Study Of The Diagnosis Of CMV Infection And Aspergillosis In Hemopathic Patients Via RQ-PCR

Objective:This study aimed at evaluating the clinical worth of plasma RQ-PCR assay for the surveillance of CMV infection and guidance of preemptive therapy in a cohort of adult Allo-HSCT recipients. Furthermore, we analyzed the risk factors for infection and the factors associated efficacy. Meanwhile, this study was conducted to analyze the clinical features of IA with haematological malignancies and set up RQ-PCR for diagnosis of Aspergillus, which was used to clinical diagnosed test, and investigate the value of Aspergillus RQ-PCR test for the early diagnosis of IA.Patients and Methods:we retrospectively reviewed the charts and CMV detected datas of 141 adult patients undergoing allo-HSCT in our hematology department between January 2008 and June 2010. All recipients were followed up for 180 days post-HSCT, and then we analyzed the clinical characteristics, results of RQ-PCR detecting plasma CMV-DNA, informations of CMV infection and antiviral therapy, related risk factors and so on.Specific target sequence for Aspergillus was screened in NCBI Gene Bank. Genomic DNA from Aspergillus, Candida and bacteria were extracted and performed RQ-PCR for validation of the target sequence. Recombinant plasmid was constructed using to prepare for RQ-PCR standard preparation. The standard curve was drawn and RQ-PCR was performed with DNA extracted from strains. EDTA-anticoagulated blood samples were collected from 52 patients with haematological malignancies suffering fever, then fungus genomic DNA was extracted from the plasma and whole blood used to perform Aspergillus RQ-PCR, respectively. Results of RQ-PCR were analyzed by ROC. Meanwhile, the clinical informations were collected dynamically for one month, then all cases were evaluated by diagnostic criteria according to the standard of diagnosis for invasive Aspergillosis. Aggregate analysis were done with datas from RQ-PCR and clinical, and then the "cut-off" value for IA diagnosis by RQ-PCR was defined.Results:The incidence of CMV infection and CMV pneumonia of 141 HSCT recipients was 81.5% and 2.9%, respectively. The median time of positive plasma CMV-DNA detection was 33 days post-HSCT, while the incidence rate of CMV infection was 34.0%,66.7%,9.7%,7.1% within the first, second, third month and beyond 90 days after HSCT, respectively, and the difference was significant (P=0.000).CMV infection was more frequent in female patients (90.4% vs 76.4%, P=0.044), with aGVHD (100% vs 73.4%, P=0.043),using ATG or basiliximab in conditioning regimens (88.0% vs 74.2%, P=0.049),and more earlier in patients using ATG or basiliximab or with aGVHD (median:28d vs 29d,P=0.007;33d vs 46d, P=0.000).All recipients were routinely adopted ganciclovir as prophylaxis for CMV infection, and single-therapy with ganciclovir (GCV) or foscarnet were performed on 63 patients and 6 patients for preemptive therapy, respectively. The total success efficacy was 87.8% and aGVHD,maximum load, positive times of CMV-DNA detection and therapy duration were in association with the efficacy.RQ-PCR detecting for 28S-ITS2 target sequence was positive in 15 Aspergillus and negative in 24 Candida and 10 bacteria, which confirmed that the RQ-PCR for Aspergillus detection was reliable and stable.The sensitivity and specificity were 93.75% and 100% in detection of strains.The DNA copies tested by RQ-PCR from whole blood were significantly highter than that from plasma(P=0.000).The result is informed as negative, suspected positive and positive when the Aspergillus-DNA detected by RQ-PCR is<10 copies/μl,10-20 copies/μl,=20 copies/μl. And two suspected positive was considered positive. Based on the cut-off value of≧20 copies/μ1,the sensitivity, specificity, PPV and NPV for diagnosis of IA by 28S-ITS2 RQ-PCR was 90%,68%,77% and 85%,respectively. The positive of RQ-PCR was ealier than Aspergillus specific appearance in lung CT and culture by a median time of 6 days,11 days, respectively.Conclusions:Plasma RQ-PCR assay is a very useful method and to be applicable to be a suitable guidance for management of CMV infection in HSCT, which has made considerable clinical worth for early diagnosis and in time therapy of CMV, and the incidence of CMV pneumonia was decline resulting in better prognosis for patients. Female recipients, with aGVHD, using ATG or basiliximab in conditioning regimens, maximum load, positive times of CMV-DNA detection and therapy duration were associated with the incidence of CMV infection and therapy efficacy.Whole blood RQ-PCR assay is a very useful method and to be applicable to diagnose IA in patients with haematological malignancies, with better sensitivity, specificity and value of early diagnosis.