Validation of a New Duplex Real-Time Polymerase Chain Reaction for Chlamydia trachomatis DNA Detection in Ocular Swab Samples.

Trachoma is the world-leading infectious cause of preventable blindness and is caused by the bacteria Chlamydia trachomatis. In developing countries, diagnosis is usually based on clinical evaluation. Serological-based tests are cheaper than molecular-based ones, but the latter are more sensitive and specific. The present study developed a new duplex qPCR which concomitantly detects the C. trachomatis cryptic plasmid and the human 18S rRNA gene, with an LOD95% for C. trachomatis DNA of 13.04 genome equivalents per reaction. The new qPCR was tested using 50 samples from an endemic area and 12 from a non-endemic area that were previously characterized using direct immunofluorescence assay (DFA) and clinical evaluation. Among the 50 endemic samples, 3 were found to be positive by clinical evaluation (6%), 18 were found to be positive by DFA (36%), and 48 were found to be positive by qPCR (96%). Next, the new duplex qPCR was validated using 50 samples previously characterized by qPCR. Validation was carried out on a benchtop instrument (ABI7500) or on a portable point-of-care instrument (Q3-Plus), showing 95% specificity and 100% sensitivity. The ubiquitous presence of C. trachomatis DNA in samples from the endemic region confirms that constant monitoring is of paramount importance for the effective measurement of the elimination of trachoma. The newly developed duplex qPCR presented in this study, along with its validation in a portable qPCR system, constitutes important tools toward achieving this goal.

Raiva em equino na região Sul do estado do Piauí / Equine rabies in the Southern region of Piauí state

Background: Rabies is an infectious disease that is important in the “One Health” worldwide with high lethality rate. The etiological agent is a neurotropic virus, genus Lyssavirus, transmitted mainly through the saliva of infected animals. For equines, the bite of hematophagous bats is the main source of infection. Piauí is an important state for equestrian sports and the increase in the number of horses with neurological clinical signs without diagnosis has increased in recent years. In this context, the aim of this study is to report to the scientific community a confirmed case of equine rabies in the Santa Luz county, Southernmost state of Piauí, Brazil. Case: A 3-year-old female non-defined breed horse, was admitted to the Hospital Veterinário da Universidade Federal do Piauí (UFPI/CPCE). The equine had difficulty walking 2 days ago, in the panoramic inspection was restless and disoriented in the paddock. Rectal temperature of 38.2°C, heart rate of 60 bpm, respiratory rate of 40 mpm, congested mucosa and dyspnea were verified. With the progression of the neurological signals, it positioned itself in a lateral decubitus with pedaling movements, hyperesthesia, dysphagia and paralysis of the hindlimbs. The clinical suspicion was rabies and the Agência de Defesa Agropecuária do Piauí (ADAPI) was communicated to euthanize the animal and collect samples for diagnosis in accordance with official standards of the Ministério da Agricultura, Pecuária e Abastecimento (MAPA). At necropsy, there was slight brain hyperemia, with no other significant organ changes. Fragments of the cerebellum, cortex, hippocampus and spinal cord were collected and sent at a temperature of 4°C to perform the Direct Immunofluorescence (DIF) assay. Samples for histopathology were not collected because they do not include assay for confirmatory diagnosis of rabies. The DIF technique with...(AU)

Raiva em equino na região Sul do estado do Piauí / Equine rabies in the Southern region of Piauí state

Background: Rabies is an infectious disease that is important in the “One Health” worldwide with high lethality rate. The etiological agent is a neurotropic virus, genus Lyssavirus, transmitted mainly through the saliva of infected animals. For equines, the bite of hematophagous bats is the main source of infection. Piauí is an important state for equestrian sports and the increase in the number of horses with neurological clinical signs without diagnosis has increased in recent years. In this context, the aim of this study is to report to the scientific community a confirmed case of equine rabies in the Santa Luz county, Southernmost state of Piauí, Brazil. Case: A 3-year-old female non-defined breed horse, was admitted to the Hospital Veterinário da Universidade Federal do Piauí (UFPI/CPCE). The equine had difficulty walking 2 days ago, in the panoramic inspection was restless and disoriented in the paddock. Rectal temperature of 38.2°C, heart rate of 60 bpm, respiratory rate of 40 mpm, congested mucosa and dyspnea were verified. With the progression of the neurological signals, it positioned itself in a lateral decubitus with pedaling movements, hyperesthesia, dysphagia and paralysis of the hindlimbs. The clinical suspicion was rabies and the Agência de Defesa Agropecuária do Piauí (ADAPI) was communicated to euthanize the animal and collect samples for diagnosis in accordance with official standards of the Ministério da Agricultura, Pecuária e Abastecimento (MAPA). At necropsy, there was slight brain hyperemia, with no other significant organ changes. Fragments of the cerebellum, cortex, hippocampus and spinal cord were collected and sent at a temperature of 4°C to perform the Direct Immunofluorescence (DIF) assay. Samples for histopathology were not collected because they do not include assay for confirmatory diagnosis of rabies. The DIF technique with...

Clinicopathologic correlation of 282 leukocytoclastic vasculitis cases in a tertiary hospital: a focus on direct immunofluorescence findings at the blood vessel wall.

This is the largest direct immunofluorescence (DIF) analysis of patients with histology-proven cutaneous leukocytoclastic vasculitis (LCV). To establish the correlation of deposition of immune complexes at the blood vessel walls with underlying causes and prognosis of LCV, we performed a retrospective study from January 2007 to December 2014. The patients are followed at the Department of Dermatology, Hospital Das Clínicas da Faculdade de Medicina da Universidade de São Paulo, a tertiary hospital at São Paulo, Brazil. We reviewed the data of 282 biopsy-proven LCV cases with DIF performed. For the statistical analysis, we included only patients with positive DIF exclusively in vessel walls (235/282 patients). We planned to find a correlation between the DIF profiles of LCV patients and the epidemiology data, underlying causes and prognosis. Ages ranged from five to 87 years old (yo), median age of 45 and 191/282 (67.73 %) were female individuals. DIF analysis showed positivity in 70.21 % of the samples, and C3 was the most frequent immunoreactant. Immunoglobulin A (IgA) deposition at the blood vessel wall was related to age and absence of autoimmune/inflammatory diseases. Immunoglobulin M (IgM) deposition at the blood vessel wall was related to females, autoimmune/inflammatory disorders, C3 and C4 consumption and antinuclear antibody and anti-SSA/anti-SSB positivity. Immunoglobulin G (IgG) deposition at the blood vessel wall was associated with age and positive ANCA; finally, C3 deposition at the blood vessel wall was associated with hematuria and renal involvement. Systemic involvement was present in 12.5 % cases of LCV patients. C3 deposits, the most frequent finding of this study, were related to renal involvement; IgA deposits to absence of autoimmune or inflammatory diseases; IgM deposition to the presence of autoimmune or inflammatory diseases and IgG deposits were associated with positive ANCA. DIF seems to be an important method to establish the prognosis and underlying etiology of LCV. Characterization of the immune complex at the blood vessel wall by DIF is relevant to determine underlying conditions related to LCV.

Correlation between histopathological findings and results of direct immunofluorescence in the diagnosis of canine localized cutaneous chronic lupus erythematosus

Background: Localized chronic cutaneous lupus erythematosus (CCLE), also known as discoid lupus erythematosus, is one of the most prevalent canine autoimmune skin diseases. Histopathology is considered the gold standard for the diagnosis of CCLE and the accuracy of which can be increased by use of direct immunofluorescence (DIF). This study aimed to investigate the fluorescence pattern revealed by DIF in cases of canine localized CCLE and to establish and compare its effectiveness with that obtained from histopathology.Materials, Methods & Results: Eleven dogs suspicious to localized CCLE, i.e., those animals that presented mucocutaneous lesions as erythema, leucoderma, erosive-ulcerative lesions, and loss of nasal planum architecture and its transition to the haired skin underwent medical physical and laboratory examinations (blood count, platelet count, determination of alanine transaminase, alkaline phosphatase, total protein, serum albumin, urea, creatinine). Only those animals that proved to be normal across both the physical and laboratorial evaluation were included in Group I. Animals belonging to this group were submitted to general anesthesia to biopsy two samples of lesioned skin from nasal planum to histopathologic examination and DIF test. Five dogs with no skin lesions were included in Group II as negative control to the DIF assay. Two samples of no lesioned skin from nasal planum were biopsied to histopathologic and DIF evaluation. The kappa (k) coefficient was used to determine the degree of agreement and reliability of the results of both tests. A P-value < 5% was considered to be statistically significant. In Group I, all animals were normal across both the physical and laboratorial evaluation. A diagnosis of canine CCLE was established in 81.8% (9/11) of the animals based on histopathology analysis.[...]

Correlation between Histopathological Findings and Results of Direct Immunofluorescence in the Diagnosis of Canine Localized Cutaneous Chronic Lupus Erythematosus

Background: Localized chronic cutaneous lupus erythematosus (CCLE), also known as discoid lupus erythematosus, is one of the most prevalent canine autoimmune skin diseases. Histopathology is considered the gold standard for the diagnosis of CCLE and the accuracy of which can be increased by use of direct immunofluorescence (DIF). This study aimed to investigate the fluorescence pattern revealed by DIF in cases of canine localized CCLE and to establish and compare its effectiveness with that obtained from histopathology.Materials, Methods & Results: Eleven dogs suspicious to localized CCLE, i.e., those animals that presented mucocutaneous lesions as erythema, leucoderma, erosive-ulcerative lesions, and loss of nasal planum architecture and its transition to the haired skin underwent medical physical and laboratory examinations (blood count, platelet count, determination of alanine transaminase, alkaline phosphatase, total protein, serum albumin, urea, creatinine). Only those animals that proved to be normal across both the physical and laboratorial evaluation were included in Group I. Animals belonging to this group were submitted to general anesthesia to biopsy two samples of lesioned skin from nasal planum to histopathologic examination and DIF test. Five dogs with no skin lesions were included in Group II as negative control to the DIF assay. Two samples of no lesioned sk

Correlation between Histopathological Findings and Results of Direct Immunofluorescence in the Diagnosis of Canine Localized Cutaneous Chronic Lupus Erythematosus

Background: Localized chronic cutaneous lupus erythematosus (CCLE), also known as discoid lupus erythematosus, is one of the most prevalent canine autoimmune skin diseases. Histopathology is considered the gold standard for the diagnosis of CCLE and the accuracy of which can be increased by use of direct immunofluorescence (DIF). This study aimed to investigate the fluorescence pattern revealed by DIF in cases of canine localized CCLE and to establish and compare its effectiveness with that obtained from histopathology.Materials, Methods & Results: Eleven dogs suspicious to localized CCLE, i.e., those animals that presented mucocutaneous lesions as erythema, leucoderma, erosive-ulcerative lesions, and loss of nasal planum architecture and its transition to the haired skin underwent medical physical and laboratory examinations (blood count, platelet count, determination of alanine transaminase, alkaline phosphatase, total protein, serum albumin, urea, creatinine). Only those animals that proved to be normal across both the physical and laboratorial evaluation were included in Group I. Animals belonging to this group were submitted to general anesthesia to biopsy two samples of lesioned skin from nasal planum to histopathologic examination and DIF test. Five dogs with no skin lesions were included in Group II as negative control to the DIF assay. Two samples of no lesioned sk

Correlation between Histopathological Findings and Results of Direct Immunofluorescence in the Diagnosis of Canine Localized Cutaneous Chronic Lupus Erythematosus

Background: Localized chronic cutaneous lupus erythematosus (CCLE), also known as discoid lupus erythematosus, is one of the most prevalent canine autoimmune skin diseases. Histopathology is considered the gold standard for the diagnosis of CCLE and the accuracy of which can be increased by use of direct immunofluorescence (DIF). This study aimed to investigate the fluorescence pattern revealed by DIF in cases of canine localized CCLE and to establish and compare its effectiveness with that obtained from histopathology.Materials, Methods & Results: Eleven dogs suspicious to localized CCLE, i.e., those animals that presented mucocutaneous lesions as erythema, leucoderma, erosive-ulcerative lesions, and loss of nasal planum architecture and its transition to the haired skin underwent medical physical and laboratory examinations (blood count, platelet count, determination of alanine transaminase, alkaline phosphatase, total protein, serum albumin, urea, creatinine). Only those animals that proved to be normal across both the physical and laboratorial evaluation were included in Group I. Animals belonging to this group were submitted to general anesthesia to biopsy two samples of lesioned skin from nasal planum to histopathologic examination and DIF test. Five dogs with no skin lesions were included in Group II as negative control to the DIF assay. Two samples of no lesioned sk

Correlation between histopathological findings and results of direct immunofluorescence in the diagnosis of canine localized cutaneous chronic lupus erythematosus

Background: Localized chronic cutaneous lupus erythematosus (CCLE), also known as discoid lupus erythematosus, is one of the most prevalent canine autoimmune skin diseases. Histopathology is considered the gold standard for the diagnosis of CCLE and the accuracy of which can be increased by use of direct immunofluorescence (DIF). This study aimed to investigate the fluorescence pattern revealed by DIF in cases of canine localized CCLE and to establish and compare its effectiveness with that obtained from histopathology.Materials, Methods & Results: Eleven dogs suspicious to localized CCLE, i.e., those animals that presented mucocutaneous lesions as erythema, leucoderma, erosive-ulcerative lesions, and loss of nasal planum architecture and its transition to the haired skin underwent medical physical and laboratory examinations (blood count, platelet count, determination of alanine transaminase, alkaline phosphatase, total protein, serum albumin, urea, creatinine). Only those animals that proved to be normal across both the physical and laboratorial evaluation were included in Group I. Animals belonging to this group were submitted to general anesthesia to biopsy two samples of lesioned skin from nasal planum to histopathologic examination and DIF test. Five dogs with no skin lesions were included in Group II as negative control to the DIF assay. Two samples of no lesioned skin from nasal planum were biopsied to histopathologic and DIF evaluation. The kappa (k) coefficient was used to determine the degree of agreement and reliability of the results of both tests. A P-value < 5% was considered to be statistically significant. In Group I, all animals were normal across both the physical and laboratorial evaluation. A diagnosis of canine CCLE was established in 81.8% (9/11) of the animals based on histopathology analysis.[...](AU)

Correlation between Histopathological Findings and Results of Direct Immunofluorescence in the Diagnosis of Canine Localized Cutaneous Chronic Lupus Erythematosus

Background: Localized chronic cutaneous lupus erythematosus (CCLE), also known as discoid lupus erythematosus, is one of the most prevalent canine autoimmune skin diseases. Histopathology is considered the gold standard for the diagnosis of CCLE and the accuracy of which can be increased by use of direct immunofluorescence (DIF). This study aimed to investigate the fluorescence pattern revealed by DIF in cases of canine localized CCLE and to establish and compare its effectiveness with that obtained from histopathology.Materials, Methods & Results: Eleven dogs suspicious to localized CCLE, i.e., those animals that presented mucocutaneous lesions as erythema, leucoderma, erosive-ulcerative lesions, and loss of nasal planum architecture and its transition to the haired skin underwent medical physical and laboratory examinations (blood count, platelet count, determination of alanine transaminase, alkaline phosphatase, total protein, serum albumin, urea, creatinine). Only those animals that proved to be normal across both the physical and laboratorial evaluation were included in Group I. Animals belonging to this group were submitted to general anesthesia to biopsy two samples of lesioned skin from nasal planum to histopathologic examination and DIF test. Five dogs with no skin lesions were included in Group II as negative control to the DIF assay. Two samples of no lesioned sk

Correlation between Histopathological Findings and Results of Direct Immunofluorescence in the Diagnosis of Canine Localized Cutaneous Chronic Lupus Erythematosus

Background: Localized chronic cutaneous lupus erythematosus (CCLE), also known as discoid lupus erythematosus, is one of the most prevalent canine autoimmune skin diseases. Histopathology is considered the gold standard for the diagnosis of CCLE and the accuracy of which can be increased by use of direct immunofluorescence (DIF). This study aimed to investigate the fluorescence pattern revealed by DIF in cases of canine localized CCLE and to establish and compare its effectiveness with that obtained from histopathology.Materials, Methods & Results: Eleven dogs suspicious to localized CCLE, i.e., those animals that presented mucocutaneous lesions as erythema, leucoderma, erosive-ulcerative lesions, and loss of nasal planum architecture and its transition to the haired skin underwent medical physical and laboratory examinations (blood count, platelet count, determination of alanine transaminase, alkaline phosphatase, total protein, serum albumin, urea, creatinine). Only those animals that proved to be normal across both the physical and laboratorial evaluation were included in Group I. Animals belonging to this group were submitted to general anesthesia to biopsy two samples of lesioned skin from nasal planum to histopathologic examination and DIF test. Five dogs with no skin lesions were included in Group II as negative control to the DIF assay. Two samples of no lesioned sk

Correlation between Histopathological Findings and Results of Direct Immunofluorescence in the Diagnosis of Canine Localized Cutaneous Chronic Lupus Erythematosus

Background: Localized chronic cutaneous lupus erythematosus (CCLE), also known as discoid lupus erythematosus, is one of the most prevalent canine autoimmune skin diseases. Histopathology is considered the gold standard for the diagnosis of CCLE and the accuracy of which can be increased by use of direct immunofluorescence (DIF). This study aimed to investigate the fluorescence pattern revealed by DIF in cases of canine localized CCLE and to establish and compare its effectiveness with that obtained from histopathology.Materials, Methods & Results: Eleven dogs suspicious to localized CCLE, i.e., those animals that presented mucocutaneous lesions as erythema, leucoderma, erosive-ulcerative lesions, and loss of nasal planum architecture and its transition to the haired skin underwent medical physical and laboratory examinations (blood count, platelet count, determination of alanine transaminase, alkaline phosphatase, total protein, serum albumin, urea, creatinine). Only those animals that proved to be normal across both the physical and laboratorial evaluation were included in Group I. Animals belonging to this group were submitted to general anesthesia to biopsy two samples of lesioned skin from nasal planum to histopathologic examination and DIF test. Five dogs with no skin lesions were included in Group II as negative control to the DIF assay. Two samples of no lesioned sk

Concordancia entre las técnicas de IFD, PCR y ELISA para determinar la frecuencia de Bordetella parapertussis y Bordetella pertussis en un brote de tos ferina en el departamento de Antioquia (Colombia) en 2013 / Correlation among the DIF, PCR and ELISA techniques for determining the frequency of Bordetella parapertussis and Bordetella pertussis in an outbreak of whoopingcough in the department of Antioquia, Colombia, 2013

Objetivo: Obtener la concordancia de técnicas diagnósticas y confirmar el diagnóstico de los casos probables de tos ferina captados por inmunofluorescencia directa (IFD) durante un brote en 2013 en el departamento de Antioquia. Materiales y métodos: Se analizaron los datos demográficos, clínicos, epidemiológicos y de resultados de laboratorio de casos probables de tos ferina confirmados por IFD en un pico de tos ferina en 2013 en el departamento de Antioquia. Las muestras de aspirado nasofaríngeo y suero fueron recolectadas y recibidas entre los periodos epidemiológicos IV - VII de 2013. Todos los pacientes confirmados por IFD fueron confirmados por PCR o ELISA. El análisis de concordancia se realizó por índice kappa. Resultados: De las 180 muestras procesadas en el LSP de Antioquia, 134 (74%) fueron positivas por la técnica de IFD, de las cuales se confirmaron por PCR 109 muestras con 24 (22%) positivas para B. parapertussis , 3 (2,8%) para B. pertussis , 17 (15,6%) para Bordetella spp. y 18 (16,5%) con infección mixta por B. pertussis y B. parapertussis . De 81 casos que se confirmaron por ELISA, 31 (38,3%) fueron positivos. En el municipio de La Estrella la edad media de los casos confirmados fue de 6,6 años y la mediana de 3 años (rango: 2-4 años). Con respecto a los casos del municipio de Medellín, la edad media fue de 28,7 años y la mediana de 25 años (rango: 12-42 años). En su mayoría, en los síntomas no hubo diferencias significativas, excepto para la tos paroxística entre los casos confirmados de B. parapertussis y B. pertussis (p = <0,04) del municipio de La Estrella. De acuerdo con el índice kappa, los resultados mostraron una fuerza de concordancia pobre y sin grado de acuerdo con los resultados de las pruebas de PCR y ELISA comparados con IFD, índice kappa: (IFD/PCR: K = 0,0944) y (IFD/ELISA: K = - 0,4533). Conclusiones: Durante este análisis, en el 2013 la población de Antioquia fue afectada por la circulación de B. parapertussis y B. pertussis en población adolescente y adulta en Medellín y en la población de 2-4 años en La Estrella. Actualmente, la PCR y la ELISA son las técnicas adecuadas para el diagnóstico de tos ferina. La IFD por su subjetividad y baja concordancia se encuentra en desuso.

Objective: To determine the correlation between diagnostic techniques and to confirm the diagnosis of probable cases of whooping cough captured by direct immunofluorescence (DIF) during an outbreak in 2013 in the department of Antioquia. Materials and methods: We analysed the demographic, clinical, and epidemiological data and the laboratory results of probable cases of whooping cough confirmed by DIF at a peak of whooping cough in 2013 in the department of Antioquia. The nasopharyngeal aspirate and serum samples were collected and received between the epidemiological periods IV - VII of 2013. All patients confirmed by DIF were confirmed by polymerase chain reaction (PCR) and/or enzyme-linked immunosorbent assay (ELISA). The analysis of agreement was performed using the kappa index. Results: Of the 180 samples processed in the public health laboratory of Antioquia, 134 (74%) were positive using the DIF technique of which 109 samples were confirmed by PCR, with 24 (22%)samples positive for B. parapertussis , 3 (2.8%) for B. pertussis , 17 (15.6%) for Bordetella spp. and18 (16.5%) for mixed infection by B. pertussis and B. parapertussis . Of the 81 cases confirmed by ELISA, 31 (38.3%) were positive. In the municipality of La Estrella, the mean age of the confirmed cases was 6.6 years, and the median was 3 years (range, 2-4 years). For the municipality of Medellin, the mean age was 28.7 years, and the median was 25 years (range, 12-42 years). For most of the symptoms, there were no significant differences, except for paroxysmal cough among the confirmed cases of B. parapertussis and B. pertussis ( p = <.04) in the municipality of La Estrella. According to the kappa index, the results showed poor correlation strength and no agreement with the results of the PCR and ELISA tests compared with DIF, kappa index: (DIF/PCR: K = 0.0944) and (DIF/ELISA: K = - 0.4533). Conclusions: During this analysis in 2013, Antioquia was affected by the circulation of B. parapertussis and B. pertussis in the adolescent and adult population in Medellin and the 2-4 year-old population in La Estrella. Currently, PCR and ELISA are the recommended techniques for diagnosing whooping cough. Due to its subjectivity and low correlation, DIF is in disuse.

Evaluation of RT-PCR and hemi-nested RT-PCR in brain samples from dogs with neurologic signs compatible with distemper / Avaliação das técnicas de RT-PCR e heminested RT-PCR em cérebros de cães com sinais neurológicos compatíveis com cinomose

The diagnostic value of RT-PCR and hemi-nested RT-PCR (hnRT-PCR) was compared in brain samples of dogs presenting neurological signs compatible with canine distemper. Samples of central nervous system (CNS) were collected from 68 dogs and tested by direct immunofluorescence test (RFID) and, independent of the results, they were stored at -20°C for at least three years. They were submitted to the RT-PCR and hnRT-PCR techniques aiming to determine the gene responsible for the viral nucleoprotein decoding. Fifty-nine samples were positive for RIFD, 40 for RT-PCR (Kappa = 0.358) and 54 for hnRT-PCR (Kappa = 0.740). All nine RIFD negative samples were also negative for RT-PCR and hnRT-PCR. In spite of the storage duration and proper sample conditions, the estimated accordance between hnRT-PCR and RIFD demonstrated that hnRT-PCR technique can be applied in retrospective studies...

Foi comparado o valor diagnóstico das técnicas de RT-PCR e heminested RT-PCR (hnRT-PCR) em amostras de cérebro de cães com sintomatologia nervosa compatível com cinomose. Fragmentos do sistema nervoso central (SNC) colhidos de 68 animais foram testados pela Imunofluorescência direta (IFD) e, independentemente do resultado, foram armazenados a -20°C por pelo menos três anos. Após esse período, foram submetidos a RT-PCR e a hnRT-PCR com oligonucleotídeos iniciadores direcionados ao gene codificador da nucleoproteína N. As proporções de resultados positivos/examinados foram: 59/68 para a IFD, 40/68 para a RT-PCR (Kappa = 0,358) e 54/68 quando associada à heminested PCR (Kappa = 0,740). Houve nove resultados negativos nas três técnicas empregadas. Os resultados do coeficiente Kappa entre a IFD e hnRT-PCR demonstram que apesar das condições de armazenamento, a hnRT-PCR pode ser utilizada em estudos retrospectivos...

Performance of direct immunofl uorescence assay for the detection of human metapneumovirus under clinical laboratory settings

Introduction: Human metapneumovirus (hMPV) is an emergent human respiratory pathogen. This study aimed to evaluate the performance of direct immunofl uorescence (DIF) to detect hMPV in a clinical laboratory setting. Methods: Nasopharyngeal aspirate samples (448) of children and adults with respiratory illness were used to detect hMPV by using DIF and real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. Results: In all, 36 (8%) samples were positive by DIF and 94 (21%) were positive by qRT-PCR. Direct immunofl uorescence specifi city was 99% and sensitivity was 38%. Conclusions: DIF is not very sensitive under clinical laboratory settings. (AU)

Evaluation of RT-PCR and hemi-nested RT-PCR in brain samples from dogs with neurologic signs compatible with distemper / Avaliação das técnicas de RT-PCR e heminested RT-PCR em cérebros de cães com sinais neurológicos compatíveis com cinomose

The diagnostic value of RT-PCR and hemi-nested RT-PCR (hnRT-PCR) was compared in brain samples of dogs presenting neurological signs compatible with canine distemper. Samples of central nervous system (CNS) were collected from 68 dogs and tested by direct immunofluorescence test (RFID) and, independent of the results, they were stored at -20°C for at least three years. They were submitted to the RT-PCR and hnRT-PCR techniques aiming to determine the gene responsible for the viral nucleoprotein decoding. Fifty-nine samples were positive for RIFD, 40 for RT-PCR (Kappa = 0.358) and 54 for hnRT-PCR (Kappa = 0.740). All nine RIFD negative samples were also negative for RT-PCR and hnRT-PCR. In spite of the storage duration and proper sample conditions, the estimated accordance between hnRT-PCR and RIFD demonstrated that hnRT-PCR technique can be applied in retrospective studies(AU)

Foi comparado o valor diagnóstico das técnicas de RT-PCR e heminested RT-PCR (hnRT-PCR) em amostras de cérebro de cães com sintomatologia nervosa compatível com cinomose. Fragmentos do sistema nervoso central (SNC) colhidos de 68 animais foram testados pela Imunofluorescência direta (IFD) e, independentemente do resultado, foram armazenados a -20°C por pelo menos três anos. Após esse período, foram submetidos a RT-PCR e a hnRT-PCR com oligonucleotídeos iniciadores direcionados ao gene codificador da nucleoproteína N. As proporções de resultados positivos/examinados foram: 59/68 para a IFD, 40/68 para a RT-PCR (Kappa = 0,358) e 54/68 quando associada à heminested PCR (Kappa = 0,740). Houve nove resultados negativos nas três técnicas empregadas. Os resultados do coeficiente Kappa entre a IFD e hnRT-PCR demonstram que apesar das condições de armazenamento, a hnRT-PCR pode ser utilizada em estudos retrospectivos(AU)

Enfermedades ampollosas autoinmunes del grupo de los pénfigos / Autoimmune blistering diseases of the pemphigus group

En muchas enfermedades dermatológicas se presentan ampollas, pero no todas son de etiología autoinmune. Para el estudio de las enfermedades ampollosas se deben tener en cuenta las manifestaciones clínicas, la historia de cómo y cuándo empezaron las ampollas, las características epidemiológicas e histológicas (por ejemplo, el nivel de la piel en el que se producen las ampollas) y la presencia o no de infiltrados inflamatorios. Para corroborar la etiología autoinmune de la enfermedad ampollosa es importante contar con los resultados de pruebas como la inmunofluorescencia directa e indirecta, el inmunoblotting, el ensayo inmunoenzimático (ELISA), la inmunoprecipitación y la microscopía electrónica. La información sobre los títulos séricos de autoanticuerpos ayuda a orientar mejor el tratamiento inmunosupresor.

Blisters may appear in many dermatological diseases, but they are not necessarily of autoimmune etiology. For the study of blistering diseases, it is necessary to take into account the clinical aspects, the history of when and how blisters appeared, the epidemiological and histological information (for instance, the skin level at which blisters are located), and whether inflammatory infiltrates are present. In order to corroborate the autoimmune etiology of blisters, it is important to have the results of confirmatory tests such as direct and indirect immunofluorescence, immune blotting, enzyme-linked immune-assay (ELISA), immune precipitation, and electronic microscopy. Information on autoantibodies serum titers may help to conduct a more precise immunosuppressive therapy.

Identificación de virus respiratorios en pacientes pediátricos / Identification of respiratory viruses in pediatric patients

Los virus son la principal causa de infecciones respiratorias agudas. Para la detección de los virus respiratorios se emplea la Inmunofluorescencia directa (IFD). Objetivos: Describir los resultados de las IFD positivas según edad de los pacientes y estacionalidad y determinar el porcentaje de positividad. Material y métodos: Estudio descriptivo retrospectivo. Se incluyeron todas las IFD realizadas en pacientes <15 años en el Hospital de Niños de Viña del Mar entre enero 2012 y noviembre 2013. Resultados: Se realizaron 32015 IFD en 9012 pacientes en ambos años. El virus más frecuente es el Virus Respiratorio Sincicial (VRS) (51 por ciento 2012, 63 porciento 2013), obteniendo un mayor número de casos en pacientes menores de 1año. El Adenovirus (ADV) ocupa el segundo lugar con una incidencia uniforme a lo largo del año. Se observó una disminución de los casos de Influenza A y B desde el año 2012 al 2013. El porcentaje de positividad global de los exámenes es muy bajo (4,25 por ciento 2012 y 3,19 por ciento 2013). El VRS alcanza la mayor positividad en invierno (31,8 por ciento 2012 y24,7 por ciento 2013) y llega al 0 por ciento durante el verano. Conclusiones: Nuestros resultados se asemejan a la epidemiología nacional principalmente en la distribución de los virus según edad y estacionalidad. El virus más frecuentemente encontrado es el VRS. Hubo menos casos de Influenza A durante el 2013 en comparación con los resultados en el país. Existe una solicitud aumentada de exámenes lo que lleva a un porcentaje de positividad muy bajo...

The main cause of acute respiratory infections is viral. The direct immunofluorescence (IF) is used in the detection of respiratory viruses. Objectives: To describe the results of positive IF in relationship to the patient´s age and seasonality and to determine the percentage of positive results. Material and Methods: A descriptive retrospective study that includes all IF performed in patients <15 years old in a private clinic in the V region from January 2012 to November 2013. Results: 32015 IF were conducted in 9012 patients during both years. The Respiratory Syncytial Virus (RSV) is the most common virus (51 percent 2012, 63 percent 2013), obtaining a larger number of cases in patients under the age of 1. The second most common virus is Adenovirus (ADV) with a uniform incidence during the year. A decrease in cases of Influenza A and B was observed from 2012 to 2013. The percent positivity of the tests is very low (4.25 percent and 3.19 percent 2012 2013). The RSV positive testing is higher during the winter (31.8 percent and 24.7 percent 2012 2013) and 0 percent during the summer. Conclusions: Our results are similar to the ones obtained by the national epidemiology in terms of age and season relationship. The RSV is the most common virus. There were fewer cases of influenza A in 2013 compared with results in the country. Due to high demand in IF testing, a very low percentage of positive results is observed...