| Objective:The incidence of invasive fungal infections(IFIs)has recently increased,and early and accurate diagnosis of IFIs is important for the rational selection of antifungal drugs with high efficacy.We developed a method for rapid and accurate clinical diagnosis of IFIs and provide a reference for personalized drug treatment.Methods:1.We designed and screened fungal internal transcribed spacer regions with universal primers and designed 8 TaqMan detection probes to establish a multi-channel real-time fluorescent PCR melting curve analysis(MCA)method.The sensitivity and specificity of this method were investigated using fungal strains and clinical isolates.2.We collected qualified sputum samples,and analyzed and compared the fungal detection rate of MCA technology and culture methods to evaluate the clinical application value of MCA technology in invasive pulmonary fungal infection(IPFI).Three pretreatment methods were designed to explore the effects of different pretreatment methods on MCA detection of sputum samples.3.Six clinically isolated Candida suspensions were prepared at different concentrations to prepare simulated Candidemia samples,which were detected and identified by MCA technology to evaluate the clinical application value of MCA technology in Candidemia.Results:1.The limit of detection and assay cut-off(melting temperature(Tm))for Candida albicans were 0.05 pg/?L and 66.50?;C.glabrata were 0.1 pg/?L and 66.25?;C.tropicalis were 0.1 pg/?L and 60.15?;C.krusei were 0.1 pg/?L and 72.15?;C.parapsilosis were 0.2 pg/?L and 63.10?;C.guilliermondii were 0.1 pg/?L and 61.84?;Cryptococcus neoformans were 0.1 pg/?L and 65.50?;Aspergillus flavus were 0.05 pg/?L and 71.50?;A.terreus,A.fumigatus and A.niger were 0.05 pg/?L and 76.80?.Analytical specificity was evaluated using 13 clinical pathogens including Streptococcus pneumoniae,Staphylococcus aureus,and Haemophilus influenzae,etc.No false-positive results were obtained for any of these samples.2.Among the sputum samples,the detection rates of total fungi,Candida albicans,non-Candida albicans,Aspergillus and mixed fungi by MCA technique were 58.5%,30.5%,7.8%,3.7%and 16.5%,respectively.In the culture method,the detection rates of total fungi,Candida albicans,non-Candida albicans,Aspergillus and mixed fungi were 20.7%,17.7%,1.2%,1.2%and 0.6%,respectively.In 59 sputum samples detected by MCA technology,the total detection rate of fungi in 4%NaOH pretreatment group was 50.8%,66.1%in 0.1%DTT group,and 62.7%in 1%trypsin group.3.Among the candidemia simulations.The limit detection concentration of simulated C.albicans sample was 44cfu/mL,C.glabrata was 73cfu/mL,C.tropicalis was 29cfu/mL,C.parapsilosis was 21cfu/mL,C krusei was 71cfu/mL,and C.guilliermondii was 53cfu/mL.Conclusions:1.The multi-channel real-time fluorescence PCR melting curve analysis displayed high sensitivity and specificity in detecting-various clinically invasive fungi.Furthermore,it simultaneously detected the genera Candida,Cryptococcus,and Aspergillus and identified Candida at the species level.2.Compared with the fungal culture method,MCA technology had higher sensitivity for detecting fungi in sputum samples,which was faster(about 3?4h)and more convenient.MCA technology could realize the early identification of fungal species.And the detection and identification of non-candida and mixed fungal infections,MCA technology also had certain advantages,which could provide a certain reference for the clinical drug treatment program of IPFI.Compared with 1%trypsin and 4%NaOH,the 0.1%DTT liquefaction method had certain significance for improving the sensitivity of MCA detection of fungi.3.MCA technology had a high sensitivity for detecting simulated IC,which limit detection concentration is 20-80cfu/mL.The identification results could be obtained within 4?5 h,which was fast,accurate and highly sensitive.MCA technology could provide a certain reference for the early diagnosis and medication of IC. |
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