Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva

Abstract

Background Pulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears promising but has been discouraged due to low bacterial load and poor sensitivity. Here, we present a novel and rapid method to enrich Mycobacterium tuberculosis (Mtb) from saliva, which may serve as a basis for a diagnostic saliva test. Methods Lipobiotin-functionalized magnetic beads (LMBs) were incubated with Mtb-spiked PBS and saliva from healthy donors as well as with saliva from TB patients. Flow cytometry was used to evaluate the capacity of the beads to bind Mtb, while real-time quantitative polymerase chain reaction (qPCR) was utilized to detect Mtb and determine the amount of mycobacterial DNA in different sample types. Results We found that LMBs bind Mtb efficiently when compared to non-functionalized beads. The development of an qPCR assay based on the use of LMBs (LMB assay) allowed us to enrich mycobacterial DNA in spiked sample types, including PBS and saliva from healthy donors (enrichment of up to ~8.7 fold). In Mtb-spiked saliva samples, we found that the LMB assay improved the detection rate of 102 bacteria in a volume of 5 ml from 0 out of 15 (0%) to 6 out of 15 (40%). Consistent with that, the LMB assay increased the rate of correctly identified saliva samples from TB patients in two independent cohorts. Conclusions Implementation of the principle of the LMB-based assay may improve the sensitivity of existing diagnostic techniques, e.g. by functionalizing materials that facilitate Mtb sampling from the oral cavity.

Accuracy of upper respiratory tract samples to diagnose Mycobacterium tuberculosis: a systematic review and meta-analysis

“…From included manuscripts (appendix pp 26-33 for study characteristics; appendix p 5 includes full methodological details and results by study), we classified types of upper respiratory tract sampling into four groups: laryngeal swabs (32 studies ), nasopharyngeal aspirate (ten studies [43][44][45][46][47][48][49][50][51][52] ), oral swabs (18 studies [53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70] ), and other (mouthwash three studies, [71][72][73] nasal swabs one study, 74 saliva four studies, [75][76][77][78] and other mucosa or dental samples three studies [79][80][81] ). Studies were published between May 13, 1933, and Dec 19, 2022, from South Africa (11 studies), Norway (seven studies), UK (seven studies), Peru (four studies), Uganda (seven studies), Canada (three studies), India (three studies), USA (three studies), Australia (two studies), Germany (two studies), Kenya (two studies), and one study from each of Brazil, Chile, China, Slovakia and Czech Republic, Denmark, Finland, France, Hungary, Italy, Japan, Malawi, Moldova, Mozambique, South Korea, southeast Asia (individual countries not specified), Spain, Sweden, Taiwan, Turkey, and Yemen.…”

The Role of Next-Generation Sequencing (NGS) in the Management of Tuberculosis: Practical Review for Implementation in Routine

“…Future challenges are to optimize existing methods in order to improve the signal, either by enriching the DNA amount or by eliminating the background noise from clinical samples. Thus, some papers have recently proposed DNA enrichment on clinical specimens by capture methods––either by microfluidics-based cell capture [ 39 , 40 ] or by magnetic bead [ 41 ]. Capture methods may be promising for enrichment in clinical sputum samples, which could facilitate culture-free MTB WGS.…”

Advances in understanding immune homeostasis in latent tuberculosis infection

“…Presently, specimens employed in the exploration of LTBI diagnostic biomarkers using transcriptomics, proteomics, metabolomics, and single‐cell transcriptomics predominantly originate from patients' blood. In the foreseeable future, the detection of dormant Mtb in saliva may be achieved through technologies like Xpert MTB‐RIF Ultra (Tang et al, 2023) and Lipobiotin‐functionalized magnetic beads (LMBs; Hansen et al, 2022). Additionally, Digital PCR, utilized for pathogenic DNA detection in saliva, may have implications in diagnosing ATB (Cassinari et al, 2021; Wang et al, 2021).…”