Complementary Nonsputum Diagnostic Testing for Tuberculosis in People with HIV Using Oral Swab PCR and Urine Lipoarabinomannan Detection.

Testing for mycobacterial lipoarabinomannan (LAM) in urine is a practical but insensitive alternative to sputum testing to diagnose tuberculosis (TB) in people with HIV (PWH). Here, we evaluated urine LAM testing alongside PCR-based tests for Mycobacterium tuberculosis (MTB) DNA in tongue swabs. We hypothesized that the two nonsputum samples would deliver complementary, not redundant, results. The study included 131 South African patients of whom 64 (48.1%) were confirmed to have TB by GeneXpert MTB/RIF Ultra (Xpert Ultra) or culture analysis of sputum. A total of 120 patients (91.6%) were coinfected with HIV and 130 yielded a valid urine LAM result (Alere DETERMINE LAM Ag). Tongue swab samples were tested by IS6110-targeted qPCR with a quantification cycle (Cq) cutoff of 32. Relative to reference sputum testing (TB culture and Xpert Ultra), combined urine LAM and oral swab testing (either sample positive) was significantly more sensitive than either nonsputum sample alone (57% sensitivity for combined testing versus 35% and 39% sensitivity for urine LAM and tongue swabs; P = 0.01 and 0.04, respectively). Specificity of combined testing (neither sample positive) was 97%. On average, tongue swab-positive participants had higher sputum signal strength than urine-LAM positive participants, as measured by sputum Xpert Ultra Cq value (P = 0.037). A subset of tongue swabs (N = 18) was also tested by using Xpert Ultra, which reproduced true positive and true negative IS6110 qPCR results and resolved the two false-positive tongue swabs. With further development, tongue swabs and urine may feasibly serve as complementary nonsputum samples for diagnosis of TB in PWH.

Diagnosing Pulmonary Tuberculosis by Using Sequence-Specific Purification of Urine Cell-Free DNA.

Transrenal urine cell-free DNA (cfDNA) is a promising tuberculosis (TB) biomarker, but is challenging to detect because of the short length (<100 bp) and low concentration of TB-specific fragments. We aimed to improve the diagnostic sensitivity of TB urine cfDNA by increasing recovery of short fragments during sample preparation. We developed a highly sensitive sequence-specific purification method that uses hybridization probes immobilized on magnetic beads to capture short TB cfDNA (50 bp) with 91.8% average efficiency. Combined with short-target PCR, the assay limit of detection was ≤5 copies of cfDNA in 10 ml urine. In a clinical cohort study in South Africa, our urine cfDNA assay had 83.7% sensitivity (95% CI: 71.0 to 91.5%) and 100% specificity (95% CI: 86.2 to 100%) for diagnosis of active pulmonary TB when using sputum Xpert MTB/RIF as the reference standard. The detected cfDNA concentration was 0.14 to 2,804 copies/ml (median 14.6 copies/ml) and was inversely correlated with CD4 count and days to culture positivity. Sensitivity was nonsignificantly higher in HIV-positive (88.2%) compared to HIV-negative patients (73.3%), and was not dependent on CD4 count. Sensitivity remained high in sputum smear-negative (76.0%) and urine lipoarabinomannan (LAM)-negative (76.5%) patients. With improved sample preparation, urine cfDNA is a viable biomarker for TB diagnosis. Our assay has the highest reported accuracy of any TB urine cfDNA test to date and has the potential to enable rapid non-sputum-based TB diagnosis across key underserved patient populations.

Real-world diagnostic accuracy of lipoarabinomannan in three non-sputum biospecimens for pulmonary tuberculosis disease.

BACKGROUND: Development of a non-sputum test using readily-obtainable biospecimens remains a global priority for tuberculosis (TB) control. We quantified lipoarabinomannan (LAM) concentrations, a pathogen biomarker for Mycobacterium tuberculosis, in urine, plasma and serum for real-world diagnostic accuracy of pulmonary TB among people living with and without HIV. METHODS: We conducted a prospective diagnostic study among adults with TB symptoms in South Africa. We measured LAM concentrations in time-matched urine, plasma and serum with an electrochemiluminescence immunoassay using two capture antibodies (FIND 28 and S4-20). From the completed cohort, we randomly selected 210 participants (2 cases: 1 control) based on sensitivity estimates, and we compared diagnostic accuracy of LAM measurements against the microbiological reference standard. FINDINGS: Urine and blood specimens from 210 of 684 adults enrolled were tested for LAM. Among 138 TB-positive adults (41% female), median urine LAM was 137 pg/mL and 52 pg/mL by FIND 28 and S4-20, respectively. Average LAM concentrations were highest in HIV-positive participants with CD4+ T cells <200 cells/mm3. Urine LAM by S4-20 achieved diagnostic sensitivity of 62% (95% CI: 53%-70%) and specificity of 99% (95% CI: 96%-100%). Plasma and serum LAM by FIND 28 showed similar sensitivity (70%, 95% CI: 62%-78%) and comparable specificities (90%, 95% CI: 82%-97%; 94%, 95% CI: 88%-99%). Diagnostic sensitivity of urine LAM by S4-20 was higher among participants without HIV (41%, 95% CI: 24%-61%) compared to HIV-positive participants with CD4 ≥200 cells/mm3 (20%, 95% CI: 8%-39%). INTERPRETATION: Detection of LAM was achievable in non-sputum specimens for pulmonary TB, but additional analyte concentration or signal amplification may be required to achieve diagnostic accuracy targets. FUNDING: Bill and Melinda Gates Foundation.