Quantiative Pcr For Early Dlagnosis Of Invasive Fungal Infections In Patients With Hematologic Malignances

Objective:Invasive fungal infection (IFI) remains a significant cause of morbidity and mortality in immunocompromised patients. In recent years the frequency of IFI has clearly increased. Early treatment of IFIs are crucial to decrease the mortality rate and the clinically economic burden. During the earliest phase of infection, clinical signs and other diagnostic methods are typically non-specific and similar to those of other infections. This causes delays or missed diagnosis increasing mortality and an lead to inappropriate therapy. Real-time PCR has high sensitivity and high specificity compared with other diagnostic methods. Real-time platforms enable quantification of fungal load in the clinical specimen, which may provide information about burden or progression of disease. This study was aimed to establish the approach of real-time quantitative PCR (q-PCR) and GM assay for early diagnosis of invasive fungal infections in patients with hematologic malignancies.Methods:1.40serum specimens were selected from patients with high invasive fungal infections risk of hematology in Shandong provincal hospital (from February2010to November2010). We also collect the patients’data of clinical、pathological、 laboratory manifestation and radiologic imagines. According to the EORTC/MSG and Chinese diagnostic criteria of invasive fungal infections, the research objects will be divided into:proven, probable, possible and non-invasive fungal infections four groups.2. The study chooses2Aspergillus stains from Bacteria room in Shandong provincal hospital as positive control. We get Aspergillus DNA from samples using Qiagen genomic DNA extracted kit from200μl Fungi fluid. The fungal template DNA was times diluted, determine the concentrartion and then conduct Real-time quantitative PCR detection. We can get the standard curve equation by using quantitative PCR instrument analysis software and standard curve according standard curve equation.3. Fngal DNA was extracted from200μl coagutant venous serum which was drawn from patients with high invasive fungal infections risk using Qiagen genomic DNA extracted kit. We next determine the concentrartion, conduct Real-time quantitative PCR detection and collect fluorescent signals. The Ct value of Real-time quantitative PCR detection in the15-35range was determined as positive standard, more than35for negative.4. Take GM from from standard threshold serum2copies, positive and negative control serum for each a copy using Platelia Aspergillus kit of Bio-rad company for quality control. Two patients serum GM was determined. We define that it is up to standard when the mean A value of standard threshold serum was in0.3-0.8, A value of positive control/A value of standard serum was graeater than2, and A value of negative control/A value of standard serum was less than0.5. The study sets GM test results to a single I value greater than0.7or Two successive greater than0.5for the positive standard. Collect experimental data, analyse and evaluate the clinical applied meaning of Real-time quantitative PCR detection and GM assay for early diagnosis of invasive fungal infection.Results:1. The positive results of quantitative PCR detection:5cases of proven invasive fungal infections have5cases,13cases of probable invasive fungal infections have11cases,9cases of possible invasive fungal infections have8cases,13cases of non-invasive fungal infections have2cases. The proven and probable invasive fungal infections were choused as positive control and non-invasive fungal infections as negative control. Real-time quantitative PCR sensitivity, specificity, positive and negative predictive value were0.89,0.85,0.89,0.85. The positive rates of Real-time quantitative PCR for different groups are diverse, specific as follows:sensitivity of proven groups is100%(5/5); sensitivity of probable groups is84.6%(11/13); sensitivity of possible groups is88.8%(8/9); sensitivity of non-invasive fungal infections groups is15.4%(2/13).2. The positive results of GM assay:32caes have20positive GM assay cases which are4cases of proven invasive fungal infections,11cases of probable invasive fungal infections,3cases of possible invasive fungal infections,2cases of non-invasive fungal infections. The proven and probable invasive fungal infections were choused as positive control and non-invasive fungal infections as negative control. GM assays sensitivity, specificity, positive and negative predictive value were0.83,0.80,0.88,0.73. The positive rates of GM assays for different groups are diverse, specific as follows:sensitivity of proven groups is80%(4/5); sensitivity of probable groups is84.6%(11/13); sensitivity of possible groups is75%(3/4); sensitivity of non-invasive fungal infections groups is20%(2/10).3. The results of combined application of quantitative PCR detection and GM assay:5cases of proven invasive fungal infections have4cases GM (+),5cases PCR (+);13cases of probable invasive fungal infections have11cases GM (+),11cases PCR (+);4cases of possible invasive fungal infections have3cases GM (+),3cases PCR (+);10cases of non-invasive fungal infections have2cases GM (+),1cases PCR (+). Analyse the data of quantitative PCR detection and GM test for the same specimen, we conclde:sensitivity of proven groups for PCR is100%(5/5), GM test is80%(4/5); sensitivity of probable groups for PCR is84.6%(11/13), GM test is84.6%(11/13); sensitivity of possible groups for PCR is75%(3/4), GM test is75%(3/4); sensitivity of non-invasive fungal infections groups for PCR is10%(1/10), GM test is is20%(2/10).4. Comprehensive analysis of the results of quantitative PCR detection or GM test in40cases:The proven and probable invasive fungal infections were choused as positive control and non-invasive fungal infections as negative control. Real-time quantitative PCR or GM assays sensitivity, specificity, positive and negative predictive value were0.94,0.85,0.89,0.92.Conclusion:1. Real-time quantitative PCR combined with GM assay can be used for early diagnosis for invasive fungal infections with hematologic malignancies.2. The sensitivity of combined application of PCR detection or quantitative GM detection is greater than Using alone one of them.