title: Development and evaluation of enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification for diagnosis of invasive aspergillosis.
abstract: Invasive aspergillosis (IA) is a life-threatening infection in immunocompromised patients, rapid and sensitive detection of Aspergillus from clinical samples has been a major challenge in the early diagnosis of IA. An enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification (NASBA-ELISA) was developed to fulfil the need for the efficient diagnosis of these infections. The primers targeting 18S rRNA were selected for the amplification of Aspergillus RNA by the isothermal digoxigenin (DIG)-labeling NASBA process. The DIG-labeled RNA amplicons were hybridized with a specific biotinylated DNA probe immobilized on streptavidin-coated microtiter plate. The hybrids were colorimetrically detected by the addition of an anti-DIG antibodies linked to ALP and substrate (disodium 4-nitrophenyl phosphate). The detection limit of the Aspergillus NABSA-ELISA system was 1 CFU and the RNA in non-target bacteria or fungus was not amplified. The performance of this NASBA-ELISA compared to RT-PCR and galactomannan (GM) was evaluated by testing blood samples from 86 patients at high risk for IA. The sensitivity of NASBA-ELISA, RT-PCR and GM-ELISA was 80.56 % (95 % CI 63.98-91.81), 72.22 % (95 % CI 54.81-85.80), 58.33 % (95 % CI 40.76-74.49), respectively, and the specificity was 80.00 % (95 % CI 66.28-89.97), 84.00 % (95 % CI 70.89-92.83), 82.00 % (95 % CI 68.56-91.42). The efficiency of the three methods in various combinations was also evaluated. Combination of NASBA-ELISA and GM-ELISA testing achieved perfect specificity (100 %; 95 % CI 92.89-100) and perfect positive predictive value (100 %; 95 % CI 83.16-100). The best sensitivity (97.22 %; 95 % CI 85.47-99.93) and the highest Youden index (0.652) were obtained by testing with both NASBA and RT-PCR in parallel. In conclusion, the NASBA-ELISA assay consists of an alternative process for large-scale samples detection with semi-quantitative results and provides good clinical performance without resorting to expensive equipment. This assay makes it possible for the NASBA based RNA diagnosis to become a routine work in laboratories in less developed countries with fewer resources.
authors.affiliation: Department of Laboratory Medicine, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Du
authors.affiliation: Department of Laboratory Medicine, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People's Republic of China. xiayun12cn@aliyun.com.
authors.forename: Yun
authors.initials: Y
authors.lastname: Xia
authors.affiliation: Department of Laboratory Medicine, Chongqing People's Hospital, Chongqing, 400016, People's Republic of China.
authors.forename: Yunyan
authors.initials: Y
authors.lastname: He
authors.affiliation: Department of Laboratory Medicine, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People's Republic of China.
authors.forename: Qingquan
authors.initials: Q
authors.lastname: Pu
authors.affiliation: Department of Laboratory Medicine, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People's Republic of China.
authors.forename: Ruoyi
authors.initials: R
authors.lastname: Hua
authors.affiliation: Department of Laboratory Medicine, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People's Republic of China.
authors.forename: Wenyao
authors.initials: W
authors.lastname: Wu
keywords: GM-EIA; Invasive aspergillosis; NASBA-ELISA; RT-PCR