Diagnostic Value Of Plasma Mycobacterium Tuberculosis Cell-free DNA Detecting For Tuberculosis Patients

Background and objectiveThe global incidence of tuberculosis(TB)is high,and the problem of TB diagnosis is an important reason hindering the prevention and control work in the world,especially in developing countries.Conventional diagnostic methods are difficult to meet clinical needs,so rapid and accurate diagnostic techniques are the key to TB control and treatment.The purpose of this study is to verify the feasibility of detecting Mycobacterium tuberculosis cell-free DNA(MTB cfDNA)in the plasma of TB patients by real-time fluorescence quantitative PCR(Q-PCR),analyze the diagnostic efficacy of this new molecular biology method for different types of TB patients,and clarify its clinical application value in TB diagnosis.At the same time,the problems and deficiencies of plasma MTB cfDNA detection in clinical practice were summarized,so as to provide a convenient and reliable new technology for TB diagnosis.MethodsFrom July 2018 to June 2019,118 patients with clinically confirmed TB admitted to Guangdong Second Provincial General Hospital and Guangzhou Chest Hospital,including 79 patients with pulmonary tuberculosis(PTB)and 39 patients with extrapulmonary tuberculosis(EPTB),were selected as the research objects(study group),and 61 patients with non-TB and other pulmonary diseases admitted in the same period were selected as controls(control group).MTB cfDNA in the plasma of all enrolled patients were detected by Q-PCR,the results were further verified by sequencing and BLAST analysis.MTB genomic DNA in the sputum of all enrolled patients were detected by Gene Xpert Mycobacterium tuberculosis/Rifampicin(Xpert MTB/RIF).The results of T cell spot test(T-SPOT.TB)and traditional bacteriological examination were recorded.The McNemar-Bowker test was used to compared the sensitivity and specificity of MTB cfDNA assay with Xpert MTB/RIF assay and T-SPOT.TB test for TB diagnosis,and Kappa test was used to compared the consistency of the above three diagnostic methods with the clinical diagnosis results.Then,analyzed the value of clinical routine method and MTB cfDNA assay in the combined diagnosis of TB.Results1.MTB cfDNA in plasma of 94 clinically diagnosed TB patients were positive by Q-PCR(verified by sequencing and BLAST),and Ct values were distributed at(23.91-37.25).2.The sensitivity and specificity of T-SPOT.TB test,Xpert MTB/RIF assay,and MTB cfDNA assay in TB diagnosis were 81.36%and 40.98%,61.02%and 96.72%,79.66%and 95.08%,respectively.The sensitivity of MTB cfDNA assay was higher than that of Xpert MTB/RIF assay(P<0.05),with no statistical difference in specificity(P>0.05).The specificity of MTB cfDNA assay was higher than that of T-SPOT.TB test(P<0.05),and there was no statistical difference in sensitivity(P>0.05).3.The agreement of MTB cfDNA assay,Xpert MTB/RIF assay,and T-SPOT.TB test with clinical diagnosis results were 84.92%,73.18%,and 67.60%,respectively.Kappa correlation coefficients were 0.77,0.61,and 0.43,respectively.4.The positive rates of T-SPOT.TB test,Xpert MTB/RIF assay,andMTB cfDNA assay for PTB and EPTB patients were 83.54%and 76.92%,69.62%and 43.59%,79.75%and 79.49%,respectively.The positive rate of MTB cfDNA assay for PTB and EPTB patients both higher than that of Xpert MTB/RIF assay(P<0.05).Compared with T-SPOT.TB test,there was no statistical difference(P>0.05).5.The positive rates of sputum smear staining microscopy,sputum MTB culture,T-SPOT.TB test,and Xpert MTB/RIF assay for TB diagnosis alone/combined with MTB cfDNA assay for TB diagnosis were 33.94%/73.39%,40.32%/70.97%,81.36%/88.14%,and 61.02%/81.36%,respectively.Conclusions1.It is feasible to detect MTB cfDNA in the plasma of TB patients by Q-PCR.2.The sensitivity of MTB cfDNA assay to the diagnosis of TB is superior to the Xpert MTB/RIF assay,which can significantly improve the diagnostic specificity of TB compared with T-SPOT.TB test.MTB cfDNA assay has the best consistency with the clinical diagnosis results,which is more reliable than the other two methods.3.MTB cfDNA assay has a high positive rate for both PTB and EPTB patients,which is expected to be a new target for rapid diagnosis of EPTB.4.Conventional clinical method combined with plasma MTB cfDNA detection can effectively improve the positive rate of TB diagnosis.5.The optimization of detection quality is the primary problem to be solved,and the evaluation of the concentration range of MTB cfDNA in plasma should be an important target for future research.