IgG Avidity in Samples Collected on Filter Paper: Importance of The Early Diagnosis of Congenital Toxoplasmosis / Avidez de IgG em amostras coletadas em papel filtro: Importância no diagnóstico precoce da toxoplasmose congênita

Abstract Objective The purpose of the present study is to standardize and evaluate the use of the immunoglobulin G (IgG) antibody avidity test on blood samples from newborns collected on filter paper to perform the heel test aiming at its implementation in ongoing programs. Methods Blood samples from newborns were collected on filter paper simultaneously with the heel prick test. All samples were subjected to immunoglobulin M IgM and IgG enzyme-linked immunosorbent assays (ELISA). Peripheral blood was collected again in the traditional way and on filter paper from newborns with high IgG levels (33). Three types of techniques were performed, the standard for measuring IgG in serum, adapted for filter paper and the technique of IgG avidity in serum and on filter paper. The results of the avidity test were classified according to the Rahbari protocol. Results Among the 177 samples, 17 were collected in duplicate from the same child, 1 of peripheral blood and 1 on filter paper. In this analysis, 1 (5.88%) of the 17 samples collected in duplicate also exhibited low IgG avidity, suggesting congenital infection. In addition, the results obtained from serum and filter paper were in agreement, that is, 16 (94.12%) samples presented high avidity, with 100% agreement between the results obtained from serum and from filter paper. Conclusion The results of the present study indicate that the avidity test may be another valuable method for the diagnosis of congenital toxoplasmosis in newborns.

Resumo Objetivo O objetivo do presente estudo é padronizar e avaliar a utilização do teste de avidez de anticorpos imunoglobulina G (IgG) em amostras de sangue de recémnascidos (RNs) coletadas em papel filtro para a realização do teste do pezinho visando a implementação nos programas já vigentes. Métodos Foram coletadas amostras de sangue de recém-nascidos em papel filtro simultaneamente ao teste do pezinho. Em todas as amostras, foram realizados os testes imunoenzimáticos (ELISA) imunoglobulina M (IgM) e IgG. Dos RNs que apresentaram altos índices de IgG (33), foi novamente coletado sangue periférico da forma tradicional e em papel filtro. Foram realizadas técnicas padrão para a dosagem de IgG em soro, adaptadas para papel filtro, e a técnica de avidez de IgG em soro e em papel filtro. Os valores obtidos para o teste de avidez foram classificados de acordo com o protocolo de Rahbari. Resultados Dentre as 177 recoletas, em 17 amostras foi realizada a coleta simultânea de sangue periférico e papel filtro da mesma criança. Nesta análise, 1 (5,88%) das 17 amostras coletadas em duplicata obteve também baixa avidez de IgG, sugerindo infecção congênita da criança, e houve concordância entre os resultados obtidos em soro e em papel filtro: 16 (94,12%) das amostras apresentaram alta avidez, com concordância de 100% entre os resultados obtidos em soro e em papel filtro. Conclusão Os dados do presente trabalho evidenciam que o teste de avidez poderá ser mais um método valioso a ser utilizado no diagnóstico da toxoplasmose congênita em RNs.

Evaluation and application of an efficient plant DNA extraction protocol for laboratory and field testing / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Nucleic acids in plant tissue lysates can be captured quickly by a cellulose filter paper and prepared for amplification after a quick purification. In this study, a published filter paper strip method was modified by sticking the filter paper on a polyvinyl chloride resin (PVC) sheet. This modified method is named EZ-D, for EASY DNA extraction. Compared with the original cetyl trimethylammonium bromide (CTAB) method, DNA extracted by EZ-D is more efficient in polymerase chain reaction (PCR) amplification due to the more stable performance of the EZ-D stick. The EZ-D method is also faster, easier, and cheaper. PCR analyses showed that DNA extracted from several types of plant tissues by EZ-D was appropriate for specific identification of biological samples. A regular PCR reaction can detect the EZ-D-extracted DNA template at concentration as low as 0.1 ng/μL. Evaluation of the EZ-D showed that DNA extracts could be successfully amplified by PCR reaction for DNA fragments up to 3000 bp in length and up to 80% in GC content. EZ-D was successfully used for DNA extraction from a variety of plant species and plant tissues. Moreover, when EZ-D was combined with the loop-mediated isothermal amplification (LAMP) method, DNA identification of biological samples could be achieved without the need for specialized equipment. As an optimized DNA purification method, EZ-D shows great advantages in application and can be used widely in laboratories where equipment is limited and rapid results are required.

Serum Analysis of Hydro-pericardium Syndrome (HPS) by Filter-Paper Blood Sampling Technique: As Substitute to Serum Collection Technique

Background: The study aimed to assess the filter paper blood sampling technique for sero- monitoring against Hydro-pericardium syndrome (HPS). In view of the fact, dried blood samples don’t require immediate refrigeration, occupy little space and are easily transported. Methods: Whole blood paired with serum samples were collected from 100 broiler chickens on filter paper strips, dried for 2hrs at 37oC, stored in polythene bag and then eluated in normal saline at 4oC for overnight. Antigen of HPS was isolated, purified and confirmed by agar gel precipitation test (AGPT) with raised hyper-immune serum. Eluates of whole blood dried on filter paper with corresponding serum samples were tested for antibody activity by indirect heam-agglutination (IHA) test. Results: ‘The IHA titers of eluates were similar to titers obtained with serum diluted as 1:10. Normal saline and phosphate buffered saline did not influence the antibody stability, when used as eluating fluid. Whole blood dried on filter paper could be stored sealed in plastic bag at 4oC or ambient temperature for at least one week with no appreciable loss of antibody titers. A strong correlation (r = 0.900) exist between the titers obtained with two methods of blood sampling. Conclusion: This study demonstrated that recovery of antibodies from blood dried on filter paper after eluation produces results comparable to those obtained by recovering antibodies from serum. Based on above findings it is concluded that filter paper blood sampling could serve as a cost effective and convenient tool for HPS sero-monitoring.

Exploration and application of a new method for rapid extraction of DNA from Chinese medicinal materials / 中草药

Objective To build an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA from Chinese medicinal materials in 30 s. Methods A new rapid DNA extraction method which was suitable for traditional Chinese medicine, with equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA from Chinese medicinal materials in 30 s was verified. The filter paper strip or disk for the adsorbed nucleic acid can be directly amplified by the reaction system. Results This method can extract the nucleic acid successfully. The extracted DNA was amplified, and the results were consistent with the traditional extraction methods. Conclusion The new rapid DNA extraction method is simple, fast, and low cost, which will make nucleic acid extraction become more and more popular, and no longer confined to professional and laboratory environment. This study provides a new idea for the application and development of molecular pharmacology.

La impronta como método no invasivo de toma de muestra para el diagnóstico molecular de la leishmaniosis cutánea en población militar de colombia / The imprint as a non-invasive method of sampling for the molecular diagnosis of cutaneous leishmaniasis in colombia's military population / In print como método não invasivo de coleta de amostra para o diagnóstico molecular da leishmaniose cutânea em população militar da colômbia

Resumen Teniendo en cuenta que los métodos tradicionales de toma de muestra y diagnóstico para la leishmaniosis cutánea presentan limitaciones, como el frotis directo, cuya sensibilidad depende de la pericia del profesional, el aspirado de lesión que puede ser usado para detección de parásitos en lámina, su ADN o para cultivos es demorado y exigente, y la biopsia de la lesión que es invasiva y dolorosa se comparó con el método de impronta en papel filtro de la lesión ulcerativa contra el método tradicional de aspirado mediante la técnica de POR convencional utilizando como blanco una región del ADN del kinetoplasto del parásito. En el presente trabajo, la por obtuvo una sensibilidad para impronta del 90,07% comparado con el aspirado, el 86,3%, que, además, por ser un método de toma de muestra no invasivo, con pocas exigencias para el transporte, se puede tomar directamente en el área de operaciones a muy bajo costo, resulta ser beneficioso para ser usado en los pacientes con leishmaniosis cutánea del Ejército Nacional de Colombia, que se encuentran en las diferentes áreas de operaciones.

Abstract Taking into account that the traditional methods of sampling and diagnosis for cutaneous leishmaniasis , have limitations, such as direct smear, whose sensitivity depends on the professional's expertise, the lesion aspiration that can be used to detect parasites in the lamina, DNA or cultures takes a long time and is demanding, and the biopsy of the lesion that is invasive and painful were compared with the imprinting method on the filter paper of the ulcerative lesion against the traditional method of aspiration by means of the conventional PCR technique using as a target a DNA region of the parasite kinetoplast. In this present work, PCR obtained an imprinting sensitivity of 90.07% compared to the aspirate of 86.3%, which, besides being a non-invasive sampling method, with few transport requirements, it can be taken directly in the area of operations at a very low cost, which turns out to be beneficial to be used in patients with cutaneous leishmaniasis of the National Army of Colombia, who are in different operations areas.

Resumo Considerando que os métodos tradicionais de coleta de amostra e diagnóstico para a leishmaniose cutânea apresentam limitações, como exame direto de esfregaços, cuja sensibilidade depende da perícia do profissional, o raspado de lesão que pode ser usado para a detecção de parasitas em lâmina, seu DNA ou para culturas é demorado e exigente, e a biopsia da lesão que é invasiva e dolorosa, comparou-se com o método in print em papel filtro da lesão ulcerativa contra o método tradicional de aspirado mediante a técnica de PCR convencional utilizando como alvo uma região do DNA do cinetoplasto do parasita. No presente trabalho, a PCR obteve uma sensibilidade para in print de 90,07% comparado com o aspirado, 86,3%, que, além disso, por ser um método de coleta de amostra não invasivo, com poucas exigências para o transporte, pode ser coletado diretamente na área de operações a muito baixo custo, resulta ser benéfico para ser usado nos pacientes com leishmaniose cutânea do Exército Nacional da Colômbia, que se encontram nas diferentes áreas de operações.

Effects of drying and storage conditions on the stability of TSH in blood spots

ABSTRACT Objective To evaluate the influence of sample drying and storage temperature on TSH stability in neonatal screening. Subjects and methods Blood samples from 29 adult volunteers as a surrogate for neonatal blood (10 with normal TSH, 9 with overt hypothyroid and 10 with subclinical hypothyroidism) were spotted on filter paper and dried at 22°C or 35°C for 3 hours. The samples were then stored at 22°C, -4°C, or -20°C, and TSH measurements were performed at day 0 (D0), D7, D30, D60, D180, and D360 of storage. Results The drying temperature did not interfere with TSH measurement on D0. TSH values remained stable up to D30 when stored at 22°C and were stable up to D60 when stored in a refrigerator or freezer. Samples stored at 22°C had a greater decrease in TSH values than samples stored in a refrigerator or a freezer. Conclusions Freezer storage is not advantageous compared to storage in the refrigerator. At the end of one year, if confirmation of the initial result is required, a reduction of TSH concentrations should be taken into account.

Significance of bacteria detection with filter paper method on diagnosis of diabetic foot wound infection / 中华烧伤杂志

Objective@#To evaluate the significance of bacteria detection with filter paper method on diagnosis of diabetic foot wound infection.@*Methods@#Eighteen patients with diabetic foot ulcer conforming to the study criteria were hospitalized in Liyuan Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from July 2014 to July 2015. Diabetic foot ulcer wounds were classified according to the University of Texas diabetic foot classification (hereinafter referred to as Texas grade) system, and general condition of patients with wounds in different Texas grade was compared. Exudate and tissue of wounds were obtained, and filter paper method and biopsy method were adopted to detect the bacteria of wounds of patients respectively. Filter paper method was regarded as the evaluation method, and biopsy method was regarded as the control method. The relevance, difference, and consistency of the detection results of two methods were tested. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of filter paper method in bacteria detection were calculated. Receiver operating characteristic (ROC) curve was drawn based on the specificity and sensitivity of filter paper method in bacteria detection of 18 patients to predict the detection effect of the method. Data were processed with one-way analysis of variance and Fisher's exact test. In patients tested positive for bacteria by biopsy method, the correlation between bacteria number detected by biopsy method and that by filter paper method was analyzed with Pearson correlation analysis.@*Results@#(1) There were no statistically significant differences among patients with wounds in Texas grade 1, 2, and 3 in age, duration of diabetes, duration of wound, wound area, ankle brachial index, glycosylated hemoglobin, fasting blood sugar, blood platelet count, erythrocyte sedimentation rate, C-reactive protein, aspartate aminotransferase, serum creatinine, and urea nitrogen (with F values from 0.029 to 2.916, P values above 0.05), while there were statistically significant differences among patients with wounds in Texas grade 1, 2, and 3 in white blood cell count and alanine aminotransferase (with F values 4.688 and 6.833 respectively, P<0.05 or P<0.01). (2) According to the results of biopsy method, 6 patients were tested negative for bacteria, and 12 patients were tested positive for bacteria, among which 10 patients were with bacterial number above 1×105/g, and 2 patients with bacterial number below 1×105/g. According to the results of filter paper method, 8 patients were tested negative for bacteria, and 10 patients were tested positive for bacteria, among which 7 patients were with bacterial number above 1×105/g, and 3 patients with bacterial number below 1×105/g. There were 7 patients tested positive for bacteria both by biopsy method and filter paper method, 8 patients tested negative for bacteria both by biopsy method and filter paper method, and 3 patients tested positive for bacteria by biopsy method but negative by filter paper method. Patients tested negative for bacteria by biopsy method did not tested positive for bacteria by filter paper method. There was directional association between the detection results of two methods (P=0.004), i. e. if result of biopsy method was positive, result of filter paper method could also be positive. There was no obvious difference in the detection results of two methods (P=0.250). The consistency between the detection results of two methods was ordinary (Kappa=0.68, P=0.002). (3) The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of filter paper method in bacteria detection were 70%, 100%, 1.00, 0.73, and 83.3%, respectively. Total area under ROC curve of bacteria detection by filter paper method in 18 patients was 0.919 (with 95% confidence interval 0-1.000, P=0.030). (4) There were 13 strains of bacteria detected by biopsy method, with 5 strains of Acinetobacter baumannii, 5 strains of Staphylococcus aureus, 1 strain of Pseudomonas aeruginosa, 1 strain of Streptococcus bovis, and 1 strain of bird Enterococcus. There were 11 strains of bacteria detected by filter paper method, with 5 strains of Acinetobacter baumannii, 3 strains of Staphylococcus aureus, 1 strain of Pseudomonas aeruginosa, 1 strain of Streptococcus bovis, and 1 strain of bird Enterococcus. Except for Staphylococcus aureus, the sensitivity and specificity of filter paper method in the detection of the other 4 bacteria were all 100%. The consistency between filter paper method and biopsy method in detecting Acinetobacter baumannii was good (Kappa=1.00, P<0.01), while that in detecting Staphylococcus aureus was ordinary (Kappa=0.68, P<0.05). (5) There was no obvious correlation between the bacteria number of wounds detected by filter paper method and that by biopsy method (r=0.257, P=0.419). There was obvious correlation between the bacteria numbers detected by two methods in wounds with Texas grade 1 and 2 (with r values as 0.999, P values as 0.001). There was no obvious correlation between the bacteria numbers detected by two methods in wounds with Texas grade 3 (r=-0.053, P=0.947).@*Conclusions@#The detection result of filter paper method is in accordance with that of biopsy method in the determination of bacterial infection, and it is of great importance in the diagnosis of local infection of diabetic foot wound.

The Challenge of Diagnosis and Indication for Treatment in Fabry Disease

Abstract Fabry disease, caused by deficient alpha-galactosidase A lysosomal enzyme activity, remains challenging to health-care professionals. Laboratory diagnosis in males is carried out by determination of alpha-galactosidase A activity; for females, enzymatic activity determination fails to detect the disease in about two-thirds of the patients, and only the identification of a pathogenic mutation in the GLA gene allows for a definite diagnosis. The hurdle to be overcome in this field is to determine whether a mutation that has never been described determines a ''classic'' or ''nonclassic'' phenotype, because this will have an impact on the decision-making for treatment initiation. Besides the enzymatic determination and GLA gene mutation determination, researchers are still searching for a good biomarker, and it seems that plasma lyso-Gb3 is a useful tool that correlates to the degree of substrate storage in organs. The ideal time for treatment initiation for children and nonclassic phenotype remains unclear.

Molecular diagnosis of dengue virus infections in samples collected on filter paper / Diagnóstico molecular de infecciones por virus Dengue en muestras colectadas en papel de filtro

Dengue virus infections (DENV) are a severe public health problem due to the high rates of morbidity and mortality involved, and the fact that no clinical treatment or vaccines are available. In order to strengthen the laboratory diagnosis for surveillance systems in tropical countries with low resources, we report an optimized method using filter paper for blood spotting and subsequent molecular diagnosis of DENV serotypes. Control strains of all serotypes, as well as 35 whole blood patient samples dispensed on filter paper, were stored at room temperature for as long as 36 months. RT-PCR of 5’UTR-C fragment was amplified through adapted protocols to diagnose all dengue serotypes. Results showed amplification for all four viral serotypes, including control viral strains and 88.6 % of the samples. These results allowed determining the utility of filter paper for the preservation of samples regularly obtained from patients with clinical suspicion of dengue in settings where low resources do not permit an immediate analysis of the samples. Likewise, this study evidence the possibility of molecular diagnosis of DENV from multiple areas of the world where there are no laboratories with the capacity to confirm DENV cases.

Las infecciones por virus Dengue (DENV) representan un grave problema de salud pública debido a las altas tasas de morbilidad y mortalidad que causan, además no cuentan con tratamiento clínico específico, ni vacuna. Con el fin de reforzar el diagnóstico de laboratorio para los sistemas de vigilancia epidemiológica en países tropicales con recursos económicos limitados, se optimizó una metodología utilizando papel de filtro para la recolección de muestras y el subsiguiente diagnóstico molecular de los serotipos de DENV. Se emplearon cepas controles correspondientes a todos los serotipos virales, así como 35 muestras de sangre total dispensadas en papel de filtro que fueron mantenidas a temperatura ambiente por 36 meses. Las muestras fueron analizadas mediante RT-PCR para la amplificación de la región del genoma correspondiente a 5´UTR-C de los DENV. Los resultados mostraron la amplificación de los mencionados fragmentos en 88,6% de las muestras analizadas, así como de las cepas controles. Estos resultados evidenciaron la utilidad del papel de filtro para conservación de muestras obtenidas de pacientes con sospecha clínica de dengue ubicados en zonas donde no es posible realizar análisis de laboratorio de forma inmediata, así como su uso para el diagnóstico molecular. De este modo se reforzaría la vigilancia en áreas, donde no hay laboratorios con capacidad para confirmar casos de DENV.

Blood collection on filter paper for HIV antibodies detection: experience of SampaCentro Project / Colheita de sangue em papel filtro para detecção de anticorpos anti-HIV: experiência do Projeto SampaCentro

Introduction: Blood samples collected on filter paper (dried blood spot [DBS]) is an immunoassay that has been used for antibodies screening. Objective: To evaluate the strategy of DBS blood collection for detection of HIV antibodies, evaluation of Q-Preven HIV 1 + 2 - DBS kit lot, and to analyze the stability of DBS samples. Methods: Blood collection on DBS was performed according to World Health Organization (WHO) recommendations. The evaluation of the kit lot for HIV antibodies detection was performed using delta (d) values from the results of 774 DBS samples from volunteers men who have sex with men (MSM) recruited in the central region of São Paulo city, Brazil. Results: DBS blood collection was performed without complications. The positive (5.26) and negative (5.23) delta values allowed to clearly differentiate HIV antibodies reactive and non-reactive samples. We observed good performance of the kit lot and samples were stable on DBS form. Conclusion: Blood collection on DBS is feasible for the study of MSM population and is suitable for laboratory routine. The overall performance of Q Preven HIV-1 + 2 - DBS kit was satisfactory, having reached the quality levels required for the development of this study. .

Introdução: As amostras de sangue colhidas em papel filtro (DBS) têm sido utilizadas na triagem de anticorpos por meio de imunoensaios. Objetivos: Avaliar a estratégia de colheita de sangue em DBS para detecção de anticorpos contra o vírus da imunodeficiência humana (HIV), verificar o lote do kit Q-Preven HIV 1+2 - DBS e analisar a estabilidade das amostras DBS. Métodos: A colheita de sangue em DBS foi realizada conforme recomendações da Organização Mundial da Saúde (OMS). A avaliação do lote do kit para detecção de anticorpos anti-HIV foi feita por meio do valor de delta a partir dos resultados das 774 amostras DBS provenientes de voluntários homens que fazem sexo com homens (HSH) recrutados na região central da cidade de São Paulo, Brasil. Resultados: A colheita de sangue em DBS foi realizada sem intercorrências. O indicador delta positivo (5,26) e negativo (5,23) permitiu discriminar com clareza amostras anti-HIV reagentes e não reagentes. O lote do kit apresentou bom desempenho e as amostras permaneceram estáveis na forma de DBS. Conclusão: A colheita de sangue em DBS mostrou-se factível para o estudo realizado com a população HSH e foi adequada para a rotina laboratorial. O desempenho global do kit Q-Preven HIV 1+2 - DBS foi satisfatório, com a qualidade requerida para o desenvolvimento deste estudo. .

Comparison of sxx commercially-available dna polymerases for direct ccr / Comparacao de seis polimerases de DNA disponiveis comercialmente para o PCR direto

SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens. .

RESUMO O propósito deste estudo foi avaliar 6 polimerases de DNA disponíveis comercialmente que são resistentes aos inibidores do PCR para uma amplificação potencial de DNA de amostras de sangue total. O DNA genômico do parasita humano da malária, Plasmodium falciparum, foi analisado sob condições que incluíram os componentes inibidores do sangue extraído de sangue ressacado em papel de filtro. Nossos resultados sugerem que a polimerase KOD FX DNA é superior a outras polimerases. .

Production of cold-adapted cellulase by Verticillium sp. isolated from Antarctic soils

Background: Cellulose can be converted to ethanol by simultaneous saccharification and fermentation (SSF). The difference between the optimal temperature of cellulase and microbial fermentation, however, has been identified as the critical problem with SSF. In this study, one fungal strain (AnsX1) with high cellulase activity at low temperature was isolated from Antarctic soils and identified as Verticillium sp. by morphological and molecular analyses. Results: The biochemical properties of crude AnsX1 cellulase samples were studied by filter paper cellulase assay. The maximum cellulase activity was achieved at low temperature in an acidic environment with addition of metal ions. Furthermore, AnsX1 cellulase demonstrated 54-63% enzymatic activity at ethanol concentrations of 5-10%. AnsX1 cellulase production was influenced by inoculum size, carbon and nitrogen sources, and elicitors. The optimal culture conditions for AnsX1 cellulase production were 5% inoculum, wheat bran as carbon source, (NH4)2SO4 as nitrogen source, and sorbitol added in the medium. Conclusions: Our present work has potential to enable the development of an economic and efficient cold-adapted cellulase system for bioconversion of lignocellulosic biomass into biofuels in future.

Analysis of the Stability of Urea in Dried Blood Spots Collected and Stored on Filter Paper

The ability to use dry blood spots (DBSs) on filter paper for the analysis of urea levels could be an important diagnostic tool for areas that have limited access to laboratory facilities. We developed a method for the extraction and quantification of urea from DBSs that were stored on 3M Whatman filter paper and investigated the effect of long-term storage on the level of urea in DBSs. DBSs of 4.5 mm in diameter were used for our assay, and we determined the urea levels in blood using a commercially available enzymatic kit (UV GLDH-method; Randox laboratories Ltd., UK). The DBSs on filter discs were stored at 4degrees C or at 37degrees C for 120 days. The mean intra- and inter-assay coefficient of variance for our method of urea extraction from dried blood was 4.2% and 6.3%, respectively. We collected 75 fresh blood samples and compared the urea content of each fresh sample with the urea content of DBSs taken from corresponding fresh blood samples. Regression analysis reported a regression coefficient (r) value of 0.97 and a recovery of urea from dried spots was 102.2%. Urea concentrations in DBSs were stable for up to 120 and 90 days when stored at 4degrees C and 37degrees C, respectively. Our results show that urea can be stored and quantitatively recovered from small volumes of blood that was collected on filter paper.

Analysis of the Stability of Urea in Dried Blood Spots Collected and Stored on Filter Paper

The ability to use dry blood spots (DBSs) on filter paper for the analysis of urea levels could be an important diagnostic tool for areas that have limited access to laboratory facilities. We developed a method for the extraction and quantification of urea from DBSs that were stored on 3M Whatman filter paper and investigated the effect of long-term storage on the level of urea in DBSs. DBSs of 4.5 mm in diameter were used for our assay, and we determined the urea levels in blood using a commercially available enzymatic kit (UV GLDH-method; Randox laboratories Ltd., UK). The DBSs on filter discs were stored at 4degrees C or at 37degrees C for 120 days. The mean intra- and inter-assay coefficient of variance for our method of urea extraction from dried blood was 4.2% and 6.3%, respectively. We collected 75 fresh blood samples and compared the urea content of each fresh sample with the urea content of DBSs taken from corresponding fresh blood samples. Regression analysis reported a regression coefficient (r) value of 0.97 and a recovery of urea from dried spots was 102.2%. Urea concentrations in DBSs were stable for up to 120 and 90 days when stored at 4degrees C and 37degrees C, respectively. Our results show that urea can be stored and quantitatively recovered from small volumes of blood that was collected on filter paper.

Diagnóstico inmunológico de la Enfermedad de Chagas a partir de muestras colectadas en papel de filtro / Immunological diagnosis of Chagas disease from samples collected on filter paper

La Enfermedad de Chagas, es producida por el parásito Trypanosoma cruzi. Los métodos de diagnóstico inmunológico son los más utilizados, pero presentan problemas de reacciones cruzadas con Leishmaniasis y Rangeliosis, por lo cual la Organización Mundial de la Salud (OMS) sugiere la ejecución de tres pruebas inmunológicas diferentes. En Venezuela la disponibilidad de las tres pruebas sólo se encuentra en centros de investigación y en laboratorios de referencia. Debido a esto, las muestras deben ser transportadas bajo estrictas condiciones de conservación y aún así pueden llegar deterioradas. El uso del papel de filtro podría ser una alternativa para la recolección y transporte de las muestras. El objetivo del presente trabajo fue comparar los resultados de las técnicas de ELISA, HAI e IFI utilizando muestras de sangre colectadas en tubo y en papel de filtro. Las muestras se procesaron mediante las técnicas de Inmunoensayo Enzimático (ELISA), Hemaglutinación Indirecta (HAI) e Inmunofluorescencia Indirecta (IFI). Para las muestras de papel se hicieron eluciones de 1/50 a diferentes tiempos para ELISA e IFI, y de 1/25 para HAI. Se obtuvo una sensibilidad de 91% y una especificad de 100% en el diagnóstico de las muestras colectadas en papel de filtro a través de las tres técnicas inmunológicas. Al compararlo con los resultados de las muestras colectadas en tubo se encontró un índice de kappa 0,91 (P<0,001), lo que indica una alta concordancia entre las dos técnicas de recolección de muestras y la confiabilidad de los resultados.

Chagas disease is produced by the parasite Trypanosoma cruzi. Immunological diagnosis methods are the most used, but they present problems of cross reactions with Leishmaniasis and Rangeliosis. For this reason, the World-Health-Organization (WHO) suggests the use of three different immunological tests. In Venezuela, the three tests are only available in research centers and laboratories of reference. Consequently, many samples must be transported under strict conditions of conservation, and despite this, they may become spoiled before arriving at the mentioned centers. The use of the filter paper could be an alternative for the collection and transportation of the samples. The aim of the present work was to compare the results of ELISA, HAI and IFI techniques using blood samples collected in tubes and in filter paper. The samples collected in tubes and filter paper were processed by ELISA, HAI and IFI techniques. For the filter paper samples elutions of 1/50 were done at various times for ELISA and IFI, and of 1/25 for HAI. Was obtained a sensibility of 91 % and specificity of 100 % in the final diagnosis of the samples collected in filter paper using the three techniques before mentioned, finding a kappa index of 0.91 (P <0.001) in both types of samples, indicating a high concordance between the two sample collection techniques and thus the reliability of the results obtained by means of three immunological techniques.

Apo E genotyping from blood stored on filter paper.

Background & objectives: Dried blood spotted on to filter paper has been found suitable for a large number of studies. In tropical countries with varying temperature conditions the use of dried blood needs to be validated. We carried out this study to assess the use of blood spotted filter paper as a transport system to study genotyping of Apo E gene. Methods: Fifty five patients visiting Cardiothoracic Neuroscience Centre (CNC) OPD at the All India Institute of Medical Sciences (AIIMS), New Delhi, and referred for lipid investigations to Cardiac Biochemistry Laboratory were selected at random. Blood was spotted on to Whatman 3 MM filter paper, dried and stored at room temperature. Genomic DNA was extracted and genotyping was carried out at the end of 0, 3 and 12 months. The study was further validated using samples collected on to filter paper from four centres and stored for eight years at room temperature. The temperature and humidity conditions of the centre varied widely. Results: Fifty five samples collected on to filter paper showed exact match of the genotyping when compared to fresh blood. In dried blood samples collected and stored for 1 yr at room temperature DNA extraction and apo E genotyping was done successfully. Interpretation & conclusions: The present results showed the feasibility of using dried blood samples on filter paper for apo E genotyping in tropical temperature. The findings need to be validated on a large sample before being recommended for use.

Comparação da coleta das amostras de sangue na triagem pré-natal, utilizando o papel de filtro e punção venosa na técnica de ELISA para detecção de HIV 1+2 / Comparison of the collection of blood samples in prenatal screening, using the filter paper and venipuncture technique ELISA for the detection of HIV 1+2

Apesar de o papel de filtro (PF) ter sido introduzido desde o século passado, (1963) por Guthrie e Susi, nas triagens neonatais, como alternativa para a coleta da amostra do sangue, seus benefícios na aplicação da prática médica laboratorial não foram aproveitados com eficiência nestes anos, comexceção dos recentes resultados obtidos nos trabalhos para a triagem pré-natal. O diagnóstico precoce do HIV em mulheres grávidas é de fundamental importância, sendo o aporte mais destacado para neutralizar a transmissão vertical (TV) do HIV-aids, pois ao ser detectada a presença de anticorpos anti--HIV, aplica-se o Protocolo ACTG 076 (Pediatric AIDS Clinical Trials Group Study Group), que é o tratamento das gestantes HIV-positivo no pré, intra e pós-parto. Objetivo: comparar a coleta das amostras de sangue no papel de filtro (PF) e no plasma (padrão-ouro) na triagem pré-natal, utilizando anticorposanti-HIV 1+2 no procedimento imunoquímico ELISA (Imunoscreen HIV 1+2 SS), da firma Mbiolog. Métodos: foram estudadas 1.142 grávidas de quatromunicípios do Estado do Rio de Janeiro: Itaboraí (N = 131), Itaguaí (N = 597), Niterói (N = 377) e São João de Meriti (N = 37), a partir do mês de novembro de 2008 até fevereiro de 2009. As grávidas foram submetidas a punção digital e venosa para a rotina da triagem pré-natal, sendo a primeira aplicada em PF. Foram calculados os limites de especificidade, sensibilidade, e valores preditivos positivos e negativos para o estudo. Resultados: os resultados da absorbância (homogeneização das variâncias) encontrados para as amostras não reativas e reativas, em ambas as técnicas, não mostraram diferenças significativas (P > 0,05) quando aplicado o teste T de Student no estudo de repetitividade. Os estudos de reprodutibilidade em ambas as técnicas não resultaram coeficientes de variação superiores a 10%. Conclusão: os resultados da sorologia para HIV no sangue seco coletado em PF foram semelhantes aos da coleta por punção venosa, validando esta técnica.

Although the filter paper (FP) have been introduced since the last century (1963) by Guthrie and Susi, in neonatal screening as an alternative to blood sample collection, its benefits in the application of medical laboratory practice not were used effectively in recent years, with the exception of the recent results obtained in the works for the prenatal screening. Early diagnosis of HIV in pregnant women is of paramount importance, being the most out standing support to counteract the vertical transmission (VT) of HIV-AIDS, because the determination of the presence of HIV antibodies, it applies to the ACTG 076 Protocol (Pediatric AIDS Clinical Trials Group Study Group), which is the treatment of pregnant HIV-positive in the pre-,intra and post-parto.Objetive: compare the collection of blood samples on filter paper (FP) and plasma (standard gold) in prenatal screening, using antibodies against HIV 1+2 ELISA immunochemical procedure (Imunoscreen HIV 1 +2 SS), the firm Mbiolog. Methods: we studied 1,142 pregnant women in four counties of the State of Rio de Janeiro: Itaboraí (N = 131), Itaguaí (N = 597), Niterói (N = 377) and St. John Meriti (N = 37), from November 2008 until February 2009.The pregnant women underwent fingerstick and venous for routine prenatal screening, the first being applied in PF. We calculated the limits of sensitivity,specificity and positive predictive value and negative for the study. Results: the results of absorbance (homogeneity of variances) for the samples foundnon-reactive and reactive in both techniques, showed no significant differences (P > 0.05) when using the Student's T test in the study of repeatability.Reproducibility studies for both techniques did not result in coefficients of variation above 10%. Conclusion: the results of serological tests for HIV in driedblood collected in PF were similar to the collection by venipuncture, validating this technique.

Comparação da coleta das amostras de sangue na triagem pré-natal, utilizando o papel de filtro e punção venosana técnica de ELISA para detecção de sífilis / Comparison of the collection of blood samples in prenatal screening, using the filter paperand venipuncture technique ELISA for the detection of syphilis

A sífilis é uma doença infecciosa sistêmica, de evolução crônica e causada pelo Treponema pallidum, um espiroqueta de transmissão sexuale vertical, que pode produzir, respectivamente, as formas adquirida e congênita da doença. No Brasil, segundo o Ministério da Saúde (MS), embora a subnotificação de casos de sífilis seja alta, alguns dados disponíveis indicam a elevada magnitude deste problema infeccioso. Objetivo: comparar a coletadas amostras de sangue no papel de filtro (PF) e no plasma (padrão-ouro) na triagem pré-natal, utilizando anticorpos antitreponêmicos totais (IgG + IgM) no procedimento imunoquímico ELISA, registrado pelo Imunoscreen, da firma MBiolog. Métodos: foram estudadas 1.142 grávidas de quatro municípiosdo Estado de Rio de Janeiro: Itaboraí (N = 131), Itaguaí (N = 597), Niterói (N = 377) e São João de Meriti (N = 37) a partir do mês de novembro de 2008 até fevereiro de 2009. As grávidas foram submetidas a punção venosa e digital para a rotina da triagem pré-natal, sendo a última aplicada em PF. Foram calculados os limites de especificidade, sensibilidade e valores preditivos positivo e negativo para o estudo. Resultados: os resultados da sorologia parasífilis nas amostras do município de Itaboraí apresentaram ELISA positivo em 4,58%, os municípios de Itaguaí, Niterói e São João de Meriti mostraram positividade em 3,18%, 2,65% e 0%, respectivamente. Os procedimentos realizados tiveram uma sensibilidade e especificidade de 100% e os critérios preditivos positivos e negativos para todas as grávidas, estudados nas 1.142 amostras, foram de 100%. Conclusão: os resultados da sorologia para sífilis no sangue seco coletado em PF foram semelhantes aos da coleta por punção venosa, validando esta técnica.

Syphilis is a systemic disease of chronic evolution and caused by Treponema pallidum, a spirochete of sexual and vertical transmission,which can produce, respectively, the form of acquired and congenital disease. In Brazil, according to the Ministry of Health (MoH), although the underreporting of cases of congenital syphilis is high, some available data indicate the high magnitude of this problem that especially affects the weakness of pregnant women. Congenital syphilis causes great social impact, which results in deterioration of quality of life on a important stratum of the population,and indirect costs to the economy of the country, which, added to the direct costs resulting from hospitalizations and procedures for the treatment of its complications, increasing the total costs of care of public health. Objective: to compare the collection of blood samples on filter paper (FP) and plasma(gold standard) in prenatal screening, using anti-treponema total (IgG + IgM) in ELISA immunochemical. Methods: we studied 1,142 pregnant of the following cities: Itaboraí (N = 131), Itaguaí (N = 597), Niterói (N = 377) and St. João de Meriti (N = 37). Blood samples were collected from the finger and venipuncture of pregnant women in stations of collection of these counties, calculating elapsed time from sample collection to delivery of the report to thecouncil. We calculated the limits of sensitivity, specificity, positive predictive value and negative for the study. Results: Itaboraí showed positive ELISA in4.58%, in Itaguai, Niterói and St. João de Meriti showed, respectively, 3.18%, 2.65% and 0%. The procedures performed had a sensitivity and specificity of 100%, and the positive and negative predictive criteria for all pregnant women studied in 1,142 samples were 100%. All positive cases were reported to the Municipality within 10 days of sample collection. Conclusion: we conclude that the implementation of collection of dried blood on filter paper in pregnantwomen screening, was similar to that collected by venipuncture, validating this technology.

Comparación de calidade del pepel de filtro hahnemuehle 2992 con el schleicher & schuell 903 en la conservación de la sangre seca para el estudio poblacional de anticuerpos igm e igg de la sífilis / Comparison of quality between hahnemuehle 2992 and schleucher & schuell 903 filter paper, in the preservation of dried blood for the population study of igm and igg antbodies to syphilis

Introducción: la alternativa para la colecta de las muestras de sangre en el papel de filtro (PF) muestra cada día mayor interés en los investigadores yasistentes de salud que buscan mejorar los programas de pesquisaje masivo de la población. Muchos autores desde la apertura esta posibilidad en el siglo pasado iniciaron sus proyectos con la conservación de la muestra de sangre seca para estudios masivos neonatales. . Objetivo: valorar la calidad del papel de filtro (Hahnemuehle, 2992) para la colecta de muestras de sangre para el tamisaje de sífilis, como alternativa en la conservación de sangre seca en los laboratorios que usan esta alternativa y estrategia en los estudios de terreno. Métodos: fue realizadoun diseño para el estudio comparativo de calidad entre el PF Schleicher & Schuell 903 que sirvió como patrón de oro y el PF Hahnemuehle 2992, para a coleta e almacenamiento de muestras de sangre para el estudio de anticuerpos totales (IgM e IgG) contra el Treponema pallidum con una técnica ELISA tipo Sándwich. Se compararon 140 señales de absorbancia del mismo número de muestras de mujeres embarazadas, previo consentimiento escrito, para registrar su semejanza estadísticamente según los procedimientos estadísticos del Test de Fisher y Student (Test T) con un valor de seguridad de P > 0,05. Resultados: se encontró correspondencia entre los valores de especificidad, sensibilidad y valor predictivo positivo entre el procedimiento usando con los dos PF en el universo estudiado, así como los valores de T = 0,344331 para el análisis de comparación de medias entre la absorbancia obtenida con los dos PF para de P < 0,001. Conclusión: se concluye que la comparación del PF Hahnemuehle 2992 con el Schleicher & Schuell 903 para el estudio de muestras de sangre seca para la detección de anticuerpos totales del Treponema pallidum por ensayo ELISA es significativamente semejante y cuentan con la misma calidad.

Introduction: the filter paper (dried blood spot DBS) as an alternative of blood sample collection shows an increased interest of scientific investigators and health assistants that search to improve massive research programs of the population. Since the opening of this possibility in the last century, many authors started their projects using dried blood spots for massive neonatal studies. Objective: to assess the filter paper quality (Hahnemuehle, 2992) for collecting blood samples for sifilis screening as an alternative to the conservation of dry blood in the laboratory using this alternative strategyand in camp studies. Methods: a design for a comparative study of quality between Schleicher and Schuell 903 paper used as golden standard- andHahnemuehle 2992 paper was realized, for the collection and storage of dried blood for the totals antibodies (IgM and IgG) against the Treponema pallidum study, using ELISA assay (sandwich type). A hundred and forty (140) signals of absorbance of the same number of pregnant samples, with previous written consent, to register their similarity, using statistic procedures of the Test of Fisher and Student (test T) with a security value of P > 0,05. It was also compared the absorbance values of the positive cases (according a settled cut off for each plate) and sensitivity,specificity and predictive value criteria were calculatedon a total of 604 pregnant of the study. Results: it was detected correspondence of sensitivity, specificity and positive predictive values, in the procedures using the two types of fi lter papers, as well as the value of T = 0,344331 for the analysis of the average comparison between the obtained absorbance with the two types to P < 0,001. Conclusion: it was concluded that Hahnemuehle 2992 fi lter paper compared with Schleicher and Schuell 903 shows signifi cantly similar and have the same quality for the detection of totals antibodies Treponema pallidum of using ELISA test.

In Vitro Screening of Anti-lice Activity of Pongamia pinnata Leaves

Growing patterns of pediculocidal drug resistance towards head louse laid the foundation for research in exploring novel anti-lice agents from medicinal plants. In the present study, various extracts of Pongamia pinnata leaves were tested against the head louse Pediculus humanus capitis. A filter paper diffusion method was conducted for determining the potential pediculocidal and ovicidal activity of chloroform, petroleum ether, methanol, and water extracts of P. pinnata leaves. The findings revealed that petroleum ether extracts possess excellent anti-lice activity with values ranging between 50.3% and 100% where as chloroform and methanol extracts showed moderate pediculocidal effects. The chloroform and methanol extracts were also successful in inhibiting nymph emergence and the petroleum ether extract was the most effective with a complete inhibition of emergence. Water extract was devoid of both pediculocidal and ovicidal activities. All the results were well comparable with benzoyl benzoate (25% w/v). These results showed the prospect of using P. pinnata leave extracts against P. humanus capitis in difficult situations of emergence of resistance to synthetic anti-lice agents.