From genomics to proteomics: beacon-based fluorescence in situ hybridization (bbFISH) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology in the clinical microbiology laboratory

The rapid and accurate identification of infectious agents and the early introduction of an appropriate antimicrobial therapy is of crucial importance in the clinical practice, mainly in serious infections such as blood stream infection (BSI), which may progress to sepsis /septic shock with high morbidity and mortality rate. The delay in an appropriate antibacterial treatment has led to increases non only in morbidity and mortality rates as well as in hospitalization time, in nosocomial-acquired infections and make it challenging to implement the back-end approach of the antimicrobial stewardship program, which has shown rewarding results in patient management and the fight against the antimicrobial resistance. Moreover, the emergence of resistant and multidrug- or even pandrug-resistant pathogens, especially the carbapenemaseproducing Enterobacteriacea (CPE) such as Klebsiella pneumoniae carbapenemase-producing (KPC) K. pneumoniae strains, is increasingly reported worldwide and is becoming an important issue in health care systems. Therefore, there is an urgent requirement to provide timely information on infection status, which allow an optimal patient’s management and treatment. Clinical microbiology has developed continuously and there has been a constant search for new techniques and diagnostic methods with the aim to optimize the identification of pathogens, their antimicrobial susceptibility profile and improve the patient’s management and treatment reducing the social and economic impact on the health care system. Molecular methods seem to be an appropriate choice, they are widely used in the diagnosis of BSIs, alongside to the conventional culture-based methods. However, these techniques involve significantly increased cost and technical complexity, both of which are likely to hamper their adoption in the laboratory routine in the clinical setting. In this paper are reported two novel technologies in clinical microbiology focused on the rapid diagnosis of BSIs directly from clinical material in order to reduce the Turn Around Time (TAT) for bacterial identification and improve patient’s management and treatment. In the first part of this study has been evaluated the performance of a new molecular assay, the beacon-based fluorescent in situ hybridization (bbFISH) in comparison to the conventional growthbased phenotypic identification of bacteria from positive blood culture vials in febrile patients. It has been also examined the bbFISH and the conventional identification assay’s total turnaround time (TAT) performance. Considering the good sensitivity and specificity of the hemoFISH® assays as well as the significant time saving in obtaining the final results (p-value 0.0001), the introduction of this assay could be reliable in the microbiology laboratories, supplementing traditional approaches, speeding up the diagnosis of bloodstream infections and identifying the majority of most important sepsis pathogens also in the case of polymicrobial infection. This assay has the potential to provide timely and cost effective information on infection status, thus allowing clinicians to make more informed decisions on appropriate antibiotic therapy at an earlier stage than is possible with culture-based approaches. hemoFISH® provides a same-day identification of the majority of microorganisms thus the turnaround time is considerably lower than microbiological culture. The second part of the present work is instead dedicated to another novel proteomic assay, the Matrix-Assisted Laser Desorption Ionization-Time of flight Mass Spectrometry (MALDI-TOF MS). The application of this technology for the identification of pathogens in blood culture (BCs) has been significantly improved the time required to obtain results with an excellent sensitivity and specificity. Results were available on average after only a few minutes, representing a significant difference in comparison with the time required for the results of conventional methods. Moreover the accuracy of this identification method seems to be considerable, with higher performance and cost effectiveness comparing with traditional methods. In addition, has been evaluated the applicability of this technology in order to predict susceptibility or resistance profiles against antibiotics, such as the carbapenems, on BCs derived directly from patients with Gram-negative bacteremia, in particular with carbapenemaseproducing Klebsiella pneumonia strains. The results of the present study have demonstrated that MALDI-TOF MS technology is a relevant tool also for the detection of antibiotic resistance. The enzymatic hydrolysis of the amide bond into the β-lactam ring of carbapenems, like ertapenem, by β-lactamases enzymes could be monitored by MALDI-TOF MS assay; hydrolyzed and nonhydrolyzed substances differ in their molecular weights and these differences can be detected by this assay within 1 to 3 hours with a great accuracy and high sensitivity. Compared to other alternative approaches like molecular genetics techniques this method is cost effective and easy to perform. The rapid detection of clinically important β-lactamases in routine diagnostic laboratories may be crucial for initial antibiotic therapy as well as for the prevention of the speed of β- lactamase-producing bacteria in health care setting. Combined with a proactive antimicrobial stewardship program of carbapenems this assay can improve patients outcomes. In order to provide even more timely information on infectious status, which allow an optimal patient’s management and treatment, always in the second part of this study has been also demonstrated the reliability of a rapid antibiotic susceptibility test (AST) performed directly from BCs flagged as positive to Gram-negative rods. The comparison of the antibiotic susceptibility data obtained by the standard method and by the direct method has showed a concordance of 100% while these data were obtained in a shorter time by the direct method than the traditional culture-based susceptibility testing. Thus, this methodological approach has great potential to become a routine method in any clinical microbiology laboratory while both this one method and MALDI-TOF MS technology can likely modified the workflow of BCs providing a same-day final report available to clinicians and hospital epidemiologists.