Abstract

BackgroundInterferon-γ release assays (IGRA) with Resuscitation promoting factor (Rpf) proteins enhanced tuberculosis (TB) screening and diagnosis in adults but have not been evaluated in children. Children often develop paucibacillary TB and their immune response differs from that of adults, which together affect TB disease diagnostics and immunodiagnostics. We assessed the ability of Rpf to identify infection among household TB-exposed children in The Gambia and investigated their ability to discriminate Mycobacterium tuberculosis complex (MTBC) infection from active TB disease in children.MethodsDetailed clinical investigations were done on 93 household TB-exposed Gambian children and a tuberculin skin test (TST) was administered to asymptomatic children. Venous blood was collected for overnight stimulation with ESAT-6/CFP-10-fusion protein (EC), purified protein derivative and RpfA, B, C, D and E. Interferon gamma (IFN-γ) production was measured by ELISA in supernatants and corrected for the background level. Infection status was defined by IGRA with EC and TB disease by mycobacterial confirmation and/or clinical diagnosis. We compared IFN-γ levels between infected and uninfected children and between infected and TB diseased children using a binomial logistic regression model while correcting for age and sex. A Receiver Operating Characteristics analysis was done to find the best cut-off for IFN-γ level and calculate sensitivity and specificity.ResultsInterferon gamma production was significantly higher in infected (IGRA+, n = 45) than in uninfected (IGRA-, n = 20) children after stimulation with RpfA, B, C, and D (P = 0.03; 0.007; 0.03 and 0.003, respectively). Using RpfB and D-specific IFN-γ cut-offs (33.9 pg/mL and 67.0 pg/mL), infection was classified with a sensitivity-specificity combination of 73–92% and 77–72% respectively, which was similar to and better than 65–75% for TST. Moreover, IFN-γ production was higher in infected than in TB diseased children (n = 28, 5 bacteriologically confirmed, 23 clinically diagnosed), following RpfB and D stimulation (P = 0.02 and 0.03, respectively).ConclusionRpfB and RpfD show promising results for childhood MTBC infection screening, and both performed similar to and better than the TST in our study population. Additionally, both antigens appear to discriminate between infection and disease in children and thus warrant further investigation as screening and diagnostic antigens for childhood TB.

Highlights

  • Interferon-γ release assays (IGRA) with Resuscitation promoting factor (Rpf) proteins enhanced tuberculosis (TB) screening and diagnosis in adults but have not been evaluated in children

  • The interferon gamma (IFN-γ) release assay (IGRA) –employing antigens 6 kDa early secretory antigenic target (ESAT-6) and 10 kDa culture filtrate (CFP-10) (EC) as peptide pool or as fusion protein—is less sensitive for detection of M. africanum (Maf) infection compared to the classical M. tuberculosis (Mtb) sensu stricto strains infection [6]; important for countries like The Gambia where up to half of Mycobacterium tuberculosis complex (MTBC) infections are caused by Maf strains [7]

  • IGRA results and MTBC infection To define MTBC infection status of the study participants, the ESAT-6/CFP-10-fusion protein (EC)-fusion protein IGRA result was taken as gold standard, with IFN-γ cut-off 124.2 pg/mL

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Summary

Introduction

Interferon-γ release assays (IGRA) with Resuscitation promoting factor (Rpf) proteins enhanced tuberculosis (TB) screening and diagnosis in adults but have not been evaluated in children. Screening for Mycobacterium tuberculosis complex (MTBC) infection is done by the widely implemented tuberculin skin test (TST) or the interferon gamma (IFN-γ) release assay (IGRA), both assessing the host’s cell mediated immune response to tuberculous antigens [3]. The IGRA –employing antigens 6 kDa early secretory antigenic target (ESAT-6) and 10 kDa culture filtrate (CFP-10) (EC) as peptide pool or as fusion protein—is less sensitive for detection of M. africanum (Maf) infection compared to the classical M. tuberculosis (Mtb) sensu stricto strains infection [6]; important for countries like The Gambia where up to half of MTBC infections are caused by Maf strains [7] Both the TST and IGRA cannot distinguish between infection and TB disease or individuals with high risk of progressing towards TB disease [8, 9]. 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

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