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Local immunodiagnosis of pulmonary TB: ELISPOT or flow cytometry, PPD or ESAT-6?

¹ Dept Respiratory Medicine, Queen Alexandra Hospital, Portsmouth, ² Depts of Respiratory Medicine, ³ Clinical Immunology, and ⁴ Respiratory Medicine, Royal Free Hospital, London, UK.

To the Editors:

Immunodiagnosis is a significant addition to the diagnostic armamentarium in tuberculosis (TB) infection and disease. For some time it has been known that cells secreting type-1 cytokines, such as interferon (IFN)- and tumour necrosis factor-, selectively accumulate in the lung in higher frequencies than in the blood 1, as would be expected in a disease dominated by pulmonary pathology.

In a recent issue of the European Respiratory Journal, Jafari et al. 2 detailed the TB-specific response in bronchoalveolar lavage (BAL) and blood from 12 patients with culture-positive but smear negative disease. Jafari et al. 2 used a combination of flow cytometry to determine the phenotype of the CD4 and CD8 lymphocytes concentrating on memory and activation markers, and enzyme-linked immunospot (ELISPOT), to measure the response to the TB-specific proteins early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10. This study is similar in design and findings to another paper by Jafari et al. 3.

One of the disadvantages of ELISPOT when compared with flow cytometry is that it cannot identify the phenotype of the cells secreting the measured cytokines (usually IFN-). Hence in the study by Jafari et al. 2 it is through association, rather than direct measurement, that the TB-specific lymphocytes are characterised as predominantly memory phenotype, as evidenced by CD45RO expression. Indeed the percentage of CD45RO+ CD4+ lymphocytes in TB (50%) and the control group (35%) appeared to be surprisingly low. True memory CD4+ lymphocytes, determined by the absence of both CD45RA and CD27 expression, were 93% in BAL in 46 controls and patients with a variety of different pathologies, compared with 23% in blood samples 4.

In the study by Jafari et al. 2, the region of difference-1 antigens, ESAT-6 and CFP-10, were used as the stimulatory antigens. Jafari et al. 2 noted that the TB-specific responses to these peptides in BAL were 10 times those in peripheral blood. However, ESAT-6 and CFP-10 only encompass a small part of the TB genome and larger proteins, such as purified protein derivative (PPD), have been shown to generate much larger responses. For example, in a previous study 5 the median CD4 T cell IFN- response to PPD in BAL was 24% but only 0.07% in blood, a 300-fold difference. More importantly, ESAT-6 responses in BAL in a large cohort of patients were negative in 19% of cases with culture confirmed TB 6.

Most enthusiasts for using region of difference-1 antigens in TB immunodiagnosis point out the difficulties of using PPD on blood samples when patients have received the bacilli Calmette–Guerin (BCG) vaccination. However, BCG vaccination has no influence on lung antigen-specific responses when PPD is used as the stimulatory antigen 5, 6. This is probably due to lymphocytes that are primed in dendritic cells in the skin re-circulating to local lymph nodes and the spleen, but not the lung.

Therefore, we suggest that flow cytometry provides more relevant information regarding lung lymphocyte responses and that purified protein derivative has several advantages over RD-1 antigens in this context.

Statement of interest

None declared.

REFERENCES

1. Schwander SK, Torres M, Sada E, et al. Enhanced responses to Mycobacterium tuberculosis antigens by human alveolar lymphocytes during active pulmonary tuberculosis. J Infect Dis 1998;178:1434–1445.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
2. Jafari C, Ernst M, Strassburg A, et al. Local immunodiagnosis of pulmonary tuberculosis by enzyme-linked immunospot. Eur Respir J 2008;31:261–265.[Abstract/Free Full Text]
3. Jafari C, Ernst M, Kalsdorf B, et al. Rapid diagnosis of smear-negative tuberculosis by bronchoalveolar lavage enzyme-linked immunospot. Am J Respir Crit Care Med 2006;174:1048–1054.[Abstract/Free Full Text]
4. Barry SM. Lymphocyte Responses in the Lung in Patients with Respiratory Disease. PhD thesis. University of London, London, UK, 2003.
5. Barry SM, Lipman MC, Bannister B, Johnson MA, Janossy G. Purified protein derivative-activated type 1 cytokine-producing CD4+ T lymphocytes in the lung: a characteristic feature of active pulmonary and nonpulmonary tuberculosis. J Infect Dis. 2003;187:243–250.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
6. Breen RA, Barry SM, Smith CJ, et al. Clinical application of a rapid lung-orientated immunoassay in individuals with possible tuberculosis. Thorax 2008;63:67–71.[Abstract/Free Full Text]

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