Description

Overview

Sepsis, a life-threatening immune response to an infection, claims the lives of 11 million people every year—accounting for 20% of all global deaths [1]. Rapid and accurate diagnosis of sepsis is crucial: every hour of delayed treatment decreases the chance of survival by 7.6% [1]. The current gold standard of sepsis diagnostics—blood cultures—can take a minimum of 72 hours to provide results and are often prone to contamination [2]. Furthermore, about one third of blood cultures are unusable for diagnostics due to undetectable pathogen concentrations [3]. Surviving Sepsis Guidelines (2021) advises that every possible septic shock patient should receive antibiotic therapy within one hour of onset, but physicians are often reluctant to administer the drugs for fear of generating antimicrobial resistance (AMR) [4].

Nucle.io is a rapid diagnostic device that merges the power of DNA computing and CRISPR to identify the pathogens and antimicrobial resistance markers driving sepsis.

Nucle.io catalyzes the detection of low-concentration pathogen RNA, computes the bacterial strains most likely responsible, and classifies any relevant AMR genes. Bacterial RNA is first amplified through adjacent strand displacement reactions (SDR) and CRISPR-CasX cascades. An outline of the system set up is as follows:

Input: mRNA biomarkers of pathogens are present at low concentrations in blood. These biomarkers must be amplified.

By leveraging the kinetics of nucleotide binding through toehold-mediated strand displacement (TMSD) reactions, RNA detection is multiplied and analyzed to a detectable signal.

1. Catalyze: The CasX (DpbCas12e) protein identifies the bacterial antimicrobial resistance mRNAs and forces it to become a guideRNA [5]. Upon activation, the enzyme cuts a target dsDNA and turns it into a computable DNA gate, allowing the detection of AMR genes.
2. Compute: Strand displacement reactions identify and amplify target regions of the 16S ribosomal bacterial gene to identify pathogens.
3. Classify: These readouts are then fed into a DNA Winner-Take-All neural network, which computes the most likely bacterial strain and AMR genes responsible for the patient’s sepsis and generates a sequence-specific output [6].

Output: The fluorophore corresponding to the pathogen of interest and the AMR marker is released after several hours, allowing physicians to accurately identify the pathogen and its possible resistance genes.

Nucle.io employs a novel approach that allows for amplification and analysis to occur in one reaction. The isothermal, portable, and modular nature of Nucle.io cuts out the lengthy middle step of sending samples away to the lab. This point-of-care diagnostic has the potential to change patient outcomes and tackle AMR by guiding clinical decision making and allowing for more rapid, targeted therapies to be administered. Nucle.io’s highly modular nature allows for the reprogrammability of which bacterial strains and AMR genes are detected by the system, accounting for regional variances in pathogen prevalence. This also enables Nucle.io to be used in fields beyond healthcare, such as in agriculture or in environmental health.

Read more about the design of our diagnostic and how it works.

Sepsis: A Case Study

Sepsis is a dysregulated response by the host organism to infectious agents within the bloodstream. When progressing to states of severe sepsis and septic shock, mortality rates have been recorded to be 21.9% and 45.6% respectively [7]. These progressions can occur in just hours. Within this short time frame, the risks of lasting effects such as brain damage, limb loss, and severe organ damage increase at a staggering rate [8]. With time to diagnose sepsis falling between 48-72 hours, physicians are often forced to treat with reactive measures, which are broad stroke methods that do not always combat the infecting agent. Furthermore, hospital associated infections (HAI) acquired after patient admittance compound the number of sepsis cases in the US by approximately 4% each year [9]. An additional complication to sepsis treatment is antimicrobial resistance, and the threat it poses to the standardized care methods used. Strains causing sepsis were found to be resistant to common antimicrobials in over 50% of the tested patients [10]. This issue becomes increasingly grave in impoverished areas and HAIs as discussed before. As more resistant forms of bacteria spread, the greater the risk of complications for sepsis patients, as there is a decrease in the power of the treatments being employed [10]. The dual threat of sepsis’ overwhelming nature and spreading antimicrobial resistance create the need for a tool like Nucle.io, that can detect and characterize sample components to promote more informed treatment plans.

Current Diagnostic Methods

Typical methods of sepsis diagnosis consist of blood cultures as well as polymerase chain reaction (PCR), followed by sequencing [4]. However, there are issues with both of these methods as blood cultures take over 48 hours to return results and PCR requires technical expertise to carry out. In addition, from a consultation with Sayonara Mato, we were informed that PCR results from even the most established sampling-to-lab systems can take 7 days to return results. The problem with these diagnostic procedures is that they are not feasible at point-of-care, and they are both laborious and cost intensive. In a case like sepsis, this is inherently not ideal, as the demand for test results is at a timescale a fraction of the size of the current testing timeline. This makes sepsis diagnosis and treatment a reactionary procedure, where tests can become obsolete as the body’s state of immune response progresses.

Current Treatments

Often, physicians will administer broad spectrum antibiotics within 1 hour of the suspicion that a patient has sepsis [8]. Early, thorough treatment raises the likelihood of recovery without side effects. However, the use of broad spectrum antibiotics can increase the risk of development of antimicrobial resistance, and lead to worse patient outcomes in morbidity and mortality when compared to targeted treatments [10].

Our Project: Nucle.io

Nucle.io detects sepsis early through our rapid, panel-based diagnostic, providing physicians with test results faster. Nucle.io harnesses a platform of unfolding reactions to catalyze, compute, and classify the biomarkers of sepsis-based bacterial infection.

• S. aureus, S. pyogenes, and E. coli.
• Resistance to vancomycin, methicillin, carbapenem, and tigecycline.

Our team has designed a tool for use on both gram positive and gram negative bacterium to emphasize the diagnostic power of the Nucle.io platform and help guide physician antibiotic choice. Moreover, the characterization of these bacterium by the identification of antimicrobial resistance genes they contain offers physicians an enhanced view of the patient’s case. These properties of the infection are read out in a quantifiable fluorescent manner.

With this information, doctors can select the most appropriate antibiotic for use in patient treatment, optimizing treatment courses and avoiding the use of broad-spectrum antibiotics. Furthermore, Nucle.io can also be used as a prognostic tool to see the onset of sepsis before symptoms appear or before rapid patient deterioration. This tool will avert both morbidity and mortality in patients.

Nucle.io is modular, multiplexable, isothermal, and lab-free. The diagnostic can be easily and simply reprogrammed to detect almost any mRNA biomarkers; sepsis is a case study to emphasize Nucle.io’s diagnostic strengths and showcase how it can cover a wide range of diagnostic fields on multiple levels of global infrastructure.

Impact on Sepsis Patients

Throughout the treatment process for sepsis, patients and their families are most often left uninformed, and without reassurance. As a result of treating the condition without point-of-care diagnostic results, the understanding that physicians are able to provide to them is often not complete, making the situation all the more overwhelming to the family sitting bedside. Nucle.io aims to change this. With our diagnostic kit, used upon patient admittance, the patient’s sepsis-causative pathogen can be quickly determined. This allows physicians the chance to design personalized treatment plans to tackle the condition early, leading to improvements in patient outcomes. In addition, Nucle.io being used at bedside will allow for patients and families to be informed throughout the entire treatment process.

Moreover, paired with the Nucle.io kit will be the McGill iGEM Sepsis Handbook, developed in partnership with Sepsis Canada. This information package aims to fill in the blanks that the physician cannot for families, by providing them with a detailed information package with each test that will allow for complete understanding of their loved-ones situation at bedside. Nucle.io aims to pair point-of-care testing with the Sepsis Handbook to shift the entirety of sepsis care to a more informed and confident process.

Sepsis and bacterial infections are extremely significant issues where healthcare resources are scarce. Therefore, we considered ease of transport, cost of manufacture, shelf life, thermostability and ease of use as key factors in deciding how we wanted to engineer our diagnostic.Depending on the needs of the user as well as the resources available, the choice between using the CasX amplifier or the strand-displacement based amplifier can be made. In a similar sense, the Winner-Take-All system can be joined with either amplifier for improved analytical power.

Modularity

Given the speed of evolution of antimicrobial resistance as well as changes in pathogen incidence variant by region, we also ensured that our diagnostic was as re-engineerable as possible so it could be rapidly modified and changed. Most of the diagnostic relies on established base modules (CasX amplifier design as well as DNA computing standard analytic parts) with only superficial changes in RNA and DNA sequences. We wanted to create a diagnostic platform that could be used beyond sepsis. Bacterial classification is also needed in environmental and agricultural applications of pathogen detection (refer to our Integrated Human Practices page and our Impact Report for more information). In addition, the establishment of these modules enables Nucle.io to be adapted widely to viral classification as shown on our Parts page. This allows Nucleio to evolve alongside the issues it aims to tackle. We chose to make the Nucle.io project as dynamic as the issues of antimicrobial resistance and sepsis infection that form the core of our project.