We found that Rpf-specific IFN-γ responses are associated with MTBC infection status in household-exposed children in The Gambia. This suggests that Rpf are attractive antigens to consider for childhood TB screening and diagnostics. Screening for infection and diagnosing TB disease represent core challenges in paediatric TB, particularly in low- and middle-income countries, where TB prevalence is high and other paediatric illnesses with TB-resembling symptoms are common. Given the worldwide shortage of PPD for tuberculin skin testing and its inherent lack of sensitivity and specificity , it is essential to develop new methods for MTBC infection screening, particularly for the high-risk group of household-exposed children of infectious adult TB patients, as recommended by the WHO.
We stimulated children’s venous blood samples in vitro with Rpf antigens and showed that RpfB and D induce lower IFN-γ responses in uninfected children compared to infected children. In our study population, RpfB and RpfD -based IGRAs identified infection with higher sensitivity than a TST. This is in line with previous studies in adults. In a multi-side study in adults including The Gambia, Sutherland et al showed that soluble IFN-γ responses after seven days whole blood stimulation with RpfA and RpfE but not RpfB were higher in TST + than TST- participants without TB disease . Similarly, Huang et al reported that RpfA and RpfD induced higher IFN-γ responses in “latent tuberculosis infected” (LTBI) household adult contacts compared to community exposed infected participants and healthy controls . Commandeur et al also found that RpfA and RpfD induced different immune responses in vitro in infected adults than in healthy controls . There are clear differences in the discriminatory capacity of the different Rpf proteins in the highlighted previous studies in adults. However, RpfA-induced responses consistently differ between infected and uninfected adults across multiple studies. An explanation for the absence of strong discriminatory RpfA-induced IFN-γ responses in our experiments could be given by the experimental set-up. Our study that focussed on exploring the possibility of integrating Rpf in TB screening, investigated a short-term stimulation of ~ 20 h, contrary to previous studies, which used several days stimulation and added costimulatory molecules such as CD28 and CD49d. In addition, the participants’ younger age likely affected our observed results. Because of the age-dependent maturation of the immune system, children’s IFN-γ production tends to be lower and the contribution of T-cells producing IFN-γ is thought to be less compared to adults [4, 27, 28].
We additionally explored whether Rpf could discriminate between infected and TB diseased children. Resuscitation promoting factor B and D also induced a significantly higher IFN-γ response in infected children compared to TB diseased children. Similar results were previously observed in adults [14, 16, 29]. In vitro, RpfA-, B-, D-, and E-specific IFN-γ producing CD4+ T-cells are less abundant in TB diseased patients compared to infected individuals [29, 30]. These observations support the hypothesis that a Rpf-specific IFN-γ response can discriminate infected individuals from TB diseased cases. In our study population, Rpf responses did not differ between those with TB disease and those without TB disease (irrespective of infection status), due to the similar Rpf response in children with TB disease compared to uninfected children. Thus, our study does not show discriminatory power of Rpf for TB disease per se, it only shows a difference between children with infection and TB disease. This difference further confirms the pattern of Rpf production during infection, which specifically increase as the bacteria transit from latency to active replication associated with progression to TB disease [15, 16, 26]. Interferon gamma responses to Rpf might indicate whether individuals are at risk of developing TB disease in the near future following infection. Generally, the background IFN-γ production was significantly higher in TB diseased children compared to children without TB disease. The ROC-curve analysis at 33.3 pg/mL cut-off could have identified children with TB disease from children without TB disease with a sensitivity of 92.9%, but with a specificity of 63.1%. Of note, this cut-off is relatively low, in particular when compared to the median (IQR) background IFN-γ level of 23.3 (5.0-37.2) pg/mL. The higher IFN-γ background response in TB diseased children might reflect the disease exacerbation profile that is accompanied by non-specific inflammation and deserves further attention [28, 31].
Our study has some clear limitations with regard to infection classification and the study population. We defined MTBC infection by a single test, i.e., IGRA with EC-fusion protein antigen. Although globally recognized as a standard for TB screening, the EC-based IGRA test is known to have lower sensitivity for M. africanum infection  and in children below 5 years of age . In fact, our study of Rpf-induced IFN-γ production levels in IGRA + and IGRA- children compares the immunogenicity of the EC and Rpf antigens. Therefore, our strict separation of infected versus uninfected children purely based on IGRA results should be considered with caution, as of course, there is no gold standard for MTBC infection. However, assuming that our IGRA results reflected the participants’ infection status more accurately than the TST results, RpfB and D still hold promise for TB screening in children. Another limitation of our study is the limited sample size. This influenced the ROC-analyses, which in turn had impact on the cut-off for IFN-γ response. Another IFN-γ cut-off for RpfB and D would have resulted in a different specificity and sensitivity, which is why we strictly adhered to one method for selecting the ideal cut-off: the Youden Index. Furthermore, all participants were part of a childhood TB contact study, meaning that they had been exposed to an adult smear-positive index TB case. Consequently, no TB-unexposed negative controls were included. Future work should include a larger control group consisting of children with TB-like symptoms, but who do not have TB disease and with confirmed alternative diagnostics. This group of children is the biggest confounder of TB disease diagnosis in children based on clinical symptoms as these symptoms resemble that of other paediatric illnesses occurring in endemic regions . Moreover, our study did not include follow-up data on the progression from infection towards TB disease. Explicitly in the case of Rpf responses, this shift warrants further investigation, because these antigens could offer new possibilities for the prediction of progression to TB disease after exposure .