The importance of direct immunofluorescence in pemphigus herpetiformis diagnosis

Abstract Pemphigus herpetiformis is an autoimmune bullous disease, that combines clinical features of dermatitis herpetiformis and linear IgA bullous dermatosis and immunological characteristics of pemphigus, which makes this disease peculiar and this diagnosis rarely suspected in the first evaluation of the patient. The reported case is of a patient with clinically bullous disease similar to dermatitis herpetiformis, whose multiple biopsies were inconclusive, and only after direct immunofluorescence with a pemphigus pattern (intraepidermal intercellular pattern) the confirmation of the diagnosis was possible.

Valoración de exposición y endemicidad de Toxoplasma gondii en donantes de sangre en la provincia de Holguín, Cuba / Assessment of exposure and endemicity of Toxoplasma gondii among blood donors from the province of Holguín, Cuba

INTRODUCCIÓN: la toxoplasmosis es la enfermedad parasitaria más difundida en el mundo que afecta al hombre, descrita hace poco más de 100 años y producida por Toxoplasma gondii. Una de las formas que el hombre adquiere la enfermedad es a través de la placenta, de órganos trasplantados y por transfusiones de sangre; la forma infectante del parásito denominada "taquizoitos" es la responsable de este tipo de infección y ocurre durante la fase hematógena del mismo en un individuo seropositivo a T gondii. En Cuba al igual que en otras partes del mundo se ha demostrado la presencia de este parásito; en la provincia de Holguín su circulación se ha confirmado en receptores de trasplante renal. OBJETIVOS: conocer la seroprevalencia en donantes de sangre que motivó el interés para la realización de este trabajo. MÉTODOS: se evaluaron 892 muestras de sueros de donantes de los 14 municipios de la provincia de Holguín, el comportamiento serológico se determinó por la Técnica de Inmunofluorescencia Indirecta. RESULTADOS: la seropositividad para Inmunoglobulina G, anti Toxoplasma gondii de un 38,2 %, CONCLUSIONES: los donantes de la provincia de Holguín, están expuesto al Toxoplasma gondii, donde existe endemicidad del parásito en todo sus municipios y queda demostrado que los individuos procedentes de áreas rurales, tienen mayor incidencia de seropositivos al Toxoplasma gondii que los de áreas urbanas.

INTRODUCTION: Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii. First described a little over 100 years ago, it is the most widely distributed parasitic disease affecting humans. The disease may be acquired from the placenta, from transplanted organs or from blood transfusions. The infecting form of the parasite, known as "tachyzoite", is responsible for this type of infection, which occurs during the hematogenous stage in a T. gondii-seropositive individual. The presence of this parasite has been demonstrated both in Cuba and in other regions of the world. In the province of Holguín its circulation has been confirmed in renal transplant recipients. OBJECTIVES: The purpose of the present study was to determine the seroprevalence of toxoplasmosis among blood donors. METHODS: An evaluation was conducted of 892 serum samples from donors from the 14 municipalities in the province of Holguín. Serological behavior was determined by indirect immunofluorescence technique. RESULTS: Seropositivity for anti-Toxoplasma gondii Immunoglobulin G was found to be 38.2 %. CONCLUSIONS: Blood donors from the province of Holguín are exposed to Toxoplasma gondii. The parasite is endemic in all municipalities, and it has been shown that Toxoplasma gondii seropositivity is higher in rural areas than in urban areas.

Eficiencia del cultivo bacteriológico y de la inmunofluorescencia en la detección de Campylobacter fetus en fluidos genitales bovinos / Efficiency of bacteriological culture and the immunofluorescent assay to detect Campylobacter fetus in bovine genital fluids

La campilobacteriosis genital bovina es una enfermedad reproductiva que afecta la producción bovina. Es causada por las subespecies de Campylobacter fetus, C. fetus fetus (Cff) y C. fetus venerealis (Cfv). El objetivo de este estudio fue identificar la presencia de C. fetus en fluidos genitales mediante cultivo bacteriológico e inmunofluorescencia directa (IFD) y comparar los resultados. Se conformaron 2 grupos de 6 vaquillonas y 5 toros cada uno. Uno se infectó con Cff (grupo Cff) y el otro con Cfv (grupo Cfv). Dos vaquillonas y 2 toros sin infectar conformaron el grupo control. Periódicamente se tomaron muestras de mucus cervicovaginal y fluido prepucial, las que se procesaron por cultivo e IFD. En el grupo Cff se infectó el 100 % de las vaquillonas y el 80 % de los toros, mientras que en el grupo Cfv se infectó el 50 y el 60 %, respectivamente. Los valores de concordancia (Kappa) obtenidos al comparar las técnicas diagnósticas fueron de 0,57 para las vaquillonas del grupo Cff y 0,52 para las del grupo Cfv, y para los toros fueron de 0,17 y 0,27, respectivamente. En las vaquillonas, la IFD arrojó más resultados positivos que el cultivo, un 5,6 % más para el grupo Cff y un 7,4 % más para el grupo Cfv. El menor porcentaje de resultados positivos por IFD en los toros, un 40 % menos que por cultivo para el grupo Cff y un 5,3 % menos para el grupo Cfv, podría deberse a un muestreo incorrecto. Los valores de Kappa indican una concordancia moderada en las vaquillonas y baja en los toros.

Bovine genital campylobacteriosis is a reproductive disease that affects cattle production. It is caused by Campylobacter fetus subspecies, C. fetus fetus (Cff) and C. fetus venerealis (Cfv). The aim of this study was to identify the presence of C. fetus in genital fluids by bacteriological culture and direct immunofluorescence (DIF) and to compare the results. Two groups of 6 heifers and 5 bulls, one infected with Cff (Cff group) and the other with Cfv (Cfv group) were formed. Two heifers and 2 bulls, all of them uninfected, made up the control group. Samples of cervicovaginal mucus and preputial fluid were processed by culture and DIF. In the Cff group, 100 % of the heifers and 80 % of the bulls were infected, while in the Cfv group, 50 % of the heifers and 60 % of the bulls were infected. The degree of agreement (Kappa values) from benchmarking diagnostic techniques were 0.57 for heifers in the Cff group and 0.52 for heifers in the Cfv group, whereas the values for bulls were 0.17 and 0.27, respectively. Heifers yielded more positive results in the DIF assay than in the culture, exhibiting 5.6 % increase in the Cff group and 7.4 % in the Cfv group. The lowest percentage of positive results for DIF in bulls, 40 % less for the Cff group and 5.2 % for the Cfv group, could be due to improper sampling. Kappa values showed moderate agreement for the heifers and low for the bulls.

Evaluación de la sensibilidad diagnóstica de tres técnicas de laboratorio para la infección por influenza A: inmunocromatografía, IFD e IFD con citocentrifugado versus RPC-TR / Evaluation of three laboratory methods diagnostic sensitivity in influenza A infection: RIDT, DFA and DFA with cytocentrifugation versus RT-PCR

Introduction: The specific diagnosis of influenza A infection makes it possible to control its spread, decreases the unnecessary use of antibiotics, clinical procedures and laboratory test, and allows early recognition of outbreaks. Different technologies are currently available in Chile for this purpose. Objective: The study presented here compares the sensitivity for influenza A virus detection of immunocromatography (RIDT), direct fluorescent antibodies-DFA and DFA with cytocentrifugation against the gold standard, RT-PCR. Material and Methods: In 175 nasal swab samples influenza A RIDT and RT-PCR were performed. Another 1689 nasal swab samples were tested by DFA and RT-PCR for influenza A. Finally, 29 nasal swab samples confirmed as Influenza A positive by RT-PCR were tested by DFA with cytocentrifugation. Results: The RIDT, DFA and DFA + cytocentrifugation sensitivity was 47,3%, 57,2% and 72,4%, respectively. Discussion and Conclusion: Their lower cost and faster turnaround time when compared to PCR make RIDT and DFA the tests of choice in diagnostic laboratories in Chile. However, their low sensitivity and NPV, especially during low season, makes more sensitive diagnostic tools necessary to confirm the results. In our study cytocentrifugation increased DFA sensitivity from 57% to 72%.

Introducción: El diagnóstico específico de influenza permite controlar la diseminación de la enfermedad, disminuir el uso de antimicrobianos, procedimientos clínicos y exámenes, e identificar rápidamente brotes. Diferentes tecnologías están actualmente disponibles en Chile para este propósito. Objetivo: Comparar la sensibilidad diagnóstica para la infección por el virus influenza A de las técnicas inmunocromatografía, inmunofluorescencia directa-IFD e IFD con citocentrifugado contra el estándar de oro, RPC-TR. Materiales y Método: En 175 muestras de hisopado nasofaríngeo se realizó inmunocromatografía y RPC-TR para influenza A. Otras 1.689 muestras de hisopado nasofaríngeo fueron procesadas mediante IFD y RPC-TR para influenza A. Finalmente, en 29 muestras de hisopado nasofaríngeo, confirmadas positivas para influenza A mediante RPC-TR, se realizó IFD con citocentrifugado. Resultados: La sensibilidad de la inmunocromatografía, IFD e IFD + citocentrifugado fue de 47,3%, 57,2% y 72,4%, respectivamente. Discusión y Conclusión: El menor costo y tiempo de respuesta de las técnicas rápidas (inmunocomatografía e IFD) en relación a la RPC-TR hacen que se mantengan como exámenes de rutina en los laboratorios diagnósticos del país. Sin embargo, su baja sensibilidad y VPN, especialmente durante períodos de baja prevalencia, obligaría a confirmar los resultados negativos con técnicas más sensibles. En nuestra comparación la citocentrifugación mejoró la sensibilidad de la IFD de 57% a 72%.

Tools to Detect Influenza Virus

In 2009, pandemic influenza A (H1N1) virus (H1N1 09) started to spread quickly in many countries. It causes respiratory infection with signs and symptoms of common infectious agents. Thus, clinicians sometimes may miss the H1N1 patient. Clinical laboratory tests are important for the diagnosis of the H1N1 infection. There are several tests available, however, the rapid test and direct fluorescence antigen test are unable to rule out the influenza virus infection and viral culture test is time consuming. Therefore, nucleic acid amplification techniques based on reverse transcription polymerase chain reaction assays are regarded as a specific diagnosis to confirm the influenza virus infection. Although the nucleic acid-based techniques are highly sensitive and specific, the high mutation rate of the influenza RNA-dependent RNA polymerase could limit the utility of the techniques. In addition, their use depends on the availability, cost and throughput of the diagnostic techniques. To overcome these drawbacks, evaluation and development of the techniques should be continued. This review provides an overview of various techniques for specific diagnosis of influenza infection.

Evaluación de la microscopía en fresco en el diagnóstico de neumocistosis pulmonar / Microscopio examination in fresh mount preparation for the diagnosis of pulmonary pneumocystosis / Avaliagáo da microscopia a fresco no diagnóstico de pneumocistose pulmonar

Para evaluar la utilidad de la microscopia en fresco en el diagnóstico de la neumocistosis pulmonar (PCP), se aplicó una técnica de inmunofluorescencia directa (IFD) con anticuerpos monoclonales a 50 secreciones respiratorias obtenidas por lavado broncoalveolar y procesadas en forma consecutiva en el Laboratorio de Parasitología, entre el 19 de enero y el 25 de febrero de 2011. Las mismas pertenecían a pacientes con SIDA y diagnóstico presuntivo de PCP, y en todas ellas la investigación de la presencia de exudados espumosos por microscopia en fresco fue negativa. Ninguna de las muestras procesadas resultó positiva para Pneumocystis jiroveci con la IFD. En base a los resultados obtenidos se concluyó que la microscopia en fresco permanece como un método rápido, económico, sencillo y seguro para el diagnóstico de la PCP en los pacientes con SIDA internados en diferentes Salas del Hospital Muñiz. Al igual que en un estudio previo, reveló poseer una sensibilidad similar a la IFD en los pacientes evaluados.

To evaluate the usefulness of fresh microscopy for the diagnosis of pulmonary pneumocystosis (PCP), direct immunofluorescence (DIF) with monoclonal antibodies technique was applied to 50 respiratory secretions obtained by bronchoalveolar lavage and consecutively processed in the Laboratory of Parasitology from January 19, 2011 to February 25, 2011. The samples belonged to AIDS patients with presumptive diagnosis of PCP, and all of them were negative for the search of foamy exudates by wet mountmicroscopy. No positive results were obtained for Pneumocystis jiroveci with the DIF. According to the results obtained, it was concluded that fresh microscopy remains being a rapid, economic, simple and accurate method for the diagnosis of PCP in AIDS patients assisted in different Wards of the Muñiz Hospital. As in a previous study, performed in a similar cohort of patients, fresh microscopy revealed a sensitivity similar to that of DIF when applied to the diagnosis of PCP.

Para avaliar a utilidade da microscopia a fresco no diagnóstico da pneumocistose pulmonar (PCP), foi aplicada uma técnica de imunofluorescencia direta (IFD) com anticorpos monoclonais em 50 secregóes respiratórias obtidas por lavagem broncoalveolar e processadas de forma consecutiva no Laboratório de Parasitologia, entre os dias 19 de janeiro de 2011 e 25 de fevereiro de 2011. As mesmas pertenciam a pacientes com AIDS e diagnóstico presuntivo de PCP, e em todas elas a pesquisa da presenga de esfregagos espumosos por microscopia a fresco foi negativa. Nenhuma das amostras processadas resultou positiva para Pneumocystis jiroveci com a IFD. Com base nos resultados obtidos foi concluido que a microscopia a fresco permanece como um método rápido, económico, simples e seguro para o diagnóstico da PCP nos pacientes com AIDS internados em diferentes. Salas do Hospital Muñiz. Do mesmo modo que num estudo prévio, revelou possuir uma sensibilidade similar a IFD nos pacientes avaliados.

Púrpura de Schõnlein- Henoch en adultos / Henoch-Schõnlein purpura in adults

La púrpura de Schõnlein-Henoch es una vasculitis sistémica mediada por inmunoglobulina A, caracterizada clínicamente por púrpura palpable, dolor abdominal, artralgias y hematuria o proteinuria en el laboratorio. La inmunofluorescencia directa de las lesiones cutáneas, así como la del riñón, revelan la presencia de depósitos perivasculares de este anticuerpo, dato de valor para confirmar el diagnóstico. Se presentan cinco pacientes adultos con PSH, insuficiencia renal aguda y neoplasias malignas en tres de ellos.

Henoch-Schönlein Purpura is a systemic vasculitis mediated by Ig A, clinically characterizedby non-thrombocytopenic palpable purpura, abdominal pain, arthritis and proteinuria orhematuria. Histologically, it is characterized by deposition of immunoglobulin A in the skinand kidneys, being these features essential for the diagnosis. We report five adult patientswith Henoch-Schönlein purpura with acute kidney failure, and malignant neoplasms onthree of them.

The role of a commercial enzyme immuno assay antigen detection system for diagnosis of C. trachomatis in genital swab samples.

In the present pilot study, endocervical and urethral swabs collected from 100 patients attending sexually transmitted disease (STD) clinics and regional centre for STD in two referral hospitals in New Delhi were analyzed by enzyme immune assay (EIA), polymerase chain reaction (PCR) and direct fluorescent antibody (DFA) for detection of C. trachomatis. It was found that EIA could detect a very low number of cases (3/100) as against DFA (11/100) and PCR (9/100). Thus, in spite of the widespread availability, lower cost and ease of performance of the enzyme-linked-immunosorbent serologic assay, the present study highlights the need to employ sophisticated diagnostic tools like DFA and PCR for detection of Chlamydia trachomatis in STD patients.

Diagnóstico de neumocistosis en pacientes oncológicos: por la técnica de inmunofluorescencia directa / Neumocistosis in oncological patients: diagnoses for immunofluorescense direct technique

El paciente con enfermedades oncológicas tiene un alto riesgo para desarrollar infecciones respiratorias, y neumonía por Pneumocystis jirovecii. En Venezuela existen pocos estudios sobre la neumocistosis en pacientes oncológicos. El objetivo de este trabajo fue detectar la presencia de Pneumocystis jirovecii en pacientes oncológicos a través de la técnica de inmunofluorescencia directa. Se recibieron, durante 10 meses, 31 muestras respiratorias (lavado broncoalveolar, esputo espontáneo e inducido, aspirados traqueales), de ellas 8 (25,5 por ciento) resultaron positivas. La distribución por tipo de cáncer fue la siguiente: 18 tumores sólidos y 13 leucemias y linfomas. La positividad entre los grupos estudiados no fue estadísticamente significativa (P>0,05). Los exámenes de laboratorio complementarios, relacionados tampoco fueron estadísticamente significativos (P>0,05). Es necesario incluir este diagnóstico en estudio microbiológico diferencial de infecciones del tracto respiratorio inferior en pacientes con cáncer, estos pacientes cursan con una sintomatología general inespecífica y tendrán una alta posibilidad de desarrollar neumocistosis.

The patient with malignancy disease has a high risk to develop respiratory infections for Pneumocystis jirovecii pneumonia. Investigations about pneumocystosis in oncological patients in Venezuela are scarce. The objective of this work was to detect Pneumocystis jirovecii in oncological patients by the method of direct immunofluorescence technique. Thirty one respiratory specimens (bronchoalveolar lavage, spontaneous and induced sputum, and tracheal aspirates) received in 10 months, 8 specimens of them (25.5) were positive the distribution by malignancy disease was the following: 18 solid tumors, and 13 leukemias, and lymphomas. No statistically significant differences were found between the studied groups and positive results (P>0.05). The complementary laboratory tests, related to the presence of Pneumocystis, were not statistically significant either P>0.05). Is necessary to include this diagnosis in the microbiological differential study of low respiratory tract infections in oncological patients, since these patients show unspecific symptoms, and have a high possibility to develop pneumocystosis.

American cutaneous leishmaniasis in dogs from an endemic urban area in Cianorte municipality, Paraná State, Brazil

American cutaneous leishmaniasis (ACL) was investigated in dogs from an urban endemic area in Cianorte, Paraná state, Brazil. Of 169 studied dogs, none presented suspected ACL lesions. Eleven animals (6.6 percent) had anti-Leishmania braziliensis antibodies (titers > 40) detected by the immunofluorescent antibody test (IFAT) while four (2.4 percent) showed L. braziliensis-complex DNA by the polymerase chain reaction (PCR). Although no associations were found between IFAT or PCR results and age, sex, origin, free-roaming animals or length of residence at the address, the majority of IFAT- or PCR-positive dogs were from the urban area of the city and were allowed to roam freely beyond their neighborhood. The presence of anti-Leishmania braziliensis antibodies and L. braziliensis-complex DNA in dogs from this urban area near a native-forest park indicates the importance of following up on these dogs to confirm the ACL diagnosis.

Etiología viral de las infecciones respiratorias agudas / Viral etiology of acute respiratory infections

Las infecciones respiratorias agudas (IRA) son patologías que afectan el tracto respiratorio desde la faringe proximal hasta los pulmones, con una evolución de menos de 15 días y constituyen la causa más frecuente de morbimortalidad en el mundo. Con la finalidad de identificar los agentes virales asociados a este tipo de infecciones en pacientes del estado Zulia, Venezuela, entre febrero 2005 y julio de 2006, se estudiaron un total de 102 muestras provenientes del tracto respiratorio (hisopado nasal, faríngeo y/o nasofaríngeo, esputo y lavado broncoalveolar) de pacientes con clínica de IRA. El aislamiento viral se realizó a través del cultivo celular y la identificación del agente patógeno por la técnica de inmunofluorescencia directa. Se obtuvieron 46 muestras positivas (45%), la incidencia estuvo homogéneamente distribuida en todos los grupos de estudio aun cuando se observó predominio en el grupo de 41 a 64 años, seguido de los lactantes. No hubo diferencias significativas en cuanto al sexo. Dentro de los patógenos virales aislados el Virus Sincicial Respiratorio (VSR) fue el agente con mayor frecuencia (32,6%) (p<0,05), seguido de Adenovirus (28,2%), Parainfluenza (23,9%) e Influenza (15,2%). Las infecciones respiratorias del tracto inferior fueron las más frecuentes (67,4%). Se demuestra una alta incidencia de agentes virales asociados a IRA en el estado Zulia. Se destaca la alta frecuencia en pacientes adultos y la mayor positividad para VSR.

Acute respiratory infections (ARI) are pathologies that affect the respiratory tract from the proximal pharynx to the lungs, with an evolution of less than 15 days. They constitute the most frequent cause of morbimortality in the world. With the purpose of identifying the viral agents associated to this type of infections in patients of Zulia State, Venezuela, between February 2005 and July 2006, a total of 102 samples of the respiratory tract (oropharyngeal swab, nasopharyngeal swab, nasopharyngeal washing, spit and bronchoalveolar lavage) were studied. Viral isolation was made through a cellular culture and the identification of the pathogenic agents by the technique of direct immunofluorescence. Forty six positive samples were obtained (45%). The greater incidence was found in the groups of 41 to 64 years old patients, followed by lactanting babies (1 to 23 months). There were no significant differences between sexes. Within the isolated viral pathogens, Respiratory Syncytial Virus (RSV) was the agent most frequently found (32.6% p<0.05), followed by Adenovirus (28.2%), Parainfluenza (23.9%) and Influenza (15.2%). The respiratory infections of the low respiratory tract were the most frequent (67.4%). A high incidence of associated viral agents to ARI in Zulia State is demonstrated. A high incidence in adult patients and the greatest positivity was found for RSV.

Direct Immunofluorescence in Behcet's Disease: A Controlled Study with 108 Cases

PURPOSE: Behcet's disease (BD) is a disease of unknown etiology, which has multisystemic involvement. This multisystemic involvement might be the clue for an autoimmune pathogenesis. In order to evaluate an autoimmune pathogenesis, we examined immunoreactans depositions in the skin of BD patients. MATERIALS AND METHODS: The skin samples of 108 BD patients (28 perilesional skin, 44 positive pathergy test site, 22 negative pathergy test site, 14 normal skin) were examined for the depositions of immunoglobulin (Ig)M, IgG, IgA, complement 3 (C3), and fibrinogen (F) using direct immunofluorescence (DIF). The data were statistically compared to the DIF of 36 systemic lupus erythematosus (SLE) patients and 20 healthy controls using chi-square Fisher exact test. RESULTS: Highly significant immunoreactans depositions were obtained in BD (deposition rates: IgM 70.3%, IgG 0%, IgA 20.3%, C3 62.9%, F 83.3%). The comparison with SLE revealed no differences in IgM, IgA, and C3. However, IgG deposition was higher in SLE while F deposition was higher in BD. In both BD and SLE, the Ig depositions were highly significant when the data were compared with the healthy controls. CONCLUSION: The significant deposition of immunoreactans in BD, especially in the negative pathergy and the normal skin sites, were observed. This study is the first controlled study revealing positive Ig depositions in BD, and it is expected to help us to reconsider the autoimmune pathogenesis in BD.

Direct Immunofluorescence in Behcet's Disease: A Controlled Study with 108 Cases

PURPOSE: Behcet's disease (BD) is a disease of unknown etiology, which has multisystemic involvement. This multisystemic involvement might be the clue for an autoimmune pathogenesis. In order to evaluate an autoimmune pathogenesis, we examined immunoreactans depositions in the skin of BD patients. MATERIALS AND METHODS: The skin samples of 108 BD patients (28 perilesional skin, 44 positive pathergy test site, 22 negative pathergy test site, 14 normal skin) were examined for the depositions of immunoglobulin (Ig)M, IgG, IgA, complement 3 (C3), and fibrinogen (F) using direct immunofluorescence (DIF). The data were statistically compared to the DIF of 36 systemic lupus erythematosus (SLE) patients and 20 healthy controls using chi-square Fisher exact test. RESULTS: Highly significant immunoreactans depositions were obtained in BD (deposition rates: IgM 70.3%, IgG 0%, IgA 20.3%, C3 62.9%, F 83.3%). The comparison with SLE revealed no differences in IgM, IgA, and C3. However, IgG deposition was higher in SLE while F deposition was higher in BD. In both BD and SLE, the Ig depositions were highly significant when the data were compared with the healthy controls. CONCLUSION: The significant deposition of immunoreactans in BD, especially in the negative pathergy and the normal skin sites, were observed. This study is the first controlled study revealing positive Ig depositions in BD, and it is expected to help us to reconsider the autoimmune pathogenesis in BD.

Direct immunofluorescence in cutaneous vesiculobullous lesions.

Direct Immunofluorescence (DIF) is invaluable in the diagnosis of cutaneous vesiculobullous lesions (VBL). It is limited by technical factors and disease nature. 1) To record the sensitivity of DIF in VBL 2) To correlate DIF with clinical, histologic findings and analyse discrepancies. Material and Methods: A retrospective study of 100 DIFs on suspected VBL of skin. DIF, histology and clinical data were reviewed. 73/100 cases showed DIF patterns concordant with clinical/histologic diagnosis. The sensitivity of DIF was 88% in Pemphigus group (39/ 44), 82% in Bullous Pemphigoid (BP) (23/28), and 20% in Dermatitis Herpetiformis (DH) (1/5).18 cases of histologically proven VBL were negative and of these, 4 had no epidermis. The remaining 9 cases were discordant with clinical/histologic features, including 4 BP and 5 DH, whose histology was non-specific and will be discussed in detail. One case of DH showed an aberrant vasculitic pattern. DIF is of great value in the diagnosis of VBL, specially in clinical/histologic dilemmas. In DH, neither biopsy nor DIF were very useful and response to therapy was the standard. Sampling errors contributed to false negative results. Proper selection of cases and judicious use are mandatory to optimize its' utility.

Aborto ovino associado com infecção por Sarcocystis sp / Ovine abortion associated with Sarcocystis sp. infection

Infecções por protozoários têm distribuição mundial e podem causar aborto, nascimentos prematuros e ou morte fetal em diversas espécies animais. Em julho de 2004, oito ovinos Corriedale apresentaram problemas reprodutivos caracterizados por aborto e natimortalidade no terço final da gestação. Dessas oito perdas, um natimorto macho foi enviado ao Setor de Patologia Veterinária para necropsia. Alterações macroscópicas não foram observadas durante a necropsia. Lesões histológicas foram observadas principalmente no cérebro e coração e se caracterizaram por encefalite não-supurativa multifocal acentuada associada à presença de protozoários no interior de células endoteliais e vasos sanguíneos e miocardite não-supurativa focal leve. Alguns desses organismos apresentaram formato de roseta. O teste de imunoistoquímica anti-Toxoplasma gondii foi negativo, mas houve reação cruzada com anticorpo anti-Neospora caninum. O exame de imunofluorescência direta para Leptospira sp. foi negativo. A bacteriologia aeróbica e micro-aeróbica não revelou crescimento significativo. Esses achados foram compatíveis com o diagnóstico de Sarcocystis sp.

Protozoal infection has worldwide distribution and may cause abortion, premature parturition or fetal death in almost all domestic animals. In July 2004, eight Corriedale sheep showed abortion and stillbirth in the third trimester of gestation. Of these reproductive losses, one stillborn male was submitted to the Laboratory of Veterinary Pathology for necropsy investigation. The direct immunofluorescence test for Leptospira sp. was negative. No significant bacteria was isolated from lung and liver by aerobic and microaerobic cultures. Macroscopic lesions were not found in any fetal tissue. The histological lesions were observed mainly in the brain and heart and consisted primarily of severe multifocal nonsupurative encephalitis and nonsuppurative myocarditis. Schizonts of a protozoan parasite consistent with Sarcocystis sp. were found in the endothelial cells and vascular endothelium in several organs. Many schizonts with merozoites arranged in a rosette-like pattern were observed in brain and kidney tissues. In sections stained with periodic acid-Schiff (PAS), the limiting membrane of some schizonts appeared to be weakly PAS-positive. Merozoites and nuclei were PAS-negative. Protozoa did not react immunohistochemically to the antibody anti-Toxoplasma gondii; however, cross-reactivity was observed with Neospora caninum antibody. These findings were consistent with the diagnosis of Sarcocystis sp.

Detection of several clostridia by a direct fluorescent antibody test in formalin-fixed, paraffin-embedded tissues

Descreve-se a aplicabilidade de uma técnica de imunofluorescência direta, para o diagnóstico de mionecroses causadas por clostrídios, a partir de tecidos fixados em formol e incluídos em parafina. Essa técnica pode auxiliar no diagnóstico do carbúnculo sintomático e da gangrena gasosa, contribuindo para determinar a real prevalência dessas doenças no país

Perdas reprodutivas associadas com infecção por Toxoplasma gondii em caprinos no sul do Brasil / Reproductive losses linked to Toxoplasma gondii infection in goats in southern Brazil

Infecção por Toxoplasma gondii foi associada com perdas reprodutivas em um rebanho caprino no Rio Grande do Sul, Brasil. Lesões macroscópicas foram observadas em dois de seis produtos caprinos enviados para diagnóstico e incluíram linfonodos mesentéricos pálidos e aumentados e pulmões com consistência firme e áreas claras intercaladas com vermelhas. Lesões histológicas, especialmente caracterizadas por infiltrados linfoplasmocitários no cérebro e pulmões, foram observadas em todos os fetos. Nefrite intersticial linfoplasmocitária, linfadenite necrosante e hepatite periportal linfoplasmocitária também foram observadas. Enquanto tanto o exame bacteriológico quanto o teste de imunofluorescência direta para Leptospira sp. foram negativos em todos os casos, a PCR e a imunoistoquímica resultaram positivamente para T. gondii em quase todas as amostras testadas. Anticorpos anti-T.gondii, em titulações de 1:512 a 1:2048, foram detectados nas amostras de soro sangüíneo das cabras que pariram natimortos (3), abortaram (1) ou cujos neonatos morreram (2). Este trabalho descreve os achados clínicos, patológicos, sorológicos, imunoistoquímicos e de PCR observados em um rebanho caprino infectado por T. gondii.

Toxoplasma gondii was implicated with reproductive losses in a goat herd in Rio Grande do Sul, Brazil. Gross changes were present in 2 out of 6 fetuses/offsprings submitted for diagnosis and included enlarged, pale mesenteric lymph nodes, and edematous, mottled red and tan lungs. Microscopic changes were observed in most fetuses and were especially characterized by lymphoplasmacytic infiltration in the brain and lungs. Other histological changes included lymphoplasmacytic interstitial nephritis, necrotizing lymphadenitis and periportal lymphoplasmacytic cell infiltrates. While bacteriological and direct immunofluorescent antibody tests for Leptospira sp. were negative in samples from all cases, immunohistochemical and PCR procedures for Toxoplasma gondii reacted positively in most of them. Antibody titles against T. gondii varying from 1:512 to 1:2048 were detected in serum samples from the mother goats of these aborted (1), stillborn (3) or dead newborn (2) kids. This paper describes the clinical, pathological, serological, molecular and immunohistochemical findings of a Toxoplasma gondii-infected goat flock.

Simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis by PCR in genitourinary specimens from men and women attending an STD clinic.

Neisseria gonorrhoeae and Chlamydia trachomatis are the two most common bacterial sexually transmitted infections that manifest primarily as urethritis in males and endocervicitis in females, though the infection may be asymptomatic especially in women. Since complications may occur in untreated symptomatic and asymptomatic infected individuals, early diagnosis and treatment of infected individuals is required to prevent severe sequelae and spread of these diseases. Recently molecular amplification assays like Polymerase Chain Reaction (PCR) and Ligase Chain Reaction (LCR) have been found to be highly sensitive and specific methods for detection of N. gonorrhoeae and C. trachonmatis not only in urethral and cervical specimens but also in urine. The objective of this study was to screen male and female Sexually Transmitted Disease (STD) clinic attenders, with and without symptoms suggestive of urethritis and cervicitis for presence of N. gonorrhoeae and C. trachomatis using a multiplex PCR based assay, to compare its performance with culture for N. gonorrhoeae and Direct Fluorescent Antibody (DFA) staining for C. trachomatis and also to compare the efficacy of PCR test performed on urine and genital swab specimens collected from this high risk group. Genital specimens and urine was collected from STD clinic attenders. N. gonorrhoeae and C. trachomatis was detected in genital specimens by culture and DFA respectively. Multiplex PCR was used to detect N. gonorrhoeae and C. trachomatis infection in both genital and urine specimens. Among men with urethritis, N. gonorrhoeae was detected in 70% by culture and 77% by PCR, while C. trachomatis as detected in 7.5% by DFA and 17.5% by PCR. Among females with endocervicitis, N. gonorrhoeae was detected in 7.7% by culture and 30.7% by PCR, while C. trachomatis was detected in 7.7% by DFA and in 15.4% by PCR. None of the asymptomatic males were positive for N. gonorrhoeae and C. trachomatis by conventional methods, while 43.9% were positive for N. gonorrhoeae and 7.5% for C. trachomatis by PCR. Fifty per cent of asymptomatic women were positive for C. trachomatis by PCR alone. We encountered PCR positive but culture/DFA negative results and also PCR negative but culture/DFA positive results. In view of this a single PCR test cannot be used for diagnosis and treatment of N. gonorrhoeae and C. trachomatis infection unless confirmed by a second test.

Usefulness of the recombinant liver stage antigen-3 for an early serodiagnosis of Plasmodium falciparum infection

In order to develop tools for an early serodiagnosis of Plasmodium falciparum infection, we evaluated the usefulness of P. falciparum liver stage antigen-3 (LSA-3) as a serodiagnostic antigen. A portion of LSA-3 gene was cloned, and its recombinant protein (rLSA-3) was expressed in Escherichia coli and purified by column chromatography. The purified rLSA-3 and 120 test blood/serum samples collected from inhabitants in malaria-endemic areas of Mandalay, Myanmar were used for this study. In microscopic examinations of blood samples, P. falciparum positive rate was 39.1% (47/120) in thin smear trials, and 33.3% (40/120) in thick smear trials. Although the positive rate associated with the rLSA-3 (30.8%) was lower than that of the blood stage antigens (70.8%), rLSA-3 based enzyme-linked immunosorbent assay could detect 12 seropositive cases (10.0%), in which blood stage antigens were not detected. These results indicate that the LSA-3 is a useful antigen for an early serodiagnosis of P. falciparum infection.

Vigilância epidemiológica da presença do vírus da raiva a partir do exame de imunofluorescência direta aplicada ao tecido cerebral de uma amostra da população canina do município de Mogi-Guaçu, SP, Brasil. Ensaio probabilístico a partir de série histórica de 1989 a 1999 / Epidemiologic surveillance of the rabies virus using the brain immunoflorescence test on a sample of a canine population. Probality an analysis with actual cases from Mogi-Guaçu, SP, Brazil, During the 1989-1999 period

Foi analisada a validade do exame laboratorial sistemático do sistema nervoso de uma amostra da população canina de uma dada área, como estratégia destinada a vigilância epidemiológica da circulação do vírus da raiva. Foi empregado o banco de dados do município de Mogi-Guacú, SP, Brasil, referente a série histórica compreendida entre janeiro de 1989 a dezembro de 1999. Neste período foram examinados 1167 animais dos quais 130 (11,2%) foram positivos ao teste de imunofluorescência aplicada a raiva. O tamanho da amostra para a detecção de pelo menos um animal positivo foi calculado pela fórmula n = {1-(1-alfa)1/d} (N - d/2} + 1. No período de 1989 a 1994 o tamanho da amostra foi calculado a partir do número real de casos registrados. Nos anos de 1995 a 1999 como não houve novos casos de raiva canina, a análise considerou hipoteticamente a presença de um caso confirmado. Também foi efetuada a simulação do número de casos de raiva que deveriam ocorrer para que a amostra efetivamente utilizada pelo Serviço de controle da raiva fosse capaz de revelar a presença de pelo menos um animal positivo. Os resultados obtidos demonstraram que no período de 1989 a 1994 em que a freqüência anual de casos de raiva canina variou de 5 a 75 o tamanho das amostras ideal seria de 12.400 a 12.922; já no período de 1995 a 1999, em que não foram diagnosticados casos de raiva canina, se ocorresse pelo menos um registro, o tamanho da amostra seria de 13.257 a 14.698. Do exposto depreende-se que em termos probabilisticos, a estratégia proposta não é indicada para a vigilância epidemiológica da presença do vírus da raiva, quando em situação de controle, pois o número de animais a serem examinados é inviável para situações concretas.