The tuberculin skin test (TST) is affected by recent BCG vaccination but not by exposure to non-tuberculosis mycobacteria (NTM) during early life

Abstract

The tuberculin skin test (TST) is widely used in TB clinics to aid Mycobacterium tuberculosis (M.tb) diagnosis, but the definition and the significance of a positive test in very young children is still unclear. This study compared the TST in Gambian children at 4(1/2) months of age who either received BCG vaccination at birth (Group 1) or were BCG naïve (Group 2) in order to examine the role of BCG vaccination and/or exposure to environmental mycobacteria in TST reactivity at this age. Nearly half of the BCG vaccinated children had a positive TST (>or=5 mm) whereas all the BCG naïve children were non-reactive, confirming that recent BCG vaccination affects TST reactivity. The BCG naïve children demonstrated in vitro PPD responses in peripheral blood in the absence of TST reactivity, supporting exposure to and priming by environmental mycobacterial antigens. Group 2 were then vaccinated at 4(1/2) months of age and a repeat TST was performed at 20-28 months of age. Positive reactivity (>or=5 mm) was evident in 11.1% and 12.5% infants from Group 1 and Group 2 respectively suggesting that the timing of BCG vaccination had little effect by this age. We further assessed for immune correlates in peripheral blood at 4(1/2) months of age. Mycobacterial specific IFNgamma responses were greater in TST responders than in non-responders, although the size of induration did not correlate with IFNgamma. However the IFNgamma: IL-10 ratio positively correlated with TST induration suggesting that the relationship between PPD induced IFNgamma and IL-10 in the peripheral blood may be important in controlling TST reactivity. Collectively these data provide further insights into how the TST is regulated in early life, and how a positive response might be interpreted.

Figure 1. BCG vaccine schedule of study subjects.

Babies were recruited at birth and randomised into either Group 1 (vaccinated at birth) or Group 2 (vaccinated at 4½ months of age) and followed up for 9 months. For all subjects a blood sample was collected at birth (50 mL cord blood), 4½- and 9- months of age (5 mL). A tuberculin skin test (TST) was performed at 4½ and at 20 – 28 months of age. Each child was followed up monthly for 9 months and then again at 20–28 months of age with a TB questionnaire (TBQ) and a general health questionnaire.

Figure 2. Tuberculin Skin Test (TST) and BCG scar results.

Two T.U. PPD were injected into the forearm of each individual. The average length x width (mm) of the induration was measured 48 – 72 hours later at (A) 4½ months of age (Group 1, n = 51, Group 2, n = 39) and (B) 20– 28 months of age (Group1, n = 45, Group 2, n = 40). The black line represents a 5 mm ‘cut off’ for positive reactivity. (C) The relationship between scar and gender (male n = 42, female n = 43). Group 1 - vaccinated at birth, Group 2 - vaccinated at 4½ months of age.

Figure 3. Relationship between TST and cytokine production.

At 4½ months of age, whole blood was incubated with PPD for 5 days. (A) IFNγ and (B) IL-10 cytokine production and (C) the ratio of IFNγ: IL-10 was measured in the supernatants of the PPD cultures (left). Unstimulated control was subtracted from the PPD stimulated values, black bar represents the median value. A Mann-Whitney U test at 5% significance was applied to compare the cytokine production between groups (left graphs). Correlation between the TST induration of the responders and IFNγ, IL-10 and the ratio of IFNγ: IL-10 was calculated using Spearmans correlation coefficient at 5% significance (right graphs), dark grey line represents line of regression. R = responder n = 23, NR = non responder n = 22, unvaccinated n = 38.

Figure 4. Relationship between TST and T cell phenotype in response to PPD.

At 4½ months of age, whole blood was cultured with PPD for 5 days. (A) frequencies of activated T cells (CD4⁺CD25⁺) and (B) naturally occurring Tregs (CD4⁺CD25⁺FOXP3⁺) were measured with respect to TST reactivity. Unstimulated control was subtracted from the PPD stimulated values, black bar represents the median value. Comparisons between responders (R), non responders (NR) and unvaccinated individuals were calculated using a Mann-Whitney U test at 5% significance. R = responder n = 22, NR = non responder n = 21, unvaccinated n = 34.