Cytodiagnosis of herpes simplex keratitis by means of an immunoperoxidase technique. A case report

Typical herpes simplex keratitis that developed in a 5-year-old boy was initially diagnosed cytologically in Papanicolaou-stained samples. Subsequently, an immunoperoxidase staining technique was used to identify the specific type of herpes simplex virus (HSV) in the destained cellular samples. The positive staining helped to establish the diagnosis of a type 1 HSV infection, permitting early treatment with acyclovir and subsequent complete recovery from the ocular herpetic infection. Emphasis is placed on the value of the immunoperoxidase technique for the rapid and specific diagnosis of cases of suspected HSV infection.     

Rapid detection of HSV from cytologic specimens collected into ThinPrep fixative

OBJECTIVE: Herpes simplex virus (HSV) infection is associated with substantial morbidity and mortality in neonates. A diagnosis of HSV on cervical cytologic studies could lead to a cesarean section, with an increase in the risk of maternal morbidity. The identification of viral lesions in sexually active women has medical and social implications. There have been reports of false positive diagnoses of HSV in patients with altered endocervical cells and with cervical intraepithelial neoplasia 3. We evaluated a polymerase chain reaction (PCR)-based assay to detect HSV-1 and HSV-2 in routinely collected cervical cytology specimens in ThinPrep fixative (Cytyc Corp., Marlborough, Massachussets, U.S.A.). STUDY DESIGN: DNA was extracted from five cases that demonstrated cytologic changes suggestive of an HSV infection. PCR amplification with subsequent gel electrophoresis was performed to detect the presence of HSV. RESULTS: HSV DNA was detected in three of five cases that were cytologically diagnosed as suspicious or strongly suspicious for HSV infection. CONCLUSION: The combination of the ThinPrep liquid-based method for cervical cytology with PCR allows prompt confirmation of the diagnosis of HSV without sacrificing the diagnostic morphology on the slide.     

Pitfalls in the diagnosis of herpes simplex infection in respiratory cytology

OBJECTIVE: To evaluate the prevalence and potential pitfalls in making an accurate diagnosis of respiratory herpetic infection. STUDY DESIGN: Eighteen cases with the diagnosis of herpes simplex virus (HSV) infection were identified from a total of 7,501 (0.24%) respiratory specimens. All cases were evaluated for classic cytomorphologic features of HSV infection and associated cytologic findings. The parameters studied included number of cells with HSV cytopathic effect, intranuclear inclusions, multinucleation, presence of atypical squamous cells, reparative changes, presence and degree of inflammation and associated obscuring factors. RESULTS: Only a minority of cases (28%) had numerous cells with classic viral cytopathic change. Four (22%) of 18 cases showed atypical squamous cells, and 5 (28%) revealed reparative changes. The majority of the cases were associated with inflammation, which was severe in 4 cases (22%). Blood and degenerative changes obscured the cytologic findings in 3 cases (17%). One case showed a necrotic background. CONCLUSION: Due to the low prevalence of HSV infection in respiratory cytology, a high index of suspicion is necessary for an HSV diagnosis. Pitfalls for a false negative diagnosis include limited number of cells with viral cytopathic change, only mononuclear cells with viral changes and obscuring inflammation or blood. Pitfalls for a false positive diagnosis of malignancy include atypical keratinized squamous cells, atypical repair, cellular degeneration and necrotic background.     

Diagnostics for herpes simplex virus: is PCR the new gold standard?

Herpes simplex virus (HSV) is one of the most common, yet frequently overlooked, sexually transmitted infections. Since the type of HSV infection affects prognosis and subsequent counseling, type-specific testing to distinguish HSV-1 from HSV-2 is recommended. Although PCR has been the diagnostic standard for HSV infections of the central nervous system, until now viral culture has been the test of choice for HSV genital infection. However, HSV PCR, with its consistently and substantially higher rate of HSV detection, will likely replace viral culture as the gold standard for the diagnosis of genital herpes in people with active mucocutaneous lesions, regardless of anatomic location or viral type. Alternatively, type-specific serologic tests based on glycoprotein G should be the test of choice to establish the diagnosis of HSV infection when no active lesion is present. Given the difficulty in making the clinical diagnosis of HSV, the growing worldwide prevalence of genital herpes and the availability of effective antiviral therapy, there is an increased demand for rapid, accurate laboratory diagnosis of patients with HSV.     

Diagnosis of herpes simplex virus in routine smears by an immunoperoxidase technique

Routine Papanicolaou-stained cervicovaginal smears from 59 patients were cytologically screened for herpetic infection. Forty-one of the smears were positive for herpes, 2 were suspicious and 16 were negative. All 59 slides were then destained and restained by a commercial immunoperoxidase kit for the detection of herpes simplex virus (HSV). The immunoperoxidase stain was positive in 23 of the 41 cytologically positive slides. One of the 2 cytologically suspicious slides was also immunoperoxidase positive, as was 1 of the 16 cytologically negative slides. This study indicates that immunoperoxidase staining is very specific but not quite as sensitive as routine Papanicolaou-stained smears in the detection of HSV. The immunoperoxidase method is thus recommended for the confirmation of HSV cases rather than for the routine diagnosis of HSV infection.     

Modulation of acute and latent herpes simplex virus infection in C57BL/6 mice by adoptive transfer of immune lymphocytes with cytolytic activity

The ability of highly lytic herpes simplex virus (HSV) cytolytic T lymphocytes to modulate the interaction between the murine host (adult C57BL/6 [H-2b] mice) and HSV type 1 Patton resulting in acute infection in the footpad and latent infection in the sensory lumbosacral dorsal root ganglia (L6, L5, L4, and L3) innervating the footpad was investigated. Results indicated that a critical threshold level of infectious HSV was required to establish infection. The adoptive transfer of cytolytic T lymphocytes derived from in vitro cultures after restimulation with HSV-infected, syngeneic stimulator cells exhibiting class I H-2-restricted, L3T4- Lyt-2+ HSV-specific cytolytic activity immediately before infection with a high dose of HSV reduced the levels of infectious HSV recovered from the footpad tissue during acute infection and the levels of latent HSV reactivated from the dorsal root ganglia to levels expected from mice infected with a low dose. Depletion of Lyt-2+ cells from the transferred population abrogated the protective ability, while depletion of L3T4+ cells had little effect. These results suggest that functionally lytic HSV-specific cytolytic T lymphocytes present at the time of HSV infection have the potential to participate in the control of the acute infection and in the subsequent establishment of latent infection.     

Confirmation of genital herpes simplex viral infection by an immunoperoxidase technique

Over the 12-month period from April 1984 to April 1985, 512,000 gynecologic (Papanicolaou) smears were examined in the Provincial Screening Program in British Columbia. During this time, 307 patients were found to have smears that contained cells consistent with, or suggestive of, a herpes simplex viral (HSV) infection. The Papanicolaou-stained smears from these 307 cases were subsequently restained, without prior destaining, using an immunoperoxidase technique specific for type 2 HSV (HSV-2) and cross reactive with HSV-1. Of the 205 smears containing cells considered to be consistent with a herpes infection, 187 were positive using the immunoperoxidase technique. Of the 102 smears showing reactive cell changes though unlikely to be causes by an HSV infection, only 5 were positive using the immunoperoxidase technique. The results show that the immunoperoxidase technique is a rapid and reliable method of confirming a suspected diagnosis of herpetic infection and that it is particularly useful in those patients in whom the Papanicolaou smear findings are equivocal.     

Comparison of real-time and nested PCR assays for detection of herpes simplex virus DNA

We performed a real-time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real-time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real-time assay. The real-time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real-time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.     

Herpes simplex-RAJI(A44), a new cell line for serologic testing by immunofluorescence.

An IF technique for the detection of HSV antibodies by using a lymphoblastoid cell line, Raji(A44), which continuously produce a constant amount of HSV antigen, is described. IF and IH tests were found to be similar with respect to sensitivity and reproducibility. Sero-conversions detected by the CF test were detected with this cell line. The Raji(A44) line (cells in suspension) can be routinely used for the diagnosis of HSV infection.     

Trigeminal ganglion infection by thymidine kinase-negative mutants of herpes simplex virus after in vivo complementation.

Infection of trigeminal ganglion by herpes simplex virus (HSV) thymidine kinase-negative (TK-) mutants was investigated in mixed infection studies in mice. Mice were corneally inoculated with TK- HSV alone or with mixtures of TK- HSV-TK+ HSV. When inoculated alone, an arabinosylthymine-selected HSV type 1 TK- mutant and a HSV type 2 TK- deletion mutant infected mouse ocular tissues but rarely infected ganglion tissues. However, both TK- mutants readily infected ganglion tissues when they were inoculated in mixtures with TK+ HSV. By means of mixed infection studies, it was demonstrated that TK- HSV could readily establish acute and latent ganglion infections. It was thought that the frequent infection of trigeminal ganglion tissue by both TK- mutants after mixed TK(-)-TK+ HSV infection was the result of in vivo complementation. After mixed TK(-)-TK+ HSV infection and subsequent cultivation of ganglion explants in arabinosylthymine, results supported the conclusion that when TK- was present in ganglia it was in the same neurons that contained TK+ HSV.     

Delayed hypersensitivity and immune protection against herpes simplex virus: suppressor T cells that regulate the induction of delayed hypersensitivity effector T cells also regulate the induction of protective T cells

We have been studying delayed hypersensitivity (DH) to herpes simplex virus (HSV) in order to examine the role of this response in host defense against acute and recurrent HSV infections. In previous reports the basic parameters of DH to HSV have been characterized by using a murine ear swelling model, and also the regulation of DH to HSV induced by i.v. injection of the virus. In this paper, we describe a murine protection system and our use of the ability to specifically regulate DH to HSV to examine the correlation between T cells that transfer DH (TDH) and cells that transfer protection from acute HSV infection. Both DH and protection can be transferred with lymph node cells from mice immunized subcutaneously 4 days previously. The effector cell appears to be a T cell, because serum from these donors confers no protection and treatment of immune cells with anti-Thy-1.2 plus complement reduced their ability to protect. Tolerance of DH to HSV was induced by i.v. injection 7 days before subcutaneous immunization. Tolerized mice were unable to generate protective cells. Furthermore, tolerized mice contained suppressor T cells that suppressed not only DH but also the development of protective cells. Regulation of protective cells was shown to be virus specific, because mice tolerized with vesicular stomatitis virus (VSV) were not impaired in their ability to generate T cells that protected from HSV infection. The correlation between the TDH cell and cells that transfer protection from acute HSV infection is discussed.     

Passive immunization of mice with monoclonal antibodies to glycoprotein gB of herpes simplex virus

To investigate the protective ability of monoclonal antibodies (MCAs) to viral glycoprotein in herpes simplex virus (HSV) infection, athymic nude mice were inoculated intracutaneously with HSV type-1 (HSV-1) in the midflank. Three hours after inoculation, one group of mice was passively immunized with one of a series of MCAs to glycoprotein gB of HSV-1, and a control group of mice was given phosphate buffered saline alone. The control mice died within 16 days after infection, whereas the mice passively immunized with any of the MCA showed suppressed development of skin lesions. Three of six mice given MCA failed to develop any visible lesions and no HSV could be isolated from the lumbar dorsal root ganglia of these mice 60 days after the challenge. BALB/c mice were also protected from infection with HSV type 2 by passive immunization with MCA to HSV-1 gB.     

HSV neutralization by the microbicidal candidate C5A

Genital herpes is a major risk factor in acquiring human immunodeficiency virus type-1 (HIV-1) infection and is caused by both Herpes Simplex virus type 1 (HSV-1) and HSV-2. The amphipathic peptide C5A, derived from the non-structural hepatitis C virus (HCV) protein 5A, was shown to prevent HIV-1 infection but neither influenza nor vesicular stomatitis virus infections. Here we investigated the antiviral function of C5A on HSV infections. C5A efficiently inhibited both HSV-1 and HSV-2 infection in epithelial cells in vitro as well as in an ex vivo epidermal infection model. C5A destabilized the integrity of the viral HSV membrane. Furthermore, drug resistant HSV strains were inhibited by this peptide. Notably, C5A-mediated neutralization of HSV-1 prevented HIV-1 transmission. An in vitro HIV-1 transmigration assay was developed using primary genital epithelial cells and HSV infection increased HIV-1 transmigration. Treatment with C5A abolished HIV-1 transmigration by preventing HSV infection and by preserving the integrity of the genital epithelium that was severely compromised by HSV infection. In conclusion, this study demonstrates that C5A represents a multipurpose microbicide candidate, which neutralizes both HIV-1 and HSV, and which may interfere with HIV-1 transmission through the genital epithelium.     

Use of acyclovir in the prevention of herpes infections after allogenic bone marrow grafts

In a double-bind controlled study, oral Acyclovir has been compared to a placebo in a series of 39 consecutive patients undergoing bone marrow transplantation. A dose of 200 mg was given every 6 h from day 8 to day 35 after transplantation. Pharmacokinetic studies have shown the good absorption of the drug despite intestinal damage related to chemoradiotherapy or gut graft-versus-host disease (GVHD), there was no sign of toxicity. The protection against herpes simplex virus (HSV) infection was complete in the treated group when compared to the control group even in patients with high anti-HSV antibody titres. The same protection was observed against cytomegalovirus (CMV) infection. The incidence of HSV and CMV was the same in both groups after treatment ended. This study confirms the efficacy of Acyclovir against HSV infection and possibly against CMV infection when it is given prophylactically after bone marrow transplantation.     

Replication of herpes simplex virus and cytomegalovirus in human leukocytes.

Human peripheral blood leukocytes, lymphocyte subpopulations, and hemic cell lines were examined for their ability to supprot HSV and CMV replication. Mitogen-stimulated mononuclear leukocytes, B lymphocytes, and T lymphcytes supported the replication of HSV to high titers over 3 to 5 days of infection. HSV replicated in unstimulated mononuclear leukocyte cultures of one of five donors, and to a limited degree in untreated B lymphocytes of three of five donors; HSV replication was not detected in unstimulated T lymphocytes (five donors). There was no evidence of enhanced uptake of 3H-thymidine in the untreated donor cells that replicated HSV. CMV replication was not detected during 9 to 10 days of infection in untreated or mitogen-treated mononuclear leukocytes and lymphocyte subpopulations from the same adult donors or in neonatal cord blood leukocytes. The ability of the cells to support HSV or CMV replication did not correlate with the presence of specific antiviral antibodies in the donor serum. HSV replication in B, T, and myeloid cell lines to high titers over 5 days of infection, whereas CMV failed to replicate in any of the hemic cell lines. A persistent HSV infection has been established in a T cell line (CEM) with high titers of infectious virus being produced concurrently with growth of the cells over the first 11 weeks of infection.     

HSV hepatitis in the mouse: a light and electron microscopic study with immunohistology and in situ hybridization

In order to characterize better the morphology and immune response in acute necrotizing HSV infection, murine HSV hepatitis was examined. BALB/c mice were inoculated intraperitoneally with 10(6) plaque-forming units (PFU) of HSV-1 (Lenette) and HSV-2 (D316). In both groups half the animals were pretreated with silica particles to block macrophage function. Up to 6 days after infection four mice from each group were sacrificed at daily intervals and the livers were examined by light and electron microscopy, immunohistology, in situ hybridization, combined immunohistology/in situ hybridization and titration of viral PFU. HSV-2 infected mice developed severe necrotizing hepatitis with persistence of HSV in the liver tissue until the end of the study. HSV-1 infected mice rapidly eliminated the virus and revealed only small necrotic foci. Early phase alterations and necrotic phase lesions were distinguished and characterized and morphologic evidence of a direct cytopathic effect of HSV was detected. A specific immune reaction in late stages appeared to be mediated by T4-positive T-lymphocytes. In situ hybridization and immunohistochemistry showed a close correlation with virus titration and were valuable in characterizing early phases and in the assessment of prognosis and differential diagnosis.     

Cytomegalovirus and Herpes Simplex Virus Effect on the Prognosis of Mechanically Ventilated Patients Suspected to Have Ventilator-Associated Pneumonia

Objective

Cytomegalovirus (CMV) and herpes simplex virus (HSV) are common viruses that can affect critically ill patients who are not immunocompromised. The aim of this study was to determine whether the identification of CMV and/or HSV in mechanically ventilated critically ill patients suspected of having pneumonia was associated with an increased mortality.

Design

Prospective epidemiological study.

Setting

Medical intensive care unit of a tertiary medical center.

Patients

Ninety-three patients with suspected pneumonia.

Interventions

Patients with suspected pneumonia had bronchoalveolar lavage and blood samples taken to confirm the diagnosis. Antigenemia was used to detect CMV in the blood. Bronchoalveolar lavage samples were submitted to testing using quantitative real-time Polymerase Chain Reaction.

Measurements and Main Results

We identified 22 patients with a CMV infection, 26 patients with an HSV infection and 45 patients without CMV or HSV infection (control group). Mortality at day 60 was higher in patients with a CMV infection than in patients from the control group (55% vs. 20%, P<0.01). Mortality at day 60 was not significantly increased in the group with HSV infection. Duration of ICU stay and ICU mortality were significantly higher in patients with CMV infections when compared to patients from the control group, whereas ventilator free days were significantly lower in patients with CMV infections when compared to patients from the control group.

Conclusions

In critically ill patients, a CMV infection is associated with an increased mortality. Further interventional studies are needed to evaluate whether treatment could improve the prognosis.     

18F]FHPG positron emission tomography for detection of herpes simplex virus (HSV) in experimental HSV encephalitis

Herpes simplex virus type 1 (HSV-1) is one of the most common causes of sporadic encephalitis. The initial clinical course of HSV encephalitis (HSE) is highly variable, and the infection may be rapidly fatal. For effective treatment with antiviral medication, an early diagnosis of HSE is crucial. Subtle brain infections with HSV may be causally related to neuropsychiatric disorders such as Alzheimer's dementia. We investigated the feasibility of a noninvasive positron emission tomography (PET) imaging technique using [(18)F]FHPG as a tracer for the detection of HSE. For this purpose, rats received HSV-1 (infected group) or phosphate-buffered saline (control group) by intranasal application, and dynamic PET scans were acquired. In addition, the distribution of tracer accumulation in specific brain areas was studied with phosphor storage imaging. The PET images revealed that the overall brain uptake of [(18)F]FHPG was significantly higher for the infected group than for control animals. Phosphor storage images showed an enhanced accumulation of [(18)F]FHPG in regions known to be affected after intranasal infection with HSV. High-performance liquid chromatography metabolite analysis showed phosphorylated metabolites of [(18)F]FHPG in infected brains, proving that the increased [(18)F]FHPG uptake in infected brains was due to HSV thymidine kinase-mediated trapping. Freeze lesion experiments showed that damage to the blood-brain barrier could in principle induce elevated [(18)F]FHPG uptake, but this nonspecific tracer uptake could easily be discriminated from HSE-derived uptake by differences in the tracer kinetics. Our results show that [(18)F]FHPG PET is a promising tool for the detection of HSV encephalitis.     

Role of inflammatory cytokine-induced cyclooxygenase 2 in the ocular immunopathologic disease herpetic stromal keratitis

Ocular infection with herpes simplex virus (HSV) results in a blinding immunoinflammatory stromal keratitis (SK) lesion. Early preclinical events include polymorphonuclear neutrophil (PMN) infiltration and neovascularization in the corneal stroma. We demonstrate here that HSV infection of the cornea results in the upregulation of the cyclooxygenase 2 (COX-2) enzyme. Early after infection, COX-2 was produced from uninfected stromal fibroblasts as an indirect effect of virus infection. Subsequently, COX-2 may also be produced from other inflammatory cells that infiltrate the cornea. The induction of COX-2 is a critical event, since inhibition of COX-2 with a selective inhibitor was shown to reduce corneal angiogenesis and SK severity. The administration of a COX-2 inhibitor resulted in compromised PMN infiltration into the cornea, as well as diminished corneal vascular endothelial growth factor levels, likely accounting for the reduced angiogenic response. COX-2 stimulation by HSV infection represents a critical early event accessible for therapy and the control of SK severity.