Performance of ESAT-6 and CFP-10 in the diagnosis of tubercular infection.INTRODUCTION
Tuberculosis continues to be a major scourge of mankind. Each year, approximately 8 million people develop active tuberculosis and 2 million people die of this disease. (1) Most of these people represent vaccine failures in that they have developed tuberculosis despite having been vaccinated previously with Mycobacterium bovis BCG or bacillus Calmette-Guerin. While not highly effective, BCG is the only vaccine against tuberculosis currently available, and it has been used widely. More than 4 billion doses of BCG have been administered worldwide. (2)
Although BCG has a limited preventive value and is not contributing to the elimination of the disease, its use in protecting at least children from TB is recommended by the WHO in countries where the disease is endemic (3) and where public health budgets cannot cover the high costs of antibiotic treatment. In Italy, a country with low prevalence in which the annual rate of tubercular disease is below 10 cases in 100,000, the laws currently in force make anti-tubercular vaccination compulsory for infants and children of below 5 years of age who test negative for tuberculin; persons living or having close contact with persons infected with TB in the contagious phase where the risk of contagion persists; health workers, including students of medicine and trainee nurses; and anybody who works in health environments at high risk of exposure to strains of Multidrug-Resistant M. tuberculosis or in environments at high risk of TB but cannot be given preventive therapy because they are clinically advised to avoid taking certain drugs. (4) The possibility of accurately and rapidly detecting M. tuberculosis infection is crucial for the global control of the disease. Diagnosis and treatment where indicated of latent tubercular infection are considered absolute priorities for controlling and eliminating tuberculosis. Specifically, early identification of infected subjects at high risk of clinical progression and of those with active pulmonary tuberculosis makes it possible to reduce the sources of contagion in the population. The diagnosis of the active disease is based essentially on the assessment of the risk of infection, the clinical-radiological picture, and the direct examination of samples, although the reference for diagnosis remains the identification of M. tuberculosis in culture. In contrast, in latent tubercular infection, in which the mycobacteria are in a state of quiescence, microbiological isolation is not possible, and diagnosis is therefore based entirely on the demonstration of an immunological response to mycobacterial antigens (5). Traditionally, the identification of latent tubercular infection has always been performed by the cutaneous tuberculin test, also known as the Intradermal (Mantoux) test. Although it remains the test of reference, it has certain limitations that partially reduce its clinical utility. In the first place, it is a test carried out in vivo which requires the patient to return to the clinic after three days for the interpretation of the results. It also suffers from poor standardisation in both execution and interpretation by health workers. Furthermore, it suffers from low specificity due to antigenic cross-reactivity between PPD, BCG and non-tubercular myco-bacteria, including environmental ones. In addition, it is affected by low sensitivity in the case of immunodepressed subjects. Lastly, it does not distinguish between latent infection and active disease and plays no role in the monitoring of the therapy. Recently introduced in vitro assays make use of the characteristics of M. tuberculosis, an intracellular pathogen which is able, if adequately stimulated with the specific proteins ESAT6 and CFP-10, to evoke an in vivo a cell-mediated Th1-type immunity response characterised by the production of [gamma]-interferon. The aim of this study is thus to highlight any cases of tubercular infection in order to measure the performance of two tests: Mantoux test and an in vitro assays (T-SPOT-TB).
PATIENTS & METHODS
The study was carried out between October 2007 and June 2008 on 452 subjects, 128 women and 224 men (age 21-86 years; median 43; DS 14.4). Of the subjects included in the study, 150 were healthy volunteers (control groups) with no known history of exposure to tuberculosis, the remaining 302 subjects belonged to a group of patients who came to the Cardinal Panico Hospital in Tricase, hospitalised with clinical features associated with fever of unknown origin. Each of the subjects in the study were subjected to the Mantoux test, administering 0.1 ml of PPD suspension at a concentration of 5 UI/ml (Biocine Test PPD, Chiron, SI, Italy) following the protocols of consent for administration and reading/interpretation.
The transverse diameter of induration (mm) at the TST (Tuberculin Skin Test) site was measured 72 hours later. TST results were interpreted according to American Thoracic Society (ATS/ Centre for Disease Control (CDC) guidelines). (6)
A 5 mm cut-off point was adopted for a positive test result in subjects with active disease; for other persons with an increased probability of recent infection or with other clinical conditions that increase the risk for progression to active TB, > 10 mm of induration is considered positive for persons at low risk for TB, for whom tuberculin testing is not generally indicated, > 15 mm of induration is considered positive. Immediately before the TST, subjects gave 10 mL of venous blood, which was collected into a heparinized tube. The blood was used to perform the T-SPOT TB test (Oxford Immu notec), an in vitro assays in order to determine the response of effector lymphocytes to stimulation with ESAT-6 and CFP-10 antigens in terms of production of [gamma]-interferon. Spots can be visualised using an ELISPOT plate reader.
To interpret the tuberculin skin test appropriately, one must understand the sensitivity and specificity of the test as well as the positive predictive value of the test. The sensitivity of a test is the percentage of people with the condition who have a positive test. If false-negative results are uncommon, the sensitivity is high.
The specificity of the test is the percentage of people without the condition who have a negative test. False-positive results decrease the specificity of a test. False positive tuberculin tests occur in individuals who have been infected with other mycobacteria, including vaccination with BCG. Some antigens in PPD are shared with the other mycobacteria and thus can elicit a skin test response.
Of the 150 control subjects (Table 1) 0, were found to be positive for both the Mantoux test and the in vitro lymphocyte stimulation assay, 23 (i.e. 15,3%) tested positive in the Mantoux test, but were negative for the in vitro assay. No subjects tested positive for the in vitro assay and negative in the Mantoux test. Lastly, 127 subjects (i.e. 84.7%) were negative for both tests (Table 1).
Of the group of 302 patients with unidentified fever, 126 (41.7%) were positive for both tests. 25 subjects were positive in the Mantoux test and negative in the in vitro assay (Table 1). These patients were diagnosed as having tubercular spondylodiscitis, a very severe extrapulmonary condition. 25 patients (Table 1), of non-European origin, were observed to be negative in the Mantoux test and positive in the in vitro assay and to have clinical respiratory symptoms indicative of pulmonary tubercular infection.
Lastly, 126 subjects (41.7%) were found to be negative for both anti-tubercular assays under study. In these patients the fever was attributed to other non-tubercular infections (Table 1). All tuberculosis patients were HIV seronegative at the time of their diagnosis with M. tuberculosis disease.
A comparison of the extent of cutaneous reaction in the Mantoux test with the results of the in vitro assay (Graph 1-2) shows that the greater the diameter of induration the higher the possibility of finding effector T-cells secreting [gamma]-interferon. The specificity of a positive ESAT-6/CFP-10 response was 100% and its sensitivity was 92,30%. The specificity of a positive Mantoux test response was 86,95% and its sensitivity was 92,30%. The positive predictive value in Mantoux test was 66,7%, while in T-SPOT TB test was 100% (Table 2).
Tuberculosis (TB) is the infectious disease with the highest mortality in the world, representing one of the biggest health problems in developing countries. However, as a result of the spread of the human immunodeficiency virus (HIV), growing immigration from countries with endemic tuberculosis, and the emergence of Multidrug-Resistant strains of M. tuberculosis (MTB), it is also beginning to cause concern for the public health authorities in industrialised countries. (6) The identification and treatment of cases of the active disease is the basic strategy for TB control programmes in all countries. However, since the transmission of the infection occurs at an early stage, i.e. before it can be correctly diagnosed, new cases of the active disease, especially if not recognised promptly, represent the most important factor in the persistence of the tubercular epidemic. (7)
The consequences of tubercular infection depend mostly on the immune response of the host organism. Its clinical manifestations include the state of latent infection (LTBI) in the absence of any microbiological or radiological evidence of the active disease, the milder forms of the disease (i.e. those characterised by few symptoms and a low mycobacterial count), and the more severe forms of pulmonary TB (with a high mycobacterial count). In immunocompetent subjects with LTBI the risk of developing the active disease is 10% in the course of a lifetime, usually occurring in the first 2-5 years following exposure/infection. In contrast, the risk is significantly higher in immunocompromised subjects, 5-10% per year of life in individuals co-infected with HIV. (8) Vaccination or chemoprophylaxis in certain categories of high-risk subject would be of great benefit for public health in that it would entail a significant reduction not only of the individual risk, but would contribute to reducing the population of infected subjects, which represent the main source for the potential development of new cases of active TB in the future. (9)
The Mantoux test is the most traditional of the diagnostic techniques used in clinical practice. Ideally, it represents an optimal approach in that it is simple and safe to administer. However, it suffers from low specificity due to the antigenic cross-reaction of the Purified Protein Derivative (PPD) with vaccinal strains of M. bovis BCG. It should be stressed that the sensitivity of the test is low in any case in immunocompromised subjects.
Other drawbacks of the test include the need for a return visit 72 hours afterwards to check the results and the variability of the reading of those results. (10) In addition, successive tests for tuberculin in the same subject can yield inconsistent results; indeed, the diameter of induration may increase, either because of the presence of a new infection (conversion) or as a result of the serial repetition of the test in subjects with a previous positive response (boosting). In addition, the diameter of induration can also diminish (reversion) both due to physiological phenomena, such as aging, and to concomitant infections that entail some lessening of the immune response. (10)
Based on this small study, the resulting test characteristics for in vitro IFN-[gamma] responses to PPD for the detection of disease due to M. tuberculosis are a sensitivity of 92,30%, a specificity of 100% and a positive predictive value of 100%. The false positive rate for Mantoux test in the healthy control subjects, however, was 15%.
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The sensitivity of responses to the recombinant ESAT6/CFP-10 was lower than its specificity.
The small group of patients with clinical symptoms were observed to be positive for both tests in 41.7% of cases. Surprisingly, in 25 cases (8.3%) weak positivity were seen in the Mantoux test and negativity in the in vitro for synthesis of [gamma]-interferon by effector cells stimulated with ESAT/6/CFP-10. These patients were clinically and analytically diagnosed as having tubercular spondylodiscitis, a severe form of extra-pulmonary tuberculosis plausibly associated with poor antigen-specific release of [gamma]-interferon by immunocompetent cells. The literature describes cases of false negativity in extra-pulmonary forms of tuberculosis.11 Interestingly, 25 patients, of non-European origin, were negative in the Mantoux test but positive in the in vitro test, probably due to a previous pulmonary tubercular infection. In conclusion, less than half the test subjects showed positive on the test.
For tubercular infection screening programmes to become more efficient, clinical practices must include diagnostic techniques that are both more sensitive and more specific, which will make it possible to provide suitable treatment of this condition in selected high-risk populations. This issue is particularly pressing in developed countries, where more than half of the cases of active TB (and their relative contacts) are immigrants from countries with high rates of TB, where vaccination with BCG and environmental exposure to non-tubercular mycobacteria are common phenomena.
(1.) Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. Consensus statement. Global Burden of tuberculosis: estimated incidence, prevalence, and mortality by contry. Who global surveillance and monitoring project. JAMA 1999; 282: 677686.
(2.) Horwitz MA, Harth G, Dillon BJ, Maslesa-Galic S. Enhancing the protective efficacy of Mycobacterium bovis vaccination against tuberculosis by boosting with the Mycobacterium tuberculosis major secretory protein. Infection and Immunity 2005;73: 4676-4683.
(3.) Frieden TR, Sterling TR, Munsiff SS, Watt CJ, Dye C. Tuberculosis. Lancet 2003; 362: 887-899.
(4.) D.P.R. n. 465 del 7 novembre 2001. Regolamento che stabilisce le condizioni nelle qualii obbligatoria la vaccinazione antitubercolare, a norma dell'articolo 93, comma 2, della legge 23 dicembre 2000, n. 388. Gazzetta Ufficiale n. 7 del 9/1/2002.
(5.) Diagnostic standards and classification of tuberculosis in adults and children. Am J Respir Crit Care Med 2000; 161: 1376-1395.
(6.) Barnes PF. Diagnosing latent tuberculosis infection: turning glitter to gold. Am J Respir Crit Care Med. 2004;170:5-6.
(7.) Small PM, Hopewell PC, Singh SP, Paz A, Parsonnet J, Ruston DC, Schecter GF, Daley CL, Schoolnik GK. The epidemiology of tuberculosis in San Francisco: a population-based study using conventional and molecular methods. N Engl J Med. 1994; 330:1703-1709.
(8.) Parrish NM, Dick JD, Bishai WR. Mechanisms of latency in Mycobacterium tuberculosis. Trends Microbiol. 1998; 6: 107-112.
(9.) Centers for Disease Control and Prevention. Targeted tuberculin testing and treatment of latent tuberculosis infection. MMWR Morb Mortal Wkly Rep 2000; 49: (RR-6):1-5.
(10.) Menzies, D. Interpretation of repeated tuberculin tests. Boosting, conversion, and reversion. Am J Respir Crit Care Med. 1999;159:15-21.
(11.) Lein AD, Von Reyn CF, Ravn P, Horsburgh CR, Alexander LN, Andersen P. Cellular immune responses to ESAT-6 discriminate between patients with pulmonary disease due to Mycobacterium avium complex and those with pulmonary disease due to Mycobacterium tuberculosis. Clin Diagn Lab Immunol. 1999;6:606-609.
Corresponding author: Dr Manuela Quattrocchi, Laboratory of Hygiene, Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento Via Prov.le Lecce-Monteroni, 73100--Lecce (Italy) Phone: 0039 832 298687 Fax: 0039 832 298686/298626 e-mail: email@example.com
Manuela Quattrocchi [1,2], Giambattista Lobreglio , Pierangelo Errico , Giancarlo Pasanisi , Antonella De Donno   Ce.S.E.V.A (Center for Surveillance, Epidemiology and Advanced Virology) AO 'Card. G Panico' Hospital, Tricase, Lecce/DiSTeBA, Faculty of Sciences, University of Salento, Lecce, Italy  Laboratory of Hygiene, Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Lecce, Italy
Table 1. Comparison of Mantoux test and in vitro ESAT-6/CFP-10 assay in studied population Mantoux(+) Mantoux (+) Mantoux (-) Mantoux (-) ESAT-6/ ESAT-6/ ESAT-6/ ESAT-6/ CFP-10 (+) CFP-10 (-) CFP-10 (+) CFP-10 (-) n. (%) n. (%) n. (%) n. (%) Control subjects 0(0,0) 23(15,3) 0(0,0) 217(84,7) Patients 126(41,7) 25(8,3) 25(8,3 ) 126(41,7) No. of subjects n. 452 Control subjects 150 Patients 302 Table 2. Sensitivity and specificity of Mantoux test and in vitro assay. Performance Performance TB spot Mantoux test (ESAT-6 and CFP10) Sensitivity 92,30% 92,30% Specificity 86,95% 100% Positive predictive value 66,7% 100%