Resumo O vírus herpes simples (HSV) é um dos agentes causadores de uma doença grave no sistema nervoso central (SNC) em humanos. A detecção de anticorpos específicos no líquido cefalorraquidiano (LCR) pode contribuir para o diagnóstico neurológico retrospectivo. Entretanto, os testes imunológicos comerciais são para uso em amostras de soro. Objetivo: Adaptar um kit comercial sorológico anti-HSV IgG para ser utilizado no de LCR. Metodos: Quarenta amostras de LCR de 38 pacientes com suspeita de infecção por HSV no SNC foram diluídas pesquisa de anticorpos anti-HSV IgG pelo método imunoenzimático (EIA). Além disso, as mesmas amostras também foram analisadas por reação em cadeia da polimerase (PCR). Resultados: A sensibilidade do teste EIA para o HSV consistiu em 5% (diluição 1:40) e 65% (diluição 1:2) no LCR, e o PCR do DNA do HSV, 15%. A análise combinada de EIA (diluição 1:2) e PCR aumentou a sensibilidade para 72,5%. Houve associação entre presença do LCR inflamatório e PCR positiva para HSV. Conclusões: Demonstramos a importância na adaptação previa do teste sorológico anti-HSV IgG EIA para ensaios do no LCR, a fim de aumentar a acuracia da análise, considerando a baixa concentração de anticorpos específicos no LCR.

Abstract Herpes simplex virus (HSV) is a cause of a severe disease of the central nervous system (CNS) in humans. The demonstration of specific antibodies in the cerebrospinal fluid (CSF) may contribute to the retrospective neurological diagnosis. However, the commercial immunological tests for HSV infection are for use in serum samples. Objective: The aim of the present study was to adapt a commercial kit anti-HSV IgG used for serum samples to be performed with a CSF sample. Methods: Forty CSF specimens from 38 patients with suspected CNS HSV infection were serially diluted for detecting anti-HSV IgG by enzyme immunoassay (EIA). The same samples were also analyzed with the polymerase chain reaction (PCR). Results: The sensitivity of EIA test for HSV was 5% (dilution 1:40) and 65% (dilution 1:2) in CSF, and HSV DNA PCR was 15%. The combined analysis of EIA (dilution 1:2) and PCR increased the sensitivity up to 72.5%. The inflammatory CSF was associated with positive HSV PCR. Conclusions: We demonstrated the importance to adapt serological anti-HSV IgG EIA test for CSF assays to increase the accuracy of the analysis, considering the low concentration of specific antibodies in CSF.

ABSTRACT Pemphigus foliaceus (PF) is an autoimmune disease characterized by blistering of the skin. Infections caused by members of the herpesviridae family have been suggested as a possible triggering factor for pemphigus vulgaris (PV), but not for PF. The present study aimed to investigate the presence of Human herpesvirus (types 1, 2, 3) in corticosteroid refractory skin lesions from a patient with PF, by a Polymerase chain reaction (PCR) assay. The sample collected from cutaneous blisters has tested positive for herpes simplex virus type 1 (HSV1) after sequence analysis of the amplified viral genomic segment. The study concluded that when PF patients present corticosteroid or immunosuppressants refractory lesions, herpetic infection should be considered.

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 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.

INTRODUÇÃO: O vírus herpes simples (HSV) é dividido em dois sorotipos (HSV-1 e HSV-2) responsáveis, respectivamente, pelos herpes labial e genital. Embora a infecção pelo HSV tenha um curso rápido, esse agente está freqüentemente relacionado a complicações no tratamento de pacientes imunocomprometidos, como indivíduos transplantados, na condição de agente oportunista. OBJETIVOS: Comparar e avaliar o uso de três técnicas atuais para diagnóstico de HSV em pacientes transplantados e não-transplantados. MATERIAL E MÉTODOS: Oitenta e quatro amostras clínicas consecutivas provenientes de 47 indivíduos transplantados e 37 não-transplantados foram coletadas de junho de 2001 a julho de 2002, sendo, simultaneamente, submetidas a nested multiplex reação em cadeia da polimerase (PCR) (nmPCR), multiplex PCR (mPCR) e isolamento viral (IV) em células vero. RESULTADOS: Das amostras, 33,3% (28/84) foram positivas para o HSV por IV, 34,5%(29/84) por mPCR e 42,8% (36/84) por nmPCR. Pela técnica de imunofluorescência direta (IFD), 85,7% (24/28) das amostras foram caracterizadas como HSV-1, 86,2% (25/29) pelo mPCR e 88,9%(32/36) pelo nmPCR. Foram caracterizadas como HSV-2 pelas três técnicas empregadas 4,8%(4/84) das amostras. Não houve diferença significante de detecção entre as técnicas de diagnóstico do HSV (p = 0,38), embora o nmPCR tenha detectado mais amostras de pacientes transplantados (p = 0,05). CONCLUSÃO: Apesar do desempenho similar entre as técnicas, o nmPCR se mostrou ferramenta útil para pacientes transplantados ou para aqueles sob tratamento antiviral, onde é esperada baixa carga viral em suas amostras.

BACKGROUND: Herpes simplex virus (HSV) is divided in two serotypes (HSV-1 and HSV-2) responsible for labial end genital herpes, respectively. Although the infection caused by HSV has a rapid course, this agent is frequently related to complications in immunocompromised patient’s treatment, like transplanted individuals as an opportunistic agent. OBJECTIVES: To compare and evaluate three current diagnosis techniques for HSV diagnosis in transplanted and non-transplanted patients. Material and methods: 84 consecutive clinical samples from 47 transplanted and 37 non-transplanted individuals were collected from June 2001 to July 2002, being simultaneously submitted to nested multiplex PCR (nmPCR), multiplex PCR (mPCR) and viral isolation (VI) in Vero cells. RESULTS: 33.3%(28/84) samples were HSV-positive by VI, 35.4%(29/84) by mPCR and 42.8%(36/84) by nmPCR. 85.7% (24/28) samples were characterized as HSV-1 by the direct immunofluorescence technique (dIF), 86.2%(25/29) by mPCR and 88.9%(32/36) by nmPCR. 4.8%(4/84) samples were characterized as HSV-2 by the three techniques. There was no significant difference regarding HSV diagnosis among the techniques (p = 0.38), although nmPCR detected more samples from transplanted patients (p = 0.05). CONCLUSION: although the three techniques presented similar performances, the nmPCR revealed to be an useful tool for transplanted patients or those under antiviral treatment, where a low viral load in their samples is expected.

The reactivation of Herpes Simplex virus (HSV) occurs in 70% to 80% of patients submitted to autologous stem cell transplantation (ASCT); it increases the severity of chemotherapy-induced mucositis. Therefore, the use of acyclovir in ASCT patients is considered standard practice. However, the minimum dose needed to prevent reactivation is a matter of debate. We compared two doses of acyclovir in a non-randomized fashion in 59 patients submitted to ASCT: 32 patients received a dose of 125 mg/m² IV every six hours and the subsequent 27 patients received a dose of 60 mg/m² IV every six hours. Viral excretion was evaluated through weekly viral culture of oral swabs. Grade 4 mucositis was more frequent in Group 1 (p= 0.03). The reactivation rates in Groups 1 and 2 were 9% and 4%, respectively (p= 0.62, 95% confidence interval -7 - 18). Prophylaxis with reduced doses of intravenous acyclovir seems to be as effective as a higher dose in inhibiting HSV reactivation, with a significant reduction in cost. Prospective randomized studies are needed to confirm our conclusions.

Two different procedures for inoculation of HSV on corneas of BALB/c mice were evaluated. The first was by the use of HSV suspensions directly on the corneas and the other was after corneal scarification. Animals by this later method presented greater morbidity and mortality than those of first group, suggesting that inoculation of HSV without scarification of the cornea should be the method of choice for the study of HSV ophthalmic infection. This model showed also be an efficient experimental system to testing antiviral drugs.

This paper reports the first isolation of a spotted fever group rickettsia from an Amblyomma cooperi ixodid collected from a capybara (Hydrochaeris hydrochaeris) in an endemic area of spotted fever in the County of Pedreira, State of São Paulo, Brazil. Isolation was performed in Vero cell culture and submitted to immunofluorescence, using antibody from Rickettsia rickettsii-positive human serum.