inc.)

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen. While there has been extensive research into the evolutionary relationships among herpesviruses, there is little data on the evolutionary relationship of HSV-1 based on sequence analysis of clinical isolates. The present study aims to be the first to document the molecular epidemiology and genetic diversity and frequency of recombination of HSV-1 (n = 42) clinical isolates in Ireland. The entire 1,171 bp of the gI-1 gene and 717 bp of the gG-1 gene of 42 clinical Irish isolates were amplified, sequenced and the phylogenies reconstructed. Putative recombinants were examined using bootscan analysis. Phylogenetic reconstruction of the nucleotide sequence alignments of the entire genes of amplified glycoproteins gI and gG suggested that three distinct HSV-1 genogroups were circulating in the Irish population. At least 15 HSV-1 intergenic recombinants with a recombination point between gI and gG, and 11 HSV-1 intragenic recombinants were detected. There was no evident association between genetic group and gender, disease recurrence or anatomical site of infection. Genital isolates (n = 30) belonged to all genogroups. However, two HSV-1 isolates, Irl 31 and Irl32, from a patient with severe mucocutaneous infection nonresponsive to acyclovir and isolated over a prolonged period were both intragenic and intergenic recombinants. The detection of variability and recombination in gG and gI genes of both HSV-1 may provide a mechanism to evade the host immune response thereby maintaining the viral genome. The variability and recombination detected may also have implications for the detection, diagnosis and treatment of HSV., (Copyright © 2013 Wiley Periodicals, Inc.)

Human herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) were isolated in the bilateral trigeminal ganglia of 12 human cadavers with no history of herpes-related symptoms within 1-5 days of death. Sixteen trigeminal ganglia were subjected to explant culture by using Vero cells, but no cytopathogenic effects (CPE) were observed. However, when another eight trigeminal ganglia were placed in a cell strainer and kept from direct contact with Vero cells during culture, CPE were clearly apparent in all cultures. The amount of DNA in the culture supernatants of 16 trigeminal ganglia decreased over time; 12 and 9 of these samples were PCR-positive for HSV-1 and VZV, respectively. In new Vero cells inoculated with supernatants collected 2 days after culture initiation, immunofluorescence staining revealed HSV-1 and VZV in 6 and 5 of 8 trigeminal ganglia, respectively. HSV-1 and VZV DNA was detected in supernatants collected 3 and 7 days after culture initiation and in Vero cells collected after culture completion, but real-time PCR revealed the DNA amounts decreased over time. There was less VZV DNA than HSV-1 DNA. These results demonstrate that infective HSV-1 and VZV can be isolated in culture, and confirm that viable HSV-1 and VZV persist in human trigeminal ganglia for some time after death., (Copyright © 2013 Wiley Periodicals, Inc.)

Herpes simplex virus (HSV) infection of the corneal stroma is the most prominent cause of scar formation impairing visual acuity and HSV keratitis is the leading cause of corneal opacity throughout the world. Suriname lacked test systems for microbial causes of ocular disease, therefore a polymerase chain reaction-based Herpes virus assay was introduced, enabling prompt recognition, and timely treatment, preventing progressive eye damage. The incidence and epidemiology of Herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), and varicella zoster virus (VZV) in ocular disease in Suriname was assessed. In a cross-sectional prospective study, ocular swabs were collected from 91 patients with a presumptive α-Herpes virus ocular infection attending the Academic Hospital between November 2008 and August 2010 and were tested by a PCR-based α-Herpes virus assay. Alpha-Herpes virus ophthalmic infections were caused predominantly by HSV-1 with a prevalence of 31%. The prevalences of VZV, HSV-2, and a mixed HSV-1/HSV-2 infection were 4%, 3%, and 2%, respectively. The first reported annual incidence of herpetic induced ocular disease in Suriname was estimated at 11.4 per 100,000 person-years (95% CI, 4.8-18.1). No clear age, ethnic or gender dependent difference in incidence was observed. The information obtained on α-Herpes virus positive ocular infections and the distribution of subtypes provided the first insight in the South American situation of α-Herpes virus induced ocular disease., (Copyright © 2012 Wiley Periodicals, Inc.)

The objective of this study was to genotype 375 clinical herpes simplex virus type 1 (HSV-1) isolates collected from the German Reference Laboratory of HSV and VZV between 1973 and 2010. The method is based on the amplification and the restriction fragment length polymorphism analysis of the glycoprotein G (gG) and gI. 45.1% of isolates were classified as genotype A, 28.5% as B, and 4.3% as C. 22.1% presented different cleavage patterns for gG and gI suggesting intergenic recombinants A/B in 7.7%, A/C in 0.5%, B/A in 9.3%, B/C in 1.9%, C/A in 1.6%, and C/B in 0.5% of isolates. Two isolates from 1982 and 2010 presented atypical gI cleavage pattern consistent with novel intragenic recombination between genotypes A and C. There were no significant differences of the prevalence of genotypes A, B as well as the recombinants A/B, B/A dependent on the age/gender of patients and the time period in which the strains were isolated. Likewise, there were no significant differences in the distribution of the genotypes A and B as etiological agents of eczema herpeticum, herpes labialis, herpes genitalis, and herpetic gingivostomatitis. The number of recombinants was not different significantly in the groups of the distinct herpetic diseases. In conclusion, the study confirms the high prevalence of recombinants in clinical HSV-1 strains. HSV-1 infections result in clinical manifestations which are independent of the gG/gI genotype and recombinants are not associated with special herpetic diseases., (Copyright © 2012 Wiley Periodicals, Inc.)

Herpes simplex virus (HSV) infection is rarely considered in the differential diagnosis of severe acute hepatitis and disseminated infection in immunocompetent adults. A case of disseminated HSV-1 infection in an 82-year-old woman initially presenting with neurological problems, signs of meningitis and prominent hepatitis was investigated. Initial diagnosis, monitoring, and follow-up were based on the application of molecular methods to cerebrospinal fluid, serum, and liver tissue samples from this patient. Following an initial full recovery, the patient presented delayed intracerebral haemorrhage and diffuse arthralgia. This atypical case, with delayed secondary progression, highlights the wide range of clinical features of HSV infection and the benefits of monitoring viral load by quantitative real-time polymerase chain reaction (PCR) during patient management., (Copyright © 2012 Wiley Periodicals, Inc.)

Enteroviruses (EVs) and human herpesviruses (HHVs) are involved frequently in acute neurological disorders of viral etiology. This study aimed to investigate the incidence of herpes simplex virus types-1 (HSV-1) and 2 (HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6) and human enteroviruses (EVs) in cerebrospinal fluid (CSF) samples of Tunisian immunocompetent patients with neuromeningeal disorders. The patients had been hospitalized at the Fattouma Bourguiba University Hospital (Monastir, Tunisia) between September 2007 and June 2009. At least one viral genome was detected in 58 (46%) out of 126 CSF samples collected. Enterovirus was detected in 31 of the positive samples (53.4%), CMV in 20 (34.5%), HSV-1 in 3 (5.2%), HSV-2 in 6 (10.3%), VZV in 4 (6.9%), HHV-6 in 2 (3.4%). More than one viral genome was detected in seven CSF samples, including CMV DNA in six of the samples. The high frequency of enteroviral infections in aseptic meningitis was confirmed. The detection of CMV DNA only suggests a direct role of this virus in the etiology of acute neuromeningeal disorder., (Copyright © 2011 Wiley Periodicals, Inc.)

The aim of the study was to develop a multiplex PCR (mPCR) for a rapid and simultaneous detection of herpes simplex 1 (HSV-1), herpes simplex 2 (HSV-2), and human cytomegalovirus (HCMV) DNA in squamous oral cells obtained from adolescents. Accuracy of the method was tested in a group of 513 adolescents, almost 11% of subjects were positive for infection with herpes viruses. Correlations with gender, age, and place of residence were sought. A similar incidence of HSV-2 and HCMV was found (4.3% and 5.4%, respectively) and the incidence of HSV-1 was the lowest (1%) in the study group. Conversely to HSV-2, HCMV was detected mostly in the youngest individuals. The same occurrence of all viruses was observed in boys and girls. The mPCR method described is suggested as a useful tool for epidemiologic studies of active herpes infections., (2010 Wiley-Liss, Inc.)

Viral loads of herpes simplex virus (HSV) are not monitored usually for the effective clinical management of HSV-related diseases. However, recently, there has been more interest about the typical HSV levels in clinical specimens, and how such data may improve understanding of the behavior of this virus in such clinical presentations, particularly in immunocompromised patients, where more prolonged therapy using higher doses of antiviral drugs may be required. Using an in-house quantitative HSV-1/HSV-2 polymerase chain reaction assay, an observational, retrospective 5-year analysis of diagnostic, quantitative HSV-1 and HSV-2 DNA levels was conducted. The results (all in median log(10) DNA copies/ml), including perhaps the first quantitative comparison of cerebrospinal fluid (CSF) HSV viral loads, were as follows: CSF: HSV-1, 3.40 (range 2.30-8.98) versus HSV-2, 3.60 (range 2.31-6.86) (P=0.559); plasma: HSV-1, 3.20 (range 2.23-5.51) versus HSV-2, 3.20 (range 3.18-3.41) (P=0.905); genital swabs: HSV-1, 6.79 (range 2.28-8.48) versus HSV-2, 6.97 (range 3.40-9.66) (P=0.810); oral swabs: HSV-1, 7.28 (range 2.46-10.04) versus HSV-2, 5.62 (range 4.60-6.63) (P=0.529). Note that with the samples usually collected for HSV testing (i.e., CSF, plasma, oral, and genital swabs) there was no significant difference in the viral loads between HSV-1 and HSV-2 types, nor between immunocompetent and immunocompromised patients for each of these different HSV types. Indeed, even between immunocompromised patients with similar diseases, for these samples, the HSV loads were found to vary considerably. These findings may therefore limit the usefulness of monitoring HSV loads in everyday clinical practice., (© 2010 Wiley-Liss, Inc.)

Idiopathic peripheral facial nerve palsy has been associated with the reactivation of herpes simplex virus type 1 (HSV-1) or varicella zoster virus (VZV). In recent studies, detection rates were found to vary strongly which may be caused by the use of different oral fluid collection devices in combination with molecular assays lacking standardization. In this single-center pilot study, liquid phase-based and absorption-based oral fluid collection was compared. Samples were collected with both systems from 10 patients with acute idiopathic peripheral facial nerve palsy, 10 with herpes labialis or with Ramsay Hunt syndrome, and 10 healthy controls. Commercially available IVD/CE-labeled molecular assays based on fully automated DNA extraction and real-time PCR were employed. With the liquid phase-based oral fluid collection system, three patients with idiopathic peripheral facial nerve palsy tested positive for HSV-1 DNA and another two tested positive for VZV DNA. All patients with herpes labialis tested positive for HSV-1 DNA and all patients with Ramsay Hunt syndrome tested positive for VZV DNA. With the absorption-based oral fluid collection system, detections rates and viral loads were found to be significantly lower when compared to those obtained with the liquid phase-based collection system. Collection of oral fluid with a liquid phase-based system and the use of automated and standardized molecular methods allow early and reliable detection of HSV-1 and VZV DNAs in patients with acute idiopathic peripheral facial nerve palsy and may provide a valuable decision support regarding start of antiviral treatment at the first clinical visit.

Investigation of varicella-zoster virus (VZV) is important epidemiologically, and determination of its prevalence rate in human trigeminal ganglia is important to provide surveillance data. To date, studies on VZV detection in trigeminal ganglia have used specimens obtained from a relatively limited number of cadavers. This study attempted to detect VZV DNA as well as Herpes simplex virus type 1 (HSV-1) DNA by the polymerase chain reaction (PCR) from 414 samples of trigeminal ganglia obtained from 207 cadavers selected at random. The detection rate was examined to determine whether there were significant differences in the positive rate between the left and right trigeminal ganglia, males and females, and among age groups. A relationship was found between the positive rates for VZV and HSV-1. VZV DNA was detected in 391 of the trigeminal ganglia (94.4%) and 201 of the cadavers (97.1%) in 121/124 males and 80/83 females. HSV-1 DNA was detected in 251 of the samples (60.6%) and 134 of the cadavers (64.7%) in 72/124 males and 62/83 females. There was no significant difference for either virus in the detection rates between the left and right trigeminal ganglia and males and females. Age and positivity for HSV-1, but not VZV, showed a significant relationship. All 134 cadavers positive for HSV-1 were also positive for VZV. VZV and HSV-1 become latent in bilateral trigeminal ganglia, and are not affected by gender. The prevalence of HSV-1 was greater in advanced age, and the HSV-1-positive rate was correlated with the VZV-positive rate., ((c) 2009 Wiley-Liss, Inc.)

Herpes simplex virus type 1 (HSV-1) has been reported increasingly as a cause of genital herpes, although HSV-1 is usually associated with oro-labial herpes. In the present study, serum specimens and materials for viral isolation were obtained serially from two patients with recrudescent HSV-1 genital infections to study serology and molecular epidemiology. Recurrent episodes, during which HSV-1 was isolated, were followed by an increase in the level of anti-HSV-1 antibody, suggesting a booster effect from re-exposure to viral antigens and the possible usefulness of the variation in the level of anti-HSV-1 antibody to diagnose recurrence. While genotypes of HSV-1 isolates obtained from one patient were different from those from the other patient, genotypes of sequential HSV-1 isolates obtained from the same patient were the same, implying that the recrudescent genital lesions of the two patients could be attributed to endogenous recurrence of a latent virus. Sera from one patient neutralized HSV-1 isolates obtained from the other patient as well as HSV-1 isolates obtained from the same patient. An HSV-1 isolate obtained during a later episode in one patient was neutralized by sera taken before/during the later episode of the same patient, as effectively as an HSV-1 isolate obtained during an earlier episode in the same patient; thus, in these two cases, HSV-1 was assumed to have multiplied during recurrence despite the presence of an anti-HSV-1 antibody that could neutralize experimentally HSV-1.

A quantitative polymerase chain reaction (PCR) assay was evaluated retrospectively on 92 cerebrospinal fluid (CSF) samples from 29 patients with herpes simplex virus (HSV) encephalitis with the aim to study if the concentration of HSV genomes can be used as a prognostic marker and for monitoring of antiviral therapy. The results were compared to those obtained previously by nested PCR, and the numbers of HSV genomes/ml were evaluated in correlation to patient outcome and treatment. The aims were to compare the sensitivity of a conventional nested PCR to a quantitative PCR, to investigate the range of HSV genome concentration in initial samples and to evaluate possible relationships between the HSV DNA concentrations in CSF, neopterin levels, and outcome of disease. The 29 initial samples contained between 2 x 10(2) and 42 x 10(6) HSV genomes/ml. There was no apparent correlation between the amount of HSV DNA in the initial samples and income status, initial neopterin levels, or prognosis. The number of HSV genomes/ml declined after treatment in all patients, but HSV DNA was still detectable after day 20 in 3 out of 16 patients. A long duration of genome detectability was found to correlate with poor outcome. There was no difference in sensitivity between the nested PCR and the quantitative PCR. While the quantitative PCR is more rational than a nested PCR, the quantitation of HSV genomes does not seem very useful as a prognostic marker in HSV encephalitis., (2009 Wiley-Liss, Inc.)

Varicella zoster virus (VZV) causes varicella (chickenpox), after which virus becomes latent in ganglia along the entire neuraxis. Virus reactivation produces zoster (shingles). Infectious VZV is found in vesicles of patients with zoster and varicella, but virus shed in the absence of disease has not been documented. VZV DNA was previously detected in saliva of astronauts during and after spaceflight, a uniquely stressful environment in which cell mediated immunity (CMI) is temporally dampened. The decline in CMI to VZV associated with zoster led to the hypothesis that infectious VZV would also be present in the saliva of astronauts subjected to stress of spaceflight. Herein, not only was the detection of salivary VZV DNA associated with spaceflight validated, but also infectious virus was detected in saliva from 2 of 3 astronauts. This is the first demonstration of shed of infectious VZV in the absence of disease.

Herpes simplex virus type 1 (HSV-1) is isolated principally from the upper half of the body innervated by the trigeminal ganglia whereas herpes simplex virus type 2 (HSV-2) is generally isolated from the lower half of the body innervated by the sacral ganglia. However, recent reports suggest that HSV-1 and HSV-2 can each infect both the upper and lower half of the body causing a variety of symptoms and there is a possibility that HSV-1 and HSV-2 infections can occur simultaneously with both causing symptoms. HSV type in clinical isolates from 87 patients with genital herpes and 57 with ocular herpes was determined by the polymerase chain reaction (PCR), and six cases of mixed infection with both HSV-1 and HSV-2 were identified. Of the six cases, three were patients with genital herpes and three were ocular herpes patients. Analysis of the copy number of the HSV-1 and HSV-2 genome by a quantitative real time PCR demonstrated that HSV-1 was dominant at a ratio of approximately 100:1 in the ocular infections. In contrast, the HSV-2 genome was present at a 4-40 times higher frequency in isolates from genital herpes patients. There was no obvious difference between the clinical course of mixed infection and those of single HSV-1 or HSV-2 infections. This study indicated that the frequency of mixed infection with both HSV-1 and HSV-2 is comparatively higher than those of previous reports. The genome ratio of HSV-1 and HSV-2 reflects the preference of each HSV type for the target organ.

Herpes simplex virus type 1 (HSV-1) DNA was extracted from human trigeminal ganglia of 121 cadavers aged between 3 months and 94 years, and its PCR amplification was performed for the RL2 HSV-1 sequence using two pairs of primers. The HSV-1 DNA was detected in 74 of 121 patients (61%); 70/74 bilaterally, 3/74 only on the left side, and 1/74 only on the right side. Although the PCR-positive rate was significantly higher in advanced age, the correlation between the PCR-positive rate and gender was unclear. Additionally, HSV-1 DNA was not detected in any of the 50 optic nerve samples., ((c) 2006 Wiley-Liss, Inc.)

While the clinical manifestations of HSV-1 and -2 overlap, the site of CNS infection, complications, response to antivirals, frequency of antiviral resistance, and reactivation rate on mucosal surfaces varies between HSV-1 and -2. Detection of HSV DNA by PCR has been shown to be the most sensitive method for detecting HSV in clinical samples. As such, we developed a PCR-based assay to accurately distinguish HSV-1 from HSV-2. Our initial studies indicated the assay using type specific primers was slightly less efficient for detecting HSV-1 and -2 DNA than the high throughput quantitative PCR assay we utilize that employs type common primers to gB. We subsequently evaluated the type specific assay on 3,131 specimens that had HSV DNA detected in the type common PCR assay. The typing results of these specimens were compared with the monoclonal antibody staining results of culture isolates collected from the same patients at the same time, and the HSV serologic status of the patient. The typing assay accurately identified both HSV-1 and -2 with a specificity of >99.5% and was significantly more sensitive than typing by culture and subsequent monoclonal antibody assays. Complete concordance was seen between the typing assay and HSV serologic status of the patient. Dual (HSV-1 and -2) infection in clinical samples was recognized in 2.6% of clinical samples using the new typing assay. This assay, when used in combination with the type common assay, can now accurately type almost all mucosal and visceral HSV isolates by molecular techniques., (Copyright (c) 2005 Wiley-Liss, Inc.)

This study compares herpes simplex virus (HSV) type-specific loop-mediated isothermal amplification (LAMP) with virus isolation and real-time PCR. Genital tract specimens were obtained from 25 patients with genital lesions; two swab samples were collected from the vulva and cervix of each patient, for a total of 50 specimens. After culturing, 10 of 50 (20%) samples were positive for HSV-1 and 12 of 50 (24%) samples were positive for HSV-2. None of the patients excreted both HSV-1 and HSV-2 virus. An original HSV type-specific LAMP assay (30 min reaction) was compared with virus isolation and HSV type-specific real-time PCR. Viral DNA was detected by LAMP in 9 of 10 HSV-1 isolated samples and 11 of 12 HSV-2 isolated samples. No viral DNA was detected in samples without virus isolation. Thus, if virus isolation was used as the standard method, the LAMP protocol was highly sensitive and specific. In comparing LAMP to real-time PCR, viral DNA was detected by the LAMP method in 9 of 12 HSV-1 DNA positive samples and 11 of 18 HSV-2 DNA positive samples. If real-time PCR was used as the standard method, then, sensitivity of the LAMP method (in particular, for HSV-2) was low. Taking this into consideration, the LAMP reaction was extended to 60 min. This led to an increase in sensitivity, resulting in an additional one and three samples testing positive for HSV-1 LAMP and HSV-2 LAMP, respectively, compared to the original LAMP protocol. Therefore, the sensitivity of the LAMP method increased to about 80%., ((c) 2005 Wiley-Liss, Inc.)

Herpes simplex viruses (HSV)-1 and -2 isolated from genital lesions were examined for cutaneous pathogenicity and its correlation with cellular tropism. HSV-1 caused vesiculation, erosion/ulcer, and zosteriform lesions successively, but skin lesions of HSV-2 developed without vesiculation in some mice, and with statistically significantly less frequent vesiculation than HSV-1. Thus, the virological type of HSV was correlated with its cutaneous pathogenicity. The growth characteristics of HSV-1 and -2 were compared in cultured human embryonic lung (HEL) fibroblasts, human lung cancer A549 cells, human neonatal epidermal keratinocytes, human neonatal dermal fibroblasts, HeLa cells, and Vero cells. HSV-2 produced plaques that were 72% times the size of HSV-1 plaques in epidermal keratinocytes but 230%-500% the size in the other cells. The difference between HSV-1 and -2 in the ratio of plaque size to virus yield in epidermal keratinocytes was much larger (502 times) than the ratio of the other cells (5.57-28.8 times). Keratinocytes are the major constituent of the epidermal layer of the skin and the cells in which vesiculation and erosion/ulceration occur histologically. Therefore, the smaller spread of HSV-2 in keratinocytes of the epidermal layer and the greater spread in other cells of the dermal layer might reflect its lesser invasiveness in the epidermal layer despite larger invasiveness in the dermal layer, which is reflected in the low incidence of erosion/ulcer of the skin compared to HSV-1. Thus, the growth of HSV in epidermal keratinocytes appeared to correlate with the cutaneous pathogenicity causing vesiculation in the skin., (2005 Wiley-Liss, Inc.)

It was previously shown that herpes simplex virus type 1 (HSV1) DNA resides latently in a high proportion of aged brains and that in carriers of the type 4 allele of the apolipoprotein E gene (APOE-epsilon4), it confers a strong risk of Alzheimer's disease. It was suggested that initial entry of brain by HSV1 and any subsequent reactivation(s) would cause a type of limited encephalitis, the resulting damage being more harmful in APOE-epsilon4 carriers. Reactivation(s) would induce synthesis of intrathecal antibodies; these are long-lived after herpes simplex encephalitis so they were sought in cerebrospinal fluid (CSF) of Alzheimer's disease patients and age-matched normal subjects. Intrathecal antibodies to human herpesvirus 6 (HHV6) were also sought as DNA of this virus has been detected previously in a high proportion of Alzheimer's disease brains. Antibody indices for HSV and HHV6 were measured using indirect ELISA for IgG antibody, and single radial immunodiffusion was used for albumin, in serum and CSF. A raised antibody index (>1.5) indicative of virus-specific intrathecal HSV1 IgG synthesis was found in 14/27 (52%) Alzheimer's disease patients and 9/13 (69%) age-matched normals (difference non-significant). A raised antibody index to HHV6 was detected in 22% of the Alzheimer's disease patients and in no normals, so presumably this virus either did not reactivate in brain or it elicited only short-lived intrathecal antibodies. The HSV1 results confirm the original PCR findings that show the presence of HSV1 DNA sequences in many elderly brains, and indicate also that the whole functional HSV1 genome is present, and that the virus has replicated.

Herpes simplex virus infections may be diagnosed by several techniques, including conventional cell culture and the polymerase chain reaction (PCR). This prospective study compares the analytical performances and usefulness of an in-house real-time PCR method and the Light Cycler HSV (1/2) detection kit (Roche Diagnostics, Mannheim, Germany). The results of both PCRs were then compared to those obtained by conventional cell culture. A total of 313 samples were tested (70 dermal samples, 81 cerebrospinal fluids (CSF), 47 ocular, 42 anogenital, 34 throat swabs, and 33 oral samples, 3 whole blood, 2 biopsies, and 1 bronchoalveolar lavage). Samples for molecular assays were extracted twice with the MagNa Pure instrument (Roche Molecular Biochemicals, Mannheim, Germany) and tested blind in parallel by the two PCR methods. Most (226) samples were also examined by cell culture. Forty three samples were found positive by both PCRs, whereas 267 were negative. The HSV-1 and -2 typing of positive samples was identical. Three of the samples were positive in the in-house PCR and negative in the Light Cycler HSV (1/2) detection kit. There was no statistically significant difference between the two tests. Only one sample gave an invalid result due to negative PCR and negative internal control result. Seven samples were positive by both real-time PCRs and negative in conventional culture. The PCRs were significantly (P < 0.05) more sensitive. The results show good agreement between the two real-time PCR methods, with the molecular tests being more sensitive than cell culture.

The etiology of idiopathic cranial nerve palsies often remains unresolved. It has been hypothesised that viral reactivation of herpesviruses in the corresponding nuclei in the brainstem is the cause. We investigated the distribution of herpes simplex virus type 1 (HSV-1) and varicella zoster virus (VZV) in nuclei that are associated with peripheral sensory ganglia [oculomotor (nIII), facial (nVII) nuclei] and in nuclei that are not associated with peripheral sensory ganglia [trochlear (nIV), abducens (nVI), and hypoglossal (nXII) nuclei] of five human brainstems. Samples of the cranial nerve nuclei and adjacent control tissue were taken from histological sections after precise identification of every single nucleus and control tissue. DNA and RNA amplification methods were used to determine the prevalence and distribution of HSV-1 and VZV. The distribution of human herpes virus type 6 (HHV-6) was also determined and served as a control, since HHV-6 infection has never been associated with idiopathic cranial nerve palsies. HSV-1 was distributed at random in all cranial nerve nuclei and control tissue, whereas VZV DNA was not detected in any of the samples examined. Surprisingly, HHV-6 was present in almost all samples where HSV-1 was also present, however, the latency associated transcript (LAT) of HSV-1 was not found in any of the samples positive for HSV-1 DNA. The absence of LAT in the samples positive for HSV-1 and the distribution of HSV-1 and HHV-6 do not support the hypothesis that idiopathic cranial nerve palsies result from viral reactivation in the brainstem nuclei.

Clinical recurrences of Herpes simplex virus type 1 (HSV-1)-associated genital herpes are thought to be caused by reactivation of latent endogenous HSV-1. However, the possibility of reinfection with exogenous HSV-1 cannot be excluded. This study aimed to determine the incidence of genital HSV-1 superinfection in patients by investigating the genotype of sequential HSV-1 isolates obtained from the same anatomical site of patients with clinical recurrences of genital HSV-1 recurrent genital herpes. Sequential genital HSV-1 isolates were genotyped by PCR amplification of the hypervariable regions located within the HSV-1 genes US1 and US12. Whereas the sequential HSV-1 isolates in 11 of the 13 patients studied had the same genotypes, the sequential isolates of 2 patients showed a different genotype. The data suggest that HSV-1-induced recurrent genital herpes can be associated with genital reinfection with an exogenous HSV-1 strain., (Copyright 2004 Wiley-Liss, Inc.)

Herpes simplex virus type 1 (HSV-1) has been proposed as an environmental risk factor for sporadic Alzheimer's disease, although this issue is still in dispute. The involvement of HSV-1 in the pathogenesis of familial Alzheimer's disease, the uncommon type of Alzheimer's disease, has not been addressed yet. We investigated formalin-fixed, paraffin-embedded, postmortem brain tissue sections of three patients with familial Alzheimer's disease for the presence of HSV-1 DNA. The nested polymerase chain reaction (PCR) detected the HSV-1 glycoprotein D gene in the brain of all three patients with familial Alzheimer's disease preferentially in the frontal and temporal cortices, whereas only one case out of six age-matched, non-Alzheimer's disease individuals could disclose the presence of HSV-1 gene. The PCR detected HSV-1 DNA in the frontal cortex of the two patients with sporadic Alzheimer's disease. The presence of HSV-1 was associated with beta-amyloid deposition in the cerebral cortex. To clarify the localization of HSV-1 in the brain tissue of patients with familial Alzheimer's disease, the in situ hybridization of the tyramide signal amplification system was used. It detected the HSV-1-specific signals predominantly in the cytoplasm of cortical neurons in a dot-like staining fashion. In addition, high-sensitivity immunohistochemistry revealed the existence of HSV-1 antigens in the cytoplasm of cortical neurons. This report provides the first evidence of reactivation of HSV-1 in the brain of patients with familial Alzheimer's disease, associated with beta-amyloid deposition, and suggests the possible involvement of HSV-1 together with genetic factors in the pathogenesis of familial Alzheimer's disease., (Copyright 2004 Wiley-Liss, Inc.)

Here, we studied the role of nitric oxide (NO) production during the first steps of the respiratory infection of BALB/c mice with herpes simplex virus type 1 (HSV-1), strain F. Nitric oxide synthase II (NOS-II) mRNA and protein were detected by reverse transcription (RT)-PCR and dot blot, respectively in samples of lungs and turbinates early post-infection (p.i.). Immunohistochemical analysis revealed pulmonar macrophages and PMN expressing NOS-II in the lungs of infected animals. Animals intranasally treated with aminoguanidine (AG), a NOS inhibitor, during the first steps of infection, showed a dose-dependent increase in pneumonitis compared to controls. Viral titres in turbinates, lungs, and brains were higher in AG treated mice. Finally, histopathology studies revealed a stronger inflammation in eyes, and lungs of these animals. Taken together, these results suggest a role of NO in controlling primary HSV intranasal infection., (Copyright 2004 Wiley-Liss, Inc.)

The impact of shedding of herpes simplex virus type 1 (HSV-1) on hospital survival of patients receiving assisted ventilation in an adult tertiary referral, acute trauma intensive care unit was assessed. The study was designed to address a clinical impression linking HSV-1 recovery with poor survival. Two hundred and forty-one males and 152 females were enrolled into a longitudinal cohort study. Combined throat swabs and tracheal secretions were tested for HSV-1 shedding using a nested nucleic acid amplification protocol; patients were ranked as nonshedders, shedders, and high-level shedders. Nonparametric analysis assessed the impact of shedding on hospital survival and logistic regression measured the confounding influence of sex, age, and the Acute Physiology, Age and Chronic Health Evaluation (APACHE II) score. Linear-by-linear association determined the influence of the level of shedding on hospital survival. The observed mortality rate was 113/393 (28.8%). Patients shedding HSV-1 106/393 (27%) had a significant reduction in hospital survival 66/106 (62%) in HSV-1 shedders compared with 217/287 (75.6%) in nonshedders (P = 0.002). This difference remained significant when adjusted for age and sex (P = 0.026). Respective mortality figures for HSV-1 shedders and nonshedders were 43/106 (40.6%) and 70/287 (24.4%) (P = 0.002). HSV-1 shedding was associated with a significant reduction in hospital survival amongst patients receiving assisted ventilation. Hospital mortality in HSV-1 shedders was increased by 16.2% over nonshedders. The role of HSV-1 in this setting needs to be addressed., (Copyright 2004 Wiley-Liss, Inc.)

One hundred forty-nine specimens were tested in a LightCycler nested multiplex polymerase chain reaction (LCnmPCR) for Herpes simplex virus (HSV)1, HSV2, and VZV. Eighty-one were from genitourinary medicine (GUM) patients and the other 68 specimens were from other patients with skin lesions. The results were compared to a conventional multiplex nested PCR (nmPCR) using agarose gel electrophoresis. Twenty-five specimens were positive in both assays for HSV1 and 29 were positive for VZV. For HSV2 there were 27 positive in the LCnmPCR and 26 positive in the nmPCR assay. The melting temperatures (Tms) of each target were different with a mean of 84.75 degrees C for HSV1, 88.57 degrees C for HSV2, and 83.62 degrees C for VZV. The melting curves of positive specimens directly overlaid the melting curves of the positive controls in the assay. The LCnmPCR assay is a convenient alternative to conventional PCR using agarose gel electrophoresis. It improves specimen turnaround time by eliminating the need for gel electrophoresis, transillumination, and gel photography. It also shows increased sensitivity for HSV2 over our standard assay. This LCnmPCR reduces further the possibility of amplicon contamination with nested PCR protocols., (Copyright 2003 Wiley-Liss, Inc.)

A subset of erythema multiforme (erythema multiforme) is associated with herpes simplex virus (HSV) infection; viral cultures of erythema multiforme lesions are, however, usually negative and viral antigens difficult to identify. Polymerase chain reaction (PCR) has been used to demonstrate the association, hence, is currently the only available sensitive diagnostic means for HSV-associated erythema multiforme. A nested PCR, which could simultaneously detect and genotype HSV in erythema multiforme lesions and in clinical swab specimen was developed using the DNA polymerase gene of HSV as target gene because it is the only detectable HSV gene in a high proportion of erythema multiforme lesions. The PCR has demonstrated its robust sensitivity on swab samples by being able to detect further 45.3% HSV cases in comparison with virus isolation with 100% specificity in both detection and genotyping confirmed by virus isolation and DNA sequencing. This study represents the first investigation of typing HSV virus in HSV-associated erythema multiforme patients, and the finding that 66.7% of the patients was attributed to HSV1, 27.8% to HSV2, and 5.6% to HSV1 and 2 co-infection may reflect the distribution of HSV1 and 2 in local general population., (Copyright 2003 Wiley-Liss, Inc.)

Herpes simplex virus type 1 (HSV-1) is the etiological agent of herpetic keratitis. The epithelial form ("epithelial keratitis") is attributed mainly to destruction of the epithelium through active viral replication within the epithelium. The stromal form ("stromal keratitis") is associated with immune reactions within the stroma and is the common cause of human blindness. In the present study, 29 HSV-1 strains isolated from human ocular materials of herpetic keratitis were classified into 14 genotypes on the basis of DNA polymorphisms. Twenty-one of 29 (72%) strains from eyes examined in the present study were of genotypes that were shown previously to be present in strains from non-ocular lesions (including genital herpes). Five of nine (56%) strains from eyes related to stromal keratitis were of the F1 genotype, while four of twenty (20%) strains from eyes not related to stromal keratitis were of the F1 genotype. Thus, the proportion of F1 genotype was assumed to be larger in the group of strains related to stromal keratitis than in that not so related, suggesting an association of the F1 genotype with stromal keratitis. A connection of F1 genotype with recurrence was proposed previously; hence, F1 genotype seems to be associated to both stromal keratitis and the recurrence, thereby supporting the relationship between stromal keratitis and recurrence., (Copyright 2003 Wiley-Liss, Inc.)

Herpesviruses are involved in the pathogenesis of acute retinal necrosis syndrome (ARN). A rapid and accurate diagnosis of herpetic infections is crucial for prompt administration of a specific antiviral therapy. The purpose of this study was to evaluate a polymerase chain reaction (PCR)-based assay to detect herpesvirus DNA in the aqueous humor of clinical samples from ten patients with uveitis and clinical suspicion of ARN. Samples were assayed for herpes simplex virus type 1-2 (HSV 1-2), varicella zoster virus (VZV) and cytomegalovirus (CMV). Clinical suspicion of ARN was confirmed for four patients. Two patients (one with bilateral ARN) tested PCR-positive for VZV DNA and the other two were positive for HSV 1-2 DNA. CMV DNA was not detected in any of the samples, and no sample was positive for DNA from more than one virus. The remaining patients did not show any evidence of herpesvirus DNA in their aqueous samples. Our findings demonstrate that the use of PCR for detecting herpesvirus DNA in aqueous humor of uveitic subjects may be a valuable tool for early diagnosis of acute retinal necrosis syndrome and for timely administration of a suitable therapy., (Copyright 2003 Wiley-Liss, Inc.)

The aims of this study were to investigate the prevalence of herpes simplex virus (HSV) types 1 and 2 in the study population and correlate the results with clinical and demographic details. Consecutive HSV isolates from 334 clinic attendees were typed by immunofluorescence. Patient information was collected from the case notes. Overall, HSV-1 was isolated from 48 and HSV-2 from 287 samples, respectively. There was no significant difference in isolation rates according to gender. However, 33% of white patients' isolates typed as HSV-1, while only 6% of the isolates from the black population were HSV-1 (P < 0.001). Initial infections were seen in 81% of HSV-1 infections and 48% of HSV-2 infections, respectively. A wide discrepancy was observed in the prevalence of HSV-1 and HSV-2 infections between the ethnic groups in this population, which was not explained in terms of gender or age. This may reflect different exposure to HSV-1 in childhood or different sexual practices. The increased prevalence in genital HSV-1 reported in recent studies was not seen in this population. However, the differing proportions of primary and first episode infections may reflect a changing epidemiology., (Copyright 2003 Wiley-Liss, Inc.)

A cell line modified genetically (Vero-ICP10-SEAP) that responds to infection by herpes simplex virus (HSV) was established. The cell line was constructed by stable transfection of Vero cell with a plasmid encoding the secreted alkaline phosphatase (SEAP) driven by the promoter of the HSV-2 ICP10 gene. Following infection with HSV, the stable line secretes a high level of the SEAP in the supernatants as measured by a chemiluminescence-based assay. The detection system is sensitive to an HSV titer as low as a single plaque-forming unit (PFU), with a linear range up to the equivalent of 2.5 x 10(4) PFU inoculum after infection for 24 h. There was no detectable enhancement in SEAP activities following inoculations with several viruses other than HSV. The Vero-ICP10-SEAP cell line was also utilized to develop an assay for determination of antiviral susceptibility given that the induced SEAP activity appeared to reflect the numbers of plaque. Evaluations of the stable line with representative acyclovir (ACV)-sensitive and-resistant HSV isolates demonstrated that their drug susceptibilities were determined accurately. In summary, this novel SEAP reporter system is a sensitive means for rapid diagnosis, quantitation, and drug susceptibility testing for HSV, with potential to the development of an automated assay., (Copyright 2002 Wiley-Liss, Inc.)

The aim was to carry out a population-based sero-prevalence survey of Epstein-Barr virus (EBV) across a wide age range in England and Wales and to identify any associations between EBV and herpes simplex virus types one and two (HSV-1 and 2). Sera from an age-stratified sample of 2,893 individuals, submitted for diagnostic purposes to 15 public health laboratories in England and Wales in 1994, were tested for immunoglobulin G (IgG) antibody to EBV. The samples had been tested previously for IgG antibody to HSV-1 and HSV-2. The serological profile of EBV was consistent with an endemic infection with peaks in transmission in those less than 5 years old and in young adults. An age adjusted analysis found a significant association between EBV and HSV-1 seropositivity that is most likely explained by similarities in their mode of transmission. The very low seroprevalence of HSV-2 in this sample complicated the comparisons of EBV and HSV-1 with HSV-2. Any associations were most likely explained by chance. Given the association between EBV and HSV-1, it is likely that recently documented epidemiological changes in HSV-1 also apply to EBV. Continuing surveillance of these herpes viruses is necessary as the predicted changes could have a significant public health impact, especially in the young adult population., (Copyright 2002 Wiley-Liss, Inc.)

Neonatal herpes simplex virus (HSV) infection is a severe disease with high mortality and morbidity in spite of the development of effective anti-viral therapies. The viral load in neonatal herpes simplex virus (HSV) infection was measured retrospectively in 37 patients. HSV DNA copy numbers in serum and cerebrospinal fluid (CSF) were quantified using a real-time PCR assay. Patients with disseminated infection had a higher viral load in their sera. whereas patients with central nervous system (CNS) infection exhibited a higher viral load in the CSF. The viral load was significantly higher in the serum of patients who died later. Interestingly, patients with HSV type-2 infection exhibited more CNS involvement and neurological impairment, together with a high viral load in the CSF, than did HSV type-1 patients. These results suggest that quantitation of HSV viral load may be useful for assessing the prognosis, and may provide additional information for the management of neonatal HSV infection., (Copyright 2002 Wiley-Liss, Inc.)

Sequential herpes simplex virus (HSV) isolates from AIDS patients receiving foscarnet therapy were evaluated for susceptibility to adefovir. Foscarnet-resistant isolates with DNA polymerase mutations in regions II, VI, and between I and VII were also associated with an important decrease in susceptibility to adefovir (mean IC50 increase: 4.6-fold compared to pre-foscarnet or wild-type isolates) suggesting that adefovir-resistant HSV could be selected in vivo by foscarnet therapy., (Copyright 2002 Wiley-Liss, Inc.)

A Herpes Consensus allows the simultaneous detection of 6 human herpesviruses: herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), and human herpes virus 6 (HHV-6). This technique was used first to examine retrospectively 100 DNA extracts from 95 CSF and 5 aqueous fluids, prepared by treatment by saturated NaCl followed by ethanol precipitation (n = 63) or by simple boiling (n = 37) and stored at -80 degrees C, and secondly to test prospectively 38 CSF samples for which two DNA extracts were prepared with commercially available DNA extraction kits. In all cases, the results were compared with those of an "in-house" PCR. Concordant results between both PCR and the Herpes Consensus techniques were obtained in 61 of 63 DNA extracts prepared by treatment by saturated NaCl (97%) and in only 31 of 37 boiled samples (84%). Both commercially available methods of DNA extraction examined appear to be suitable for Herpes Consensus PCR, although they cannot remove completely PCR inhibitors that must be sought in case of negative results. This preliminary study shows that the Herpes Consensus method should be of value for rapid diagnosis of herpesvirus infections on condition that it is performed on purified DNA extracts.

Herpes simplex viruses (HSV) are responsible for neurological disorders that require rapid diagnostic methods and specific antiviral therapy. During 1997, 1431 cerebrospinal fluid samples (CSF) collected from 1339 patients with neurological disorder presentations were processed for HSV detection. Eleven patients were positive for HSV, seven presenting with encephalitis (6/7 due to HSV1) and 4 with aseptic meningitis (4/4 due to HSV2). The incidence of HSV encephalitis was 2.33 cases / 10(6) inhabitants/year. Among encephalitis (HSV encephalitis) cases, 1 patient died due to the late implementation of antiviral therapy, and sequelae were observed in 4 cases. No sequelae were observed in aseptic meningitis cases. Four HSV encephalitis cases were monitored by PCR detection in CSF. Despite acyclovir therapy, PCR remained positive in CSF up to 20 days in 2 cases. This result suggest that the antiviral treatment for HSV encephalitis should be monitored by PCR detection of HSV in CSF., (Copyright 2000 Wiley-Liss, Inc.)

Herpetic stromal keratitis is caused by ocular infection with herpes simplex virus type 1 (HSV-1) and constitutes a leading cause of human blindness. The effect of meliacine, an antiviral compound isolated from leaves of Melia azedarach L. that inhibits HSV-1 replication in vitro, was examined on experimental corneal HSV-1 inoculation in Balb/c mice. Mice were inoculated with HSV-1 strain KOS at their corneas after abrasion. Meliacine was administered topically 3 times a day for 4 days beginning 1 day before inoculation. Infected animals treated or not with meliacine were observed carefully for the development of stromal keratitis and the clinical scoring was done 14 days post-infection. Histological examination of corneas and viral isolation from eyes from HSV-1 infected mice treated or not with meliacine were also carried out. It was found that the treatment of HSV-1-induced ocular disease in Balb/c mice with meliacine reduced significantly the development of clinical disease, as well as the histological damage in corneas. The viral titers detected in eyes of infected and treated mice were 2-orders-of-magnitude lower than those corresponding to HSV-1 infected control animals. Mock-infected and treated mice did not reveal any corneal alteration due to the administration of the compound. Meliacine was found to exert a strong antiviral action on HSV-1-induced ocular disease in mice with no evidence of toxic effects., (Copyright 2000 Wiley-Liss, Inc.)

Recurrent acyclovir (ACV)-resistant (ACV-r) herpes simplex virus type 1 (HSV-1) infections occurred in a patient with Wiskott-Aldrich syndrome, an X-linked recessive immunodeficiency syndrome composed of three clinical characteristics of immunodeficiency, thrombocytopenia, and an eczematous dermatitis. The patient had severe and recurrent ACV-r herpes simplex and was treated with vidarabine in a satisfactory manner from 1993 to 1997. During the 4-year observation period, two ACV-sensitive (ACV-s) HSV-1 isolates and five ACV-r HSV-1 isolates were recovered. The nucleotide sequence of the thymidine kinase (TK) gene from these sequential ACV-r isolates was compared with the ACV-s isolates. A single nucleotide deletion of cytosine (C) from homopolymer stretch of four C residues between nucleotide 1061 and 1064 of the open reading frame was found in all ACV-r isolates. No other differences were observed in the TK nucleotide sequence between ACV-s and ACV-r isolates. The TK nucleotide sequences of the two ACV-s isolates were identical to each other and those of the five ACV-r isolates were identical to one another. These results suggest that the ACV-r HSV-1 might have derived from the ACV-s strain in the patient body and that TK-associated ACV-r HSV-1 can reactivate from latency.

Drug-resistance of herpes simplex virus (HSV) is caused most frequently by mutation of the viral thymidine kinase (TK) gene. To elucidate the significance of detecting nucleotide changes of the TK gene for screening drug-resistant viruses, the frequency and variation of the genetic polymorphisms in the whole coding region of the TK gene were studied in 14 acyclovir-susceptible HSV type 1 (HSV-1) clinical isolates from 14 patients with epithelial herpetic keratitis. Two reference HSV-1 laboratory strains, McKrae and PH, and two acyclovir-resistant variants of the PH strain were also studied as controls. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing detected nucleotide differences at 24 positions, and amino acid substitutions at 12 codons in the TK gene of the examined viruses. Nucleotide diversity of 0.0029 per base (the average number of nucleotide substitutions of 3.3 per 1,131 base pairs) in the TK gene in the clinical isolates was comparable to 0.0037 per base of the whole HSV-1 genome in Japanese isolates reported previously. PCR-SSCP analysis of the acyclovir-resistant strains easily detected aberrantly shifted bands by comparing them with those of the parental strain, followed by the quick determination of mutated sequences. These results suggest that detection of nucleotide changes of the TK gene is useful for serial observation of persistent or recurrent HSV infection as observed in immunocompromised hosts, but that it is not useful for screening drug-resistant viruses from nonepidemic clinical isolates because of the comparable genetic polymorphisms in the TK gene as in the whole HSV-1 genome.

Patients attending the genitourinary medicine clinic at Watford General Hospital, UK, were examined for clinical signs of genital herpes infection. Genital swabs were taken from 194 patients (126 female, 68 male) who presented with genital ulceration or symptoms which were suggestive of genital herpes infection. Swabs from these patients were tested by three methods: (i) Detection of herpes simplex virus (HSV) antigen by direct HSV enzyme immunoassay (EIA), (ii) HSV isolation in Vero cell culture and (iii) HSV polymerase chain reaction (PCR). HSV was detected in 76 patients (39%) by EIA, in 93 (48%) by isolation in cell culture, and in 115 (59%) by PCR. Isolation by cell culture has been considered as the "gold standard" for the detection of HSV in genital lesions, but in this study HSV PCR was significantly more sensitive. Comparison of the three methods was as follows: Cell culture vs. PCR: Sensitivity 93/115 (80.9%), Specificity 79/79 (100%). HSV EIA vs. PCR: Sensitivity 75/115 (65.2%), Specificity 78/79 (98.7%). HSV EIA vs. Cell culture: Sensitivity 75/93 (80.7%), Specificity 100/101 (99%). EIA was less effective in detecting HSV among recurrent than among first episode infections, in comparison to culture or HSV PCR. This is the first comparison of HSV PCR with two other routine diagnostic methods for confirming genital herpes infection in a symptomatic population. The infecting HSV type was identified by restriction digestion of 108 HSV amplicons: HSV-1:37/108 (34%), HSV-2:71/108 (66%). In this population HSV-1 causes a significant proportion of genital herpes cases, and HSV-1 genital infection was detected in significantly more first episode infections (40.3%) than among recurrent infections (22.2%).

The incidence of herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) in herpes simplex encephalitis (HSE) was investigated using cerebrospinal fluid (CSF) samples from sixty-four cases of HSE. A polymerase chain reaction (PCR) employing primers flanking a region of the HSV thymidine kinase gene common to both HSV-1 and HSV-2 was used to detect HSV in the CSF. HSV-1 and HSV-2 were differentiated by digestion with restriction enzymes. Two enzymes were employed; Aval which cleaved only the HSV-2 gene product and Avall which cleaved only the HSV-1 gene product. Sixty-three cases of HSE were found to be due to HSV-1; one case due to HSV-2. These data confirm previous observations that HSV-2 is a rare cause of post-neonatal herpes encephalitis but indicates that a PCR procedure capable of detection of both viruses is essential for efficient diagnosis of HSE.

Clinical data indicate that the recurring herpes simplex virus (HSV) from oro-labial lesions is HSV subtype 1 and that the virus from genital lesions is HSV-2. This suggests that HSV-1 and HSV-2 reside in latent forms in the trigeminal ganglia and sacral ganglia, respectively. However, the distribution of latent HSV-1 and HSV-2 infections in human spinal ganglia has not been fully examined. This report concerns the application of polymerase chain reaction (PCR) and in situ hybridization (ISH) to such a study. By using PCR and employing the respective primers, HSV-1 and HSV-2 DNAs were detected in 207 of 524 samples from 262 spinal ganglia (from the cervical to the sacral ganglia) examined on both sides. The percentages of HSV-1 and HSV-2 detected in a given set of ganglia were similar, indicating an absence of site preference. By ISH, few but positive hybridization signals were detected evenly in sacral ganglia sections. The data suggest that regional specificity of recurrent HSV infections is not due to regional distribution of latent virus, but that local host factors may be important for recurrences.