Background Antigen specific release of IP-10 is an established marker for infection with M.tuberculosis . Compared to IFN-γ, IP-10 is released in 100-fold higher concentrations enabling the development of novel assays for detection. Dried blood spots are a convenient sample for high throughput newborn screening. Aim To develop a robust and sensitive ELISA-based assay for IP-10 detection in plasma, dried blood spots (DBS) and dried plasma spots (DPS); to validate the ELISA in clinically relevant samples; and to assess the performance of the assay for detection of Cytomegalovirus (CMV) and M.tuberculosis specific immune responses. Method We raised mice and rat monoclonal antibodies against human IP-10 and developed an ELISA. The assay was validated and applied to the detection of CMV and M.tuberculosis specific responses in 18 patients with immune reactivity towards M.tuberculosis and 32 healthy controls of which 22 had immune reactivity towards CMV and none towards M.tuberculosis . We compared the performance of this new assay to IFN-γ. Results The ELISA was reliable for IP-10 detection in both plasma and filter paper samples. The linear range of the ELISA was 2.5–600 pg/ml. IFN-γ was not readily detectable in DPS samples. IP-10 was stabile in filter paper samples for at least 4 weeks at 37°C. The correlation between IP-10 detected in plasma, DPS and DBS samples was excellent (r 2 >0.97). Conclusions This newly developed assay is reliable for IP-10 quantification in plasma, DBS and DPS samples from antigen stimulated and non-stimulated whole blood. The filter paper assays enable easy sample acquisition and transport at ambient temperature e.g. via the postal system. The system can potentially simplify diagnostic assays for M.tuberculosis and CMV infection.

In the absence of a validated correlate of protection or robust animal models for human tuberculosis, Mycobacterial growth inhibition assays (MGIAs) aim to assess vaccines ability to inhibit mycobacterial growth in-vitro. We optimised a reproducible murine splenocyte MGIA based on in-vitro infection with virulent Mycobacterium tuberculosis (M.tb) Erdman. We identified splenocyte viability as a problem in state-of-art MGIA protocols, which can be improved by simple changes in culture conditions (viability increase from 21% to 46% at last day of culture). The growth inhibitory potential in mice immunised with either BCG, H56:CAF01 or H56:CAF01 administered side-by-side with BCG was significantly better compared to placebo in all groups (0.3 log10 CFU [±0.2, p = 0.049], 0.5 [±0.2, p = 0.016] and 0.6 [±0.1, p = 0.0007], respectively) corresponding to the levels of in-vivo protection. Unexpectedly the CAF01 adjuvant control group also induced significant growth inhibition of 0.3 log10 CFU (±0.2, p = 0.047). Finally, we explored vaccine-associated T cell effector functions. Despite presence of high levels of vaccine-specific T cells, we found no increase in CD4+ T cell number or cytokine expression profile, nor a difference in cytokine levels in the supernatant after four days culture with or without M.tb. Spontaneous IFN-γ release correlated with growth inhibition levels (p = 0.02), however the cellular source was not found.

WHAT'S KNOWN ON THIS SUBJECT: IP-10 is a novel immunologic marker for tuberculosis (TB) infection. It has been suggested that IP-10 may perform better in children compared with the QuantiFERON test, but only a few studies have investigated IP-10 for diagnosing active TB in children. WHAT THIS STUDY ADDS:This study is the first to investigate IP-10 and QuantiFERON for diagnosing TB in children by using consensus classifications. Both IP-10 and QuantiFERON exhibited poor performance in children from a high-burden setting, and performance was especially compromised in young children. METHODS:Hospitalized Tanzanian children with symptoms of TB were tested with the QFT-IT and IP-10 tests and retrospectively classified into diagnostic groups. Adults with confirmed TB were assessed in parallel. RESULTS:A total of 203 children were included. The median age was 3.0 years (interquartile range: 1.2-7.0), 38% were HIV infected, 36% were aged ,2 years, and 58% had a low weight-for-age. IP-10 and QFT-IT test performance was comparable but sensitivity was low: 33% (1 of 3) in children with confirmed TB and 29% (8 of 28) in children with probable TB. Rates of indeterminate responders were high: 29% (59 of 203) for IP-10 and 26% (53 of 203) for QFT-IT. Age ,2 years was associated with indeterminate test outcome for both IP-10 (adjusted odds ratio [aOR]: 2.2; P = .02) and QFT-IT (aOR: 2.4; P = .01). TB exposure was associated with positive IP-10 test outcome (aOR: 3.6; P = .01) but not with positive QFT-IT outcome (aOR 1.4; P = .52). In 102 adults, test sensitivity was 80% for both tests (P = .248).

BackgroundEarly secretory antigenic target-6 (ESAT-6) is an immunodominant Mycobacterium tuberculosis (M.tb) antigen included in novel vaccines against tuberculosis (TB) and in interferon-gamma (IFN-γ) release assays (IGRAs). Therefore, the availability of an ESAT-6–free IGRA is essential to determine M.tb infection status following vaccination with ESAT-6–containing vaccines. We aimed to qualify a recently developed ESAT-6–free IGRA and to assess its diagnostic performance in comparison to QuantiFERON-TB Gold In-tube (QFT).MethodsParticipants with different levels of M.tb exposure and TB disease were enrolled to determine the ESAT-6–free IGRA cutoff, test assay performance in independent cohorts compared to standard QFT, and perform a technical qualification of antigen-coated blood collection tubes.ResultsESAT-6–free IGRA antigen recognition was evaluated in QFT-positive and QFT-negative South African adolescents. The ESAT-6–free IGRA cutoff was established at 0.61 IU/mL, based on receiver operating characteristic analysis in M.tb-unexposed controls and microbiologically confirmed pulmonary TB patients. In an independent cohort of healthy adolescents, levels of IFN-γ released in QFT and ESAT-6–free IGRA were highly correlated (P < .0001, r = 0.83) and yielded comparable positivity rates, 41.5% and 43.5%, respectively, with 91% concordance between the tests (kappa = 0.82; 95% confidence interval, 0.74–0.90; McNemar test P = .48). ESAT-6–free IGRA blood collection tubes had acceptable lot-to-lot variability, precision, and stability.ConclusionsThe novel ESAT-6–free IGRA had diagnostic accuracy comparable to QFT and is suitable for use in clinical trials to assess efficacy of candidate TB vaccines to prevent established M.tb infection.