Elsevier

The Lancet

Volume 357, Issue 9273, 23 June 2001, Pages 2017-2021
The Lancet

Articles
Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells

https://doi.org/10.1016/S0140-6736(00)05115-1Get rights and content

Summary

Background

Identification of individuals latently infected with Mycobacterium tuberculosis is an important part of tuberculosis control. The current method, the tuberculin skin test (TST), has poor specificity because of the antigenic cross-reactivity of purified protein derivative (PPD) with M bovis BCG vaccine and environmental mycobacteria. ESAT-6 is a secreted antigen that is highly specific for M tuberculosis complex, but is absent from M bovis BCG. With an enzyme-linked immunospot (ELISPOT) assay for interferon gamma, we have identified ESAT-6-specific T cells as an accurate marker of M tuberculosis infection.

Methods

We did a prospective, masked study of 50 healthy contacts, with varying but well defined degrees of exposure to M tuberculosis, who attended an urban contact-tracing clinic. We assessed and compared the efficacy of our assay and TST for detection of symptomless infected individuals by correlation of test results with the degree of exposure to an infectious index case.

Findings

The ESAT-6 ELISPOT assay results had a strong positive relation with increasing intensity of exposure (odds ratio=9·0 per unit increase in level of exposure [95% CI 2·6–31·6], p=0·001), whereas TST results had a weaker relation with exposure (1-9 [1·0–3·5], p=0·05). By contrast, ELISPOT results were not correlated with BCG vaccination status (p=0·7), whereas TST results were significantly more likely to be positive in BCG-vaccinated contacts (12·1 [1·3–115·7], p=0·03).

Interpretation

This new antigen-specific T cell-based assay could allow more accurate identification of symptom-free individuals recently exposed to M tuberculosis, and thereby help to improve tuberculosis control.

Introduction

Control of the global tuberculosis epidemic could be enhanced by identification and treatment of symptom-free people who are latently infected with Mycobacterium tuberculosis, as well as those with active disease. Even in countries with a low prevalence of tuberculosis, 30–40% of new cases are probably caused by recent transmission of M tuberculosis from infectious cases.1 Immunocompetent individuals with M tuberculosis infection have a 10% risk of developing active disease in their lifetime; half this risk is in the first 1-2 years after exposure.2 Chemoprophylaxis of recently infected individuals prevents the development of active tuberculosis,3 and isoniazid preventive therapy is highly cost effective. It is therefore important to identify recently infected contacts and infected individuals at greatest risk of progression—eg, intravenous drug users, HIV-1 infected individuals, and children.4, 5

The tuberculin skin test (TST) for diagnosis of latent M tuberculosis infection has remained almost unchanged for a century. Intradermal inoculation of purified protein derivative (PPD), which is a crude precipitate of more than 200 M tuberculosis antigens that are common to M bovis BCG and environmental mycobacteria, elicits a local cutaneous delayed-type-hypersensitivity response in sensitised individuals. The broad antigenic cross-reactivity of PPD causes the poor specificity of TST; a positive reaction can be a response not only to M tuberculosis infection, but also to BCG vaccination or exposure to environmental mycobacteria.6, 7, 8 Because a third of the world's population is thought to be infected with M tuberculosis,9 and most people have been vaccinated with BCG, accurate identification of those infected with M tuberculosis for targeted chemoprophylaxis is difficult.5, 6, 8

M tuberculosis infection evokes a strong cell-mediated immune response, and detection of T cells that are specific to this bacterium might be a means to detect infection. We therefore selected ESAT-6, a secreted antigen that is expressed in M tuberculosis complex10 (M tuberculosis, M bovis, and M africanum), but is absent from all strains of M bovis BCG vaccine11, 12 and most environmental mycobacteria.13 ESAT-6 is highly immunogenic in animal models of tuberculosis,14, 15 tuberculosis patients,16, 17, 18, 19 and people exposed to tuberculosis bacteria (contacts).20, 21 Cellular immune responses to ESAT-6 have been detected by standard in-vitro assays in 60-80% of tuberculosis patients.22 We used a highly sensitive assay to detect ESAT-6-specific T cells, the ex-vivo ELISPOT assay for interferon gamma.23 The ELISPOT assay is based on the principle of a sandwich-capture ELISA. The assay detects interferon-gamma molecules in the immediate vicinity of the T cell from which they were secreted, while still at a high concentration. Thus, each spot represents the footprint of an antigen-specific T cell that secretes interferon gamma (spot-forming cell). ELISPOT assay can detect antigen-specific T cells from blood without an in-vitro stimulation step,23 hence incubation periods are short. We reported that ESAT-6-specific T cells were an accurate marker of M tuberculosis infection in 45 (96%) of 47 patients with culture-confirmed active disease.21 We also noted that 22 (85%) of 26 TST-positive household contacts of patients with sputum smear-positive pulmonary tuberculosis had circulating ESAT-6-specific T cells.21 These results suggested a new approach for the detection of recent tuberculosis infection.24

Our aim was to assess whether this approach could be used to identify individuals in routine clinical practice, who were at high risk of recent infection and to compare the results with those for TST. Unfortunately, no test can reliably confirm latent M tuberculosis infection in symptom-free individuals—ie, there is no gold standard for comparison—hence measurement of accuracy of a new assay is difficult. However, airborne transmission of M tuberculosis is promoted by close and prolonged contact with an infectious person,25, 26, 27 and a key factor is the amount of time a contact spends sharing room air with an infectious individual.28 We postulated that the ESAT-6 based ex-vivo ELISPOT assay would correlate better with intensity of exposure to M tuberculosis than TST, but would be independent of BCG-vaccination status. Therefore, we investigated people attending a contact-tracing clinic who had well defined but differing amounts of exposure to M tuberculosis.

Section snippets

Participants

We prospectively recruited healthy adult contacts from the Contact Tracing Clinic, Northwick Park Hospital, North West London Hospitals NHS Trust, London, UK. This clinic is held once a week and is attended by individuals thought to have been in contact with a newly identified case of tuberculosis. There were no exclusion criteria. We consecutively recruited 50 contacts of index cases who had sputum-smear-positive pulmonary tuberculosis. We identify cases in this report by number (HC 1–HC 54).

Results

20 participants were contacts of an index case (IC). Figure 1 shows the proximity of these contacts to IC, and results of ESAT-6 ELISPOT and TST. IC shared a house with two cohabitants, including a work colleague, and worked consecutively in two separate offices within a large university building. He had a productive cough throughout the last 3 months that he worked in office A (figure 1) and during the next 2 months when he worked in office B (figure 1), before his admission to hospital.

The

Discussion

We have shown that the ESAT-6 ELISPOT assay identifies individuals at high risk of M tuberculosis infection more accurately than the TST. The strong positive relation between ESAT-6 ELISPOT results and M tuberculosis exposure, and the lack of relation with BCG vaccination status, allow symptomless M tuberculosis infection to be distinguished from BCG vaccination, thereby avoiding unnecessary chemoprophylaxis in uninfected individuals.

Although the numbers of individuals in some exposure

References (30)

  • P Andersen et al.

    Specific immune-based diagnosis of tuberculosis

    Lancet

    (2000)
  • PM Small et al.

    The epidemiology of tuberculosis in San Francisco: a population-based study using conventional and molecular methods

    N Engl J Med

    (1994)
  • GW Comstock

    Frost revisited: the modern epidemiology of tuberculosis

    Am J Epidemiol

    (1975)
  • International Union Against Tuberculosis Committee on Prophylaxis

    Efficacy of various durations of isoniazid preventive therapy for tuberculosis: five years of follow-up in the IUAT trial

    Bull World Health Organ

    (1982)
  • American Thoracic Society

    Targeted tuberculin testing and treatment of latent tuberculosis infection

    MMWR Morb Mortal Wkly Rep

    (2000)
  • British Thoracic Society

    Control and prevention of tuberculosis in the United Kingdom: code of practice 2000

    Thorax

    (2000)
  • PE Fine et al.

    Tuberculin sensitivity: conversions and reversions in a rural African population

    Int J Tuberc Lung Dis

    (1999)
  • DO Kwamanga et al.

    Effect of non-tuberculous Mycobacteria infection on tuberculin results among primary school children in Kenya

    East Afr Med J

    (1995)
  • RE Huebner et al.

    The tuberculin skin test

    Clin Infect Dis

    (1993)
  • C Dye et al.

    Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country: consensus statement. WHO global surveillance and monitoring project

    JAMA

    (1999)
  • AL Sorensen et al.

    Purification and characterization of a low-molecular-mass T-cell antigen secreted by

    Mycobacterium tuberculosis. Infect Immun

    (1995)
  • GG Mahairas et al.

    Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent

    M bovis. J Bacteriol

    (1996)
  • MA Behr et al.

    Comparative genomics of BCG vaccines by whole genome DNA microarray

    Science

    (1999)
  • M Harboe et al.

    Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG

    Infect Immun

    (1996)
  • L Brandt et al.

    Key epitopes on the ESAT-6 antigen recognized in mice during the recall of protective immunity to

    Mycobacterium tuberculosis. J Immunol

    (1996)
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