Evaluación de la calidad del diagnóstico de malaria en la red local de laboratorios y en los laboratorios intermedios en el contexto de la eliminación de la enfermedad en Ecuador / Quality evaluation of malaria diagnosis in the local laboratories network and in intermediate laboratories in a setting towards the disease elimination in Ecuador

Resumen Introducción. El cumplimiento de la meta de eliminación de la malaria en Ecuador en el 2020 exige contar con la capacidad requerida para el diagnóstico microscópico ajustado a los estándares de calidad de la Organización Mundial de la Salud (OMS) y de la Organización Panamericana de la Salud (OPS) y proveer el tratamiento adecuado a los pacientes. Objetivo. Conocer la idoneidad o competencia de los microscopistas de la red pública local para el diagnóstico parasitológico de la malaria y el desempeño de los laboratorios intermedios de referencia. Materiales y métodos. Se hizo un estudio descriptivo de corte transversal a partir de la información obtenida en los talleres de evaluación de idoneidad en el diagnóstico microscópico de la red de laboratorios en las coordinaciones zonales de salud utilizando un panel de láminas para evaluar la concordancia del diagnóstico. Además, se calificó el desempeño de los laboratorios intermedios en el diagnóstico en el marco del programa de evaluación externa del desempeño. Los resultados se compararon con los obtenidos por el laboratorio supranacional de Perú. Resultados. En los 11 talleres realizados, se evaluó la idoneidad de 191 microscopistas, de los cuales 153 (80,1 %) aprobaron las pruebas. Las medianas de los indicadores fueron las siguientes: concordancia entre la detección y el resultado, 100 % (Q1- Q3: 96-100); concordancia en la especie, 100 % (Q1- Q3: 93-100); concordancia en el estadio, 93,0 % (Q1- Q3: 86-95) y concordancia en el recuento, 77 % (Q1- Q3: 71-82). En el programa de evaluación externa de desempeño, los tres laboratorios intermedios obtuvieron una concordancia del 100 % en el resultado y una del 96 % en la especie. Conclusiones. Los indicadores de competencia de la red local y de desempeño de los laboratorios intermedios alcanzaron altos estándares de calidad acordes con el proceso de entrenamiento implementado en el país.

Abstract Introduction: To reach the goal of malaria elimination in Ecuador for the year 2020, it is necessary to have a laboratory network with the capacity to perform microscopic diagnosis according to the WHO/PAHO quality standards and to provide the adequate treatment of cases. Objective: To determine the level of competence for parasitological diagnosis of the microscopists from the local public network and the performance of intermediate reference laboratories. Materials and methods: We conducted a cross-sectional study based on the information collected in workshops carried out to appraise the competence for microscopic diagnosis of the local laboratory network (zonal health coordinating offices 1 to 8) using a slide panel to evaluate diagnosis agreement, as well as the diagnostic performance of the intermediate laboratories using an external quality assessment program. The results were compared against the reference standards of the supranational laboratory in Perú. Results: We evaluated the competencies of 191 microscopists in 11 workshops and 153 (80.1%) of them were approved. The medians of the indicators were the following: concordance for parasite detection, 100% (Q1- Q3: 96-100), concordance for species identification, 100% (Q1- Q3: 93-100), and concordances for stage identification, 93.0% (Q1- Q3: 86-95) and parasite counting, 77.0% (Q1- Q3: 71-82). In the external quality assessment, the three intermediate laboratories obtained 100% in parasite detection concordance and 96% for species detection concordance. Conclusions: The results for the primary network and the performance indicators for the intermediate laboratories showed the high-quality standards of the training program implemented in the country.

Ultrastructural changes of erythrocytes in whole blood after exposure to prospective in silico-designed anticancer agents: a qualitative case study

BACKGROUND: Novel, in silico-designed anticancer compounds were synthesized in our laboratory namely, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). These compounds were designed to have improved bioavailability when compared to their source compound, 2-methoxyestradiol. This theoretically would be due to their increased binding affinity to carbonic anhydrase II, present in erythrocytes. Since the novel compounds under investigation are proposed to be transported within erythrocytes bound to carbonic anhydrase II, the morphological effect which they may exert on whole blood and erythrocytes is of great significance. A secondary outcome included revision of previously reported procedures for the handling of the whole blood sample. The purpose of this study was twofold. Firstly, the ultrastructural morphology of a healthy female's erythrocytes was examined via scanning electron microscopy (SEM) after exposure to the newly in silico-designed compounds. Morphology of erythrocytes following exposure to ESE-15-ol and ESE-16 for 3 minutes and 24 hours at 22°C were described with the use of SEM. The haemolytic activity of the compounds after 24 hours exposure were also determined with the ex vivo haemolysis assay. Secondly, storage conditions of the whole blood sample were investigated by determining morphological changes after a 24 hour storage period at 22°C and 37°C. RESULTS: No significant morphological changes were observed in the erythrocyte morphology after exposure to the novel anticancer compounds. Storage of the whole blood samples at 37°C for 24 hours resulted in visible morphological stress in the erythrocytes. Erythrocytes incubated at 22°C for 24 hours showed no structural deformity or distress. CONCLUSIONS: From this research the optimal temperature for ex vivo exposure of whole blood samples to ESE-15-ol and ESE-16 for 24 hours was determined to be 22°C. Data from this study revealed the potential of these compounds to be applied to ex vivo study techniques, since no damage occurred to erythrocytes ultrastructure under these conditions. As no structural changes were observed in erythrocytes exposed to ESE-15-ol and ESE-16, further ex vivo experiments will be conducted into the potential effects of these compounds on whole blood. Optimal incubation conditions up to 24 hours for whole blood were established as a secondary outcome.

[Morphology of red blood cells for differentiating between the upper and the lower urinary tract infection]

Accurate differentiation between upper and lower urinary tract infection [UTI] requires invasive produces. The objective of this study was to determine the morphology of RBC that originates from kidney or bladder for differentiating between upper and lower urinary tract infection. UTI was induced by the injection of 0.5ml of normal saline containing human E coli [10[4]/cc] into the bladder of rats [n=50]. After 48 hours, the rat urine was drained from the bladder and analyzed for the morphology of RBC, culture and cell counter. For diagnosing pyelonephritis, both bilateral kidneys and bladder were biopsied for histopathology and culture. Dysmorphic RBC was detected in 33% [10/30] of the subjects with upper UTI and 13% [4/30] with lower UTI, which showed no significant difference [x[2] =0.04, P=0.63 Fisher Exact Test]. The mean and SD of percentage of dysmorphic RBC in the subjects with upper and lower UTI were 35 +/- 34% and 44 +/- 43%, respectively, which revealed no significant difference [t=0.55, P=0.25]. In this study, dysmorphic RBC showed rates of 45% sensitivity and 50% specificity for upper UTI diagnosis. Urine osmolality may influence the results of RBC morphology for differentiating between upper and lower UTI

Observation the electroporation of blood cells in vitro by scanning electron microscope / 法医学杂志

OBJECTIVE@#To observe changes on cell membrane in blood cells after they were been electrified.@*METHODS@#Blood were electrified for 5, 10, 20, 30 s, 1 min respectively, and Scanning electron microscope was used to detect the changes on their cell membranes.@*RESULTS@#Pores were detected both on electrified erythrocytes and leukocytes with round or ellipse shapes. The erythrocytes often have one or more pores while the leukocytes often have more pores looked like cribble. The rates of perforated cells were increased with the prolonging time of electrification, 5 s with 6% and 1 min increased to 40%.@*CONCLUSIONS@#Alternating current can cause the cell perforating, and the rates of perforated cell were increased with the prolonging time of electrification.

Evaluation of red blood aggregation in diabetes by computarized image analisis

Red blood cell (RBC) aggregation has been widely studied and its importance is well established in the rheology of microcirculation. RBC aggregation is a major factor responsible for the flow properties of blood. Increased RBC aggregation has been observed in several pathological states. Therefore, the measurement of erythrocyte aggregation is rhealogically important for quantifying flow abnormality in pathological conditions. Normal RBC under low flow or at rest form rouleaux aggregates, while abnormal RBC aggregation may lead to the formation of irregular aggregate structures, which may be induced by cell-associated factors (reduced membrane sialic acid levels) but also by extracellular factors. The main objective of the present investigation was to study RBC aggregate morphology in diabetic patients, using direct microscopic observation and numerical processing of recorded digitized images. Blood samples were obtained from 20 diabetic patients and from 15 normal control subjects. The aggregate morphology was quantified by the so-called Aggregate Shape Parameter (ASP) defined as the ratio of the aggregate projected area to its square perimeter. ASP appeared higher (p<10 - 5) in diabetic patients (0.65 +/- 0.18) than in normal controls (0.28 +/- 0.15). This rheo-optical method based on the theoretical model for rouleaux aggregates provides a useful reference for measuring deviations of RBC aggregate morphology. Increased aggregation of RBC resulting from a decreased sialylation of glycophorins may be an important factor in the development of vascular diseases and in the microcirculation impairment.

Evaluation of red blood cell aggregation in diabetes by computerized image analysis

Red blood cell (RBC) aggregation has been widely studied and its importance is well established in the rheology of microcirculation. RBC aggregation is a major factor responsible for the flow properties of blood. Increased RBC aggregation has been observed in several pathological states. Therefore, the measurement of erythrocyte aggregation is rheologically important for quantifying flow abnormality in pathological conditions. Normal RBC under low flow or at rest form rouleaux aggregates, while abnormal RBC aggregation may lead to the formation of irregular aggregate structures, which may be induced by cell-associated factors (reduced membrane sialic acid levels) but also by extracellular factors. The main objective of the present investigation was to study RBC aggregate morphology in diabetic patients, using direct microscopic observation and numerical processing of recorded digitized images. Blood samples were obtained from 20 diabetic patients and from 15 normal control subjects. The aggregate morphology was quantified by the so-called Aggregate Shape Parameter (ASP) defined as the ratio of the aggregate projected area to its square perimeter. ASP appeared significantly higher (p < 10(-5)) in diabetic patients (0.65 +/- 0.18) than in normal controls (0.28 +/- 0.15). This rheo-optical method based on the theoretical model for rouleaux aggregates provides a useful reference for measuring deviations of RBC aggregate morphology. Increased aggregation of RBC resulting from a decreased sialylation of glycophorins may be an important factor in the development of vascular diseases and in the microcirculation impairment.

Soluble transferrin receptor in sickle cell diseases: correlation with spleen function

OBJECTIVE: To correlate spleen function with soluble transferrin receptor (sTfR) levels and red cell ferritin (RCF) values in patients with sickle cell diseases. DESIGN: Prospective study. LOCATION: University Hospital, School of Medical Sciences, State University of Campinas; a tertiary hospital. PARTICIPANTS: 60 patients with sickle cell diseases, in a steady state, who had not received blood transfusions for 3 months; 28 normal individuals with no clinical or laboratory signs of anemia. MEASUREMENTS: Determination of serum iron, transferrin iron-binding capacity, serum ferritin, RCF and sTfR. Evaluation of spleen function: erythrocytes with pits were quantified. RESULTS: Patients with sickle cell anemia had sTfR levels significantly higher than in normal individuals or those with HbSC (p=0.0001) and there was an inverse correlation between sTfR and fetal Hb (p=0.0016). RCF values were significantly higher in sickle cell anemia patients than in normal individuals or those with HbSC (p=0.0001), and there was a correlation between RCF and pitted erythrocytes (p=0.0512). CONCLUSION: The association between sTfR and fetal Hb confirms the contribution of fetal Hb to improving the hemolytic state by minimizing the consequent reactive erythrocyte expansion. High sTfR levels are not related to the degree of spleen function deficiency seen in sickle cell disease patients. The deficiency in the exocytosis process of the spleen occurring in sickle cell anemia patients may contribute to their accumulation of RCF

Hemócitos presentes no basitarso de operárias de Melipona quadrifasciata anthidioides Lepeletier (Hymenoptera, Apidae, Meliponinae) / Haemocytes found in the basitarsus of workers of Melipona quadrifasciata anthidioides Lepeletier (Hymenoptera, Apidae, Meliponinae)

É relatada a presença de hemócitos no basitarso de Melipona quadrifasciata anthidioides. Dois tipos de hemócitos estão presentes; plasmatócitos e granulócitos. São descritos aspectos ultraestruturais bem como alguns comentários sobre suas possíveis funções

Freeze-etch replication of PNH erythrocytes for transmission electron microscopy

Freeze-etch replication of paroxysmal nocturnal hemoglobinurea [PNH] erythrocytes by a new freeze-fracture technique for transmission electron microscopy revealed two types of cells: 1. Macrocytes with round pits. 2. Microcytes with a single elongated spiculum and well defined wart-like spherical projections about 100 nm in diameter and intramembranous particles. A ridge separating the fracture face from the etched face should be achieved by proper etching to avoid ice crystallization

Estudos sobre a preservacao de eritrocitos / Studies about eritrocits preservation

Estudou-se a preservacao de eritrocitos in vitro, mantidos em ADSOL (AD), em meio CE com vitamina E (CE) e em meio CE com vitamina E apos fixacao parcial com GL utaraldeido (GACE), a 4'graus'C por 14 a 22 semanas. A morfologia eritrocitaria foi melhor preservada em amostras GACE, onde observou-se, apos 10 semanas, mais de 35 por cento de discocitos, e menos de vinte por cento de esferocitos - forma pre-hemolitica. Nestas amostras os parametros eritrocitarios apresentaram-se mais estaveis durante 14 semanas, com menores indices de hemolise (1 por cento), em comparacao as CE (2 por cento) e as AD (sete por cento), em 12 semanas. O consumo de glicose, o PH e os niveis de ATP diminuiram exponencialmente. O ATP atingiu quarenta por cento dos valores iniciais apos 6 semanas, enquanto PH e o consumo de glicose apresentaram queda mais rapida nas 3 primeiras semanas. As concentracoes de 2,3-DPG reduziram-se rapidamente apos a primeira semana, com valores mais baixos em amostras AD na segunda e terceira semanas, exaurindo-se em ate 5 semanas. As atividades das enzimas HX, PFKM PK, GAPD, G6-PD, GSHPX, 6-PGD e GR diminuiram exponencialmente, mantendo-se em patamares de quarenta por cento a oitenta por cento das iniciais apos a quinta semana, geralmente menores em GACE. As proteinas da membrana eritrocitaria, avaliadas por eletroforese em gel de poliacrilamida-DODEC il-sulfato de sodio apresentaram a formacao de oligomeros em GACE. Amostras GACE sao adequadas a estudos de eritrocitos

Mechanism of anti-stress activity of Ocimum sanctum Linn, eugenol and Tinospora malabarica in experimental animals.

Effects of restraint stress (RS) and its modulation by O. sanctum (Os), eugenol and T. malabarica (Tm) were evaluated on some biochemical and biophysical parameters in rats. RS induced elevations in blood glucose and urea levels, were unaffected by either Os, eugenol or Tm pretreatment. However, both Os and eugenol lowered RS-induced cholesterol levels. RS also caused a generalized increase in enzyme activity and Os, eugenol or Tm effectively lowered the RS-induced elevations in lactate dehydrogenase (LDH) and alkaline phosphatase. RS also induced (a) increased membrane protein clusterization, (b) increased membrane fluidity and (c) reduced membrane thickness--in RBC membrane, whereas, the effects on the synaptosomal membrane were less marked. The RS-induced changes in RBC membrane dynamics were attenuated/reversed by Os, eugenol or Tm, in a differential manner. These biochemical and membrane changes during Rs and their modulation by the adaptogens are discussed in light of the possible mechanisms of action of these agents, during such aversive stimuli.

Effect of inactivated viral vaccines (human) on frequency of micronuclei in bone marrow erythrocytes of mice.

Cytogenetic effects of the two inactivated viral vaccines (polio and antirabies) were studied in adult male mice by the micronucleus test. Polio salk vaccine did not induce micronuclei formation at both human (0.5 ml) and 1/5th human doses. Antirabies vaccine induced micronuclei in poly and total erythrocytes only at human dose of 2 ml. Beta-propiolactone (BPL) induced micronuclei at higher dose of 5.7 mg, but not at 0.57 mg (approximate concentration present in 2 ml of rabies vaccine). The P/N ratio was not affected in vaccinated and BPL inoculated animals. Antirabies vaccine induced micronuclei percentage was more than the BPL value.

Hematúria recorrente na infância: estudo de 250 casos / Recurrent hematuria in children: a study of 250 cases

Para avaliarmos a prevalência de determinadas patologias e esquematizar protocolo de investigaçäo pouco invasivo, estudamos 250 crianças com hematúria, de seis meses a 17 anos, acompanhadas ambulatorialmente, sendo 102 do sexo feminino e 148 do sexo masculino, submetendo-se aos seguintes exames: urina tipo I, urocultura, uréia, creatinina e complemento sérico total e fraçöes, eletroforese de hemoglobina, proteinúria em volume urinário de 24h, pesquisa de dismorfismo eritrocitário nas hemácias da urina, uricosúria e calciúria em urina de 24h (três amostras) e prova de sobrecarga oral de cálcio nas crianças com hipercalciúria (HCa). Realizamos avaliaçäo radiológica e biópsia renal quando necessário. O diagnóstico etiológico foi possível em 83% das crianças, sendo: 1) glomerulopatia em 79 casos (31%), 45 casos de outras glomerulopatias (48%), 19 casos de doença de Alport (7%) e 15 casos de doença de Berger (6%); 2) hipercalciúria em 67 casos (27%); 3) calculose renal em 27 pacientes (11%); 4) infecçäo urinária (ITU) em 14 casos (6%); 5) malformaçöes renais em oito casos (3%). Permanecem ainda em estudo 43 criançs (17%). Podemos concluir, através desses resultados, que, uma vez estabelecida metodologia de investigaçäo adequada, nos casos de hematúria recorrente, poderemos obter diagnóstico etiológico, utilizando métodos invasivos em apenas alguns casos, quando houver suspeita de glomerulopatia

Luminescence in erythrocytes of different amphibian larvae

Con el microscopio de fondo oscuro los eritrocitos de larvas de Telmatobius laticeps, Telmatobius pisanoi, Hyla pulchella andina y Bufo arenarum, presentan cuadros de luminiscencia dque difieren según la especie y el estadio; el fenómeno disminuye en los estadios cercanos a la metamorfosis hasta desaparecer en el adulto. La luminiscencia, visible en granulaciones ubicadas tanto en el núcleo como en el citoplasma, se vincularía con los diferentes tipos de hemoglobina dependientes, a su vez, del sitio eritopoyético

Distribución espacial de la hemoglobina en los hematies de pacientes asmáticos / Spatial distribution of hemoglobins in the hematies of patients with asthma

El objetivo de este estudio fue demostrar si los hematíes de los asmáticos presentan alteraciones morfológicas. Los resultados demostraron que en los pacientes asmáticos la hemoglobina pierde su distribución anular para dar lugar a una formación homogénea que borra la cavidad eritrocino, dándole el aspecto de un flan. Estas alteraciones no se observaron en el grupo control

Efecto de Ca²+ sobre la invasión del Plasmodium falciparum a los eritrocitos humanos / Ca²+ effect on Plasmodium falciparum invasion of human erythrocytes

Se demostró que el Ca²+ es un requerimento indispensable para el proceso de invasión del Plasmodium falciparum a los eritrocitos humanos. La incubación de los esquizontes en un medio de cultivo libre de Ca²+ inhibió la reinvasión. Se produjo el mismo efecto al disminuír la concentración de iones Ca²+ en el medio extracelular. El Laü+ no remplaza al Ca²+ para restablecer la invasión, por el contrario actúa como un inhibidor del proceso. El contenido normal de Ca²+ en el eritrocito previo a la invasión no es un factor importante puesto que el tratamiento con el ionósforo A23187 y EGTA 1mM (ácido etilenglicolbis (ß -amino-etileter) N, N'tetraacético) no impidió que las células tratadas fueran invadidas. Después de modificar el contenido intracelular de Ca²+ en los esquizontes se inhibió dramáticamente la reinvasión de eritrocitos normales sugiriendo que el Ca²+ localizado dentro del parásito es usado para la invasión. Las mediciones efectuadas con superíndice 45Ca²+ revelaron que durante la reinvación, el parásito induce un influjo de iones Ca²+ en los eritrocitos invadidos y no invadidos, elevandose su contenido de Ca²+ 13 veces por encima del nivel normal (0.14 pmol/10superíndice 7 células). Las evidencias ultraestructurales aclararon que el Ca²+ esta involucrado durante la secuencia de eventos de la invasión. Cuando existe depleción de Ca²+ en el medio por la presencia del quelator EGTA se impidió la reorientación e interiorización del merozoíto en el eritrocito aún después de que se hubiera efectuado el reconocimiento inicial entre ambas células. A pesar de que se indujo invaginación en la membrana del eritrocito, no fue posible que se produjera la fusión. Se discuten las implicaciones de los efectos del Ca²+ durante el mecanismo de invasión