A CRISPR fingerprint of pathogenic C. auris fungi

(BOSTON) — Infection with the pathogenic yeast fungus Candida auris (C. auris) can wreak havoc on the health of hospital patients and residents of nursing homes, especially those who are already weakened by other illnesses. The pathogen easily spreads and colonizes surfaces and objects where it can survive for weeks to months, and is often resistant to standard disinfectants. C. auris infections are especially problematic for patients who receive organ transplants or chemotherapy, and whose immune systems are compromised, such as by HIV. Infections also are a threat to patients who are at high risk of infection, such as those requiring invasive devices, like breathing or feeding tubes, or different types of catheters. Once C. auris infections reach the bloodstream or vital organs, they become life-threatening with symptoms and immune-reactions similar to those caused by bacterial and viral pathogens.

Although C. auris infections, in principle, can be treated with several antifungal medications, strains of the pathogen that have developed antimicrobial resistance (AMR) against those drugs have emerged fast and become a difficult challenge for hospital physicians. It means that some infections have to be treated with a different drug than the initially chosen antifungal agent or, in the worst-case scenario, are impossible-to-treat with any of the available drugs, which, if still administered, pose an unnecessary additional burden on patients’ bodies.

“Clinicians need a much more effective diagnostic approach to accurately quantify the abundance of the pathogen in patients and assess its antifungal resistance in order to better respond to C. auris infections in their patients and help prevent future hospital-associated outbreaks,” said Justin Rolando, Ph.D., a first author on a new study that addresses this challenge head-on. “Current diagnostic methods for detecting C. auris are too costly, slow, and dependent on complex equipment and trained personnel in order to effect real change.”

The new study offers a solution to this problem with a new precision diagnostic approach that for the first time enables fast and accurate quantification of C. auris strains from easily obtained swab samples, as well as the quantification of AMR-causing mutations in fungal populations with mixed antifungal susceptibility. The next-generation test builds on previous diagnostic accomplishments of the groups of Wyss Institute Core Faculty members David Walt, Ph.D. and James Collins, Ph.D., who led the effort, and was greatly facilitated by the team’s collaboration with the Wadsworth Center Mycology Lab at New York State Department of Health, which provided a first cohort of patient samples (surveillance swabs) for the team’s initial technology validation.

The research team integrated SHERLOCK technology, a CRISPR-based diagnostic method pioneered by Collins’ group that allows the detection of pathogen-derived (or other) nucleic acid sequences with single nucleotide precision with ultra-sensitive single-molecule microarray technology advanced in Walt’s group. By monitoring the development of finely tuned fluorescent signals produced by thousands of parallel single-molecule assays in real-time, and analyzing the signals using a machine learning-based artificial intelligence method, the team created a fast and quantitative approach, named dSHERLOCK (short for digital SHERLOCK), that measures the degree of fungal colonization of C. auris in patient samples and pinpoints the presence of mutations that cause specific antimicrobial resistances (AMRs). The findings are published in Nature Biomedical Engineering.

A new case for SHERLOCK

In 2019, after having experienced several outbreaks of C. auris infection caused by strains that had become treatment-resistant, the NY State Department of Health released an urgent call to accelerate diagnostic developments that could help control future outbreaks. Collins, along with co-authors Helena de Puig, Ph.D. and Xiao Tan, M.D., Ph.D., submitted a proposal to leverage the group’s SHERLOCK system for the cause, which the Center funded. Co-first author Nicole Weckman, Ph.D., who joined the team as a postdoctoral fellow in 2020, then took a deeper dive into CRISPR technology to design C. auris-detecting assays and started to collaborate with Rolando and other member of Collins’ and Walt’s groups with the common aim to turn the detection system into a quantitative and clinically useful diagnostic tool.

Using dSHERLOCK, the researchers were able to reliably detect C. auris in swab samples, which they obtained from their collaborators at the Wadsworth Center. The assay is completed within 20 minutes and can accurately quantify how much of the pathogen the samples contained within 40 minutes. Current clinical practice requires that samples obtained from patients in hospitals are sent to one of seven central laboratories, like the Wadsworth Center Mycology Lab, whose process to determine the presence of the pathogen and AMR can take up to a week; while infected patients need to be treated immediately.

Importantly, by tweaking the CRISPR-mediated detection mechanism, the researchers managed to amplify C. auris targets that contained mutations associated with AMR and showed these two common antifungal drugs displayed different “kinetics.” According to Weckman, who now is an Assistant Professor and Paul Cadario Chair in Global Engineering at University of Toronto, dSHERLOCK’S single-molecule detection assays are designed such that positive fluorescent signals produced from distinct targets are generated at different rates. This allowed the team to identify sequence-specific fluorescence signatures that corresponded to defined AMRs against the often-used azole and echinocandin antifungal drugs. They were able to trace several of these signatures in an individual sample, which is key to optimizing treatments since existing diagnostics only pick up one strain of C. auris in an all-or-nothing fashion, preventing them from giving true guidance.

Devising digital diagnostics

Rolando, Weckman and the team further streamlined the assay’s reaction conditions to greatly simplify its multistep process, basically converting it into a “one-pot-reaction” that proceeds autonomously from start to finish. But to realize dSHERLOCK’s full potential they needed to enhance its usefulness with a computational analytical pipeline that was spearheaded by co-first author Anton Thieme, who had joined Walt’s group as a master’s student at the time of the study. "One of our microarrays contains about 18,000 individual compartments many of which contain a single C.auris target molecule – essentially the 1s in ‘digital’ SHERLOCK. Performing dSHERLOCK assays across all compartments provides us with an extraordinary large amount of fluorescent data that represent the pathogen’s presence, extent of fungal infection, as well as the pathogen’s genetic variability,” said Rolando. Thieme added that “translating these complex data into clinically actionable results is crucial. We developed a tailored computational solution that creates output that can be easily interpreted by trained hospital staff,” said Thieme. “The machine learning algorithm that we devised decodes the developing fluorescence signatures in dSHERLOCK assays and determines the presence and quantity of both the pathogen and specific AMR strains.”

“The capabilities that we are introducing with dSHERLOCK satisfy the major clinical requirements for a next-generation assay to rapidly identify and quantify the C. auris burden in easily obtained patient samples and produce a quantitative snapshot of the AMR landscape in individual samples,” said Wyss Founding Core Faculty member and co-senior author Collins. “This has not been possible using previous diagnostic methods and is a technological feat that, in addition to CRISPR engineering, required us to deeply integrate the SHERLOCK technology with the Walt group’s cutting-edge single molecule detection technology and a tailored machine learning approach.” Collins is also the Termeer Professor of Medical Engineering & Science at MIT.

The other senior author, Walt, who leads the Wyss Institute’s Diagnostics for Human and Planetary Health platform, highlighted: “Through this convergence of breakthrough technologies initiated by the pull of an acute clinical need, we engineered a compelling solution. But the dSHERLOCK platform has much broader utility beyond the C. auris threat: by allowing us to refit the specifics of the CRISPR-based detection machinery it can be relatively easily adopted to detect, quantify, and characterize multiple other pathogens that pose serious health problems. This is exactly what we are striving to do at the Wyss’ Diagnostics platform.” Walt is also the Hansjörg Wyss Professor of Biologically Inspired Engineering at Harvard Medical School and Professor of Pathology at Brigham and Women’s Hospital in Boston.

“This study beautifully shows the power of collaboration at the Wyss Institute and how disparate cutting-edge technologies can converge to solve pressing unmet medical needs that could have a huge impact on patients and our health care system. The potential ripple effects of dSHERLOCK technology can’t be overestimated,” said Wyss Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and the Hansjörg Wyss Professor of Biologically Inspired Engineering at Harvard John A. Paulson School of Engineering and Applied Sciences.

Other authors on the study were Nayoung Kim at the Wyss Institute, and Emily Cotnoir and Vishnu Chaturvedi at the Wadsworth Center Mycology Lab. The study was funded by the Wyss Institute at Harvard University, Health Research Inc. (grant #GR110013801), New York State Department of Health, and Wadsworth Center Division of Infectious Diseases (grant #WC-20190-01).

PRESS CONTACT

Wyss Institute for Biologically Inspired Engineering at Harvard University
Benjamin Boettner, benjamin.boettner@wyss.harvard.edu

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The Wyss Institute for Biologically Inspired Engineering at Harvard University (www.wyss.harvard.edu) is a research and development engine for disruptive innovation powered by biologically-inspired engineering with visionary people at its heart. Our mission is to transform healthcare and the environment by developing ground-breaking technologies that emulate the way Nature builds and accelerate their translation into commercial products through formation of startups and corporate partnerships to bring about positive near-term impact in the world. We accomplish this by breaking down the traditional silos of academia and barriers with industry, enabling our world-leading faculty to collaborate creatively across our focus areas of diagnostics, therapeutics, medtech, and sustainability. Our consortium partners encompass the leading academic institutions and hospitals in the Boston area and throughout the world, including Harvard’s Schools of Medicine, Engineering, Arts & Sciences and Design, Beth Israel Deaconess Medical Center, Brigham and Women’s Hospital, Boston Children’s Hospital, Dana–Farber Cancer Institute, Massachusetts General Hospital, the University of Massachusetts Medical School, Spaulding Rehabilitation Hospital, Boston University, Tufts University, Charité – Universitätsmedizin Berlin, University of Zürich, and Massachusetts Institute of Technology.

 

 

 

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