Dynamic antigen-specific T-cell responses after point-source exposure to Mycobacterium tuberculosis.

RATIONALE: The kinetics of Mycobacterium tuberculosis-specific Th1-type T-cell responses after M. tuberculosis infection are likely to be important in determining clinical outcome. OBJECTIVE: To investigate the kinetics of T-cell responses, in the context of a point-source school tuberculosis outbreak, in three groups of contacts who differed by preventive treatment status and tuberculin skin test (TST) results: 38 treated TST-positive students, 11 untreated TST-positive staff, and 14 untreated students with negative or borderline TST results. METHODS: We used the ex vivo IFN-gamma enzyme-linked immunospot assay (ELISpot) to track T cells specific for two region of difference 1 (RD1) antigens, early secretory antigenic target 6 and culture filtrate protein 10, for 18 mo after cessation of tuberculosis exposure. MAIN RESULTS: The treated TST-positive students had an average 68% decline in frequencies of RD1-specific IFN-gamma-secreting T cells per year (p < 0.0001) and 6 of 38 students had no detectable RD1-specific T cells by 18 mo. No change in frequencies of these cells was observed in the untreated TST-positive staff (p = 0.38) and none were ELISpot-negative at 18 mo. Of the 14 untreated students, 7 were persistently ELISpot-positive (all of whom had borderline TST results), and 7 became ELISpot-negative (all but one had negative TST results) during follow-up. CONCLUSIONS: The decrease in M. tuberculosis-specific T cells and their disappearance in a proportion of treated students likely reflect declining antigenic and bacterial load in vivo induced by antibiotic treatment. The observed disappearance of M. tuberculosis-specific T cells in the untreated TST-negative contacts suggests that an acute resolving infection may occur in some contacts.

Use of genome level-informed PCR as a new investigational approach for analysis of outbreak-associated Mycobacterium tuberculosis isolates.

Mycobacterium tuberculosis strain CH, the index isolate linked to a major tuberculosis outbreak associated with high levels of transmissibility and virulence, was characterized by microarray analysis by use of a PCR product array representative of the genome of M. tuberculosis strain H37Rv. Seven potential genomic deletions were identified in CH, five of which were confirmed by PCR analysis across the predicted deletion points. The panel of five PCRs required to individually interrogate these loci was collectively referred to as the genome level-informed PCR (GLIP) assay. GLIP analysis was performed with CH, 12 other epidemiologically linked isolates, and 43 recent, non-outbreak-associated isolates derived from patients within the local area. All 13 outbreak-linked isolates showed a profile corresponding to the presence of all five deletions. These 13 isolates were also found to share common variable-number tandem repeat and mycobacterial interspersed repetitive unit profiles. None of the 43 non-outbreak-associated isolates exhibited the five-deletion profile. Although three individual deletions were present in upwards of 44% of the non-outbreak-associated isolates, no single-deletion isolates were detected. Interestingly, none of these deletions had been previously recognized, and sequence analysis of the immediate flanking regions in CH failed to identify a likely mechanism of deletion for four of the five loci. The GLIP assay also proved valuable in ongoing surveillance of the outbreak, rapidly identifying a further two outbreak-associated cases months after the initial cluster and, importantly, dismissing a further 12 epidemiologically suspect cases, which allowed the optimum deployment of public health resources.

Comparison of T-cell-based assay with tuberculin skin test for diagnosis of Mycobacterium tuberculosis infection in a school tuberculosis outbreak.

BACKGROUND: The diagnosis of latent tuberculosis infection relies on the tuberculin skin test (TST), which has many drawbacks. However, to find out whether new tests are better than TST is difficult because of the lack of a gold standard test for latent infection. We developed and assessed a sensitive enzyme-linked immunospot (ELISPOT) assay to detect T cells specific for Mycobacterium tuberculosis antigens that are absent from Mycobacterium bovis BCG and most environmental mycobacteria. We postulated that if the ELISPOT is a more accurate test of latent infection than TST, it should correlate better with degree of exposure to M tuberculosis. METHODS: A large tuberculosis outbreak in a UK school resulted from one infectious index case. We tested 535 students for M tuberculosis infection with TST and ELISPOT. We compared the correlation of these tests with degree of exposure to the index case and BCG vaccination. FINDINGS: Although agreement between the tests was high (89% concordance, kappa=0.72, p<0.0001), ELISPOT correlated significantly more closely with M tuberculosis exposure than did TST on the basis of measures of proximity (p=0.03) and duration of exposure (p=0.007) to the index case. TST was significantly more likely to be positive in BCG-vaccinated than in non-vaccinated students (p=0.002), whereas ELISPOT results were not associated with BCG vaccination (p=0.44). INTERPRETATION: ELISPOT offers a more accurate approach than TST for identification of individuals who have latent tuberculosis infection and could improve tuberculosis control by more precise targeting of preventive treatment.