Study participant demographics, TST screening and TB disease diagnosis
A total of 93 children all tested HIV-negative were included in this study with a median (IQR) age of 8.0 (4.7-10.9) years and 47 (50.5%) were female. Forty-three (43; 46.2%) children were symptomatic with a median (IQR) age of 9.7 (6.2-12.0). A BCG scar was present in 61 out of 66 (92.4%) assessed children. TB disease was diagnosed in 28 (30.1%) children (5 cases were bacteriologically confirmed TB all of the M. tuberculosis Euro-American lineage also called Mtb-lineage 4 and clinically diagnosed TB were 23 cases) (Figure 1). The remaining 15 children were deemed to have other respiratory diseases. Fifty children (all asymptomatic) received the Mantoux test (tuberculin skin test; TST) and 26 (52.0%) were TST+.
IGRA results and MTBC infection
To define MTBC infection status of the study participants, the EC-fusion protein IGRA result was taken as gold standard, with IFN-g cut-off 124.2 pg/mL. Overall, EC-fusion protein induced a high IFN-g response in all study participants (median (IQR) 275.5 (74.7-1435.0) pg/mL). Among the 65 children without TB disease (asymptomatic or symptomatic), 45 (70.3%) were IGRA+.
Comparison of TST and IGRA results, and age within the subgroups
Among the 50 children who received a TST, 22 (44.0%) were TST+IGRA+, 12 (24.0%) were TST-IGRA- and 16 children had discordant results (Figure 1). Tuberculin skin test and IGRA results (in mm skin induration and IFN-γ levels, respectively) were correlated (Spearman’s ρ 0.4; P < 0.001) [see Additional file 1, Supplementary Figure 1]. Tuberculin skin test+ children were older than TST- children (median (IQR) 7.5 (5.0-8.8) years versus 4.0 (3.0-7.3) years; P = 0.05) [see Additional file 1, Supplementary Figure 2]. There was a borderline age difference between IGRA+ (infected) and IGRA- (uninfected) children (median (IQR) 8.0 (5.0-10.0) years versus 4.5 (3.0-8.0) years; P = 0.06) [see Additional file 1, Supplementary Figure 3]. TB diseased children were significantly older than children without TB disease (irrespective of infection status) (median (IQR) 9.7 (6.0-12.2) years versus 7.0 (4.0-9.0) years, P = 0.02). There was no difference in age between children with bacteriologically confirmed and clinically diagnosed TB disease. We observed a trend of TB disease and infection in older children compared to younger children, which aligns with the slow progression to nature of MTBC infection. Resuscitation promoting factor-specific IFN-γ responses did not differ between children age below 5 years and above 5 years, and there was no correlation between Rpf-specific IFN-γ levels and age in years (data not shown).
IFN-γ response to RpfA-D in MTBC infected compared to uninfected children without TB disease
Whole blood stimulation with Rpf induced significantly higher IFN-g responses in the infected (IGRA+) compared to the uninfected (IGRA-) children among those without TB disease. The medium (IQR) IFN-g level of infected versus uninfected children was 34.6 (5.2-73.0) pg/mL versus 3.0 (0.0-11.1) pg/mL (P = 0.03) for RpfA, 77.4 (21.3-235.7) pg/mL versus 13.6 (0.0-31.6) pg/mL (P = 0.007) for RpfB, 45.0 (25.9-115.8) pg/mL versus 24.9 (0.0-30.4) pg/mL (P = 0.03) for RpfC, and 138.0 (67.5-340.5) pg/mL versus 49.3 (16.3-105.1) pg/mL (P = 0.004) for RpfD (see Figure 2). Purified protein derivative induced a higher IFN-γ response in infected children (medium (IQR) 1078.9 (113.1-3937.8) pg/mL versus 92.7 (3.8-350.1) pg/mL, P = 0.01).
Performance of RpfB and D in an IGRA-based test for screening MTBC infection
In our quest for a suitable alternative screening antigen, RpfB and D were found to induce the most differential IFN-γ response (P < 0.01; Figure 2). This result motivated our decision to further analyse their discriminatory capacity using ROC-curves. The area under the curves (AUC) were 0.77 and 0.74 for RpfB and RpfD, respectively (Figure 3). Based on the Youden Index, a cut-off at 33.9 pg/mL for RpfB-specific IFN-γ levels was derived, leading to 73% (24/33) sensitivity and 92% (12/13) specificity (Figure 3). Similarly, for RpfD, a cut-off of 67.0 pg/mL led to 77% (33/43) sensitivity and 72% (13/18) specificity (Figure 3). For the TST, these figures were 65% (22/34) sensitivity and 75% (12/16) specificity (Figure 1).
In contrast, when using TST results as standard only the stimulations with EC-fusion protein and PPD induced a significantly different IFN-g response between infected and uninfected groups (P=0.004 and 0.003 respectively [see Additional file 1, Supplementary Figure 4]).
IFN-γ response to RpfB and D in MTBC infected compared to diseased children
Since RpfB and D responses could discriminate between infected and uninfected children, we evaluated the diagnostic performance of TB disease. Resuscitation promoting factor B and D induced a significantly higher IFN-γ response in infected compared to TB diseased children (medium (IQR) 47.1 (7.1-129.8) pg/mL versus 9.1 (0.0-75.3) pg/mL, P = 0.02 and 91.1 (40.9-284.5) pg/mL versus 60.1 (11.0-114.2) pg/mL, P = 0.03) respectively (Figure 2). EC-fusion protein induced higher IFN-γ responses in infected compared to TB diseased children (P=0.0002), but no significant difference was obtained with PPD stimulation between these groups (Figure 2). There was no significant difference in IFN-γ response between uninfected and TB diseased children for any of the Rpf antigens, EC or PPD (Figure2).
Background IFN-γ response in TB diseased children compared to children without TB disease
In general, TB diseased children had significantly higher background IFN-γ responses (whole blood stimulated with medium alone) compared to the other children without TB disease (irrespective of their infection status) (medium (IQR) 52.6 (42.0-78.4) pg/mL versus 18.6 (0.0-41.7) pg/mL, P = 0.0003) (Figure 2). Using an ROC-curve, we further analysed the medium background response as a screening and/or diagnosis tool in our study setting. The AUC was 0.77, and at optimal cut-off 33.3 mg/mL (based on the Youden Index), sensitivity and specificity were 92.9% and 63.1%, respectively [see Additional file 1, Supplementary Figure 5].