| Chapter 1: Global Case Characteristics of Pathogenic Fonsecaea spp.Aims: To study and elucidate the epidemiological and clinical characteristics,potential susceptibility factors of patients with pathogenic Fonsecaea spp.to inform the clinical management of DFD caused by Fonsecaea spp.Methods: DFD cases reported in different languages were collected by retrieval,Continuous variables(age and time,etc.)were expressed as mean ± standard deviation;categorical variables(including sex,etc.)were expressed as counts and percentages and analyzed by unpaired two-sample t-test with uneven variance,and Fisher’s exact probability method was used to compare the study variables between the two groups.Results: It was found that the number of cases of infection with four pathogenic bacteria(Fonsecaea pedrosoi,F.monophora,F.nubica,F.pugnacius)increased over time during 2000-2021.cases of Fonsecaea spp.were highly prevalent in southeastern China,the coastal region of southern China,and the middle and lower reaches of the Yangtze and Yellow rivers.The mean age of patients with Fonsecaea spp.infection was 54.07±12.57,male:female=3:1.Skin infections caused by Fonsecaea spp.were mostly in the exposed and vulnerable upper and lower extremities.The frequency of infection sites in male patients was lower limbs(thighs,calves)> upper limbs(forearms,upper arms)> feet > hands > ankles > face > buttocks > brain > ears > eyes;in female patients,lower limbs > upper limbs > hands > face > ankles > feet > brain >eyes > buttocks.Female patients had a clear history of no trauma more than a history of trauma.For the identification of Fonsecaea spp.at the species level,ITS sequencing is currently widely used.Conclusions: In recent years,the cases of Fonsecaea spp.(four pathogenic strains)infections with isolated pathogenic strains have shown a gradual increase.China has become one of the regions with the highest number of Fonsecaea spp..The clinical manifestations of Fonsecaea spp.are diverse,and accurate diagnosis of Fonsecaea spp.is the key to improve treatment.For the identification of Fonsecaea spp.at the species level,ITS sequencing is currently widely used.Chapter 2: In vitro antifungal drug susceptibility test of Fonsecaea spp.Aims: To study the susceptibility characteristics of Fonsecaea spp.strains to existing antifungal drugs by in vitro drug sensitivity testing and to provide scientific reference for rational clinical use of drugs.Methods: Fonsecaea spp.isolates were subjected to in vitro susceptibility testing to 10 clinical antifungal drugs using the broth microdilution method(CLSI M38-A2 method)recommended by the American Committee for Clinical Laboratory Standardization for filamentous fungi,and resistant strains were screened with a view to discovering the drug resistance characteristics of Fonsecaea spp.Results: Among the common antifungal agents,Fonsecaea spp.showed the highest antifungal activity against posaconazole,esaconazole,and voriconazole,while amphotericin B,fluconazole,and echinocandins showed lower antibacterial activity against Fonsecaea spp.The three common pathogenic species of Fonsecaea spp.were ranked in order of susceptibility to the drugs and clinical treatment efficiency Fonsecaea monophora,Fonsecaea nubica,and Fonsecaea pedrosoi in descending order.The mean MIC50 values of in vitro sensitization results: terbinafine 7.47 ug/ml;amphotericin B 5.84 ug/ml;5-fluorocytosine 5.96 ug/ml;posaconazole(0.331 ug/ml)> esaconazole(0.341 ug/ml)> voriconazole(0.348 ug/ml)> itraconazole(6.717 ug/ml)> Fluconazole 7.429 ug/ml;Anidulafungin(5.596 ug/ml)> Caspofungin(6.281 ug/ml)> Micafungin(7.395 ug/ml).Conclusions: The sensitivity to drugs and the efficiency of clinical treatment were ranked in descending order: Fonsecaea monophora,Fonsecaea nubica,Fonsecaea pedrosoi.For the administration of drugs against Fonsecaea spp.preference was given to azoles(posaconazole,esaconazole,voriconazole,etc.),followed by terbinafine,itraconazole,caspofungin,and amphotericin B.There is a trend towards a decrease in the antibacterial activity of most antifungal drugs against Fonsecaea spp.Combination drug sensitivity studies are urgently needed but still not systematic and the progress of related studies is slow.It is worthy of vigilance and further monitoring by subsequent related studies.Chapter 3: Identification,molecular typing,and phylogenetic tree genetics of Fonsecaea spp.Aims: To clarify the population composition and intraspecific genetic diversity characteristics of Fonsecaea spp.through molecular identification and genetic diversity analysis of Fonsecaea spp.strains.At the same time,the strain of Fonsecaea spp.was isolated from mainland China to enrich the hypothesis of natural environmental origin of the pathogenic fungus.Methods: We collected Fonsecaea spp.31 strains for culture,identification and genotyping at the species level to reveal their population genetic characteristics;ITS,BT2,Cdc42,Lac,PKS1,ESP,FOMO,FOUN,FOPE and FOPU were sequenced by nucleic acid amplification technology.BLAST comparison with NCBI database for preliminary identification to species level.The sequences of these 10 single gene fragments were also compared for their superiority and inferiority in the identification of Fonsecaea spp.Analysis of the collected strain genetic data analysis,temporal phylogeny and ribosomal internal transcribed spacer(ITS)sequences were performed using MEGA v.11,sequences were compared using the Clustal W option,and neighbor joining trees were estimated using the Kimura 2 parameter model plus Gamma distribution correction(K2+G: 0.96)with 500 Bootstrap values.All temporal phylogenetic trees were built using the Rel Time-ML method.Results: Nine strains of Fonsecaea pedrosoi(29.03%),18 strains of F.monophora(58.06%),and 6 strains of F.nubica(19.35%)were collected.ITS sequences classified Fonsecaea spp.to species level,41.9% as Fonsecaea pedrosi,35.4% as Fonsecaea monophora,16.1% as Fonsecaea monophora/Fonsecaea pedros,and 3.2% as Fonsecaea pedrosi/Fonsecaea nubica,and BT2 identified all of Fonsecaea spp.as Fonsecaea monophora.Cdc42 identified 74.2% of Fonsecaea spp.as Fonsecaea monophora vs.25.8% as Fonsecaea nubica.LACL identified 93.5% of Fonsecaea spp.as Fonsecaea monophora vs.6.5% as Fonsecaea nubica.PKS1 identified Fonsecaea spp.as 100% as Fonsecaea pedrosi.ESP identified Fonsecaea spp.as 58.0% as Fonsecaea monophora,6.5% as Fonsecaea nubica,16.1% as Cladophialophora immunda,19.4% non-specific amplification.FOMO identified Fonsecaea spp.as 83.8% as Fonsecaea monophora with 12.9% as Fonsecaea nubica and 3.2% as Fonsecaea pedrosi/ Fonsecaea nubica.FOUN identified 64.5% as Fonsecaea spp.as Fonsecaea monophora with 35.5% as Fonsecaea nubica.FOPU identified 29.0% as Fonsecaea spp.as Fonsecaea pedrosi,6.5% as Fonsecaea monophora and 6.5% as Fonsecaea pedrosi/ Fonsecaea nubica;FOPU identified 29.0% as Fonsecaea spp.6.5% Fonsecaea monophora,6.5% Fonsecaea nubica,41.9% Fonsecaea monophora/Fonsecaea pedrosi,and 16.1% non-specific amplification.the chronological order of strain differentiation of Fonsecaea spp.was probably F.brasiliensis(about 38.27 Ma),F.multimorphosa(about 21.30 Ma),F.minima(around 20.01 Ma),F.erecta(about 19.11 Ma),Fonsecaea pedrosoi(~17.41 Ma)F.pugnacious(~8.23Ma),F.nubica(~1.64 Ma,),F.monophora(~ 0.08 Ma).three globally prevalent pathogenic strains of Fonsecaea pedrosoi,F.monophora,and F.nubica accounted for the largest number of strains.Fonsecaea spp.strains may be derived from environmental strains and are closely related to environmental nonpathogenic strains.The addition of evolutionary time indicators in the study of constructing the taxonomic system allows the new taxonomic system to reflect the temporal dimension in the evolution of taxa,which can reflect the evolutionary history of taxa more comprehensively and promote the scientificity and stability of the taxonomic system.Conclusions: ITS sequencing remains the main reference standard for the identification of Fonsecaea spp.The evolutionary order of differentiation of Fonsecaea spp.strains may be F.brasiliensis,F.multimorphosa,F.minima,F.erecta,F.pedrosoi,F.pugnacius,F.nubica,F.monophora.Fonsecaea spp.strains may be derived from environmental strains and are closely related to environmental nonpathogenic strains.Chapter 4: Global transmission mode and transmission pattern hypothesis of Fonsecaea spp strainsAims: To analyze more comprehensively the population structure,genetic evolution,dispersal pathways,transmission routes and to reveal the population genetics of Fonsecaea spp.Methods: Dna SP v.6(http://www.ub.edu/dnasp/)was used to determine DNA polymorphisms.All haplotype network models were constructed using Pop ART 1.7(http://popart.otago.ac.nz/index.shtml).Comparisons of categorical variables were performed using chi-square tests.Differences between groups were considered statistically significant if the P value was ≤0.05.Divergence times were measured in millions of years ago(Ma).Results: Fonsecaea pedrosoi,F.monophora,and F.nubica are the three globally prevalent pathogenic strains that account for the largest number of strains,and each species is closely related to other countries.In Fonsecaea pedrosoi,a total of 9 haploids were produced.Fonsecaea pedrosoi cluster type(Hap1)is prevalent in Brazil,Colombia,Puerto Rico,Venezuela,Argentina,Uruguay,Mexico,Cuba,Mozambique,Congo,Libya,Netherlands,USA,Costa Rica,Australia,Vietnam,Thailand,China and Japan.Fonsecaea pedrosoi in Brazil has the richest and most diverse haploid diversity.In Fonsecaea monophora,19 haplotypes were produced.subpopulation of Fonsecaea monophora(Hap 1)is endemic in Congo,USA,Guinea,Netherlands,Mexico,Cuba,Costa Rica,Brazil,Malasia,India,Thailand,China(Nanjing,Shanghai,Guangzhou,Taiwan),and Korea.Fonsecaea monophora in China has the richest and most diverse haploid diversity.In Fonsecaea nubica,a total of 15 haploid subtypes were produced.Fonsecaea nubica population in Madagascar region had the richest and most diverse haplotype diversity.Conclusions:In this study,a systematic analysis of global Fonsecaea spp.cases with collectible strains was conducted.This study found that the global epidemic of Fonsecaea spp.may originate from marine fungi and has three major centers of transmission origin.Fonsecaea pedrosoi variant originated in(the northern part of the Pacific Ocean,with the northern part of the Atlantic Ocean near the North Pacific Ocean near Costa Rica,Colombia,Venezuela,Brazil,Mexico,and other waters where typhoons are frequent in the North Atlantic Ocean).Fonsecaea monophora variant originated from the area near the South China Sea.Fonsecaea nubica variant originating near Madagascar.Fonsecaea pugnacius originated in the Atlantic Ocean near Brazil.The mode and pattern of transmission is driven by frequent marine typhoons in summer and autumn.We propose the hypothesis that non-pathogenic environmental strains(Fonsecaea brasiliensis,F.multimorphosa,F.minima and F.erecta)evolve into pathogenic strains(Fonsecaea pedrosoi,F.pugnacius,F.nubica and F.monophora)and spread globally through global tropical cyclones and oceanic current pathways.F.multimorphosa is potentially infectious to humans and warrants vigilance and further monitoring in subsequent related studies.The results of this study deepen the understanding of the modes and patterns of transmission of Fonsecaea spp.and will greatly contribute to the prevention,treatment and therapy of Fonsecaea spp.infections,and provide insights and references for researchers in the field of fungal research,especially in other pathogenic and environmental fungi. |
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