Detection of herpes simplex-1 and -2 and varicella zoster virus by quantitative real-time polymerase chain reaction in corneas from patients with bacterial keratitis / Detecção de vírus herpes simplex tipo 1 e 2 e varicella zoster por reação em cadeia de polimerase em tempo real em córneas de pacientes com ceratites bacterianas

ABSTRACT Objective: Bacterial keratitis occurs worldwide, and despite recent developments, it remains a potentially blinding condition. This study assesses the presence of herpes simplex virus (HSV-1 and -2) and varicella zoster virus (VZV) by quantitative real-time polymerase chain reaction (qPCR) in corneal scrapings from patients with bacterial keratitis. Methods: A total of 65 patients with clinical diagnoses of infectious corneal ulcers prospectively underwent clinical eye examinations. Corneal scrapings were investigated by Gram staining, Giemsa staining, culture, and qPCR (the study group). Risk factors and epidemiological data were recorded. The control group comprising 25 eyes with typical herpes dendritic keratitis was also analyzed by qPCR. Results: From the study group (n=65), nine patients (13.8%) had negative smears, cultures, and qPCR findings. Fifty-six (86.2%) patients had positive cultures: 51 for bacteria, 4 for fungi, and 1 for amoebae. Of the patients who had positive bacterial cultures, qPCR identified 10 patients who were also positive for virus: one for VZV and nine for HSV-1. Of the 25 patients in the control group, 21 tested positive for HSV-1 by qPCR analysis. Conclusions: Herpes may be present in patients with bacterial corneal ulcers, and qPCR may be useful in its detection.

RESUMO Objetivo: Ceratites bacterianas ocorrem mundialmente e apesar dos novos desenvolvimentos permanece como uma condição que pode levar à cegueira. Avaliar a presença de herpes simples (-1 e -2) e vírus varicella zoster (VZV) por reação em cadeia quantitativa de polimerase em tempo real (qPCR) em raspados corneanos de pacientes com ceratite bacteriana. Métodos: Sessenta e cinco pacientes com ceratite infecciosa foram submetidos a raspados corneanos estudados para gram, Giemsa, cultura e qPCR (grupo de estudo). Foram avaliados fatores de risco e epidemiológicos. O grupo controle foi composto por 25 casos de úlcera dendrítica típica por herpes analisados por qPCR. Resultados: Do grupo de estudo (n=65), nove pacientes (13,8%) apresentaram cultura, qPCR e raspado negativos. Cinquenta e seis (86,2%) pacientes apresentaram cultura positiva, 51 para bacteria, 4 para fungo e 1 para ameba. A qPCR identificou 10 pacientes do grupo de cultura positiva para bactéria que também foram positivos para vírus, um VZV e 9 para HSV-1. Dos 25 pacientes que compunham o grupo controle, 21 apresentaram qPCR positivo para HSV-1. Conclusão: Herpes pode estar presente em pacientes com úlceras de córnea bacterianas e a qPCR pode ser útil na sua detecção.

Macrophages are important determinants of acute ocular HSV-1 infection in immunized mice.

PURPOSE: To determine the effect of macrophage depletion on herpes simplex virus type (HAV)-1 replication in the eye and on the establishment of latency in trigeminal ganglia (TG) of immunized and ocularly infected mice. METHODS: BALB/c mice were immunized with five HSV-1 glycoprotein DNA genes or were sham immunized. The virulent HSV-1 strain KOS was used as a positive vaccine control. Immunized mice were depleted of their macrophages by dichloromethylene diphosphonate (Cl(2)MDP) injection. After ocular infection with the HSV-1 strain McKrae, virus replication in the eye, blepharitis, corneal scarring, and dermatitis were determined. Finally, the copy numbers of latency-associated transcript (LAT) and CD4(+) and CD8(+) T-cell transcripts in the TGs of surviving mice 30 days after infection were determined by RT-PCR. RESULTS: Depletion of macrophages in immunized mice increased HSV-1 replication in the eye of infected mice between days 1 and 5 after ocular infection. Depletion of macrophages did not alter the HSV-1-induced death or corneal scarring in immunized mice. Macrophage depletion, however, resulted in increased blepharitis in immunized mice. Finally, macrophage depletion had no effect on the establishment of latency in immunized mice, as the TGs from both depleted and mock-depleted mice were negative for the presence of the LAT transcript. CONCLUSIONS: In immunized mice during primary HSV-1 ocular infection, macrophages play an important role in vaccine efficacy against HSV-1 replication in the eye and blepharitis in infected mice. During the latent stage of HSV-1 infection, however, macrophage depletion failed to have any observable effect on HSV-1 latency in the TGs of infected mice.

"Steel wool keratopathy": a clinical sign of chronic inflammation.

PURPOSE: To introduce into the clinical nomenclature a sign frequently observed in our patients with persistent corneal inflammation associated with herpetic stromal keratitis. METHODS: Case reports and review of the literature. RESULTS: Four representative patients with herpesvirus stromal keratitis are presented. Herpes simplex virus-1 (HSV-1) was confirmed by culture in 1 case and by polymerase chain reaction in a second case. In the remaining 2 cases, the diagnosis was made based on characteristic clinical findings for herpes simplex virus and varicella zoster virus (VZV). On clinical examination, all 4 representative cases of stromal keratitis revealed a well-defined, localized region of intertwined, metallic-like, polychromatic material in the corneal stroma, a sign we have termed steel wool keratopathy. We have only rarely observed this finding in patients with stromal keratitis not caused by a herpesvirus. CONCLUSION: Steel wool keratopathy seems to represent a focal region of stromal degeneration or deposition associated with chronic inflammation. Although we most often observe this finding in patients with stromal keratitis secondary to HSV or VZV, we cannot exclude the possibility that this sign represents the sequelae of chronic/recurrent inflammation rather than a specific pathologic response to herpetic antigens.

Improved protection from primary ocular HSV-1 infection and establishment of latency using multigenic DNA vaccines.

PURPOSE: To compare the effectiveness of immunization with "naked" DNA corresponding to the genes encoding five HSV-1 glycoproteins, gB, gC, gD, gE, and gI (5gP DNA), with immunization with the five glycoproteins (5gP protein). Also, to compare immunization of 5gP protein in Montanide ISA 720 (SEPPIC, Paris, France), an adjuvant recently approved for use in humans, with immunization of 5gP protein in Freund's adjuvant. METHODS: BALB/c mice were vaccinated with 5gP DNA or 5gP protein emulsified in ISA 720 or Freund's adjuvant. Neutralizing antibody titers were determined by plaque-reduction assays. IL-2, -4, and -12 and IFN-gamma levels were determined by ELISA after in vitro stimulation of spleen cells. After ocular challenge with 2 x 10(5) plaque-forming units [pfu] per eye of HSV-1 strain McKrae, virus replication in the eye, survival, blepharitis, corneal scarring, and latency were determined. RESULTS: Neutralizing antibody titers (approximately 1:800-1:1200), corneal scarring (trace) and survival (100%) were similar for all vaccine groups, including 5gP DNA. Compared with the other vaccine groups, the 5gP DNA group had less ocular virus replication, as judged both by maximum virus titer and time of viral clearance. ISA 720 appeared more effective than Freund's against ocular virus replication and eye disease. The 5gP DNA-vaccinated mice had less blepharitis and latency than any other group and had the highest levels of IL-12 and IFN-gamma. All vaccine groups had similar levels of IL-2. CONCLUSIONS: The 5gP DNA vaccine appeared to be more effective than the corresponding protein subunit vaccine, regardless of adjuvant. Emulsification of the 5gP protein in ISA 720 appeared to be more effective than emulsification in Freund's adjuvant.

Estudio clínico y virológico en un grupo de niños chilenos con herpes ocular: análisis genómico de las cepas / Clinical-virological study in chilean children with ocular herpes: genomic analysis of strains

Objetivo: Caracterizar el diagnóstico de infección ocular por virus herpes simples (HSV) en un grupo de niños chilenos, mediante el estudio clínico y de laboratorio virológico. Métodos: La población estudiada comprendió niños menores de 15 años, con diagnóstico clínico de herpes ocular, que fueron atendidos por los autores y un grupo de oftalmólogos entrenados especialmente para el estudio. Junto con detallar el tipo de infección herpética, a todos los pacientes se les tomaron muestra para estudio virológico que incluyó estudio de cultivos celulares y posteriormente técnica de reacción en cadena de polimerasa (PCR), con el fin de tipificar las cepas y características genómicas del virus infectante. Resultados: El estudio enroló 18 niños, cuyas edades fluctuaron entre los 40 días y 13 años, con una media de 6 años. De las formas clínicas observadas, la más frecuentes fueron la blefaritis y la queratitis dendrítica constituyendo en 27 y 22 por ciento de los casos, respectivamente. El diagnóstico de HSV fue confirmado en 15 de 18 pacientes, constituyendo un 83 por ciento de positividad. 14 de 15 casos correspondieron a HSV tipo 1, y en un niño se diagnóstico infección por HSV tipo 2. Los antecedentes clínicos de este caso confirmaron que se trataba de una infección perinatal, lo que permitió instaurar el tratamiento en forma oportuna. El estudio permitió identificar un caso de excreción ocular viral asintomática, lo que sumando a un cuadro de recurrencias múltiples obligó a indicar terapia profiláctica permanente con aciclovir. Conclusiones: La blefaritis y queratitis herpética constituyeron en conjunto el 70 por ciento de los casos. El rendimiento celular y PCR fue elevado en los casos con alto índice de replicación viral, como la queratitis y blefaritis. En los casos con menor replicación, como queratitis estromal o conjuntivitis, el estudio PCR demostró una mayor sensibilidad que el estudio en cultivo celular. La presencia de un caso de infección perinatal por HSV-2 pudiera ser indicativo de un aumento en la frecuencia de esta forma de presentación.

A therapeutic vaccine that reduces recurrent herpes simplex virus type 1 corneal disease.

PURPOSE: To investigate the therapeutic efficacy of periocular vaccination with herpes simplex virus (HSV) recombinant glycoprotein D from HSV-1 (gD1) or HSV-2 (gD2) in decreasing HSV-induced recurrent dendritic keratitis and HSV-induced recurrent ocular shedding in rabbits latently infected with HSV-1. METHODS: Rabbits latently infected with HSV-1 were vaccinated periocularly (by subconjunctival injection) with gD1 and adjuvant, gD2 and adjuvant, or adjuvant alone. Eyes were examined daily for 49 days for recurrent herpetic keratitis and for recurrent infectious HSV-1 shedding. RESULTS: In both vaccinated groups, a significantly decreased number of eyes exhibited recurrences of herpetic keratitis compared with recurrences in adjuvant-treated control eyes (gD1 group, 27/1372, [2%]; gD2 group, 24/1274, [2%]; and control, 54/1274 [4%]; P < 0.005). Eyes in the gD1-vaccinated group (44/1308 [3.4%]; P = 0.01), but not those in the gD2-vaccinated group (71/1274 [5.6%]; P = 0.93), had significantly decreased viral shedding (positive cultures compared with total cultures) compared with eyes in the adjuvant-treated control group (69 of 1275 [5.4%]). CONCLUSIONS: Recurrent HSV-1 corneal disease was significantly reduced by therapeutic local periocular vaccination. The vaccine may be more efficacious against HSV-1-induced recurrent corneal disease than against recurrent HSV-1 ocular shedding. Its efficacy against corneal disease appeared to be longer lasting than its efficacy against recurrent spontaneous shedding. The heterotypic gD2 vaccine was as efficacious as the homotypic gD1 vaccine against recurrent corneal disease, whereas the homotypic vaccine was much more efficacious than the heterotypic vaccine against recurrent HSV-1 shedding. This is the first report in any animal model of a successful therapeutic vaccine against recurrent HSV-1-induced corneal disease. These results support the concept that development of a therapeutic vaccine for ocular HSV-1 recurrence in humans may be possible.

Newly acquired herpes simplex virus keratitis after penetrating keratoplasty.

BACKGROUND: After penetrating keratoplasty for reasons unrelated to herpes simplex virus (HSV) keratitis, any nonspecific epithelial defect may still be caused by HSV. The purpose of this study is to determine the incidence of newly acquired herpetic keratitis and to assess contributing factors. METHODS: The authors retrospectively studied the results of 2398 penetrating keratoplasties performed between 1980 and 1995. Three typical case histories are discussed. RESULTS: Of 2112 patients in whom the primary diagnosis was not related to HSV keratitis, 18 presented with epithelial herpetic keratitis in their corneal graft. The incidence of newly acquired herpetic keratitis after penetrating keratoplasty was 1.2 per 1000 person-years. In most cases, the infection occurred in the first 2 years after the transplantation. Most often, well-known reactivating stimuli could have caused the HSV infection. CONCLUSIONS: Herpes simplex virus keratitis may develop after penetrating keratoplasty even without a clinical history of HSV in the host. Thus, HSV should be considered in the differential diagnosis of a postpenetrating keratoplasty epithelial defect. The high incidence of this infection in the first 2 years after such surgery suggests a causal relation between corneal transplantation and the HSV infection.

Estudio clínico virológico en un grupo de niños chilenos con herpes ocular: análisis genómico de las cepas / Clinic-virological study in chilean children with ocular herpes: genomic analysis of strains

Se estudiaron todos los niños menores de 15 años, con diagnóstico clínico de herpes ocular, efectuado por oftalmólogos de servicios oftalmológicos de la Facultad de Medicina de la Universidad de Chile y de un centro oftalmológico privado, desde abril de 1995 a abril de 1996, en Santiago de Chile. Se consignó información clínico epidemiológica, examen clínico y se obtuvieron mediante torulado muestras de conjuntiva y/o córnea del ojo afectado, conjuntiva del ojo sano y mucosa oral, utilizando una técnica estándar. Se efectuó aislamiento viral en células VERO (ATCCL81). En caso de detectarse efecto citopático, el aislado viral se propagó y almacenó para su estudio. Se efectuó identificación y tipificación viral con anticuerpos tipo específicos marcados con fluoresceína (DAKO). Se efectuó amplificación génica y análisis con enzimas de restricción. Resultados: se determinó que 14/15 aislados correspondían a herpes simplex tipo I (HSV-I) y uno a HSV-2, el cual muy probablemente correspondió a una infección perinatal por este virus. Las cepas de HSV-I presentaron patrones genómicos distintos entre sí y con la cepa de referencia norteamericana HSV-IF. La forma de presentación más frecuente fue de blefaritis herpética, seguida por la dendrita corneal, tanto en el primer espisodio clínico como en las recurrencias y en estas últimas, tiende a presentarse la misma forma clínica. La tasa de recurrencia es mayor que en la población adulta. La infección fue unilateral con la excepción de un caso con distintas formas de presentación en cada ojo y aislamiento simultáneo bilateral. En dos casos fue posible detectar excreción oral del virus subclínica