The Establishment Of A Detection Method For Tuberculosis-biomarker CFP-10 Based On Mass Spectrometry And Its Application In The Diagnosis Of Tuberculosis Disease

[Objective]The limitations of existing laboratory diagnostic methods are an important cause for the high incidence and mortality of Tuberculosis(TB).The establishment of a direct detection method for tuberculosis-biomarker is of great significance for the diagnosis,treatment,prevention and control of TB.Culture filtrate protein 10(CFP-10)is recognized as a representative biomarker of Mycobacterium Tuberculosis(Mtb)infection.However,there is currently a lack of a direct detection method for CFP-10 with high sensitivity and specificity.The purposes of this study are to evaluate the possibility of mass spectrometry for the quantitative detection of CFP-10 in serum samples,to establish a method based on mass spectrometry for the detection of tuberculosis biomarker CFP-10,to detect the blood samples of infants,childhood tuberculosis and human immunodeficiency virus(HIV)-TB co-infectors and to evaluate the diagnostic performance of serum CFP-10 detection in TB.[Methods]1.3-4 months old children with maternal HIV exposure from South Africa were included and evaluated for TB disease,during a follow-up of up to 4 years.Children were classified as Confirmed,clinical-diagnosed,or Unlikely TB based on clinical,laboratory,histopathological,and radiological evaluations.At each visit,blood samples of young children were collected and stored in the form of cryopreserved sera.Finally,660 serum samples from 519 HIV-exposed children(284 HIV-infected,235 HIV-uninfected)were evaluated for the quantitation of CFP-10 peptide using MALDI-TOF and Nano Disk.2.High performance liquid chromatography-tandem mass spectrometry/Mass spectrometry(LC-MS/MS)technology and multiple reaction monitoring(MRM)technology were applied to this research.A new LC-MS/MS-MRM-based serum CFP-10 detection method was established by optimizing the pre-processing steps,chromatographic and mass spectrometry conditions.Methodological evaluations containing linear range,limit of detection,limit of quantification,inter-day/intraday precision,accuracy,standard recovery rate,matrix effect and residual effect were conduct for new established method.3.Children less than 13 years of age with suspected TB were enrolled,with a follow-up of up to 6 months.Children were classified as Confirmed,clinical-diagnosed,or Unlikely TB based on clinical,laboratory,histopathological,and radiological evaluations.The blood samples at enrollment,2 months and 6 months after anti-TB treatment were collected for the quantitation of CFP-10 peptide expression applying newly established mass spectrometry method.Compared with culture,Xpert MTB/RIF,TST and CXR,we evaluate the diagnostic accuracy of CFP-10 assay applying LC-MS/MS-MRM in children with TB.4.In this study,the capsid protein p24 was selected as the biomarker of HIV-1 infection,and the high-HIV-1-specific p24 peptide was screened and determined.A LC-MS/MSMRM-based serum multiple detection assay was established by combining Mtb virulence factor CFP-10 specific peptides and p24 peptides,which analyzes CFP-10 and p24 peptides from the same serum sample to simultaneously diagnose TB and HIV infection.Methodological evaluations containing linear range,limit of detection,limit of quantification,inter-day/intraday precision,accuracy,standard recovery rate and matrix effect were conduct for new established method.56 adult patients(31 HIV-1 positive,25 HIV-1 negative)and 26infants(16 HIV-1 positive,25 HIV-1 negative)were included to evaluate the diagnostic accuracy of this assay.Enzyme-linked immunosorbent assay(ELISA)and Real-time Quantitative polymerase chain reaction(q PCR)were compared with this new method.Twenty patients(7 HIV/TB co-infections,4 TB infections,1 HIV infection,and 8 control patients)were included to evaluate the diagnostic performance of serum multiple detection assay in HIV-TB co-infectors.[Results]1.Detection of serum CFP-10 peptide exhibited 100% sensitivity for Confirmed(5/5,95%confidence interval [CI],47.8–100)and 83.7% sensitivity for Unconfirmed(36/43,95% CI,69.3–93.2)TB cases in HIV-infected children,with 93.1%(203/218,95% CI,88.9–96.1)specificity.In HIV-uninfected children,serum CFP-10-positivity detected the single Confirmed TB case and 15 of 20 Unconfirmed TB cases(75.0%;95% CI,50.9–91.3),with96.2%(177/184,95% CI,92.3–98.5)specificity.CFP-10 peptide signal was detected in serum up to 60 weeks before TB diagnosis,and its diagnostic sensitivity reached 76.5%(13/17,95%CI,50.1-93.2%)at ≤ 24 weeks before diagnosis.CFP-10 peptide positivity and expression levels declined following anti-TB therapy initiation.2.A new LC-MS/MS-MRM-based serum CFP-10 detection assay was successfully established in this study.The standard curve of this new assay performed a good linear relationship with the range of 6.25-200 f M.The limit of detection of CFP-10 was 2f M and the limit of quantification was 7f M within 100 u L serum sample.The intra-day/inter-day precision of serum CFP-10 detection were less than 20%.The accuracy was between 95-108%.Standard recovery rate ranged from 79.9 to 100%.Both matrix effect and residual effect of this assay are in line with the requirement of quantitative detection with biological samples.3.In this study,a new LC-MS/MS-MRM-based serum CFP-10 detection assay was used to quantitatively analyze CFP-10 in 270 serum samples from 160 children.For smear-positive confirmed TB,CFP-10 exhibited same sensitivity(6/6,100.0%;95%CI,54.1-100.0%)with culture and Xpert.In smear-negative confirmed TB,serum CFP-10 positivity detected 78.4%(29/37;61.8-90.2%)TB cases,higher than the sensitivity of Xpert(21/37,56.8%,39.5-72.9%).CFP-10 assay performed a highest sensitivity of 81.0%(34/42;65.9-91.4%)than all other methods(culture,Xpert,0%;TST,17.9-54.3%;CXR,13.0-42.1%)for diagnosing unconfirmed TB,with 84.0%(73.7-91.5%)specificity.Different from other diagnostic methods with the sensitivities(culture: 83.3%;Xpert: 55.6%;TST: 66.7%;CXR: 66.7%)for diagnosing pediatric TB with age > 5 years old higher than the sensitivities of children aged<5 years(95%CI: Culture: 34.8-38.1%;Xpert: 30.4-38.1%;TST: 47.6-66.7%;CXR: 38.1-47.8%),the sensitivities of CFP-10 diagnosing TB in children with different ages were stable(71.4-88.9%).CFP-10,showed decreasing sensitivities after treatment for TB cases with positive response,but CFP-10 assay exhibited raised sensitivities and increased signal for patients with negative response.4.HIV-1 specific p24 peptide(ETINEEAAEWER,1462.83 m/z)was screened as the target peptide in this study,and a new LC-MS/MS-MRM serum p24 detection was successfully established combing with TB specific CFP-10 peptide.After methodological evaluations,we found the intra-day/inter-day precision of serum CFP-10 detection were less than 20%.The accuracy was between 94.1-109.4%.Standard recovery rate ranged from 89.6 to 99.8%.Matrix effect of this new assay are in line with the requirement of quantitative detection with biological samples.For the preliminary clinical validation,we found that among adults,the sensitivity of the p24 peptide to diagnose HIV-1 virus infection was 90.3%(95%CI,74.3-98.0%),and the specificity was 100 %(95%CI,86.3-100%);among infants group,the sensitivity was 87.5%(95%CI,61.7-98.5%)and the specificity was 100%(95%CI,69.2-100%).Applying q PCR as gold standard,the sensitivity of p24 peptide was 88.9%,which was hither than the sensitivities of ELISA tests(55.6-66.7%).Simultaneously detecting CFP-10 and p24 peptides,the sensitivity of multiple serum detections to diagnose HIV-TB coinfected patients was 85.7%(6/7,95%CI,42.1-99.6%),the overall specificity was 100%(13/13,75.3-100.0%).Most importantly,the alternation tendencies of these two peptides at different time points during ART and anti-TB treatment could reflect therapeutic efficacy,distinguish treatment failure and indicate the potential Immune Reconstitution Inflammatory Syndrome(IRIS)cases.[Conclusion]1.Detection of CFP-10 peptide via MALDI-TOF and Nano Disk in sera demonstrated high sensitivity and specificity for rapid TB diagnosis in HIV-infected and-uninfected young children,suggesting its potential utility for early TB detection and monitoring of anti-TB treatment response.2.A new LC-MS/MS-MRM-based serum CFP-10 detection assay was first established in this study,allowing quantitative detection of the tuberculosis biomarker CFP-10 in blood samples.3.LC-MS/MS-MRM-based serum CFP-10 detection assay,which would be not affected by age and HIV status,could effectively diagnose TB in children,especially for clinicaldiagnosed TB cases readily missed by sputum-based methods.We also found that CFP-10 peptide detection displayed the potential of monitoring anti-TB treatment responses.4.A new LC-MS/MS-MRM-based serum multiple detection assay was first established in this study,allowing simultaneously quantitative detection of the tuberculosis biomarker CFP-10 and HIV-1biomarker p24 in blood samples.Serum multiple detection assay were demonstrated to diagnose HIV-TB co-infected patients and performed the potential of both antiretroviral therapy and anti-TB therapy monitoring in patients.