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1.
Braz. J. Pharm. Sci. (Online) ; 57: e19073, 2021. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1345463

RESUMO

A reversed-phase high performance liquid chromatography (RP-HPLC) method with ultraviolet detection was developed and validated for the simultaneous quantification of antiretroviral drugs lamivudine (3TC), stavudine (d4T), and zidovudine (AZT) in perfusate samples obtained from the Single-Pass Intestinal Perfusion studies. The chromatographic analysis was performed using a Gemini C18 column and didanosine as internal standard (IS). The following parameters were considered for the validation procedure: system suitability, accuracy, precision, linearity and selectivity. The limits of detection were 0.32 µg/mL for 3TC, 0.11 µg/mL for d4T and 0.45 µg/mL for AZT and the limits of quantification were 1.06 µg/mL for 3TC, 0.38 µg/mL for d4T and 1.51 µg/mL for AZT. Repeatability and intermediate precision ranged from 1.05 to 1.31 and 1.50 to 1.87, respectively, and are expressed as percent of relative standard deviation (RSD). Based on these results, the developed and validated RP-HPLC method can be used for simultaneous determination of 3TC, d4T, and AZT in perfusate samples. Furthermore, this method is simple and adequate for measurements of the antiretroviral drugs in the same sample, since those compounds are mostly co-administered. Besides, this work can be used as an initial base for the development of similar methods in the same conditions presented in our study.


Assuntos
Zidovudina/farmacologia , Cromatografia Líquida de Alta Pressão/métodos , Lamivudina/farmacologia , Estudo de Validação , Antirretrovirais/farmacologia , Perfusão/instrumentação , Permeabilidade , Preparações Farmacêuticas/administração & dosagem , Limite de Detecção
2.
Braz. J. Pharm. Sci. (Online) ; 57: e18899, 2021. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1339302

RESUMO

Microbiological quality of pharmaceuticals is fundamental in ensuring efficacy and safety of medicines. Conventional methods for microbial identification in non-sterile drugs are widely used; however they can be time-consuming and laborious. The aim of this paper was to develop a chemometric-based rapid microbiological method (RMM) for identifying contaminants in pharmaceutical products using Fourier transform infrared with attenuated total reflectance spectrometry (FTIR-ATR). Principal components analysis (PCA) and linear discriminant analysis (LDA) were used to obtain a predictive model capable of distinguishing Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), Enterococcus faecium (ATCC 8459), Escherichia coli (ATCC 8739), Micrococcus luteus (ATCC 10240), Pseudomonas aeruginosa (ATCC 9027), Salmonella typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 6538), and Staphylococcus epidermidis (ATCC 12228) microbial growth. FTIR-ATR spectra provide data on proteins, DNA/RNA, lipids, and carbohydrates constitution of microbial growth. Microbial identification provided by PCA/LDA based on FTIR-ATR method were compatible with those obtained using traditional microbiological methods. The chemometric-based FTIR-ATR method for rapid identification of microbial contaminants in pharmaceutical products was validated by assessing the sensitivity (93.5%), specificity (83.3%), and limit of detection (17-23 CFU/mL of sample). Therefore, we propose that FTIR-ATR spectroscopy may be used for rapid identification of microbial contaminants in pharmaceutical products and taking into account the samples studied


Assuntos
Análise Espectral/instrumentação , Preparações Farmacêuticas/análise , Análise Discriminante , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Análise de Fourier , Pseudomonas aeruginosa/classificação , Bacillus subtilis/classificação , Candida albicans/classificação , Limite de Detecção
3.
Con-ciencia (La Paz) ; 7(1): 67-77, abr. 2019.
Artigo em Espanhol | LILACS | ID: biblio-1178663

RESUMO

El presente estudio describe la validación del método para la cuantificación de Ciclosporina A en sangre total por Cromatografía Líquida de Alta Resolución (HPLC) en Fase Reversa con Detector de Arreglo de Diodos. La Ciclosporina A es un fármaco inmunosupresor con un estrecho margen terapéutico además de su amplia variabilidad en los procesos farmacocinéticos, justifican su monitorización de dosis a pacientes con trasplantes de órganos para evitar los efectos secundarios y el posible rechazo del órgano trasplantado. La separación de la Ciclosporina A de una matriz compleja como la sangre fue llevada a cabo de manera exitosa utilizando como fase estacionaria una columna C8 5um (250 mm x 4,6mm) o equivalente a L7 según la USP 37, la fase móvil fue una mezcla de acetonitrilo y agua en gradiente, flujo 1.4 ml/min, temperatura de la columna 75ºC y detección con Arreglo de Diodos a 210nm. El método fue validado con los siguientes parámetros: especificidad, linealidad, precisión, exactitud, límite de detección y límite de cuantificación. También se realizó la prueba de aptitud del sistema. El método fue específico para la Ciclosporina A, la respuesta fue lineal en el rango de 100 a 1000 ng/mL de concentración del analito. El valor del coeficiente de variación o desviación estándar relativa (C.V. o DSR) para la precisión fue óptimo. La recuperación media fue de 103,06%. Y el límite de detección y cuantificación resultaron óptimos para la cuantificación de Ciclosporina en sangre total en el rango definido. Finalmente el método cromatográfico nos permitió separar y cuantificar a la Ciclosporina A presente en las muestras de sangre de pacientes con transplante renal.


This study describe the validation method for the quantification of Cyclosporin A in Whole Blood by Liquid Chromatography High Efficiency (HPLC) in reverse phase. Cyclosporine A is immunosuppressant United Nations UN the narrow scam therapeutic margin: In addition to its extensive ¿variability in pharmacokinetic processes justify their monitoring dose Patients with organ transplants paragraph Avoid Side Effects and poaible organ rejection transplanted. Separation of Cyclosporine A of a matrix complex as the blood was carried out successfully using as the stationary phase C8 column, the mobile phase is a mixture of acetonitrile and water gradient, flow 1.4 ml / min, Temperature column was 75 ° C detection 210 nm in one. The method was validated with the following parameters: specificity, linearity, precision, accuracy, limit of detection and limit of quantification. Aptitude Test System was also performed. Was Method Specific for Cyclosporin A, the response was linear in the range 100 a 1000 ng/mL concentration of the analyte. The coefficient of variation or relative standard deviation (RSD or CV) for the precision was optimal. Recovery media WAS 103,06%. And the limit of detection and quantification were optimal for the quantification of total Cyclosporine in blood in the range defined. Finally Chromatographic Method allowed us to separate and quantify Cyclosporin A in samples of patients with renal transplantation.


Assuntos
Ciclosporina , Dosagem , Limite de Detecção , Sensibilidade e Especificidade , Transplante de Rim , Padrões de Referência
4.
Braz. j. med. biol. res ; 52(3): e8186, 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-989465

RESUMO

Klebsiella pneumoniae is one of the main pathogenic bacteria that causes nosocomial infections, such as pneumonia, urinary tract infection, and sepsis. Therefore, the rapid and accurate detection of K. pneumoniae is important for the timely treatment of infectious patients. This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of K. pneumoniae-specific gene ureR_1 (Gene ID: 11847803). The ureR_1 gene was obtained through local and online BLAST, and the specific primers were designed for its detection. Positive reactions were observed on all 140 K. pneumoniae clinical isolates while all the 82 non-K. pneumoniae clinical isolates were negative. Plasmids with the specific gene and the mouse blood with K. pneumoniae were used for sensitivity analysis. The detection limit of the LAMP was 1 bacterium/reaction. The results showed that the LAMP targeted to ureR_1 is a fast, specific, sensitive, inexpensive, and suitable method for the detection of K. pneumoniae.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Genes Bacterianos , Klebsiella pneumoniae/genética , Plasmídeos/isolamento & purificação , Plasmídeos/genética , Temperatura , Fatores de Tempo , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Primers do DNA/isolamento & purificação , Primers do DNA/genética , Limite de Detecção , Klebsiella pneumoniae/isolamento & purificação
5.
Mem. Inst. Invest. Cienc. Salud (Impr.) ; 16(3): 6-12, dic. 2018. tab, ilus
Artigo em Espanhol | LILACS, BDNPAR | ID: biblio-998219

RESUMO

El cáncer de cuello uterino es el segundo cáncer femenino más común a nivel mundial. El agente causal es el virus de papiloma humano (VPH). Se han identificado 13 tipos de virus de papiloma humano de alto riesgo oncogénico (VPH-AR), entre los cuales el VPH 16 y VPH 18 son los más frecuentemente detectados en cáncer de cuello uterino, siendo en Paraguay detectados en el 70% de casos de cáncer invasor. Por ello, el objetivo fue estandarizar y determinar el límite de detección de una técnica de PCR convencional para la detección de VPH 16 y 18. Para la detección de ADN de VPH 16 y 18, se observaron mejores resultados con 2mM de MgCl2 y 60°C para la temperatura de alineamiento. El límite de detección para las PCR fue de 14,6x10-11ng/µL para VPH 16 y 21,7x10-12ng/µL para VPH 18. Este trabajo servirá de base a otros estudios de detección e identificación de estos tipos virales por PCR, con miras a identificar un grupo de mujeres positivas para VPH-AR que poseen mayor riesgo de desarrollo de lesión y cáncer de cuello uterino y precisan de un seguimiento más cercano(AU


Cervical cancer is the second most common female cancer worldwide. It is caused by the human papilloma virus (HPV). Thirteen genotypes of high oncogenic risk human papilloma viruses (HPV-HR) have been identified, among which types 16 and 18 are the most frequently detected in cervical cancer. In Paraguay, they are detected in 70% of the invasive cancer cases. Therefore, the objective was to standardize and determine the detection limit of a conventional PCR technique for the detection of HPV 16 and 18. Better results were observed with 2mM MgCl2 and 60°C for the alignment temperature in detection of HPV 16 and 18 DNA. The limit of detection was 14.6x10-11ng/µL for HPV 16 and 21.7x10-12ng/µL for HPV 18. This work will help other studies for the detection and identification of these viral types by PCR in order to identify a group of HPV-HR positive women who have higher risk for the development of lesions and cervical cancer and need a closer follow-up(AU)


Assuntos
Humanos , Feminino , Neoplasias do Colo do Útero/virologia , Reação em Cadeia da Polimerase/métodos , Infecções por Papillomavirus/virologia , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Sequência de Bases , Genoma Viral , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Limite de Detecção
6.
Con-ciencia (La Paz) ; 6(1): 15-25, jun. 2018. ilus., tab.
Artigo em Espanhol | LILACS, LIBOCS | ID: biblio-1178720

RESUMO

Staphylococcus aureus puede contaminar una gran gama de alimentos, siendo los quesos frescos un medio diferencial y selectivo para el desarrollo de este microorganismo, llegando a la producción de enterotoxina termoestable ocasionando una intoxicación estafilocóccica trasmitida por alimentos. El empleo del método 3M Petrifilm Staph Express (STX) supone considerables ventajas frente al método convencional o de referencia, facilitando y favoreciendo muchos aspectos técnicos y económicos, dentro de ellos tenemos la optimización de tiempo en cuanto se refiere a la preparación de medios de cultivo, necesidad de equipamiento, requerimiento de infraestructura, menor requerimiento de personal y la disminuye los costos del uso de consumo eléctrico, disminuye la generación de residuos sólidos, entre otros. La comparación entre el método alternativo frente al método convencional fue hecha por medio de la contaminación artificial de una matriz libre de S. aureus y un recuento bajo de microbiota acompañante, donde se establecieron diferentes niveles de contaminación, evaluándose los indicadores de desempeño (exactitud relativa, precisión relativa, linealidad, curva de calibración y límite de cuantificación). En indicador de exactitud relativa se obtuvo un 95,6% de recuperación para el nivel más bajo (aproximadamente 8 UFC/g) y 98,7% de recuperación para el nivel de contaminación más alto (aproximadamente 5300 UFC/g), teniendo un rango de 70 a 120% de aceptación. Para la precisión relativa se calcularon la RSD de todos los niveles ensayados, obteniéndose datos que aceptan la precisión relativa del método de placa seca rehidratable porque los valores fueron menores al RSD teórico calculado. El límite de detección fue ensayado con <10UFC/g obteniéndose un CV de 1,16%. Por los resultados obtenidos podemos concluir que el método 3M Petrifilm Staph Express (STX) cumple con los indicadores de desempeño frente a la norma ISO 6888-1:2003.


Staphylococcus aureus can contaminate a wide range of foods, being fresh cheeses a differential and selective medium for the development of this microorganism, reaching the production of thermostable enterotoxin causing a staphylococcal intoxication transmitted by food. The use of the 3M Petrifilm Staph Express (STX) method has considerable advantages over the conventional or reference method, facilitating and favoring many technical and economic aspects, within them we have the optimization of time as regards the preparation of culture media, need for equipment, infrastructure requirements, lower personnel requirements and decreases the costs of the use of electricity consumption, decreases the generation of solid waste, among others. The comparison between the alternative method and the conventional method was made by means of the artificial contamination of a free matrix of S. aureus and a low accompanying microbiota count, where different levels of contamination were established, evaluating the performance indicators (relative accuracy, relative precision, linearity, calibration curve and limit of quantification). In the relative accuracy indicator, 95.6% recovery was obtained for the lowest level (approximately 8 CFU / g) and 98.7% recovery for the highest level of contamination (approximately 5300 CFU / g), with a range of 70 to 120% acceptance. For the relative precision, the RSD of all the tested levels was calculated, obtaining data that accept the relative accuracy of the dry rehydratable plate method because the values were lower than the calculated theoretical RSD. The limit of detection was tested with <10 CFU /g, obtaining a CV of 1.16%. Based on the results obtained, we can conclude that the 3M Petrifilm Staph Express (STX) method complies with the performance indicators against the ISO 6888-1: 2003 standard.


Assuntos
Staphylococcus aureus , Queijo , Calibragem , Limite de Detecção , Alimentos
7.
Biomédica (Bogotá) ; 37(supl.2): 187-192, jul.-set. 2017. tab, graf
Artigo em Espanhol | LILACS | ID: biblio-1038791

RESUMO

Resumen Introducción. Las técnicas de biología molecular han permitido ampliar el conocimiento sobre las fuentes de ingestión de sangre de los insectos vectores. Sin embargo, la utilidad de estas técnicas depende de la cantidad de sangre ingerida y del proceso de digestión en el insecto. Objetivo. Determinar el tiempo límite de detección del gen citocromo b (Cyt b) de humanos en hembras de Lutzomyia evansi alimentadas experimentalmente. Materiales y métodos. Se evaluaron ocho grupos de hembras de L. evansi alimentadas con sangre humana, las cuales fueron sacrificadas en intervalos de 24 horas desde el momento de la ingestión sanguínea. Se extrajo el ADN total de cada hembra y se amplificó un segmento de 358 pb del gen Cyt b. Los productos amplificados fueron sometidos a un análisis de polimorfismos en la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism, RFLP), con el fin de descartar falsos positivos. Resultados. El segmento del gen Cyt b de humanos fue detectado en 86 % (49/57) de las hembras de L. evansi a partir de las 0 horas y hasta 168 horas después de la ingestión de sangre. En 7 % (4/57) de los individuos se amplificó el ADN del insecto y en el 7 % restante no se amplificó la banda de interés. No se encontraron diferencias estadísticas en cuanto a la amplificación del segmento del gen Cyt b de humanos ni al número de muestras amplificadas entre los grupos de hembras sacrificadas a distintas horas después de la ingestión. Conclusión. El segmento del gen Cyt b de humanos fue detectable en hembras de L. evansi hasta 168 horas después de la ingestión de sangre.


Abstract Introduction: Molecular biology techniques have allowed a better knowledge of sources of blood meals in vector insects. However, the usefulness of these techniques depends on both the quantity of ingested blood and the digestion process in the insect. Objective: To identify the time limit for detection of the human cytochrome b (Cyt b) gene in experimentally fed females of Lutzomyia evansi. Materials and methods: Eight groups of L. evansi females were fed on human blood and sacrificed at intervals of 24 hours post-ingestion. Total DNA was extracted from each female and a segment of 358 bp of Cyt b was amplified. In order to eliminate false positives, amplification products were subjected to a restriction fragment length polymorphism (RFLP) analysis. Results: The human Cyt b gene segment was detected in 86% (49/57) of the females of L. evansi, from 0 to 168 hours after blood ingestion. In 7% (4/57) of the individuals we amplified insect DNA, while in the remaining 7%, the band of interest was not amplified. We did not find any statistical differences between groups of females sacrificed at different times post-blood meal regarding the amplification of the human Cyt b gene segment or the number of samples amplified. Conclusion: The human Cyt b gene segment was detectable in L. evansi females up to 168 hours after blood ingestion.


Assuntos
Animais , Feminino , Humanos , Psychodidae/fisiologia , Proteínas Sanguíneas/análise , Citocromos b/análise , Insetos Vetores/fisiologia , Fatores de Tempo , Simulação por Computador , Polimorfismo de Fragmento de Restrição , DNA/análise , Proteínas Sanguíneas/farmacocinética , Citocromos b/farmacocinética , Digestão , Comportamento Alimentar , Limite de Detecção , Genes
8.
Rev. argent. endocrinol. metab ; 54(3): 101-108, set. 2017. tab
Artigo em Espanhol | LILACS | ID: biblio-957975

RESUMO

El cáncer diferenciado de tiroides (CDT) es el cáncer endocrinológico más frecuente y en las últimas décadas su incidencia ha aumentado. El seguimiento de la enfermedad se efectúa con la medición de tiroglobulina (Tg) sérica, ecografía cervical y barrido corporal total diagnóstico. Los métodos de Tg han evolucionado a través del tiempo. Actualmente, los ensayos inmunométricos de Tg se clasifican en 1.ª y 2.ª generación (1.ª G y 2.ª G). Comprobamos que los ensayos de 2.ª G alcanzan una precisión adecuada para medir valores del orden de 0,1 ng/ml y los de 1.ª G de 1 ng/ml. La bibliografía señala que en el caso de los pacientes de bajo riesgo, una Tg bajo levotiroxina indetectable por un método de 2.ª G puede evitar la realización de Tg estimulada, sea por la suspensión de la terapia hormonal como por el empleo de la TSH recombinante humana, debido a su mayor sensibilidad. Sin embargo, por su menor especificidad, un valor detectable no asegura la presencia de enfermedad, y debería confirmarse. Para optimizar la utilidad clínica de dicha medición se podrían emplear valores de cortes de acuerdo con la población y el método en lugar de la sensibilidad funcional o límite de cuantificación del mismo. Se señalan también otros aspectos críticos en la medición de Tg como son la discordancia entre distintas metodologías y las interferencias en su medición, principalmente por anticuerpos antitiroglobulina. En presencia de interferencias pierden utilidad los ensayos de Tg de 1.ª y 2.ª G. El seguimiento de los pacientes con Tg interferida tiene limitaciones todavía no resueltas. Es importante consensuar entre médicos y bioquímicos las dificultades técnicas y los criterios de interpretación de los valores de Tg en el seguimiento de los pacientes con CDT.


Differentiated thyroid cancer (DTC) is the most common endocrine cancer (tumour) and its incidence has risen in the past decades. Its follow-up includes measuring serum thyroglobulin (Tg), performing neck ultrasound and a diagnostic whole-body scan. Tg assays have evolved with time. At present immunoassays for Tg are classified as 1 st and 2 nd generation assays (1 st G and 2 nd G). 2 nd G assays show an adequate (good) precision at levels close to 0.1 ng/ml and 1 st G assays at levels close to 1 ng/ml. The literature shows that for low risk patients on levothyroxine treatment, who undetectable levels by 2 aG assays can avoid the stimulation test performed by thyroid hormone withdrawal or after recombinant human TSH, due to better sensitivity. However, due to lower specificity, detectable levels do not confirm the presence of disease (tumour), and should be confirmed. To optimise the clinical usefulness of the test, cut-off values specific for population and method should be used, instead of functional sensitivity or quantification limit. Critical issues for measuring Tg are discussed, such as non-harmonisation of methods, and interferences, mainly by antithyroglobulin antibodies (ATg). 1 st and 2 nd G assays are less useful in presence of ATg, and follow up of such patients is limited. Consensus between physicians and the laboratory on technical issues and interpretation criteria of Tg values is of outmost importance in the follow-up of DTC patients.


Assuntos
Humanos , Tireoglobulina/análise , Testes de Função Tireóidea/métodos , Neoplasias da Glândula Tireoide/diagnóstico , Sensibilidade e Especificidade , Limite de Detecção , Razão Sinal-Ruído
9.
Braz. j. med. biol. res ; 48(2): 146-153, 02/2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-735854

RESUMO

Although 17β-estradiol (E2) deficiency has been linked to the development of osteoarthritis (OA) in middle-aged women, there are few studies relating other estrogens and estrogen metabolites (EMs) to this condition. We developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method to measure the levels of six EMs (i.e., estrone, E2, estriol, 2-hydroxyestrone, 2-hydroxyestradiol, and 16a-hydroxyestrone) in healthy pre- and postmenopausal women and women with OA. This method had a precision ranging from 1.1 to 3.1% and a detection limit ranging from 10 to 15 pg. Compared to healthy women, serum-free E2 was lower in the luteal and postmenopausal phases in women with OA, and total serum E2 was lower in postmenopausal women with OA. Moreover, compared to healthy women, total serum 2-hydroxyestradiol was higher in postmenopausal women with OA and total serum 2-hydroxyestrone was lower in both the luteal and follicular phases in women with OA. In conclusion, our HPLC-ESI-MS/MS method allowed the measurement of multiple biochemical targets in a single assay, and, given its increased cost-effectiveness, simplicity, and speed relative to previous methods, this method is suitable for clinical studies.


Assuntos
Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Cromatografia Líquida de Alta Pressão/métodos , Estrogênios/sangue , Osteoartrite/sangue , Pós-Menopausa/sangue , Pré-Menopausa/sangue , Espectrometria de Massas por Ionização por Electrospray/métodos , Estradiol/análogos & derivados , Estradiol/sangue , Estriol/sangue , Estrogênios/metabolismo , Estrona/sangue , Fase Folicular/sangue , Hidroxiestronas/sangue , Limite de Detecção , Fase Luteal/sangue , Osteoartrite/metabolismo , Pós-Menopausa/metabolismo , Pré-Menopausa/metabolismo , Estatísticas não Paramétricas
10.
Mem. Inst. Oswaldo Cruz ; 109(8): 978-983, 12/2014. graf
Artigo em Inglês | LILACS | ID: lil-732610

RESUMO

The Global Program for the Elimination of Lymphatic Filariasis (GPELF) aims to eliminate this disease by the year 2020. However, the development of more specific and sensitive tests is important for the success of the GPELF. The present study aimed to standardise polymerase chain reaction (PCR)-based systems for the diagnosis of filariasis in serum and urine. Twenty paired biological urine and serum samples from individuals already known to be positive for Wuchereria bancrofti were collected during the day. Conventional PCR and semi-nested PCR assays were optimised. The detection limit of the technique for purified W. bancrofti DNA extracted from adult worms was 10 fg for the internal systems (WbF/Wb2) and 0.1 fg by using semi-nested PCR. The specificity of the primers was confirmed experimentally by amplification of 1 ng of purified genomic DNA from other species of parasites. Evaluation of the paired urine and serum samples by the semi-nested PCR technique indicated only two of the 20 tested individuals were positive, whereas the simple internal PCR system (WbF/Wb2), which has highly promising performance, revealed that all the patients were positive using both samples. This study successfully demonstrated the possibility of using the PCR technique on urine for the diagnosis of W. bancrofti infection.


Assuntos
Adolescente , Adulto , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , DNA de Helmintos/isolamento & purificação , Filariose Linfática/diagnóstico , Microfilárias/isolamento & purificação , Wuchereria bancrofti/isolamento & purificação , Antígenos de Superfície/sangue , Antígenos de Superfície/urina , Filariose Linfática/sangue , Filariose Linfática/urina , Limite de Detecção , Microfilárias/genética , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Sensibilidade e Especificidade , Wuchereria bancrofti/genética
11.
Electron. j. biotechnol ; 17(4): 183-188, July 2014. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-719110

RESUMO

Background A simple, rapid, low-cost and environmentally friendly method was developed to determine dopamine (DA) in the presence of ascorbic (AA) and uric acid (UA) based on a novel technique to prepare a graphene-chitosan (GR-CS) nanocomposite and modify it on the surface of carbon paste electrode (CPE). For our design, CS acts as a media to disperse and stabilize GR, and then GR plays a key role to selective and sensitive determination of DA. Results Under physiological conditions, the linear range for dopamine was determined from 1 × 10- 4 to 2 × 10- 7 mol/L with a good correlation coefficient of 0.9961 in the presence of 1000-fold interference of AA and UA. The detection limit was estimated to be 9.82 × 10- 8 mol/L (S/N = 3). In order to study the stability and reproducibility, GR/CS/CPE underwent successive measurements in 10 times and then tested once a d for 30 d. The result exhibited 98.25% and 91.62% activities compared with the original peak current after 10-time measurements and 30-d storage. Conclusion The GR/CS/CPE has wide linear concentration range, low detection limit, and good reproducibility and stability, which suggests that our investigations provide a promising alternative for clinic DA determination.


Assuntos
Carbono/química , Dopamina/análise , Quitosana/química , Grafite/química , Eletrodos , Nanocompostos/química , Técnicas Eletroquímicas , Limite de Detecção , Técnicas de Química Sintética , Concentração de Íons de Hidrogênio
12.
Rev. Inst. Adolfo Lutz ; 73(2): 148-157, abr.-jun. 2014. tab, graf
Artigo em Português | LILACS, Sec. Est. Saúde SP | ID: lil-782598

RESUMO

Neste trabalho são apresentadas planilhas eletrônicas construídas em software Microsoft Excel® quepossibilitam avaliar as estimativas de limite de decisão (CCα) e capacidade de detecção (CCβ) nas regiõesdo limite de detecção e do Valor Máximo Permitido (VMP). As estimativas são realizadas a partir decurvas analíticas lineares e homoscedásticas construídas em procedimentos de calibração segundo asnormas ISO e recomendações IUPAC. Após a validação por processamento manual dos dados, as planilhaseletrônicas foram utilizadas nas determinações de nitrito em águas envasadas (VMP = 0,02 mg/L) e defluoreto em águas de abastecimento público (intervalo de conformidade = 0,6 a 0,8 mg/L). Na análise defluoreto, em que existe um valor mínimo requerido (0,6 mg/L) e um valor máximo aceitável (0,8 mg/L)para a concentração, a planilha calcula a concentração crítica em ambos os limites com uma probabilidadede erro tipo I igual a 0,05. Desta forma, as planilhas eletrônicas permitem efetuar a rápida decisão entreconforme e não conforme na interpretação dos resultados...


Assuntos
Humanos , Microbiologia da Água , /métodos , Limite de Detecção , Estatística como Assunto , Validação de Programas de Computador
13.
Biol. Res ; 47: 1-8, 2014. graf, tab
Artigo em Inglês | LILACS | ID: biblio-950718

RESUMO

BACKGROUND: Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India. RESULTS: Linear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p<0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p=0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000 - 25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians. CONCLUSION: The HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.


Assuntos
Humanos , RNA Viral/sangue , Infecções por HIV/diagnóstico , HIV-1/isolamento & purificação , Carga Viral/métodos , Compostos Orgânicos , Kit de Reagentes para Diagnóstico/economia , Sequência de Bases/genética , Genes gag/genética , Modelos Lineares , Sensibilidade e Especificidade , HIV-1/classificação , Estatísticas não Paramétricas , Gerenciamento Clínico , Limite de Detecção , Reação em Cadeia da Polimerase em Tempo Real , Invenções , Índia
14.
Clin. biomed. res ; 34(3): 260-265, 2014. ilus, tab
Artigo em Português | LILACS | ID: biblio-834468

RESUMO

Introduction: Polyomaviruses (BKV and JCV) cause infection mainly in immunocompromised adults. A sensitive and specific diagnosis tool is fundamental to demonstrate the BKV and JCV infections. Nowadays many laboratories are using a PCR technique for detecting polyomaviruses genome in clinical samples. In this context, the purpose of this study is to determine the threshold of detection of the nested-PCR for polyomaviruses JC and BK. Methods: Serial dilutions of the samples of BKV and JCV of known concentration (100 copies/mL, 50 copies/mL, 25 copies/mL, 10 copies/mL, 5 copies/mL, and 1 copy/ml) were subjected to the technique of nested-PCR. All dilutions were tested 11 times to determine the minimum detection limit. Results: The minimum detection limit of the nested-PCR for JC and BK viruses was 25 copies/mL. This dilution (25 copies/mL) showed 100% PCR positive reaction. Furthermore, we found that weak positive results were obtained at dilutions of 1,5 and 10 copies/mL in some repetitions. Dilutions of 25, 50, and 100 copies/mL always had very positive results. Conclusions: These values are similar to those reported in other studies, contributing to the indication of this PCR for potential diagnostic purposes.


Introdução: Os poliomavírus (JCV e BKV) causam infecções principalmente em adultos imunocomprometidos. Um diagnóstico sensível e específico é de fundamental importância para os pacientes portadores de JCV e BKV. Atualmente alguns laboratórios têm utilizado a técnica de PCR para a detecção do material genético destes vírus em amostras clínicas. Assim, o objetivo deste estudo é determinar o limite mínimo de detecção da técnica de nested-PCR para os poliomavírus JC e BK. Métodos: Diluições seriadas (100 cópias/mL; 50 cópias/mL; 25 cópias/mL; 10 cópias/mL; 5 cópias/mL e 1 cópia/mL) de controles positivos comerciais de JCV e BKV com concentrações conhecidas foram submetidas à técnica de nested-PCR semi-duplex. Todas as diluições foram testadas 11 vezes para determinação do limite mínimo de detecção. Resultados: O limite mínimo de detecção da reação de nested-PCR para os vírus JC e BK foi de 25 cópias/mL para ambos, com 100% de positividade das diluições testadas na reação de PCR. Ainda, pudemos observar que resultados positivos fracos foram obtidos nas diluições de 1, 5 e 10 cópias/mL em algumas das repetições realizadas. As diluições de 25, 50 e 100 cópias/mL sempre obtiveram resultado rancamente positivo. Conclusões: Estes valores são semelhantes aos relatados em outros estudos, contribuindo para a indicação desta reação de PCR para potenciais fins diagnósticos.


Assuntos
Humanos , Vírus BK , Infecções por Polyomavirus/diagnóstico , Vírus JC , Limite de Detecção , Reação em Cadeia da Polimerase , Terapia de Imunossupressão , Manejo de Espécimes/normas
15.
Rev. Soc. Bras. Med. Trop ; 46(5): 625-628, Sept-Oct/2013. tab, graf
Artigo em Inglês | LILACS | ID: lil-691413

RESUMO

Introduction Herpes simplex virus (HSV) and varicella zoster virus (VZV) are responsible for a variety of human diseases, including central nervous system diseases. The use of polymerase chain reaction (PCR) techniques on cerebrospinal fluid samples has allowed the detection of viral DNA with high sensitivity and specificity. Methods Serial dilutions of quantified commercial controls of each virus were subjected to an in-house nested-PCR technique. Results The minimum detection limits for HSV and VZV were 5 and 10 copies/µL, respectively. Conclusions The detection limit of nested-PCR for HSV and VZV in this study was similar to the limits found in previous studies. .


Assuntos
Humanos , DNA Viral/análise , Herpes Simples/diagnóstico , Herpes Zoster/diagnóstico , /genética , Reação em Cadeia da Polimerase , Simplexvirus/genética , Limite de Detecção , Sensibilidade e Especificidade
16.
Rev. Inst. Adolfo Lutz ; 72(3): 234-238, 2013. tab
Artigo em Português | LILACS, Sec. Est. Saúde SP, SESSP-CTDPROD, Sec. Est. Saúde SP, SESSP-IALPROD, Sec. Est. Saúde SP | ID: lil-742468

RESUMO

Os métodos microscópicos para detecção de Cryptosporidium e Giardia são dispendiosos e apresentam reprodutibilidade variável e baixa recuperação, porém a rigorosa padronização da técnica e o controle de qualidade nos testes de recuperação permitem sua utilização em amostras ambientais. Neste estudo foi determinada a sensibilidade do ELISA, analisando-se o limite de detecção. Ademais, foi verificada aviabilidade de utilizá-lo como teste de triagem nos locais em que os métodos microscópicos foram aplicados para confirmar a veiculação de oocistos e cistos íntegros, e que podem representar risco de transmissão da criptosporidiose eou giardíase. Os limites de detecção foram altos: 98 cistos e 3.492 oocistos. Do total de amostras ambientais analisadas, 20 % foram positivas para Cryptosporidium spp. e nenhuma para Giardia spp.. Concluiu-se que ELISA não apresenta sensibilidade adequada como teste de triagem para a finalidade deste estudo, pois as amostras ambientais geralmente apresentam números de cistos e oocistos abaixo daqueles observados em amostras fecais infectadas, para as quais os kits de reagente ELISA foram validados. Além disso, as diluições das soluções estoques dos cistos e oocistos em água destilada podem ter resultado em condições de reações desfavoráveis e ter influenciado na sensibilidade do ELISA.


The microscopic methods for detecting Cryptosporidium and Giardia are expensive, and a variablereproducibility and a low recovery are found. Nevertheless, a rigorous technical standardization and aquality control in the recovery testing give support to use them for analyzing the environmental samples.This study determined the sensitivity of ELISA for analyzing Giardia spp. and Cryptosporidium spp.in raw water samples. Also, it was investigated whether this assay could be used as a screening test fordetecting Giardia cysts and Cryptosporidium oocysts in raw water samples; and to deduce the potentialtransmission of giardiasis and cryptosporidiosis from these specimens. The detection limits were high,being 98 cysts and 3.492 oocysts. Of the analyzed environmental samples, 20 % showed ELISA positivefor Cryptosporidium spp., and none for Giardia spp.. Therefore, ELISA showed to be unsuitable to beused as screening assay, because in the environmental samples usually occur a lower numbers of oocystsand cysts than those regularly found in infected stool samples, for which these kits have been validated.Furthermore, the dilutions prepared from the stock solutions of cysts and oocysts in distilled water couldhave resulted in unfavorable reaction conditions, and it has affected the sensitivity of ELISA.


Assuntos
Cryptosporidium , Giardia , Limite de Detecção , Água Bruta/análise , Ensaio de Imunoadsorção Enzimática
17.
Investig. segur. soc. salud ; 5: 165-178, 2003. ilus
Artigo em Espanhol | LILACS, COLNAL | ID: biblio-1400008

RESUMO

Objetivo Planear, diseñar y ejecutar la estandarización de las metodologías analíticas cuyo montaje se ha realizado con anterioridad. Métodos Una vez establecido el método de ensayo se realiza un análisis preliminar de reproducibilidad y un análisis de interferencia por matriz, para luego realizar la validación y calcular los atributos del método. Resultados En el Laboratorio de Salud Pública se estandarizaron trece métodos analíticos a los cuales se les calcularon los atributos de: límite de detección, límite de cuantificación, exactitud, precisión, sensibilidad y rango útil. Conclusiones Se determinó la alta confiabilidad de las pruebas analíticas del Laboratorio de Salud Pública para la generación de intervenciones sanitarias.


Objective To plan, design and execute the standardization of analytical methodologies that have been previously assembled. Methods Once the test method has been established, a preliminary reproducibility analysis and a matrix interference analysis are performed, and then the validation and calculation of the method attributes are carried out. Results Thirteen analytical methods were standardized in the Public Health Laboratory and the following attributes were calculated: limit of detection, limit of quantification, accuracy, precision, sensitivity and useful range. Conclusions The high reliability of the analytical tests of the Public Health Laboratory for the generation of health interventions was determined.


Assuntos
Humanos , Masculino , Feminino , Padrões de Referência , Laboratórios , Métodos de Análise Laboratorial e de Campo , Saúde , Saúde Pública , Limite de Detecção , Métodos
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