When the COVID-19 pandemic was at its peak, and multiple variants were threatening lives around the world, scientists relied on a process called ‘tiled amplicon sequencing’ to track the virus’s spread.
Now, an international team of scientists led by researchers at the Yale School of Public Health (YSPH) is applying those same techniques to more rapidly sequence tuberculosis.
In a new study appearing in the Journal of Clinical Microbiology, YSPH researchers report that they successfully adapted the tiled amplicon sequencing method to accurately read the genome of Mycobacterium tuberculosis — the bacteria that causes tuberculosis — without needing to culture the bacteria first. This procedure is attractive to scientists and clinicians because it reduces the time it takes to scan tuberculosis samples from weeks to days and lowers testing costs from hundreds of dollars per sample to less than $20.
Tiled amplicon sequencing was first developed to read the genomes of viruses like Zika that were found in very low concentrations in patient samples. Under this approach, scientists prepare small pieces of DNA that can copy specific parts of a genome — amplicons. Like linking pieces in a puzzle, with enough overlapping amplicons, researchers can create a complete picture of the genome.
This approach proved especially useful during the COVID-19 pandemic, when researchers and public health officials were able to quickly and reliably sequence virus samples to track emerging variants and speed up the development of vaccines to fight them.
“Our goal was to piggyback on the success of this method for viruses by applying it to bacteria with the hope that TB sequencing could become similarly accessible,” said Chaney Kalinich, co-lead author of the study and a current emergency medicine resident at Mass General Brigham. However, a bacterial pathogen like M. tuberculosis has a genome more than a hundred times the size of SARS-CoV2, the virus that causes COVID-19.
“Our protocol uses 5,128 primers,” Kalinich said. “It took me two days of pipetting them by hand just to make the primer pools.”
M. tuberculosis is often found at low concentrations and is notoriously slow to grow. Researchers typically analyze TB samples by collecting patient sputum and then incubating the sample in the lab for up to six weeks until there are enough bacteria to sequence. Although this approach can identify drug-resistant TB, the long delays between sample collection and genome analysis make it less likely that clinicians can use the genetic data to guide patient treatment.
Using samples from tuberculosis patients in Moldova and Peru, the Yale team demonstrated that the tiled amplicon sequencing method could read an entire genome of a TB sample in a matter of days, even samples that were low concentration, or contained other bacteria. This ability to sequence the whole genome of a TB sample accurately and reliably has the potential to be a transformative improvement in tuberculosis surveillance and care, the researchers said.
“It could improve clinical and public health outcomes while enhancing surveillance and control efforts by providing valuable insights into TB epidemiology and resistance patterns,” said Dr. Ted Cohen, MD, DPH, MPH, a professor of epidemiology (microbial diseases) at YSPH who worked on the study.
Tuberculosis is the world’s leading cause of death from a single infectious agent, resulting in more than a million deaths a year. Worldwide cases have been increasing over the past two years. The researchers believe that the availability of a cheap, reliable sequencing method could significantly improve tuberculosis surveillance in low- and middle-income countries, where the disease burden is highest. India, Bangladesh, Indonesia, and the Democratic Republic of the Congo account for more than half of all global tuberculosis deaths — and all four of these countries have already used tiled amplicon sequencing during the COVID-19 pandemic.
Dr. Seth Redmond, BSc, MSc, PhD, an associate research scientist at YSPH and the study’s senior author, stressed that more research is needed to see how well the study’s results translate to real-world settings.
“We can sequence brilliantly in New Haven. We can sequence brilliantly in London. We can sequence in the places where we have all the resources, but the drug-resistant TB is in other places,” he said. “It’s in South Africa. It’s in Moldova. It’s in India. These are the places where we need to make sure this works.”
The researchers are currently working with colleagues in the Republic of Moldova to implement the approach in real-world settings. They are also seeking funding to further optimize the protocol, formally compare it to culture-based whole genome sequencing, and assess its costs and potential health impacts relative to the current standard of care.
Scientists from University College London, Universidad Peruana Heredia in Peru, and the Chiril Draganiuc Institute of Pneumology in Moldova contributed to the study.