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CRISPR-based malaria testing on-the-fly

CRISPR-based malaria testing on-the-fly

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A Plasmodium species-specific, ultrasensitive, nucleic acid detection assay to enable fast and inexpensive testing of symptomatic and asymptomatic carriers in low-resource settings

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Credit: Peter Nguyen / Wyss Institute at Harvard University

(BOSTON) — To achieve the goal of eradicating malaria set by the World Health Organization (WHO)’s Global Malaria Control Programme, it is critical that all local transmission of malaria parasites in defined geographic areas is eliminated. One important cornerstone on this path is the development of rapid, sensitive and species-specific diagnostic capabilities that are useful in the low-resource settings (LRSs) of many areas with endemic malaria.

Currently, the presence of the four major malaria-causing Plasmodium species P. falciparum, P. vivax, P. ovale, and P. malariae is determined by microscopic analysis of blood samples in which parasites can be detected in red blood cells, or with so-called rapid diagnostic tests for specific Plasmodium proteins (antigens).

“Unfortunately, available rapid diagnostic approaches cannot distinguish all four Plasmodium species from one other, which can be important to initiate the definitive course of treatment, and, most importantly, they are ineffective for detecting low numbers of Plasmodium parasites in asymptomatic individuals,” said Nira Pollock, M.D., Ph.D., Associate Medical Director of Boston Children’s Hospital’s Infectious Diseases Diagnostic Laboratory and Associate Professor of Pathology and Medicine at Harvard Medical School. “These “asymptomatic carriers” are silent reservoirs for ongoing transmission by malaria-spreading mosquitoes and extremely important for ongoing global efforts to eradicate malaria,” added Jeffrey Dvorin, M.D., Ph.D., Associate Professor of Pediatrics at Harvard Medical School and Senior Associate Physician in Infectious Diseases at Boston Children’s Hospital.

Now, a multi-disciplinary research collaboration which was led by Wyss Core Faculty member James Collins, Ph.D. at Harvard’s Wyss Institute for Biologically Inspired Engineering and the Massachusetts Institute of Technology (MIT), and assembled by clinical fellow Rose Lee, M.D., MSPH, which also included Pollock and Dvorin, created a field-applicable, ultrasensitive diagnostic assay that specifically detects DNA sequences from all Plasmodium species in symptomatic and asymptomatic malaria. The new malaria diagnostic method combines an optimized 10-minute rapid sample preparation protocol with the CRISPR-based SHERLOCK system to enable highly specific and sensitive Plasmodium detection in another 60 minutes in simple reporter devices. It is published in PNAS.

“This field-ready SHERLOCK diagnostic malaria assay surpasses the sensitivity and specificity requirements set by the WHO for a desired test that can be used to detect low parasite density in asymptomatic carriers of all major Plasmodium species,” said Wyss Founding Core Faculty member James Collins, Ph.D. “Its highly streamlined design could provide a viable solution to the present diagnostic bottleneck on the path to eliminate malaria, and more generally enabling malaria surveillance in low-resource settings.” Collins is a lead of the Institute’s Living Cellular Devices Focus Area, and also the Termeer Professor of Medical Engineering & Science at MIT.

The research team demonstrated their engineered SHERLOCK (short for Specific High-sensitivity Enzymatic Reporter unLOCKing) assay to be capable of detecting less than two parasites per microliter of blood, the WHO’s suggested “limit of detection” (LOD) for a test with broad utility in endemic areas. Showing the assay’s clinical potential by analyzing clinical samples containing P. falciparum and P. vivax species, they called out them out with 100% sensitivity, by correctly identifying true positive samples, and 100% specificity, by also correctly identifying samples lacking a certain Plasmodium species in true negative samples. Near 100% sensitivity and specificity are key attributes of diagnostic assays to be used in real-world testing. Moreover, the assay is designed so that it can also determine the presence of frequently mutated P. falciparum strains that have lost their HRP2 antigen and thus escape the detection by common rapid diagnostic tests.

Collins’ group at the Wyss Institute and MIT co-developed the SHERLOCK technology with Feng Zhang’s group at the Broad Institute. It was licensed to Sherlock Biosciences, a startup that used it to create a rapid molecular diagnostic for other disease applications, and recently received an emergency use authorization from the FDA for its COVID-19 rapid diagnostic.

Other methods have been developed that, like the new SHERLOCK assay, amplify and detect the DNA (or RNA) nucleic acid material of Plasmodium species. However, to date, these methods remain limited by their need for expensive laboratory equipment, as in the case of polymerase chain reaction (PCR)-based methods, complicated sample preparation techniques, and trained personnel or, as in the case of simpler “isothermal” amplification methods performed at a single temperature, they have not shown the desired sensitivities in the field.

The SHERLOCK malaria assay takes advantage of the CRISPR-Cas12a enzyme which can be programmed to become active with a so-called guide RNA that binds to a specific target nucleic acid target sequence, in this case a sequence from one of the four Plasmodium species. Activated Cas12a then non-specifically cleaves any single-stranded DNA strand in its vicinity with an extremely high turn-over rate of about 1,250 collateral cleavage reactions per second. The researchers leveraged this amplifying activity in their assay by integrating it with an optimized sample preparation, which does not require a specific nucleic extraction step like some other nucleic acid amplification tests (NAATs), and isothermal amplification of specific Plasmodium DNA and RNA sequences at the front end. Guide RNAs that recognize species-specific motifs in the amplified Plasmodium sequences then unleash Cas12a activity, which collaterally attacks single stranded DNA reporter sequences whose cleavage products help signal the presence of the pathogen-specific nucleic acids. At the backend of the assay, the signal is engineered to either cause a change in fluorescence in a hand-held device, or specific band on a lateral flow strip commonly used in clinical point-of-care devices.

“Importantly, the assay is compatible with different sample types, such as whole blood, plasma, serum, and dried blood, and all components required for amplification, Cas12a activation and signal generation can be lyophilized in a single test tube to work together in a one-pot-reaction after they are reconstituted and mixed with patient sample,” said first-author Rose Lee, a clinical fellow in Collins’ group and Boston Children’s Hospital with a strong interest in infectious disease diagnostics and was instrumental in assembling the multi-disciplinary team together with Collins. “This avoids having to depend on a functional cold-chain and allows testing to be performed in low-resource settings with minimal expertise.”

“The collaboration’s molecular assay for malaria diagnosis points the way in which Wyss Institute capabilities in the synthetic biology field, when paired with expertise in infectious disease biology and epidemiology, could change the course of truly debilitating diseases that paralyze large populations around the globe,” said the Wyss Institute’s Founding Director Don 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 Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

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By Benjamin Boettner

Other authors on the study are other members of Collins’ group, including Helena De Puig, Ph.D., Peter Nguyen, Ph.D., Nicolaas Angenent-Mari, Nina Donghia; and additional collaborators, including James McGee at Boston Children’s Hospital; and Catherine Klapperich, Ph.D., Professor and Director of the Precision Diagnostics Center at Boston University. The study was supported by the Wyss Institute for Biologically Inspired Engineering.

PRESS CONTACT

Wyss Institute for Biologically Inspired Engineering at Harvard University

Benjamin Boettner, benjamin.boettner@wyss.harvard.edu, +1 617-432-8232

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The Wyss Institute for Biologically Inspired Engineering at Harvard University (http://wyss.harvard.edu) uses Nature’s design principles to develop bioinspired materials and devices that will transform medicine and create a more sustainable world. Wyss researchers are developing innovative new engineering solutions for healthcare, energy, architecture, robotics, and manufacturing that are translated into commercial products and therapies through collaborations with clinical investigators, corporate alliances, and formation of new startups. The Wyss Institute creates transformative technological breakthroughs by engaging in high risk research, and crosses disciplinary and institutional barriers, working as an alliance that includes Harvard’s Schools of Medicine, Engineering, Arts & Sciences, Design, and Education, and in partnership with 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 Zurich and Massachusetts Institute of Technology.

Boston Children’s Hospital (http://www.childrenshospital.org) is ranked the #1 children’s hospital in the nation by U.S. News & World Report and is the primary pediatric teaching affiliate of Harvard Medical School. Home to the world’s largest research enterprise based at a pediatric medical center, its discoveries have benefited both children and adults since 1869. Today, 3,000 researchers and scientific staff, including 9 members of the National Academy of Sciences, 21 members of the National Academy of Medicine and 12 Howard Hughes Medical Investigators comprise Boston Children’s research community. Founded as a 20-bed hospital for children, Boston Children’s is now a 415-bed comprehensive center for pediatric and adolescent health care. For more, visit our Discoveries blog and follow us on social media @BostonChildrens, @BCH_Innovation, Facebook and YouTube.

Media Contact
Benjamin Boettner
Benjamin.Boettner@wyss.harvard.edu

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Health Officials: Man Dies From Bubonic Plague In New Mexico

Health Officials: Man Dies From Bubonic Plague In New Mexico

Authored by Jack Phillips via The Epoch Times (emphasis ours),

Officials in…

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3 hours ago

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March 13, 2024

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Health Officials: Man Dies From Bubonic Plague In New Mexico

Authored by Jack Phillips via The Epoch Times (emphasis ours),

Officials in New Mexico confirmed that a resident died from the plague in the United States’ first fatal case in several years.

A bubonic plague smear, prepared from a lymph removed from an adenopathic lymph node, or bubo, of a plague patient, demonstrates the presence of the Yersinia pestis bacteria that causes the plague in this undated photo. (Centers for Disease Control and Prevention/Getty Images)

The New Mexico Department of Health, in a statement, said that a man in Lincoln County “succumbed to the plague.” The man, who was not identified, was hospitalized before his death, officials said.

They further noted that it is the first human case of plague in New Mexico since 2021 and also the first death since 2020, according to the statement. No other details were provided, including how the disease spread to the man.

The agency is now doing outreach in Lincoln County, while “an environmental assessment will also be conducted in the community to look for ongoing risk,” the statement continued.

“This tragic incident serves as a clear reminder of the threat posed by this ancient disease and emphasizes the need for heightened community awareness and proactive measures to prevent its spread,” the agency said.

A bacterial disease that spreads via rodents, it is generally spread to people through the bites of infected fleas. The plague, known as the black death or the bubonic plague, can spread by contact with infected animals such as rodents, pets, or wildlife.

The New Mexico Health Department statement said that pets such as dogs and cats that roam and hunt can bring infected fleas back into homes and put residents at risk.

Officials warned people in the area to “avoid sick or dead rodents and rabbits, and their nests and burrows” and to “prevent pets from roaming and hunting.”

“Talk to your veterinarian about using an appropriate flea control product on your pets as not all products are safe for cats, dogs or your children” and “have sick pets examined promptly by a veterinarian,” it added.

“See your doctor about any unexplained illness involving a sudden and severe fever, the statement continued, adding that locals should clean areas around their home that could house rodents like wood piles, junk piles, old vehicles, and brush piles.

The plague, which is spread by the bacteria Yersinia pestis, famously caused the deaths of an estimated hundreds of millions of Europeans in the 14th and 15th centuries following the Mongol invasions. In that pandemic, the bacteria spread via fleas on black rats, which historians say was not known by the people at the time.

Other outbreaks of the plague, such as the Plague of Justinian in the 6th century, are also believed to have killed about one-fifth of the population of the Byzantine Empire, according to historical records and accounts. In 2013, researchers said the Justinian plague was also caused by the Yersinia pestis bacteria.

But in the United States, it is considered a rare disease and usually occurs only in several countries worldwide. Generally, according to the Mayo Clinic, the bacteria affects only a few people in U.S. rural areas in Western states.

Recent cases have occurred mainly in Africa, Asia, and Latin America. Countries with frequent plague cases include Madagascar, the Democratic Republic of Congo, and Peru, the clinic says. There were multiple cases of plague reported in Inner Mongolia, China, in recent years, too.

Symptoms

Symptoms of a bubonic plague infection include headache, chills, fever, and weakness. Health officials say it can usually cause a painful swelling of lymph nodes in the groin, armpit, or neck areas. The swelling usually occurs within about two to eight days.

The disease can generally be treated with antibiotics, but it is usually deadly when not treated, the Mayo Clinic website says.

“Plague is considered a potential bioweapon. The U.S. government has plans and treatments in place if the disease is used as a weapon,” the website also says.

According to data from the U.S. Centers for Disease Control and Prevention, the last time that plague deaths were reported in the United States was in 2020 when two people died.

Tyler Durden Wed, 03/13/2024 - 21:40

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Riley Gaines Explains How Women’s Sports Are Rigged To Promote The Trans Agenda

Riley Gaines Explains How Women’s Sports Are Rigged To Promote The Trans Agenda

Is there a light forming when it comes to the long, dark and…

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7 hours ago

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March 13, 2024

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Riley Gaines Explains How Women's Sports Are Rigged To Promote The Trans Agenda

Is there a light forming when it comes to the long, dark and bewildering tunnel of social justice cultism?  Global events have been so frenetic that many people might not remember, but only a couple years ago Big Tech companies and numerous governments were openly aligned in favor of mass censorship.  Not just to prevent the public from investigating the facts surrounding the pandemic farce, but to silence anyone questioning the validity of woke concepts like trans ideology. 

From 2020-2022 was the closest the west has come in a long time to a complete erasure of freedom of speech.  Even today there are still countries and Europe and places like Canada or Australia that are charging forward with draconian speech laws.  The phrase "radical speech" is starting to circulate within pro-censorship circles in reference to any platform where people are allowed to talk critically.  What is radical speech?  Basically, it's any discussion that runs contrary to the beliefs of the political left.

Open hatred of moderate or conservative ideals is perfectly acceptable, but don't ever shine a negative light on woke activism, or you might be a terrorist.

Riley Gaines has experienced this double standard first hand.  She was even assaulted and taken hostage at an event in 2023 at San Francisco State University when leftists protester tried to trap her in a room and demanded she "pay them to let her go."  Campus police allegedly witnessed the incident but charges were never filed and surveillance footage from the college was never released.  

It's probably the last thing a champion female swimmer ever expects, but her head-on collision with the trans movement and the institutional conspiracy to push it on the public forced her to become a counter-culture voice of reason rather than just an athlete.

For years the independent media argued that no matter how much we expose the insanity of men posing as women to compete and dominate women's sports, nothing will really change until the real female athletes speak up and fight back.  Riley Gaines and those like her represent that necessary rebellion and a desperately needed return to common sense and reason.

In a recent interview on the Joe Rogan Podcast, Gaines related some interesting information on the inner workings of the NCAA and the subversive schemes surrounding trans athletes.  Not only were women participants essentially strong-armed by colleges and officials into quietly going along with the program, there was also a concerted propaganda effort.  Competition ceremonies were rigged as vehicles for promoting trans athletes over everyone else. 

The bottom line?  The competitions didn't matter.  The real women and their achievements didn't matter.  The only thing that mattered to officials were the photo ops; dudes pretending to be chicks posing with awards for the gushing corporate media.  The agenda took precedence.

Lia Thomas, formerly known as William Thomas, was more than an activist invading female sports, he was also apparently a science project fostered and protected by the athletic establishment.  It's important to understand that the political left does not care about female athletes.  They do not care about women's sports.  They don't care about the integrity of the environments they co-opt.  Their only goal is to identify viable platforms with social impact and take control of them.  Women's sports are seen as a vehicle for public indoctrination, nothing more.

The reasons why they covet women's sports are varied, but a primary motive is the desire to assert the fallacy that men and women are "the same" psychologically as well as physically.  They want the deconstruction of biological sex and identity as nothing more than "social constructs" subject to personal preference.  If they can destroy what it means to be a man or a woman, they can destroy the very foundations of relationships, families and even procreation.  

For now it seems as though the trans agenda is hitting a wall with much of the public aware of it and less afraid to criticize it.  Social media companies might be able to silence some people, but they can't silence everyone.  However, there is still a significant threat as the movement continues to target children through the public education system and women's sports are not out of the woods yet.   

The ultimate solution is for women athletes around the world to organize and widely refuse to participate in any competitions in which biological men are allowed.  The only way to save women's sports is for women to be willing to end them, at least until institutions that put doctrine ahead of logic are made irrelevant.          

Tyler Durden Wed, 03/13/2024 - 17:20

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Congress’ failure so far to deliver on promise of tens of billions in new research spending threatens America’s long-term economic competitiveness

A deal that avoided a shutdown also slashed spending for the National Science Foundation, putting it billions below a congressional target intended to…

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15 hours ago

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March 13, 2024

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Science is again on the chopping block on Capitol Hill. AP Photo/Sait Serkan Gurbuz

Federal spending on fundamental scientific research is pivotal to America’s long-term economic competitiveness and growth. But less than two years after agreeing the U.S. needed to invest tens of billions of dollars more in basic research than it had been, Congress is already seriously scaling back its plans.

A package of funding bills recently passed by Congress and signed by President Joe Biden on March 9, 2024, cuts the current fiscal year budget for the National Science Foundation, America’s premier basic science research agency, by over 8% relative to last year. That puts the NSF’s current allocation US$6.6 billion below targets Congress set in 2022.

And the president’s budget blueprint for the next fiscal year, released on March 11, doesn’t look much better. Even assuming his request for the NSF is fully funded, it would still, based on my calculations, leave the agency a total of $15 billion behind the plan Congress laid out to help the U.S. keep up with countries such as China that are rapidly increasing their science budgets.

I am a sociologist who studies how research universities contribute to the public good. I’m also the executive director of the Institute for Research on Innovation and Science, a national university consortium whose members share data that helps us understand, explain and work to amplify those benefits.

Our data shows how underfunding basic research, especially in high-priority areas, poses a real threat to the United States’ role as a leader in critical technology areas, forestalls innovation and makes it harder to recruit the skilled workers that high-tech companies need to succeed.

A promised investment

Less than two years ago, in August 2022, university researchers like me had reason to celebrate.

Congress had just passed the bipartisan CHIPS and Science Act. The science part of the law promised one of the biggest federal investments in the National Science Foundation in its 74-year history.

The CHIPS act authorized US$81 billion for the agency, promised to double its budget by 2027 and directed it to “address societal, national, and geostrategic challenges for the benefit of all Americans” by investing in research.

But there was one very big snag. The money still has to be appropriated by Congress every year. Lawmakers haven’t been good at doing that recently. As lawmakers struggle to keep the lights on, fundamental research is quickly becoming a casualty of political dysfunction.

Research’s critical impact

That’s bad because fundamental research matters in more ways than you might expect.

For instance, the basic discoveries that made the COVID-19 vaccine possible stretch back to the early 1960s. Such research investments contribute to the health, wealth and well-being of society, support jobs and regional economies and are vital to the U.S. economy and national security.

Lagging research investment will hurt U.S. leadership in critical technologies such as artificial intelligence, advanced communications, clean energy and biotechnology. Less support means less new research work gets done, fewer new researchers are trained and important new discoveries are made elsewhere.

But disrupting federal research funding also directly affects people’s jobs, lives and the economy.

Businesses nationwide thrive by selling the goods and services – everything from pipettes and biological specimens to notebooks and plane tickets – that are necessary for research. Those vendors include high-tech startups, manufacturers, contractors and even Main Street businesses like your local hardware store. They employ your neighbors and friends and contribute to the economic health of your hometown and the nation.

Nearly a third of the $10 billion in federal research funds that 26 of the universities in our consortium used in 2022 directly supported U.S. employers, including:

  • A Detroit welding shop that sells gases many labs use in experiments funded by the National Institutes of Health, National Science Foundation, Department of Defense and Department of Energy.

  • A Dallas-based construction company that is building an advanced vaccine and drug development facility paid for by the Department of Health and Human Services.

  • More than a dozen Utah businesses, including surveyors, engineers and construction and trucking companies, working on a Department of Energy project to develop breakthroughs in geothermal energy.

When Congress shortchanges basic research, it also damages businesses like these and people you might not usually associate with academic science and engineering. Construction and manufacturing companies earn more than $2 billion each year from federally funded research done by our consortium’s members.

A lag or cut in federal research funding would harm U.S. competitiveness in critical advanced technologies such as artificial intelligence and robotics. Hispanolistic/E+ via Getty Images

Jobs and innovation

Disrupting or decreasing research funding also slows the flow of STEM – science, technology, engineering and math – talent from universities to American businesses. Highly trained people are essential to corporate innovation and to U.S. leadership in key fields, such as AI, where companies depend on hiring to secure research expertise.

In 2022, federal research grants paid wages for about 122,500 people at universities that shared data with my institute. More than half of them were students or trainees. Our data shows that they go on to many types of jobs but are particularly important for leading tech companies such as Google, Amazon, Apple, Facebook and Intel.

That same data lets me estimate that over 300,000 people who worked at U.S. universities in 2022 were paid by federal research funds. Threats to federal research investments put academic jobs at risk. They also hurt private sector innovation because even the most successful companies need to hire people with expert research skills. Most people learn those skills by working on university research projects, and most of those projects are federally funded.

High stakes

If Congress doesn’t move to fund fundamental science research to meet CHIPS and Science Act targets – and make up for the $11.6 billion it’s already behind schedule – the long-term consequences for American competitiveness could be serious.

Over time, companies would see fewer skilled job candidates, and academic and corporate researchers would produce fewer discoveries. Fewer high-tech startups would mean slower economic growth. America would become less competitive in the age of AI. This would turn one of the fears that led lawmakers to pass the CHIPS and Science Act into a reality.

Ultimately, it’s up to lawmakers to decide whether to fulfill their promise to invest more in the research that supports jobs across the economy and in American innovation, competitiveness and economic growth. So far, that promise is looking pretty fragile.

This is an updated version of an article originally published on Jan. 16, 2024.

Jason Owen-Smith receives research support from the National Science Foundation, the National Institutes of Health, the Alfred P. Sloan Foundation and Wellcome Leap.

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