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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0266-0) contains supplementary material, which is available to authorized users.
Highlights
Invasive aspergillosis (IA), an opportunistic fungal infection, is increasingly recognized as a major cause of morbidity and mortality in immunocompromised patients, including those receiving aggressiveDu et al AMB Expr (2016) 6:91Boutboul et al 2002) and is included as an important component of microbiological factors in the diagnostic criteria devised by the Mycology Study Group of the European Organization for Research and Treatment of Cancer (EORTC/MSG) (De Pauw et al 2008)
The Nucleic acid sequence-based amplification (NASBA)-ELISA can be performed without special equipment and reagents for detection of amplified product compared to NASBAelectrophoresis
Analytical sensitivity of the NASBA process and analytical sensitivity of the NASBA‐ELISA with DIG detection system Analytical sensitivity of NASBA by using 1 % agarose gel electrophoresis was obtained by 10-fold serial dilutions of genomic RNA (Fig. 1)
Summary
Invasive aspergillosis (IA), an opportunistic fungal infection, is increasingly recognized as a major cause of morbidity and mortality in immunocompromised patients, including those receiving aggressiveDu et al AMB Expr (2016) 6:91Boutboul et al 2002) and is included as an important component of microbiological factors in the diagnostic criteria devised by the Mycology Study Group of the European Organization for Research and Treatment of Cancer (EORTC/MSG) (De Pauw et al 2008). The advantages of NASBA have promoted interest in evaluating its application to detection of Aspergillus RNA in clinical samples. Conventional NASBA requires several steps after RNA amplification, including electrophoresis or blotting and hybridization, which are inconvenient for detecting a large number of samples in clinical routine. The NASBAenzyme-linked immunosorbent assay (NASBA-ELISA) consisting of an alternative process for large-scale screening allows for application in daily life. This technique combines an immunological method to quantify the NASBA product directly after immobilization of biotinylated DNA on a microplate. The NASBA-ELISA can be performed without special equipment and reagents for detection of amplified product compared to NASBAelectrophoresis. NASBA-ELISA allows the using of RNA based diagnosis for routine purposes in poor developed laboratories
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