Establishment And Clinical Application Of Real-time PCR For Invasive Aspergillosis Detection In Blood

Background:Invasive aspergillosis(IA) has become a major cause of death due to infectionin immunocompromised patients.It remains a high mortality(40%～100%) amongthose treated for haematological malignancy and those chemotherapy and receiving asolid organ transplant. The high mortality is due in part to difficulties in diagnosisbecause the traditional techniques in laboratory, lack sensitivity and waste time. Inrecent years a new diagnositic assay has focus on the detection of circulatingmarkers including genomic fungal DNA and fungal antigen.Objective:To develop a real-time PCR assay for rapid detecting aspergillus in bloodusing self-designed primers and probe.To establish a standardized method for theextraction of fungal DNA from clinical specimens.There is a need to develop astandard protocol for the detection of Aspergillus spp.The test will be evaluated byDNA isolation from patients who were at high risk of invasive aspergillosisinfection. The result of clinical study supposed to be the guidline for drawing blood.Methods:①Based on the 18s RNA, 5.8s RNA, 28s RNA, ITSⅠ,ITSⅡgene sequenceof aspergillus,several relative fungies and human being in the genebank. One pair of primers and one probe were designed using ABI Primer Express 2.0.We designedanother pair of primer near the target amplification and obtain a nucleotidesequence,the length of which was 200～300bp. then, the nucleotide sequence wascloned into the vector pMD19-T. The recombinant vector was used to establish thestandard curve which can quantify the initial fungal burden of the sample. The studyalso included the approach to find the best way of extracting the fungal DNA fromclinical sample, the sensitivity, specificity and reproducibility.②We collected 232 samples of 71 patients at high risk of IA who were once inNanFang hospital from April. 2006 to February.2007.The sensitivity, specificity,accuracy,positive predictive value,negative predictive value of real-time PCR assaywere given after detecting samples.The diagnosing result by real-time PCR andELISA method was compared by x~2 test.Analysing the different performance ofreal-time PCR assay in one patient during his antifungal therapy can help doctorsdiagnose and treat the patient correctly.Results:①Methodogy analysis showed that the real-time PCR for diagnosing IA wassuccessfully established. The priemers and probe target the internal transcribedspacer regionsⅠfrom fungal rRNA gene complex. The homology of the recombinedplasmid was up to 99% to the NCBI gene sequence examed by sequencealigment. The standard curve of real-time PCR was completed: Y=-0.37X+16.05,R~2=0.997 The CV in the experiment is 1.89%,1.56%,2.57%.The CV betweenthe experiments is 3.32%,3.58%,4.50%.Real-time PCR procedure was able todectect at least 10 copies of ITSⅠgene equivalent to 5～10 CFU/ml and had nocross-reaction with human genomic DNA, other fungus and bacterium.②The 71 patients consisted of 10 patients with proven IA, 30 patients withprobable IA, 8 patients with possible IA and 23 patients with no evidence of IA. The sensitivity, specificity, accuracy, positive predictive value,negative predictive value ofreal-time PCR assay were 80%～87.5%, 95.6%(22/23), 90.1%～90.7%,88.8%～97.6%and 75.8 %～91.7%. Compared to the real-time PCR,the statistic data for ELISA assayindicated a less specific rate of dectection of IA in high-riskpatients(P=0.021(2-sided),P＜0.05). The real-time PCR result turned from positive tonegative after 2～3 days antifungal treatment and the sensitivity rate delined to30%～40%. 6 of 8 patients died while their PCR result remained positive afterantifungal treatment.Conclusions:The detection of IA by real-time PCR was successfully established with highsensitivity, specificity and accuracy. Applied in clinical experiments,real-time PCRmethod shows many advantages than commercial ELISA assay. Standardizedclinical criteria and standardized approach to detect fungal DNA in IA patientsamples,may permit a more reliable comparison of future studies.This technologyhas promising future of its application and great research value.