CRISPR detection of circulating cell-free Mycobacterium tuberculosis DNA in adults and children, including children with HIV: a molecular diagnostics study

Abstract

Background: Tuberculosis remains a leading cause of global mortality, especially for adults and children living with HIV (CLHIV) underdiagnosed by sputum-based assays. Non-sputum-based assays are needed to improve tuberculosis diagnosis and tuberculosis treatment monitoring. Our aim in this study was to determine whether ultrasensitive detection of Mycobacterium tuberculosis cell-free DNA (Mtb-cfDNA) in blood can diagnose tuberculosis and evaluate tuberculosis treatment responses.

Methods: In this molecular diagnostics study we analysed archived serum from two patient populations evaluated for tuberculosis in Eswatini and Kenya to detect Mtb-cfDNA, analysing serum from all individuals who had both sufficient serum volumes and clear diagnostic results. An optimised CRISPR-mediated tuberculosis (CRISPR-TB) assay was used to detect Mtb-cfDNA in serum at enrolment from adults and children with presumptive tuberculosis and their asymptomatic household contacts, and at enrolment and during tuberculosis treatment from a cohort of symptomatic CLHIV at high risk for tuberculosis, who provided longitudinal serum at enrolment and during tuberculosis treatment.

Findings: CRISPR-TB identified microbiologically and clinically confirmed tuberculosis cases in the predominantly HIV-negative Eswatini adult cohort with 96% sensitivity (27 [96%] of 28, 95% CI 80-100) and 94% specificity (16 [94%] of 17, 71-100), and with 83% sensitivity (5 [83%] of 6, 36-100) and 95% specificity (21 [95%] of 22, 77-100) in the paediatric cohort, including all six cases of extrapulmonary tuberculosis. In the Kenyan CLHIV cohort, CRISPR-TB detected all (13 [100%] of 13, 75-100) confirmed tuberculosis cases and 85% (39 [85%] of 46, 71-94) of unconfirmed tuberculosis cases diagnosed by non-microbiological clinical findings. CLHIV who were CRISPR-TB positive at enrolment had a 2·4-times higher risk of mortality by 6 months after enrolment. Mtb-cfDNA signal decreased after tuberculosis treatment initiation, with near or complete Mtb-cfDNA clearance by 6 months after tuberculosis treatment initiation.

Interpretation: CRISPR-mediated detection of circulating Mtb-cfDNA shows promise to increase the identification of paediatric tuberculosis and HIV-associated tuberculosis, and potential for early diagnosis and rapid monitoring of tuberculosis treatment responses.

Funding: US Department of Defense, National Institute of Child Health and Human Development, National Institute of Allergy and Infectious Diseases, University of Washington Center for AIDS Research, and the Weatherhead Presidential Endowment fund.

Figure 1:. Study participants

CRISPR-TB diagnostic performance was evaluated by blind analysis of serum before tuberculosis treatment from a cohort of adults and children with presumptive tuberculosis and their asymptomatic household contacts (Eswatini cohort), and longitudinal serum collected at enrolment and during tuberculosis treatment in a cohort of children living with HIV at high risk for tuberculosis (PUSH cohort). Mtb-cfDNA=Mycobacterium tuberculosis cell-free DNA. *Adults were classified using WHO guidelines (appendix p 22). †Children were classified using modified NIH classification criteria (appendix p 23). ‡Children with missing chest x-ray results were excluded because they could not be classified as having unconfirmed or unlikely tuberculosis. §Triplicate CRISPR-TB results that had coefficients of variation of 20% or higher were considered invalid. ¶142 children had serum available at baseline; 11 children had their first serum sample available at a later visit. ||People without a confirmed tuberculosis diagnosis who initiated tuberculosis treatment were considered to have a clinical tuberculosis diagnosis. People without a confirmed tuberculosis who did not initiate tuberculosis treatment were considered to have no clinical tuberculosis diagnosis. **NIH tuberculosis symptoms suggestive of tuberculosis (appendix p 23).

Figure 2:. Analytical validation of the CRISPR-TB assay

Three CRISPR-TB target sequences mapped in the genome of Mycobacterium tuberculosis H37Ra (A), and their signal detected in serial dilutions of extracted M tuberculosis (H37Ra) gDNA (B) and in serum samples of patients with culture-confirmed tuberculosis (C; n=7), indicating two-sided p values in the Wilcoxon signed-rank tests. (D) CRISPR-TB signal detected with healthy human plasma spiked with or without gDNA from the indicated M tuberculosis or NTM species. (E) CRISPR-TB standard curve, in which shading denotes the 95% CI of the fitted line. CRISPR-TB serum values (F) and clinical and assay data for 45 adults and 28 children (E; Eswatini cohort), categorised as confirmed tuberculosis, clinically diagnosed tuberculosis, unconfirmed tuberculosis, and non-tuberculosis control by study physicians with expertise in pulmonary medicine using results of standardised tuberculosis tests, indicating two-sided p values for Wilcoxon rank-sum tests. Red squares indicate EPTB cases. The mean (SD) of three technical replicates for each sample (B–E) or the median (IQR; F) are presented. The blue dashed line indicates the lowest limit of positive signal cutoff threshold. au=arbitrary units. CT=confirmed tuberculosis. CDxT=clinically diagnosed tuberculosis. EPTB= extrapulmonary tuberculosis. gDNA=genomic DNA. NT=non-tuberculosis control. NTC=no template control. NTM=non-tuberculous mycobacteria. PL intensity= photoluminescence intensity. UT=unconfirmed tuberculosis. Xpert=Gene Xpert MTB/RIF Ultra.

Figure 3:. CRISPR-TB evaluation in children with HIV at high risk of tuberculosis (PUSH cohort)

(A) Clinical and assay data for children with HIV who are immunocompromised and symptomatic classified with confirmed, unconfirmed, and unlikely tuberculosis by NIH consensus diagnostic criteria with the addition of death possibly or likely to be related to tuberculosis, as judged by an expert review panel (appendix p 23). Urine LAM and serum Mtb-cfDNA results were not used for classification. Subgroup A (cases 79–90) included children who had symptom improvement after antiretroviral therapy initiation without tuberculosis treatment initiation (appendix p 33). Red squares indicate EPTB cases. (B) CRISPR-TB agreement with microbiological and clinical data of confirmed and unconfirmed tuberculosis cases. Grey circle indicates 13 children classified as having unconfirmed tuberculosis with negative CRISPR-TB, Xpert, and culture. (C) CRISPR-TB signal in children with confirmed, unconfirmed, and unlikely tuberculosis, with the number of signal positive and total cases in each group, and the two-sided p values from the Wilcoxon rank-sum test. (D) Survival curve for children segregated by high and low CRISPR-TB signal, HRs from a Cox proportional hazard model before and after adjustment for age and CD4 percentage differences. (E) CRISPR-TB signal initiation in 51 children who received tuberculosis treatment and had two or more longitudinal serum samples available for analysis (appendix p 16), with two-sided p values from the Wilcoxon signed rank test. (C, E) Median (IQR) presented for each group. The blue dashed line indicates the lowest limit of positive signal cutoff threshold. au=arbitrary units. aHR=adjusted hazard ratio. EPTB=extrapulmonary tuberculosis. HR=hazard ratio. LAM=mycobacterial lipoarabinomannan. Mtb-cfDNA=Mycobacterium tuberculosis cell-free DNA. NIH=National Institutes of Health. TST=tuberculin skin test. Xpert=Gene Xpert MTB/RIF.