Novel interferon-gamma assays for diagnosing tuberculosis in young children in India

Abstract

Setting: The tuberculin skin test (TST) and interferon-gamma release assays (IGRAs) are used as supportive evidence to diagnose active tuberculosis (TB). Novel IGRAs could improve diagnosis, but data are lacking in young children.

Design: Children (age 5 years) with suspected TB were prospectively screened at a tertiary hospital in Pune, India; the children underwent TST, and standard (early secretory antigenic target 6 and culture filtrate protein 10) and enhanced (five additional novel antigens) enzyme-linked immunospot (ELISpot) assays.

Results: Of 313 children (median age 30 months) enrolled, 92% had received bacille Calmette-Guérin vaccination, 53% were malnourished and 9% were coinfected with the human immunodeficiency virus (HIV); 48 (15%) had TB, 128 (41%) did not, and TB could not be ruled out in 137 (44%). The sensitivity of enhanced (45%) and standard (42%) ELISpot assays for diagnosing TB was better than that of TST (20%) (P  0.03); however, enhanced ELISpot was not more sensitive than the standard ELISpot assay (P = 0.50). The specificity of enhanced ELISpot, standard ELISpot and TST was respectively 82% (95%CI 74-89), 88% (95%CI 81-94) and 98% (95%CI 93-100). Rv3879c and Rv3615c, previously reported to be promising antigens, failed to improve the diagnostic performance of the ELISpot assay.

Conclusion: The TST and the standard and novel ELISpot assays performed poorly in diagnosing active TB among young children in India.

Figure A1

TB diagnostic categories (adapted from Jain et al.). TB = tuberculosis; CSF = cerebrospinal fluid; WBC = white blood cell; CT = computed tomography; MRI = magnetic resonance imaging.

Figure A2

Median SFU response for children who had blood collected within 3 days of TST placement vs. those who had blood collected beyond 3 days. TB = tuberculosis; SFU = spot-forming unit; PPD = purified protein derivative; ESAT-6 = early secretory antigenic target 6; CFP-10 = culture filtrate protein 10.

Figure 1

Consort diagram. Of the 302 (96%) children who had blood available for ELISpot assays,* 30 (9%) had invalid ELISpot results; 303 (97%) TSTs were placed and read. ‘Definite TB’ included children with any clinical specimen positive for mycobacterial culture. ‘Probable TB’ included children without microbiological confirmation but with a clinical picture suggestive of active TB. ‘No TB’ included children with negative mycobacterial cultures and complete resolution of symptoms without TB treatment. ‘Possible TB’ comprised all other children in whom TB could be neither confirmed nor excluded. * ELISpot includes standard and enhanced IGRA results. TB = tuberculosis; TST = tuberculin skin test; ELISpot = enzyme linked immunospot-based assay; IGRA = interferon-gamma release assay.

Figure 2

A) ROC for standard ELISpot, enhanced ELISpot, standard ELISpot combined with Rv3615c antigens and standard ELISpot combined with Rv3879c antigens for the diagnosis of definite TB. AUCs for standard ELISpot = 0.54 (95% CI 0.37–0.72, P = reference), enhanced ELISpot = 0.52 (95% CI 0.35–0.69, P = 0.67), standard ELISpot with Rv3615c = 0.57 (95% CI 0.40–0.74, P = 0.53), standard ELISpot with Rv3879c = 0.52 (95% CI 0.35–0.70, P = 0.07). P values are for differences between AUCs with standard ELISpot as reference. B) ROC for standard ELISpot, enhanced ELISpot, standard ELISpot combined with Rv3615c antigens and standard ELISpot combined with Rv3879c antigens for the diagnosis of active TB. AUCs for standard ELISpot = 0.52 (95% CI 0.41–0.63, P = reference), enhanced ELISpot = 0.48 (95% CI 0.38–0.59, P = 0.18), standard ELISpot with Rv3615c = 0.53 (95%CI 0.42–0.63, P = 0.77), standard ELISpot with Rv3879c = 0.50 (95% CI 0.39–0.60, P = 0.05). P values are for differences between AUCs with standard ELISpot as reference. ELISpot = enzyme linked immunospot-based assay; ROC = receiver operating characteristic; TB = tuberculosis; AUC = area under the curve; CI = confidence interval. This image can be viewed online in colour at http://www.ingentaconnect.com/content/iuatld/ijtld/2017/00000021/00000004/art00010

Figure 3

A) ELISpot assay median SFUs in response to recombinant Mycobacterium tuberculosis antigens among children with and those without active TB. Median SFUs in response to PPD were significantly higher than those in response to ESAT-6, CFP-10, Rv3878c, Rv3878, Rv3873, Rv2654 and Rv3615c antigens (P < 0.001) among children with and those without active TB. B) ELISpot assay median SFUs in response to recombinant M. tuberculosis antigens among children in the no TB category and those with definite TB. Median SFUs in response to PPD were significantly higher than those in response to ESAT-6, Rv3878c, Rv3878, Rv3873, Rv2654 and Rv3615c antigens (P < 0.01), but not CFP-10 (P = 0.10) among children with definite TB. TB = tuberculosis; SFU = spot-forming unit; PPD = purified protein derivative; ESAT-6 = early secretory antigenic target 6; CFP-10 = culture filtrate protein 10. This image can be viewed online in colour at http://www.ingentaconnect.com/content/iuatld/ijtld/2017/00000021/00000004/art00010