| BackgroundDuring the past 4 decades,with an increased number of immunocompromised hosts,Trichosporon species have emerged as important human pathogens.Invasive trichosporonosis(IT)is an emerging fatal opportunistic infection.Due to delayed diagnosis and the lack of an optimal treatment strategy,especially for the Trichosporon fungemia(TF),the mortality remains high(53%-76%).Invasive trichosporonosis can involve most organs of the human body,Trichosporon fungemia,including catheter-related fungemia,represents the main type of this opportunistic infection,which accounts for 58.8%–74.7% of infections.Trichosporon spp.currently represents the second most common yeast fungemia in patients with HMDs.Although European guideline(2014)recommend voriconazole or fluconazole for the treatment of IT;due to the low incidence,these recommendations are mainly based on low-level evidence obtained from in vitro susceptibility tests,animal models,and case reports/case series rather than clinical data from multi-centers randomized control trials.Information on the use of antifungals for TF and other related clinical issues,such as the management of vascular catheters and neutrophil recovery,is still lack.In recent years,in vitro susceptibility tests on clinical isolates of Trichosporon spp.have found that it has decreased susceptibility to fluconazole.Currently,only voriconazole and fluconazole are recommended for clinical application in the treatment of TF,once the resistance to fluconazole occurs and voriconazole is not available,it will inevitably affect the clinical prognosis of patients.Thus,selecting the comprehensive and appropriate strategy of managing TF remains difficult.ObjectiveDue to lake of comprehensive analysis or case-control study with large patient sample,no related data has been reported to guide our understanding of the epidemiology,the prognostic factors and therapeutic aspects of TF;in particular,catheter management in patients with TF is currently lacking in definite clinical recommendations.Therefore,to improve our knowledge of this uncommon infection,we reviewed the English-language literature of TF cases that have been reported over the past 4 decades and electronic medical record system in our hospital,helping us to guide the evaluation of clinical prognosis and the making the treatment plan.Secondly,in view of catheter-related TF(common type of this infection,belong to the biofilm-related infection),we evaluated the in vitro activity of the antifungal combinations against clinical isolatesof T.asahii in biofilm and planktonic forms,to guide the choice of antifungal lock therapy for the catheter-related TF.Finally,by the in vitro and in vivo induction method and clinical case analysis,we explore the effect of calcineurin inhibitor on the acquired fluconazole resistance of T.asahii.Based on the above clinical and basic research results,it provides suggestions and basis for the first-line doctors to develop a more comprehensive clinical treatment plan for TF.Methods and ResultsPart One Clinical Study on the Outcome Analysis and Therapeutic Strategy of Trichosporon FungemiaBased on the electronic medical record data system of our hospital,and all cases in English literature related to TF,which were included in PubMed database from 1975 to 2014,a total of 185 cases were included in the study.The important clinical information of each patient was collected,and the correlation between prognostic factors/ clinical treatment regimens and patient outcomes was analyzed.The the number of reported cases generally increased during this period.Cases were reported in 4 continents and had been changed from mainly in North America and Europe to Asia and South America.The mean age of the patients was 47 years.Most cases were male.Hematological diseases(with acute leukemia 77.36% in HMDs)were the most common underlying diseases or conditions.Most patients had a history of neutropenia,chemotherapy,antimicrobial use,prophylactic/empirical antifungal therapy,or central venous catheter(CVC)use before or at the onset(present on admission [POA])of TF.59.46% cases involved single invasive tissue infection or disseminated infections.The most commonly used method to identify Trichosporon species was combined the morphological method with the biochemical test,and molecular method was used more in the resent 10-year period.The most commonly isolated Trichosporon species was Trichosporon asahii.A total of 109 patients(58.92%)experienced a negative outcome(death/worsening),and 76 patients(41.08%)experienced a positive outcome(cure/improvement).The prognosis was not improved obviously in last 10-year period compared with other periods.In a univariate analysis,a significantly greater proportion of patients with negative outcome had a history of using antimicrobials,prophylactic/empirical antifungal therapy,CVC use or bacterial bloodstream co-infection POA of TF.In addition,a significantly greater proportion of patients with a positive outcome had fungemia without invasive tissue infection than with invasive tissue infection.Among the patients experiencing a positive outcome,a significantly greater proportion followed neutrophil recovery or catheter removal.Amphotericin B/liposomal amphotericin B(AmB)were the most frequently used monotherapies in the early stage.The therapy with a single triazole has increased to become the most frequently used drug during 2005-2014.A significantly greater proportion of patients experiencing a negative outcome received the antifungal regimen using AmB.However,a significantly greater proportion of patients experiencing a positive outcome received an antifungal regimen using voriconazole or a regimen using an AmB-triazole combination.Among the 4 commonly used antifungal compounds,significant differences in positive outcome rate were found between regimens using voriconazole and AmB,between fluconazole and AmB,and between the combination of AmB-triazole and AmB;differences in outcome between other antifungal treatments were not statistically significant.Part Two In Vitro Activities of Antifungal Combinations Against Biofilms and Planktonic Forms of Trichosporon asahii Clinical IsolatesWe evaluated the in vitro activity of the combinations of three common antifungals(voriconazole,caspofungin and amphotericin B)against 16 clinical isolates of T.asahii in biofilm and planktonic forms by a broth microdilution checkerboard method.Trichosporon biofilms were prepared according to the 96-well plate-based method.Under planktonic conditions,the amphotericin B-caspofungin combination showed the highest percentage of synergistic effects(81.25%).Under biofilm conditions,the voriconazole-amphotericin B combination showed the highest percentage of synergistic effects(87.5%),and the SMIC90 for these two drugs obviously decreased from ?1,024 μg/mL to 64 μg/mL for voriconazole and from 1,024 μg/mL to 32 μg/mL for amphotericin B,respectively.Part Three Study of Function of Calcineurin in Acquired Fluconazole Resistance of Trichosporon asahiiThe standard strain T.asahii CBS2479 was induced by gradient concentration of fluconazole combined with cyclosporin A,gradient concentration of fluconazole and cyclosporin A respectively;meanwhile,a blank control was set.The MIC value of fluconazole was detected during the induction stage,and in vitro susceptibility of the common triazole antifungals was detected by the end point of induction by a broth microdilution checkerboard method(CLSI M27-A3).After the induction,the first Group obtained fluconazole-resistant strains,the MIC of fluconazole increased by 8 to 16 times,the MIC of itraconazole increased 4 to 8 times,and the MIC of voriconazole increased 2 times.The second Group also obtained fluconazole-resistant strains,the MIC of fluconazole increased 32 times,the MIC of itraconazole increased 8 to 16 times,and the MIC of voriconazole increased 2 to 4 times.After that,fluconazole-resistant strains cultured on non-drug medium for 20 generations,and the stability of drug resistance phenotype was observed.After culturing on non-drug medium,the fluconazole resistance in the first Group was unstable and the MIC of fluconazole,itraconazole and voriconazole was recovered or near the initial state;while the resistance of second Group to fluconazole,itraconazole and voriconazole was stable.Cyclophosphamide was used to conduct periodic immunosuppression in experimental mice,and CBS2479 were prepared for intravenous injection of rat tail,and a model of persistent infection under the intervention of fluconazole was constructed.The inoculated mice were were randomly divided into 4 groups,and intraperitoneal drug injection was performed daily: fluconazole combined with cyclosporine A,fluconazole,cyclosporine A and blank control.The survival of the mice was recorded.The hearts,lungs,liver and kidneys of the dead and non-dead mice were dissected for culture,routine histopathological detection(HE and bright green stainings)at the end point.The isolated strains were tested for fluconazole susceptibility.After the in vivo intervention of fluconazole,the MIC value of fluconazole in 90% isolated strains of fluconazole group was increased to different degrees(by 2-4 times),while the MIC value of fluconazole-cyclosporin A combined group was only increased by 2 times in 60% isolated strains.The mice were inoculated in a randomly assigned immunosuppressed mouse with MIC increased strains induced in vivo one by one.During the inoculation,the strains were isolated from the tissue again and tested with fluconazole.The MIC value of combined group was not stable,and eventually the MIC value of fluconazole was restored to the initial state after in vivo passage,while the MIC value of fluconazole drug group was basically stable.The source of cases was the same as that of the first part of this study,and a summary analysis was conducted on the patients who were clearly diagnosed with hematological diseases and developed TF after receiving bone marrow transplantation.TF occurred in 19 patients with haematological diseases during the course of anti-rejection therapy after bone marrow transplantation,and all the 14 patients who did not receive fluconazole or voriconazole died.Of the 5 patients treated with fluconazole or voriconazole,4 were treated with calcineurin inhibitors(cyclosporine A3 and tacrolimus 1)in the anti-rejection regimen,and all 4 were eventually cured.Patients who were treated with cyclophosphamide combined with fludarabine died.ConclusionsPart OneThe patient 1 continues to receive high doses of glucocorticoid therapy,and there are other risk factors that lead to secondary TF.Due to receiving the empirical antifungal treatment drug(caspofungin),and the application of voriconazole was not timely.At the same time,the patient was considered to be catheter-related TF,and no targeted measures were given for catheter management,resulting in poor prognosis of the patient.This suggests that there are still a lot of problems to be solved in the current clinical treatment of TF,including: evaluation of patients’ prognosis,selection of drug therapy and specific measures of catheter management.Due to the presence of a special genetic background,the patient 2 received fluconazole and itraconazole treatment not regularly over the next 14 years,which eventually made the isolate decreased susceptibility to azole drugs,even appeared the fluconazole resistance.This indicates T.asahii is also can appear the risk of azole drug resistance.For long-term use of azole drug treatment of trichosporonosis,we need to develop a more comprehensive and scientific treatment strategies,so as to avoid or reduce the occurrence of induced resistance.Since the first report,the number of cases of TF has been increasing year by year.The reported cases spread gradually from North America and Europe to Asia and South America.With the wide application of triazole antifungals,the overall prognosis of the patients was not improved obviously.Most cases of TF occur in patients with certain underlying diseases,especially patients with hematologic malignancies or neutropenia who are treated with broad-spectrum antibiotics or central venous catheterization.Neutropenia,central venous catheterization,antibiotic use,chemotherapy,and prophylactic/ empirical antifungal therapy are the main risk factors for the development of TF.Molecular methods have gradually been applied in clinical application for identification.T.asahii is the most common clinical isolates.TF patients often have a poor prognosis when antibiotics,prophylactic/empirical antifungal therapy,central venous catheterization,bacterial bacteremia or invasive tissue/organ infection are combined.Voriconazole was expected to improve patient outcome.If voriconazole cannot be obtained clinically,treatment with fluconazole-amphotericin B combination can be considered,and fluconazole monotherapy can be used after disease control.For the patients with neutropenia,if the clinical condition permits,adjust the treatment regimen of immunosuppressive agents,and give granulocyte colony stimulating factor or granulocyte-macrophage colony stimulating factor,so as to promote the recovery of peripheral blood granulocyte level in patients.For the patients with catheter-related TF,central venous catheterization should be removed as soon as possible if the condition permits.Part Two Based the first part of the clinical study,Trichosporon fungemia was mainly catheter-related bloodstream infections(CR-BSIs),which is biofilm-related infections.We also suggested for Trichosporon CR-BSIs to improve patient’s prognosis when feasible,according to the result of first part.However,if the patient’s existing catheter cannot be removed,high concentration of antifungal lock therapy need to be considered.In this part of the study,it was confirmed that all single antifungals were not strong enough to inhibit the T.asahii biofilm(the SMICs of single antifungals have been demonstrated to be higher than concentrations for antifungal lock therapy),so do not be suitable for use as a lock solution in antifungal lock therapy for T.asahii CR-BSIs.However,after the combination,both SMIC levels of the voriconazole and amphotericin B were significantly decreased(reaching the recommended concentrations of lock solution).Therefore,this combination is a potential option as a lock solution for antifungal lock therapy of catheter-related TF,especially for patients who are catheter dependent or have risks associated with catheter removal(like case 1 in our hospital).Part Three Based the first part of the clinical study,fluconazole is the most commonly used triazole drug.This part’s in vitro and in vivo study found that calcineurin plays a certain role in the acquired resistance to fluconazole by T.asahii.The occurrence of acquired resistance can be partially blocked by the application of calcineurin inhibitor,and even if resistance occurs,the phenotype can be converted after the release of drug stress.Analysis of a series of relevant cases found that for some specific patients had the needs to use the calcineurin inhibitors.So,for the patients receiving anti-rejection treatment after bone marrow transplantation,calcineurin inhibitors can be considered for combination use.Or for other patients who need long-term treatment with fluconazole(like case 2 in our hospital),further screening of the fungi-specific calcineurin inhibitors for clinical application is required,so as to reduce the clinical fluconazole resistance in long-term application.The summaryAs a doctoral student for clinical study,I started from clinical problems and aimed to solve tough clinical problems.Based on the in-depth analysis of the disease course of the case 1 in our hospital,it aims at the deficiencies of the existing medical guideline(2014 ESCMID and ECMM joint clinical guidelines for the diagnosis and management of rare invasive yeast infections is the only one involving the treatment of trichosporonosis in the world.There are 15 pages in the text,but only 2 pages are involved in trichosporonosis).A systematic retrospective case-control study and related basic research were conducted for the selected the important clinical problems which were not be solved in the TF treatment process.At the same time,according to case 2,acquired drug resistance appear after a long time period of fluconazole treatment t(first 14 years follow-up patient),and the global trend of clinical fluconazole resistance,in vitro and in vivo experiments and clinical case series analysis were done,to explore clinical value of calcineurin inhibitor in blocking fluconazole acquired resistance of T.asahii.Based on the in-depth analysis of the medical records of 185 cases of TF reported worldwide over a 40-year period,it was found that although the disease was rare since it was first reported in 1975,the number of reported cases in the world gradually increased from North America and Europe to Asia and South America.It gradually extends from adults to newborns,children and the elderly.T.asahii is the most common clinical isolate of TF,and the prognosis of the patients has not improved significantly in the past 40 years.Blood system diseases are the most common basic diseases,but the proportion of other basic diseases is increasing.Risk factors include: neutropenia,chemotherapy,central venous catheterization,use of antibacterial agents or a history of prophylactic/ empirical antifungal therapy antifungal therapy,and a growing variety of patient conditions.Most patients are associated with a single deep tissue or disseminated infection,most commonly involving the lungs,skin,liver,kidneys and spleen.Based on the results of this study,a relatively comprehensive evaluation of the prognosis of clinical patients can be conducted: if patients have a history of using antibacterial drugs,prophylactic/ empirical antifungal therapy,a history of bacterial bacteremia or central venous catheterization,and the strains have not been identified,the clinical prognosis is generally poor.The clinical prognosis is better if the patient is simply fungemia and is not associated with other invasive tissue or organ infections(not covered by current guideline).Suggestions for the formulation of a comprehensive and comprehensive treatment plan for patients with TF are proposed.1.Voriconazole is the preferred antifungal treatment for TF.If voriconazole cannot be obtained clinically,the treatment regimen of fluconazole combined with amphotericin B can be considered not covered by current guideline),and fluconazole monotherapy can be used after disease control.2.Patients with hyponeutropenia: if the clinical condition permits,adjust the treatment regimen of immunosuppressive agents,and give granulocyte colony stimulating factor or granulocyte-macrophage colony stimulating factor,so as to promote the recovery of peripheral blood granulocyte level in patients(not covered by current guideline).3.Patients with catheter-related TF: central venous catheterization should be removed as soon as possible if the condition permits.If the condition does not permit,it is recommended to consider the combination of voriconazole and amphotericin B for catheter lock therapy(not covered by current guideline).The use of calcineurin inhibitors combined with fluconazole is recommended for those infected patients who need to receive anti-rejection treatment after bone marrow transplantation.For other patients needed to treat with long-term fluconazole,we need further screening for fungi-specific calcineurin inhibitors in clinical application,the combined clinical strategies may reduce long-term fluconazole usage caused T.asahii acquired drug resistance of,but need further clinical data to support. |
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