Use of enzyme linked immunospot assay (ELISpot) for monitoring treatment response of pulmonary tuberculosis patients

Background Tuberculosis (TB) remains one of the major causes of death from a single infectious agent worldwide. The rapid emergence of drug resistant mycobacteria has strengthened the demand for rapid methods for detection of mycobacteria in clinical samples. As prevention of tuberculosis relies on the early detection and cure of the infectious cases, current efforts are focused upon improving the rapidity of identification of Mycobacterium tuberculosis, allowing prompt initiation of appropriate therapy. The rapid enzyme linked immunospot assay (ELISpot) method was developed in the late 1990s based on the numbers of spots made by interferon gamma producing T cells stimulated by culture filtrate protein-10 (CFP-10) or early secretory antigenic target-6 (ESAT-6). Therefore, a T-cell response to these antigens could in theory serve as a specific marker of M. tuberculosis infection. Aim of the work Is to assess the potential utility of ELISpot assay for monitoring treatment response of pulmonary tuberculosis patients. Patients and methods The study was done on 30 patients diagnosed as pulmonary tuberculosis on clinical, radiological and bacteriological bases. They were collected from Zagazig Chest Hospital and Zagazig University Hospitals from January 2010 to January 2011. A total of 15 healthy volunteers were enrolled in this study as control subjects. The following were performed for all patients before treatment initiation: full history taking, complete clinical examination, chest X-ray, postero-anterior and lateral views, tuberculin skin test (TST) by Mantoux technique, and routine laboratory investigations. Three successive sputum samples for sputum smear Ziehl–Neelsen (Z–N) staining and sputum collection for Mycobacterium culture on Lowenstein–Jensen media (L.J. media) were done. Collection of 2 ml heparinized blood for enzyme linked immunospot assay (ELISpot) was done. All patients received four antituberculous drugs, isoniazid, rifampicin, pyrazinamide and ethambutol, for the initial 2 months. After 2 months of therapy; another three successive sputum samples for sputum smear Ziehl–Neelsen (Z–N) staining and sputum collection for Mycobacterium culture on Lowenstein–Jensen media were done. Collection of 2 ml heparinized blood for enzyme linked immunospot assay (ELISpot) was done. Results The results of this study showed that all patients were complaining of cough and expectoration. Tuberculin skin test was positive in 18 patients (60%). Most patients (46.6%) had moderately advanced disease as regards the radiological extent. It was found that the median INF-γ ELISpot response to ESAT-6 was significantly decreased after 2 months of antituberculosis therapy. The number of pre-treatment ESAT-6 ELISpot count in patients with positive tuberculin skin test was significantly higher than those with negative tuberculin skin test (P < 0.01). As regards bacillary load, a statistical significant difference between patients with AFB+++ and patients with (AFB+, AFB++) as regards pre-treatment ELISpot count was recorded. Higher statistical significant difference in patients with AFB+, AFB++ and AFB+++ pre and post treatment ESAT-6 ELISpot count was found. It was found that the number of pre-treatment ESAT-6 ELISpot count in patients with cavitary lesion was higher than those without cavitary lesion and the difference was highly significant (P = 0.01). As regards radiological extent, it was found that the number of pre-treatment ESAT-6 ELISpot count in patients with far advanced disease was higher than patients with minimal or moderately advanced disease. Also, after 2 months of therapy the number of ESAT-6 ELISpot count in patients with far advanced disease showed more decline than patients with minimal or moderately advanced disease. It was found that ELISpot assay sensitivity, specificity, positive predictive value and negative predictive value in relation to L.J. media were 93.3%, 100%, 100% and 88.2%, respectively. Conclusion ELISpot assay may be used as a useful tool in the diagnosis of pulmonary tuberculosis. The decrease in the M. tuberculosis-specific T cell responses following 2 months of successful antituberculosis therapy may have a clinical value as a supplemental tool for the monitoring treatment response of pulmonary tuberculosis patients