| Objective:To understand the clinical epidemiological characteristics of filamentous fungi in China,and explore the application efficiency of molecular sequencing technology and MALDI-TOF mass spectrometry in the identification and diagnosis of clinical filamentous fungi.Methods:Collect 566 filamentous fungi from 35 hospitals in 8 provinces collected by China Hospital Infection invasive fungi monitoring project(CHIF-NET)from 2018 to 2019.Based on the comprehensive results of mass spectrometry identification and molecular identification,clinical epidemiological analysis and discussion were carried out,and yeastone in vitro drug sensitivity test was carried out on Aspergillus in ear samples.566 filamentous fungi were used to evaluate the application of two protein extraction methods:conventional method and silicon bead method in the identification of filamentous fungi by Vitek MS mass spectrometry.178 representative filamentous fungi were selected to evaluate the performance of ITS,β-tubulin and calmodulin sequences in the molecular identification of filamentous fungi.The typical strains found in this study were observed morphologically.The effectiveness of 178 original strains of filamentous fungi was evaluated.Finally,the identification methods of various filamentous fungi were comprehensively analyzed.Results:566 strains of filamentous fungi were used to evaluate the application of two protein extraction methods of conventional method and silicon bead method in the identification of filamentous fungi by Vitek MS mass spectrometry.Only 15 strains(2.7%)had no identification results using the two protein extraction methods.551 strains(97.3%)obtained comprehensive identification results through cross integration of one or two protein extraction methods,and species or complex or section could be identified,of which 526 strains(95.5%)could be identified by conventional method,512 strains(92.9%)were identified by silicon bead method,and 487 strains(88.4%)were identified by the two methods.517 strains(93.8%)of Aspergillus,18 strains(3.3%)of Fusarium and 16 strains(2.9%)of other filamentous fungi were isolated.In Aspergillus,the identification rates of the two methods were higher than 93.8%,and the consistency was 89.9%.In Fusarium and other filamentous fungi,the consistency of the two methods was 61.1%and 68.8%,respectively.The average identification rate of Fusarium by conventional method was 94.4%,which was better than silicon bead method;The average identification rate of other filamentous fungi by silicon bead method was 93.8%,which was better than the conventional method.For 178 strains of filamentous fungi,the identification efficiency of VITEK MS mass spectrometry was:43 strains(24.2%)identified species,117 strains(65.7%)identified complex or section,3 strains(1.7%)identified incorrectly,and 15 strains(8.4%)had no identification results.Aspergillus:the identification efficiency of 141 strains of Aspergillus was:29 strains(20.6%)were identified as species,109 strains(77.3%)were identified as complex or section,3 strains(2.1%)had no identification results,and no strains were identified incorrectly.Fusarium:the identification efficiency of 17 strains of Fusarium:7 strains(41.2%)identified species,8 strains(47.1%)identified complex or section,2 strains(11.8%)had no identification results and no strains were identified incorrectly.The identification efficiency of 20 other fungi was:7 strains(35.0%)were identified to species,7 strains(35.0%)had no identification results,and 3 strains(15%)were identified incorrectly.For 178 strains of filamentous fungi,ITS can identify filamentous fungi as genus or complex group,β-tubulin can identify Aspergillus,Penicillium and Talaromyces as complex or species,and calmodulin can identify Aspergillus,Penicillium and Talaromyces as species.Aspergillus:ITS can identify all strains to groups,and even Aspergillus fumigatus to species.β-tubulin can directly identify 5 of the 11 Aspergillus species to species,including Aspergillus fumigatus,Aspergillus terreus,Aspergillus tubingensis and Aspergillus luchuensis in the Aspergillus Section Nigri and Aspergillus nomius in the Aspergillus Section Flavi.Except Aspergillus flavus/Aspergillus oryzae,calmodulin can directly identify the remaining 10 of the 11 Aspergillus species to species.Fusarium:for Fusarium,ITS sequence alignment combined with phylogenetic tree analysis can identify species of Fusarium keratoplasticum,Fusarium falciforme and Fusarium chlamydosporum,and complex or section can be identified for three species of Fusarium:Fusarium verticillioides,Fusarium proliferatum and Fusarium solani.Other fungi:for Penicillium,ITS can identify all strains to genera,and even Penicillium georgiense to species.β-tubulin could not distinguish Penicillium chrysogenum from ITS relatives,and identified Penicillium oxalicum and Penicillium georgiense as species.calmodulin can directly identify three kinds of Penicillium to species.For Talaromyces marneffei and Talaromyces funiculosus,ITS can identify genera and β-tubulin and calmodulin can identify species.In addition,ITS can identify four species of Rhizopus,Scedosporium and Alternaria,while Trichoderma can only identify genera.Among 178 strains of filamentous fungi,the original identification results of the source unit of the strain were as follows:49 strains(27.5%)were identified as species,72 strains(40.4%)were identified as complex or section,30 strains(16.9%)were identified as genera,and 27 strains(15.2%)were identified incorrectly.Aspergillus:the identification efficiency of 141 strains of Aspergillus is:44 strains(31.2%)are identified as species,72 strains(51.1%)are identified as groups,and 116 strains(82.3%)can be identified as species or groups.Among the Aspergillus that can be identified as species,Aspergillus niger(91.7%)and Aspergillus fumigatus(87%)account for a relatively high proportion.10 strains(7.1%)were identified as genus,and 15 strains(10.6%)were identified incorrectly.From January 2018 to December 2019,more than 600 filamentous fungal strains were collected from 35 hospitals in 8 provinces in the North of China.A total of 566 strains were included in this study after eliminating incomplete information and non filamentous fungi,involving 10 genera and 24 species or complex or section.Among the 10 genera,Aspergillus spp.was the main one,520 strains accounted for 91.9%;46 strains of Non Aspergillus spp.(8.1%)involved 9 genera,including 20 strains of Fusarium(3.5%),5 strains of Penicillium(0.9%),5 strains of Scedosporium(0.9%),5 strains of Trichoderma(0.9%),4 strains of Talaromyces(0.7%)and 7 strains of other fungi(1.2%).Among the 24 strains,the top five in the total isolation rate are Aspergillus,from high to low:268 strains of Aspergillus fumigatus(47.3%),1 12 strains of Aspergillus niger complex(19.8%),78 strains of Aspergillus flavus(13.8%),36 strains of Aspergillus terreus complex(6.2%),23 strains of Aspergillus nidulans(4.1%),etc.Based on the analysis of basic diseases of patients with five sources of Aspergillus spp.,the top three diseases were respiratory diseases accounting for 62.6%(323/516),ear canal diseases accounting for 15.3%(79/516)and cardiovascular diseases accounting for 4.1%(21/516).The lower respiratory tract was the most common site for Aspergillus isolation.Sputum in the lower respiratory tract accounted for 75.4%(389/516)of all samples,followed by ear swabs accounting for 15.5%(80/516),alveolar lavage fluid accounting for 5.2%(27/516)and other types of samples accounting for 3.9%(20/516).The age range of patients with Aspergillus infection was 4-94 years,with an average of 61.4 years and a median of 64 years.Aspergillus fumigatus,Aspergillus flavus,Aspergillus terreus and Aspergillus nidulans were most distributed in the elderly group(61～80 years old),accounting for 45.7%to 60.9%;For Aspergillus niger complex,the distribution of middle-aged group(41～60 years old)was the most,accounting for 41.1%.CONCLUSION:This study clarified the epidemiological trend of large samples of filamentous fungi isolated from clinical centers in the North of China,compared the application efficiency of mass spectrometry identification and molecular identification on common and rare clinical filamentous fungi in detail,observed the morphology of representative strains found in this study,and analyzed the current situation of filamentous fungi identification ability in many hospitals,It provides data support for the monitoring of clinical invasive fungal diseases and the improvement of fungal laboratory ability in China. |
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