Rapid Detection and Quantification of Mycobacterium tuberculosis DNA in Paraffinized Samples by Droplet Digital PCR: A Preliminary Study

Abstract

Background: Rapid and reliable diagnosis of tuberculosis (TB) represents a diagnostic challenge in compartmentalized extrapulmonary TB infection because of the small number of mycobacteria (MTB) and the frequent lack of fresh samples to perform culture. Here, we estimate the performances of homemade droplet digital PCR (ddPCR)-based assays against culture in 89 biopsies, for those fresh and formalin-fixed and paraffin-embedded (FFPE) subsamples were available.Methods: MTB diagnosis in fresh subsamples was performed by culture. Fresh subsamples were also analyzed for acid-fast bacilli smear-microscopy (AFB) and Xpert® MTB/RIF (Xpert). MTB examination was repeated in blind in the 89 FFPE subsamples by in-house ddPCR assays targeting the IS6110 and rpoB. Analytical sensitivity of ddPCR assays was evaluated using serial dilution of H37Rv strain. Limit of detection (LOD) was calculated by probit analysis. Results were expressed in copies/106 cells.Results: IS6110 and rpoB ddPCR assays showed a good linear correlation between expected and observed values (R2: 0.9907 and 0.9743, respectively). Probit analyses predicted a LOD of 17 and 40 copies/106 cells of MTB DNA for IS6110 and rpoB, respectively. Of the 89 biopsies, 68 were culture positive and 21 were culture negative. Considering mycobacterial culture as reference method, IS6110 assay yielded positive results in 67/68 culture-positive samples with a median interquartile range (IQR) of 1,680 (550–8,444) copies/106 cells (sensitivity: 98.5%; accuracy: 98.9). These performances were superior to those reported by the rpoB assay in FFPE subsamples (sensitivity: 66.20%; accuracy: 74.1) and even superior to those reported by Xpert and AFB in fresh subsamples (sensitivity: 79.4 and 33.8%, respectively; accuracy: 84.3 and 49.4, respectively). When Xpert and AFB results were stratified according to mycobacterial load detected by rpoB and IS6110 ddPCR, bacterial load was lower in Xpert and AFB negative with respect to Xpert and AFB-positive samples (p = 0.003 and 0.01 for rpoB and p = 0.01 and 0.11 for IS6110), confirming the poor sensitivity of these methods in paucibacillary disease.Conclusion: ddPCR provides highly sensitive, accurate, and rapid MTB diagnosis in FFPE samples, as defined by the high concordance between IS6110 assay and culture results. This approach can be safely introduced in clinical routine to accelerate MTB diagnosis mainly when culture results remain unavailable.

Diagnosing osteo‐articular tuberculosis and multidrug resistance using real‐time polymerase chain reaction and high‐resolution melt‐curve analysis

“…Even in those studies, the diagnosis of OATB was limited to either a single case or a single gene target, 16,17 or studies where OATB specimens formed a small chunk of the total EPTB specimens. 6 Our observation on performance of different gene targets is in contrast to a recent study by Antonello et al, 18 wherein in-house droplet-digital PCR (on 89 Italian EPTB specimens including four OATB specimens) reported a sensitivity of 66.2% for rpoB and 98.5% for IS6110. These differences in performance of rpoB could be attributed to different molecular chemistries used and copy numbers of the two genes (rpoB single copy number, IS6110 upto 25).…”

“…The Xpert MTB/RIF assay may have revolutionized diagnosis of PTB and RifR, but performance of Xpert was inferior to qPCR in diagnosing OATB. 18 Even the WHO guidelines of 2020 that recommended Xpert Ultra as the initial test for diagnosing EPTB and detecting RifR, cautioned that the recommendation for OATB (synovial fluid) was more conditional as the evidence for test accuracy was of "low" certainty. 10 Not only this, Xpert and Xpert ultra are limited in their performance as they detect only RifR and miss InhR.…”

Advances in droplet digital polymerase chain reaction on microfluidic chips

“…132 In terms of common pathogenic bacteria, ddPCR can be used to detect Mycobacterium tuberculosis, which is difficult to diagnose because of the small number of Mycobacterium tuberculosis and the difficulty of culturing it. 133 The ability to detect Mycobacterium tuberculosis DNA in classroom air, which was counted using the Poisson distribution and compared to sputum samples from patients, suggests that the risk of exposure to air containing Mycobacterium tuberculosis in classrooms is close to that of exposure in public clinics, which is undoubtedly another major health risk. Detection of enteric microorganisms in aerosol samples and wastewater using ddPCR allows investigation and traceability of disease transmission in cities with poor sanitation, as well as identification of colonized DNA and RNA in aquatic sediments to obtain information on changes in colonization.…”

Diagnostic Modalities of Tuberculosis- Then and Now

“…Thus, it enables absolute gene quantification (exact nucleic acid targets) rather than relative one. This method can detect single copy of DNA [59]. It offers a higher sensitivity than qPCR and can be used to identify Mtb in sputum [60] and blood samples [61].…”