Rapid tuberculosis diagnosis from respiratory or blood samples by a low cost, portable lab-in-tube assay

Rapid portable assays are needed to improve diagnosis, treatment, and reduce transmission of tuberculosis (TB), but current tests are not suitable for patients in resource-limited settings with high TB burden. Here we report a low complexity, lab-in-tube system that is read by an integrated handheld device that detects <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) DNA in blood and respiratory samples from a variety of clinical settings. This microprocessor-controlled device uses an LCD user interface to control assay performance, automate assay analysis, and provide results in a simple readout. This point-of-care single-tube assay uses a DNA enrichment membrane and a low-cost cellulose disc containing lyophilized recombinase polymerase amplification and CRISPR-Cas12a reagents to attain single-nucleotide specificity and high sensitivity within 1 hour of sample application, without a conventional DNA isolation procedure. Assay results obtained with serum cell-free DNA isolated from a cohort of children aged 1 to 16 years detected pulmonary and extrapulmonary TB with high sensitivity versus culture and GeneXpert MTB/RIF results (81% versus 55% and 68%) and good specificity (94%), meeting the World Health Organization target product profile criteria for new nonsputum TB diagnostics. Changes in assay results for serum isolated during treatment were also highly predictive of clinical response. Results obtained with noninvasive sputum and saliva specimens from adults with bacteriologically confirmed pulmonary TB were also comparable to those reported for reference methods. This rapid and inexpensive lab-in-tube assay approach thus represents one means to address the need for point-of-care TB diagnostics useable in low-resource settings.