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Clinical Laboratories and Pathology Groups

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Clinical Laboratories and Pathology Groups

Hosted by Robert Michel
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At-Home Paper Influenza Test Differentiates Strains, Gives Hope for Improved Screening and Surveillance of Viral Outbreaks

Researchers used CRISPR-based assays to develop new clinical laboratory point-of-care blood test which boasts accuracy, affordability, and accessibility

Here’s a novel use of paper as clinical laboratory test media. Researchers at Princeton University in New Jersey, the Massachusetts Institute of Technology’s Broad Institute, and Harvard University have developed an at-home paper-strip test that can not only identify the presence of influenza, but it can also differentiate between different strains of the flu bug.

According to UPI, the test can “distinguish between influenza A and influenza B—the two main types of seasonal flu—as well as identifying more virulent strains like H1N1 and H3N2.”

Many research teams are working to develop paper-based diagnostic screening tests because of their lower cost to produce and usefulness in remote locations. Should this near-patient point-of-care test become clinically viable, it could mean shorter times to answer, enabling speedier diagnoses and earlier start of treatment.

It also means patient specimens do not have to be transported to a clinical laboratory for testing. And reduced cost per test makes it possible to test more people. This serves the public health aspect of monitoring outbreaks of influenza and other diseases and gives hope for improved treatment outcomes.

“Being able to tease apart what strain or subtype of influenza is infecting a patient has repercussions both for treating them and public health interventions, said Jon Arizti Sanz, PhD, co-lead study author and postdoctoral researcher at the Broad Institute of Harvard and MIT, in a Broad Institute news release.

The researchers published their findings in The Journal of Molecular Diagnostics titled, “CRISPR-Based Assays for Point-of-Need Detection and Subtyping of Influenza.”

“Ultimately, we hope these tests will be as simple as rapid antigen tests, and they’ll still have the specificity and performance of a nucleic acid test that would normally be done in a laboratory setting,” Cameron A. Myhrvold, PhD (above), Assistant Professor of Molecular Biology at Princeton University in New Jersey, told CIDRAP. Influenza tests that can be performed at the point of care and in remote locations may reduce the number of screening tests performed by clinical laboratories. (Photo copyright: Michael James Butts/Hertz Foundation.)

Inspiration from Prior COVID-19 Test

According to an article published by the Center for Infectious Disease Research and Policy Research and Innovation Office (CIDRAP) at the University of Minnesota, the original test was developed in 2020 in a Harvard laboratory led by computational geneticist Pardis Christine Sabeti, MD, PhD, professor, Department of Organismic and Evolutionary Biology, and co-senior author of the study.

Her team developed their tests using Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), “a clustered regularly interspaced short palindromic repeats (CRISPR)-based RNA detection platform,” the researchers wrote in their Journal of Molecular Diagnostics paper.

“SHINE has a runtime of 90 minutes, can be used at room temperature and only requires an inexpensive heat block to heat the reaction. The SHINE technology has previously been used to identify SARS-CoV-2 and later to distinguish between the Delta and Omicron variants,” Bioanalysis Zone reported.

“The test uses genetically engineered enzymes to identify specific sequences of viral RNA in samples,” the researchers told UPI. Originally designed to detect COVID-19, the team adapted the technology to detect influenza in 2022 “with the aim of creating a screening tool that could be used in the field or in clinics rather than hospitals or high-tech diagnostic labs,” they said.

Influenza A and B as well as H1N1 and H3N2 subtypes were the targets of the four SHINE assays. “When tested on clinical samples, these optimized assays achieved 100% concordance with quantitative RT-PCR. Duplex Cas12a/Cas13a SHINE assays were also developed to detect two targets simultaneously,” the researchers wrote in their paper.

The team used “20 nasal swabs from people with flu-like symptoms during the 2020-2021 flu season, nasal fluid from healthy people as the control, and 2016-2021 influenza sequences downloaded from the National Center for Biotechnology Information Influenza (NICB) database. They compared the results with those from quantitative reverse transcription-polymerase chain reaction (RT-PCR) tests,” CIDRAP reported.

The original 2020 test (shown above) takes 90 minutes to develop at room temperature. The test developers aim to drop this down to 15 minutes. In comparison, typical polymerase chain reaction (PCR) testing requires medical laboratories to have specialized equipment, trained staff, and prolonged processing times, the Broad Institute news release notes. (Photo copyright: Broad Institute.)

Implications of the New Tests

The ease of the new tests is an important development since approximately only 1% of individuals who come down with the flu see doctors for testing, according to the news release. And researchers had this in mind, looking at speed, accuracy, and affordability as a means to “improve outbreak response and infection care around the world,” UPI reported.

There are great benefits to strain differentiation that be achieved with the new test. Doctors are hopeful the test will help dial in the best treatment plans for patients since some strains are resistant to the antiviral medication oseltamivir (Tamiflu), UPI noted. This is significant since Tamiflu “is a common antiviral,” said Sanz in the Broad Institute news release.

“These assays have the potential to expand influenza detection outside of clinical laboratories for enhanced influenza diagnosis and surveillance,” the Journal of Molecular Diagnostics paper noted. This allows for more strategic treatment planning.

“Using a paper strip readout instead of expensive fluorescence machinery is a big advancement, not only in terms of clinical care but also for epidemiological surveillance purposes,” said Ben Zhang, an MD candidate in the Health Sciences and Technology at Harvard and co-first author of the study, in the Broad Institute news release.

Future Plans for Tests

“With further development, the test strip could be reprogrammed to distinguish between SARS-CoV-2 and flu and recognize swine flu and avian flu, including the H5N1 subtype currently causing an outbreak in US dairy cattle,” the study authors told CIDRAP.

The team is also looking at ways to help prevent H5N1 from crossing into human contamination, Sanz told UPI.

The new Princeton/MIT/Harvard tests echo the trend to bring in affordability and ease-of-use with accurate results as an end goal. Faster results mean the best treatments for each person can start sooner and may render the transport of specimens to a clinical laboratory as a second step unnecessary.

As research teams work to develop paper-based viral tests for their plethora of benefits, clinical laboratories will want to pay close attention to this development as it can have a big implication on assisting with future outbreaks.

Additional research is needed before these tests are going to be commonplace in homes worldwide, but this first step brings inspiration and hope of what’s to come. 

—Kristin Althea O’Connor

Related Information:

Simple Test for Flu Could Improve Diagnosis and Surveillance

Simple Paper-Strip Test Might Spot Flu, Identify Strain

CRISPR-Based Assays for Point-of-Need Detection and Subtyping of Influenza

Paper Strip Test Can Identify Flu Subtypes, May Have Other Applications, Scientists Say

Streamlined Inactivation, Amplification, and Cas13-based Detection of SARS-Cov-2

Paper Strip Test Using CRISPR and SHINE Technology Has Been Developed for Rapid Influenza Diagnosis

CRISPR-Related Tool Set to Fundamentally Change Clinical Laboratory Diagnostics, Especially in Rural and Remote Locations

SHERLOCK makes accurate, fast diagnoses for about 61-cents per test with no refrigeration needed; could give medical laboratories a new diagnostic tool

Genetics researchers have been riveted by ongoing discoveries related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) for some time now and so have anatomic pathology laboratories. The diagnostic possibilities inherent in CRISPR have been established, and now, a new diagnostic tool that works with CRISPR is set to change clinical laboratory diagnostics in a foundational way.

The tool is called SHERLOCK, which stands for (Specific High-sensitivity Enzymatic Reporter unLOCKing). And it is causing excitement in the scientific community for several reasons:

  • It can detect pathogens in extremely small amounts of genetic matter;
  • Tests can be performed using urine and/or saliva rather than blood;
  • The tests are extremely sensitive; and they
  • Cost far less than the diagnostic tests currently in use.

In an article published in Science, researchers described SHERLOCK tests that can distinguish between strains of Zika and Dengue fever, as well as determining the difference between mutations in cell-free tumor DNA.

How SHERLOCK and CRISPR Differ and Why That’s Important

Scientists have long suspected that CRISPR could be used to detect viruses. However, far more attention has been given to the its genome editing capabilities. And, there are significant differences between how CRISPR and SHERLOCK work. According to the Science article, when CRISPR is used to edit genes, a small strip of RNA directs an enzyme capable of cutting DNA to a precise location within a genome. The enzyme that CRISPR uses is called Cas9 (CRISPR associated protein 9). It works like scissors, snipping the strand of DNA, so that it is either damaged or replaced by a healthy, new sequence.

SHERLOCK, however, uses a different enzyme—Cas13a (originally dubbed C2c2 by the researchers who discovered it). Cas13a goes to RNA, rather than DNA, and once it starts cutting, it doesn’t stop. It chops through any RNA it encounters. The researchers who developed SHERLOCK describe these cuts as “collateral cleavage.” According to an article published by STAT, “All that chopping generates a fluorescent signal that can be detected with a $200 device or, sometimes, with the naked eye.”

 

The screenshot above is from a video in which Feng Zhang, PhD (center), a Core Member of the Broad Institute at MIT and one of the lead researchers working on SHERLOCK, and his research team, explain the difference and value SHERLOCK will make in the detection of diseases like Zika. Click on the image above to watch the video. (Video copyright: Broad Institute/MIT.)

Early Stage Detection in Clinical Laboratories

A research paper published in Science states that SHERLOCK can provide “rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity.” Attomolar equates to about one part per quintillion—a billion-billion. According to the article on the topic also published in Science, “The detection sensitivity of the new CRISPR-Cas13a system for specific genetic material is one million times better than the most commonly used diagnostic technique.” Such sensitivity suggests that clinical laboratories could detect pathogens at earlier stages using SHERLOCK.

The Stat article notes that, along with sensitivity, SHERLOCK has specificity. It can detect a difference of a single nucleotide, such as the difference between the African and Asian strains of Zika (for example, the African strain has been shown to cause microcephaly, whereas the Asian strain does not). Thus, the combination of sensitivity and specificity could mean that SHERLOCK would be more accurate and faster than other diagnostic tests.

Clinicians in Remote Locations Could Diagnose and Treat Illness More Quickly

Perhaps one of the most important aspects of SHERLOCK is the portability and durability of the test. It can be performed on glass fiber paper and works even after the components have been freeze dried. “We showed that this system is very stable, so you can really put it on a piece of paper and it will survive. You don’t have to refrigerate it all the times,” stated Feng Zhang, PhD, in an interview with the Washington Post. Zhang is a Core Member of the Broad Institute at MIT and was one of the scientists who developed CRISPR.

The researchers note that SHERLOCK could cost as little as 61-cents per test to perform. For clinicians working in remote locations with little or no power, such a test could improve their ability to diagnose and treatment illness in the field and possibly save lives.

“If you had something that could be used as a screening test, very inexpensively and rapidly, that would be a huge advance, particularly if it could detect an array of agents,” stated William Schaffner, MD, Professor and Chair of the Department of Preventive Medicine at Vanderbilt University Medical Center, in the Post article. Schaffner describes the Broad Institute’s research as being “very, very provocative.”

The test could radically change the delivery of care in more modern settings, as well. “It looks like one significant step on the pathway [that] is the Holy Grail, which is developing point-of-care, or bedside detection, [that] doesn’t require expensive equipment or even reliable power,” noted Scott Weaver, PhD, in an article on Big Think. Weaver is a Professor and Director at the Institute for Human Infections and Immunity University of Texas Medical Branch in Galveston, Texas.

Just the Beginning

Anatomic pathologists and clinical laboratories will want to follow SHERLOCK’s development. It could be on the path to fundamentally transforming the way disease gets diagnosed in their laboratories and in the field.

According to the Post article, “The scientists have filed several US patent applications on SHERLOCK, including for uses in detecting viruses, bacteria, and cancer-causing mutations.” In addition to taking steps to secure patents on the technology, the researchers are exploring ways to commercialize their work, as well as discussing the possibility of launching a startup. However, before this technology can be used in medical laboratory testing, SHERLOCK will have to undergo the regulatory processes with various agencies, including applying for FDA approval.

—Dava Stewart

 

Related Information:

New CRISPR Tool Can Detect Tiny Amounts of Viruses

CRISPR Cousin SHERLOCK May Be Able to Track Down Diseases, Scientists Say

Nucleic Acid Detection with CRISPR-Cas13a/C2c2

A New CRISPR Breakthrough Could Lead to Simpler, Cheaper Disease Diagnosis

Meet CRISPR’s Younger Brother, SHERLOCK

Trends in Genomic Research That Could Impact Clinical Laboratories and Anatomic Pathology Groups Very Soon

Pathologists and Clinical Laboratories May Soon Have a Test for Identifying Cardiac Patients at Risk from Specific Heart Drugs by Studying the Patients’ Own Heart Cells

Patent Dispute over CRISPR Gene-Editing Technology May Determine Who Will Be Paid Licensing Royalties by Medical Laboratories

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