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

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

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

Evaluación del riesgo ambiental de carbofurano en bioensayos con organismos no blanco / Environmental risk assessment of carbofuran in bioassays with non-target organisms

El carbofurano es uno de los plaguicidas más empleado en la agricultura peruana. El objetivo de este trabajo fue evaluar el riesgo ambiental del carbofurano en bioensayos de toxicidad, sobre once especies no destinatarias. La siguiente secuencia relativa de mayor a menor cocientes de riesgo (CR) del carbofurano para los 19 puntos finales de efecto en once especies fue encontrada: Daphnia magna (mortalidad) > Lemna minor (inhibición de formación de las hojas) > Paracheirodon innesi (nado extraño) = P. innesi (incremento del movimiento opercular) > Chironomus calligraphus (mortalidad) > L. minor (necrosis) > P. innesi (mortalidad) > L. minor (clorosis) > Tetrapygus niger (inhibición de la fecundación) > Coturnix japonica (mortalidad) > Oncorhynchus mykiss (incremento de coloración) > Ceraeochrysa cincta (no eclosión de huevos) > O. mykiss (mortalidad con oxígeno) > O. mykiss (mortalidad sin oxígeno) > Trichogramma pretiosum (mortalidad) > C. cincta (mortalidad) > T. pretiosum (no emergencia de adultos) > Trichogramma pintoi (mortalidad) > Beauveria bassiana (inhibición del crecimiento). Los cocientes de riesgo (CR) indicaron en todos los casos un alto riesgo del carbofurano principalmente en el ambiente acuático en comparación con el terrestre.

Carbofuran is one of the most employed pesticides in Peruvian agriculture. This research aimed to evaluate the risk assessment in bioassays of carbofuran on eleven non-target species. The following relative sequence in decreasing ecotoxicity order in terms of RQ (Risk quotient) to carbofuran to 19 end points on 11 species was found: Daphnia magna (mortality) > Lemna minor (inhibition of new fronds) > Paracheirodon innesi (strange swim) = P. innesi (increase of opercula movement) > Chironomus calligraphus (mortality) > L. minor (necrosis) > P. innesi (mortality) > L. minor (chlorosis) > Tetrapygus niger (inhibition of fertilization)> Coturnix japonica (mortality) > Oncorhynchus mykiss (increase of coloration) > Ceraeochrysa cincta (not hatch of eggs) > O. mykiss (mortality without oxygen) > O. mykiss (mortality with oxygen) > Trichogramma pretiosum (mortality) > C. cincta (mortality) > T. pretiosum (not emergence of adults) > Trichogramma pintoi (mortality) > Beauveria bassiana (inhibition of growth). Risk quotient (RQ) indicated that carbofuran is highly risky mainly to aquatic environment than terrestrial.

Diferencias en la susceptibilidad a la deltametrina en poblaciones de Aedes aegypti de Trujillo, Venezuela / Different susceptibility to deltamethrin in Aedes aegypti populations from Trujillo state, Venezuela

El dengue es un importante problema de salud pública en Venezuela, donde aedes aegypti es el principal vector. El propósito de este estudio fue determinar el estado de la susceptibilidad a la deltametrina en nueve poblaciones naturales de A. aegypti del estado Trujillo, en comparación con la cepa susceptible Rockefeller. Los bioensayos fueron llevados a cabo siguiendo la metodología de la Organización Mundial de la Salud. Las poblaciones Trujillo, Pampán, Pampanito, Flor de Patria, Motatán, Tres Esquinas y Cubita mostraron valores de mortalidades comprendidas entre 89% y 97%, y KDT50 entre 15,7 min y 24,1 min, sugiriendo la posibilidad de resistencia la cual debe ser confirmada. Las poblaciones Monay y Filo fueron susceptibles, con KDT50 de 15,5 y 20,2 min respectivamente, y mortalidades a las 24 horas de 99 y 98%. Estos resultados deben ser considerados al momento de diseñar el programa de control del vector para asegurar la efectividad del mismo.

Dengue is an important public health problem in Venezuela, where Aedes aegypti is the main vector. The purpose of this study was to determine the status of susceptibility to deltamethrin in nine natural populations of A. aegypti from Trujillo state compared with the susceptible Rockefeller strain. Bioassays were carried out following the methodology of the World Health Organization. The values of mortalities were found between 89% and 97%, allowing categorize the populations from Trujillo, Pampan, Pampanito, Flor de Patria, Motatan, Tres Esquinas and Cubita as resistant under verification, with values KDT50 between 15.7 min and 24.1 min, suggesting the possibility of resistance which must be confirmed and the populations from Monay and Filo as susceptible, with a KDT50 of 15.5 and 20.2 min, respectively, and 99 and 98% mortality at 24 hours. These results should be considered when designing programs for vector control to ensure those are effective to control A. aegytpti populations.

Methods of statistical analysis of quantitative data obtained by toxicological bioassays using rodents in Japan: historical transition of the decision tree.

Massive amounts of quantitative data are being obtained by toxicity tests with time. These data sometimes include the data with a different distribution pattern or with different numbers of animals in each group. The tree-type algorithm has long been used commonly to promptly analyze the difference between the control group and dosage groups. In Japan, the decision tree has been used since 1982 with several modifications of the initial procedure. Recently, the method has been revised to have a high power. The changes have been made in two directions; one is a sophisticated method and the other is a simplified method. In this paper, the historical transition of the decision tree is described and the future of the decision tree forecasted.

Selección de una cepa de Culex quinquefasciatus resistente a lambdacialotrina y su espectro de resistencia cruzada a otros insecticidas

Se seleccionó una cepa de campo de Culex quinquefasciatus resistente al insecticida piretroide lambdacialotrina, con el objetivo de poder utilizarla como cepa de referencia en el laboratorio, en los estudios de bioquímica y genética de la resistencia; evaluar la utilidad de este insectivida para el control de mosquitos en Cuba y determinar la resistencia cruzada a insectividas organofosforados, piretroides y un carbamato. Se obtuvo una alta resistencia a lambdacialotrina después de 6 generaciones de presión de selección. Se observó baja o ninguna resistencia cruzada a otros piretroides (deltametrina y cipermetrina), a un carbamato (propoxur) y a los insecticidas organofosforados (clorpirifos y metil-pirimifos); sin embargo, se observó una alta resistencia cruzada a malatión (organofosforado).

A field strain of Culex quinquefasciatus resistant to pyrethroid lambda-cyhalothrin insecticide was selected to be used as a reference strain in the laboratory for conducting studies of biochemistry and genetics of resistance, to evaluate the utility of this insecticide for the control of mosquitoes in Cuba, and to determine the cross resistance to organophosphate insecticides, pyrethroids and a carbamate. A hig resistance to lambdacyhalothrin was obtained after 6 generations of selective pressure. Lowe or no cross resistance to other pyrethroids (deltamethrin and cyper-methrin), to a carbamate (propoxur) and to the organophosphate insecticides (clorpirifos and methyl-pyrimifos) was observed. A high cross resistance to malathion (organophosphate) was detected.

Estudio de la resistencia en una cepa de Culex quinquefasciatus, procedente de Medellín, Colombia

Para conocer el estado de la resistencia en una cepa de Culex quinquefasciatus, procedente de una localidad de la ciudad de Medellín, Colombia, se determinaron los niveles de susceptibilidad y/o resistencia a 5 insecticidas organofosforados (malatión, metil-pirimifos, clorpirifos, temefos y fentión), 4 piretroides (cipermetrina, deltamemetrina, permetrina y lambdacialotrina) y un carbamato (propoxur). Se observó resistencia a todos los insecticidas organofosforados, aunque con valores relativamente menores para metil-pirimifos y fentión. No se encontró resistencia a los piretroides lambdacialotrina y cipermetrina, ni al carbamato propoxur, insecticidas que pueden ser muy útiles para el control de mosquitos de Colombia. Se demostró mediante el uso del sinergista piperonil butóxico que las oxidasas de función múltiple desempeñaron una función importante en la resistencia a los insecticidas organofosforados y piretroides. El uso del S.S.S. tributil fosfotritiado reveló que la sobreproducción de esterasas inespecíficas constituyó un mecanismo de resistencia para los insecticidas organofosforados, excepto metil-pirimifos y para los piretroides excepto lambdacialotrina. Este resultado debe tenerse en cuenta en las estrategias que se vayan a usar para el control de Culex quinquefasciatus de Colombia. Estos 2 mecanismos de resistencia no son responsables de la resistencia al carbamato propoxur. El análisis electroforético reveló la presencia de las esterasas B1, A6 y B6, que presumimos tienen una función importante en la resistencia.

The levels of susceptibility and/or resistance to 5 organophosphate insecticides (malathion, methyl-pyrimifos, clorpirifos, temephos and fenthion), 4 pyrethroids (cypermethrin, deltamethrin, permethrin and lambda-cyhalothrin), and a carbamate (propoxur) were deternmined in order to know the state of resistance in a strain of Culex quinquefasciatus from a locality of the city of Medellín, Colombia. Resistance to all organophosphate insecticeides, though with relatively lower values for methyloirimifos and fenthion, was observed. No resistance to lambda-cyhalothrin and cypermethrin or to propoxur was found. These insecticides may be useful for the control of mosquitoes in Colombia. It was demonstrated by using the piperonil butoxide sinergist that the oxidases of multiple function played an important role in the resistance to organophosphate insecticides and pyrethroids. The utilization of S.S.S. tributyl phosphotritiate revealed that the superproduction of unspecific esterases was a mechanism of resistance to organophosphate insecticides, except methyl-pirimifos and for perythroids, exceptlambda-cyhalothrin. This result should be taken into consideration for the strategies to be used to control Culex quinquefasciatus in Colombia. These two mechanism of resistance are not responsible for the resistance to propoxur. The electrophoretic analysis showed the presence of esterases B1, A6 and B6, which seem to have an important function in resistance.