Until now, the detection of both recent infection and LTBI essentially relies on the tuberculin skin test (TST) results. In Europe, TST is based on the intra-dermal injection of tuberculin units (TU) of PPD (PPD RT Statens Seruminstituut;
Copenhague Denmark) in the forearm of the subjects. Other countries, such as the USA and Canada, recommend to use another PPD (Tubersol®). Tubersol®
is used with a dose of 5 TU, but its re-activity was shown to be equivalent to the injection of TU of RT PPD (75).
Finally, determination of the inflamma-tory response to PPD is performed after 48-7h to 10h by the measurement of the diameter of induration at the site of the injection.
In most countries, including Belgium, an induration equal to > 18 mm or an increase of > 10 mm between two TST separated by less than years strongly suggest a TB infection. However, several factors interfere with the interpretation.
These factors are: 1) extreme age (<5 yrs and > 65 yrs), ) immunosuppression, ) identified contact with a TB patient, ) the prevalence of TB within the tar-get population, 4) the prevalence of mycobacteria other than TB (MOTT) 5) a recent vaccination with live-attenu-ated virus vaccines and 6) a recent BCG vaccination. As a consequence, precise guidelines for a correct interpretation of the TST have been proposed (table n°3, adapted from reference (76)).
This oldest diagnostic test in use of M.
tuberculosis infection suffers from many drawbacks because:
1) It requires skilled workers, and some variability during the pro-cedure is observed,
) Two visits are required,
) There is a risk of false positive re-sults mainly due to MOTT and BCG vaccination,
4) There is a risk of false negative re-sults, secondary to an immune suppression (i.e. iatrogenic or infectious), recent vaccination with live-attenuated virus or the use of expired tuberculin, 5) There is a risk of boosting effect
due to the repeated injections that will elicit immune respons-es to PPD and finally,
6) TST relies on the injection of ma-terial.
Immunodiagnostic
TST is an immune diagnosis, as it relies on the importance of the delayed-type hypersensitivity (DTH) induced by the injection of mycobacterial antigens (fig-ure 10). PPD-specific DTH is not possible in the absence of PPD-specific memory T cells because they orchestrate this im-mune reaction. However, PPD is a crude extract of unspecific mycobacterial antigens that do therefore not afford enough specificity in the diagnosis of TB infection. In addition, drawbacks of the PPD TST have to be taken in account (see above).
Alternatively, as T cells from a M. tu-berculosis-infected individual produce IFN-γ when they are in vitro stimu-lated with mycobacterial antigens, it has recently been suggested that in vitro measurement of IFN-γ levels may replace TST (figure 11, adapted from reference (60)). The diagnostic value of the in vitro release of significant levels of IFN-γ secretion will be linked to the
Table n°3 – Results of TST and interpretation Threshold
(mm of induration) Result Remark
< 5 mm Negative False negative results have to be ruled out
5-9 mm Negative In general
Positive Pronounced immunodeficiency, HIV infection
Suspect
- household contact with proven TB patient - children < 5 yrs
- elderly > 65 yrs
10-17 mm Positive - household contact with proven TB patient - and/or increased risk of TB infection or disease Suspect - absence of associated risk factors
- and/or recent BCG vaccination (< 5 yrs)
> 18 mm Positive
specificity of the antigen used for the stimulation. Therefore, different com-mercial tests using PPD or ESAT-6 and CFP-10 as major antigens have been proposed. These are the QuantiFERON-TB (QFT) (PPD), the QFT-QuantiFERON-TB Gold (ESAT-6/CFP-10/7.7kDa), the QFT-TB Gold in Tube (ESAT-6/CFP-10/7.7kDa) (Cellestis International, Melbourne, VIC, Australia) and the T-SPOT.TB (ESAT-6/CFP-10) (Ox-ford Immunotec Ltd, Ox(Ox-ford UK). Those test, used to track recent contacts of TB index cases, have brought a innovative and interesting approach for the diag-nosis of recent M. tuberculosis infections (60). In contrast, it remains difficult to accurately determine the sensitivity and specificity of IFN-γ release assays (IGRA) for the diagnosis of LTBI, because a gold standard for this diagnosis is missing, so that we are currently unable to defi-nitely confirm the presence of most M.
tuberculosis infections.
Therefore, several authors have com-pared the global agreement between TST and IGRA, and the results are highly variable with either satisfactory (60) or poor agreement (77, 78). In addition to
these commercial tests, other in-house assays using other antigens, such as HspX, HBHA or Ag85 are described with sometimes promising results (see be-low). Taken as a whole, detecting TB in-fection by the induction of IFN-γ secre-tions is a promising immunodiagnostic tool that could avoid some limitations of TST.
Advantages of IGRA are numerous:
1) Single visit required for the pa-tient,
) No injection of material, ) Simple procedure based on
pe-ripheral venopuncture, 4) Rapid procedure,
5) Objective readout relying on fixed cut-off,
6) No risk of immune boost, 7) According to the antigen used in
the assay, no or little influence of a BCG vaccination or from MOTT infection.
Figure 10. The delayed-type hypersensitivity reaction induced by the intradermal injection of tuberculinintheforearmofaLTBIsubject.The first phase includes the capture and the processing of the antigens which are presented by professional antigen presenting cells to memory T cells. This step is followed by the release of cytokines that activate endothelial cells, recruit inflammatory cells (i.e. monocytes) with a subsequent fluid and protein accumulation. The lesion then becomes visible; TU
= tuberculin unit. Adapted from: Janeway CA, Travers P, Walport M and Schlomchik MJ. Immunobiologie. Le système immunitaire fondamental et pathologique. e edition, ed : de boeck 00. p. 471-500.
However, these procedures are more ex-pensive than TST and require laboratory infrastructures, and these factors could limit the spread of IGRA use in endemic areas.