Repeated tuberculin testing does not induce false positive ELISPOT results

The Enzyme Linked ImmunoSpot (ELISPOT) is a new rapid T cell based blood test (otherwise known as an interferon‐γ assay) for the diagnosis of latent tuberculosis infection.¹,²,³ The commercially available form of the assay, T‐SPOT® TB (Oxford Immunotec, Abingdon, UK) has European regulatory approval as an in vitro diagnostic test and is increasingly being used in clinical practice. The test is based on the enumeration of interferon‐γ producing T cells which are specific for two highly antigenic proteins, early secretory antigenic target‐6 (ESAT‐6) and culture filtrate protein 10 (CFP‐10).¹ These proteins are expressed by Mycobacterium tuberculosis but are absent from M bovis BCG vaccine. Hence, the test does not give false positive results in BCG vaccinated individuals.¹,²,³

ESAT‐6 and CFP‐10 are, however, contained within tuberculin purified protein derivative (PPD). Since ELISPOT is a highly sensitive method for measuring even low numbers of antigen specific T cells,⁴ concerns have been raised as to whether repeated tuberculin skin tests might induce T cell responses to these specific antigens, resulting in false positive ELISPOT results.

As T‐SPOT® TB enters clinical practice, it may initially be used by some people in conjunction with the tuberculin skin test. It is therefore important to know whether false positive ELISPOT results are induced by tuberculin testing. The following results strongly suggest that this is not the case.

The results reported here are from a 2 year follow up of a group of people with potential point source exposure to multidrug resistant tuberculosis on a maternity unit in Modena University Hospital, Italy.⁵ Forty four BCG unvaccinated subjects were negative at initial screening by tuberculin skin test and ELISPOT, 3 months after the point source exposure ceased. All participants had negative results on serological testing for HIV infection. Tuberculin skin tests were administered and read by two experienced chest physicians using 5 units of PPD‐S injected intradermally about 2 hours after blood was drawn for ELISPOT assays. The ELISPOT assays were performed and scored, as previously described,⁵ by two technicians without knowledge of personal identifiers. All these individuals underwent repeated testing by skin test and ELISPOT at 9, 15 and 24 months after the point exposure. At 24 months all 44 individuals remained ELISPOT negative, although three had become positive with the tuberculin skin test (fig 1). Thus, inoculation of three PPD skin tests over a 21 month period in 44 initially ELISPOT negative individuals did not induce any false positive ELISPOT results.

Figure 1 Time course of development of positive Mantoux results in the three participants who became tuberculin skin test (TST) positive as a result of repeated skin testing.

These results show that repeated tuberculin skin testing over time does not induce a T cell response to ESAT‐6 or CFP‐10 resulting in false positive ELISPOT results. Our findings suggest that this new interferon‐γ blood assay could be used in association with the standard PPD skin test without any reduction in its high diagnostic specificity. Given the high sensitivity of the ELISPOT assay for detecting even low numbers of antigen specific T cells, the absence of a detectable response to ESAT‐6 and CFP‐10 suggests that T cells specific for these antigens were not induced by repeated inoculation of PPD. This is consistent with the observation that ESAT‐6 has very poor immunogenicity when administered as a candidate vaccine, unless inoculated with powerful adjuvants.⁶ This is in stark contrast to its potent immunogenicity when presented to the immune system during natural M tuberculosis infection; indeed, ESAT‐6 is the strongest known target of T cell responses during tuberculosis infection.

Our results also suggest that T‐SPOT® TB could be especially useful in distinguishing true latent tuberculosis infection from false positive tuberculin skin test results that have arisen through “boosting”. Boosting occurs in people who undergo repeated tuberculin skin tests (such as healthcare workers) and causes false positive skin test results in uninfected people. This phenomenon is a major problem in tuberculosis screening programmes for healthcare workers, prisoners, and other groups at persistent risk of tuberculosis exposure, and was almost certainly the reason why three individuals in our study developed positive skin test results after repeated testing. Our findings suggest that T‐SPOT® TB will maintain its high specificity even in individuals with false positive skin test results due to boosting from repeated tuberculin testing. Thus, use of T‐SPOT® TB could enhance our ability to screen for latent tuberculosis infection even in populations who have already been repeatedly screened by the skin test.