Detection Of Herpes Simplex Virus Research Articles

Diagnosis of genital herpes by real time PCR in routine clinical practice

Background: Virus isolation in cell culture is the recognised diagnostic gold standard for genital herpes. Although increasing evidence indicates that polymerase chain reaction (PCR) provides a more rapid and sensitive...

Detection of herpes simplex virus, cytomegalovirus and Epstein-Barr virus DNA in atherosclerotic plaques and in unaffected bypass grafts

Herpes virus infections are suspected to be involved in the pathogenesis of atherosclerosis. Viral DNA of herpes simplex virus (HSV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) was analyzed by real-time PCR on 48 biopsies from atherosclerotic plaques extracted by end-arterectomy (46 coronary arteries, 2 carotid arteries), and in tissue from non-atherosclerosis vessels from the same patient as controls (23 internal mammary arteries, 43 saphenous veins). HSV-1 DNA was detected significantly more frequently in plaques (35%) than in control veins (9%, P = 0.006). However, the frequency of HSV-1 DNA detection in the internal mammary artery grafts was as high as in plaques (22%, P = 0.28). CMV and EBV DNA were exclusively found in plaques but not in controls, with 10% for CMV (P = 0.06 versus veins, P = 0.17 versus graft arteries) and 2% for EBV (P = 1.0), respectively. HSV-2 was neither detected in plaques nor in controls. Herpes viral DNA was significantly associated only with arterial hypertension but not with other classical risk factors (P = 0.02), in accordance with the hypothesis that herpes viral infection may alter the vessel wall. We conclude that herpes viral infections may have a role in atherosclerosis and that the presence of herpes viral DNA in the grafts used for bypass surgery might constitute a potential risk for atherosclerosis or restenosis.

Evaluation of a new assay for detection of herpes simplex virus type 1 and type 2 DNA by real-time PCR/Evaluierung eines neuen Tests zur Detektierung von Herpes simplex Virus Typ 1 und Typ 2 DNA mittels Real-time PCR

Abstract Abstract Molecular detection of herpes simplex virus (HSV) DNA is recognized as reference standard assay for the sensitive and specific diagnosis of central nervous system and genital infections caused by HSV. In this study, a qualitative molecular assay based on automated DNA extraction on the MagNA Pure LC (Roche Applied Science, Mannheim, Germany) and a commercially available kit, the LightCycler – HSV 1/2 Detection Kit (Roche), were evaluated. This kit includes a homologous internal control. The accuracy and the detection limit of the new molecular assay were determined with samples from European Union Concerted Action HSV Proficiency Panels. A total of 153 clinical specimens including cerebrospinal fluids, genital swabs, and oral swabs were investigated and the results were compared to those obtained from a home-brew molecular assay based on manual DNA extraction and real-time polymerase chain reaction (PCR). When the accuracy of the new molecular assay was tested, all positive samples except for the one containing 3.0 × 102–9.0 × 102 HSV type 1 (HSV-1) genome equivalents (GE) per ml could be detected. All negative samples tested negative. When the detection limit was determined, 6.0 × 102–1.8 × 103 HSV-1 GE per ml, i.e. 12 to 36 GE per LightCycler (LC) capillary and 2.0 × 102–6.0 × 102 HSV type 2 (HSV-2) GE per ml, i.e. 4 to 12 GE per LC capillary could consistently be detected. Results obtained from clinical specimens corresponded to 100% to those obtained by the molecular assay based on manual DNA extraction and real-time PCR. In seven clinical samples, an unexpected melting peak was detected. In conclusion, the new molecular assay allows rapid detection of HSV-1 and HSV-2 DNA in the routine diagnostic laboratory. The inclusion of a homologous internal control ensures accurate interpretation of negative results.

Evaluation of the ELVIS plate method for the detection and typing of herpes simplex virus in clinical specimens

This study was conducted to assess the reliability of a commercial enzyme-linked viral inducible system (ELVIS) (Diagnostic Hybrids, Inc., Athens, OH) for rapid detection and typing of herpes simplex virus (HSV). Results using ELVIS were compared to those of shell vial culture (SVC) and HSV detection with monoclonal antibodies and an immunoperoxidase stain plus typing with MicroTrak direct fluorescent antibodies (Trinity Biotech PLC, Wicklow, Ireland). Specimens yielding discrepant HSV results were tested by polymerase chain reaction (PCR); those with discrepant typing results were stained with Simulfluor (Chemicon, Temecula, CA). Of the 206 samples tested, 144 were negative and 54 were HSV-positive by both methods (agreement, 96.1%). Five specimens were positive by ELVIS but negative by SVC; 3 of these were positive and 2 were negative by HSV PCR. Both of the latter were the result of mechanical problems early in the study. Three specimens were positive by SVC but negative by ELVIS; all 3 were positive by HSV PCR. After resolution of discrepancies, the sensitivity and specificity for detection of HSV were 95.0% and 100% for SVC, respectively, and 95.0% and 98.6% for ELVIS. Of the 46 HSV-positive samples that were typed, 26 were called type 2 and 18 were type 1 by both methods (agreement, 95.7%). The 2 specimens with discrepant results were called HSV-2 by SVC, staining with MicroTrak, and HSV-1 with ELVIS; both of these were type 2 when stained with the Simulfluor reagent. ELVIS is a reliable alternative to SVC for rapid detection and typing of HSV.

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

Utilidad de la reacción en cadena de la polimerasa en el diagnóstico de las infecciones herpéticas del sistema nervioso

To assess the performance of a polymerase chain reaction (PCR) method in cerebrospinal fluid (CSF) for the diagnosis of nervous system infections caused by herpesvirus, and to estimate the incidence of encephalitis due to herpes simplex virus type 1 in the adult population of the island of Gran Canaria. We studied 330 CSF specimens from 312 patients (281 HIV-negative and 31 HIV-positive) remitted to investigate clinically suspected encephalitis or meningitis, or to study neuropathy or demyelinating disease. A multiplex PCR technique was used to detect herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (CMV), Epstein-Barr virus and human herpesvirus type 6. The patients' clinical records were reviewed to establish the definite diagnosis. Nine samples from eight patients (2.6%) showed positive results (9.7% of patients with pathological CSF and none with normal CSF). The eight patients had clinical and analytic findings of herpesvirus nervous system infection: HSV-1 DNA in four patients with encephalitis, HSV-2 DNA in one patient with meningitis, VZV DNA in two patients with meningitis and CMV DNA in one HIV-positive patient with encephalitis. Herpesvirus was the cause of 50% of encephalitis cases and 10% of meningitis cases. The incidence of HSV-1 encephalitis was five cases per million inhabitants per year. Diagnosis of herpesvirus nervous system infections by PCR in CSF is not appropriate when CSF parameters are normal. We found a higher incidence of herpesvirus encephalitis than has been reported in other studies.

Herpes simplex virus acute retinal necrosis during pregnancy.

As pregnancy is liable to modify immune response, the authors explored the immune functions of a pregnant patient with acute retinal necrosis (ARN) to ascertain whether pregnancy may promote the onset of infection. Polymerase chain reaction (PCR) was used for the detection of herpes simplex virus (HSV) DNA in ocular, uterus cervix, and cerebrospinal fluid samples. Peripheral blood mononuclear cells were cultured for 72 hours with mitogens and cellular proliferation was assessed using (methyl-3H) thymidine incorporation. Flow cytometry was performed for T, B, and NK cell count using CD2, CD3, CD4, CD8 (T cells), CD19, CD20 (B cells), and a combination of CD3-CD16 and CD56 monoclonal antibodies (NK cells). Unilateral ARN, with a confluent peripheral necrotizing retinitis extending throughout the entire retina, was diagnosed clinically. The herpetic infection (herpes simplex virus 1) was confirmed using PCR of aqueous humor specimen. The immunologic study performed during and after pregnancy showed that T and B lymphocytes were quantitatively normal and responses to concanavalin A, phytohemagglutinin, and pokeweed mitogens were weaker during pregnancy. A reduced response to mitogens, with postdelivery normalization, was noted in a pregnant woman with an ARN syndrome. Further studies are needed to explore the antigen-specific immune deviation in pregnant patients with ARN.

Herpes simplex virus DNA in corneal transplants: prospective study of 38 recipients.

Herpes simplex virus (HSV) infection of the eye can induce epithelial and stromal keratitis and may also lead to postoperative endothelial failure in keratoplasty. Clinical symptoms and/or virus culture of corneal scrapings most frequently provide the basis for diagnosis of ocular HSV infection, and although HSV DNA has been shown to be present in the cornea, its role in success or failure of corneal grafts remains unclear. In this study, a PCR assay was used to detect HSV DNA in corneal buttons of 38 corneal graft recipients and in donor scleral remnants, retaining one-half of each sample for subsequent viral isolation. Recipients were followed up clinically for a period of 6 months after keratoplasty. All recipients but three were found to be HSV seropositive. Eight recipient corneal buttons contained detectable HSV DNA (7 HSV-1, 1 HSV-2, the latter case confirmed by viral culture). Two donor corneas were found positive for HSV-1 DNA, with negative cultures, and endothelial graft failure occurred in one of the matching recipients after 4 months. One recipient with no history of herpes contracted herpetic keratitis 4 months after keratoplasty, even though the corneal button and donor scleral remnants contained no detectable HSV DNA. The study confirms previous observations of HSV DNA in the corneal tissue of HSV seropositive patients apparently unrelated to any clinical manifestation of herpes infection. However, as demonstrated by culture, HSV remains infectious and may therefore induce donor-to-host infection in corneal recipients.

Detection of herpes simplex virus, cytomegalovirus, and Epstein-Barr virus in the semen of men attending an infertility clinic

Objective To investigate the prevalence of herpes simplex virus (HSV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) in the semen of men with fertility problems. Design A descriptive clinical study. Setting Outpatient infertility clinic of a private hospital. Patient(s) One hundred thirteen men attending an infertility clinic in Athens. Intervention(s) Semen samples were collected by masturbation. Main outcome measure(s) Detection of HSV, CMV, and EBV in semen by a nested polymerase chain reaction technique. Complete spermogram including sperm count, motility, pH, viscosity, and morphology. Result(s) Viral DNA was detected by the nested polymerase chain reaction technique in 64 (56.6%) of 113 semen samples. Specifically, HSV DNA was detected in 56 (49.5%) semen samples, EBV DNA in 19 (16.8%) semen samples, and CMV DNA in 8 (7.1%) semen samples. HSV was significantly related to low sperm count and poor motility. In contrast, CMV and EBV did not show any association with sperm concentration and motility. Conclusion(s) Herpes simplex virus seems to play a significant role in male infertility, and its early detection by the nested polymerase chain reaction technique will permit successful antiviral therapy to increase the possibility for fertility restoration and long-term protection of the sperm quality. Finally, the detection of herpes viruses within semen will allow better control of the transmission of these viruses.

Erythema multiforme in a neonate

We describe a case of erythema multiforme in a 2-week-old boy. He had no remarkable antecedents, and a polymerase chain reaction-based technique failed to detect herpes simplex virus DNA in the skin biopsy specimen. To our knowledge, only one previous biopsy-proven case of erythema multiforme during the neonatal period has been reported. (J Am Acad Dermatol 2003;48:S78-9.)

Cytologic, colposcopic, and virologic detection of cervical herpes simplex virus

Studies on cervical herpes simplex virus (HSV) in Egypt are scant. The clinical symptoms of genital herpes are a poor indicator of infection w1x. The exact prevalence of genital herpes is unknown because not all patients with active infections seek medical care and because the proportion of asymptomatic disease is high. The principal site of HSV infection in the female is the cervix. Initial genital infection involves the cervix in more than 80% of cases and both the ectocervix and the endocervix may be involved. Herpes infection of the lower genital tract has been associated with cervical cancer although a causative role remains unproven. Several adverse outcomes of pregnancies complicated by gestational HSV infections have been described. These include spontaneous abortion prematurity intrauterine growth retardation congenital infection and neonatal infection. The objectives of this study were: (1) to determine the isolation rate of cervical HSV in relation to different clinical conditions and colposcopic findings; and (2) to determine the sensitivity and specificity of the Papanicolaou (Pap) smear direct fluorescence antibody (DFA) stain and tissue culture in patients attending the outpatient clinic of Benha University Hospital. (excerpt)

A double-blind, randomized, placebo-controlled trial of acyclovir in late pregnancy for the reduction of herpes simplex virus shedding and cesarean delivery

Objective: The purpose of this study was to assess the efficacy of acyclovir in the reduction of herpes simplex virus culture and polymerase chain reaction positivity and cesarean delivery. Study Design: Women with recurrent genital herpes simplex virus were randomized to acyclovir 400 mg three times daily or placebo from 36 weeks of gestation until delivery. A subset of daily specimens for herpes simplex virus culture and DNA polymerase chain reaction was self-collected. Analyses used χ2, Fisher exact, and Mann-Whitney U tests. Results: Lesions occurred at delivery among 11 of 78 women (14%) who received placebo and 4 of 84 women (5%) who received acyclovir (P =.08). Herpes simplex virus culture and polymerase chain reaction positivity near delivery occurred in 7% and 34% women in the placebo group and 0 and 2% in the acyclovir group (P =.03 and <.01, respectively). Cesarean delivery for herpes simplex virus occurred in 8 of the women (10%) in the placebo group and in 3 of the women (4%) in the acyclovir group (P =.17). Despite reductions in herpes simplex virus detection, 6% of the women who received acyclovir had herpes simplex virus detected by polymerase chain reaction on >20% of days. Neonatal outcomes were similar between groups. Conclusion: Acyclovir significantly reduced, but did not eliminate, herpes simplex virus lesions and detection in late pregnancy. (Am J Obstet Gynecol 2003;188:836-43.)

Detection of Herpes simplex Virus Genomic DNA in Various Subsets of Erythema multiforme by Polymerase Chain Reaction

Background: The wide variation in the detection of herpes simplex virus (HSV) DNA (36–75%) by polymerase chain reaction (PCR) in erythema multiforme (EM) may be partly attributed to differences in case selection in terms of subsets of EM studied. Objective: To determine the frequencies of detection of HSV DNA in specific subsets of EM. Methods: Nested PCR was used to detect HSV DNA in skin biopsies with histologically proven EM. Results: PCR was performed on skin biopsies from 63 patients with EM. HSV DNA was detected in 3/11 (27.2%) of single-episode HSV-associated EM (HAEM), 6/10 (60%) of recurrent HAEM, 1/4 (25%) of single-episode idiopathic EM and 6/12 (50%) of recurrent idiopathic EM. HSV DNA was not detected in atypical EM (0/11), suspected drug-induced EM (0/9) or Stevens-Johnson syndrome (0/6). Conclusion: The overall PCR positive rates of HAEM (42.9%) and idiopathic EM (43.8%) were comparable suggesting that idiopathic EM is likely to be related to a subclinical HSV infection.

Multiplex polymerase chain reaction for the detection of herpes simplex virus, varicella-zoster virus and cytomegalovirus in ocular specimens

Purpose. A majority of ocular viral diseases are caused by herpes group of viruses. Such infections, especially atypical herpetic keratitis, iridocyclitis and intra-ocular inflammations, can often present with overlapping clinical manifestations misleading the diagnosis. Molecular techniques are most useful in such instances for an accurate and rapid diagnosis since conventional methods are time consuming and less sensitive. A multiplex PCR was developed and used for the detection of herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV) in ocular samples. Methods. One hundred and forty six ocular samples (corneal scrapings – 52, aqueous fluid – 36, vitreous fluid – 31, tissues – 26, skin vesicle scraping – 1) were included in the study. The sensitivity of the assay was determined using serial dilutions of standard strains of HSV, VZV, and CMV vis-à-vis plaque forming assay. Results. The sensitivity of the assay was 4, 4 and 12PFU/ml or 20, 20 and 60 genome copy numbers of HSV, VZV and CMV respectively. Using DNA from various sources (fungal, bacterial, human leukocytes, tissues) along with standard positive controls, the assay was found to be highly specific. HSV DNA was detected in majority of the clinical samples (33.6%), most frequent being corneal samples. Comparatively, VZV and CMV infections were detected in small number of samples (VZV-3, CMV-2). Conclusions. We found the assay very useful in our set-up whenever a differential diagnosis of herpetic infections was suggested by the ophthalmologist. The multiplex PCR we have described here can be of greater value in clinics with larger number of patients suspected of having HSV, VZV or CMV infections.

Detection of herpes simplex virus in pseudoexfoliation syndrome and exfoliation glaucoma.

The pathogenesis of pseudoexfoliation syndrome (PEX) remains unknown. An infection, possibly viral, is one of the proposed pathogenetic mechanisms. This study examines the presence of herpes simplex virus (HSV) and varicella-zoster virus (VZV) in iris and anterior capsule specimens of PEX and non-PEX patients. Iris and anterior capsule specimens were obtained from 64 patients with PEX (study group, SG) and 61 patients without PEX (control group, CG). The presence of HSV and VZV DNA was evaluated with a polymerase chain reaction (PCR). Herpes simplex virus type I was detected significantly more often in iris specimens from the SG (13.79%), compared to those from the CG (1.75%). Varicella-zoster virus DNA was not detected in any of the examined specimens. Results imply a possible relationship between HSV type I and PEX, although no aetiological role of HSV infection in PEX pathogenesis can be established. Results also advocate against any association between VZV and PEX.

Prevalence and risk factors associated with perianal ulcer in advanced acquired immunodeficiency syndrome

Background: The aims of this study were to evaluate the prevalence of perianal ulcer in AIDS patients with advanced disease, and to investigate risk factors associated with these lesions. Methods: A cross-sectional study was conducted to determine the prevalence and risk factors associated with the presence of perianal ulcer in AIDS patients. A type-specific polymerase chain reaction (PCR) assay was carried out for detection of herpes simplex virus (HSV) DNA on swabs obtained from the ulcerative lesions. Results: In total, 272 hospitalized AIDS patients were included in the study, for evaluation of the risk factors associated with the lesion. Perianal ulceration was found in 25 of 272 patients (prevalence= 9.2%).The presence of HSV DNA was shown by type-specific PCR in 22 of 23 (95.6%) patients. Multivariate analysis revealed that a history of esophageal candidiasis (odds ratio (OR)=15.1; 95% confidence interval (CI)=3.8–59.1) and a history of perianal ulcer (OR=19.2; 95% CI 6.4–58.1) were significant risk factors for the presence of perianal ulcer. Conclusion: We conclude that a history of perianal ulcer and a history of esophageal candidiasis were risk factors independently associated with perianal ulcer in AIDS patients with advanced disease.

Use of a fragment of glycoprotein G-2 produced in the baculovirus expression system for detecting herpes simplex virus type 2-specific antibodies.

Fragments of glycoprotein G (gG-2(281-594His)), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1(t26-189His)), and glycoprotein D of HSV-1 (gD-1(1-313)), were expressed in the baculovirus expression system to develop an assay for the detection of HSV-1 and HSV-2 type-specific antibodies. The expression of the gG-1(t26-189His) and gG-2(281-594His) fragments was analyzed by Western blotting using monoclonal antibodies LP10 and AP1, respectively. The molecular masses of the major products of gG-1(t26-189His) and the fragment of gG-2(281-594His) were 36 to 39 kDa and 64 to 72 kDa, respectively. Human sera positive for HSV-1 reacted with gG-1(t26-189His), sera positive for HSV-2 reacted with the gG-2(281-594His) fragment, and sera positive for both types reacted with gG-1(t26-189His) and gG-2(281-594His) in Western blotting. The human sera recognized polypeptides of gG-2(281-594His) with molecular masses of 57 to 67 and 120 to 150 kDa and additional faint bands of 21, 29, and 45 kDa. The recombinant gG-1(t26-189His) and the recombinant gG-2(281-594His) fragment were used as type-specific antigens for the detection of HSV-1- and HSV-2-specific antibody responses in human sera, respectively. As type-common antigens, an extract of HSV-1-infected Vero cells and recombinant gD-1(1-313) were used. An enzyme-linked immunosorbent assay to detect type-specific antibodies was developed, and the sensitivity and specificity were evaluated by comparison with commercial tests by using sera obtained from different sources. The sensitivity and specificity were 91.5 and 95.5%, respectively, compared to the Gull assay. The gG-2(281-594His) fragment can be obtained in relatively large quantities at low cost.

A prospective study of hormonal contraceptive use and cervical shedding of herpes simplex virus in human immunodeficiency virus type 1-seropositive women.

Cross-sectional analyses have demonstrated an association between use of hormonal contraceptives and shedding of herpes simplex virus (HSV). This prospective study evaluated the effect of initiating use of hormonal contraception on cervical HSV detection. Two hundred women who were seropositive for HSV-2 and human immunodeficiency virus (HIV) type 1 were examined for cervical mucosal HSV by use of quantitative DNA polymerase chain reaction before and after beginning the use of hormonal contraceptives. Cervical HSV was detected in 32 women (16.0%) before initiating and in 25 women (12.5%) after initiating use of hormonal contraception (P=.4). There were no significant differences in HSV shedding among the subgroups of women starting combination oral contraceptives containing both estrogen and progesterone or progesterone-only contraceptives. Among the 54 women who shed HSV at least once, the median change in cervical HSV after initiation of hormonal contraception was -313 copies/swab. In this prospective study, use of hormonal contraceptives did not increase detection of cervical HSV.

Rapid detection of herpes simplex virus DNA in genital ulcers by real-time PCR using SYBR green I dye as the detection signal.

We have evaluated a real-time PCR procedure based on the LightCycler technology for rapid detection of herpes simplex virus (HSV) in genital lesions. Two sets of primers, corresponding to the thymidine kinase and DNA polymerase regions, were used for the amplification reactions in separate capillaries containing the SYBR Green I dye as detection signal. In 28 of 118 samples (24%), HSV was isolated by conventional cell culture. All cell culture-positive samples were also positive by real-time PCR. Six additional cell culture-negative samples were positive by PCR with both sets of primers. Total processing time was less than 3 h. Real-time PCR using SYBR Green I as detection signal is a sensitive procedure for the rapid diagnosis of HSV in genital lesions.

Polymerase chain reaction for diagnosis of genital herpes in a genitourinary medicine clinic

Background: Polymerase chain reaction (PCR) has well established advantages over culture for diagnosis of herpes viruses, but its technical complexity has limited its widespread application. However, recent methodological advances have...