ABSTRACT
@#Introduction: Carpanenamase-producing Enterobacteriaceae (CPE) has emerged as a threat to hospitalized patients. Phenotypic test such as Modified hodge test was less sensitive and specific especially to detect blaNDM-1 which is the most predominant genotype in this region. Nucleic acid amplification technology offers improved specificity and sensitivity. Failed amplification due to the presence of inhibitors is a limitation. In this study, we tried to use previous method described by Villumseen et al with some modification using another DNA extraction kit. Methods: Ten mls of sterile whole blood taken from nearly expired blood bag from blood bank was spiked with 200 µl of 0.5mcFarland bacterial suspension from thirty-six confirmed isolates of blaNDM-1 carbapenamase-producing Klebsiella pneumoniae in an aerobic Bactec Plus and incubated until the growth was detected. The blood specimen was subjected to DNA extraction method using Macherey-Nachel, Nucleospin® Blood QuickPure followed with multiplex PCR. Results: Out of the 36 isolates, 12 isolates revealed blaNDM-1 , 9 isolates revealed blaNDM-1 and blaOXA-48, 7 isolates revealed blaNDM-1, blaVIM and blaKPC genotypes that were amplified at cycle threshold of less than 30. Another 8 isolates could not pick up any genotypes possibly due to pipetting error as all the internal control were amplified. Eight true negative gram negative isolates underwent same procedure and none amplified at a cycle threshold less than 30. Conclusion: This modified method was proved to give a high yield of CPE genotypes with the cycle threshold was set at less than or equal to 30 and able to overcome the presence of PCR inhibitors
ABSTRACT
@#Introduction: Carpanenamase-producing Enterobacteriaceae (CPE) has emerged as a threat to hospitalized patients. Phenotypic test such as Modified hodge test was less sensitive and specific especially to detect blaNDM-1 which is the most predominant genotype in this region. Nucleic acid amplification technology offers improved specificity and sensitivity. Failed amplification due to the presence of inhibitors is a limitation. In this study, we tried to use previous method described by Villumseen et al with some modification using another DNA extraction kit. Methods: Ten mls of sterile whole blood taken from nearly expired blood bag from blood bank was spiked with 200 µl of 0.5mcFarland bacterial suspension from thirty-six confirmed isolates of blaNDM-1 carbapenamase-producing Klebsiella pneumoniae in an aerobic Bactec Plus and incubated until the growth was detected. The blood specimen was subjected to DNA extraction method using Macherey-Nachel, Nucleospin® Blood QuickPure followed with multiplex PCR. Results: Out of the 36 isolates, 12 isolates revealed blaNDM-1 , 9 isolates revealed blaNDM-1 and blaOXA-48, 7 isolates revealed blaNDM-1, blaVIM and blaKPC genotypes that were amplified at cycle threshold of less than 30. Another 8 isolates could not pick up any genotypes possibly due to pipetting error as all the internal control were amplified. Eight true negative gram negative isolates underwent same procedure and none amplified at a cycle threshold less than 30. Conclusion: This modified method was proved to give a high yield of CPE genotypes with the cycle threshold was set at less than or equal to 30 and able to overcome the presence of PCR inhibitors.
ABSTRACT
ABSTRACT The indiscriminate use of pesticides on grape crops is harmful for consumers´ healthin "in natura" consumption and in the ingestion of wine and grape juice. During winemaking, a rapid and efficient fermentation stage is critical to avoid proliferation of contaminating microorganisms and to guarantee the product´s quality. Polymerase chain reaction (PCR) has the advantage of detecting these contaminants in the early stages of fermentation. However,this enzymatic reaction may also be susceptible to specific problems, reducing its efficiency. Agricultural practices, such as fungicide treatments, may be a source of PCR inhibiting factors and may also interfere in the normal course of fermentation.The action of the pesticides captan and folpet on PCR and on yeast metabolism was evaluated, once these phthalimide compounds are widely employed in Brazilian vineyards. DNA amplification was only observed at 75 and 37.5 µg/mL of captan concentrations, whereas with folpet, amplification was observed only in the two lowest concentrations tested (42.2 and 21.1µg/mL).Besides the strong inhibition on Taq polymerase activity, phthalimides also inhibited yeast metabolism at all concentrations analyzed.Grape must containing captan and folpet residues could not be transformed into wine due to stuck fermentation caused by the inhibition of yeast metabolism. Non-compliance with the waiting period for phthalimide fungicides may result in financial liabilities to the viticulture sector.The use of yeasts with high fungicide sensitivity should be selected for must fermentation as a strategy for sustainable wine production and to assure that products comply with health and food safety standards.
ABSTRACT
Objetivo: Demostrar la presencia de Rotavirus en las diferentes fases de un proceso de compostaje: matrices utilizadas como materia prima, mezcla a compostar y producto terminado. Materiales y métodos. El Rotavirus se determinó durante los tres procesos de compostaje. La detección viral se realizó por inmunocromatografía, ELISA y RT- PCR. Resultados. Se evidenció presencia viral en el primer proceso de compostaje, ausencia viral en el segundo y en el tercer proceso de compostaje, se presentaron interferencias que dificultaron interpretar los resultados de la PCR, lo cual impidió llegar a un resultado concluyente de su presencia en el abono. Conclusiones. Los abonos orgánicos pueden ser portadores de virus motivo por el cual se deben implementar pruebas de calidad para evitar que este material contribuya con la diseminación viral. Dentro de estos abonos existen sustancias capaces de interferir en las pruebas de detección.
Objective. To show the presence of rotavirus in different stages of a composting process: matrices used as raw material, mixture to be composted and the final product. Materials and methods. Immunochromatography, ELISA and RT-PCR were used for viral detection. Results. Rotavirus was found in the first composting step, no virus was found in the second step, and some inhibitory substances were found in the third step that posed difficulties in interpreting the PCR results and therefore providing a concluding result on rotavirus presence in the final product. Conclusions. Organic fertilizers can be vectors of human pathogenic viruses; therefore quality control tests must be implemented to avoid further viral dissemination. There are inhibitory substances present in organic fertilizers capable of interfering with the detection tests.
Objetivo. Demonstrar a presença de Rotavírus nas diferentes fases de um processo de compostagem: matrizes utilizadas como matéria-prima, mistura de composto e produto final. Materiais e métodos: A determinação do Rotavírus foi realizada nos três processos de compostagem. A detecção viral foi realizada por imunocromatografia, ELISA e RT-PCR. Resultados. Evidenciou-se a presença viral no primeiro processo de compostagem, ausência de vírus no segundo, e no terceiro processo de compostagem, apresentaram-se interferências que dificultaram a interpre tação dos resultados da PCR, tornando impossível chegar a um resultado conclusivo de sua presença no adubo. Conclusões. Os adubos orgânicos podem abrigar o vírus é por isso que se devem implementar provas de qualidade para evitar que esse material contribuía com a disseminação viral. Dentro desses fertilizantes existem substâncias capazes de interferir nas provas de detecção.