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Consortium Tests Nanopore Sequencing for Tuberculosis Dx in Madagascar

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SAN FRANCISCO (GenomeWeb) – A team of researchers is testing Oxford Nanopore Technologies' MinIon device for diagnosing tuberculosis in Madagascar.

The collaboration, which includes researchers from the Institut Pasteur Madagascar, the University of Oxford, the European Bioinformatics Institute, and Stony Brook University, aims to develop a protocol for diagnosing Mycobacterium tuberculosis and drug resistance profiling in rural areas of Madagascar with limited laboratory facilities and equipment.

The University of Oxford's Modernising Medical Microbiology laboratory is spearheading the technology development. In 2015, the group developed a diagnostic tuberculosis test, which Public Health England now uses for all cases of suspected tuberculosis in the UK. That test is based on microbial whole-genome sequencing and runs on an Illumina sequencer.

"We've shown with Public Health England that it's possible to use sequencing in this way, and we routinely use the test," said Sarah Hoosdally, a project manager in the lab. "The challenge is whether this technology can be translated to a place with a high burden of tuberculosis, such as Madagascar, where the infrastructure and lab technology is very different."

Tuberculosis is one of the top 10 causes of death worldwide and the main cause of death related to antimicrobial resistance. Only around 54 percent of drug-resistant tuberculosis cases are successfully treated, according to the World Health Organization. In Madagascar, tuberculosis has a higher-than-average incidence rate, at 237 per 100,000 individuals, versus 140 per 100,000 globally. In 2016, there were around 59,000 cases and 13,000 deaths due to tuberculosis in Madagascar.

Appropriate diagnosis, which includes drug resistance profiling, is critical for reducing the spread of the disease and getting patients the appropriate treatment, but can be challenging in rural environments with limited resources and infrastructure.

Currently, testing consists of first culturing the sample and then testing its growth in the presence of a variety of antibiotics. The entire process can take nearly a month since tuberculosis grows slowly. "By the time a patient is diagnosed, treatment has been delayed and the disease could have spread," Hoosdally said. For people who live in rural locations, getting to and from a healthcare clinic can be challenging, so ideally, there would be a way to do the testing, diagnosis, and antibiotic resistance profiling the same day — the ultimate goal of the project.

The team turned to the MinIon because it is portable and does not need lots of lab equipment to run. Hoosdally said that the first step was to run the sequencer in Madagascar's national reference laboratory. After that, they moved out to a local, rural laboratory. A key component that is still being developed is the ability to test directly from patient samples, rather than having to culture the sample first.

Currently, the NGS-based tuberculosis diagnostic run in the UK relies on a shortened culturing step to ensure there is enough bacterial DNA to sequence. The culture step is significantly shorter, a few days to a week, than culture-based diagnostics, which takes at least three weeks, but Hoosdally said that the goal is to be able to extract DNA directly from patient sputum and sequence it, which would enable a same-day diagnosis.

The team has tested the method for extracting DNA directly from patient sputum samples and "while it still needs a lot of development, it is possible to sequence DNA [with it]," she said.

The main hurdle is the high level of background human DNA and DNA from other bacteria that are in the sample. "When you extract DNA, you extract DNA from everything that's in the sample," Hoosdally said, as opposed to enriching for bacterial DNA by culturing the sample in the lab.

Simon Grandjean Lapierre, a postdoctoral research fellow at Stony Brook University, is based in Madagascar and heads the program there. He said that the team is coordinating with Madagascar's Ministry of Public Health and testing the protocol in the field on patient samples. The test is accurate enough to give a yes/no result for whether a patient has TB, but is not yet accurate enough for drug resistance profiling. Thus, in parallel, the researchers are also sending samples to the reference lab for a more comprehensive drug resistance profiling workup and to better understand phylogeny for public health surveillance.

Understanding the phylogeny is critical for understanding transmission and how to best deploy preventative resources, Lapierre said. Tuberculosis is airborne, and once someone contracts it, they can experience symptoms right away, or the disease can sit latent for a few months.

The goal is to have a nanopore sequencing-based diagnostic that can be run in the field, diagnose TB, do drug resistance profiling, and be used for epidemiological purposes, Lapierre said. Continued methods development, as well as anticipated improvements to Oxford Nanopore's software and chemistry that increase its accuracy, will be key to making that possible, he added.

Another component of the diagnostic strategy involves using drones to transport both patient samples and medications between patients and the laboratories or clinics.

Of the approximately 25 million people who live in Madagascar, around 70 percent live in remote rural areas, Lapierre said. "We can use drones to move samples directly from the community to the lab, and make sure that medicines go back, leapfrogging over the impediments of needing to walk for a few days to get to a clinic," he said.

Aside from developing strategies to improve diagnostics, the team is also conducting research, both to establish performance metrics for the nanopore-based diagnostic test and also to better understand drug resistance and TB epidemiology in Madagascar. For instance, the researchers are conducting a retrospective trial, analyzing a collection of multidrug-resistant TB samples from patients that have been diagnosed in Madagascar along with a set of controls, Lapierre said. The team is sequencing those samples on both the MinIon and other sequencing instruments to better understand the MinIon's performance. In addition, he said, the group is doing epidemiological research on previously collected samples from all over the country to "assess questions that are more fundamental science questions," such as how TB genomics can shed light on population migration and resistance emergence, particularly in low-incidence settings.

The work in Madagascar adds to a number of efforts worldwide seeking to develop better and faster methods for diagnosing TB. The University of Oxford, for instance, is also spearheading another consortium — Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) — which aims to sequence the genomes of 60,000 TB isolates and test them for susceptibility to 14 drugs. Another group, the Sharing Mycobacterial Analytic Capacity project, which involves the University of British Columbia and PHE, involves developing informatics and better end-user reports of genomic information.

These efforts are all necessary to meet the WHO's goal of ending the TB epidemic, specifically, reducing new TB cases by 80 percent and TB-caused deaths by 90 percent by 2030. According to the WHO, there was a 22 percent drop in TB deaths between 2000 and 2016, however, multidrug resistance continues to be particularly problematic, with only one in five people with MDR-TB receiving appropriate treatment and a $2.3 billion funding shortfall to implement TB strategies.

Lapierre said new technologies, such as nanopore-based diagnostics and the use of drones for transporting samples and medicines, can play an important role in reaching the remote areas where there is a lot of unmet need, while also helping to reduce costs. "The objective is that through this research, within a few years, doing DNA sequencing is going to be cost effective and more widely available," he said.