Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens

Abstract

Background: When available, nucleic acid tests (NATs) offer powerful tools to strengthen the potential of tuberculosis (TB) diagnosis assays. The sensitivity of molecular assays is critical for detection of Mycobacterium tuberculosis (MTB) in paucibacillary sputum.Materials and Methods: The impact of targeting repetitive IS6110 sequences on the PCR sensitivity was evaluated across mycobacterium strains and reference material. Six lysis-extraction protocols were compared. Next, 92 clinical sputum specimens including 62 culture-positive samples were tested and the results were compared to sputum-smear microscopy, culture, and Xpert MTB/RIF test. Finally, the capacity to detect low MTB DNA concentrations was assessed in 40 samples containing <1.5 × 102 copies/ml ex vivo or after dilution.Results: The lower limit of detection (LOD) using the IS6110 PCR was 107 genome copies/ml (95% CI: 83–130) using MTB H37Rv as a reference strain, versus 741 genome copies/ml (95% CI: 575–1094) using the senX3 PCR. The proportion of recovered MTB DNA after lysis and extraction ranged from 35 to 82%. The Chelex® method appeared as a more efficient protocol among the six different protocols tested. The sensitivity and specificity in clinical sputum samples were 95.1% (95% CI: 90.7–99.6) and 100% (95% CI: 96.2–100.8), respectively. Among 40 samples with low MTB DNA concentration, 75% tested positive for IS6110 PCR, versus 55% using the Xpert MTB/RIF assay (p = 0.03).Conclusion: Laboratory assays based on an efficient MTB lysis and DNA extraction protocols combined with amplification of IS6110 repeat sequences appear as a sensitive diagnostic method to detect MTB DNA in sputum with low bacterial load.

Combined testing for herpes simplex virus and Mycobacterium tuberculosis DNA in cerebrospinal fluid of patients with aseptic meningitis in Burkina Faso, West Africa

“…Furthermore, in this study a Chelex®‐based method was used for extraction of HSV and MTB DNA. This method is rapid, efficient with a low risk for laboratory‐induced contamination since all the procedure can be carried out in a single tube as recently reported . The expense for Chelex® method is very low as compared to the commercial methods based on silica columns or magnetic beads costing from 2 to 6$ per test, which makes it particularly advantageous in low‐income countries.…”

Demonstration of a fast and easy sample-to-answer protocol for tuberculosis screening in point-of-care settings: A proof of concept study

“…Although some studies have shown M. tuberculosis isolates with lower number of IS6110 copies, this genomic marker is considered one of the best options to increase the sensibility of diagnostic tests. Indeed, the inclusion of the genomic sequences IS6110 and S1081 in the new generation of the GenXpert MTB/Rif test resulted in increased sensibility [31][32][33][34][35][36].…”

CRISPR-based biosensing is prospective for rapid and sensitive diagnosis of pediatric tuberculosis

“…Although the CRISPR platform does not seem to solve the problem of specimen collection and the paucibacillary feature, its superior sensitivity and low LOD make this novel technology promising in detecting trace MTB in pediatric specimens. Combined with reasonable sample-pre-processing methods (Kolia-Diafouka et al, 2018), CRISPR platforms make it possible to reduce the volume of specimen that needs to be collected, and use noninvasive, easily accessible specimens instead of conventional ones to detect TB. Another research (Xiao et al, 2020) using Cas12a trans-cleavage has successfully identified MTB and six major nontuberculous mycobacteria (NTM) species, allowing the application of this method in the field of rapid MTB identification.…”