Performance of a stool-based quantitative PCR assay for the diagnosis of tuberculosis in adolescents and adults: a multinational, prospective diagnostic accuracy study

Abstract

Background: Despite increasing availability of rapid molecular tests for the diagnosis of tuberculosis in high-burden settings, many people with tuberculosis are undiagnosed. Reliance on sputum as the primary specimen for tuberculosis diagnostics contributes to this diagnostic gap. We evaluated the diagnostic accuracy and additive yield of a novel stool quantitative PCR (qPCR) assay for the diagnosis of tuberculosis in three countries in Africa with high tuberculosis burdens.

Methods: We undertook a prospective diagnostic accuracy study in Eswatini, Mozambique, and Tanzania from Sept 21, 2020, to Feb 2, 2023, to compare the diagnostic accuracy for tuberculosis of a novel stool qPCR test with the current diagnostic standard for Mycobacterium tuberculosis DNA detection from sputum and stool, Xpert-MTB/RIF Ultra (Xpert Ultra). Sputum, stool, and urine samples were provided by a cohort of participants, aged 10 years or older, diagnosed with tuberculosis. Participants with tuberculosis (cases) were enrolled within 72 h of treatment initiation for tuberculosis diagnosed clinically or following laboratory confirmation. Participants without tuberculosis (controls) consisted of household contacts of the cases who did not develop tuberculosis during a 6-month follow-up. The performance was compared with a robust composite microbiological reference standard (CMRS).

Findings: The cohort of adolescents and adults (n=408) included 268 participants with confirmed or clinical tuberculosis (cases), 147 (55%) of whom were living with HIV, and 140 participants (controls) without tuberculosis. The sensitivity of the novel stool qPCR was 93·7% (95% CI 87·4-97·4) compared with participants with detectable growth on M tuberculosis culture, and 88·1% (81·3-93·0) compared with sputum Xpert Ultra. The stool qPCR had an equivalent sensitivity as sputum Xpert Ultra (94·8%, 89·1-98·1) compared with culture. Compared with the CMRS, the sensitivity of the stool qPCR was higher than the current standard for tuberculosis diagnostics on stool, Xpert Ultra (80·4%, 73·4-86·2 vs 73·5%, 66·0-80·1; p=0·025 on paired comparison). The qPCR also identified 17-21% additional tuberculosis cases compared to sputum Xpert Ultra or sputum culture. In controls without tuberculosis, the specificity of the stool qPCR was 96·9% (92·2-99·1).

Interpretation: In this study, a novel qPCR for the diagnosis of tuberculosis from stool specimens had a higher accuracy in adolescents and adults than the current diagnostic PCR gold standard on stool, Xpert-MTB/RIF Ultra, and equivalent sensitivity to Xpert-MTB/RIF Ultra on sputum.

Funding: National Institutes of Health (NIH) Allergy and Infectious Diseases, and NIH Fogarty International Center.

Figure 1:. Study profile

STARD algorithm for inclusion of participants with tuberculosis and controls without signs and symptoms of tuberculosis who did not develop tuberculosis during a 6-month follow-up. (A) Participants in the stool diagnostics analysis. (B) Participants in the stool qPCR and reference standard analysis. qPCR=quantitative PCR. Xpert=Xpert Ultra assay. CMRS=composite microbiological reference standard. CCRS=clinical composite reference standard. Complete MRS=complete microbiological reference standard. LAM=lipoarabinomannan. *Participants living with HIV (n=147) who qualified by national guidelines (n=122) had a urine LAM test, which was included in these reference standards.

Figure 2:. Alluvial plot of semiquantitative values of qPCR, sputum Xpert Ultra, and stool Xpert Ultra

Alluvial plot showing the relationship between semiquantitative measures of the cycle threshold recorded in stool by a novel qPCR and by Xpert Ultra. Included are 148 participants treated for tuberculosis who had a sputum Xpert Ultra, stool Xpert Ultra, and a stool qPCR undertaken and had a positive result for at least one test. qPCR=quantitative PCR.

Figure 3:. Diagnostic accuracy of the stool qPCR (A and B) and stool Xpert Ultra (C and D) undertaken on stool specimens for the diagnosis of tuberculosis

(A) Sensitivity of the stool qPCR test compared with the CMRS and CCRS in participants with tuberculosis. (B) Specificity of the stool qPCR in the control population without tuberculosis. (C) Sensitivity of the stool Xpert test compared with the CMRS and CCRS in participants with tuberculosis. (D) Specificity of the stool Xpert test in the control population without tuberculosis. The analysis for both assays was completed in the entire cohort and restricted to people living with HIV, HIV-non-reactive participants, adolescents, and adults with a valid stool qPCR or stool Xpert result, respectively. qPCR=quantitative PCR. CMRS=composite microbiological reference standard. CCRS=clinical composite reference standard. PLHIV=people living with HIV. HIV NR=non-reactive HIV. TP=true positive. FN=false negative. FP=false positive. TN=true negative.