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Non-Sputum CRISPR-Based Tuberculosis Test Shows High Sensitivity in HIV-Infected Children

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NEW YORK – Despite decades of intensive efforts, tuberculosis is a global scourge that has proven difficult to stamp out. Rapid diagnosis is a key to bringing rates down, but traditional sputum-based testing is particularly challenging in children, people who are HIV positive, and cases where the bacteria set up shop outside of the lungs. 

To fill this void, researchers at Tulane University have developed a CRISPR-based test that detects cell-free TB DNA found at low levels in blood samples, including extrapulmonary infections in kids who are HIV positive.

Sputum, or phlegm, is typically used for TB diagnostics. Children are incapable of producing the amount of sputum required for PCR-based TB testing, however, and HIV infection hampers sputum production even further. In cases of extra-pulmonary TB, sputum samples would also be lacking in bacteria, also termed paucibacillary. Without a positive diagnostic test, TB drug treatments are not usually initiated because they can be toxic.

Overall, children make up about 10 percent of TB cases globally, according to the World Health Organization. The WHO also estimates that global healthcare challenges related to the coronavirus pandemic led new TB diagnoses to decline from 7.1 million in 2019 to 5.8 million in 2020 and the number of undiagnosed cases to rise from 2.9 million to 4.1 million. In 2020, more than 1.5 million people died of TB.

In a study published last month in The Lancet Microbe, the Tulane team validated the CRISPR test in pediatric patients infected with TB, as well as in children with both TB and HIV infection. Tony Hu, the corresponding author on the study and a molecular biologist at Tulane, said his team was motivated to find new solutions to the age-old problem of TB.

"Unlike COVID, we've known this pathogen for centuries, so why is TB diagnosis still so challenging?" he said in an interview.

The team considered biomarkers in urine or other bodily fluid, but decided that blood is a sample type that is most routinely collected and handled in clinical labs. Previous groups have described PCR-based detection of cfDNA from TB, but Hu noted that the sensitivity of these tests was quite low because the target DNA is only present at very low concentrations.

Hu's team wondered whether CRISPR might help. He had reviewed a 2019 publication of a CRISPR-based TB assay by a team at Vision Medicals in China, but noted that this work was done primarily on sputum samples, not blood.

The team was able to modify the CRISPR method to improve sensitivity and enable detection of the minute amount of cell-free TB DNA that circulates in the blood due to the host immune response.

Specifically, the team came up with a number of notable innovations to a previously described CRISPR-Cas12a-powered fluorescence assay. For example, it used a multicopy gene called IS6110 to improve sensitivity, as well as an optimized Quick-cfDNA Serum & Plasma from Zymo Research extraction protocol to increase the concentration of cfDNA derived from blood samples.

It also used a thermostable protein called AccuPrime to enhance nucleic acid amplification by improving complimentary base pairing and tweaked the CRISPR step to improve reaction kinetics.

In the Lancet Microbe study, Hu and his team evaluated samples from Eswatini, formerly known as Swaziland, and Kenya. Eswatini has one of the highest rates of HIV infection in the world, and 66 percent of TB patients were also HIV positive as of 2019, according to a report from the US Centers for Disease Control and Prevention.

In a cohort of 73 HIV-negative TB-infected adults and controls from Eswatini, the test showed 96 percent sensitivity and 94 percent specificity. It also showed 83 percent sensitivity and 95 percent specificity in a cohort of 28 children, including six cases of extrapulmonary TB.

The Kenya study consisted of 153 children with a median age of 2 years old who were living with HIV and had suspected TB. The CRISPR-TB assay detected all confirmed TB cases, and 85 percent of the unconfirmed cases that had been diagnosed by non-microbiological clinical findings.

The team now expects the technology to be used for both diagnosis as well as treatment monitoring after antimicrobial treatment, as cfDNA can be an indicator of bacterial death. The assay can also be adapted to detect drug resistance genes, Hu said.

Hu and his team had previously developed a mass spec test for paucibacillary TB that has since been licensed and is being commercialized by Nanopin Tech, he said.

Going forward, Hu and his team are in conversations with a startup industry partner as well as an investor to commercialize the CRISPR-based test as well, he said, and hopes to make an announcement in a few weeks.

Regarding the two tests, Hu said that nucleic acids and antigens represent different pathological processes, and thus the tests could potentially be used for different purposes in clinical diagnosis. 

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