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Malaria Assays Use CRISPR for Point-of-Care Multispecies Detection

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NEW YORK – Newly designed CRISPR-based malaria diagnostic assays have the potential to become ultra-fast, ultra-cheap point-of-care tools for the highly sensitive detection of multiple malaria parasite species in low-resource settings.

The assays, which were described in a study published on Monday in the Proceedings of the National Academy of Sciences, use the SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) CRISPR platform for ultrasensitive detection and differentiation of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae.

The diagnostics comprise a 10-minute parasite rapid extraction protocol, followed by a 60-minute reaction that results in Plasmodium species-specific detection via fluorescent or lateral flow strip readout. The assays are capable of detecting fewer than two parasites per microliter of blood, and would cost an estimated $.61 per test, the researchers said.

SHERLOCK has been used a number of times as the basis for viral diagnostics since it was first invented by members of Feng Zhang's lab at the Broad Institute in 2017 and then further developed in 2018. In April 2018, researchers in the lab of Pardis Sabeti at the Broad published a paper in Science demonstrating that SHERLOCK could detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations down to 1 copy per μl, and that it could distinguish between the four DENV serotypes as well as region-specific strains of ZIKV from the 2015 to 2016 pandemic.

More recently, Broad researchers used SHERLOCK combined with microfluidics technology to develop a molecular diagnostics platform for the detection of more than 160 different viruses — including SARS-CoV-2 — in human samples. And genetic engineering company Sherlock Biosciences, which counts SHERLOCK as one its foundational technologies, is using the platform to create SARS-CoV-2 diagnostics for multiple settings, including high-volume CLIA laboratories and hospitals, and the point of care. Sherlock Bio's SARS-CoV-2 test received Emergency Use Authorization from the US Food and Drug Administration in May.

In the new study in PNAS, researchers led by James Collins of the Broad, MIT, and the Wyss Institute for Biologically Inspired Engineering noted that achieving the eradication of malaria requires several things, including ultrasensitive assays capable of detecting infections at fewer than 100 parasites per microliter of blood in order to find asymptomatic carriers of Plasmodium parasites; diagnostics that work in resource-limited settings; and sensitive point-of-care diagnostics for non-falciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure.

Molecular methods, such as PCR, have high sensitivity and specificity, but are too complex to work in the field and require too much expensive instrumentation to be practical in low-resource settings, the researchers said. Further, light microscopy remains the gold standard for distinguishing Plasmodium species, but it requires a skilled technician for interpretation and is time-intensive.

The SHERLOCK-based method, on the other hand, uses CRISPR-Cas12a to address the problems of ultrasensitive detection, multispecies detection, non-falciparum detection, and practicality for low-resource settings. The researchers optimized the SHERLOCK parameters, including the reaction temperature, recombinase polymerase amplification primer concentration, and the single-stranded DNA reporter concentration, and importantly, they improved the limit of detection by increasing sample input volume.

The P. falciparum and P. vivax assays exhibited 100 percent sensitivity and specificity on five and 10 clinical samples, respectively, the researchers said, establishing their potential use as field-applicable diagnostics for ultrasensitive detection of asymptomatic carriers as well as rapid point-of-care clinical diagnostics for non-falciparum malaria species and low parasite density P. falciparum infections.

Further, the workflow was simplified to include sample preparation without nucleic acid extraction, isothermal assay conditions that don't require a thermocycler, and field-applicable readouts, including use of a handheld fluorimeter or lateral flow strip. They also lyophilized the reaction into a pellet so it can be easily used in the field.

According to Wyss Institute researcher Rose Lee, who was first author on the PNAS paper, either readout option would be viable in the field. With the handheld fluorimeter option, a user would do the reaction in a tube, and then transfer that tube into a fluorimeter and read it.

"There are certainly fluorimeters that you could buy that are not expensive to do that," she said. "Within the field of low-resource setting engineering, people even talk about how you can outfit a smartphone, or something like that, to actually function as a fluorimeter. That technology is kind of already there. And there are certainly other papers out there that talk about creating fluorimeters for low-resource settings from just basic things that you can pull together."

The other readout, she added, would be a lateral flow strip. A user would only have to put the paper strip in the reaction tube and then read the signal to interpret the results — it's "super easy," Lee noted.

The method is reminiscent of the SARS-CoV-2 diagnostic that startup company Caspr Biotech is developing, which is based out of a lyophilized format of isothermal amplification combined with CRISPR detection. Caspr's test is also meant to be portable, low-cost, and used with minimal external equipment in low-resource settings.

The SHERLOCK-based method combines the best of many worlds, Lee said. While it has the sensitivity of PCR-based methods, it also has the simplicity of isothermal amplification technology. She also emphasized the importance of testing for multiple targets at once, as the specific diagnosis can affect the choice of treatment a patient will receive.

"Plasmodium vivax and ovale, in particular — these are the non-falciparum species — are the less deadly species of malaria," she said. "Falciparum is for sure the most common one in Africa, but vivax and ovale are actually just as common, or even more common, in parts of the world like South America or Southeast Asia. And the thing about those diseases is they can relapse. So, you can get a mild fever and infection, and people see some parasites when they look at it underneath the microscope, and they think you have falciparum, and you do get better with the short-term therapy treatment that's usually given. But you can, without giving a longer therapy, relapse even years afterwards."

Indeed, the researchers noted in the paper that Plasmodium vivax and Plasmodium ovale uniquely require an 8-aminoquinolone therapy such as primaquine or tafenoquine to prevent relapse.

The assays reached the World Health Organization's goal for limit of detection for asymptomatic carriers, the investigators also noted. Further, they found that SHERLOCK was capable of attomolar to subattomolar detection in the absence of commercial kit nucleic acid extraction and sample nucleic acid concentration.

Their CRISPR diagnostic was also able to detect clinically relevant levels of parasitemia in 40 minutes or less from unextracted blood samples with better sensitivity than existing point-of-care antigen-based rapid diagnostic tests, filling a clinical diagnostic gap for non-falciparum malaria and P. falciparum whose HRP2 gene has been deleted — HRP2 is the most common target for rapid malaria antigen tests, and HRP2 deletions have risen in the past two decades, rendering many of these tests obsolete, the researchers said.

There's still more work to be done on the assays before they can be used in the field or at the point of care, Lee said. The researchers had originally planned to team up with investigators in Kenya to test the workflow in the field, but those plans fell through because of the COVID pandemic. They're still planning to undertake that field-testing pilot, but the timeline is uncertain.

"Another goal for the project moving forward is trying make the assay multiplexed and then also potentially adding detection of resistance," Lee added.

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