Evaluation of Xpert® MTB/RIF assay in induced sputum and gastric lavage samples from young children with suspected tuberculosis from the MVA85A TB vaccine trial

Objective Diagnosis of childhood tuberculosis is limited by the paucibacillary respiratory samples obtained from young children with pulmonary disease. We aimed to compare accuracy of the Xpert ® MTB/RIF assay, an automated nucleic acid amplification test, between induced sputum and gastric lavage samples from young children in a tuberculosis endemic setting. METHODS: We analyzed standardized diagnostic data from HIV negative children younger than four years of age who were investigated for tuberculosis disease near Cape Town, South Africa [2009-2012]. Two paired, consecutive induced sputa and early morning gastric lavage samples were obtained from children with suspected tuberculosis. Samples underwent Mycobacterial Growth Indicator Tube [MGIT] culture and Xpert MTB/RIF assay. We compared diagnostic yield across samples using the two-sample test of proportions and McNemar's χ 2 test; and Wilson's score method to calculate sensitivity and specificity. RESULTS: 1,020 children were evaluated for tuberculosis during 1,214 admission episodes. Not all children had 4 samples collected. 57 of 4,463[1.3%] and 26 of 4,606[0.6%] samples tested positive for Mycobacterium tuberculosis on MGIT culture and Xpert MTB/RIF assay respectively. 27 of 2,198[1.2%] and 40 of 2,183[1.8%] samples tested positive [on either Xpert MTB/RIF assay or MGIT culture] on induced sputum and gastric lavage samples, respectively. 19/1,028[1.8%] and 33/1,017[3.2%] admission episodes yielded a positive MGIT culture or Xpert MTB/RIF assay from induced sputum and gastric lavage, respectively. Sensitivity of Xpert MTB/RIF assay was 8/30[26.7%; 95% CI: 14.2-44.4] for two induced sputum samples and 7/31[22.6%; 11.4-39.8] [p = 0.711] for two gastric lavage samples. Corresponding specificity was 893/893[100%;99.6-100] and 885/890[99.4%;98.7-99.8] respectively [p = 0.025]. CONCLUSION: Sensitivity of Xpert MTB/RIF assay was low, compared to MGIT culture, but diagnostic performance of Xpert MTB/RIF did not differ sufficiently between induced sputum and gastric lavage to justify selection of one sampling method over the other, in young children with suspected pulmonary TB. Trial Registration ClinicalTrials.gov NCT0095392

Demographic and clinical characteristics recorded after long-term follow-up of previously MVA85A-vaccinated individuals.

@<p>Dose of MVA85A vaccine administered intradermally; pfu, plaque forming units.</p>*<p>We applied TST cut-offs consistent with the original trial protocols, namely 15 mm in TB008, and 10 mm in TB014 and TB011.</p><p>N, participants in the original clinical trial; n, participants re-enrolled.</p

Longitudinal tracking of Ag85A-specific IFN-γ ELISPOT responses in <i>M.tb</i>-infected and/or HIV-infected subjects, who were vaccinated with MVA85A 2 to 6 years ago as part of the TB011 trial [24].

<p>(<b>A</b>) <i>M.tb</i>-infected, HIV-negative individuals (n = 11); (<b>B</b>) <i>M.tb</i>-uninfected, HIV-infected individuals (n = 8); (<b>C</b>) <i>M.tb</i> and HIV-co-infected individuals (n = 9); (<b>D</b>) HIV-infected individuals on successful ART (irrespective of <i>M.tb</i>-infection status, n = 9). Frequencies of IFN-γ spot forming cells (SFC) detected before vaccination and at the last time point after MVA85A vaccination were compared using the Wilcoxon signed rank test. Median and IQR IFN-γ ELISPOT response values are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087340#pone.0087340.s004" target="_blank">Table S1</a></b>.</p

Memory phenotype of highly persistent Ag85A-specific CD4 T cells.

<p>(<b>A</b>) Representative flow cytometry overlay plots, from a MVA85A-vaccinated child, showing CD45RA and CCR7 co-expression patterns by the entire CD4 T cell subset (blue) and Ag85A-specific CD4 T cells co-expressing IFN-γ, TNF-α and IL-2 (black dots) after in vitro stimulation with Ag85A peptides, live BCG or PHA. (<b>B</b>–<b>D</b>) Proportions of cytokine-expressing Ag85A-specific CD4 T cells expressing the indicated combination of CD45RA and CCR7, measured 3–5 years after MVA85A vaccination in adolescents (<b>B</b>, n = 9), children (<b>C</b>, n = 16) and infants (<b>D</b>, n = 15, 11 and 12 for groups 1, 2 and 3, respectively). (<b>E</b>) Proportions of cytokine-expressing BCG-specific CD4 T cells expressing the indicated combination of CD45RA and CCR7 in infants (n = 15, 11 and 12 for groups 1, 2 and 3, respectively). For all box and whisker plots, horizontal lines represent medians, boxes represent the IQR and whiskers represent the range for each group of participants.</p

Characterization of Ag85A-specific CD4 T cell Th1 cytokine expression patterns after long-term follow-up, 3–5 years after MVA85A vaccination.

<p>(<b>A</b>) Representative gating of CD4 T cell expression of IFN-γ, IL-2, TNF-α and/or IL-17 in blood left unstimulated, or stimulated with BCG or Ag85A peptide pool, collected 1,187 days post-vaccination from a single participant who was 5 months old when originally vaccinated. (<b>B–E</b>) Frequencies of cytokine-expressing Ag85A or BCG-speciific CD4 T cells were measured by whole blood intracellular cytokine staining assay in adolescents (<b>B</b>), children (<b>C</b>) and infants (<b>D</b> and <b>E</b>). Cytokine expression patterns in adolescents and children who acquired <i>M.tb</i>-infection and those who remained uninfected were not different (data not shown); all individuals, irrespective of <i>M.tb</i>-infection status are shown in <b>B</b> (n = 9) and <b>C</b> (n = 16). Cytokine expression patterns in all infants (<b>D</b> and <b>E</b>, n = 15, 11, 12 and 11 for groups 1, 2, 3 and placebo respectively) were also not significantly different. For all box and whisker plots, horizontal lines represent medians, boxes represent the IQR and whiskers represent the range for each group of participants.</p

Early MVA85A-induced Ag85A-specific T cell responses predict the level of persisting Ag85A-specific T cell responses.

<p>(<b>A</b>) Representative Spearman correlation analysis of the frequencies of Ag85A-specific T cells detected by IFN-γ ELISpot assay on day 84 post-vaccination and more than 3 years post-vaccination in infants who received 5×10⁷ pfu of MVA85A. (<b>B</b>–<b>D</b>) Plots showing the Spearman r values obtained from correlation analyses between the frequencies of Ag85A-specific T cell responses detected after long-term follow-up (more than 1000 days post-vaccination), and those detected before MVA85A vaccination (day 0), or at 7, 28, 84 and 168 days MVA85A vaccination. Blue bars denote Spearman r values with a p-value above 0.05, orange bars denote p-values between 0.05 and 0.01 and red bars denote p-values below 0.01. (<b>B</b>) Infants who received 2.5×10⁷ pfu of MVA85A; (<b>C</b>) Infants who received 5×10⁷ pfu of MVA85A; (<b>D</b>) Infants who received 1×10⁸ pfu of MVA85A.</p

Longitudinal tracking of Ag85A-specific IFN-γ ELISPOT responses in subjects vaccinated with MVA85A 3 to 6 years ago.

<p>Red data points denote measurements from individuals who became infected with <i>M.tb</i> since completion of the original clinical trial. <i>M.tb</i> infection was defined as conversion to a positive IFN-γ ELISpot assay response to ESAT-6/CFP-10. Frequencies of IFN-γ spot forming cells (SFC) detected before vaccination and at the last time point after MVA85A vaccination were compared using the Wilcoxon signed rank test. Blue p-values denote comparison of data from all individuals, while red p-values denote comparison of <i>M.tb</i>-uninfected (negative responders to ESAT-6/CFP-10) individuals only. (<b>A–D</b>) Longitudinal tracking of Ag85A-specific T cell responses in adults (<b>A</b>, n = 17), adolescents (<b>B</b>, n = 9), children (<b>C</b>, n = 15) and infants (<b>D</b>, n = 27), who received 5×10⁷ plaque forming units (pfu) of MVA85A. (<b>E</b>) Comparison of Ag85A-specific IFN-γ ELISPOT responses in subjects who were vaccinated more than 3 years ago with different doses of MVA85A as infants, or who received the placebo vaccine, Prevenar (n = 24 for Group 1, n = 27 for Group 2; n = 24 for Group 3 and n = 23 for the placebo Group). The overall effect was calculated using the Kruskal-Wallis test, while responses in each dose group were compared to the placebo group using the Mann-Whitney U test. Median and IQR IFN-γ ELISPOT response values are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087340#pone.0087340.s004" target="_blank">Table S1</a></b>.</p

Differences in yield of Xpert MTB/RIF assay test by sample or category of sample.

<p>Differences in yield of Xpert MTB/RIF assay test by sample or category of sample.</p

Crude yield from induced sputum and gastric lavage samples for <i>Mycobacterium tuberculosis</i>.

<p>Crude yield from induced sputum and gastric lavage samples for <i>Mycobacterium tuberculosis</i>.</p

Demographic characteristics at baseline of all admission events.

<p>Demographic characteristics at baseline of all admission events.</p