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

Abstract

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 Mycobacterium tuberculosis (Mtb) 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.

Fig. 1.. Schematic of a CRISPR-based POC TB diagnostic device that detects Mtb DNA in multiple specimen types.

(A) LIT-TB assay workflow indicating the time required for its sample lysis, lysate loading, assay incubation, and analysis steps. (B) Design and components of the LIT-TB assay tube. (C) Overview of sample inputs and assay functions of the handheld device to process the diagnostic specimen and capture and analyze the assay results. (D) A 3D rendering of the components of the POC LIT assay device.

Fig. 2.. RPA-CRISPR assay optimization and portable device development.

(A) Signal produced by RPA-CRISPR reactions using gRNAs with (gRNA1) or without consensus PAM sequences (gRNA2 and gRNA3). n = 3. *P < 0.05 and **P < 0.01 versus gRNA2 + gRNA3 by Kruskall-Wallis test with Dunn’s test for multiple comparisons. (B) Evaluation of gRNA2 + gRNA3 multiplex assay performance at different temperatures. n = 3. (C) RPA-CRISPR reaction kinetics for Mtb DNA detection when using RPA-CRISPR reagents lyophilized on different support matrices over time. n = 3. (D) Storage temperature effects on lyophilized RPA-CRISPR reagent activity. NTC, no template control. n = 3. (E) Circuit diagram of the microprocessor connections controlling image sensor, LED, heater, LCD screen, and wireless output components of the assay device. Vcc, voltage at the common collector. (F) Heating efficiency values with different insulating materials in the LIT incubator port. n = 5. **P < 0.01 versus ambient air by Kruskall-Wallis test with Dunn’s test for multiple comparisons. (G) Workflow used to optimize ROI detection. Training data images were assessed by shrinking a circle centered on the fixed position of the LIT-TB reagents to exclude mean pixel values that do not differ from background noise. Radii of varying size were compared to identify the optimal size of the ROI for analysis. I_x, intensity for a given radius x; I_x+n, intensity for a radius x plus n; I_blank, intensity of the background (no-template control); I_b, mean intensity for a given distance n radius away from the center. (H) Representative images of the detection ROI with RPA-CRISPR signal and (I) a concentration curve generated with DNA isolated from healthy serum spiked with serial dilutions of Mtb DNA. The black dotted line is the linear regression best-fit line, and red dotted lines are the 95% CI. n = 3. (J) Signal detected in RPA-CRISPR assays performed with DNA isolated from Mtb and other mycobacteria species. n = 3. (K) RPA-CRISPR assay specific for a drug-resistant mutant (rpoB S450L) performed using DNA from drug-resistant mutant Mtb, wild-type Mtb, or NTC. n = 3. Data in (A) to (D), (F), (J), and (K) are presented as means ± SD, and dots in (A), (D), (F), (J), and (K) represent replicates. a.u., arbitrary units.

Fig. 3.. LIT-TB assay performance with pediatric TB cohort serum samples.

(A) Clinical findings and LIT-TB results for different TB subgroups. N/A, not available. n = 1, PTB and EPTB; n = 12, confirmed PTB; n = 6, unconfirmed PTB; n = 8, EPTB. (B) TST and serial LIT-TB results in pediatric close contacts of the TB cohort. n = 35. (C) Serum LIT-TB signals at baseline and after treatment for all TB cases with follow-up samples (n = 26). (D) Serum LIT-TB signals over time in confirmed PTB (n = 11), unconfirmed PTB (n = 6), and EPTB (n = 8) cases before and after treatment initiation. Data in (C) and (D) are presented as means ± SD, where the dashed line denotes the positive signal threshold. *P < 0.05, **P < 0.01, and ***P < 0.001 versus baseline by Welch ANOVA with Dunnett’s T3 test for multiple comparisons. n.s., not significant. (E to H) Clinical findings and serum LIT-TB results before and after anti-TB treatment initiation for a child with EPTB who had rapid treatment responses (E), a child with PTB and EPTB who displayed EPTB symptoms through 8-week posttreatment initiation with clinical response at week 35 (F), and children with confirmed TB who had drug resistance (G) or were noncompliant with the treatment regimen between 4 and 10 weeks posttreatment initiation but exhibited treatment response after adjusted treatment or increased compliance (H). Positive signal threshold (dashed line) was calculated using mean plus SD of triplicate NTC samples. Data represent single LIT-TB results in the designated patients at the indicated time points. NIH, National Institutes of Health.

Fig. 4.. LIT-TB diagnostic performance with direct saliva and sputum specimens obtained from adult TB cohorts.

(A) Schematic of in-tube sputum and saliva liquification by DTT and heat-mediated DNAse and Mtb inactivation and Mtb lysis. (B) Sputum absorbance at 600 nm before and after a 10-min RT incubation with the indicated chemicals, where decreased absorbance indicates sputum solubilization. n = 3 replicates per condition. (C) RPA-CRISPR fluorescent signal detected after assay DNA capture discs made from different materials were incubated with Mtb DNA-spiked sputum lysates, rinsed, and added to RPA-CRISPR reactions. PC, polycarbonate. n = 3 replicates per condition. (D) Clinical findings and saliva LIT-TB results for a case-control TB cohort and (E) relative fluorescent intensity detected for these samples. **P < 0.01 by Welch’s t test. n = 15, Mtb negative; n = 15, active TB. (F) Clinical findings and sputum LIT-TB results for a cohort of individuals diagnosed with TB disease or NTM infections or with no evidence of Mtb infection and (G) relative fluorescent intensity detected for these samples. ****P < 0.0001 by Welch’s t test. n = 23, culture negative; n = 8, NTM; n = 5, Mtb. (H) Sputum LIT-TB signal detected for two patients with TB with serial samples collected at ≤2 and ≥ 6 weeks after anti-TB treatment initiation. Positive signal thresholds for saliva and sputum cohorts were calculated by the mean plus three times the SD of triplicate NTC normal saliva and artificial sputum samples. Optimization experiments were run in triplicate, and patient testing results were obtained from a single LIT-TB test. Data in (B), (C), (E), and (G) are presented as means ± SD; dots represent individual samples. Schematic illustration created with BioRender. OD, optical density.