Búsqueda de flavivirus en individuos con sospecha clínica de dengue y resultado negativo para el antígeno NS1 en Paraguay / Search of flavivirus in individuals with clinical suspicion of dengue and negative for the NS1 antigen in Paraguay

Los flavivirus son responsables de una considerable morbi-mortalidad a nivel mundial. Entre ellos, el virus del dengue (DENV) es causante de graves problemas de salud pública en Paraguay. El objetivo del estudio fue detectar infecciones por flavivirus a través de una reacción de RT-nested PCR genérica para flavivirus en 195 muestras de individuos con sospecha de dengue, negativos por el test inmunocromatográfico (antígeno NS1 ­ DENV), provenientes del área metropolitana de Asunción entre 2011 y 2013. Las muestras positivas para flavivirus fueron sometidas a dos reacciones de RT-nested PCRs específicas para DENV. El límite de detección (LD) para flavivirus fue de 0,2 UFP/reacción. En total 43/195 muestras fueron positivas para flavivirus. De estas, 38/43 (88,4%) correspondieron a DENV (6 DENV-1, 30 DENV-2 y 2 DENV-3). Además, 5/43 casos (11,6%) positivos para flavivirus fueron negativos para DENV por ambas reacciones específicas, pudiendo deberse a infecciones por otros flavivirus. Los resultados sugieren que la utilización de una reacción genérica seguida de otras reacciones específicas para DENV en casos febriles negativos para NS1 por el método inmunocromatográfico permitiría detectar más casos de infecciones por DENV y además, podría contribuir a la identificación de casos debido a infecciones por otros flavivirus.

Flaviviruses are responsible for considerable worldwide morbidity and mortality. Among them, the dengue virus (DENV) causes serious public health problems in Paraguay. The objective of the study was to detect flavivirus infections using a generic RT-nested -PCR in 195 samples of individuals with suspected dengue and negative for the inmunochromatographic test (NS1 antigen ­ DENV), from the metropolitan area of Asuncion between 2011 and 2013. The flavivirus-positive samples were subjected to two reactions of DENV-specific RT-nested PCRs. The detection limit (DL) for flavivirus was 0.2 PFU / reaction. In total, 43/195 samples were positive for flavivirus. Of them, 38/43 (88,4%) corresponded to DENV (6 DENV-1, 30 DENV-2 and 2 DENV-3). In addition, 5/43 cases (11.6%) positive for flavivirus were negative for DENV by both specific reactions, and may be infections caused by other flaviviruses. The results suggest that the use of a generic reaction followed by other DENV specific reactions in febrile negative cases for NS1 by the immunochromatographic method would allow the detection of more cases of DENV infections and could contribute to the identification of cases due to infections by others flaviviruses.

Molecular characterisation of the NSP4 gene of group A human rotavirus G2P[4] strains circulating in São Paulo, Brazil, from 1994 and 2006 to 2010

Group A human rotaviruses (HuRVA) are causative agents of acute gastroenteritis. Six viral structural proteins (VPs) and six nonstructural proteins (NSPs) are produced in RV-infected cells. NSP4 is a diarrhoea-inducing viral enterotoxin and NSP4 gene analysis revealed at least 15 (E1-E15) genotypes. This study analysed the NSP4 genetic diversity of HuRVA G2P[4] strains collected in the state of São Paulo (SP) from 1994 and 2006-2010 using reverse transcription-polymerase chain reaction, sequencing and phylogenetic analysis. Forty (97.6%) G2P[4] strains displayed genotype E2; one strain (2.4%) displayed genotype E1. These results are consistent with the proposed linkage between VP4/VP7 (G2P[4]) and the NSP4 (E2) genotype of HuRVA. NSP4 phylogenetic analysis showed distinct clusters, with grouping of most strains by their genotype and collection year, and most strains from SP were clustered together with strains from other Brazilian states. A deduced amino acid sequence alignment for E2 showed many variations in the C-terminal region, including the VP4-binding domain. Considering the ability of NSP4 to generate host immunity, monitoring NSP4 variations, along with those in the VP4 or VP7 protein, is important for evaluating the circulation and pathogenesis of RV. Finally, the presence of one G2P[4]E1 strain reinforces the idea that new genotype combinations emerge through reassortment and independent segregation.

Desenvolvimento de teste diagnóstico para a triagem sorológica de diversas infecções virais / Development of diagnostics for serological screening of various viral infections

A leishmaniose visceral canina (LVC) e uma doença parasitária causada por protozoários do gênero Leishmania, principalmente por Leishmania infantum. A epidemiologia da doença varia de região para região e o entendimento dos fatores associados à infecção em cães pode ajudar na elaboração de medidas de controle mais específicas. O diagnóstico sorológico da infecção sofreu mudanças importantes nos últimos anos com a introdução do TR-DPP® e do estabelecimento de novos critérios de diagnóstico (TR-DPP® + EIE-LVC) pelo Ministério da Saúde. Dentro desse contexto, no presente estudo objetivou-se estudar a epidemiologia da LVC no município de Goiana, estado de Pernambuco, nordeste do Brasil. Para tal, realizaram-se testes sorológicos (TR-DPP® e EIE-LVC) e análise clínico-epidemiológica em 360 cães semi e domiciliados, de ambos os sexos, raças e idades variadas, nos distritos de Atapuz, Tejucupapo e Pontas de Pedra no referido município. No TR-DPP®47 (13,1 por cento) animais foram reagentes, onde se observou associação significativa dos resultados com os seguintes sinais clínicos: alopecia, lesões na pele paresia e linfonodomegalia. Já no EIE-LVC 21 (5,8 por cento) animais foram reagentes, havendo associação significativa entre a classificação clínica dos animais, condição corporal, alopecia, lesões na pele, secreção ocular, paresia e linfonodomegalia. Já de acordo com o critério do Ministério da Saúde do Brasil, apenas 15 (4,2 por cento) animais foram classificados como positivos. De fato, verificou-se uma fraca concordância (Kappa = 0,39) entre os dois testes sorológicos. Conclui-se que a LVC encontra-se estabelecida em Goiana e que o uso do TR-DPP® como teste de triagem e do EIE-LVC como teste confirmatório pode levar a perda de cães infectados, uma vez que cães positivos do TR-DPP® são negativos no EIE-LVC e vice-versa.

Prevalência de anticorpos contra agentes virais e bacterianos em eqüídeos do município de Monte Negro, Rondônia, Amazônia ocidental brasileira / Seroprevalence of viral and bacterial agents in equids from Monte Negro municipality, State of Rondônia. brazilian western Amazon

Foram examinados 176 eqüídeos (15 muares e 161 eqüinos) do município de Monte Negro, Rondônia, Amazônia Ocidental Brasileira, frente a agentes virais e bacterianos. A amostra correspondeu ao total de eqüídeos no município, considerando um nível de confiança de 99%, prevalência esperada de 50% e erro padrão de 10%. As infecções virais foram investigadas pelas provas de Imunodifusão em gel de Agar (Anemia Infecciosa Eqüina - AIE), Inibição da hemaglutinação (Influenza eqüina tipos 1 e 2 - IE-1 e 2) e Soroneutralização em cultura celular (Arterite Viral Eqüina - AVE, Herpesvírus Eqüino tipo 1 - HVE1, Estomatite Vesicular - EV e Encefalomielite Eqüina do Leste - EEE, do Oeste - WEE e Venezuela - VEE). Para o diagnóstico da leptospirose, foi utilizada a prova de Soroaglutinação Microscópica (SAM); para o diagnóstico da brucelose, o teste do Antígeno Acidificado Tamponado (AAT) foi utilizado como teste de triagem e as provas de Soroaglutinação Lenta em Tubos (SLT) e 2- mercaptoetanol como testes diagnósticos. Foram constatados 9,6% dos eqüídeos reativos para AIE, 22,7% para HVE1, 19,9% para IE- 1, 42,0% para IE-2, 21,0% para EEE, 11,3% para VEE, 3,4% para Brucella spp. e 91,4% para Leptospira spp. Os sorovares de leptospira mais freqüentes foram Bratislava (10,5%), Icterohaemorrhagiae (8,7%) e Autumnalis (8,7%) nos eqüinos e Patoc (26,6%) nos muares. Não foram encontrados animais com anticorpos contra AVE, EV e WEE.

Sera from 174 equidaes (15 mules and 161 equines) of Monte Negro municipality, Rondônia State were analyzed against viral and bacterial agents. The serum sample corresponded the total equid population in the municipality considering a confidence interval of 99%, expected prevalence of 50% and absolute desired of 10%. For the viral agents, sera were tested by the Agar Gel Immunodiffusion Test (Equine Infection Anemia - EIA), Inhibition Haemagglutination Test (Equine Influenza 1 and 2 - EI - 1 and 2), and Virusneutralizating Tests (Equine Viral Arteritis - EVA, Equine Herpesvirus 1 - EHV1, Vesicular Stomatitis - VS, Equine Encephalitis Eastern - EEE, Western - WEE and Venezuelan VEE). The diagnosis for brucellosis was made by Agglutination Tests and the Microscopic Agglutination Test was used for leptospirosis. The results showed positivity of 9.6% for EIA, 22.7% for HVE1, 19.9% for IE-1, 42.0% for IE-2, 21.0% for EEE, 11.3% for VEE, 3.4% for brucellosis, and 91.4% for leptospirosis. The most frequent serovars detected were Bratislava (10.5%), Icterohaemorrhagiae (8.7%), Autumnalis (8.7%) for equines and Patoc (26.6%) for mules. No one of the examined samples reacted to EVA, VS, or WEE.

Caracterização de anticorpos monoclonais contra rotavírus bovino e suas aplicações como ferramenta de diagnóstico / Characterization of monoclonal antibodies against bovine rotavirus and their diagnostic applications

Anticorpos monoclonais (AcM) para rotavírus bovino foram caracterizados para sua aplicação como ferramenta de diagnóstico, utilizando-se as técnicas de isotipificação, dot-blot, western-blot, imunofluorescência indireta (IFI) e ELISA de captura. A caracterização imunoquímica demonstrou que os cinco AcM 1G5, 4F7, 1E12, 4F3 e 3C12 foram do isótipo IgG2a. Pela técnica de dot-blot, os AcM 1G5, 4F7, 1E12, 4F3 detectaram antígenos do rotavírus, em diferentes concentrações, e dois AcM (1E12 e 4F3) reconheceram proteínas virais pela técnica de western-blot. Todos os AcM reagiram positivamente na técnica de IFI em cultivo celular e foram capazes de detectar antígeno viral em amostras fecais bovinas e humanas, pela técnica de ELISA de captura. Identificaram-se dois grupos de AcM, um deles formado pelos AcM 4F7, 1E12 e 1G5, para seu possível uso na detecção de antígeno viral em fezes por meio do ELISA de captura ou dot-blot e outro pelos 4F3 e 3C12, que podem ser usados para detectar antígeno viral em culturas de células por meio de IFI.

This work was carried out to characterize and evaluate five bovine rotavirus, monoclonal antibodies (MAbs), as a diagnosis tool, by isotyping, dot-blot, western-blot, indirect immunofluorescence (IFI) and ELISA techniques. The immunochemistry characterization showed that all five MAbs (4F7, 4F3, 1G5, 1E12 and 3C12) were IgG2a isotype. The dot-blot immunoassay showed that 1G5, 4F7, 1E12 and 4F3 detected viral antigen in different concentrations and two MAbs (1E12 and 4F3) recognized viral proteins by western-blot. All MAbs detected viral antigen in bovine and human fecal samples by capture ELISA technique and viral antigen in infected MA-104 cell culture by IFI. In conclusion, two groups of Mabs were indetified: one with Mabs 4F7, 1E12 and 1G5 showed the best results to detect rotavirus antigen in fecal samples by capture ELISA or dot-blot techniques assay and other with 4F3 and 3C12 which may be used to detect rotavirus antigens in cell culture by IFI. The results showed the potential use of these MAbs as diagnosis tools in diarrheas by rotavirus in bovines.

First time detection of Japanese encephalitis virus antigen in dry and unpreserved mosquito Culex tritaeniorhynchus Giles, 1901, from Karnal district of Haryana state of India.

Japanese encephalitis virus (JEV) antigen has been detected by antigen capture enzyme linked immunosorbentassay (ELISA) in dry specimens of the mosquito Culex tritaeniorhynchus Giles, 1901, collected from Karnal district of Haryana state in northern India. These mosquitoes were stored in dry condition for 20 months, at room temperature, before processing. The procedure of detecting JEV infection in long time stored, dry vector mosquitoes, has important application in the surveillance of Japanese encephalitis.

Detection of human parvovirus B19 in a patient with hepatitis

Parvovirus B19 has been associated by some investigators with cases of severe hepatitis. The aim of the present study was to determine the presence of active parvovirus B19 infection among 129 Brazilian patients with non-A-E hepatitis. The patients were assayed for antibodies against parvovirus B19, IgM class, by ELISA. In IgM-positive cases, parvovirus B19 DNA was assayed by PCR in serum and liver tissue and parvovirus VP1 antigen in liver tissue was assayed by immunohistochemistry. Antibodies against parvovirus B19, IgM class, were detected in 3 (2.3 percent) of 129 patients with non-A-E hepatitis. Previous surgery and blood transfusions were reported by these 3 patients. One patient was a 56-year-old female with severe hepatitis, with antimitochondrial antibody seropositivity and submassive necrosis at liver biopsy, who responded to corticosteroid therapy. Strong evidence for active parvovirus B19 infection was found in this patient, with parvovirus B19 DNA being detected by PCR in liver tissue. Furthermore, parvovirus VP1 antigen was also detected in liver tissue by immunohistochemistry. The other two IgM-positive patients were chronic hepatitis cases, but active infection was not proven, since neither viral DNA nor antigen were detected in their liver tissues. This and other reports suggest a possible relation between parvovirus B19 infection and some cases of hepatitis

Rapid diagnosis of cholera by coagglutination test.

In this study the coagglutination test for the rapid diagnosis of cholera is evaluated in comparison with the conventional culture method. A total of 553 stool specimens were processed from cases of acute gastro-enteritis. The sensitivity and specificity of coagglutination test was 92.77% and 95.65% respectively. The coagglutination test is found to be simple, reliable and rapid method for the diagnosis of cholera.

Rol del virus herpes simplex en la queratitis inmune estromal / Role of herpes simplex virus in the immune stromal keratitis

Background: Herpes simplex virus (HSV) infection of the cornea is a leading cause of blindness in occidental countries and a common recurrent manifestation of it is the immune stromal keratitis (ISK). However, it is not known whether active viral replication occurs during the acute phase of the disease, because isolation of the virus by conventional culture techniques has not been accomplished. Aim: To establish the presence of HSV in patients with ISK. Material and methods : Fourteen corneal swabbing samples, from active diseased eyes of patients with clinical diagnosis of ISK, were submitted to Herpchek© and PCR for the identification of HSV antigens and genome. Results: All ISK samples were negative by both techniques. Conclusions : It was not possible to identify HSV antigens nor their genome by the methodology used. It is likely that, they can't be detected in corneal superficial layers or probably there is no viral replication at this stage of the disease, so antiviral therapy should be reconsidered

Tissue distribution of bovine viral diarrhea virus antigens in persistently infected cattle

The tissue distribution and cellular localization of viral antigens in three cattle with persistent bovine viral diarrhea virus (BVDV) infection was studied. In three cases, necropsy findings of oral ulcers, abmasal ulcers and necrosis of Peyer's patches were suspected have been caused by BVDV infection. Non-cytopathic BVDV was isolated from a tissue pool of liver, kidneys and spleen. Immunohistochemical detection of BVDV showed that BVDV antigens were detected in both epithelial and nonepithelial cells in all examined organs, including the gastrointestinal tract, liver, pancreas, lung, lymphatic organs (spleen, lymph nodes), adrenal gland, ovary, uterus, and the mammary gland. These findings support the hypothesis that animals with persistent BVDV infection spread BVDV through all routes, and that infertility in BVDV infection is associated with the infection of BVDV in the ovaries and uteri.

incorporación de la técnica de RT-PCR para la detección del virus sincitial respiratorio / Incorporation of the RT-PCR technique to the detection of respiratory syncitial virus

Objetivo: Estandarizar e incorporar técnicas de biología molecular, RT-PCR para la detección del virus sincitial respiratorio que complementen y enriquezcan el diagnóstico.Material y métodos: se utilizaron cepas de referencia del virus sincitial respiratorio de los grupos A y B (virus stock ATCC), las cuales se propagaron y titularon en células HEp-2. Se realizaron pruebas de inmunofluorescencia indirecta y de la prueba RT-PCR. Para ello se extrajo el ARN del virus con trizol, se realizó una transcripción reversa para obtener ADNc y, para la PCR se utilizaron oligonucleótidos que amplifican un fragmento del gen de la proteína G del virus sincitial respiratorio. Para comprobar la especificidad de la prueba se utilizaron virus de la misma familia (sarampión y parainfluenza) y virus de diferentes familias (influenza y adenovirus). Al evaluar la sensibilidad se utilizaron diferentes diluciones del ADNc viral.Resultados: en el cultivo, el efecto citopático se puede observar claramente del cuarto al octavo día. La prueba de inmunofluorescencia como se sabe es una técnica sensible y específica para el virus. La RT-PCR que se desarrollo, fue efectiva para la amplificación del ADNc; además, mostró ser específica para el virus sincitial respiratorio, ya que no amplifica material genético de otros virus incluyendo a aquellos que pertenecen a la misma familia. La sensibilidad de la prueba es alta, alcanzando a amplificar hasta picogramos de ADNc.Conclusiones: la rapidez de la inmunofluorescencia, permite dar un diagnóstico presuntivo que después puede ser comprobado por el aislamiento y propagación del virus en cultivo celular. La técnica de RT-PCR con los oligonucleótidos que utilizamos fue sensible, específica y rápida, por tanto debe ser tomada en cuenta para el apoyo al diagnóstico clínico. Con lo anterior, no se trata de sustituir las técnicas tradicionales, sino contar con mayores opciones y además reforzarlas; de esta manera, se podrá fundamentar mejor el diagnóstico de las infecciones virales.

Search for virulence-associated antigens of rhodococcus equi in strains isolated from patients with Acquired immunodeficiency Syndrome

Rhodococcus equi (formerly Corynebacterium equi) are known to be highly virulent, intermediate in virulence, or avirulent correlated with specific virulence-associated antigens identified immunochemically by different molecular weights. The association of virulence antigens with infection of AIDS patients by this organism has not been sufficiently evaluated in Brazil or Italy. The objective of the present study was to search for virulence-associated antigens of 15-to 17kD and 20-kD in Rhodococcus equi strains isolated from patients with rhodococcal infection and AIDS. Four Brazilian and 9 Italian strains were studied. All isolates were analyzed by gel electrophoresis followed by immunoblotting using specific monoclonal antibodies to identify virulence-associated antigens. The results obtained on gel electrophoresis analyses showed complexing of R. equi components with proteins of molecular weights ranging from 10-to 150-kD. By immunoblotting, a wide diversity in R. equi virulence-associated antigens was detected: 1 of the 4 Brazilian isolates and 2 Italian isolates had the 15-to 17-kD virulence-associated antigen, 3 Brazilian isolates and 1 Italian isolate had the 20-kD virulence-associated antigen, and the other Italian isolates had no virulence-associated antigens. These results indicate that the pathogenicity of R. equi trains for humans does not depend only on the presence of these well established virulence-associated antigens.

Western blot detection of infectious bursal disease virus infection

In order to evaluate the use of a Western blot methodology for the diagnosis of infectious bursal disease virus (IBDV) infection, chickens were experimentally infected with IBDV strains and tested for the presence of viral antigens and antibodies by a blocking Western blot test (bWB). The viral proteins obtained from the bursa of Fabricius (BF) were transferred to a nitrocellulose membrane after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the chicken sera obtained by heart puncture were used for the detection of these proteins. In order to eliminate nonspecific reactions, we used a rabbit anti-chicken serum (blocking tool). By the use of the bWB test, two distinct viral proteins of 43-kDa (VP2) and 32-kDa (VP3) were detected. We suggest the use of this methodology for the detection of IBDV infection in animals suspected of having IBDV reinfection and a chronic subclinical form of the disease. With the use of the rabbit anti-chicken sera for blocking, this method is practical, sensitive and less time consuming.

Correlación entre la presencia de anticuerpos y antígeno de citomegalovirus en los leucocitos de sangre para el diagnóstico de la infección activa primaria / Cytomegalovirus infection diagnosis measuring antibodies against the virus or the antigen presence in peripheral leukocytes

Background: The diagnosis of cytomegalovirus infections measuring IgG or IgM antibodies has a high rate of false positive or negative results, specially in immunocompromised patients. Aim: To compare the diagnostic yield of antibodies against cytomegalovirus with the measurement of the antigen in peripheral leukocytes. Material and methods: Forty three blood samples coming from pediatric patients with suspected cytomegalovirus infections were analyzed. Low affinity IgG and IgM antibodies against Epstein Barr virus and cytomegalovirus, using indirect ELISA assays, and the virus antigen in peripheral leukocytes, using a commercial immunoperoxidase assay, were measured. Results: Seven patients had positive IgM antibodies against cytomegalovirus. In five of these the viral antigen was detected in peripheral leukocytes. Twenty patients had positive antibodies against Epstein Barr virus, and in 16 patients all serologic tests were negative. Conclusions: There is not a good correlation between antibodies against cytomegalovirus and the detection of its antigen in patients with acute infections

Isolation and characterization of soluble antigens of sheep poxvirus.

A soluble antigen fraction of sheep poxvirus (SPV) isolated from infectious virus particles by ultracentrifugation and purified by subtractive immunoaffinity chromatography was characterized. Exclusion chromatography studies revealed 10 proteins of molecular weight (MW) 220, 168, 87.3, 71.5, 52.5, 36.7, 31.0, 23.4, 18.3 and 14.2 kDa. Nine of them were found to be precipitinogens and 5 were identified as structural components of the virus particles. SDS-PAGE analysis revealed a polypeptide profile of 10 bands with 2 prominent polypeptides of 64 and 42 kDa. Western blotting, however, detected 2 immunogenic polypeptides of MW 100 and 64 kDa. Moreover, crossed immunoelectrophoresis showed the presence of proteins of varied electrophoretic mobility and sharing of antigenic determinants among a few soluble antigens. Physico-chemical characterization further revealed that these precipitinogens can withstand ambient temperatures, but were sensitive to trypsin and ether whereas, chloroform had no effect on immunoprecipitation pattern of soluble antigens.

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

Detección del antígeno viral de la rabia por el método avidina - biotina - peroxidasa / Detection of the rabies viral antigen by the avidin - biotin - peroxidase method

Antecedentes: La rabia humana y animal es frecuente en Colombia. Su diagnóstico requiere inmnofluorescencia directa (IFD) e inoculación intracerebral al ratón (IIC), condiciones que alcanzan pocos laboratorios. La histopatología muestra cuerpos de Negri entre el 40 por ciento al 87 por ciento de los casos. Nuestro objetivo es desarrollar y determinar la utilidad del método avidina-biotina-peroxidasa (ABP) en tejido fijado en formol e incluido en parafina. Materiales y métodos: Se estandarizó la técnica ABP usando muestras de encéfalo con rabia confirmada por IFD, IIC al ratón o histopatología. La técnica se pudo a prueba estudiando cortes de 100 encéfalos humanos o de animales sospechosos de tener rabia. Se escogieron encéfalos incluídos en parafina o mantenidos en congelación durante años, SNC fresco de ratón inoculado con virus fijo y cerebros normales o con otras encefalitis virales. Se determinó la sensibilidad, la especificidad y la reproducibilidad del método. Resultados: La técnica de ABP muestra sensibilidad mayor o igual a .80 y especificidad mayor o igual a .97. La concordancia con respecto a la IIC y a la IFD dio resultados entre substanciales y casi perfectos, según la escala de Landys y Koch, niveles altos que también se obtienen al evaluar la reproducibilidad intraobservador e interobservador. La técnica fue siempre mejor que la HE. Conclusiones: La técnica es útil, confiable, reproducible, fácil de realizar y funciona bien inclusive en encéfalos con pobre preservación tisular. Merece incluirse entre los procedimientos de rutina o alternativos en el dignóstico de la rabia humana y animal

Neural spread of Junin virus in intraperitoneally inoculated rats

Una vez establecida la ruta neural como la seguida por el virus Junín (VJ) a partir de su inoculación intradérmica en ratas lactantes, resultaba de interes determinar cuál era la vía adoptada luego de inoculado intraperitonealmente. Desde la 2da semana se evidenció una enfermedad neurológica que a los 30 días post-infección alcanzó un 84 por ciento de mortalidad. En curso de ese período, se efectuaron cosechas de tejidos extraneurales y neurales para la marcación por inmunoperoxidasa del antígenos viral y el examen histopatológico, asi como la titulación de infectividad que también se extendió a sangre. En todas las muestras de tejido en que se detectó virus infectivo, sea por cocultivo o por aislamiento convencional, se logró la marcación del antígeno viral. El VJ estuvo presente en valores mínimos en bazo e hígado desde el día 2 al 5, y en sangre del 5 al 15. En tejidos neurales, el antígeno viral fue inicialmente revelado al día 5, tanto en ganglios raquídeos torácicos como en los segmentos medulares relacionados. A partir del día 7, la positividad se extendió a la médula espinal en toda su extensión; a la vez, ya había evidencias de presencia viral en tronco cerebral, con disfusión al resto de estructuras encefálicas desde el día 10. Pese a la presencia masiva del antígeno viral en neuromas, dichas células no mostraban cambios morfológicos aparentes. Dado que la infección de ganglios raquídeos y de médula espinal invariablemente precedió al acesso viral a encéfalo, y ello ocurrió en forma concomitante a la desaparición del virus en órganos linfo-reticulares y en sangre, la vía neural parece ser la adoptada por el VJ desde cavidad peritoneal hasta sistema nervioso central