Multiplex sequence-specific polymerase chain reaction reveals new MASP2 haplotypes associated with MASP-2 and MAp19 serum levels.

Deficiency of mannan-binding lectin-associated serine protease 2 (MASP-2) has been associated with infections, whereas high levels appear to increase the risk of inflammatory disorders. Nevertheless, MASP2 haplotypes have been poorly investigated. To overcome haplotyping cost and time consumption, we developed multiplex polymerase chain reactions with sequence-specific primers (PCR-SSP) for 8 single nucleotide polymorphisms (SNPs), reducing the number of necessary reactions from 18 to 7. SNPs were distributed from the promoter to the last exon, and a single PCR-SSP was used for p.D120G. We evaluated the phylogenetic relationships and global distribution of 10 identified haplotypes in 338 Danish individuals with known MASP-2 and MAp19 levels and 309 South Brazilians. Four haplotypes were associated with reduced MASP-2 levels in plasma (lower than 200 ng/mL). Simultaneous association with the highest MASP-2 (over 600 ng/mL) and lowest MAp19 levels (lower than 200 ng/mL) was demonstrated with the intron 9 mutation (Kruskal-Wallis p < 0.0001). Cumulative genotype frequencies predict approximately 0.4% severely deficient and 25% overproducing individuals in both populations. Rapid and low-cost screening of patients with multiplex MASP2 PCR-SSP could be used to identify clinical conditions where MASP-2 (or MAp19) levels may be disease modifying, possibly improving disease outcome through early therapeutic and preventive measures.

Inmunohistoquímica en el diagnóstico de la fiebre amarilla. Experiencia en el Dpto. de Patología, del Instituto de Investigaciones en Ciencias de la Salud y la Cátedra de Anatomía Patológica, Facultad de Ciencias Medicas, Universidad Nacional de Asunción / Immunohistochemistry in the diagnosis of yellow fever. Experience in the Department of Pathology, Health Sciences Research Institute and the Department of Pathology, Faculty of Medical Sciences, National University of Asuncion

La fiebre amarilla (FA) es una enfermedad hemorrágica aguda, inmunoprevenible producida por un arbovirus cuyo órgano blanco es el hígado. Se presenta en forma epidémica y en dos formas epidemiológicas: la urbana y la selvática, ambas con características clínicas e histopatológicas semejantes. Para el diagnóstico se utilizan distintos métodos como serología, cultivo celular, biología molecular, histopatología e inmunohistoquímica (IHQ). La IHQ identifica, en las muestras obtenidas postmortem, la presencia, a nivel tisular, del antígeno viral causante de la enfermedad mediante una unión antígeno-anticuerpo altamente específica. El objetivo de este estudio fue determinar por IHQ la presencia del antígeno del virus de la FA en tejidos hepáticos de pacientes fallecidos con sospecha de FA en el Paraguay. Estudio descriptivo en muestras de tejido hepático (bloques de parafina) obtenidos postmortem, con autorización de los familiares, de 4 pacientes fallecidos con sospecha clínica de FA. Los bloques de parafina fueron enviados, codificados para respetar la confidencialidad de los pacientes, por la Cátedra de Anatomía Patológica de la Facultad de Ciencias Médicas de la Universidad Nacional de Asunción (UNA) al Dpto. de Patología del Instituto de Investigaciones en Ciencias de la Salud (IICS-UNA) para el análisis por IHQ. Se utilizó el anticuerpo primario anti-fiebre amarilla y la técnica del complejo avidina-biotina-peroxidasa, se agregaron cortes de control positivo y negativo para cada caso. Todas fueron positivas para FA. La positividad de la tinción se evaluó por microscopía óptica y en relación a los hallazgos histopatológicos, se consideró positivos aquellos casos en los que se observó una tinción marrón en el citoplasma de las células hepáticas. La positividad se observó en todos los casos sospechosos y en los controles positivos. Los controles negativos no presentaron esta tinción. La IHQ en conjunto con otras técnicas laboratoriales y los criterios clínicos contribuye al diagnóstico postmortem en los casos de FA, y se constituye en una herramienta de diagnóstico muy útil que permite realizar el diagnóstico retrospectivo, en los casos que no ha sido posible realizar otros estudios.

Yellow fever (YF) is an acute hemorrhagic disease, which is immuno-preventable, caused by an arbovirus whose target organ is the liver. It occurs epidemically and in two epidemiological forms: urban and wild, both with clinical and histopathological similar characteristics. Several methods are used for the diagnosis such as serology, cell culture, molecular biology, histopathology and immunohistochemistry (IHC). The IHC determines the presence of the viral antigen causing the disease at tissue level in post-mortem samples through a highly specific antigen-antibody binding. The objective of this study was to determine the presence of the YF viral antigen by IHC in the liver tissue of deceased patients with suspicion of YF in Paraguay. This is a descriptive study carried out on liver tissue samples (paraffin blocks), obtained post-mortem with the permission of the relatives, of four patients that died and had clinical suspicion of YF. Paraffin blocks were sent, coded to respect the patient confidentiality, by the Department of Pathology of the Faculty of Medicine-National University of Asunción (UNA) to the Department of Pathology of the Instituto de Investigaciones en Ciencias de la Salud (IICS-UNA) to be analysed by IHC. The anti-yellow fever primary antibody and the technique of avidin-biotin-peroxidase complex were used. Also, samples of positive and negative controls for each case were added. Positive staining was evaluated by optical microscopy, and in relation to the histopathological findings those with a brown staining in the cytoplasm of the hepatic cells were considered positive. Positivity was observed in all suspected cases and positive controls. The negative controls did not show any staining. The IHC techniques together with other laboratory techniques and the clinical criteria contribute to postmortem diagnosis in YF cases and represent a useful diagnostic tool which allows the performance of retrospective diagnosis in those cases where other studies could not have been made.

Niveles de concordancia en el diagnóstico clínico – patológicode las enfermedades hematológicas / Levels of concordance in the clinical and pathological diagnosis of

En el diagnóstico hematopatológico los hallazgos clínicos (interrogatorio, examen físico,laboratorio, radiología u otros) se correlacionan con el examen morfológico cuanti-cualitativo de lascélulas sanguíneas y hematopoyéticas en el frotis de sangre periférica y aspirados medulares. Encaso de presunciones clínicas, para el diagnóstico final se tienen en cuenta los resultadosanatomopatológicos considerados de certeza. Este trabajo de corte transverso, describe y analiza laconcordancia diagnóstica entre la clínica-hematológica y la anatomía patológica en 370 pacientesprovenientes de los grandes centros médicos del Paraguay, con 679 estudios anatomopatológicoscitohistológicos analizados conjunta y sistemáticamente con técnicas especiales en la Sección deHematopatología del Instituto de Investigaciones en Ciencias de la Salud entre los años 1997 y2002. La concordancia, evaluada por el índice kappa, puede clasificarse en tres grupos: Grupo 1:concordancia buena (11 por ciento de casos) síndrome mieloproliferativo crónico (IK=0,48). Grupo 2:concordancia regular (47 por ciento) síndrome linfoproliferativo crónico (IK=022), leucemias agudaslinfoblásticas (IK=020) y mieloides (IK=0,20). Grupo 3: concordancia baja (42 por ciento) aplasiasmedulares (IK=0,14), hiperplasias reactivas (IK=0,5) y mielodisplasias (IK=0,4) demostrándose lanecesidad de estudios anatomopatológicos complementarios con técnicas especiales para llegar aldiagnóstico de certeza como para estadificar, pronosticar y subclasificar las patologíashematológicas.

Deficiency of mannan-binding lectin associated serine protease-2 due to missense polymorphisms.

Mannan-binding lectin (MBL) and ficolins distinguish between self, non-self and altered-self by recognizing patterns of ligands on the surface of microorganisms or aberrant cells. When this happens MBL-associated serine protease-2 (MASP-2) is activated and cleaves complement factors to start inflammatory actions. We examined human populations for MASP-2 levels, MASP-2 function and for the presence of mutations in coding exons of MASP2. The MASP-2 levels were lowest in Africans from Zambia (median, 196 ng/ml) followed by Hong Kong Chinese (262 ng/ml), Brazilian Amerindians (290 ng/ml) and Danish Caucasians (416 ng/ml). In the Chinese population, we uncovered a novel four amino-acid tandem duplication (p.156_159dupCHNH) associated with low levels of MASP-2. The frequency of this mutation as well as the SNPs p.R99C, p.R118C, p.D120G, p.P126L and p.V377A were analyzed. The p.156_159dupCHNH was only found in Chinese (gene frequency 0.26%) and p.D120G was found only in Caucasians and Inuits from West-Greenland. The p.P126L and p.R99Q were present in Africans and Amerindians only, except for p.R99Q in one Caucasian. The MASP-2 levels were reduced in individuals with p.V377A present. The MASP-2 present in individuals homozygous for p.377A or p.99Q had a normal enzyme activity whereas MASP-2 in individuals homozygous for p.126L was non-functional.