Diagnostic accuracy of Mycobacterium tuberculosis cell-free DNA for tuberculosis: A systematic review and meta-analysis

Abstract

Background Diagnosis of tuberculosis (TB) is still difficult. The purpose of our study was to evaluate the diagnostic accuracy of Mycobacterium tuberculosis cell-free DNA (cfDNA) for diagnosing of TB. Methods We searched relevant databases for studies that used cfDNA to diagnose TB. We evaluated the accuracy of cfDNA compared with the composite reference standard (CRS) and culture. True positive, false positive, false negative, and true negative values for cfDNA were obtained first, then the estimated pooled sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic odds ratio (DOR), and the area under the summary receiver operating characteristic (SROC) curve (AUC) of cfDNA for diagnosing TB were calculated with 95% confidence intervals (CIs). Heterogeneity was determined using the I2 statistic. When the heterogeneity was obvious, the source of heterogeneity was further discussed. Results We included 14 independent studies comparing cfDNA with the CRS, and 4 studies compared with culture. The pooled sensitivity, specificity, PPV, NPV, DOR, and AUC of the SROC were 68%, 98%,99%, 62%, 83, and 0.97 as compared with the CRS, respectively. The pooled sensitivity, specificity, PPV, NPV, DOR, and AUC of the SROC were 48%, 91%, 92%, 60%, 5, and 0.88 as compared with culture, respectively. The heterogeneity between studies was significant. Conclusions The accuracy of cfDNA testing for TB diagnosis was good compared with CRS and culture. cfDNA can be used for rapid early diagnosis of TB.

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

“…CRISPR-TB detected Mtb -cfDNA (median 0·13 copies per μL, IQR 0·07–1·57; mean 5·58 copies per μL, SD 18·25) in the earliest available serum of 102 children ( figure 3A ; appendix p 11), and about half had Mtb -cfDNA concentrations lower than the LOD of PCR (0·25 copies per μL; appendix p 10) in agreement with previously reported PCR analytical 22 and diagnostic sensitivity estimates. 9 – 11 Median CRISPR-TB signal of children with confirmed and unconfirmed tuberculosis was significantly higher than among children with unlikely tuberculosis who met no NIH tuberculosis criteria. Median CRISPR-TB among children with unlikely tuberculosis with any NIH tuberculosis criterion was higher than those with no NIH criteria, but not significantly so ( figure 3C ; table 2 ; appendix p 12).…”

“…Sensitive detection of circulating Mtb -cfDNA could potentially identify both pulmonary tuberculosis and extrapulmonary tuberculosis given that Mtb -cfDNA released during apoptosis and M tuberculosis lysis 9 , 23 should accumulate in the circulation during tissue perfusion irrespective of its sites of origin. However, previous studies have determined that Mtb -cfDNA circulates at very low concentrations, 22 resulting in poor and highly variable diagnostic sensitivity, 9 – 11 which was not improved by use of a CRISPR assay with a high LOD. 14 Here we provide evidence that the combination of an assay optimised for serum cfDNA extraction and sensitive and specific target amplification and cleavage can sensitively detect serum Mtb -cfDNA in most patients with tuberculosis, including those missed by sputum-based diagnostics.…”

“…Sensitive detection of pathogen-derived cell-free DNA (cfDNA) in the circulation has potential for diagnosis and treatment evaluation, 6 – 8 but PCR-based studies that have analysed circulating Mycobacterium tuberculosis cfDNA ( Mtb -cfDNA) have reported poor and highly variable diagnostic sensitivity. 9 – 11 Clustered regularly-interspaced short palindromic repeats (CRISPR)-Cas12a cleavage activity can be used to enhance detection sensitivity, given that binding of a Cas12a guide RNA (gRNA) complex to its target DNA sequence can be used to cleave a quenched fluorescent probe, causing an enzymatic signal amplification that can markedly enhance the detection of low-concentration target sequences. This approach has been applied to detect M tuberculosis -derived DNA targets in sputum, 12 , 13 but had poor performance when applied to detect circulating Mtb -cfDNA, 14 because of the high cfDNA limit of detection (LOD) of the employed diagnostic assay.…”

Reimagining the status quo: How close are we to rapid sputum-free tuberculosis diagnostics for all?

“…While there are no validated blood tests for active TB, there is increasing optimism regarding the detection of antigens, immune cell profiling, host transcriptomics, or cell-free Mtb DNA. 62 Several studies have demonstrated associations between host mRNA signatures and TB risk, although preventive therapy guided by one such mRNA biosignature failed to reduce TB incidence in a large randomised controlled trial. 63 Despite commercial progress to develop mRNA biosignature assays, foremost of which is the Xpert Host Response (Xpert HR) cartridge that detects a 3-gene signature in capillary blood directly using the widely-deployed GeneXpert platform, 64 independent validations 65 , 66 demonstrate most transcriptomic biomarkers will not meet the WHO diagnostic accuracy criteria for triage or confirmation.…”

“…Proof-of-concept data has been generated for Mtb DNA detection in peripheral blood mononuclear cells 80 and cell-free DNA but no prototypes with public data are available. 62 , 81 …”