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.
Highlights
Tuberculosis (TB) is a deadliest infectious disease, accounting for about 10.4 million new cases and 1.3 million deaths worldwide in 2016 (World Health Organization, 2017)
Both M. tuberculosis mc27000 and M. bovis BCG were grown in Middlebrook 7H9 medium (Difco, Detroit, MI, United States) containing 100 μg/ml pantothenic supplemented with 0.05% Tween80
The limit of detection (LOD) for M. tuberculosis H37Rv was estimated to 107 and 741 genome copies/ml using IS6110 and senX3 PCR, respectively (Figure 2A)
Summary
Tuberculosis (TB) is a deadliest infectious disease, accounting for about 10.4 million new cases and 1.3 million deaths worldwide in 2016 (World Health Organization, 2017). A major priority and a challenge for TB control are to strengthen the capacity to diagnose the disease. Mycobacterial culture remains the gold standard test for TB diagnosis in high resource settings. Culture has high sensitivity with a limit of detection (LOD) to 10–100 cfu/ml, but the time-to-result ranges from 2 to 8 weeks (American Thoracic Society, 2000) and this method requires a BSL-3 laboratory facility. In most low resource settings, bacterial culture, is unavailable, leaving sputum smear microscopy as the major direct bacteriological test for TB diagnosis (Wejse, 2014). The LOD of the unconcentrated smear test is approximately 10,000 acid-fast bacilli (AFB)/ml, and microscopy has suboptimal specificity partially due to the possible contamination by non-tuberculosis mycobacteria (American Thoracic Society, 2000). The sensitivity of molecular assays is critical for detection of Mycobacterium tuberculosis (MTB) in paucibacillary sputum
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