Evaluation of a Novel Microbiological Diagnostic Test for Latent Mycobacterium Tuberculosis Infection

Objectives:

i) To determine whether M. tuberculosis (Mtb) DNA can be detected in CD34+ peripheral blood mononuclear cells (PBMC) isolated from

* asymptomatic adults being screened for LTBI (LTBI screenees) * adults with newly-diagnosed active tuberculosis (active cases) ii) To determine whether strains of Mtb isolated from CD34+ PBMC of recent household TB contacts are genetically identical to those isolated from the sputum of index cases with pulmonary TB to whom they have been exposed iii) To characterise immunological, clinical and epidemiological correlates of ability to detect Mtb in CD34+ PBMC in a heterogeneous population of IGRA-positive and -negative LTBI screenees.

iv) To determine whether presence of Mtb DNA in CD34+ PBMC of IGRA-positive LTBI screenees associates with the presence of a peripheral blood transcriptional signature recently reported to predict risk of progression to active TB v) To assess longitudinal change in CD34+ PCR assay results in

\- LTBI screenees completing chemoprophylaxis for LTBI

* LTBI screenees not receiving chemoprophylaxis * Patients completing treatment for active TB

4.1. Background

Tuberculosis (TB) is an infectious disease caused by organisms of the M. tuberculosis complex (MTBC). The primary pathogen in humans is M.tuberculosis (Mtb). TB commonly affects the lungs and is transmitted through inhalation of infectious droplets expectorated by a patient with active pulmonary TB. Tuberculosis has been a major cause of morbidity and mortality throughout human history. In Europe and North America, a surge in TB cases in the 17th and 18th century was followed by a rapid decline primarily attributed to improved socioeconomic conditions, application of public health measures and isolation of TB patients.1 Towards the end of the 19th century, after the downward trend of TB cases and deaths in Europe and North America, two major events, the discovery of Mtb and X-ray, became key in the diagnosis and management of TB.1 Subsequently, vaccination with M. bovis bacille Calmette-Guérin (BCG) and antibiotic treatment in the first half of the 20th century led to global optimism that TB control and elimination was within reach. However, TB remains a serious public health threat to this day and it is the second leading infectious cause of death globally. 2, 3 According to the 2015 WHO report, an estimated 9.6 million new TB cases (12% of which were HIV co-infected) and 1.5 million deaths (of which 0.4 million were among HIV co-infected people) occurred globally in 2014.4 In addition, one third of the global population is estimated to have latent Mtb infection (LTBI), of whom around 10% will develop active TB in their lifetime.5

Although only 22 low or middle income countries (LMICs) account for 80% of the global burden, TB is still an important disease among immigrant populations in high income countries. In the United Kingdom (UK), reductions in TB incidence achieved in the 20th century have reversed with a 50% increase in TB notifications between 1998 and 2009.6 In 2013, a total of 7,892 TB cases equivalent to an incidence of 12.3 per 100,000 population per year, the highest among high income countries, were reported.7 A high proportion (37.8%) of TB cases in the UK is found in London, where the TB incidence rate is 35.5 per 100,000 population per year; immigrants from India (30%), Pakistan (20%) and Somalia (5%) accounted for the majority of these cases.7

Currently, the key strategies in TB control are accurate and rapid diagnosis of active disease together with effective treatment to interrupt transmission. However, delayed diagnosis is a major challenge facilitating transmission in communities.8 Generally, it takes several months from the onset of symptoms until a TB patient starts treatment. For example, in the UK, nearly a third of pulmonary TB patients start treatment 4 months after onset of symptoms.7 Such long delays, coupled with the large pool of LTBI suggests the need for interventions to identify and treat individuals with LTBI to achieve TB control. Antimicrobial treatment of LTBI (chemoprophylaxis) is effective in reducing risk of reactivation and is routinely implemented in high income, low incidence settings. However, implementation of this policy, particularly in high burden settings, is hampered by the lack of a diagnostic test to identify the sub-set of Mtb-sensitised individuals who are at risk of developing active TB, at whom chemoprophylaxis should be targeted.

Currently, the diagnosis of LTBI is made by detecting an adaptive immune response to Mtb infection, either in vivo (using the tuberculin skin test) or ex vivo (using Interferon-Gamma Release Assays \[IGRAs\]). This approach, which does not detect Mtb directly, is limited in that it cannot distinguish the 10% of sensitised individuals who are at risk of reactivation (in whom chemoprophylaxis may be of benefit) from the 90% who are immunologically sensitised but who do not progress to active disease (in whom chemoprophylaxis confers no benefit). This latter group is potentially heterogeneous: some may have eliminated the infection, while others may have LTBI but be able to contain it indefinitely.9 Tools to dissect out these possible phenotypes could allow more precise targeting of chemoprophylaxis, but they are currently lacking. In this study, the investigators will investigate whether Mtb can be detected in CD34+ cells isolated from peripheral blood of a) adults being screened for LTBI (LTBI screenees), and b) adults with newly-diagnosed active TB (active cases).

4.2. Rationale for the study

This study builds on a recent observation by one of the co-investigators (Stephen Reece) who was part of a group at the Max Planck Institute for Infection Biology, Berlin, who have detected Mtb DNA in CD34+ long-term repopulating pluripotent hematopoietic stem cells (LTpHSCs) isolated from 100 ml of peripheral blood of seven healthy adults with LTBI (positive IGRA, active TB excluded); Mtb DNA was not detected in LTpHSCs of seven IGRA-negative healthy adults. 10 If this remarkable finding can be replicated in another setting, it could lead to the development of a novel microbiological test ('the CD34+ PCR assay') with several potential applications:

a) to identify individuals who are latently infected with antibiotic-resistant Mtb (allowing selection of an appropriate chemoprophylaxis regimen), b) to monitor efficacy of chemoprophylaxis and interventions designed to prevent acquisition of LTBI, such as vaccines and programmes of micronutrient supplementation, c) to track spreading of particular strains through a population (providing a tool for public health intervention), d) to discriminate between Mtb-sensitised individuals who are at risk of progression to active disease vs. those who are not, allowing chemoprophylaxis to be targeted at populations who may benefit from it, and e) to provide new insights into the immunobiology of LTBI and the pathogenesis of human tuberculosis.

4.3. Hypotheses

i) Mtb DNA can be detected in CD34+ PBMC of asymptomatic IGRA-positive adults and active cases of TB at diagnosis, but not in IGRA-negative adults.

ii) Strains of Mtb isolated from CD34+ PBMC of asymptomatic IGRA-positive TB contacts will be genetically identical to those isolated from the sputum of index cases of pulmonary TB to whom they have been recently exposed.

iii) Ability to detect Mtb DNA in CD34+ PBMC of LTBI screenees associates with IGRA results as well as recognised clinical and epidemiological risk factors for LTBI iv) Ability to detect Mtb DNA in CD34+ PBMC of IGRA-positive LTBI screenees associates with peripheral blood transcriptomic signatures previously defined to be associated with progression to active TB v) Antimicrobial therapy is effective in eliminating Mtb from CD34+ PBMC isolated from individuals with LTBI or active TB in whom it was detectable at baseline; and that results of the CD34+ PCR assay are reproducible on repetition in LTBI screenees who have not received chemoprophylaxis

5\. Objectives

5.1. Primary Objective

To determine whether M. tuberculosis (Mtb) DNA can be detected in CD34+ peripheral blood mononuclear cells (PBMC) isolated from

* asymptomatic adults undergoing routine screening for LTBI (LTBI screenees) * adults with newly-diagnosed active tuberculosis (active cases)

5.2. Secondary Objectives

* To determine whether strains of Mtb isolated from CD34+ PBMC of TB contacts are genetically identical to those isolated from the sputum of index cases with pulmonary TB to whom they have been exposed * To characterise immunological, clinical and epidemiological correlates of ability to detect Mtb in CD34+ PBMC in a heterogeneous population of IGRA-positive and -negative LTBI screenees. * To determine whether presence of Mtb DNA in CD34+ PBMC of IGRA-positive screenees associates with the presence of a peripheral blood transcriptional signature recently reported to predict risk of progression to active TB11 * To assess longitudinal change in CD34+ PCR assay results in * LTBI screenees completing chemoprophylaxis for LTBI * LTBI screenees not receiving chemoprophylaxis * patients completing treatment for active TB

Study Design and Study Visits

The investigators will conduct a longitudinal study. Two groups of participants will be recruited: adults being screened for LTBI (LTBI screenees), and patients about to initiate treatment for active TB (active cases). LTBI screenees are those who are being screened for LTBI as part of standard care: they include recent contacts of an infectious index TB case, new entrants from high TB burden countries, health workers undergoing occupational health screening and patients who are about to undergo immunosuppressive therapy. Patients with active TB are those who have been diagnosed with acitve TB who are about to start taking anti-TB therapy.

At baseline (Visit 1), participants who fulfil inclusion criteria and give written informed consent to participate in the study will be asked to provide sociodemographic information including details of age, sex and ethnic origin as well as details of TB exposure history (including specific exposure to participants with active TB enrolled in the study) and other clinically relevant data, all of which will be recorded on a Case Report Form (CRF). An HIV serology test will be performed if this is not already being done as part of routine care. A 120 ml (117 ml if HIV test is already done as part of routine care) blood sample will be collected for IGRA (QFT-Plus), blood culture for Mtb, peripheral blood transcriptome risk signature analysis and the CD34+ PCR assay for Mtb DNA at baseline. IGRA-positive participants in whom active TB has been excluded using clinical evaluation and chest X-ray (part of standard care) will be offered a 3-month course of rifampicin and isoniazid chemoprophylaxis if they fulfil criteria for this therapy (also part of standard care).12

Communication of positive HIV test results will be handled according to the recommendations of the UK National Guidelines for HIV Testing 2008. Positive HIV test results will be communicated in a face-to-face interview with a study doctor, in the presence of a nurse from the Barts Health NHS Trust Genito-Urinary Medicine (GUM) clinic (the local HIV service). Participants who test HIV-positive will be referred to their nearest GUM clinic for confirmation of their results and further management as appropriate.

At 3 months (Visit 2), details of prescription of and adherence to chemoprophylaxis will be recorded, and a second blood sample (117 ml) will be taken for repeat IGRA (QFT-Plus), blood culture and CD34+ PCR assay. For participants who took chemoprophylaxis, a Tempus tube will also be taken at Visit 2.

At baseline (Visit 1), participants who fulfil inclusion criteria and give written informed consent to participate in the study will be asked to provide basic sociodemographic information inclu