Comparative Analysis of Polymerase Chain Reaction Assay for Herpes Simplex Virus 1 Detection in Tear

PURPOSE: To comparatively analyze the methodological efficacy of the polymerase chain reaction (PCR) assay for herpes simplex virus 1 (HSV) detection in tears. METHODS: This retrospective study reviewed the medical records of 115 patients who were clinically diagnosed with herpes keratitis, and their tear samples were collected for HSV detection. PCR positive rates were analyzed for their dependence on the PCR primers used (conventional PCR primer vs. nested PCR primer), the tear collecting method used (micropipetting vs. collection with schirmer strip), the disease manifestation and the patient's previous medication history. RESULTS: HSV DNA was detected in 23 out of 115 (20%) tear samples. The PCR positive rate in tear samples did not differ depending on the PCR primer or tear collection method used. Typical epithelial lesions showed a higher positive rate (31.4%) than atypical epithelial lesions (10.9%). The previous history of the antiviral agent seemed to affect the PCR positive rate. CONCLUSIONS: Although the PCR positive rate was not dependent on the tear collection method or primers, HSV detection in tears using PCR was shown to be a supplementary diagnostic test in typical and atypical herpes epithelitis.

Detecção do vírus herpes simples por reação em cadeia da polimerase em pacientes com ceratite herpética típica ou atípica / Molecular detection of herpes simplex virus by polymerase chain reaction in patients with typical and atypical herpetic keratitis

OBJETIVO: Avaliação dos pacientes com o quadro clínico de ceratite herpética (CH) típicas e atípicas, pela reação em cadeia da polimerase (PCR) correlacionando com o diagnóstico clínico. MÉTODOS: Foi realizada a PCR em 28 pacientes com ceratite herpética típica e atípica. RESULTADOS: A PCR foi positiva em 57,14 por cento (n=16) do total (n=28). Nos casos de CH típica a positividade foi de 60,00 por cento (n=12) em 20 casos. Para CH epitelial a positividade foi de 69,23 por cento (n=9), sendo 77,78 por cento (n=7) apenas para as lesões epiteliais dendríticas. Os casos de CH atípica apresentaram positividade de 50 por cento (n=4) em oito casos. CONCLUSÃO: Quadro clínico típico de CH teve boa correlação com o resultado positivo observado na PCR. Entretanto, metade dos pacientes com o quadro de CH atípica apresentou PCR positivo, portanto, o exame do PCR é teste importante para o auxílio e diagnóstico da CH. No caso de CH estromal, foi demonstrado que a técnica da PCR conseguiu identificar o vírus HSV.

PURPOSE: To evaluate patients with clinically typical and atypical herpetic keratitis (HK) by means of polymerase chain reaction (PCR) as compared with the clinical diagnosis. METHODS: PCR in 28 patients with clinical symptoms of typical and atypical HK was performed. RESULTS: PCR was positive in 57.14 percent (n=16) of the total cases (n=28). The test was positive in 60.0 percent (n=12) of the 20 typical HK cases. For epithelial HK, the test was positive in 69.23 percent (n=9), and 77.78 percent (n=7) only for dendritic injuries. Atypical HK presented a positive test in 50 percent (n=4) of eight cases. CONCLUSION: Clinical typical picture of HK had a good correlation with the positive result of PCR, mainly for epithelial injury of the dendritic type. However, 50 percent of the patients with atypical HK presented positive PCR. This result showed that PCR test can provide an effective HK diagnosis. In the stromal case of HK, PCR was a useful technique to identify HSV virus.

An immunofluorescence test for diagnosis of ophthalmic herpes in a mouse corneal model

Herpes simplex virus type 1 (HSV-1) ophthalmic disease is the most common cause of corneal blindness in humans world-wide. Current culture techniques for HSV take several days and commercially available HSV laboratory based diagnostic techniques vary in sensitivity. Our study was conducted to evaluate the use of a quicker and simpler method to herpes ophthalmic diagnosis. Corneal smears were made by firm imprints of infected mouse eyes to glass slides, after smears were fixated with cold acetone, and an indirect immunofluorescence (IIF) method was performed using monoclonal antibodies in a murine model of ophthalmic herpes. Eye swabs from infected mice were inoculated in Vero cells for virus isolation. Cytology and histology of the eye were also performed, using hematoxylin-eosin routine. Mouse eyes were examined by slit-lamp biomicroscopy for evidence of herpetic disease at various times postinoculation. We made a comparative evaluation of sensitivity, specificity and speed of methods for laboratory detection of HSV. Our results indicate that this IIF method is quick, sensitive, specific and can be useful in the diagnosis of ophthalmic herpes as demonstrated in an animal model.

A doença oftálmica do vírus herpes simplex do tipo 1 (HSV-1) é a causa mais comum de cegueira córnea em humanos mundialmente. Técnicas de cultura atuais para HSV levam vários dias e laboratórios de HSV comercialmente disponíveis estabelecem que as técnicas diagnósticas variam em sensibilidade. Nosso estudo foi conduzido para avaliar a aplicação prática de um método mais rápido e simples para diagnosticar o herpes oftálmico. Decalques córneos foram feitos por impressões firmes de olhos de camundongos a lâminas de vidro, depois os decalques foram fixados com acetona fria, e um método de imunofluorescência indireta (IIF) foi executado empregando anticorpos monoclonais no modelo murino de herpes oftálmico. Swabs de córnea foram inoculados em células Vero para o isolamento de vírus a partir de camundongos infectados. A citologia e a histologia do olho foi feita pela rotina de hematoxilina e eosina. Os olhos de camundongos foram examinados através de oftalmomicroscopia para evidência de doença herpética em vários tempos pós-inoculação. A avaliação comparativa da sensibilidade, especificidade e velocidade de métodos para detecção laboratorial de HSV foi feita. Nossos resultados indicam que este método de IIF é rápido, sensível, específico e pode ser útil no diagnóstico de herpes oftálmico como demonstrado no modelo animal.

Herpes simplex uveitis

BACKGROUND: Uveitis in herpes simplex virus (HSV) ocular disease is usually associated with corneal stromal disease. It has generally been believed that herpetic uveitis in the absence of corneal disease is very rare. When seen it is usually attributed to varicella zoster virus (VZV) infections. The diagnosis of uveitis caused by herpes simplex is often not diagnosed resulting in inadequate treatment and a poor visual result. METHODS: Seven patients from a large uveitis practice who presented with a clinical picture of: anterior uveitis and sectoral iris atrophy without keratitis, a syndrome highly suggestive of herpetic infection, are reported. Polymerase chain reaction (PCR) was done in the aqueous of four of them and was positive for HSV. One patient had bilateral disease. Most of the patients also had severe secondary glaucoma. RESULTS: Of the seven patients presented five had no history of any previous corneal disease. Two had a history of previous dendritic keratitis which was not active at the time of uveitis development. One patient with bilateral disease was immunosuppressed at the time when the uveitis developed. Six of the seven patients had elevated intraocular pressures at the time of uveitis and five required glaucoma surgery. Intractable glaucoma developed in two patients leading to rapid and severe visual loss despite aggressive management. CONCLUSION: Findings suggest that uveitis without corneal involvement may be a more frequent manifestation of ocular herpes simplex disease than previously thought. Absence of corneal involvement delays a correct diagnosis and may worsen visual outcome. Primary herpetic uveitis (when there is no history of previous corneal disease) seems to be more severe than the uveitis in patients with previous corneal recurrences. The associated glaucoma may be a devastating complication

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.

Ceratite herpética em transplante de córnea por causas não ligadas ao Herpes / Herpetic keratitis after penetrating keratoplasty for cases not related to herpes simplex infection

Objetivo: Alertar para a possibilidade do desenvolvimento de ceratite causada pelo HSV (Herpes Simplex Virus) no pós-operatório de ceratoplastia por causas não ligadas ao herpes. Local: Instituto de Oftalmologia Tadeu Cvintal - São Paulo - SP - Brasil. Método: Relato de casos e revisão de literatura. Resultado: Foi comprovado, através de exames complementares, a presença do HSV no transplante de córnea por causas não ligadas ao herpes Conclusão: A ceratite herpética deve constar no diagnóstico diferencial de casos de epiteliopatia persistente, úlceras de aspecto linear, dendrítico ou geográfico no pós-operatório de transplante de córnea, mesmo que a causa inicial da ceratoplastia não tenha relação com o HSV.

Detección del virus herpes simple tipo I por inmunofluorescencias directa en córneas nativas y transplantadas con leucomas postherpéticos / Detection of herpes simplex virus type 1 by direct immunofluorescence in native and transplanted corneas with postherpetic leukomas

Por muchos años se ha sabido que el virus HVS-1 se perpetúa en el ganglio trigeminal, una vez ocurrida su colonización; asimismo es ampliamente conocida la facilidad con que recidivan los cuadros herpéticos corneales. Algunos trabajos previos han demostrado la persistencia de partículas virales en córnea. En el presente trabajo se incluyeron pacientes con diagnóstico clínico de leucomas corneales (postherpéticos) inactivos y se estudiaron por técnicas de inmunofluorescencia directa bajo estrictas normas de seguridad. Se encontró 90 por ciento de positividad independientemente del tiempo transcurrido desde el último cuadro activo. El 10 por ciento de negatividad nos lo explicamos por errores en el diagnóstico inicial o por lesiones corneales secundarias a HVS-2.

Secuelas oculares debidas a infección herpética en un grupo de niños chilenos / Ocular sequels caused by herpetic infection in chilean children

Estudio prospectivo, multicéntrico, en el cual investigadores entrenados seleccionaron y siguieron clínica y virológicamente (aislamiento viral, anticuerpos monoclonales y PCR) pacientes de 0 a 18 años de edad con diagnóstico clínico de infección ocular por HSV. Se efectuó determinación de la agudeza visual (AV) por el Test de Mirada Preferencial, HOTV, E Snellen u optotipos de Snellen, con la mejor corrección óptica, examen refractivo bajo cicloplegia, estudio completo de estrabismo, topografía, paquimetría y fotografía. Se enrolaron entre 1993 a 1997 16 niños, dos de ellos con compromiso bilateral (18 ojos). En todos salvo un niño, se identificó HSV tipo I. Mediana de edad 5 años. El diagnóstico de ingreso fue el edema disciforme en 45 por ciento y en 33 por ciento de los casos una queratitis epitelial dendrítica. El 87,5 por ciento de los pacientes con edema disciforme presentó al menos una recurrencia por año. Los pacientes con compromiso epiteliar presentaron en todos los casos AV > 20/40, a diferencia de los pacientes con edema disciforme en que 71,4 por ciento presentaron AV final < de 20/50. El leucoma corneal fue severo en 62,5 por ciento de los ojos con edema disciforme y desarrollaron estrabismo 50 por ciento de los casos. En tres casos se requiere trasplante corneal. La enfermedad ocular herpética afecta principalmente a población joven y sana. El diagnóstico más frecuente fue el edema disciforme que presentó evolución crónica, requirió un gran número de consultas médicas y de tratamiento prolongado (entre 3 y 6 meses) y presentaron la mayor frecuencia de secualas. En general, el 78 por ciento de los pacientes estudiados desarrolló algún tipo de secuela visual

Utilidad del examen oftalmológico en el diagnóstico del herpes neonatal / Ophthalmologic exam is useful in the diagnosis of neonatal herpes virus infection

Con el propósito de mostrar la importancia del examen oftalmológico en niños con afecciones sistémicas y manifestaciones oculares se describe el caso de un lactante de 41 días, con antecedentes de meningoencefalitis, neumopatía, microcefalia y opacidad corneal, en cuyo examen oftalmológico con biomicroscopio se comprobó una extensa úlcera de la córnea, de la cual se identificó mediante cultivo, reacción de polimerasa en cadena y anticuerpos monoclonales, un virus herpes simplex tipo 2. El examen oftalmológico completo en niños, con alteraciones o malformaciones oculares, asociados a manifestaciones sistémicas, posiblemente causadas por infección congénita a perinatal puede ser de gran utilidad para orientar el diagnóstico y el tratamiento

Enhanced detection of herpes simplex virus from ocular specimens of herpetic keratitis patients.

Corneal scrapings collected from 70 patients were used to assess the diagnostic value of indirect immunofluorescence (indirect IF) procedure in comparison with routine virus culture (RVC) for the diagnosis of Herpes simplex virus induced keratitis (HSK). Virus specific antigen was detected by indirect IF in 22 (31.42%) cases. In contrast, only 20% (14) of the cases had positive viral isolation which sometimes took as long as a week to show a cytopathogenic effect (CPE). It is concluded that antigen detection by indirect IF is a rapid, specific and sensitive technique for demonstrating HSV-1 antigen in corneal scrapings from HSK patients and a useful laboratory tool not only for diagnosing HSK but also for monitoring efficiency of anti HSV treatment for HSK.

Queratitis herpética: correlación clínico-virológica / Herpetic keratitis: clinic-virological correlation

Patients and methods: Forty one patients with a clinical diagnosis of herpetic keratitis were studied. Viral isolation, polymerase chain reaction (PCR) and typification were done in a sample taken by swabbing the ocular lesion. Results: Twenty six patients (31 percent female) had epithelial keratitis, that was mild or moderate in 88 percent of cases and acute in 77 percent of them. In 20 patients (77 percent), viral isolation and PCR were positive (HSV-2 in one case). Fiften patients (67 percent female) had stromal keratitis, 93 percent of cases were moderate or severe and 53 percent were acute. Viral isolation was negative in all cases and in 20 percent PCR was positive. Conclusions: Viral isolation and PCR were equally sensitive in epithelial keratitis, but in stromal keratitis only PCR could detect the virus. Moderate acute dendrite was the predominant clinical manifestation. The higher proportion of women with stromal keratitis supports is possibly autoimmune etiology). HSV-2 is seldomly isolated and possibly associated to vertical transmission

Investigations in the virological etiology in acute retinal inflammation.

Virological investigations for herpes simplex virus (HSV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) were performed in nine patients of acute retinal inflammation. Serum samples of all the patients were assayed for IgG and IgM antibodies to HSV, CMV and EBV. Vitreous fluid (VF) from 5 patients was tested by immunofluorescence and culture for detection of HSV and CMV. In four patients, VF was also assayed for IgG and IgM antibodies to HSV and CMV. HSV was shown to be the etiological agent in 3 patients with acute retinal necrosis (ARN) syndrome and in one patient with multifocal retinitis. CMV was the causative agent in one patient of ARN and 1 patient with clinically diagnosed CMV retinitis. Evidences of infection with these three viral agents could not be obtained in one patient with clinical diagnosis of CMV retinitis who tested positive for antibody to human immunodeficiency virus 1. All other patients were HIV negative. Identification of the causative viral agent of acute retinal inflammations may help an ophthalmologist to institute specific therapy particularly for HSV and CMV infections.

Síndrome de vasculite retiniana hemorrágica multifocal aguda; relato de um caso / Acute multifocal hemorrhagic retinal vasculitis syndrome; report of one case

Os autores apresentam um caso de paciente com a síndrome de vasculite retiniana hemorrágica multifocal aguda, descrita por Blumenkranz e colaboradores, cuja etiologia permanece desconhecida. No caso apresentado o paciente foi tratado com a prednisona oral com alguns benefícios, com o acyclovir, sem benefício e com a fotocoagulaçäo com laser de argônio nas áreas isquêmicas

Emergencias en oftalmología: cuándo derivar al especialista? / Emergency in oftalmology: when overthrow to the specialist?

En este artículo se comentarán los cuadros de mayor frecuencia que afectan al ojo, y que pueden ser abordados directamente por el pediatra. Se analizarán tambien los signos de riesgo que permiten determinar el momento en que el paciente debe referirse, en forma mas o menos urgente, al oftalmólogo

Ojo rojo, una afección grave? / Red eye, a dangerous disease?

Al enfrentar este problema, el médico debe considerar como diagnósticos diferencial la posibilidad de conjuntivitis, iritis, glaucoma agudo, úlcera corneana, queratitis herpética o traumatismo de la cornea. En este artículo se pasa revista a las principales claves diagnósticas y a las bases de la terapéutica de cada una de estas enfermedades.