Diagnosis failure of bovine leukosis: serology variation during the peripartum period.

This study demonstrates the influence of pregnancy on serum diagnosis of enzootic bovine leukosis (EBL), emphasizing the importance of routine testing to maintain herd health. For this, 143 pregnant cows were sampled in duplicate (30 days before and 15 days after calving). For EBL diagnosis, samples were submitted to agar gel immunodiffusion testing (AGID). Different results were observed before and after delivery in seventy-six serum samples (53.15%), indicating variations in the levels of serum globulins in the blood during the peripartum period. Therefore, using a single sample for serological diagnosis during the birth season might not represent the correct infection status of animal health due to physiological variations in antibody concentrations.

Risk factors and detection of brucella ovis in naturally infected rams and ewes / Fatores de risco e detecção de brucella ovis em carneiros e ovelhas naturalmente infectados

The objective of this study was to verify the occurrence of ovine brucellosis using Agar Gel Immunodiffusion (AGID) and Polymerase Chain Reaction (PCR) techniques, as well as to identify the main risk factors associated with infection in sheep flocks belonging to municipalities in the microregion from Teresina, PI, Brazil. A total of 100 urine and blood samples were collected from sheep aged 6 months or older. The urine samples were submitted to conventional PCR and the blood samples were examined by the AGID technique. Of the 100 blood samples, 17 (17%) were reactive to the AGID test. In conventional PCR of 100 urine samples, six (6%) were positive. Risk factors associated to infection by B. ovis included the rearing system (OR=0.19), feed management (OR=0.05), presence of dystotic births (OR=4.50), miscarriages (OR=3.75) and source of water offered to the animals (OR=0.19). Thus, it was concluded that it is possible to detect the occurrence of animals with ovine brucellosis since PCR is a reliable method to confirm infection. Furthermore, there are risk factors associated to infection by B. ovis in the municipalities studied.

Objetivou-se verificar a ocorrência da brucelose ovina através das técnicas de Imunodifusão em Gel de Ágar (IDGA) e Reação em Cadeia da Polimerase (PCR), bem como identificar os principais fatores de risco associados à infecção nos rebanhos ovinos pertencentes a municípios da microrregião de Teresina, PI, Brasil. Foram colhidas 100 amostras de urina e de sangue de ovinos com idade superior ou igual a seis meses. As amostras de urina foram submetidas a PCR convencional e as amostras de sangue à técnica de IDGA. Das 100 amostras de sangue 17 (17%) foram reagentes ao teste de IDGA. Já na PCR convencional das 100 amostras de urina, seis (6%) foram positivas. Ressalta-se que três animais foram positivos em ambos os testes. Como fatores associados à infecção por B. ovis, observou-se o tipo de sistema de criação (OR=0,19), o manejo alimentar (OR=0,05), presença de partos distócicos (OR=4,50), abortamentos (OR=3,75) e a fonte de água fornecida aos animais (OR=0,19). Assim, conclui-se que foi possível detectar a ocorrência de animais com brucelose ovina, uma vez que a PCR é um método confirmatório. Além disso, há fatores de risco associados à infecção por B. ovis nos municípios estudados.

Síntesis intratecal de IgG anti-herpes virus como evidencia neuroinmunológica en pacientes pediátricos / Intrathecal synthesis of IgG against herpes virus as neuroimmunological evidence in pediatric patients

En los procesos neuroinflamatorios se produce a nivel de líquido cefalorraquídeo una activación policlonal y poliespecífica. Esta activación se produce desde los primeros días y puede permanecer por períodos prolongados. Luego por mecanismos de apoptosis los clones que no responden directamente contra los agentes biológicos involucrados no proliferan. El Reibergrama permite saber si las inmunoglobulinas presentes en el líquido cefalorraquídeo se sintetizaron o no en el sistema nervioso central (SNC) y el Índice de Anticuerpo (IA) determina la especificidad de las mismas en caso de que exista síntesis intratecal. Con estas herramientas nos propusimos identificar la respuesta neuroinmunológica frente a agentes de la familia herpesvirus en pacientes pediátricos con proceso inflamatorio del SNC a partir de sus respectivos IA. Para lograr esto se cuantificaron los niveles de IgG y albúmina en suero y líquido cefalorraquídeo (LCR) mediante inmunodifusión radial simple y por ensayo inmunoenzimático, con lo cual se construyó el Reibergrama que permitió la selección de 85 pacientes pediátricos con síntesis intratecal de inmunoglobulinas, que se diferenciaron en cuatro grupos según sus edades. Mediante ensayo inmunoenzimático se cuantificaron los niveles de IgG específica contra citomegalovirus, virus varicela zoster y virus herpes simple, tanto en suero como en LCR y se determinó el IA específico. La respuesta contra los virus estudiados fue similar para los distintos grupos de edades, lo cual nos permite afirmar la exposición temprana a los mismos(AU)

In a neuroinflammatory process a polyclonal and poly-specific activation is produced in cerebrospinal fluid. This activation starts from the first days and may persist for a long time. The clones not related directly against the biological agent do not proliferate by apoptosis. Reibergram determine if part of the immunoglobulins content in cerebrospinal fluid belongs from the blood or it is synthesized in the central nervous system. Antibody index determines if the specific antibodies was synthesized intrathecally. By these tools it can be possible to identify the humoral immune response against some herpes virus in pediatric patients suffering from a central nervous system inflammatory process. Quantification of specific IgG against citomegalovirus, varicella zoster and herpes simplex virus in serum and cerebrospinal fluid was done by ELISA. Specific Antibody index against these viruses were similar for the different age groups, which confirm the early exposure of the population(AU)

Performance assessment of imported ELISA in the serodiagnosis of the enzootic bovine leukosis in herds of Pernambuco state, Brazil / Avaliação do desempenho de ELISA importado no sorodiagnóstico da leucose enzoótica bovina em rebanhos do estado de Pernambuco, Brasil

Enzootic bovine leukosis (EBL) is an infectious disease of cosmopolitan distribution and chronic character caused by a virus of the Retroviridae family, bovine leukemia virus (BLV). The epidemiological situation of EBL in Brazil has motivated studies to improve its diagnosis, based on the recommended serological techniques: agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). This study was designed to evaluate the use of imported ELISA for the detection of BLV in dairy herds raised in Pernambuco, Brazil, comparing it to AGID. Blood serum samples from 327 dairy cattle from the state of Pernambuco were tested to AGID and the imported commercial ELISA CHEKIT-Leucose-serum, produced by the IDEXX® laboratory for the diagnosis of EBL. Discarding 25 inconclusive samples from one or both tests, 302 samples were analyzed, being 24.1% positive (73/302) in the AGID and 45% (136/302) in the ELISA, which compared to the AGID, a technique considered standard, presented sensitivity of 98.6%, specificity of 72% and Kappa coefficient of 0.55. The lack of agreement in the diagnostic methods was probably due to the high sensitivity of the ELISA, which makes it possible to detect antibodies even in situations with low serum levels. Although AGID has been shown to be an efficient test so far, in more advanced stages of an EBL control and eradication program, with low prevalence rates, ELISA will present better performance, due to its higher sensitivity, avoiding the permanence of animals that spread the disease in the herds.(AU)

A leucose enzoótica bovina (LEB) é uma doença infecciosa de distribuição cosmopolita e caráter crônico causada por um vírus da família Retroviridae, o vírus da leucemia bovina (VLB). A situação epidemiológica da LEB no Brasil vem motivando estudos para o aprimoramento do seu diagnóstico, tendo como base as técnicas sorológicas recomendadas: imunodifusão em gel de ágar (IDGA) e Enzyme-Linked Immunoabsorbent Assay (ELISA). Este estudo teve como objetivo avaliar o uso de ELISA importado para a detecção do VLB em rebanhos leiteiros criados em Pernambuco, Brasil, comparando-o ao IDGA. Amostras de soro sanguíneo de 327 bovinos leiteiros do estado de Pernambuco foram testadas para IDGA e ELISA comercial importado CHEKIT-Leucose-serum, produzido pelo laboratório IDEXX® para o diagnóstico da LEB. Descartadas 25 amostras inconclusivas de um ou ambos os testes, foram analisadas 302 amostras, sendo 24,1% positivas (73/302) na IDGA e 45% (136/302) no ELISA, que em relação à IDGA, técnica considerada padrão, apresentou sensibilidade de 98,6%, especificidade de 72% e coeficiente Kappa de 0.55. A falta de concordância entre os métodos diagnósticos deveu-se, provavelmente, à elevada sensibilidade do ELISA, que possibilita detectar anticorpos mesmo em situações com baixos teores séricos. Apesar da IDGA se mostrar até o momento um teste eficiente, em etapas mais avançadas de um programa de controle e erradicação da LEB, com baixos índices de prevalência, o ELISA apresentará melhor desempenho, por possuir maior sensibilidade, evitando-se a permanência de animais disseminadores da doença nos rebanhos.​(AU)

Neurobrucelosis evaluada por reibergrama. Presentación de un caso / Neurobrucellosis evaluated by reibergram. A case report

Introducción: La brucelosis es una enfermedad zoonótica y endémica en muchas partes del mundo. La causa principal de la infección se produce por la ingestión de leche no pasteurizada o por el contacto con animales infectados. La neurobrucelosis incluye afecciones en el sistema nervioso central y periférico. Las principales manifestaciones clínicas son la meningitis, la encefalitis, la neuritis óptica y la periférica. Objetivo: Evaluar, mediante reibergrama, la dinámica intratecal de las clases mayores de inmunoglobulinas y el estado de la barrera sangre/LCR de un paciente con neurobrucelosis. Presentación del caso: Los niveles de IgA, IgM. IgG y albúmina en suero y líquido cefalorraquídeo fueron cuantificados por inmunodifusión. Los resultados fueron colocados en el reibergrama correspondiente. El paciente mostró síntesis intratecal de las tres clases mayores de inmunoglobulinas, sin disfunción de la barrera sangre/LCR. Conclusión: El estudio neuroinmunológico del líquido cefalorraquídeo puede indicar el curso activo de la respuesta inmune intratecal contra el patógeno, donde la síntesis intratecal de inmunoglobulinas y el funcionamiento de la barrera sangre/líquido cefalorraquídeo constituyeron los principales marcadores en el diagnóstico de la neuroinflamación(AU)

Introduction: Brucellosis is a zoonotic and an endemic disease in many areas around the world. The main cause of infection is the intake of unpasteurized milk or the contact with infected animals. Neurobrucellosis includes pathologic conditions in the central and peripheral nervous systems. The main clinical manifestations are meningitis, encephalitis, optical neuritis, and peripheral neuritis. Objective: To evaluate, through reibergram, the intrathecal dynamics of the major immunoglobulin classes and the blood-CSF barrier function in one patient with neurobrucellosis. Case report: IgA, IgM, IgG and albumin levels in serum and cerebrospinal fluid were quantified by using a radial immunodiffusion technique. Results were placed in the corresponding reibergram. The patient showed evidences of intrathecal synthesis of the three major immunoglobulins without blood-CSF barrier dysfunction. Conclusion: The neuroimmunological study of cerebrospinal fluid can indicate the active course of the intrathecal immune response against this pathogen, where the intrathecal synthesis of immunoglobulins and blood-cerebrospinal fluid barrier function constitute the main markers in the diagnosis of neuroinflammation(AU)

Optimización del uso de las Placas de Inmunodifusión Radial Simple de la firma Siemens / Optimization of the usage of single radial immunodiffusion plates from Siemens

Introducción: La Inmunodifusión radial simple es una técnica con fundamento inmunológico confiable por su especificidad para la cuantificación de inmunoglobulinas principales y se emplea también para otras proteínas. Las placas de Inmunodifusión comerciales se ofertan con un número determinado de pocillos donde se coloca la muestra biológica que contiene la proteína a cuantificar. Objetivo: Evaluar la sensibilidad y la especificidad de la modificación introducida para optimizar el uso de las placas de inmunodifusión radial simple de la marca SIEMENS por aumento del número de muestras por placas. Material y Métodos: Se presenta una innovación que permite optimizar el área biológicamente activa de la placa no utilizada para emplearla para la cuantificación de otras muestras. Se realizan montajes paralelos de muestras de controles en los pocillos tradicionales y en los realizados en los espacios disponibles para cuantificar IgG y albúmina para suero y líquido cefalorraquídeo. Resultados: La sensibilidad del empleo por el método tradicional y por el nuevo no presenta diferencias significativas. En cuanto a la especificidad tampoco existen diferencias significativas menos en las placas para cuantificar albúmina en suero por lo que se recomienda diluir la muestra de suero antes de ser utilizada en el área disponible. En el caso de las placas NOR y LC Partigen® el número de muestras a ser beneficiadas con la cuantificación se duplica, pero de igual manera puede ser aplicada en otras placas de otras firmas comerciales. Conclusiones: Esta innovación permite hacer un uso óptimo de las placas de inmunodifusión con el consiguiente ahorro de material de importación y se puede aplicar fácilmente en todos los laboratorios del país(AU)

Introduction: Single radial immunodiffusion assay is a technique with immunological base, which is reliable because of its specificity in the quantification of main immunoglobulins, although it is also used for other proteins. Commercial immunodiffusion plates are offered with a determined number of holes where the biological samples containing protein to be quantified are placed. Objective: To evaluate the sensitivity and specificity of the modification implemented to optimize the usage of single radial immunodiffusion plates from Siemens by increasing the number of samples in the plates. Materials and Methods: An innovating procedure that allows to optimize the non-used biologically active area and use it in the quantification of other samples is presented. A parallel quantification of control samples from traditional holes and the other ones opened in available spaces was performed in order to quantify IgG and albumin in serum and in cerebrospinal fluid. Results: Sensitivity was not affected significantly between the normal plates and the usage of the new procedure. Regarding specificity, there are also no significant differences except in the plates used to quantify serum albumin; so, it is recommended to dilute serum samples before the application. In case of NOR and LC Partigens®, this proposed modification duplicates the number of samples to be quantified in each plate, but otherwise, it could be applied in other commercial immunoplates. Conclusions: This innovation allows to make an optimal usage of immunodiffusion plates with the consequent saving of import materials, which can be easily applied in all the laboratories of the country(AU)

Enzyme-linked immunosorbent assay and agar gel immunodiffusion assay for diagnosis of equine infectious anemia employing p26 protein fused to the maltose-binding protein.

A codon-optimized equine infectious anemia virus p26 gene was fused to a maltose-binding protein (MBP) and expressed in Escherichia coli for use as an antigen in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for diagnosis of equine infectious anemia. An analysis of analytical sensitivity and specificity showed that the antigen MBP-p26rec reacted positively with a reference World Organization for Animal Health serum and demonstrated no cross-reaction against sera from vaccinated animals in either test. The diagnostic characteristics were evaluated and presented excellent values. The AGIDrec showed 100% sensitivity and specificity, and the ELISArec showed 100% sensitivity and 99.64% specificity. In addition, MBP-p26rec was stabile after three years of storage at 4 °C, maintaining its immunoreactivity.

Absence of Bovine leukemia virus (BLV) infection in buffaloes from Amazon and southeast region in Brazil.

Enzootic bovine leucosis is an infectious disease caused by Bovine leukemia virus (BLV) and is well described in bovines. The majority of infected animals are asymptomatic, one to five percent develop lymphoma and from 30 to 50% present a persistent lymphocytosis. The virus occurs naturally in cattle and experimentally in buffaloes, capybaras and rabbits. The occurrence of lymphoma in buffaloes has been attributed to BLV infection by some authors in India and Venezuela, but not confirmed by other studies and little information on natural BLV infection in buffaloes is available. The aim of this study was to evaluate the occurrence of BLV in a sub-sample of buffalo from Amazon and southeast regions in Brazil. Three hundred and fifteen serum samples were negative using commercial AGID and ELISA (ELISA-gp51) which detect anti-BLV glycoprotein gp51 antibodies. The same samples were also evaluated for antibodies to whole virus through a commercial ELISA (ELISA-BLV) in which 77 (24.44%) were found seropositive and two (0.63%) inconclusive. On the other hand, all animals were negative by PCR to BLV targeted to the env and tax genes. These results suggest that ELISA-BLV produces false positive results in buffalo serum (p<0.001). In addition, one buffalo lymphoma sample was negative in both PCR assays used in this study. BLV was not detected in buffaloes from the Amazon basin and the southeast region of Brazil. Serological tests, like ELISA-BLV, usually used for cattle may produce false-positive results for BLV in buffaloes and direct detection tests such as PCR should be chosen in these surveys. The occurrence of lymphoma in buffalo was not associated with BLV infection in the one case analyzed in this work and the etiology and pathogenesis of this disease should be clarified.

Production of pseudorabies virus recombinant glycoprotein B and its use in an agar gel immunodiffusion (AGID) test for detection of antibodies with sensitivity and specificity equal to the virus neutralization assay.

Pseudorabies virus (PrV) causes Aujeszky's disease (AD), which affects mainly swine, but also cattle, sheep, and wild animals, resulting in substantial economic losses due to animal mortality and lost productivity worldwide. To combat PrV, eradication programs using PrV strains lacking the gene encoding glycoprotein E (gE) are ongoing in several countries. These eradication programs have generated a currently unmet demand for affordable, easy-to-use, and sensitive tests that can detect PrV infection in pigs infected with either wild-type virus or vaccine strain (gE-deleted) virus. To meet this demand, we used the baculovirus-insect cell system to produce recombinant glycoprotein B (gB) as antigen for an immune assay. The high GC-content (70% average) of the gB gene from the Argentinian PrV CL15 strain necessitated the use of betaine as a PCR enhancer to amplify the extracellular domain. Recombinant gB was expressed at high levels and reacted strongly with sera from PrV infected pigs. We used the recombinant gB to develop an agar gel immunodiffusion (AGID) test for detection of PrV antibodies. Compared to the gold standard virus neutralization (VN) assay, the AGID sensitivity and specificity were 95% and 96.6% respectively. Thus, recombinant gB produced in the baculovirus-insect cell system is a viable source of antigen for the detection of PrV antibodies in AGID tests. Considering its relatively lower cost, simplicity of use and result interpretation, our AGID is a valuable alternative tool to the VN assay.

Diagnóstico serológico de la esporotricosis mediante el empleo del antígeno de micelio de Sporothrix schenckii sensu stricto / Serological diagnosis of sporotrichosis using an antigen of Sporothrix schenckii sensu stricto mycelium

En este estudio se desarrolló y se evaluó el ensayo por inmunoabsorción ligado a enzimas (ELISA), para la detección de anticuerpos en sueros de pacientes con esporotricosis, para lo cual se empleó un antígeno crudo de Sporothrix schenckii sensu stricto obtenido a partir de la forma micelial. Los sueros positivos para esporotricosis fueron ensayados por otras técnicas serológicas: inmunodifusión doble (IDD) y contrainmunoelectroforesis (CIE). El ensayo fue validado utilizando sueros de otras patologías como histoplasmosis, paracoccidioidomicosis, tuberculosis, leishmaniasis, lupus y sueros de individuos sanos como controles negativos. Se encontró una especificidad de 100 % con las técnicas utilizadas y una sensibilidad del antígeno de S.schenckii sensu stricto, por encima del 98% para IDD, CIE y ELISA. Estos resultados demuestran la alta sensibilidad y especificidad del antígeno de S. schenckii sensu stricto, para el diagnóstico de la esporotricosis, empleando las técnicas de IDD, CIE y ELISA. Los resultados sugieren, que este antígeno podría ser usado en conjunto con otras pruebas convencionales para el diagnóstico diferencial y puede ser útil para monitorizar la evolución de la enfermedad y respuesta al tratamiento.

We developed and analyzed an Enzyme-Linked Immunosorbent Assay (ELISA) in order to detect antibodies in sera from sporotrichosis patients. We used a crude antigen of Sporothrix schenckii sensu stricto, obtained from the mycelial phase of the fungi. Positive sera were analyzed by other serological techniques such as double immunodiffusion (IGG) and counterimmunoelectrophoresis (CIE). The assay was validated by using sera from patients with other pathologies such as: histoplasmosis, paracoccidioidomycosis, tuberculosis, leishmaniasis, lupus and healthy individuals as negative controls. For the Sporothrix schenckii sensu stricto antigen, we found a 100% of specificity by every technique and sensitivity higher than 98% with IDD, CIE and ELISA. Our results show a high sensitivity and specificity for the Sporothrix schenckii sensu stricto antigen, so it can be used for IDD, CIE and ELISA. The results suggest that this antigen could be used in conjunction with other conventional tests for differential diagnosis and may be useful for monitoring the disease progression and response to treatment.

Ferramentas diagnósticas de Lentivirose de Pequenos Ruminantes: padronização da técnica de ELISA indireto / Diagnostic tools of small ruminant lentiviruses: standardization of indirect ELISA

As Lentiviroses de Pequenos Ruminantes (LVPR) incluem a Maedi-Visna (MV) em ovinos e a Artrite Encefalite Caprina (CAE). Essas enfermidades estão difundidas no mundo e são responsáveis por grandes perdas na produtividade destes animais. Os LVPR são vírus RNA da subfamília Lentivirinae que causam uma infecção persistente, sendo a detecção precoce uma das formas mais eficientes para limitar sua disseminação no rebanho. Visando contribuir com essas questões, este experimento foi realizado na Universidade Federal do Piauí (UFPI) em parceria com a Embrapa Caprinos e Ovinos, com o objetivo de padronizar a técnica de ensaio imunoenzimático indireto e compará-lo com a imunodifusão em gel de agarose no diagnóstico da CAE. Foram utilizadas 696 amostras de soros de caprinos machos e fêmeas oriundas do banco de soros da Unidade de Pesquisa de LVPR do Centro de Ciências Agrárias da UFPI. As amostras foram coletadas no período de janeiro de 2007 a março de 2010. Na padronização, verificou-se que 0,25 µg de proteína/poço, diluição de 1:200 do soro e concentração de 1:3.000 do conjugado anticorpo anti-IgG cabra apresentaram os melhores resultados. O ponto de corte obtido foi de 0,36. Na comparação, o Imunodifusão em Gel de Ágar (IDGA) detectou 128 (18,4%) amostras positivas, e o ELISA indireto (ELISA-i), 259 (37,2%). A sensibilidade e a especificidade do teste ELISA-i com relação ao IDGA foi de 94,5% e 75,7%, respectivamente. Verificou-se maior índice de positividade em caprinos acima de seis meses (p < 0,05), e nos machos obteve-se prevalência de 56,7% em comparação às fêmeas, 35,4%, (p < 0,01).(AU)

The Small Ruminant Lentiviruses (SRLVs) include Maedi-Visna (MV) of sheep and Caprine Arthritis-Encephalitis (CAE). These diseases are widespread and responsible for major production losses regarding sheep and goats. The SRLV is a RNA virus of the subfamily Lentivirus genus that causes persistent infections in goats. Early detection is one of the best ways to limit its spread in the herd. To contribute to these issues, this experiment was conducted at Universidade Federal do Piauí in partnership with Embrapa Goats and Sheep, with the objective of standardizing the technique of indirect ELISA (i-ELISA) and to compare it with Immunodiffusion in Agarose Gel to diagnose Caprine Lentiviruses (LC). Six hundred ninety six serum samples were used from the University Veterinary Hospital, Universidade Federal do Piauí, from January 2007 to March 2010. Standardization showed that 0.25 µg protein/well, a 1:200 dilution of the serum and concentration of 1:3,000 of the conjugated anti-goat IgG presented the best results. It was observed that the Agar Gel Immunodiffusion (AGID) detected 128 (18.4%) positive samples, and ELISA, 259 (37.2%). The sensitivity and specificity of i-ELISA regarding AGID were 94.5% and 75.7%, respectively. A higher prevalence was observed among animals older than six months (p < 0.05). The prevalence among males was of 56.7%, and among females, 35.4% (p < 0.01).(AU)

Peroxidase-linked assay for detection of antibodies against bovine leukosis virus.

A peroxidase linked assay (PLA) was designed to screen bovine sera for the presence of specific antibodies against bovine leukosis virus (BLV). Out of 201 samples of bovine sera analyzed, 52.2% were considered positive by PLA, 26.4% by AGID, and 38.9% by ELISA. Western blotting analyses excluded 27 samples found to be positive by PLA. PLA showed 100% of sensitivity when compared with AGID and ELISA. Specificity was 64.8% and 78%, respectively (kappa coefficients were 0.70 and 0.83). These findings indicate that PLA can be used as an alternative method for the diagnosis of BLV infection in cattle.

Técnicas electroforéticas en el HYDRASYS 2. Utilidad diagnóstica en diferentes enfermedades / Electrophoretic techniques in HYDRASYS 2. Diagnostic usability in different diseases

Se presentan los resultados de la estandarización de las técnicas de electroforesis de hemoglobina (Hb), isoenzimas de la deshidrogenasa láctica (LDH) y proteinuria en el equipo Hydrasys 2, así como el estudio de pacientes atendidos en el Instituto de Hematología e Inmunología y en otros centros hospitalarios del país. Se realizó el diagnóstico de 149 portadores de hemoglobinopatías (AS, AC, b talasemia heterocigótica, variante rápida), 60 enfermos (SS, SC, CC), 24 pacientes con a talasemia o deficiencia de hierro y se cuantificó la hemoglobina fetal a 93 casos con hemoglobinopatía S. Se determinaron los valores normales de actividad e isoenzimas de LDH en la población mediante el estudio de 50 donantes de sangre. En los pacientes con anemia drepanocítica se encontró un aumento significativo de la isoenzima 1 (p= 0,000) y disminución de isoenzimas 3 (p= 0,002). Se realizó el estudio de proteínas en orina a 8 pacientes con enfermedades hematológicas que presentaron microalbuminuria al menos en 2 ocasiones, con concentraciones ³ 0,04 g/L. En 2 pacientes el resultado fue normal; en 2 se encontraron proteínas de origen tubular; y en otros 2, proteínas de origen glomerular

We present the results of the standardization of the techniques of electrophoresis of hemoglobin (Hb), lactate dehydrogenase isoenzymes (LDH) and proteinuria in HYDRASYS 2 equipment, and the study of patients treated at the Institute of Hematology and Immunology and other hospitals in the country. 149 hemoglobinopathies carriers were diagnosed (AS, AC, b thalassemia heterozygous fast variant), 60 patients (SS, SC, CC), 24 patients with athalassemia or iron deficiency. Fetal hemoglobin was quantified in 93 cases with hemoglobinopaty S. Normal values of activity and LDH isoenzymes were determined in the population through the study of 50 blood donors. In patients with sickle cell anemia we found a significant increase in isoenzyme 1 (p=0.000) and isozyme 3 decreased (p=0.002). We performed the study of proteins in urine in 8 patients with hematologic malignancies who had microalbuminuria at least 2 times, with concentrations ³ 0.04 g / L. In 2 patients the results were normal, in 2 proteins were tubular origin, and in 2, proteins of glomerular origin

Development of agar diffusion method for dosage of gramicidin / Desenvolvimento do método de difusão em agar para dosagem de gramicidina

Gramicidin, an antimicrobial peptide active against Gram positive bacteria, is commonly used in pharmaceutical preparations for topical use. Considering that only the turbidimetric method has been described in the literature, the present study sought to develop and validate an agar diffusion method for the dosage of gramicidin. The method was developed and validated using the Kocuria rhizophila ATCC 9341 as a test microorganism. Two designs were used: a 3x3 parallel-line model, and a 5x1 standard curve. The validation demonstrated that the method follows the linear model (r²= 0.994), presenting a significant regression between the zone diameter of growth inhibition and the logarithm of the concentration within the range of 5 to 25.3 µg/mL. The results obtained for both designs were precise, having a relative standard deviation (R.S.D.) for intra-day precision of 0.81 for the 3x3 assay and 1.90 for the 5x1 assay. For the inter-day precision, the R.S.D. was 1.35 for the 3x3 and 2.64 for the 5x1. The accuracy was verified and results confirmed to be accurate, having a tolerance interval of 95 percent, which lay within permitted limits and appropriate trueness. In addition, the method was considered selective, with limit of detection and upper and lower limits of quantification of 2.00, 5.00 and 25.3 µg/mL, respectively. No difference in precision between the designs used in the agar diffusion method was evident (p>0.05). The method proved to be appropriate for the microbiological dosage of the raw material gramicidin.

A gramicidina, um peptídeo antimicrobiano ativo contra bactérias Gram positivo, é utilizada em preparações farmacêuticas de uso tópico. Neste trabalho procurou-se desenvolver e validar outro método para o doseamento de gramicidina tendo em vista que somente o método turbidimétrico é descrito. O método de difusão em ágar foi desenvolvido e validado utilizando como microrganismo teste Kocuria rhizophila ATCC 9341. Foram utilizados dois delineamentos: retas paralelas 3x3 e curva padrão 5x1. A validação demonstrou que o método segue o modelo linear (r²= 0,994) havendo regressão significativa entre o diâmetro dos halos de inibição e o logaritmo da concentração na faixa de 5,00 a 25,3 µg/mL. Os resultados obtidos por ambos os delineamentos foram precisos apresentando desvio padrão relativo (DPR) para precisão intra-dia de 0,81 para ensaio 3x3 e de 1,90 para ensaio 5x1. Para a precisão inter-dias o DPR foi de 1,35 para 3x3 e de 2,64 para 5x1. A exatidão foi verificada e os resultados foram exatos apresentando intervalo de tolerância a 95 por cento dentro dos limites permitidos e veracidade adequada. O método foi seletivo com limites de detecção e quantificação inferior e superior iguais a 2,00, 5,00 e 25,3 µg/mL, respectivamente. Não foi observada diferença entre a precisão dos delineamentos empregados no método de difusão em ágar (p>0.05). O método se mostrou adequado para a dosagem microbiológica de gramicidina matéria-prima.

Accuracy of routine diagnostic tests used in paracoccidioidomycosis patients at a university hospital.

The identification of appropriate laboratory measures to confirm clinical hypotheses is important in routine paracoccidioidomycosis medical care. The clinical records and laboratory reports of 401 paracoccidioidomycosis patients attended at the Tropical Diseases Area, Faculdade de Medicina de Botucatu, from 1974 to 2008 were reviewed. Direct mycological (DM), cell block (CB), histopathological (HP), and double immunodiffusion (DID) tests were evaluated before treatment. Typical Paracoccidioides brasiliensis yeast forms were observed in clinical specimens of 86% of the patients, but 14% were detected only by serological test. DM of 51 different tissue specimens produced 74.5% sensitivity, and 62.5% sensitivity was observed in 112 sputum samples. CB in 483 sputum samples generated 55.3% sensitivity. HP performed in 239 samples from different tissues revealed 96.7% sensitivity. Serology carried out in 351 patients and 200 healthy controls provided 90.0% sensitivity, 100.0% specificity, 100.0% positive predictive value, 85.1% negative predictive value and 93.6% accuracy. Comparisons of laboratory measurements performed in the same patient showed that sensitivity decreases from HP to DID to CB and DM, with the last two assays providing similar sensitivities. This study demonstrated that P. brasiliensis identification by HP, CB, and/or DM associated with DID is sufficient to establish the laboratorial diagnosis of paracoccidioidomycosis in practically all cases.

Diffusional restrictions in glyoxyl-agarose immobilized penicillin G acylase of different particle size and protein loading / Restricciones para la difusión de penicilina G acilasa inmovilizada en glicosil agarosa

Particle size and enzyme protein loading are design parameters of enzyme immobilization affecting biocatalyst performance that can be varied within broad margins. Their effect on mass transfer limitations at different bulk penicillin G concentrations has been studied with glyoxyl agarose immobilized penicillin G acylase biocatalysts of average particle size of 5·10-5m and 10·10-4m at protein loadings from 15 to 130 mg/g gel. Internal diffusional restrictions were evaluated for such biocatalysts: Thiele modulus varied from 1.17 for the small particles at the lower protein load to 5.84 for the large particles at the higher protein load. Effectiveness factors at different bulk substrate concentrations were determined for all biocatalysts, values ranging from 0.78 for small particle size at 25 mM penicillin G to 0.15 for large particle size at 2 mM penicillin G. Enzyme protein loading had a strong impact on the effectiveness factors of immobilized penicillin G acylase, being it more pronounced in the case of large particle size biocatalysts. At conditions in which 6-aminopenicillanic acid is industrially produced, all biocatalysts tested were mass-transfer limited, being this information valuable for reactor design and performance evaluation.

An atypical Paracoccidioides brasiliensis clinical isolate based on multiple gene analysis.

An atypical isolate of Paracoccidioides brasiliensis (IFM54648), recovered from the sputum of a Brazilian man, was not detected in immunodiffusion tests for paracoccidioidomycosis and in species-specific PCR for the major antigen 43-kDa glycoprotein coding gene (gp43). The mycological characteristics of the isolate were similar to those of a typical P. brasiliensis. A total of 8 genes were sequenced from IFM54648, and the sequences were compared between the new isolate and other reference isolates and database sequences. We analyzed fragments of the gene sequences that code for gp43, the internal transcribed spacer regions of ribosomal RNA, the D1/D2 domains of the large subunit ribosomal RNA, glucan synthase, chitin synthase, glyoxalase I mRNA, 70-kDa heat-shock protein mRNA and urease. The gene sequences were 98.9-100% identical between IFM54648 and Pb01 (another atypical isolate). When compared to the other typical isolates, the identities were generally lower than 98%. A phylogenetic tree constructed using gp43 sequences showed that IFM54648 clustered with Pb01 at a considerable distance from other isolates. Therefore, this isolate is likely related to Pb01, which has recently been shown to be genetically distinct from other isolates of this species.