Detecção laboratorial de amostras de bacilos gram negativos produtores de carpapenemases, isoladas de pacientes colonizados, internados no Hospital Universitário Antônio Pedro

A família Enterobacteriaceae abriga alguns dos gêneros mais frequentemente encontrados em amostras biológicas estão muito associados às infecções relacionadas com a assistência à saúde (IRAS). A resistência aos beta-lactâmicos tem aumentado nas últimas décadas, principalmente no que se refere à produção de enzimas, destacando as carbapenemases. No ambiente hospitalar, pacientes colonizados por bactérias portadoras dessas enzimas são os principais reservatórios desse mecanismo de resistência, de forma que a detecção laboratorial por meio das culturas de vigilância torna-se cada vez mais necessária. Os métodos fenotípicos de detecção apresentam baixo custo e simples execução. O presente estudo teve como objetivo detectar fenotipicamente a produção de carbapenemases entre 111 amostras enterobactérias associadas com quadros de colonização, obtidas a partir de pacientes atendidos no Hospital Universitário Antônio Pedro. A detecção de amostras produtoras de carbapenemases foi realizada através dos testes fenotípicos de Hodge Modificado, Inativação do Carbapenêmico Modificado e de Inativação do Carbapenêmico com EDTA. O perfil de sensibilidade aos antimicrobianos foi determinado por disco-difusão e a confirmação genotípica foi realizada por Reação em Cadeia da Polimerase (PCR). Foram identificadas Klebsiella pneumoniae (26; 65%), Escherichia coli (6; 15%), Enterobacter asburiae (2; 5%), Acinetobacter baumanni (1; 2,5%), Citrobacter farmeri (1; 2,5%), Enterobacter sp. (1; 2,5%), Klebsiella oxytoca (1; 2,5%), Leclercia sp. (1; 2,5%), Providencia rettgeri (1; 2,5%). A produção de carbapenemases foi observada em 36% (40/111) dos isolados. As amostras foram resistentes a: Amicacina (25%), Gentamicina (32%), Tetraciclina (45%), Imipenem (75%), Sulfametoxazole-Trimetoprima (87,5%), Ciprofloxacina (87,5%), Ceftazidima (92,5%), Cefepime (97,5%), Cefotaxima (100%) e Ceftriaxona (100%). Foi observado que 85% das cepas tinham o gene blaKPC, 12,5% blaNDM e nenhuma blaOXA-48. Os resultados indicam que o é uma boa alternativa ao Teste de Hodge Modificado para a detecção fenotípica de Enterobactérias Resistentes aos Carbapenêmicos. Os resultados encontrados destacam a importância da cultura de vigilância para a melhoria na qualidade da assistência hospitalar.Enterobacteriaceae family shelters some of the genus most prevalent in biological samples that are closely associated with healthcare-associated infections (HAI). Resistance to beta-lactams has increased in the last decades, mainly in what refers to the production of enzymes, emphasizing carbapenemases. In health care settings, patients colonized by bacteria carrying these enzymes are the main reservoirs of this mechanism of resistance, so that laboratory detection through surveillance cultures becomes more and more necessary. Phenotypic detection methods have low cost and simple execution. The present study aimed to detect phenotypically the production of carbapenemases among 111 enterobacterial samples associated with colonization tables, obtained from patients attending the Hospital Universitário Antônio Pedro. The detection of carbapenemase-producing samples was analyzed by phenotypic tests, like Modified Hodge Teste, modified Carbapenem Inactivation Method and Carbapenem Inactivation Method with EDTA. The antimicrobial sensitivity profile was determined by disk diffusion and the genotypic confirmation was performed by Polymerase Chain Reaction (PCR). Were identified Klebsiella pneumoniae (26; 65%), Escherichia coli (6; 15%), Enterobacter asburiae (2; 5%), Acinetobacter baumanni (1; 2,5%), Citrobacter farmeri (1; 2,5%), Enterobacter sp. (1; 2,5%), Klebsiella oxytoca (1; 2,5%), Leclercia sp. (1; 2,5%), Providencia rettgeri (1; 2,5%). The production of carbapenemases was observed in 36% (40/111) of the isolates. Samples were resistant to Amikacin (25%), Gentamicin (32%), Tetracycline (45%), Imipenem (75%), Sulfamethoxazole-Trimethoprim (87.5%), Ciprofloxacin (87,5%), Cefepime (97.5%), Cefotaxime (100%) and Ceftriaxone (100%). It was observed that 85% of the strains were positive for the genes blaKPC, 12,5% for blaNDM and none for blaOXA-48. The results indicate that Modified Carbapenemmal Inactivation Method is an alternative to Modified Hodge Test for phenotypic detection of Carbapenem-Resistant Enterobacteriaceae. The results highlight the importance of the surveillance culture for high quality hospital care

Evaluation of Molecular Test for the Discrimination of “Naked” DNA from Infectious Parvovirus B19 Particles in Serum and Bone Marrow Samples

Low levels of parvovirus B19 (B19V) DNA can be detected in the circulation and in different tissue of immunocompetent individuals for months or years, which has been linked to inflammatory diseases such as cardiomyopathy, rheumatoid arthritis, hepatitis, and vasculitis. However, the detection of B19V DNA does not necessarily imply that infectious virions are present. This study aimed to evaluate the method based on the Benzonase® treatment for differentiation between the infectious virions from “naked” DNA in serum and bone marrow (BM) samples to be useful for the B19V routine diagnosis. In addition, we estimated the period of viremia and DNAemia in the sera and bone marrow of nonhuman primates experimentally infected with B19V. Serum samples from ten patients and from four cynomolgus monkeys experimentally infected with B19V followed up for 60 days were used. Most of the human serum samples became negative after pretreatment; however, only decreased viral DNA loads were observed in four patients, indicating that these samples still contained the infectious virus. Reduced B19V DNA levels were observed in animals since 7th dpi. At approximately 45th dpi, B19V DNA levels were below 105 IU/mL after Benzonase® pretreatment, which was not a consequence of active B19V replication. The test based on Benzonase® pretreatment enabled the discrimination of “naked DNA” from B19V DNA encapsidated in virions. Therefore, this test can be used to clarify the role of B19V as an etiological agent associated with atypical clinical manifestations

Evaluation of Molecular Test for the Discrimination of “Naked” DNA from Infectious Parvovirus B19 Particles in Serum and Bone Marrow Samples

Low levels of parvovirus B19 (B19V) DNA can be detected in the circulation and in different tissue of immunocompetent individuals for months or years, which has been linked to inflammatory diseases such as cardiomyopathy, rheumatoid arthritis, hepatitis, and vasculitis. However, the detection of B19V DNA does not necessarily imply that infectious virions are present. This study aimed to evaluate the method based on the Benzonase® treatment for differentiation between the infectious virions from “naked” DNA in serum and bone marrow (BM) samples to be useful for the B19V routine diagnosis. In addition, we estimated the period of viremia and DNAemia in the sera and bone marrow of nonhuman primates experimentally infected with B19V. Serum samples from ten patients and from four cynomolgus monkeys experimentally infected with B19V followed up for 60 days were used. Most of the human serum samples became negative after pretreatment; however, only decreased viral DNA loads were observed in four patients, indicating that these samples still contained the infectious virus. Reduced B19V DNA levels were observed in animals since 7th dpi. At approximately 45th dpi, B19V DNA levels were below 105 IU/mL after Benzonase® pretreatment, which was not a consequence of active B19V replication. The test based on Benzonase® pretreatment enabled the discrimination of “naked DNA” from B19V DNA encapsidated in virions. Therefore, this test can be used to clarify the role of B19V as an etiological agent associated with atypical clinical manifestations