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Fast Assay for Pathogen Identification and Characterisation

Periodic Reporting for period 4 - FAPIC (Fast Assay for Pathogen Identification and Characterisation)

Reporting period: 2019-11-01 to 2020-10-31

Sepsis, a life-threatening organ-dysfunction caused by a dysregulated host response to infection, is life threatening with high mortality. Fast identification of patients most at risk for severe outcomes is crucial to start appropriate therapy. The precise diagnosis is also of outstanding importance because the optimal antibiotic can only be prescribed if the pathogenic agent and its susceptibility have been identified.
Bacterial pathogens frequently encountered in human infections are genetically diverse but can share phenotypic traits in common hence the need for DNA-based identification. However, the high multiplexing of DNA-based detection reactions is a challenging task because all relevant interactions of oligonucleotides cannot be computed in a reasonable time scale. Thus, the FAPIC partner Austrian Institute of Technology with the help of expertise at Univ. Warwick and AXO Science developed in silico and in vitro techniques that allowed to improve the sensitivity and specificity of DNA-based assays. We were able to provide markers for bacterial pathogens and determine if resistance genotypes were present. Universities of Warwick, Hasselt and Zagreb all contributed expertise on the key ARGs to be targeted based on experience from the study of clinically important resistance genotypes. The dissemination of pathogens into the environment is a further public health threat so an environmental assay was designed at Warwick in collaboration with Univ. Lyon and AXO, with DNA extraction developed together with Molzym.
To solve the problem of lowering the time-to-result of sepsis diagnosis Molzym developed with Bee Robotics an automated method of enrichment and extraction of the DNA from bacteria present in whole blood samples. The approach was integrated into an overall concept of a near bedside molecular device for rapid pathogen analysis. This "PathoDoc" consisted of a bench top robotic system that directed 1 ml blood samples through extraction, PCR amplification and hybridisation-based detection of pathogen DNA in a completely automated solution. The diagnostic sensitivity against blood culture was suboptimal, but provided Molzym a valuable technical basis and collaborative network of short- to mid-term development and marketing of molecular tools for rapid diagnostics in routine applications. The PathoDoc and array were subjected to detailed preclinical evaluation using spiked blood samples at Warwick which provided further data for optimisation.
The FAPIC project allowed us to perform two clinical studies at Jessa hospital. The second and largest study included approximately 2000 patients with suspected sepsis presenting at the Emergency Department. Blood samples were collected from all patients together with clinical and laboratory parameters. Risk factors and innate immune response of these patients were analysed to allow for fast identification of high-risk patients. The study enabled a clinical evaluation of the diagnostic system. Both universities (Hasselt and Radboudumc) benefitted from these studies. In addition, an extensive biobank collection of blood samples was collected allowing for future research on sepsis.
The chemical and physical conditions of pre-analytical processes and their integration into robotic functions were adapted and consolidated. In particular, processing of blood samples encompassed the optimisation of human cell lysis, human DNA degradation, filtration of the lysate, retention and on-membrane lysis of bacteria and purification of their DNA. This DNA served for further analytical processes, including project-developed hybridisation-based array identification of pathogens in both clinical and environmental samples.
The development and evaluation of an ultraplex assay targeting the genetic mechanisms of the clinically relevant antibiotic resistance genes and virulence factors has been one of the main results.
Two automated instruments were developed based on specifications and assay protocols developed during the project by the consortium and these were namely the PathoDoc and PathoRobot. The PathoDoc was developed to offer full automation for single samples providing DNA extraction, PCR cycling, microarray processing and imaging of the developed arrays at the end of the assay thus integrating four processes into one instrument. Although the instrument performs within specification some further testing is necessary to optimise the assay parameters for automation particularly reducing the overall time of the assay from start to finish to make the instrument a more viable product for the market.
Pre-clinical and clinical validations of PathoRobot and Molzym’s commercial SelectNA™plus (1 ml sample extraction) were done in comparison to culture. The main result was that the sensitivity of molecular analysis of the blood of septic patients was at an inacceptable low level. While potentially applicable to other specimens, conceptual modifications and technical adaptations of the systems are still needed to be done for their use in blood stream analysis.
The clinical validation of a new multiple pathogen detection system (PathoRobot - AXOBot) was performed using 320 fresh blood samples collected from patients with suspected sepsis/bacteraemia in Hasselt and 405 fresh blood samples in Zagreb.
An environmental assay was designed to target key pathogens surviving in water samples.
In FAPIC, we have developed tools that allow us to better understand the genetic mechanisms that bacterial pathogens use to evade an antibiotic therapy.
The progressed state of the extraction systems gives reason to continue optimisation efforts after the end of the project. Further adaptions and validations of the prototypes of PathoDoc and PathoRobot employing specimens other than blood offer the opportunity of using them as tools for routine application at hospital and privately run laboratories in a short- to mid-term time frame. Thereby, diseases such as infectious endocarditis, bacterial meningitis, joint infections and others can be diagnosed within a day when culture needs much longer times. Moreover, the results indicate that molecular analysis uncovers infections where culture is negative. Patients can thus be optimally treated by a targeted chemotherapy in the absence of positive culture results.
Both the PathoDoc and PathoRobot once fully optimised could offer useful automated techniques for single sample testing and DNA extraction from larger sample volumes. With a big emphasis now on automation for laboratories where minimal sample handling by the operator is desirable automated instruments are becoming more important. Both instruments are prototypes and together with collaboration with other consortium parties further R&D efforts would make both products viable to bring to market which would have a positive impact on jobs and increased turnover.
At the emergency department, ensuring a rapid diagnostic result in patients with life-threatening infections can help in a fast change to appropriate antibiotic therapy, thereby reducing selective pressure for AMR. Assessment of the host response and risk factors early in the disease can help in triage and isolation measurements, and in better patient management. Rapid pathogen identification directly from blood can facilitate an earlier start of more appropriate antibiotics, reducing the continuation of broad-spectrum antibiotics and thus reducing the selective pressure for AMR.
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