Molecular characterization and implementation of the loop-mediated isothermal amplification technique for the detection of carbapenemases and the fosA gene in bacterial isolates obtained from urocultures of patients treated in Basic Health Units

Abstract

Urinary Tract Infections (UTI) are among the most common types of infectious diseases, with approximately 150 million cases per year worldwide. It is estimated that annually there are about 8 million ITUs in the United States, many of which result in a visit to the doctor. The socioeconomic impact of these infections is usually high, including health care costs and time lost at work. In Brazil, ITUs are considered among the most frequent bacterial infections, responsible for 80 out of every 1,000 medical consultations, besides being a significant cause of morbidity and being associated with high health care costs. Bacterial resistance has important implications for urological practice, particularly in relation to community ITUs. Currently, in the face of a scenario of a decrease in therapeutic options favorable to the control of certain infections caused by resistant uropathogens, the use of old drugs such as phosphomycin has been increasingly used in medical practice. Thus, it is important to highlight the need for studies aimed at the phenotypic and molecular characterization of ITU isolates to elucidate the dissemination of resistance mechanisms among bacteria from community patients, in order to better understand the origin and dissemination of resistant strains aimed at the establishment of control and prevention measures of these infections. The objectives of this study are: to determine the prevalence and susceptibility profile of uropathogens isolated from patients treated in the 34 Basic Health Units (UBS) of the municipality of São Bernardo do Campo, to perform the molecular characterization of uropathogens resistant to carbapenem and phosphomycin using total sequencing technique of the bacterial genome, implement the technique of isothermal amplification mediated by loop ( LAMP) for the detection of carbapenemases and the fosA gene in phenotypically resistant bacterial isolates of community origin (urocultures). In addition, the present study aims, through the analysis of the data of the research participants, to determine the probable origin of isolates resistant to beta-lactams and phosphomycin and to describe the main risk factors associated with infections caused by resistant bacteria. For the study of prevalence, data from the results of urocultures and antibiograms of patients treated in the 34 UBS in the period of 24 months will be collected. To determine the distribution and prevalence of uropathogens, the results of laboratory tests (urocultures and antibiograms) will be obtained through consultations performed by the Matrix Diagnosis system®, a computerized system used by the Clinical Analysis Laboratory of FMABC. Uroculture will be performed by laminoculture or by conventional method and sensitivity tests will be performed by the disc-diffusion method standardized by CLSI and/or BRCAST. Phenotypic detection of ESBL beta-lactamase production will be detected by automated PhoenixÒ method and confirmed by disk approximation test. For the detection of carbapenemases of bacterial isolates, confirmatory tests such as mCIM (Modified Carbapenem Inactivation Method) or CarbapenembacÒ test and molecular tests such as the LAMP (Loop-Mediated Isothermal Amplification) technique will be performed. The isolates resistant to carbapenems and phosphomycin will be analyzed by other molecular tests such as PFGE (Pulsed Field Electrophoresis), LAMP and complete sequencing of the bacterial genome using ion Torrent Personal Genome MachineTM (Life Technologies) platform, in order to characterize the probable resistance mechanisms present in these isolates. To determine the origin of resistant isolates and determine the risk factors associated with community urinary infections, participants will be asked to complete a simple questionnaire where some data related to previous infections, pre-existing diseases, previous use of antimicrobials, hospitalization or prior performance of any hospital care or procedure wil (AU)