Research Of Nucleic Acid Sequence-based Amplification Assay For Diagnosis Of Invasive Candidiasis

Objective: Invasive candidiasis(IC)is an infection with atypical clinical symptoms and physical signs.Moreover,IC exhibits gradually increasing morbidity and mortality.Early diagnosis and treatment are beneficial to improve the survival rate and prognosis for the patients.This study intends to establish a nucleic acid sequence-based amplification(NASBA)technique to detect Candida,aiming for rapid diagnosis of IC.The results of G-test and blood culture were also investigated to find out the optimal strategy for laboratory diagnosis of IC.Methods: Firstly,the primers for highly conserved sequence of Candida 18 S rRNA were determined and then establish NASBA combined with electrophoresis assay to detect Candida albicans(ATCC14053),of which the sensitivity and specificity of the method were evaluated.Secondly,the inclusion criteria of the Case Group of IC and the Control Group without IC were determined by referencing to definitions of European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergyand Infectious Diseases Mycoses Study Group(EORTC/MSG)for invasive fungal disease.38 serum samples were collected and included in the final statistics(18 in the case group(15 proven cases,3 probable cases),20 in the control group).The serum samples of the two groups were collected and were tested by the NASBA method developed in this study.After that,the results of blood culture and G-test of all cases were collected.The diagnostic performance parameters of the three methods were evaluated.Results: The electrophoretic results of NASBA amplification products revealed seven species of Candida(Candida albicans,Candida tropicalis,Candida parapsilosis,Candida glabrata,Candida krusei,Candida dubliniensis,Candida guilliermondii)showed specific bands of RNA amplification products,while the control strains(Aspergillus fumigatus,Cryptococcus neoformans,Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa,Corynebacterium striatum,Clostridium difficile)showed no target band.The specificity of NASBA assay is fine,and detection limit of this method was up to 1CFU/ml.The sensitivity of NASBA,G-test and blood culture in diagnosis of IC was 72.22%,55.56%,72.22%,respectively,and the specificity was 90%,75%,100%.All of positive samples by NASBA assay in the case group were from patients with candidemia.Conclusion: The diagnostic performances of NASBA were better than G-test;The sensitivity of NASBA is consistent with blood culture,andother diagnostic performances were slightly inferior to blood culture.NASBA may shorten times to Candida detection compared to blood culture,NASBA method possess the advantages of high efficiency,accuracy and simplicity in the diagnosis of IC,especially of candidemia.Performing NASBA method and blood culture for patients with IC at the same time is recommended in this study.