A research team headed by scientists at the Francis Crick Institute in London, U.K., has developed and validated a 20-gene signature that can discriminate active tuberculosis (TB) from latent tuberculosis infection (LTBI) and can also predict if an asymptomatic, infected individual will go on to develop active TB. The research, including a small, prospective study involving 53 TB patients in the U.K. and their close contacts, could help develop strategies for diagnosing and treating TB-infected individuals before they develop symptoms, and before they pass the infection on to others.
“Treating active TB before symptoms start could spare patients and their families from unpleasant symptoms, reduce the spread of disease, and offer peace of mind to people who are not going to develop active TB,” explains Anne O'Garra, Ph.D., group leader at the Francis Crick Institute, and senior author of the team’s published paper in Nature Communications. “This study was a promising proof of principle, offering new insights into how to develop gene signatures for active TB. The next step will be to develop and test different gene signatures in larger groups of people, with the aim of being able to offer validated tests to patients within the next decade.”
The Francis Crick researchers, working in collaboration with scientists at the University of Leicester, U.K., BIOASTER and bioMerieux in France, and the University of Cape Town in South Africa, describe their findings in a paper entitled “A Modular Transcriptional Signature Identifies Phenotypic Heterogeneity of Human Tuberculosis Infection.”
TB is the world’s leading cause of mortality from an infectious disease, with estimates for 2016 indicating that there were 10.4 million incident and 6.3 million new cases of TB disease, and 1.67 million deaths, the researchers report. In fact, it is estimated that about a third of the world’s population has been infected with Mycobacterium tuberculosis, with the vast majority remaining asymptomatic and demonstrating no microbiological evidence for active infection. However, individuals with such LTBI are thought to represent the primary reservoir for future active TB, “with 90% of all TB cases estimated to arise from reactivation of existing infection.”
Diagnosis of active pulmonary TB requires culture of the causative bacterium, M. tuberculosis, which can take up to six weeks. And while a World Health Organization–endorsed molecular test can speed diagnosis, the assay still requires a sputum sample, which can be difficult to obtain, the authors note. “Difficulties in obtaining sputum lead to ~30% of patients in the USA and 50% of South African patients to be treated empirically.”
Demonstrating that an individual has harbored a LTBI is based on evidence of immune sensitization, using the tuberculin skin test (TST) or the M. tuberculosis antigen-specific interferon-γ (IFN-γ) release assay (IGRA), but neither test can distinguish active from latent infection. The Francis Crick team had previously identified a signature of 393 gene transcripts in whole blood that could discriminate patients with active TB from healthy individuals, patients with other chronic infections, and most patients with LTBI. A number of other studies have also reported blood signatures that discriminate active TB, they note, and while research groups around the work have tried to narrow the signature down to fewer genes, “there is a relative lack of concordance across studies that have reported a reduced and optimised diagnostic gene signature, although an agreement exists for some of the pathways they represent.”
The Francis Crick Institute–led researchers now report on RNA-sequencing studies that have validated prior microarray findings in previously published cohorts and in a new Leicester cohort of 53 TB patients. The original 393-transcript signature was characterized by overabundance of IFN-inducible genes and underabundance of B- and T-cell genes. The researchers also describe their collaboration with BIOASTER to develop a honed down panel of just 20 genes that act as a blood signature for distinguishing between active TB and LTBI, and which, they claim, unlike gene signatures developed by external groups, excludes potentially confounding active viral or bacterial infections. “…this signature did not detect influenza, representative of many viral infections that share a strong IFN-inducible signature, providing a proof of principle for the development of transcriptional biomarkers for TB as diagnostics, with the aim of obtaining the highest sensitivity, whilst maintaining specificity against LTBI and other diseases,” the authors write.
The 20-gene signature was then used to test samples from contacts of the Leicester TB patients recruited by co-author Pranab Haldar, M.D., and a team at the National Institute for Health Research (NIHR) Leicester Biomedical Research Centre. The results showed that while the gene signature wasn’t found in contacts who remained healthy, it was identified in six of the nine individuals who went on to develop active TB.
“Being able to track TB patients' contacts and take monthly blood samples gave us a unique insight into how immune responses develop,” explains Dr. Haldar, who is senior clinical lecturer in respiratory medicine at the University of Leicester. “There was a wide variation in the immune response between people, highlighting the limitations of taking a 'one size fits all' approach. The next step will be to better understand what the different responses mean, and we will need to study larger groups in detail to achieve this. The ability of our signature to distinguish from viral infections is an important step forward for developing reliable tests that we can use in the clinic.”
The researchers acknowledge that use of the 20-gene signature to predict whether asymptomatic contacts of TB patients will go to develop active disease will need validating in much larger cohorts. “In the contacts we saw, the new 20-gene signature performed better than previous signatures but there is still work to be done,” Dr. Halder notes. “We only saw a small number of contacts who developed TB in the group, so we will need to look at much larger groups to refine and develop the techniques.”