Evaluation of a serum-based antigen test for tuberculosis in HIV-exposed infants: a diagnostic accuracy study

Abstract

Background: Non-sputum methods are urgently needed to improve tuberculosis diagnosis and treatment monitoring in children. This study evaluated the ability of a serum assay quantifying a species-specific peptide of the Mycobacterium tuberculosis CFP-10 virulence factor via nanotechnology and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to diagnose tuberculosis in HIV-infected and HIV-uninfected infants.

Methods: Serum CFP-10 peptide signal was blinded evaluated in cryopreserved sera of 519 BCG-immunized, HIV-exposed infants (284 HIV-infected, 235 HIV-uninfected) from a multi-center randomized placebo-controlled isoniazid prophylaxis trial conducted in southern Africa between 2004 and 2008, who were followed up to 192 weeks for Mtb infection and TB. Children were classified as confirmed, unconfirmed, or unlikely tuberculosis cases using 2015 NIH diagnostic criteria for pediatric TB.

Results: In HIV-infected infants, CFP-10 signal had 100% sensitivity for confirmed TB (5/5, 95% CI, 47.8-100) and 83.7% sensitivity for unconfirmed TB (36/43, 95% CI 69.3-93.2), with 93.1% specificity (203/218, 95% CI 88.9-96.1). In HIV-uninfected infants, CFP-10 signal detected the single confirmed TB case and 75.0% of unconfirmed TB cases (15/20; 95% CI 50.9-91.3), with 96.2% specificity (177/184, 95% CI, 92.3-98.5). Serum CFP-10 achieved 77% diagnostic sensitivity for confirmed and unconfirmed TB (13/17, 95% CI, 50-93%) at ≤ 24 weeks pre-diagnosis, and both CFP-10-positivity and concentration declined following anti-TB therapy initiation.

Conclusions: Serum CFP-10 signal exhibited high diagnostic sensitivity and specificity for tuberculosis in HIV-infected and HIV-uninfected infants and potential utility for early TB detection and monitoring of anti-TB treatment responses.

Keywords: CFP-10; Mass spectrometry; Nanotechnology; Pediatric tuberculosis.

Fig. 1

Inclusion of P1041 parent study participants with evaluable clinical information and serum samples. All children that met inclusion criteria were classified using 2015 NIH criteria for TB diagnosis into one of three groups: (1) confirmed TB: all children with a positive Mtb culture result; (2) unconfirmed TB: all children with ≥ 2 different categories of non-bacteriological evidence of TB; and (3) unlikely TB (non-TB): all children with no evidence, or insufficient evidence, of TB or a confirmed alternative diagnosis

Fig. 2

Serum CFP-10 can distinguish TB from unlikely TB cases and the comparison with other methods. a Serum CFP-10 signal in HIV-infected and HIV-uninfected children with TB and unlikely (UL) TB, where horizontal bars indicate mean values. ***p < 0.001 by ANOVA with Tukey’s post-test. b Venn diagram of all positive CFP-10pep, and Mtb culture and smear results. Note that one child diagnosed with unconfirmed TB subsequently had a positive Mtb culture, resulting in a mismatch between the overall number of confirmed TB cases and positive Mtb cultures. The positive proportions of culture, smear and CFP-10pep among extrapulmonary TB c PTB only and d EPTB cases

Fig. 3

Serum CFP-10pep assay in early detection, recurrent TB, and TB treatment monitoring. a Distribution of CFP-10pep-positive and CFP-10pep-negative signal (above the x-axis) and culture and/or smear positive and culture + smear negative results (below the x-axis) in total TB cases, where time zero (vertical dashed line) denotes time of TB diagnosis. Only one sample per child was available and evaluated in each 12-week interval. b Recurrent TB cases. The orange vertical lines denote time of TB diagnosis relative to study enrollment, the pink box indicates the duration of an anti-TB treatment, and the vertical green lines indicate the time of TB resolution, while the red dots and blue bar connected by red lines indicate the time and intensity of serum CFP-10pep signal. c Serum CFP-10pep signal changes from pre-diagnosis (pre) to post-diagnosis (post) in TB cases with (red dots) and without (blue dots) positive anti-TB treatment responses. Connected points indicate the magnitude and time frame of changes for individual patients. **p < 0.01 by paired t test, *p < 0.05 by repeated measure mixed model to compare the paired difference between TB cases with and without positive anti-TB treatment responses