Beyond the dichotomy of latent and active tuberculosis – defining health and disease in Mycobacterium tuberculosis infection

Principle Investigator

Scientific interest within the context of the graduate college:

Health and disease is not well defined for tuberculosis, one of the most important infectious diseases. Here, we propose to build upon an existing cohort study of tuberculosis contacts in Berlin to define a recently recognized new form of TB, subclinical TB, using a novel phage-based assay. People with evidence of subclinical TB will be high-priority contacts for prophylactic anti-TB treatment to prevent progression to active TB.

Project description:

Introduction: Tuberculosis (TB) and its etiologic agent, human-adapted Mycobacterium tuberculosis (Mtb) complex, is the leading cause of death from a single infectious agent and the 13th total leading cause of death worldwide. Overall, an estimated 1.8 billion people, 20-25% of the world’s population, is infected with Mtb, yet the majority of infected individuals do not fall ill to TB. Conventionally, TB is classified into active disease vs. latent TB infection. Active disease is defined by clinical symptoms (fever, cough, weight loss), radiological signs (cavities), and microbiological proof of disease (PCR or growth in culture). Latent TB (LTBI) is defined as immunoreactivity to specific TB antigens, most commonly through Interferon Gamma Release Assays (Quantiferon Gold In Tube) or more traditionally, by tuberculin skin testing in the absence of the aforementioned signs. In recent years, there has been growing recognition that the binary classification of TB into “latent” and “active” does not accurately reflect the complex pathophysiology of Mtb infection and the processes that lead from health to disease. The binary classification may be inadequate for informing research and programmatic advances for global TB elimination.¹ In contrast, subclinical TB has been used to refer to a disease state where individuals are asymptomatic, but infectious (Figure 1).² Studies estimate that up to 68% of global TB transmissions are due to subclinical TB.³ Subclinical TB thus represents a key stage of TB that sits between health and disease and that is challenging to diagnose in the absence of clinical symptoms. Better methods are thus needed to detect individuals with subclinical TB.

The Actiphage method uses a bacteriophage that is specific for the Mycobacterium genus. The phage is used to efficiently lyse bacterial cells present in a sample to release mycobacterial DNA making it accessible for detection by PCR, using primers mycobacterial species or groups-specific primers (i.e. IS6110 insertion sequences). Importantly, the bacteriophage method only allows for the detection of viable mycobacteria, as viable bacteria are required for the phage to replicate and then to lyse the host cell at the end of its replication cycle. Therefore this combination of phage and PCR provides enhanced sensitivity and specificity and live/dead differentiation due to the efficacy of the bacteriophage host-lysis process.

To perform the assay, peripheral blood mononuclear cells (PBMCs) are isolated from 2 ml of heparinized blood. The PBMCs are lysed by osmotic shock to release any intracellular mycobacteria. The bacteriophages are added to the sample and incubated for 3.5 hours to allow the phage to complete their replication cycle and lyse the mycobacterial cells. Any remaining intact bacterial cells are separated from free genomic DNA released by the phage by filtration. The DNA is then further cleaned and concentrated before any mycobacterial genomic DNA present in the sample is detected by specific PCR.

Figure 1. Disease strata in tuberculosis.⁴

For TB, the method has not been formally validated against other direct detection methods, because there are none that are as sensitive as the Actiphage test (levels of Mycobacterium tuberculosis in blood are normally too low to detect).  In cattle, measured against the comparative skin test [SICCT], the sensitivity of the assay was shown to be 98% with 100% specificity (n = 41 SICCT-positive animals; n = 45, SICCT-negative animals).⁵ In humans, the Actiphage blood testing had a sensitivity of 73.3% (95% confidence interval [CI] 48.1-89.1%) and a specificity of 100% (95% CI 56.6-100%).⁶

The overall objective of this project is to conduct a prospective clinical cohort study of contacts of TB patients and classify these individuals into TB infection (i.e. a positive IGRA), subclinical TB (i.e. a positive Actiphage test) and no clinical symptoms, or clinical TB (symptomatic). The study builds upon existing collaborations (Prof Cath Rees, University of Nottingham, Actipahge assay) and the Berlin TB contact cohort study with the Gesundheitsamt Berlin (PI Matthias Gröschel, recruiting contacts since Q1/2024). In this study, all contacts who provide written informed consent are included and undergo blood testing as part of their routine visits to the Gesundheitsamt.

Aim 1: Establish the Actiphage test in our labs at the Department of Infectious Disease at Charité – Universitätsmedizin Berlin. This will include a 2-week visit of the doctoral candidate to the Cath Rees lab in Nottingham to get acquainted with the method.

Aim 2: Determine the proportion of subclinical TB among exposed contacts in Berlin using the Actiphage test. We hypothesize that the test will detect subclinical TB in ~20% of exposed contacts and will help to further identify contacts who require preventative treatment. We also hypothesize that these contacts are at a higher risk of progressing to active TB. TB contacts will provide an additional blood tube for PBMC isolation. Contacts for this analysis will be recruited during three months (n = ~500 contacts) and will be contacted every three months for 1 year (80-90% of exposed contacts will develop active TB within the first year to ask for symptoms suspicious of TB).⁷ The aim will be to describe what proportion of contacts is IGRA positive (test is routinely performed already), Actiphage positive, and presents with symptoms of active TB.

The study will be further embedded into a larger study of sympatry, i.e. the preferential interaction of Mtb strains and their hosts. The role of subclinical infection is unknown at this stage. The prospective students will obtain critical skills in infection epidemiology by active engagement in the Berlin TB household study. The students will further learn basic lab skills, including preparation of clinical samples, isolation of PBMC and performing of Actiphage assays, along with PCR. Finally, prospective students will become acquainted with data analysis tools and statistics.

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