Immunology And Detection Of Aspergillus Dna In Bronchoalveolar Lavage Fluid By Real-time PCR In Early Diagnosis Of Chronic Pulmonary Aspergillosis

Part Ⅰ Detection of Aspergillus Antigen and Antibody for the Diagnosis of Chronic pulmonary aspergillosisObjective: Our study aims to compare the clinical usefulness of a quantitative 1,3-beta-D-glucan,serum galactomannan,Aspergillus-specific Ig G antibodies,Aspergillus-specific Ig M antibodies,and bronchoalveolar lavage fluid galactomannan for diagnosing chronic pulmonary aspergillosis(CPA).Methods: Serum and bronchoalveolar lavage fluid(BALF)samples were obtained from patients with CPA were enrolled from The First Affiliated Hospital of Guangzhou Medical University and Guangzhou Thoracic Hospital from January 2016 to January 2017.Immunosuppressed patients were excluded.The serum BDG,galactomannan,Aspergillus-specific Ig G antibodies,Aspergillus-specific Ig M antibodies,BALF-GM were detected.We compare the sensitivity,specificity,positive predictive value,negative predictive value of the series immunological tests between CPA and control groups.Optimal cut-off level of these immunological tests were established with receiver operating characteristic curve(ROC).Based on the clinical data evaluated and the usefulness of different immunological tests methods,thus offering a better diagnostic way among CPA patients.Results: 1.79 cases of CPA and 74 control cases were enrolled.Classification of CPA cases included: 10 cases(12.66 %)of Simple aspergilloma,2 cases(2.53%)of aspergillus nodule,35 cases(44.30%)of CNPA/SAIA,28 cases of CCPA(35.44 %),and 4 cases of CFPA(5.06%).There were 63 proven pulmonary aspergillosis and 16 probable one.The mean age of CPA group was 51.34 ± 14.80 years old,including 57 males(72.15%)and 22 females(27.85%).The average age of the control group was 55.23 ± 14.57 years old,48 cases(64.68%)and 26 cases(35.14%).The most common underlying csuse of patients with CPA was bronchiectasis(67.09%)and pulmonary tuberculosis(65.82%).2.The serum BDG,serum GM,aspergillus-specific Ig M,aspergillus-specific Ig G and BALF-GM differed significantly between the two groups(P <0.05).The sensitivity,specificity of the five immunological tests methods were 49.36%,44.30%,17.72%,32.91%,45.57% and 91.89%,98.64%,94.59%,90.54%,89.74%.When i ndividual diagnosis,BALF-GM had a better diagnostic value for CPA,and the Youden index was 0.59(cut point ≥0.85μg / L),AUC = 0.94(95% CI 0.89-0.99),and the optimal cutoff was 0.44μg / L(Sen 0.98,Spe 0.82).3.In various types of CPA,the sensitivity of serum BDG,GM was similar to the total case group,while the sensitivity and specificity of aspergillus-specific Ig M antibodies were low.The sensitivity of BALF-GM(cut-off point ≥0.65μg / L)in SA,CCPA,SAIA / CNPA,CFPA was 90.00%,71.43%,42.86% and 75.00%,respectively.The sensitivity of aspergillus-specific Ig G antibody(cut point ≥80 AU / ml)in SA,CCPA,SAIA / CNPA,CFPA was 60.00%,57.14%,42.86% and 50.00%,respectively.4.Combined detection improved the diagnostic performance.The sensitivity,and specificity of the serum BDG/GM/Aspergillus-specific Ig G was 76.19%and,85.14%,respectively.The sensitivity and specificity of serum BDG/GM/Aspergillus-specific Ig G/ Aspergillus-specific Ig M was 84.13% and 78.38%,respectively.For detecting serum GM/BALF-GM sensitivity,specificity was 76.19% and 93.24%,respectively.5.In the anti-Aspergillus treatment,the aspergillus-specific Ig G levels declined inpatients whose treatment was effective.but an increase in antibody titers may signal poor prognosis.Conclusion: 1.BALF-GM is superior to serum GM,and BALF-GM has high diagnostic performance for CPA.2.Antigens combined antibody detection can reduce the risk of missed diagnosis,For serum G/GM/Ig G combined detection,BALF-GM can improve the sensitivity.3.Aspergillus-specific Ig M antibody may not be useful for the diagnosis of CPA,Ig G on the diagnosis of CNPA due to other types of CPA.4.Aspergillus-specific Ig G antibody in different classification CPA diagnostic value is various,lower cut-off point adjustable sensitivity.Monitoring the Aspergillus-specific Ig G antibody will help to evaluate the prognosis of patients with CPA.Part Ⅱ Detection of Aspergillus DNA in BALF by Real-time PCR for the Early Diagnosis of Chronic pulmonary aspergillosisObjective: Evaluation of diagnostic value of QPCR in early detection of BALF Aspergillus DNA in patients with chronic aspergillosis.Methods: The patient’s inclusion criteria is identical to those described previously(section 1).We detected Aspergillus DNA in BALF from patients with proven CPA.After centrifugation,the BALF pellets were subject to DNA extraction.Glass-bead beating with DNA Extraction Kit was used to extract the BALF DNA,which was detected by QPCR(Taqman probe).Results: 140 bronchoalveolar lavage fluid from 128 cases were collected.79 cases of CPA which including 63 proven one and 16 probable one,and 49 cases control group.The sensitivity of BALF-QPCR in CPA group was 85.15%,specificity was 89.80%,positive predictive value was 96.63%,negative predictive value was 70.59%,Youden index was 0.79,AUC = 0.87(95% CI 0.80-0.94),P <0.005.The sensitivity of the diagnosis group was 87.18%,the specificity was 89.80%,the positive predictive value was 95.77%,the negative predictive value was 78.26%,the Youden index was 0.77,AUC = 0.89(95% CI 0.82-0.95),P <0.005.BALF-QPCR was well consistent with pathologic diagnosis(Kappa 0.766,P <0.005)on a single sample basis.Conclusion: BALF-QPCR has a good early diagnostic value in patients with chronic pulmonary aspergillosis,combined with BALF-GM can reduce false positives.