[Quantitative real-time polymerase chain reaction for the diagnosis of invasive fungal disease].

OBJECTIVE: To establish the diagnostic assays of quantitative real-time polymerase chain reaction (PCR) for the diagnosis of invasive fungal disease and evaluate their accuracy in clinical samples. METHODS: Three assays have been developed for the diagnosis of clinically prevalent fungi, Aspergillus spp. and Candida spp.. Their analytical sensitivity and specificity were evaluated. Twenty-one serum samples from invasive fungal disease patients and non-invasive fungal disease patients were analyzed by the diagnostic assays and the accuracy was evaluated. RESULTS: Three assays were managed to run at the same condition. The universal fungi assay was able to detect, but not differentiate between most of the pathogenic fungi, including A. fumigatus, A. flavus, A. niger, C. albicans, C. parapsilosis, C. tropicalis, C.kruseii, C.glabrata, Cryptococcus neoformans, Rhizomucor variabilis, Rhizopus arrhizus and Penicillosis marneffei. The assays of Aspergillus spp. and Candida spp. were able to detect, but not differentiate between A. fumigatus, A. flavus, A. niger and C. albicans, C. parapsilosis, C. tropicalis. The detection limits of assays were 4 pg of A. fumigatus gDNA, 2 copies of A. fumigatus and 2 copies of C. albicans respectively. The results of quantitative real-time PCR matched well with each other and were identical to the classical procedures in all patients. Among the 11 patients with invasive fungal disease, the pathogens were identified down to a genus level in 2 invasive aspergillosis patients and 2 invasive candidiasis patients. And the other 7 patients could be diagnosed with invasive fungal disease by the universal fungi assay. All the 10 serum samples from non-invasive fungal disease patients were revealed as negative. CONCLUSION: The universal fungi assay is helpful in screening while the assays of Aspergillus spp. and the Candida spp. may identify the pathogens down to a genus level.

[Utility of galactomannan enzyme immunoassay and (1,3)beta-D-glucan assay in invasive fungal infection].

OBJECTIVE: To evaluate the performance of the galactomannan enzyme immunoassay (GM test) and (1,3)beta-D-glucan assay (G test) for the diagnosis of invasive fungal infection (IFI). METHODS: A retrospective study was performed in 115 hospitalized patients at Peking University First Hospital who were at risk of IFI. Patients were diagnosed as IFI according to revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated at different cutoff values for two assays respectively. Two tests were combined to evaluate the changes of sensitivity, specificity, PPV and NPV. RESULTS: The best sensitivity (54.5%, 63.6%) and specificity (77.9%, 69.2%) were obtained with the cutoff values of 0.5 and 20 x 10(3) pg/L in GM test and G test respectively. The PPV were 20.7% and 17.9%, and the NPV were 94.2% and 94.7% respectively. The sensitivity increased to 90.9% and the specificity was 52.9% after a combined utility of two tests. CONCLUSION: The GM test and G tests are both useful in diagnosis of IFI with the cutoff values of 0.5 and 20 x 10(3) pg/L. A better sensitivity is acquired if there is a combined utility of two tests.

Different expression of dectin-1 and Toll-like receptor 2 in the lungs of different immune status mice infected with Aspergillus fumigatus.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) is a severe and frequently fatal disease in patients receiving treatment with immunosuppressive agents such as cyclophosphamide. Aspergillus fumigatus (A. fumigatus) now is a leading cause of IPA. Dectin-1 and Toll-like receptor 2 (TLR2) are important pattern recognition receptors involved in immune responses to A. fumigatus in vitro. However, the expression of the two receptors during the infection of A. fumigatus in vivo is not completely understood. The effects of cyclophosphamide treatment on the expression of the receptors need to be further studied. METHODS: We established different immune status in mice models with or without A. fumigatus infection. On days 1, 3 and 5 post inoculation, pulmonary tissues from mice of the different groups were harvested. Dectin-1 and TLR2 mRNA expression in the lungs of the mice were investigated by real-time PCR. The pulmonary fungal burden in the mice with A. fumigatus infection was also evaluated. RESULTS: In the immunocompetent mice, three days after A. fumigatus inoculation, dectin-1 and TLR2 expression increased markedly compared with the normal control group. Cyclophosphamide inhibited the clearance of pathogens and the expression of dectin-1 with or without A. fumigatus infection in the lungs as well. There was no statistical difference in TLR2 expression between the different immune status groups. CONCLUSIONS: Our results suggest that in vivo, dectin-1 and TLR2 are activated during the experimental period which would provide a broad range of possibilities for a specific and effective inflammatory response to kill A. fumigatus. Inhibition of dectin-1 expression may be one of the mechanisms of cyclophosphamide in the development of IPA.