Dataset Information

Detection of Mycobacterium tuberculosis Complex in Sputum Samples Using Droplet Digital PCR Targeting mpt64.

ABSTRACT: Tuberculosis (TB) is one of the top 10 causes of death worldwide. It is challenging to find methods of diagnosis of active pulmonary TB that are sensitive enough to detect cases for proper treatment before unintentional transmission. Droplet digital PCR (ddPCR) is a highly sensitive method to detect genetic material of pathogens, but it has rarely been used for diagnosis of TB. This study compared the sensitivity of ddPCR with that of GeneXpert and AFB smear microscopy in 180 leftover sputum samples from patients suspected of having TB on the basis of clinical symptoms and radiography. Absolute quantification of copy numbers of MTB-specific genes was possible using ddPCR targeting the mpt64 gene. Among the 180 samples, 41.1% were diagnosed as having TB using ddPCR. The sensitivities of AFB smear microscopy, GeneXpert and ddPCR were 41.9%, 82.4% and 100%, respectively. AFB smear microscopy and GeneXpert both had a specificity of 100%, and the specificity of ddPCR was 95.3%. The accuracy of ddPCR (97.2%) is higher than that of GeneXpert (92.7%). This robust ddPCR system could potentially be used as a method for early diagnosis of TB.

SUBMITTER: Aung YW

PROVIDER: S-EPMC9966028 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Detection of <i>Mycobacterium tuberculosis</i> Complex in Sputum Samples Using Droplet Digital PCR Targeting <i>mpt64</i>.

Aung Ye Win YW Faksri Kiatichai K Sangka Arunnee A Tomanakan Kanchana K Namwat Wises W

Pathogens (Basel, Switzerland) 20230218 2

Tuberculosis (TB) is one of the top 10 causes of death worldwide. It is challenging to find methods of diagnosis of active pulmonary TB that are sensitive enough to detect cases for proper treatment before unintentional transmission. Droplet digital PCR (ddPCR) is a highly sensitive method to detect genetic material of pathogens, but it has rarely been used for diagnosis of TB. This study compared the sensitivity of ddPCR with that of GeneXpert and AFB smear microscopy in 180 leftover sputum sam ...[more]

PMID: 36839617

Similar Datasets

Project description: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 (R 2: 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.

Dataset Information

Detection of Mycobacterium tuberculosis Complex in Sputum Samples Using Droplet Digital PCR Targeting mpt64.

Publications

Detection of <i>Mycobacterium tuberculosis</i> Complex in Sputum Samples Using Droplet Digital PCR Targeting <i>mpt64</i>.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets