Infecção na corrente sanguínea : epidemiologia e diagnóstico molecular.

Abstract

Health Care-Related Infections (HAIs) are considered a public health problem, damaging the clinical situation of patients and directly affecting the high rates of morbidity and mortality and increased hospital expenses. To identify the pathogen responsible for the infectious process, many laboratories use conventional microbiological techniques, known as the "gold standard", but the release of test results is time-consuming. In order to speed up the results and contribute to the patient's clinical outcome, molecular techniques are considered promising, in which realtime Polymerase Chain Reaction (PCR) has been shown to be an important method to aid in the rapid diagnosis of pathogenic bacteria and selection of appropriate antibiotic therapy. In this way, the objective of this dissertation was to conduct an epidemiological survey of bloodstream infections (BSI) in the Intensive Care Units (ICU) of the hospital under study, as well as to develop real-time PCR methods for molecular discrimination of Gram and genes involved in antimicrobial resistance. Article I presents the results of the BSI in patients hospitalized in the neopediatric ICU, in which it was possible to analyze the epidemiological, microbiological profile and the maternal chronic disorders and/or obstetric complications. A retrospective study was carried out through the survey of the neopediatric patient (14 years) hospitalized in the ICU in the year 2016 was carried out. Patient information about the pathogenic bacteria responsible for BSI and its mechanism of phenotypic resistance was collected from hospital admission. Of the 22 patients with positive blood cultures, 59.1% were aged ≥60 years, 54.5% were male, and 90.9% had a history of previous comorbidities, in which heart disease in patients with a diagnosis of BSI was considered a risk factor for an unfavorable clinical outcome (p= 0.035). CoNS was isolated in 54.2% of blood cultures and 76.9% were resistant to oxacillin. The BSI rate was low in the study population, but it was possible to know the epidemiological and microbiological profiles, helping to monitor infection control and patient safety. Article III presents the results of PCR standardizations in real time, using the TaqMan system for molecular discrimination of bacterial Gram. Specific Gram-PCR was performed, using universal primers and specific probes, in eight clinical isolates and three standard strains. The analytical sensitivity was determined by dilutions with an amount of Staphylococcus aureus and Escherichia coli DNA ranging from 1 ng/μL to 1 pg/μL. The analytical specificity of the probes was also tested using standard strains of Candida albicans and Candida tropicalis. With the TaqMan system, through the analytical specificity, it was possible to discriminate molecularly all the bacteria tested by Gram, with fluorescence emission in Gram-positive and Gram-negative isolates for their respective probes; for fungal isolates, there was no amplification. As to analytical sensitivity, the detection limit of S. aureus and E. coli was 10 pg/μL, where the efficiency was 93.055 (R2= 0.99) and 69.592 (R2= 0.998), respectively. Thus, it was possible to standardize specific TaqMan Gram-specific real-time PCR reactions, which obtained satisfactory efficiency in the analytical sensitivity and specificity assays. Article IV presents the results of PCR standardizations in real time, using the fluorophore Syber Green for the detection of genes involved in bacterial resistance. We tested primers in uniplex reactions for two Gram-positive bacterial resistance genes (blamecA e blavanA) and seven resistance genes in Gram-negative bacteria, in which four of them were tested in uniplex reaction (blaCTX-M, blaSHV, blaTEM, blaVIM, and blaKPC) and three in multiplex reaction (blaIMP, blaSPM, and blaNDM). The melting temperature (Tm) was: blamecA (75.7 °C), blavanA (85.3 °C), blaCTX-M (88.9 °C), blaSHV (91.2 °C), blaTEM (85,5 °C), blaIMP (81.3 °C), blaSPM (84.1 °C), blaVIM (87.3 °C), blaKPC (89.8 ºC) e blaNDM (89.3 ºC). Thus, it was possible to standardize reactions, uniplex and multiplex, to detect genes involved in antimicrobial resistance using the fluorophore Syber Green. These can be considered reproducible and important to help in choosing the appropriate antibiotic therapy.